Tabla espec. clave
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|Ca, H, M, R, Rb, F, Fe, Po||ELISA, IP, ICC, IF, IHC, WB||M||Purified||Monoclonal Antibody|
|Presentation||Purified Mouse monoclonal IgG1 in PBS, pH 7.4 with 0.1% sodium azide.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Maintain at 2-8ºC in undiluted aliquots for up to 6 months after date of receipt.|
|Material Size||200 µg|
Ficha datos de seguridad (MSDS)
Referencias bibliográficas | 594 Disponible | Ver todas las referencias
|Visión general referencias||Aplicación||Especie||Pub Med ID|
|A Cre-conditional MYCN-driven neuroblastoma mouse model as an improved tool for preclinical studies. |
Althoff, K; Beckers, A; Bell, E; Nortmeyer, M; Thor, T; Sprüssel, A; Lindner, S; De Preter, K; Florin, A; Heukamp, LC; Klein-Hitpass, L; Astrahantseff, K; Kumps, C; Speleman, F; Eggert, A; Westermann, F; Schramm, A; Schulte, JH
Oncogene 34 3357-68 2015
Neuroblastoma, a childhood cancer that originates from neural crest-derived cells, is the most common deadly solid tumor of infancy. Amplification of the MYCN oncogene, which occurs in approximately 20-25% of human neuroblastomas, is the most prominent genetic marker of high-stage disease. The availability of valid preclinical in vivo models is a prerequisite to develop novel targeted therapies. We here report on the generation of transgenic mice with Cre-conditional induction of MYCN in dopamine β-hydroxylase-expressing cells, termed LSL-MYCN;Dbh-iCre. These mice develop neuroblastic tumors with an incidence of greater than 75%, regardless of strain background. Molecular profiling of tumors revealed upregulation of the MYCN-dependent miR-17-92 cluster as well as expression of neuroblastoma marker genes, including tyrosine hydroxylase and the neural cell adhesion molecule 1. Gene set enrichment analyses demonstrated significant correlation with MYC-associated expression patterns. Array comparative genome hybridization showed that chromosomal aberrations in LSL-MYCN;Dbh-iCre tumors were syntenic to those observed in human neuroblastomas. Treatment of a cell line established from a tumor derived from a LSL-MYCN;Dbh-iCre mouse with JQ1 or MLN8237 reduced cell viability and demonstrated oncogene addiction to MYCN. Here we report establishment of the first Cre-conditional human MYCN-driven mouse model for neuroblastoma that closely recapitulates the human disease with respect to tumor localization, histology, marker expression and genomic make up. This mouse model is a valuable tool for further functional studies and to assess the effect of targeted therapies.
|Comparison of the metabolic activation of environmental carcinogens in mouse embryonic stem cells and mouse embryonic fibroblasts. |
Krais, AM; Mühlbauer, KR; Kucab, JE; Chinbuah, H; Cornelius, MG; Wei, QX; Hollstein, M; Phillips, DH; Arlt, VM; Schmeiser, HH
Toxicology in vitro : an international journal published in association with BIBRA 29 34-43 2015
We compared mouse embryonic stem (ES) cells and fibroblasts (MEFs) for their ability to metabolically activate the environmental carcinogens benzo[a]pyrene (BaP), 3-nitrobenzanthrone (3-NBA) and aristolochic acid I (AAI), measuring DNA adduct formation by (32)P-postlabelling and expression of xenobiotic-metabolism genes by quantitative real-time PCR. At 2 μM, BaP induced Cyp1a1 expression in MEFs to a much greater extent than in ES cells and formed 45 times more adducts. Nqo1 mRNA expression was increased by 3-NBA in both cell types but induction was higher in MEFs, as was adduct formation. For AAI, DNA binding was over 450 times higher in MEFs than in ES cells, although Nqo1 and Cyp1a1 transcriptional levels did not explain this difference. We found higher global methylation of DNA in ES cells than in MEFs, which suggests higher chromatin density and lower accessibility of the DNA to DNA damaging agents in ES cells. However, AAI treatment did not alter DNA methylation. Thus mouse ES cells and MEFs have the metabolic competence to activate a number of environmental carcinogens, but MEFs have lower global DNA methylation and higher metabolic capacity than mouse ES cells.
|Mitochondrial ROS Induces Cardiac Inflammation via a Pathway through mtDNA Damage in a Pneumonia-Related Sepsis Model. |
Yao, X; Carlson, D; Sun, Y; Ma, L; Wolf, SE; Minei, JP; Zang, QS
PloS one 10 e0139416 2015
We have previously shown that mitochondria-targeted vitamin E (Mito-Vit-E), a mtROS specific antioxidant, improves cardiac performance and attenuates inflammation in a pneumonia-related sepsis model. In this study, we applied the same approaches to decipher the signaling pathway(s) of mtROS-dependent cardiac inflammation after sepsis. Sepsis was induced in Sprague Dawley rats by intratracheal injection of S. pneumoniae. Mito-Vit-E, vitamin E or vehicle was administered 30 minutes later. In myocardium 24 hours post-inoculation, Mito-Vit-E, but not vitamin E, significantly protected mtDNA integrity and decreased mtDNA damage. Mito-Vit-E alleviated sepsis-induced reduction in mitochondria-localized DNA repair enzymes including DNA polymerase γ, AP endonuclease, 8-oxoguanine glycosylase, and uracil-DNA glycosylase. Mito-Vit-E dramatically improved metabolism and membrane integrity in mitochondria, suppressed leakage of mtDNA into the cytoplasm, inhibited up-regulation of Toll-like receptor 9 (TLR9) pathway factors MYD88 and RAGE, and limited RAGE interaction with its ligand TFAM in septic hearts. Mito-Vit-E also deactivated NF-κB and caspase 1, reduced expression of the essential inflammasome component ASC, and decreased inflammatory cytokine IL-1β. In vitro, both Mito-Vit-E and TLR9 inhibitor OND-I suppressed LPS-induced up-regulation in MYD88, RAGE, ASC, active caspase 1, and IL-1β in cardiomyocytes. Since free mtDNA escaped from damaged mitochondria function as a type of DAMPs to stimulate inflammation through TLR9, these data together suggest that sepsis-induced cardiac inflammation is mediated, at least partially, through mtDNA-TLR9-RAGE. At last, Mito-Vit-E reduced the circulation of myocardial injury marker troponin-I, diminished apoptosis and amended morphology in septic hearts, suggesting that mitochondria-targeted antioxidants are a potential cardioprotective approach for sepsis.
|CSB interacts with SNM1A and promotes DNA interstrand crosslink processing. |
Iyama, T; Lee, SY; Berquist, BR; Gileadi, O; Bohr, VA; Seidman, MM; McHugh, PJ; Wilson, DM
Nucleic acids research 43 247-58 2015
Cockayne syndrome (CS) is a premature aging disorder characterized by photosensitivity, impaired development and multisystem progressive degeneration, and consists of two strict complementation groups, A and B. Using a yeast two-hybrid approach, we identified the 5'-3' exonuclease SNM1A as one of four strong interacting partners of CSB. This direct interaction was confirmed using purified recombinant proteins-with CSB able to modulate the exonuclease activity of SNM1A on oligonucleotide substrates in vitro-and the two proteins were shown to exist in a common complex in human cell extracts. CSB and SNM1A were also found, using fluorescently tagged proteins in combination with confocal microscopy and laser microirradiation, to be recruited to localized trioxsalen-induced ICL damage in human cells, with accumulation being suppressed by transcription inhibition. Moreover, SNM1A recruitment was significantly reduced in CSB-deficient cells, suggesting coordination between the two proteins in vivo. CSB-deficient neural cells exhibited increased sensitivity to DNA crosslinking agents, particularly, in a non-cycling, differentiated state, as well as delayed ICL processing as revealed by a modified Comet assay and γ-H2AX foci persistence. The results indicate that CSB coordinates the resolution of ICLs, possibly in a transcription-associated repair mechanism involving SNM1A, and that defects in the process could contribute to the post-mitotic degenerative pathologies associated with CS.
|HMGB1 facilitates repair of mitochondrial DNA damage and extends the lifespan of mutant ataxin-1 knock-in mice. |
Ito, H; Fujita, K; Tagawa, K; Chen, X; Homma, H; Sasabe, T; Shimizu, J; Shimizu, S; Tamura, T; Muramatsu, S; Okazawa, H
EMBO molecular medicine 7 78-101 2015
Mutant ataxin-1 (Atxn1), which causes spinocerebellar ataxia type 1 (SCA1), binds to and impairs the function of high-mobility group box 1 (HMGB1), a crucial nuclear protein that regulates DNA architectural changes essential for DNA damage repair and transcription. In this study, we established that transgenic or virus vector-mediated complementation with HMGB1 ameliorates motor dysfunction and prolongs lifespan in mutant Atxn1 knock-in (Atxn1-KI) mice. We identified mitochondrial DNA damage repair by HMGB1 as a novel molecular basis for this effect, in addition to the mechanisms already associated with HMGB1 function, such as nuclear DNA damage repair and nuclear transcription. The dysfunction and the improvement of mitochondrial DNA damage repair functions are tightly associated with the exacerbation and rescue, respectively, of symptoms, supporting the involvement of mitochondrial DNA quality control by HMGB1 in SCA1 pathology. Moreover, we show that the rescue of Purkinje cell dendrites and dendritic spines by HMGB1 could be downstream effects. Although extracellular HMGB1 triggers inflammation mediated by Toll-like receptor and receptor for advanced glycation end products, upregulation of intracellular HMGB1 does not induce such side effects. Thus, viral delivery of HMGB1 is a candidate approach by which to modify the disease progression of SCA1 even after the onset.
|Proteotoxic stress induces phosphorylation of p62/SQSTM1 by ULK1 to regulate selective autophagic clearance of protein aggregates. |
Lim, J; Lachenmayer, ML; Wu, S; Liu, W; Kundu, M; Wang, R; Komatsu, M; Oh, YJ; Zhao, Y; Yue, Z
PLoS genetics 11 e1004987 2015
Disruption of proteostasis, or protein homeostasis, is often associated with aberrant accumulation of misfolded proteins or protein aggregates. Autophagy offers protection to cells by removing toxic protein aggregates and injured organelles in response to proteotoxic stress. However, the exact mechanism whereby autophagy recognizes and degrades misfolded or aggregated proteins has yet to be elucidated. Mounting evidence demonstrates the selectivity of autophagy, which is mediated through autophagy receptor proteins (e.g. p62/SQSTM1) linking autophagy cargos and autophagosomes. Here we report that proteotoxic stress imposed by the proteasome inhibition or expression of polyglutamine expanded huntingtin (polyQ-Htt) induces p62 phosphorylation at its ubiquitin-association (UBA) domain that regulates its binding to ubiquitinated proteins. We find that autophagy-related kinase ULK1 phosphorylates p62 at a novel phosphorylation site S409 in UBA domain. Interestingly, phosphorylation of p62 by ULK1 does not occur upon nutrient starvation, in spite of its role in canonical autophagy signaling. ULK1 also phosphorylates S405, while S409 phosphorylation critically regulates S405 phosphorylation. We find that S409 phosphorylation destabilizes the UBA dimer interface, and increases binding affinity of p62 to ubiquitin. Furthermore, lack of S409 phosphorylation causes accumulation of p62, aberrant localization of autophagy proteins and inhibition of the clearance of ubiquitinated proteins or polyQ-Htt. Therefore, our data provide mechanistic insights into the regulation of selective autophagy by ULK1 and p62 upon proteotoxic stress. Our study suggests a potential novel drug target in developing autophagy-based therapeutics for the treatment of proteinopathies including Huntington's disease.
|Reliable LC3 and p62 autophagy marker detection in formalin fixed paraffin embedded human tissue by immunohistochemistry. |
Schläfli, AM; Berezowska, S; Adams, O; Langer, R; Tschan, MP
European journal of histochemistry : EJH 59 2481 2015
Autophagy assures cellular homeostasis, and gains increasing importance in cancer, where it impacts on carcinogenesis, propagation of the malignant phenotype and development of resistance. To date, its tissue-based analysis by immunohistochemistry remains poorly standardized. Here we show the feasibility of specifically and reliably assessing the autophagy markers LC3B and p62 (SQSTM1) in formalin fixed and paraffin embedded human tissue by immunohistochemistry. Preceding functional experiments consisted of depleting LC3B and p62 in H1299 lung cancer cells with subsequent induction of autophagy. Western blot and immunofluorescence validated antibody specificity, knockdown efficiency and autophagy induction prior to fixation in formalin and embedding in paraffin. LC3B and p62 antibodies were validated on formalin fixed and paraffin embedded cell pellets of treated and control cells and finally applied on a tissue microarray with 80 human malignant and non-neoplastic lung and stomach formalin fixed and paraffin embedded tissue samples. Dot-like staining of various degrees was observed in cell pellets and 18/40 (LC3B) and 22/40 (p62) tumors, respectively. Seventeen tumors were double positive for LC3B and p62. P62 displayed additional significant cytoplasmic and nuclear staining of unknown significance. Interobserver-agreement for grading of staining intensities and patterns was substantial to excellent (kappa values 0.60 - 0.83). In summary, we present a specific and reliable IHC staining of LC3B and p62 on formalin fixed and paraffin embedded human tissue. Our presented protocol is designed to aid reliable investigation of dysregulated autophagy in solid tumors and may be used on large tissue collectives.
|Milnacipran remediates impulsive deficits in rats with lesions of the ventromedial prefrontal cortex. |
Tsutsui-Kimura, I; Yoshida, T; Ohmura, Y; Izumi, T; Yoshioka, M
The international journal of neuropsychopharmacology / official scientific journal of the Collegium Internationale Neuropsychopharmacologicum (CINP) 18 2015
Deficits in impulse control are often observed in psychiatric disorders in which abnormalities of the prefrontal cortex are observed, including attention-deficit/hyperactivity disorder and bipolar disorder. We recently found that milnacipran, a serotonin/noradrenaline reuptake inhibitor, could suppress impulsive action in normal rats. However, whether milnacipran could suppress elevated impulsive action in rats with lesions of the ventromedial prefrontal cortex, which is functionally comparable with the human prefrontal cortex, remains unknown.Selective lesions of the ventromedial prefrontal cortex were made using quinolinic acid in rats previously trained on a 3-choice serial reaction time task. Sham rats received phosphate buffered saline. Following a period of recovery, milnacipran (0 or 10mg/kg/d × 14 days) was orally administered 60 minutes prior to testing on the 3-choice task. After 7 days of drug cessation, Western blotting, immunohistochemistry, electrophysiological analysis, and morphological analysis were conducted.Lesions of the ventromedial prefrontal cortex induced impulsive deficits, and repeated milnacipran ameliorated the impulsive deficit both during the dosing period and after the cessation of the drug. Repeated milnacipran remediated the protein levels of mature brain-derived neurotrophic factor and postsynaptic density-95, dendritic spine density, and excitatory currents in the few surviving neurons in the ventromedial prefrontal cortex of ventromedial prefrontal cortex-lesioned rats.The findings of this study suggest that milnacipran treatment could be a novel strategy for the treatment of psychiatric disorders that are associated with a lack of impulse control.
|Global analysis of H3K4me3 and H3K27me3 profiles in glioblastoma stem cells and identification of SLC17A7 as a bivalent tumor suppressor gene. |
Lin, B; Lee, H; Yoon, JG; Madan, A; Wayner, E; Tonning, S; Hothi, P; Schroeder, B; Ulasov, I; Foltz, G; Hood, L; Cobbs, C
Oncotarget 6 5369-81 2015
Epigenetic changes, including H3K4me3 and H3K27me3 histone modification, play an important role in carcinogenesis. However, no genome-wide histone modification map has been generated for gliomas. Here, we report a genome-wide map of H3K4me3 and H3K27me3 histone modifications for 8 glioma stem cell (GSC) lines, together with the associated gene activation or repression patterns. In addition, we compared the genome-wide histone modification maps of GSC lines to those of astrocytes to identify unique gene activation or repression profiles in GSCs and astrocytes. We also identified a set of bivalent genes, which are genes that are associated with both H3K4me3 and H3K27me3 marks and are poised for action in embryonic stem cells. These bivalent genes are potential targets for inducing differentiation in glioblastoma (GBM) as a therapeutic approach. Finally, we identified SLC17A7 as a bivalent tumor suppressor gene in GBM, as it is down-regulated at both the protein and RNA levels in GBM tissues compared with normal brain tissues, and it inhibits GBM cell proliferation, migration and invasion.
|MARK/Par1 Kinase Is Activated Downstream of NMDA Receptors through a PKA-Dependent Mechanism. |
Bernard, LP; Zhang, H
PloS one 10 e0124816 2015
The Par1 kinases, also known as microtubule affinity-regulating kinases (MARKs), are important for the establishment of cell polarity from worms to mammals. Dysregulation of these kinases has been implicated in autism, Alzheimer's disease and cancer. Despite their important function in health and disease, it has been unclear how the activity of MARK/Par1 is regulated by signals from cell surface receptors. Here we show that MARK/Par1 is activated downstream of NMDA receptors in primary hippocampal neurons. Further, we show that this activation is dependent on protein kinase A (PKA), through the phosphorylation of Ser431 of Par4/LKB1, the major upstream kinase of MARK/Par1. Together, our data reveal a novel mechanism by which MARK/Par1 is activated at the neuronal synapse.
|Epinephrine Activation of the β2-Adrenoceptor Is Required for IL-13-Induced Mucin Production in Human Bronchial Epithelial Cells. |
Al-Sawalha, N; Pokkunuri, I; Omoluabi, O; Kim, H; Thanawala, VJ; Hernandez, A; Bond, RA; Knoll, BJ
PloS one 10 e0132559 2015
Mucus hypersecretion by airway epithelium is a hallmark of inflammation in allergic asthma and results in airway narrowing and obstruction. Others have shown that administration a TH2 cytokine, IL-13 is sufficient to cause mucus hypersecretion in vivo and in vitro. Asthma therapy often utilizes β2-adrenoceptor (β2AR) agonists, which are effective acutely as bronchodilators, however chronic use may lead to a worsening of asthma symptoms. In this study, we asked whether β2AR signaling in normal human airway epithelial (NHBE) cells affected mucin production in response to IL-13. This cytokine markedly increased mucin production, but only in the presence of epinephrine. Mucin production was blocked by ICI-118,551, a preferential β2AR antagonist, but not by CGP-20712A, a preferential β1AR antagonist. Constitutive β2AR activity was not sufficient for IL-13 induced mucin production and β-agonist-induced signaling is required. A clinically important long-acting β-agonist, formoterol, was as effective as epinephrine in potentiating IL-13 induced MUC5AC transcription. IL-13 induced mucin production in the presence of epinephrine was significantly reduced by treatment with selective inhibitors of ERK1/2 (FR180204), p38 (SB203580) and JNK (SP600125). Replacement of epinephrine with forskolin + IBMX resulted in a marked increase in mucin production in NHBE cells in response to IL-13, and treatment with the inhibitory cAMP analogue Rp-cAMPS decreased mucin levels induced by epinephrine + IL-13. Our findings suggest that β2AR signaling is required for mucin production in response to IL-13, and that mitogen activated protein kinases and cAMP are necessary for this effect. These data lend support to the notion that β2AR-agonists may contribute to asthma exacerbations by increasing mucin production via activation of β2ARs on epithelial cells.
|Severe myopathy in mice lacking the MEF2/SRF-dependent gene leiomodin-3. |
Cenik, BK; Garg, A; McAnally, JR; Shelton, JM; Richardson, JA; Bassel-Duby, R; Olson, EN; Liu, N
The Journal of clinical investigation 125 1569-78 2015
Maintenance of skeletal muscle structure and function requires a precise stoichiometry of sarcomeric proteins for proper assembly of the contractile apparatus. Absence of components of the sarcomeric thin filaments causes nemaline myopathy, a lethal congenital muscle disorder associated with aberrant myofiber structure and contractility. Previously, we reported that deficiency of the kelch-like family member 40 (KLHL40) in mice results in nemaline myopathy and destabilization of leiomodin-3 (LMOD3). LMOD3 belongs to a family of tropomodulin-related proteins that promote actin nucleation. Here, we show that deficiency of LMOD3 in mice causes nemaline myopathy. In skeletal muscle, transcription of Lmod3 was controlled by the transcription factors SRF and MEF2. Myocardin-related transcription factors (MRTFs), which function as SRF coactivators, serve as sensors of actin polymerization and are sequestered in the cytoplasm by actin monomers. Conversely, conditions that favor actin polymerization de-repress MRTFs and activate SRF-dependent genes. We demonstrated that the actin nucleator LMOD3, together with its stabilizing partner KLHL40, enhances MRTF-SRF activity. In turn, SRF cooperated with MEF2 to sustain the expression of LMOD3 and other components of the contractile apparatus, thereby establishing a regulatory circuit to maintain skeletal muscle function. These findings provide insight into the molecular basis of the sarcomere assembly and muscle dysfunction associated with nemaline myopathy.
|Possible dual role of decorin in abdominal aortic aneurysm. |
Ueda, K; Yoshimura, K; Yamashita, O; Harada, T; Morikage, N; Hamano, K
PloS one 10 e0120689 2015
Abdominal aortic aneurysm (AAA) is characterized by chronic inflammation, which leads to pathological remodeling of the extracellular matrix. Decorin, a small leucine-rich repeat proteoglycan, has been suggested to regulate inflammation and stabilize the extracellular matrix. Therefore, the present study investigated the role of decorin in the pathogenesis of AAA. Decorin was localized in the aortic adventitia under normal conditions in both mice and humans. AAA was induced in mice using CaCl2 treatment. Initially, decorin protein levels decreased, but as AAA progressed decorin levels increased in all layers. Local administration of exogenous decorin prevented the development of CaCl2-induced AAA. However, decorin was highly expressed in the degenerative lesions of human AAA walls, and this expression positively correlated with matrix metalloproteinase (MMP)-9 expression. In cell culture experiments, the addition of decorin inhibited secretion of MMP-9 in vascular smooth muscle cells, but had the opposite effect in macrophages. The results suggest that decorin plays a dual role in AAA. Adventitial decorin in normal aorta may protect against the development of AAA, but macrophages expressing decorin in AAA walls may facilitate the progression of AAA by up-regulating MMP-9 secretion.
|Bacterial induction of Snail1 contributes to blood-brain barrier disruption. |
Kim, BJ; Hancock, BM; Bermudez, A; Del Cid, N; Reyes, E; van Sorge, NM; Lauth, X; Smurthwaite, CA; Hilton, BJ; Stotland, A; Banerjee, A; Buchanan, J; Wolkowicz, R; Traver, D; Doran, KS
The Journal of clinical investigation 125 2473-83 2015
Bacterial meningitis is a serious infection of the CNS that results when blood-borne bacteria are able to cross the blood-brain barrier (BBB). Group B Streptococcus (GBS) is the leading cause of neonatal meningitis; however, the molecular mechanisms that regulate bacterial BBB disruption and penetration are not well understood. Here, we found that infection of human brain microvascular endothelial cells (hBMECs) with GBS and other meningeal pathogens results in the induction of host transcriptional repressor Snail1, which impedes expression of tight junction genes. Moreover, GBS infection also induced Snail1 expression in murine and zebrafish models. Tight junction components ZO-1, claudin 5, and occludin were decreased at both the transcript and protein levels in hBMECs following GBS infection, and this repression was dependent on Snail1 induction. Bacteria-independent Snail1 expression was sufficient to facilitate tight junction disruption, promoting BBB permeability to allow bacterial passage. GBS induction of Snail1 expression was dependent on the ERK1/2/MAPK signaling cascade and bacterial cell wall components. Finally, overexpression of a dominant-negative Snail1 homolog in zebrafish elevated transcription of tight junction protein-encoding genes and increased zebrafish survival in response to GBS challenge. Taken together, our data support a Snail1-dependent mechanism of BBB disruption and penetration by meningeal pathogens.
|Hyperosmotic stress activates the expression of members of the miR-15/107 family and induces downregulation of anti-apoptotic genes in rat liver. |
Santosa, D; Castoldi, M; Paluschinski, M; Sommerfeld, A; Häussinger, D
Scientific reports 5 12292 2015
microRNAs are an abundant class of small non-coding RNAs that negatively regulate gene expression. Importantly, microRNA activity has been linked to the control of cellular stress response. In the present study, we investigated whether the expression of hepatic microRNAs is affected by changes in ambient osmolarity. It is shown that hyperosmotic exposure of perfused rat liver induces a rapid upregulation of miR-15a, miR-15b and miR-16, which are members of the miR-15/107 microRNAs superfamily. It was also identified that hyperosmolarity significantly reduces the expression of anti-apoptotic genes including Bcl2, Ccnd1, Mcl1, Faim, Aatf, Bfar and Ikbkb, which are either validated or predicted targets of these microRNAs. Moreover, through the application of NOX and JNK inhibitors as well as benzylamine it is shown that the observed response is mediated by reactive oxygen species (ROS), suggesting that miR-15a, miR-15b and miR-16 are novel redoximiRs. It is concluded that the response of these three microRNAs to osmotic stress is ROS-mediated and that it might contribute to the development of a proapoptotic phenotype.
|Foam Cell Formation In Vivo Converts Macrophages to a Pro-Fibrotic Phenotype. |
Thomas, AC; Eijgelaar, WJ; Daemen, MJ; Newby, AC
PloS one 10 e0128163 2015
Formation of foam cell macrophages, which sequester extracellular modified lipids, is a key event in atherosclerosis. How lipid loading affects macrophage phenotype is controversial, with evidence suggesting either pro- or anti-inflammatory consequences. To investigate this further, we compared the transcriptomes of foamy and non-foamy macrophages that accumulate in the subcutaneous granulomas of fed-fat ApoE null mice and normal chow fed wild-type mice in vivo. Consistent with previous studies, LXR/RXR pathway genes were significantly over-represented among the genes up-regulated in foam cell macrophages. Unexpectedly, the hepatic fibrosis pathway, associated with platelet derived growth factor and transforming growth factor-β action, was also over-represented. Several collagen polypeptides and proteoglycan core proteins as well as connective tissue growth factor and fibrosis-related FOS and JUN transcription factors were up-regulated in foam cell macrophages. Increased expression of several of these genes was confirmed at the protein level in foam cell macrophages from subcutaneous granulomas and in atherosclerotic plaques. Moreover, phosphorylation and nuclear translocation of SMAD2, which is downstream of several transforming growth factor-β family members, was also detected in foam cell macrophages. We conclude that foam cell formation in vivo leads to a pro-fibrotic macrophage phenotype, which could contribute to plaque stability, especially in early lesions that have few vascular smooth muscle cells.
|CoREST1 promotes tumor formation and tumor stroma interactions in a mouse model of breast cancer. |
Mazumdar, S; Arendt, LM; Phillips, S; Sedic, M; Kuperwasser, C; Gill, G
PloS one 10 e0121281 2015
Regulators of chromatin structure and gene expression contribute to tumor formation and progression. The co-repressor CoREST1 regulates the localization and activity of associated histone modifying enzymes including lysine specific demethylase 1 (LSD1) and histone deacetylase 1 (HDAC1). Although several CoREST1 associated proteins have been reported to enhance breast cancer progression, the role of CoREST1 in breast cancer is currently unclear. Here we report that knockdown of CoREST1 in the basal-type breast cancer cell line, MDA-MB-231, led to significantly reduced incidence and diminished size of tumors compared to controls in mouse xenograft studies. Notably, CoREST1-depleted cells gave rise to tumors with a marked decrease in angiogenesis. CoREST1 knockdown led to a decrease in secreted angiogenic and inflammatory factors, and mRNA analysis suggests that CoREST1 promotes expression of genes related to angiogenesis and inflammation including VEGF-A and CCL2. CoREST1 knockdown decreased the ability of MDA-MB-231 conditioned media to promote endothelial cell tube formation and migration. Further, tumors derived from CoREST1-depleted cells had reduced macrophage infiltration and the secretome of CoREST1 knockdown cells was deficient in promoting macrophage migration and macrophage-mediated angiogenesis. Taken together, these findings reveal that the epigenetic regulator CoREST1 promotes tumorigenesis in a breast cancer model at least in part through regulation of gene expression patterns in tumor cells that have profound non-cell autonomous effects on endothelial and inflammatory cells in the tumor microenvironment.
|Paraquat induces epithelial-mesenchymal transition-like cellular response resulting in fibrogenesis and the prevention of apoptosis in human pulmonary epithelial cells. |
Yamada, A; Aki, T; Unuma, K; Funakoshi, T; Uemura, K
PloS one 10 e0120192 2015
The aim of this study is to investigate the molecular mechanisms underlying delayed progressive pulmonary fibrosis, a characteristic of subacute paraquat (PQ) poisoning. Epithelial-mesenchymal transition (EMT) has been proposed as a cause of organ fibrosis, and transforming growth factor-β (TGF-β) is suggested to be a powerful mediator of EMT. We thus examined the possibility that EMT is involved in pulmonary fibrosis during PQ poisoning using A549 human alveolar epithelial cells in vitro. The cells were treated with various concentrations of PQ (0-500 μM) for 2-12 days. Short-term (2 days) high-dose (greater than 100 μM) treatments with PQ induced cell death accompanied by the activation of caspase9 as well as a decrease in E-cadherin (an epithelial cell marker), suggesting apoptotic cell death with the features of anoikis (cell death due to the loss of cell-cell adhesion). In contrast, long-term (6-12 days) low-dose (30 μM) treatments with PQ resulted in a transformation into spindle-shaped mesenchymal-like cells with a decrease of E-cadherin as well as an increase of α-smooth muscle actin (α-SMA). The mesenchymal-like cells also secreted the extracellular matrix (ECM) protein fibronectin into the culture medium. The administration of a TGF-β1 receptor antagonist, SB431542, almost completely attenuated the mesenchymal transformation as well as fibronectin secretion, suggesting a crucial role of TGF-β1 in EMT-like cellular response and subsequent fibrogenesis. It is noteworthy that despite the suppression of EMT-fibrogenesis, apoptotic death was observed in cells treated with PQ+SB431542. EMT-like cellular response and subsequent fibrogenesis were also observed in normal human bronchial epithelial (NHBE) cells exposed to PQ in a TGF-β1-dependent manner. Taken together, our experimental model reflects well the etiology of PQ poisoning in human and shows the involvement of EMT-like cellular response in both fibrogenesis and resistance to cell death during subacute PQ poisoning of pulmonary epithelial cells.
|DNA methyltransferase inhibitor zebularine induces human cholangiocarcinoma cell death through alteration of DNA methylation status. |
Nakamura, K; Nakabayashi, K; Htet Aung, K; Aizawa, K; Hori, N; Yamauchi, J; Hata, K; Tanoue, A
PloS one 10 e0120545 2015
Cholangiocarcinoma (CCA) is a cancer arising from the neoplastic transformation of cholangiocytes. During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Zebularine (1-(β-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one) acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. In this study, we explore the effect and possible mechanism of action of zebularine on CCA cells. We demonstrate that zebularine exerts an antitumor effect on CCA cells. Zebularine treatment decreased the concentrations of DNA methyltransferase (DNMT) proteins, and DNMT1 knockdown led to apoptotic cell death in the CCA cell lines TFK-1 and HuCCT1. DNA methylation analysis demonstrated that zebularine induced DNA demethylation, and the GO Biological Process terms "hemophilic cell adhesion", "regulation of transcription, DNA-dependent" and "Wnt signaling pathway" were found to be significantly enriched in association with demethylated genes. Furthermore, we observed that zebularine treatment decreased β-catenin protein levels in TFK-1 and HuCCT1 cells. These results suggest that zebularine alters DNA methylation status, and that some aspect of DNA demethylation by zebularine induces suppression of the Wnt signaling pathway, which leads to apoptotic cell death in CCA. We previously reported a novel mechanism of zebularine-induced cell growth arrest and apoptosis in hepatocellular carcinoma via a DNA methylation-independent pathway. Together, our present and previous studies indicate that zebularine could function as both a DNMT inhibitor and a non-DNMT inhibitor reagent, and that, while the optimal usage of zebularine may depend on cancer type, zebularine may be useful for chemotherapy against cancer.
|Inhibitory effect of statins on inflammation-related pathways in human abdominal aortic aneurysm tissue. |
Yoshimura, K; Nagasawa, A; Kudo, J; Onoda, M; Morikage, N; Furutani, A; Aoki, H; Hamano, K
International journal of molecular sciences 16 11213-28 2015
HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitors (statins) have been suggested to attenuate abdominal aortic aneurysm (AAA) growth. However, the effects of statins in human AAA tissues are not fully elucidated. The aim of this study was to investigate the direct effects of statins on proinflammatory molecules in human AAA walls in ex vivo culture. Simvastatin strongly inhibited the activation of nuclear factor (NF)-κB induced by tumor necrosis factor (TNF)-α in human AAA walls, but showed little effect on c-jun N-terminal kinase (JNK) activation. Simvastatin, as well as pitavastatin significantly reduced the secretion of matrix metalloproteinase (MMP)-9, monocyte chemoattractant protein (MCP)-2 and epithelial neutrophil-activating peptide (CXCL5) under both basal and TNF-α-stimulated conditions. Similar to statins, the Rac1 inhibitor NSC23766 significantly inhibited the activation of NF-κB, accompanied by a decreased secretion of MMP-9, MCP-2 and CXCL5. Moreover, the effect of simvastatin and the JNK inhibitor SP600125 was additive in inhibiting the secretion of MMP-9, MCP-2 and CXCL5. These findings indicate that statins preferentially inhibit the Rac1/NF-κB pathway to suppress MMP-9 and chemokine secretion in human AAA, suggesting a mechanism for the potential effect of statins in attenuating AAA progression.
|Mitofusin 2 Deficiency Affects Energy Metabolism and Mitochondrial Biogenesis in MEF Cells. |
Kawalec, M; Boratyńska-Jasińska, A; Beręsewicz, M; Dymkowska, D; Zabłocki, K; Zabłocka, B
PloS one 10 e0134162 2015
Mitofusin 2 (Mfn2), mitochondrial outer membrane protein which is involved in rearrangement of these organelles, was first described in pathology of hypertension and diabetes, and more recently much attention is paid to its functions in Charcot-Marie-Tooth type 2A neuropathy (CMT2A). Here, cellular energy metabolism was investigated in mouse embryonic fibroblasts (MEF) differing in the presence of the Mfn2 gene; control (MEFwt) and with Mfn2 gene depleted MEFMfn2-/-. These two cell lines were compared in terms of various parameters characterizing mitochondrial bioenergetics. Here, we have shown that relative rate of proliferation of MEFMfn2-/- cells versus control fibroblasts depend on serum supplementation of the growth media. Moreover, MEFMfn2-/- cells exhibited significantly increased respiration rate in comparison to MEFwt, regardless of serum supplementation of the medium. This effect was correlated with increased level of mitochondrial markers (TOM20 and NAO) as well as mitochondrial transcription factor A (TFAM) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) protein levels and unchanged total ATP content. Interestingly, mitochondrial DNA content in MEFMfn2-/- cells was not reduced. Fundamentally, these results are in contrast to a commonly accepted belief that mitofusin 2 deficiency inevitably results in debilitation of mitochondrial energy metabolism. However, we suggest a balance between negative metabolic consequences of mitofusin 2 deficiency and adaptive processes exemplified by increased level of PGC-1α and TFAM transcription factor which prevent an excessive depletion of mtDNA and severe impairment of cell metabolism.
|Impaired spatial memory and enhanced long-term potentiation in mice with forebrain-specific ablation of the Stim genes. |
Garcia-Alvarez, G; Shetty, MS; Lu, B; Yap, KA; Oh-Hora, M; Sajikumar, S; Bichler, Z; Fivaz, M
Frontiers in behavioral neuroscience 9 180 2015
Recent findings point to a central role of the endoplasmic reticulum-resident STIM (Stromal Interaction Molecule) proteins in shaping the structure and function of excitatory synapses in the mammalian brain. The impact of the Stim genes on cognitive functions remains, however, poorly understood. To explore the function of the Stim genes in learning and memory, we generated three mouse strains with conditional deletion (cKO) of Stim1 and/or Stim2 in the forebrain. Stim1, Stim2, and double Stim1/Stim2 cKO mice show no obvious brain structural defects or locomotor impairment. Analysis of spatial reference memory in the Morris water maze revealed a mild learning delay in Stim1 cKO mice, while learning and memory in Stim2 cKO mice was indistinguishable from their control littermates. Deletion of both Stim genes in the forebrain resulted, however, in a pronounced impairment in spatial learning and memory reflecting a synergistic effect of the Stim genes on the underlying neural circuits. Notably, long-term potentiation (LTP) at CA3-CA1 hippocampal synapses was markedly enhanced in Stim1/Stim2 cKO mice and was associated with increased phosphorylation of the AMPA receptor subunit GluA1, the transcriptional regulator CREB and the L-type Voltage-dependent Ca(2+) channel Cav1.2 on protein kinase A (PKA) sites. We conclude that STIM1 and STIM2 are key regulators of PKA signaling and synaptic plasticity in neural circuits encoding spatial memory. Our findings also reveal an inverse correlation between LTP and spatial learning/memory and suggest that abnormal enhancement of cAMP/PKA signaling and synaptic efficacy disrupts the formation of new memories.
|An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease. |
Kizuka, Y; Kitazume, S; Fujinawa, R; Saito, T; Iwata, N; Saido, TC; Nakano, M; Yamaguchi, Y; Hashimoto, Y; Staufenbiel, M; Hatsuta, H; Murayama, S; Manya, H; Endo, T; Taniguchi, N
EMBO molecular medicine 7 175-89 2015
The β-site amyloid precursor protein cleaving enzyme-1 (BACE1), an essential protease for the generation of amyloid-β (Aβ) peptide, is a major drug target for Alzheimer's disease (AD). However, there is a concern that inhibiting BACE1 could also affect several physiological functions. Here, we show that BACE1 is modified with bisecting N-acetylglucosamine (GlcNAc), a sugar modification highly expressed in brain, and demonstrate that AD patients have higher levels of bisecting GlcNAc on BACE1. Analysis of knockout mice lacking the biosynthetic enzyme for bisecting GlcNAc, GnT-III (Mgat3), revealed that cleavage of Aβ-precursor protein (APP) by BACE1 is reduced in these mice, resulting in a decrease in Aβ plaques and improved cognitive function. The lack of this modification directs BACE1 to late endosomes/lysosomes where it is less colocalized with APP, leading to accelerated lysosomal degradation. Notably, other BACE1 substrates, CHL1 and contactin-2, are normally cleaved in GnT-III-deficient mice, suggesting that the effect of bisecting GlcNAc on BACE1 is selective to APP. Considering that GnT-III-deficient mice remain healthy, GnT-III may be a novel and promising drug target for AD therapeutics.
|Protease Omi facilitates neurite outgrowth in mouse neuroblastoma N2a cells by cleaving transcription factor E2F1. |
Ma, Q; Hu, QS; Xu, RJ; Zhen, XC; Wang, GH
Acta pharmacologica Sinica 36 966-75 2015
Omi is an ATP-independent serine protease that is necessary for neuronal function and survival. The aim of this study was to investigate the role of protease Omi in regulating differentiation of mouse neuroblastoma cells and to identify the substrate of Omi involved in this process.Mouse neuroblastoma N2a cells and Omi protease-deficient mnd2 mice were used in this study. To modulate Omi and E2F1 expression, N2a cells were transfected with expression plasmids, shRNA plasmids or siRNA. Protein levels were detected using immunoblot assays. The interaction between Omi and E2F1 was studied using immunoprecipitation, GST pulldown and in vitro cleavage assays. N2a cells were treated with 20 μmol/L retinoic acid (RA) and 1% fetal bovine serum to induce neurite outgrowth, which was measured using Image J software.E2F1 was significantly increased in Omi knockdown cells and in brain lysates of mnd2 mice, and was decreased in cells overexpressing wild-type Omi, but not inactive Omi S276C. In brain lysates of mnd2 mice, endogenous E2F1 was co-immunoprecipitated with endogenous Omi. In vitro cleavage assay demonstrated that Omi directly cleaved E2F1. Treatment of N2a cells with RA induced marked differentiation and neurite outgrowth accompanied by significantly increased Omi and decreased E2F1 levels, which were suppressed by pretreatment with the specific Omi inhibitor UCF-101. Knockdown of Omi in N2a cells suppressed RA-induced neurite outgrowth, which was partially restored by knockdown of E2F1.Protease Omi facilitates neurite outgrowth by cleaving the transcription factor E2F1 in differentiated neuroblastoma cells; E2F1 is a substrate of Omi.
|Myocardin Family Members Drive Formation of Caveolae. |
Krawczyk, KK; Yao Mattisson, I; Ekman, M; Oskolkov, N; Grantinge, R; Kotowska, D; Olde, B; Hansson, O; Albinsson, S; Miano, JM; Rippe, C; Swärd, K
PloS one 10 e0133931 2015
Caveolae are membrane organelles that play roles in glucose and lipid metabolism and in vascular function. Formation of caveolae requires caveolins and cavins. The make-up of caveolae and their density is considered to reflect cell-specific transcriptional control mechanisms for caveolins and cavins, but knowledge regarding regulation of caveolae genes is incomplete. Myocardin (MYOCD) and its relative MRTF-A (MKL1) are transcriptional coactivators that control genes which promote smooth muscle differentiation. MRTF-A communicates changes in actin polymerization to nuclear gene transcription. Here we tested if myocardin family proteins control biogenesis of caveolae via activation of caveolin and cavin transcription. Using human coronary artery smooth muscle cells we found that jasplakinolide and latrunculin B (LatB), substances that promote and inhibit actin polymerization, increased and decreased protein levels of caveolins and cavins, respectively. The effect of LatB was associated with reduced mRNA levels for these genes and this was replicated by the MRTF inhibitor CCG-1423 which was non-additive with LatB. Overexpression of myocardin and MRTF-A caused 5-10-fold induction of caveolins whereas cavin-1 and cavin-2 were induced 2-3-fold. PACSIN2 also increased, establishing positive regulation of caveolae genes from three families. Full regulation of CAV1 was retained in its proximal promoter. Knock down of the serum response factor (SRF), which mediates many of the effects of myocardin, decreased cavin-1 but increased caveolin-1 and -2 mRNAs. Viral transduction of myocardin increased the density of caveolae 5-fold in vitro. A decrease of CAV1 was observed concomitant with a decrease of the smooth muscle marker calponin in aortic aneurysms from mice (C57Bl/6) infused with angiotensin II. Human expression data disclosed correlations of MYOCD with CAV1 in a majority of human tissues and in the heart, correlation with MKL2 (MRTF-B) was observed. The myocardin family of transcriptional coactivators therefore drives formation of caveolae and this effect is largely independent of SRF.
|Melatonin stimulates dendrite formation and complexity in the hilar zone of the rat hippocampus: participation of the Ca++/Calmodulin complex. |
Domínguez-Alonso, A; Valdés-Tovar, M; Solís-Chagoyán, H; Benítez-King, G
International journal of molecular sciences 16 1907-27 2015
Melatonin (MEL), the main product synthesized by the pineal gland, stimulates early and late stages of neurodevelopment in the adult brain. MEL increases dendrite length, thickness and complexity in the hilar and mossy neurons of hippocampus. Dendrite formation involves activation of Ca2+/Calmodulin (CaM)-dependent kinase II (CaMKII) by CaM. Previous work showed that MEL increased the synthesis and translocation of CaM, suggesting that MEL activates CaM-dependent enzymes by this pathway. In this work we investigated whether MEL stimulates dendrite formation by CaMKII activation in organotypic cultures from adult rat hippocampus. We found that the CaMKII inhibitor, KN-62, abolished the MEL stimulatory effects on dendritogenesis and that MEL increased the relative amount of CaM in the soluble fraction of hippocampal slices. Also, PKC inhibition abolished dendritogenesis, while luzindole, an antagonist of MEL receptors (MT1/2), partially blocked the effects of MEL. Moreover, autophosphorylation of CaMKII and PKC was increased in presence of MEL, as well as phosphorylation of ERK1/2. Our results indicate that MEL stimulates dendrite formation through CaMKII and the translocation of CaM to the soluble fraction. Dendritogenesis elicited by MEL also required PKC activation, and signaling through MT1/2 receptors was partially involved. Data strongly suggest that MEL could repair the loss of hippocampal dendrites that occur in neuropsychiatric disorders by increasing CaM levels and activation of CaMKII.
|Dietary Vitamin D3 Restriction Exacerbates Disease Pathophysiology in the Spinal Cord of the G93A Mouse Model of Amyotrophic Lateral Sclerosis. |
Moghimi, E; Solomon, JA; Gianforcaro, A; Hamadeh, MJ
PloS one 10 e0126355 2015
Dietary vitamin D3 (D3) restriction reduces paw grip endurance and motor performance in G93A mice, and increases inflammation and apoptosis in the quadríceps of females. ALS, a neuromuscular disease, causes progressive degeneration of motor neurons in the brain and spinal cord.We analyzed the spinal cords of G93A mice following dietary D3 restriction at 2.5% the adequate intake (AI) for oxidative damage (4-HNE, 3-NY), antioxidant enzymes (SOD2, catalase, GPx1), inflammation (TNF-α, IL-6, IL-10), apoptosis (bax/bcl-2 ratio, cleaved/pro-caspase 3 ratio), neurotrophic factor (GDNF) and neuron count (ChAT, SMI-36/SMI-32 ratio).Beginning at age 25 d, 42 G93A mice were provided food ad libitum with either adequate (AI;1 IU D3/g feed; 12 M, 11 F) or deficient (DEF; 0.025 IU D3/g feed; 10 M, 9 F) D3. At age 113 d, the spinal cords were analyzed for protein content. Differences were considered significant at P ≤ 0.10, since this was a pilot study.DEF mice had 16% higher 4-HNE (P = 0.056), 12% higher GPx1 (P = 0.057) and 23% higher Bax/Bcl2 ratio (P = 0.076) vs. AI. DEF females had 29% higher GPx1 (P = 0.001) and 22% higher IL-6 (P = 0.077) vs. AI females. DEF males had 23% higher 4-HNE (P = 0.066) and 18% lower SOD2 (P = 0.034) vs. AI males. DEF males had 27% lower SOD2 (P = 0.004), 17% lower GPx1 (P = 0.070), 29% lower IL-6 (P = 0.023) and 22% lower ChAT (P = 0.082) vs. DEF females.D3 deficiency exacerbates disease pathophysiology in the spinal cord of G93A mice, the exact mechanisms are sex-specific. This is in accord with our previous results in the quadriceps, as well as functional and disease outcomes.
|The sirtuin-2 inhibitor AK7 is neuroprotective in models of Parkinson's disease but not amyotrophic lateral sclerosis and cerebral ischemia. |
Chen, X; Wales, P; Quinti, L; Zuo, F; Moniot, S; Herisson, F; Rauf, NA; Wang, H; Silverman, RB; Ayata, C; Maxwell, MM; Steegborn, C; Schwarzschild, MA; Outeiro, TF; Kazantsev, AG
PloS one 10 e0116919 2015
Sirtuin deacetylases regulate diverse cellular pathways and influence disease processes. Our previous studies identified the brain-enriched sirtuin-2 (SIRT2) deacetylase as a potential drug target to counteract neurodegeneration. In the present study, we characterize SIRT2 inhibition activity of the brain-permeable compound AK7 and examine the efficacy of this small molecule in models of Parkinson's disease, amyotrophic lateral sclerosis and cerebral ischemia. Our results demonstrate that AK7 is neuroprotective in models of Parkinson's disease; it ameliorates alpha-synuclein toxicity in vitro and prevents 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopamine depletion and dopaminergic neuron loss in vivo. The compound does not show beneficial effects in mouse models of amyotrophic lateral sclerosis and cerebral ischemia. These findings underscore the specificity of protective effects observed here in models of Parkinson's disease, and previously in Huntington's disease, and support the development of SIRT2 inhibitors as potential therapeutics for the two neurodegenerative diseases.
|Mechanisms of hyperhomocysteinemia induced skeletal muscle myopathy after ischemia in the CBS-/+ mouse model. |
Veeranki, S; Tyagi, SC
International journal of molecular sciences 16 1252-65 2015
Although hyperhomocysteinemia (HHcy) elicits lower than normal body weights and skeletal muscle weakness, the mechanisms remain unclear. Despite the fact that HHcy-mediated enhancement in ROS and consequent damage to regulators of different cellular processes is relatively well established in other organs, the nature of such events is unknown in skeletal muscles. Previously, we reported that HHcy attenuation of PGC-1α and HIF-1α levels enhanced the likelihood of muscle atrophy and declined function after ischemia. In the current study, we examined muscle levels of homocysteine (Hcy) metabolizing enzymes, anti-oxidant capacity and focused on protein modifications that might compromise PGC-1α function during ischemic angiogenesis. Although skeletal muscles express the key enzyme (MTHFR) that participates in re-methylation of Hcy into methionine, lack of trans-sulfuration enzymes (CBS and CSE) make skeletal muscles more susceptible to the HHcy-induced myopathy. Our study indicates that elevated Hcy levels in the CBS-/+ mouse skeletal muscles caused diminished anti-oxidant capacity and contributed to enhanced total protein as well as PGC-1α specific nitrotyrosylation after ischemia. Furthermore, in the presence of NO donor SNP, either homocysteine (Hcy) or its cyclized version, Hcy thiolactone, not only increased PGC-1α specific protein nitrotyrosylation but also reduced its association with PPARγ in C2C12 cells. Altogether these results suggest that HHcy exerts its myopathic effects via reduction of the PGC-1/PPARγ axis after ischemia.
|STIM2 regulates PKA-dependent phosphorylation and trafficking of AMPARs. |
Garcia-Alvarez, G; Lu, B; Yap, KA; Wong, LC; Thevathasan, JV; Lim, L; Ji, F; Tan, KW; Mancuso, JJ; Tang, W; Poon, SY; Augustine, GJ; Fivaz, M
Molecular biology of the cell 26 1141-59 2015
STIMs (STIM1 and STIM2 in mammals) are transmembrane proteins that reside in the endoplasmic reticulum (ER) and regulate store-operated Ca(2+) entry (SOCE). The function of STIMs in the brain is only beginning to be explored, and the relevance of SOCE in nerve cells is being debated. Here we identify STIM2 as a central organizer of excitatory synapses. STIM2, but not its paralogue STIM1, influences the formation of dendritic spines and shapes basal synaptic transmission in excitatory neurons. We further demonstrate that STIM2 is essential for cAMP/PKA-dependent phosphorylation of the AMPA receptor (AMPAR) subunit GluA1. cAMP triggers rapid migration of STIM2 to ER-plasma membrane (PM) contact sites, enhances recruitment of GluA1 to these ER-PM junctions, and promotes localization of STIM2 in dendritic spines. Both biochemical and imaging data suggest that STIM2 regulates GluA1 phosphorylation by coupling PKA to the AMPAR in a SOCE-independent manner. Consistent with a central role of STIM2 in regulating AMPAR phosphorylation, STIM2 promotes cAMP-dependent surface delivery of GluA1 through combined effects on exocytosis and endocytosis. Collectively our results point to a unique mechanism of synaptic plasticity driven by dynamic assembly of a STIM2 signaling complex at ER-PM contact sites.
|Runx1 Transcription Factor Is Required for Myoblasts Proliferation during Muscle Regeneration. |
Umansky, KB; Gruenbaum-Cohen, Y; Tsoory, M; Feldmesser, E; Goldenberg, D; Brenner, O; Groner, Y
PLoS genetics 11 e1005457 2015
Following myonecrosis, muscle satellite cells proliferate, differentiate and fuse, creating new myofibers. The Runx1 transcription factor is not expressed in naïve developing muscle or in adult muscle tissue. However, it is highly expressed in muscles exposed to myopathic damage yet, the role of Runx1 in muscle regeneration is completely unknown. Our study of Runx1 function in the muscle's response to myonecrosis reveals that this transcription factor is activated and cooperates with the MyoD and AP-1/c-Jun transcription factors to drive the transcription program of muscle regeneration. Mice lacking dystrophin and muscle Runx1 (mdx-/Runx1f/f), exhibit impaired muscle regeneration leading to age-dependent muscle waste, gradual decrease in motor capabilities and a shortened lifespan. Runx1-deficient primary myoblasts are arrested at cell cycle G1 and consequently differentiate. Such premature differentiation disrupts the myoblasts' normal proliferation/differentiation balance, reduces the number and size of regenerating myofibers and impairs muscle regeneration. Our combined Runx1-dependent gene expression, ChIP-seq, ATAC-seq and histone H3K4me1/H3K27ac modification analyses revealed a subset of Runx1-regulated genes that are co-occupied by MyoD and c-Jun in mdx-/Runx1f/f muscle. The data provide unique insights into the transcriptional program driving muscle regeneration and implicate Runx1 as an important participant in the pathology of muscle wasting diseases.
|Lentivirus-mediated RNAi knockdown of the gap junction protein, Cx43, attenuates the development of vascular restenosis following balloon injury. |
Han, XJ; Chen, M; Hong, T; Zhu, LY; He, D; Feng, JG; Jiang, LP
International journal of molecular medicine 35 885-92 2015
Percutaneous coronary intervention [PCI or percutaneous transluminal coronary angioplasty (PTCA)] has been developed into a mature interventional treatment for atherosclerotic cardiovascular disease. However, the long-term therapeutic effect is compromised by the high incidence of vascular restenosis following angioplasty, and the underlying mechanisms of vascular restenosis have not yet been fully elucidated. In the present study, we investigated the role of the gap junction (GJ) protein, connexin 43 (Cx43), in the development of vascular restenosis. To establish vascular restenosis, rat carotid arteries were subjected to balloon angioplasty injury. At 0, 7, 14 and 2 days following balloon injury, the arteries were removed, and the intimal/medial area of the vessels was measured to evaluate the degree of restenosis. We found that the intimal area gradually increased following balloon injury. Intimal hyperplasia and restenosis were particularly evident at 14 and 28 days after injury. In addition, the mRNA and protein expression of Cx43 was temporarily decreased at 7 days, and subsequently increased at 14 and 28 days following balloon injury, as shown by RT-PCR and western blot analysis. To determine the involvement of Cx43 in vascular restenosis, the lentivirus vector expressing shRNA targeting Cx43, Cx43-RNAi-LV, was used to silence Cx43 in the rat carotid arteries. The knockdown of Cx43 effectively attenuated the development of intimal hyperplasia and vascular restenosis following balloon injury. Thus, our data indicate the vital role of the GJ protein, Cx43, in the development of vascular restenosis, and provide new insight into the pathogenesis of vascular restenosis. Cx43 may prove to be a novel potential pharmacological target for the prevention of vascular restenosis following PCI.
|Suppression of adrenal βarrestin1-dependent aldosterone production by ARBs: head-to-head comparison. |
Dabul, S; Bathgate-Siryk, A; Valero, TR; Jafferjee, M; Sturchler, E; McDonald, P; Koch, WJ; Lymperopoulos, A
Scientific reports 5 8116 2015
The known angiotensin II (AngII) physiological effect of aldosterone synthesis and secretion is mediated by either Gq/11 proteins or βarrestin1 (βarr1), both of which can couple to its type 1 receptors (AT₁Rs), present in adrenocortical zona glomerulosa (AZG) cell membranes. In the present study, we examined the relative potencies of all the currently used in the clinic AT₁R antagonist drugs (angiotensin receptor blockers, ARBs, or sartans) at preventing activation of these two signaling mediators (G proteins and βarrs) at the AngII-bound AT1R and, consequently, at suppression of aldosterone in vitro. All ARBs were found to be potent inhibitors of G protein activation at the AT₁R. However, candesartan and valsartan were the most potent at blocking AngII-induced βarr activation at this receptor, among the tetrazolo-biphenyl-methyl derivatives, translating into excellent efficacies at aldosterone suppression in H295R cells. Conversely, irbesartan and losartan were largely G protein-selective inhibitors at the AT₁R, with very low potency towards βarr inhibition. As a result, they were very weak suppressors of βarr1-dependent aldosterone production in H295R cells. These findings provide important pharmacological insights into the drug class of ARBs and medicinal chemistry insights for future drug development in the field of AngII antagonism.
|Augmented AMPK activity inhibits cell migration by phosphorylating the novel substrate Pdlim5. |
Yan, Y; Tsukamoto, O; Nakano, A; Kato, H; Kioka, H; Ito, N; Higo, S; Yamazaki, S; Shintani, Y; Matsuoka, K; Liao, Y; Asanuma, H; Asakura, M; Takafuji, K; Minamino, T; Asano, Y; Kitakaze, M; Takashima, S
Nature communications 6 6137 2015
Augmented AMP-activated protein kinase (AMPK) activity inhibits cell migration, possibly contributing to the clinical benefits of chemical AMPK activators in preventing atherosclerosis, vascular remodelling and cancer metastasis. However, the underlying mechanisms remain largely unknown. Here we identify PDZ and LIM domain 5 (Pdlim5) as a novel AMPK substrate and show that it plays a critical role in the inhibition of cell migration. AMPK directly phosphorylates Pdlim5 at Ser177. Exogenous expression of phosphomimetic S177D-Pdlim5 inhibits cell migration and attenuates lamellipodia formation. Consistent with this observation, S177D-Pdlim5 suppresses Rac1 activity at the cell periphery and displaces the Arp2/3 complex from the leading edge. Notably, S177D-Pdlim5, but not WT-Pdlim5, attenuates the association with Rac1-specific guanine nucleotide exchange factors at the cell periphery. Taken together, our findings indicate that phosphorylation of Pdlim5 on Ser177 by AMPK mediates inhibition of cell migration by suppressing the Rac1-Arp2/3 signalling pathway.
|Inhibitor of DNA Binding 4 (ID4) is highly expressed in human melanoma tissues and may function to restrict normal differentiation of melanoma cells. |
Peretz, Y; Wu, H; Patel, S; Bellacosa, A; Katz, RA
PloS one 10 e0116839 2015
Melanoma tissues and cell lines are heterogeneous, and include cells with invasive, proliferative, stem cell-like, and differentiated properties. Such heterogeneity likely contributes to the aggressiveness of the disease and resistance to therapy. One model suggests that heterogeneity arises from rare cancer stem cells (CSCs) that produce distinct cancer cell lineages. Another model suggests that heterogeneity arises through reversible cellular plasticity, or phenotype-switching. Recent work indicates that phenotype-switching may include the ability of cancer cells to dedifferentiate to a stem cell-like state. We set out to investigate the phenotype-switching capabilities of melanoma cells, and used unbiased methods to identify genes that may control such switching. We developed a system to reversibly synchronize melanoma cells between 2D-monolayer and 3D-stem cell-like growth states. Melanoma cells maintained in the stem cell-like state showed a striking upregulation of a gene set related to development and neural stem cell biology, which included SRY-box 2 (SOX2) and Inhibitor of DNA Binding 4 (ID4). A gene set related to cancer cell motility and invasiveness was concomitantly downregulated. Intense and pervasive ID4 protein expression was detected in human melanoma tissue samples, suggesting disease relevance for this protein. SiRNA knockdown of ID4 inhibited switching from monolayer to 3D-stem cell-like growth, and instead promoted switching to a highly differentiated, neuronal-like morphology. We suggest that ID4 is upregulated in melanoma as part of a stem cell-like program that facilitates further adaptive plasticity. ID4 may contribute to disease by preventing stem cell-like melanoma cells from progressing to a normal differentiated state. This interpretation is guided by the known role of ID4 as a differentiation inhibitor during normal development. The melanoma stem cell-like state may be protected by factors such as ID4, thereby potentially identifying a new therapeutic vulnerability to drive differentiation to the normal cell phenotype.
|Direct Exposure to Ethanol Disrupts Junctional Cell-Cell Contact and Hippo-YAP Signaling in HL-1 Murine Atrial Cardiomyocytes. |
Noritake, K; Aki, T; Funakoshi, T; Unuma, K; Uemura, K
PloS one 10 e0136952 2015
Direct exposure of cardiomyocytes to ethanol causes cardiac damage such as cardiac arrythmias and apoptotic cell death. Cardiomyocytes are connected to each other through intercalated disks (ID), which are composed of a gap junction (GJ), adherens junction, and desmosome. Changes in the content as well as the subcellular localization of connexin43 (Cx43), the main component of the cardiac GJ, are reportedly involved in cardiac arrythmias and subsequent damage. Recently, the hippo-YAP signaling pathway, which links cellular physical status to cell proliferation, differentiation, and apoptosis, has been implicated in cardiac homeostasis under physiological as well as pathological conditions. This study was conducted to explore the possible involvement of junctional intercellular communication, mechanotransduction through cytoskeletal organization, and the hippo-YAP pathway in cardiac damage caused by direct exposure to ethanol. HL-1 murine atrial cardiac cells were used since these cells retain cardiac phenotypes through ID formation and subsequent synchronous contraction. Cells were exposed to 0.5-2% ethanol; significant apoptotic cell death was observed after exposure to 2% ethanol for 48 hours. A decrease in Cx43 levels was already observed after 3 hours exposure to 2% ethanol, suggesting a rapid degradation of this protein. Upon exposure to ethanol, Cx43 translocated into lysosomes. Cellular cytoskeletal organization was also dysregulated by ethanol, as demonstrated by the disruption of myofibrils and intermediate filaments. Coinciding with the loss of cell-cell adherence, decreased phosphorylation of YAP, a hippo pathway effector, was also observed in ethanol-treated cells. Taken together, the results provide evidence that cells exposed directly to ethanol show 1) impaired cell-cell adherence/communication, 2) decreased cellular mechanotransduction by the cytoskeleton, and 3) a suppressed hippo-YAP pathway. Suppression of hippo-YAP pathway signaling should be effective in maintaining cellular homeostasis in cardiomyocytes exposed to ethanol.
|BAD-mediated apoptotic pathway is associated with human cancer development. |
Stickles, XB; Marchion, DC; Bicaku, E; Al Sawah, E; Abbasi, F; Xiong, Y; Bou Zgheib, N; Boac, BM; Orr, BC; Judson, PL; Berry, A; Hakam, A; Wenham, RM; Apte, SM; Berglund, AE; Lancaster, JM
International journal of molecular medicine 35 1081-7 2015
The malignant transformation of normal cells is caused in part by aberrant gene expression disrupting the regulation of cell proliferation, apoptosis, senescence and DNA repair. Evidence suggests that the Bcl-2 antagonist of cell death (BAD)-mediated apoptotic pathway influences cancer chemoresistance. In the present study, we explored the role of the BAD-mediated apoptotic pathway in the development and progression of cancer. Using principal component analysis to derive a numeric score representing pathway expression, we evaluated clinico-genomic datasets (n=427) from corresponding normal, pre-invasive and invasive cancers of different types, such as ovarian, endometrial, breast and colon cancers in order to determine the associations between the BAD-mediated apoptotic pathway and cancer development. Immunofluorescence was used to compare the expression levels of phosphorylated BAD [pBAD (serine-112, -136 and -155)] in immortalized normal and invasive ovarian, colon and breast cancer cells. The expression of the BAD-mediated apoptotic pathway phosphatase, PP2C, was evaluated by RT-qPCR in the normal and ovarian cancer tissue samples. The growth-promoting effects of pBAD protein levels in the immortalized normal and cancer cells were assessed using siRNA depletion experiments with MTS assays. The expression of the BAD-mediated apoptotic pathway was associated with the development and/or progression of ovarian (n=106, pless than 0.001), breast (n=185, pless than 0.0008; n=61, p=0.04), colon (n=22, pless than 0.001) and endometrial (n=33, pless than 0.001) cancers, as well as with ovarian endometriosis (n=20, pless than 0.001). Higher pBAD protein levels were observed in the cancer cells compared to the immortalized normal cells, whereas PP2C gene expression was lower in the cancer compared to the ovarian tumor tissue samples (n=76, pless than 0.001). The increased pBAD protein levels after the depletion of PP2C conferred a growth advantage to the immortalized normal and cancer cells. The BAD-mediated apoptotic pathway is thus associated with the development of human cancers likely influenced by the protein levels of pBAD.
|B-cell precursor acute lymphoblastic leukemia and stromal cells communicate through Galectin-3. |
Fei, F; Joo, EJ; Tarighat, SS; Schiffer, I; Paz, H; Fabbri, M; Abdel-Azim, H; Groffen, J; Heisterkamp, N
Oncotarget 6 11378-94 2015
The molecular interactions between B-cell precursor acute lymphoblastic leukemia (pre-B ALL) cells and stromal cells in the bone marrow that provide microenvironmentally-mediated protection against therapeutic drugs are not well-defined. Galectin-3 (Lgals3) is a multifunctional galactose-binding lectin with reported location in the nucleus, cytoplasm and extracellular space in different cell types. We previously reported that ALL cells co-cultured with stroma contain high levels of Galectin-3. We here establish that, in contrast to more mature B-lineage cancers, Galectin-3 detected in and on the ALL cells originates from stromal cells, which express it on their surface, secrete it as soluble protein and also in exosomes. Soluble and stromal-bound Galectin-3 is internalized by ALL cells, transported to the nucleus and stimulates transcription of endogenous LGALS3 mRNA. When human and mouse ALL cells develop tolerance to different drugs while in contact with protective stromal cells, Galectin-3 protein levels are consistently increased. This correlates with induction of Galectin-3 transcription in the ALL cells. Thus Galectin-3 sourced from stroma becomes supplemented by endogenous Galectin-3 production in the pre-B ALL cells that are under continuous stress from drug treatment. Our data suggest that stromal Galectin-3 may protect ALL cells through auto-induction of Galectin-3 mRNA and tonic NFκB pathway activation. Since endogenously synthesized Galectin-3 protects pre-B ALL cells against drug treatment, we identify Galectin-3 as one possible target to counteract the protective effects of stroma.
|The mTORC1/4E-BP pathway coordinates hemoglobin production with L-leucine availability. |
Chung, J; Bauer, DE; Ghamari, A; Nizzi, CP; Deck, KM; Kingsley, PD; Yien, YY; Huston, NC; Chen, C; Schultz, IJ; Dalton, AJ; Wittig, JG; Palis, J; Orkin, SH; Lodish, HF; Eisenstein, RS; Cantor, AB; Paw, BH
Science signaling 8 ra34 2015
In multicellular organisms, the mechanisms by which diverse cell types acquire distinct amino acids and how cellular function adapts to their availability are fundamental questions in biology. We found that increased neutral essential amino acid (NEAA) uptake was a critical component of erythropoiesis. As red blood cells matured, expression of the amino acid transporter gene Lat3 increased, which increased NEAA import. Inadequate NEAA uptake by pharmacologic inhibition or RNAi-mediated knockdown of LAT3 triggered a specific reduction in hemoglobin production in zebrafish embryos and murine erythroid cells through the mTORC1 (mammalian target of rapamycin complex 1)/4E-BP (eukaryotic translation initiation factor 4E-binding protein) pathway. CRISPR-mediated deletion of members of the 4E-BP family in murine erythroid cells rendered them resistant to mTORC1 and LAT3 inhibition and restored hemoglobin production. These results identify a developmental role for LAT3 in red blood cells and demonstrate that mTORC1 serves as a homeostatic sensor that couples hemoglobin production at the translational level to sufficient uptake of NEAAs, particularly L-leucine.
|TGF-Beta Blockade Increases Renal Inflammation Caused by the C-Terminal Module of the CCN2. |
Rodrigues-Díez, R; Rayego-Mateos, S; Orejudo, M; Aroeira, LS; Selgas, R; Ortiz, A; Egido, J; Ruiz-Ortega, M
Mediators of inflammation 2015 506041 2015
The CCN family member 2 (CCN2, also known as connective tissue growth factor) may behave as a risk biomarker and a potential therapeutic target for renal disease. CCN2 participates in the regulation of inflammation and fibrosis. TGF-β is considered the main fibrogenic cytokine; however, in some pathological settings TGF-β also has anti-inflammatory properties. CCN2 has been proposed as a downstream profibrotic mediator of TGF-β, but data on TGF-β role in CCN2 actions are scarce. Our aim was to evaluate the effect of TGF-β blockade in CCN2-mediated experimental renal damage. Systemic administration of the C-terminal module of CCN2 to mice caused sustained renal inflammation. In these mice, TGF-β blockade, using an anti-TGF-β neutralizing antibody, significantly increased renal expression of the NGAL (a kidney injury biomarker), kidney infiltration by monocytes/macrophages, and upregulation of MCP-1 expression. The anti-inflammatory effect of TGF-β seems to be mediated by a dysregulation of the systemic Treg immune response, shown by decreased levels of circulating CD4(+)/Foxp3(+)Treg cells. Our experimental data support the idea that TGF-β exerts anti-inflammatory actions in the kidney and suggest that it is not an optimal therapeutic target.
|Connexin43 Mediated Delivery of ADAMTS5 Targeting siRNAs from Mesenchymal Stem Cells to Synovial Fibroblasts. |
Liu, S; Niger, C; Koh, EY; Stains, JP
PloS one 10 e0129999 2015
Osteoarthritis is a joint-destructive disease that has no effective cure. Human mesenchymal stem cells (hMSCs) could offer therapeutic benefit in the treatment of arthritic diseases by suppressing inflammation and permitting tissue regeneration, but first these cells must overcome the catabolic environment of the diseased joint. Likewise, gene therapy also offers therapeutic promise given its ability to directly modulate key catabolic factors that mediate joint deterioration, although it too has limitations. In the current study, we explore an approach that combines hMSCs and gene therapy. Specifically, we test the use of hMSC as a vehicle to deliver ADAMTS5 (an aggrecanase with a key role in osteoarthritis)-targeting siRNAs to SW982 synovial fibroblast-like cells via connexin43 containing gap junctions. Accordingly, we transduced hMSCs with ADAMTS5-targeting shRNA or non-targeted shRNA, and co-cultured them with synovial fibroblasts to allow delivery of siRNAs from hMSC to synovial fibroblasts. We found that co-culture of hMSCs-shRNA-ADAMTS5 and synovial fibroblasts reduced ADAMTS5 expression relative to co-culture of hMSCs-shRNA-control and synovial fibroblasts. Furthermore, ADAMTS5 was specifically reduced in the synovial fibroblasts populations as determined by fluorescence-activated cell sorting, suggesting transfer of the siRNA between cells. To test if Cx43-containing gap junctions are involved in the transfer of siRNA, we co-cultured hMSCs-shRNA-ADAMTS5 cells with synovial fibroblasts in which connexin43 was knocked down. Under these conditions, ADAMTS5 levels were not inhibited by co-culture, indicating that connexin43 mediates the delivery of siRNA from hMSCs to synovial fibroblasts. In total, our findings demonstrate that hMSCs can function as donor cells to host and deliver siRNAs to synovial fibroblasts via connexin43 gap junction in vitro. These data may have implications in the combination of hMSCs and gene therapy to treat diseases like osteoarthritis, in vivo.
|Fibroblast-epithelial cell interactions drive epithelial-mesenchymal transition differently in cells from normal and COPD patients. |
Nishioka, M; Venkatesan, N; Dessalle, K; Mogas, A; Kyoh, S; Lin, TY; Nair, P; Baglole, CJ; Eidelman, DH; Ludwig, MS; Hamid, Q
Respiratory research 16 72 2015
Epithelial-to-mesenchymal transition (EMT), which involves changes in cellular morphology of highly polarized epithelial cells and the gain of mesenchymal cell phenotype with migratory and invasive capacities, is implicated in smoking-related chronic obstructive pulmonary disease (COPD). However, the interactions of fibroblasts and epithelial cells and the participation of fibroblasts in the EMT processes in COPD are poorly understood. Here, we investigated the hypothesis that EMT is active in human bronchial epithelial (HBE) cells of COPD patients, and that mediators secreted by lung fibroblasts from COPD patients induce EMT.Primary HBE cells from normal subjects and COPD patients were purchased from LONZA. HLFs were derived from resected lung obtained from normal (N) and COPD (D) subjects and their conditioned medium (CM) was collected after 2-day culture in serum-free medium. The expression of epithelial and mesenchymal markers as well as EMT-related transcription factors in lung biopsies, and in HBE cells following stimulation with CM from both normal human lung fibroblasts (NHLF) and COPD human lung fibroblasts (DHLF) was evaluated by immunohistochemistry, qRT-PCR and western blot.Basal mRNA expression of mesenchymal markers and EMT-related transcription factors were increased in DHBE cells compared to normal human bronchial epithelial cells (NHBE) cells as well as in COPD lungs. CM from NHLF significantly induced vimentin expression in both NHBE and COPD human bronchial epithelial cells (DHBE) cells, but only increased N-cadherin expression in DHBE cells. CM from NHLF significantly induced Twist1 and Twist2 expression in NHBE cells and increased Snai2 (Slug) expression in DHBE cells. While CM from NHLF had no effect on such EMT markers, CM from DHLF significantly increased the protein expression of E-cadherin and vimentin in NHBE cells compared to control. N-cadherin expression was upregulated to a greater degree in NHBE cells than DHBE cells. Only CM from DHLF significantly increased E-/N-cadherin ratio in DHBE cells.Our results suggest that DHBE cells have partially undergone EMT under baseline conditions. DHLF-CM promoted EMT in NHBE, suggesting that interactions between fibroblast and epithelial cells may play an important role in the EMT process in COPD.
|CD44 plays a functional role in Helicobacter pylori-induced epithelial cell proliferation. |
Bertaux-Skeirik, N; Feng, R; Schumacher, MA; Li, J; Mahe, MM; Engevik, AC; Javier, JE; Peek, RM; Ottemann, K; Orian-Rousseau, V; Boivin, GP; Helmrath, MA; Zavros, Y
PLoS pathogens 11 e1004663 2015
The cytotoxin-associated gene (Cag) pathogenicity island is a strain-specific constituent of Helicobacter pylori (H. pylori) that augments cancer risk. CagA translocates into the cytoplasm where it stimulates cell signaling through the interaction with tyrosine kinase c-Met receptor, leading cellular proliferation. Identified as a potential gastric stem cell marker, cluster-of-differentiation (CD) CD44 also acts as a co-receptor for c-Met, but whether it plays a functional role in H. pylori-induced epithelial proliferation is unknown. We tested the hypothesis that CD44 plays a functional role in H. pylori-induced epithelial cell proliferation. To assay changes in gastric epithelial cell proliferation in relation to the direct interaction with H. pylori, human- and mouse-derived gastric organoids were infected with the G27 H. pylori strain or a mutant G27 strain bearing cagA deletion (∆CagA::cat). Epithelial proliferation was quantified by EdU immunostaining. Phosphorylation of c-Met was analyzed by immunoprecipitation followed by Western blot analysis for expression of CD44 and CagA. H. pylori infection of both mouse- and human-derived gastric organoids induced epithelial proliferation that correlated with c-Met phosphorylation. CagA and CD44 co-immunoprecipitated with phosphorylated c-Met. The formation of this complex did not occur in organoids infected with ∆CagA::cat. Epithelial proliferation in response to H. pylori infection was lost in infected organoids derived from CD44-deficient mouse stomachs. Human-derived fundic gastric organoids exhibited an induction in proliferation when infected with H. pylori that was not seen in organoids pre-treated with a peptide inhibitor specific to CD44. In the well-established Mongolian gerbil model of gastric cancer, animals treated with CD44 peptide inhibitor Pep1, resulted in the inhibition of H. pylori-induced proliferation and associated atrophic gastritis. The current study reports a unique approach to study H. pylori interaction with the human gastric epithelium. Here, we show that CD44 plays a functional role in H. pylori-induced epithelial cell proliferation.
|Loss of F-box only protein 2 (Fbxo2) disrupts levels and localization of select NMDA receptor subunits, and promotes aberrant synaptic connectivity. |
Atkin, G; Moore, S; Lu, Y; Nelson, RF; Tipper, N; Rajpal, G; Hunt, J; Tennant, W; Hell, JW; Murphy, GG; Paulson, H
The Journal of neuroscience : the official journal of the Society for Neuroscience 35 6165-78 2015
NMDA receptors (NMDARs) play an essential role in some forms of synaptic plasticity, learning, and memory. Therefore, these receptors are highly regulated with respect to their localization, activation, and abundance both within and on the surface of mammalian neurons. Fundamental questions remain, however, regarding how this complex regulation is achieved. Using cell-based models and F-box Only Protein 2 (Fbxo2) knock-out mice, we found that the ubiquitin ligase substrate adaptor protein Fbxo2, previously reported to facilitate the degradation of the NMDAR subunit GluN1 in vitro, also functions to regulate GluN1 and GluN2A subunit levels in the adult mouse brain. In contrast, GluN2B subunit levels are not affected by the loss of Fbxo2. The loss of Fbxo2 results in greater surface localization of GluN1 and GluN2A, together with increases in the synaptic markers PSD-95 and Vglut1. These synaptic changes do not manifest as neurophysiological differences or alterations in dendritic spine density in Fbxo2 knock-out mice, but result instead in increased axo-dendritic shaft synapses. Together, these findings suggest that Fbxo2 controls the abundance and localization of specific NMDAR subunits in the brain and may influence synapse formation and maintenance.
|Inhibition of ERBB2-overexpressing Tumors by Recombinant Human Prolidase and Its Enzymatically Inactive Mutant. |
Yang, L; Li, Y; Bhattacharya, A; Zhang, Y
EBioMedicine 2 396-405 2015
ERBB2 is an oncogenic receptor tyrosine kinase overexpressed in a subset of human breast cancer and other cancers. We recently found that human prolidase (PEPD), a dipeptidase, is a high affinity ERBB2 ligand and cross-links two ERBB2 monomers. Here, we show that recombinant human PEPD (rhPEPD) strongly inhibits ERBB2-overexpressing tumors in mice, whereas it does not impact tumors without ERBB2 overexpression. rhPEPD causes ERBB2 depletion, disrupts oncogenic signaling orchestrated by ERBB2 homodimers and heterodimers, and induces apoptosis. The impact of enzymatically-inactive mutant rhPEPD(G278D) on ERBB2 is indistinguishable from that of rhPEPD, but rhPEPD(G278D) is superior to rhPEPD for tumor inhibition. The enzymatic function of rhPEPD stimulates HIF-1α and other pro-survival factors in tumors, which likely attenuates its antitumor activity. rhPEPD(G278D) is also attractive in that it may not interfere with the physiologic function of endogenous PEPD in normal cells. Collectively, we have identified a human protein as an inhibitory ERBB2 ligand that inhibits ERBB2-overexpressing tumors in vivo. Several anti-ERBB2 agents are on the market but are hampered by drug resistance and high drug cost. rhPEPD(G278D) may synergize with these agents and may also be highly cost-effective, since it targets ERBB2 with a different mechanism and can be produced in bacteria.
|MURC/cavin-4 Is Co-Expressed with Caveolin-3 in Rhabdomyosarcoma Tumors and Its Silencing Prevents Myogenic Differentiation in the Human Embryonal RD Cell Line. |
Faggi, F; Codenotti, S; Poliani, PL; Cominelli, M; Chiarelli, N; Colombi, M; Vezzoli, M; Monti, E; Bono, F; Tulipano, G; Fiorentini, C; Zanola, A; Lo, HP; Parton, RG; Keller, C; Fanzani, A
PloS one 10 e0130287 2015
The purpose of this study was to investigate whether MURC/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Caveolin-3 (Cav-3) in heart and muscle tissues, may be expressed and play a role in rhabdomyosarcoma (RMS), an aggressive myogenic tumor affecting childhood. We found MURC/cavin-4 to be expressed, often concurrently with Cav-3, in mouse and human RMS, as demonstrated through in silico analysis of gene datasets and immunohistochemical analysis of tumor samples. In vitro expression studies carried out using human cell lines and primary mouse tumor cultures showed that expression levels of both MURC/cavin-4 and Cav-3, while being low or undetectable during cell proliferation, became robustly increased during myogenic differentiation, as detected via semi-quantitative RT-PCR and immunoblotting analysis. Furthermore, confocal microscopy analysis performed on human RD and RH30 cell lines confirmed that MURC/cavin-4 mostly marks differentiated cell elements, colocalizing at the cell surface with Cav-3 and labeling myosin heavy chain (MHC) expressing cells. Finally, MURC/cavin-4 silencing prevented the differentiation in the RD cell line, leading to morphological cell impairment characterized by depletion of myogenin, Cav-3 and MHC protein levels. Overall, our data suggest that MURC/cavin-4, especially in combination with Cav-3, may play a consistent role in the differentiation process of RMS.
|Huntington disease iPSCs show early molecular changes in intracellular signaling, the expression of oxidative stress proteins and the p53 pathway. |
Szlachcic, WJ; Switonski, PM; Krzyzosiak, WJ; Figlerowicz, M; Figiel, M
Disease models & mechanisms 8 1047-57 2015
Huntington disease (HD) is a brain disorder characterized by the late onset of motor and cognitive symptoms, even though the neurons in the brain begin to suffer dysfunction and degeneration long before symptoms appear. There is currently no cure. Several molecular and developmental effects of HD have been identified using neural stem cells (NSCs) and differentiated cells, such as neurons and astrocytes. Still, little is known regarding the molecular pathogenesis of HD in pluripotent cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Therefore, we examined putative signaling pathways and processes involved in HD pathogenesis in pluripotent cells. We tested naïve mouse HD YAC128 iPSCs and two types of human HD iPSC that were generated from HD and juvenile-HD patients. Surprisingly, we found that a number of changes affecting cellular processes in HD were also present in undifferentiated pluripotent HD iPSCs, including the dysregulation of the MAPK and Wnt signaling pathways and the dysregulation of the expression of genes related to oxidative stress, such as Sod1. Interestingly, a common protein interactor of the huntingtin protein and the proteins in the above pathways is p53, and the expression of p53 was dysregulated in HD YAC128 iPSCs and human HD iPSCs. In summary, our findings demonstrate that multiple molecular pathways that are characteristically dysregulated in HD are already altered in undifferentiated pluripotent cells and that the pathogenesis of HD might begin during the early stages of life.
|Erythropoietin produced by genetic-modified NIH/3T3 fibroblasts enhances the survival of degenerating neurons. |
Li, YC; Chen, SJ; Chien, CL
Brain and behavior 5 e00356 2015
Erythropoietin (EPO) has potent neuroprotective effects. The short-term delivery of high-dose EPO seemed to improve patients' neuromuscular functions; however, excessive EPO resulted in systematically high hematocrit and thrombotic risk. In our study, we established a cellular material for future in vivo studies of neurodegenerative diseases based on EPO provided regionally at a nontoxic level.A mouse EPO cDNA was subcloned into the pCMS-EGFP vector and transfected into NIH/3T3 fibroblasts to design a biological provider that can regionally release EPO for the treatment of neurological diseases. After G418 selection, a stable EPO-overexpressing cell line, EPO-3T3-EGFP, was established. To further confirm the neuroprotective abilities of secreted EPO from EPO-3T3-EGFP cells, a cell model of neurodegeneration, PC12-INT-EGFP, was applied.The expression level of EPO was highly elevated in EPO-3T3-EGFP cells, and an abundant amount of EPO secreted from EPO-3T3-EGFP cells was detected in the extracellular milieu. After supplementation with conditioned medium prepared from EPO-3T3-EGFP cells, the survival rate of PC12-INT-EGFP cells was significantly enhanced. Surprisingly, a fraction of aggregated cytoskeletal EGFP-tagged α-internexin in PC12-INT-EGFP cells was disaggregated and transported into neurites dynamically. The immunocytochemical distribution of IF proteins, including NF-M, phosphorylated-NF-M, and the α-INT-EGFP fusion protein, were less aggregated in the perikaryal region and transported into neurites after the EPO treatment.The established EPO-overexpressing NIH/3T3 cell line, EPO-3T3-EGFP, may provide a material for future studies of cell-based therapies for neurodegenerative diseases via the secretion of EPO on a short-term, high-dose, regional basis.
|A Glaucoma-Associated Variant of Optineurin, M98K, Activates Tbk1 to Enhance Autophagosome Formation and Retinal Cell Death Dependent on Ser177 Phosphorylation of Optineurin. |
Sirohi, K; Kumari, A; Radha, V; Swarup, G
PloS one 10 e0138289 2015
Certain missense mutations in optineurin/OPTN and amplification of TBK1 are associated with normal tension glaucoma. A glaucoma-associated variant of OPTN, M98K, induces autophagic degradation of transferrin receptor (TFRC) and death in retinal cells. Here, we have explored the role of Tbk1 in M98K-OPTN-induced autophagy and cell death, and the effect of Tbk1 overexpression in retinal cells. Cell death induced by M98K-OPTN was dependent on Tbk1 as seen by the effect of Tbk1 knockdown and blocking of Tbk1 activity by a chemical inhibitor. Inhibition of Tbk1 also restores M98K-OPTN-induced transferrin receptor degradation. M98K-OPTN-induced autophagosome formation, autophagy and cell death were dependent on its phosphorylation at S177 by Tbk1. Knockdown of OPTN reduced starvation-induced autophagosome formation. M98K-OPTN expressing cells showed higher levels of Tbk1 activation and enhanced phosphorylation at Ser177 compared to WT-OPTN expressing cells. M98K-OPTN-induced activation of Tbk1 and its ability to be phosphorylated better by Tbk1 was dependent on ubiquitin binding. Phosphorylated M98K-OPTN localized specifically to autophagosomes and endogenous Tbk1 showed increased localization to autophagosomes in M98K-OPTN expressing cells. Overexpression of Tbk1 induced cell death and caspase-3 activation that were dependent on its catalytic activity. Tbk1-induced cell death possibly involves autophagy, as shown by the effect of Atg5 knockdown, and requirement of autophagic function of OPTN. Our results show that phosphorylation of Ser177 plays a crucial role in M98K-OPTN-induced autophagosome formation, autophagy flux and retinal cell death. In addition, we provide evidence for cross talk between two glaucoma associated proteins and their inter-dependence to mediate autophagy-dependent cell death.
|Krüppel-like factor 6 regulates mitochondrial function in the kidney. |
Mallipattu, SK; Horne, SJ; D'Agati, V; Narla, G; Liu, R; Frohman, MA; Dickman, K; Chen, EY; Ma'ayan, A; Bialkowska, AB; Ghaleb, AM; Nandan, MO; Jain, MK; Daehn, I; Chuang, PY; Yang, VW; He, JC
The Journal of clinical investigation 125 1347-61 2015
Maintenance of mitochondrial structure and function is critical for preventing podocyte apoptosis and eventual glomerulosclerosis in the kidney; however, the transcription factors that regulate mitochondrial function in podocyte injury remain to be identified. Here, we identified Krüppel-like factor 6 (KLF6), a zinc finger domain transcription factor, as an essential regulator of mitochondrial function in podocyte apoptosis. We observed that podocyte-specific deletion of Klf6 increased the susceptibility of a resistant mouse strain to adriamycin-induced (ADR-induced) focal segmental glomerulosclerosis (FSGS). KLF6 expression was induced early in response to ADR in mice and cultured human podocytes, and prevented mitochondrial dysfunction and activation of intrinsic apoptotic pathways in these podocytes. Promoter analysis and chromatin immunoprecipitation studies revealed that putative KLF6 transcriptional binding sites are present in the promoter of the mitochondrial cytochrome c oxidase assembly gene (SCO2), which is critical for preventing cytochrome c release and activation of the intrinsic apoptotic pathway. Additionally, KLF6 expression was reduced in podocytes from HIV-1 transgenic mice as well as in renal biopsies from patients with HIV-associated nephropathy (HIVAN) and FSGS. Together, these findings indicate that KLF6-dependent regulation of the cytochrome c oxidase assembly gene is critical for maintaining mitochondrial function and preventing podocyte apoptosis.
|Topotecan synergizes with CHEK1 (CHK1) inhibitor to induce apoptosis in ovarian cancer cells. |
Kim, MK; James, J; Annunziata, CM
BMC cancer 15 196 2015
Topotecan (TPT) is a therapeutic option for women with platinum-resistant or -refractory ovarian cancer. However, the dose-limiting toxicity of TPT is myelosuppression. This led us to seek a combination treatment to augment TPT anti-cancer activity in a cancer-targeted manner. Ovarian serous cancers, a major subtype, show dysregulated DNA repair pathway and often display a high level of CHEK1 (CHK1), a cell cycle regulator and DNA damage sensor. CHEK1 inhibitors are a novel approach to treatment, and have been used as single agents or in combination chemotherapy in many cancers.We evaluated the cellular effects of TPT in a panel of high grade serous (HGS) and non-HGS ovarian cancer cells. We then determined IC50s of TPT in the absence and presence of CHEK1 inhibitor, PF477736. Synergism between TPT and PF477736 was calculated based on cellular viability assays. Cytotoxic effect of the combined treatment was compared with apoptotic activities by Caspase3/7 activity assay and Western blotting of cleaved-PARP1 and γH2AX.Non-HGS ovarian cancer cells were generally more sensitive to TPT treatment compared to HGS ovarian cancer cells. When combined with CHEK1 inhibitor, TPT potently and synergistically inhibited the proliferation of HGS ovarian cancer cells. This dramatic synergism in cellular toxicity was consistent with increases in markers of apoptosis.Our findings suggest that the addition of CHEK1 inhibitor increases the response of ovarian cancer cells to TPT. Furthermore, reduced dosages of both drugs achieved maximal cytotoxic effects by combining TPT with CHEK1 inhibitor. This strategy would potentially minimize side effects of the drugs for extended clinical benefit.
|Interferon-β suppresses murine Th1 cell function in the absence of antigen-presenting cells. |
Boivin, N; Baillargeon, J; Doss, PM; Roy, AP; Rangachari, M
PloS one 10 e0124802 2015
Interferon (IFN)-β is a front-line therapy for the treatment of the relapsing-remitting form of multiple sclerosis. However, its immunosuppressive mechanism of function remains incompletely understood. While it has been proposed that IFN-β suppresses the function of inflammatory myelin antigen-reactive T cells by promoting the release of immunomodulatory cytokines such as IL-27 from antigen-presenting cells (APCs), its direct effects on inflammatory CD4+ Th1 cells are less clear. Here, we establish that IFN-β inhibits mouse IFN-γ+ Th1 cell function in the absence of APCs. CD4+ T cells express the type I interferon receptor, and IFN-β can suppress Th1 cell proliferation under APC-free stimulation conditions. IFN-β-treated myelin antigen-specific Th1 cells are impaired in their ability to induce severe experimental autoimmune encephalomyelitis (EAE) upon transfer to lymphocyte-deficient Rag1-/- mice. Polarized Th1 cells downregulate IFN-γ and IL-2, and upregulate the negative regulatory receptor Tim-3, when treated with IFN-β in the absence of APCs. Further, IFN-β treatment of Th1 cells upregulates phosphorylation of Stat1, and downregulates phosphorylation of Stat4. Our data indicate that IFN-γ-producing Th1 cells are directly responsive to IFN-β and point to a novel mechanism of IFN-β-mediated T cell suppression that is independent of APC-derived signals.
|The p53R172H mutant does not enhance hepatocellular carcinoma development and progression. |
Ahronian, LG; Driscoll, DR; Klimstra, DS; Lewis, BC
PloS one 10 e0123816 2015
Hepatocellular carcinoma is a highly deadly malignancy, accounting for approximately 800,000 deaths worldwide every year. Mutation of the p53 tumor suppressor gene is a common genetic change in HCC, present in 30% of cases. p53R175H (corresponding to p53R172H in mice) is a hotspot for mutation that demonstrates "prometastatic" gain-of-function in other cancer models. Since the frequency of p53 mutation increases with tumor grade in HCC, we hypothesized that p53R172H is a gain-of-function mutation in HCC that contributes to a decrease in tumor-free survival and an increase in metastasis. In an HCC mouse model, we found that p53R172H/flox mice do not have decreased survival, increased tumor incidence, or increased metastasis, relative to p53flox/flox littermates. Analysis of cell lines derived from both genotypes indicated that there are no differences in anchorage-independent growth and cell migration. However, shRNA-mediated knockdown of mutant p53 in p53R172H-expressing HCC cell lines resulted in decreased cell migration and anchorage-independent growth. Thus, although p53 mutant-expressing cells and tumors do not have enhanced properties relative to their p53 null counterparts, p53R172H-expressing HCC cells depend on this mutant for their transformation. p53 mutants have been previously shown to bind and inhibit the p53 family proteins p63 and p73. Interestingly, we find that the levels of p63 and p73 target genes are similar in p53 mutant and p53 null HCC cells. These data suggest that pathways regulated by these p53 family members are similarly impacted by p53R172H in mutant expressing cells, and by alternate mechanisms in p53 null cells, resulting in equivalent phenotypes. Consistent with this, we find that p53 null HCC cell lines display lower levels of the TA isoforms of p63 and p73 and higher levels of ΔNp63. Taken together these data point to the importance of p63 and p73 in constraining HCC progression.
|Mesenchymal stem cells in rabbit meniscus and bone marrow exhibit a similar feature but a heterogeneous multi-differentiation potential: superiority of meniscus as a cell source for meniscus repair. |
Ding, Z; Huang, H
BMC musculoskeletal disorders 16 65 2015
The restoration of damaged meniscus has always been a challenge due to its limited healing capacity. Recently, bone marrow-derived mesenchymal stem cells (BMSCs) provide a promising alternative to repair meniscal defects. However, BMSCs are not ideal chondroprogenitor cells for meniscus repair because they have a high propensity for cartilage hypertrophy and bone formation. Our hypothesis is that mesenchymal stem cells (MSCs) reside in meniscus maintain specific traits distinct from others which may be more conducive to meniscus regeneration.MSCs were isolated from bone marrow and menisci of the rabbits. The similarities and differences between BMSCs and MMSCs were investigated in vitro by a cell culture model, ex vivo by a rabbit meniscus defect model and in vivo by a nude rat implantation model using histochemistry, immunocytochemistry, qRT-PCR and western blotting.Our data showed that two types of MSCs have universal stem cell characteristics including clonogenicity, multi-potency and self-renewal capacity. They both express stem cell markers including SSEA-4, Nanog, nucleostemin, strol-1, CD44 and CD90. However, MMSCs differed from BMSCs. MMSC colonies were much smaller and grew more slowly than BMSC colonies. Moreover, fewer MMSCs expressed CD34 than BMSCs. Finally, MMSCs always appeared a pronounced tendency to chondrogenic differentiation while BMSCs exhibited significantly greater osteogenic potential, whatever in vitro and in vivo.This study shows the similarities and differences between MMSCs and BMSCs for the first time. MMSCs are a promising source of mesenchymal stem cells in repairing meniscus defect.
|Pathophysiological role of microRNA-29 in pancreatic cancer stroma. |
Kwon, JJ; Nabinger, SC; Vega, Z; Sahu, SS; Alluri, RK; Abdul-Sater, Z; Yu, Z; Gore, J; Nalepa, G; Saxena, R; Korc, M; Kota, J
Scientific reports 5 11450 2015
Dense fibrotic stroma associated with pancreatic ductal adenocarcinoma (PDAC) is a major obstacle for drug delivery to the tumor bed and plays a crucial role in pancreatic cancer progression. Current, anti-stromal therapies have failed to improve tumor response to chemotherapy and patient survival. Furthermore, recent studies show that stroma impedes tumor progression, and its complete ablation accelerates PDAC progression. In an effort to understand the molecular mechanisms associated with tumor-stromal interactions, using in vitro and in vivo models and PDAC patient biopsies, we show that the loss of miR-29 is a common phenomenon of activated pancreatic stellate cells (PSCs)/fibroblasts, the major stromal cells responsible for fibrotic stromal reaction. Loss of miR-29 is correlated with a significant increase in extracellular matrix (ECM) deposition, a major component in PDAC stroma. Our in vitro miR-29 gain/loss-of-function studies document the role of miR-29 in PSC-mediated ECM stromal protein accumulation. Overexpression of miR-29 in activated stellate cells reduced stromal deposition, cancer cell viability, and cancer growth in co-culture. Furthermore, the loss of miR-29 in TGF-β1 activated PSCs is SMAD3 dependent. These results provide insights into the mechanistic role of miR-29 in PDAC stroma and its potential use as a therapeutic agent to target PDAC.
|Regulation of NKT cell-mediated immune responses to tumours and liver inflammation by mitochondrial PGAM5-Drp1 signalling. |
Kang, YJ; Bang, BR; Han, KH; Hong, L; Shim, EJ; Ma, J; Lerner, RA; Otsuka, M
Nature communications 6 8371 2015
The receptor-interacting protein kinase 3 (RIPK3) plays crucial roles in programmed necrosis and innate inflammatory responses. However, a little is known about the involvement of RIPK3 in NKT cell-mediated immune responses. Here, we demonstrate that RIPK3 plays an essential role in NKT cell function via activation of the mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5). RIPK3-mediated activation of PGAM5 promotes the expression of cytokines by facilitating nuclear translocation of NFAT and dephosphorylation of dynamin-related protein 1 (Drp1), a GTPase is essential for mitochondrial homoeostasis. Ripk3(-/-) mice show reduced NKT cell responses to metastatic tumour cells, and both deletion of RIPK3 and pharmacological inhibition of Drp1 protects mice from NKT cell-mediated induction of acute liver damage. Collectively, the results identify a crucial role for RIPK3-PGAM5-Drp1/NFAT signalling in NKT cell activation, and further suggest that RIPK3-PGAM5 signalling may mediate crosstalk between mitochondrial function and immune signalling.
|Behavior-linked FoxP2 regulation enables zebra finch vocal learning. |
Heston, JB; White, SA
The Journal of neuroscience : the official journal of the Society for Neuroscience 35 2885-94 2015
Mutations in the FOXP2 transcription factor cause an inherited speech and language disorder, but how FoxP2 contributes to learning of these vocal communication signals remains unclear. FoxP2 is enriched in corticostriatal circuits of both human and songbird brains. Experimental knockdown of this enrichment in song control neurons of the zebra finch basal ganglia impairs tutor song imitation, indicating that adequate FoxP2 levels are necessary for normal vocal learning. In unmanipulated birds, vocal practice acutely downregulates FoxP2, leading to increased vocal variability and dynamic regulation of FoxP2 target genes. To determine whether this behavioral regulation is important for song learning, here, we used viral-driven overexpression of FoxP2 to counteract its downregulation. This manipulation disrupted the acute effects of song practice on vocal variability and caused inaccurate song imitation. Together, these findings indicate that dynamic behavior-linked regulation of FoxP2, rather than absolute levels, is critical for vocal learning.
|A plastic relationship between vinculin-mediated tension and adhesion complex area defines adhesion size and lifetime. |
Hernández-Varas, P; Berge, U; Lock, JG; Strömblad, S
Nature communications 6 7524 2015
Cell-matrix adhesions are central mediators of mechanotransduction, yet the interplay between force and adhesion regulation remains unclear. Here we use live cell imaging to map time-dependent cross-correlations between vinculin-mediated tension and adhesion complex area, revealing a plastic, context-dependent relationship. Interestingly, while an expected positive cross-correlation dominated in mid-sized adhesions, small and large adhesions display negative cross-correlation. Furthermore, although large changes in adhesion complex area follow vinculin-mediated tension alterations, small increases in area precede vinculin-mediated tension dynamics. Modelling based on this mapping of the vinculin-mediated tension-adhesion complex area relationship confirms its biological validity, and indicates that this relationship explains adhesion size and lifetime limits, keeping adhesions focal and transient. We also identify a subpopulation of steady-state adhesions whose size and vinculin-mediated tension become stabilized, and whose disassembly may be selectively microtubule-mediated. In conclusion, we define a plastic relationship between vinculin-mediated tension and adhesion complex area that controls fundamental cell-matrix adhesion properties.
|Prenatal ethanol exposure alters adult hippocampal VGLUT2 expression with concomitant changes in promoter DNA methylation, H3K4 trimethylation and miR-467b-5p levels. |
Zhang, CR; Ho, MF; Vega, MC; Burne, TH; Chong, S
Epigenetics & chromatin 8 40 2015
Maternal consumption of alcohol during pregnancy is associated with a range of physical, cognitive and behavioural outcomes in the offspring which are collectively called foetal alcohol spectrum disorders. We and others have proposed that epigenetic modifications, such as DNA methylation and post-translational histone modifications, mediate the effects of prenatal alcohol exposure on gene expression and, ultimately, phenotype. Here we use an inbred C57BL/6J mouse model of early gestational ethanol exposure equivalent, developmentally, to the first 3-4 weeks of pregnancy in humans to examine the long-term effects on gene expression and epigenetic state in the hippocampus.Gene expression analysis in the hippocampus revealed sex- and age-specific up-regulation of solute carrier family 17 member 6 (Slc17a6), which encodes vesicular glutamate transporter 2 (VGLUT2). Transcriptional up-regulation correlated with decreased DNA methylation and enrichment of histone H3 lysine 4 trimethylation, an active chromatin mark, at the Slc17a6 promoter. In contrast to Slc17a6 mRNA levels, hippocampal VGLUT2 protein levels were significantly decreased in adult ethanol-exposed offspring, suggesting an additional level of post-transcriptional control. MicroRNA expression profiling in the hippocampus identified four ethanol-sensitive microRNAs, of which miR-467b-5p was predicted to target Slc17a6. In vitro reporter assays showed that miR-467b-5p specifically interacted with the 3'UTR of Slc17a6, suggesting that it contributes to the reduction of hippocampal VGLUT2 in vivo. A significant correlation between microRNA expression in the hippocampus and serum of ethanol-exposed offspring was also observed.Prenatal ethanol exposure has complex transcriptional and post-transcriptional effects on Slc17a6 (VGLUT2) expression in the mouse hippocampus. These effects are observed following a relatively moderate exposure that is restricted to early pregnancy, modelling human consumption of alcohol before pregnancy is confirmed, and are only apparent in male offspring in adulthood. Our findings are consistent with the idea that altered epigenetic and/or microRNA-mediated regulation of glutamate neurotransmission in the hippocampus contributes to the cognitive and behavioural phenotypes observed in foetal alcohol spectrum disorders. Although further work is needed in both mice and humans, the results also suggest that circulating microRNAs could be used as biomarkers of early gestational ethanol exposure and hippocampal dysfunction.
|Neurological effects of inorganic arsenic exposure: altered cysteine/glutamate transport, NMDA expression and spatial memory impairment. |
Ramos-Chávez, LA; Rendón-López, CR; Zepeda, A; Silva-Adaya, D; Del Razo, LM; Gonsebatt, ME
Frontiers in cellular neuroscience 9 21 2015
Inorganic arsenic (iAs) is an important natural pollutant. Millions of individuals worldwide drink water with high levels of iAs. Chronic exposure to iAs has been associated with lower IQ and learning disabilities as well as memory impairment. iAs is methylated in tissues such as the brain generating mono and dimethylated species. iAs methylation requires cellular glutathione (GSH), which is the main antioxidant in the central nervous system (CNS). In humans, As species cross the placenta and are found in cord blood. A CD1 mouse model was used to investigate effects of gestational iAs exposure which can lead to oxidative damage, disrupted cysteine/glutamate transport and its putative impact in learning and memory. On postnatal days (PNDs) 1, 15 and 90, the expression of membrane transporters related to GSH synthesis and glutamate transport and toxicity, such as xCT, EAAC1, GLAST and GLT1, as well as LAT1, were analyzed. Also, the expression of the glutamate receptor N-methyl-D-aspartate (NMDAR) subunits NR2A and B as well as the presence of As species in cortex and hippocampus were investigated. On PND 90, an object location task was performed to associate exposure with memory impairment. Gestational exposure to iAs affected the expression of cysteine/glutamate transporters in cortex and hippocampus and induced a negative modulation of NMDAR NR2B subunit in the hippocampus. Behavioral tasks showed significant spatial memory impairment in males while the effect was marginal in females.
|Ageing is associated with deterioration of calcium homeostasis in isolated human right atrial myocytes. |
Herraiz-Martínez, A; Álvarez-García, J; Llach, A; Molina, CE; Fernandes, J; Ferrero-Gregori, A; Rodríguez, C; Vallmitjana, A; Benítez, R; Padró, JM; Martínez-González, J; Cinca, J; Hove-Madsen, L
Cardiovascular research 106 76-86 2015
Ageing-related cardiac disorders such as heart failure and atrial fibrillation often present with intracellular calcium homeostasis dysfunction. However, knowledge of the intrinsic effects of ageing on cellular calcium handling in the human heart is sparse. Therefore, this study aimed to analyse how ageing affects key mechanisms that regulate intracellular calcium in human atrial myocytes.Whole membrane currents and intracellular calcium transients were measured in isolated human right atrial myocytes from 80 patients with normal left atrial dimensions and no history of atrial fibrillation. Patients were categorized as young (less than 55 years, n = 21), middle aged (55-74 years, n = 42), and old (≥75 years, n = 17). Protein levels were determined by western blot. Ageing was associated with the following electrophysiological changes: (i) a 3.2-fold decrease in the calcium transient (P less than 0.01); (ii) reduction of the L-type calcium current (ICa) amplitude (2.4 ± 0.3 pA/pF vs. 1.4 ± 0.2 pA/pF, P less than 0.01); (iii) lower levels of L-type calcium channel alpha-subunit (P less than 0.05); (iv) lower rates of both fast (14.5 ± 0.9 ms vs. 20.9 ± 1.9, P less than 0.01) and slow (73 ± 3 vs. 120 ± 12 ms, P less than 0.001) ICa inactivation; and (v) a decrease in the sarcoplasmic reticulum calcium content (10.1 ± 0.8 vs. 6.4 ± 0.6 amol/pF, P less than 0.005) associated with a significant decrease in both SERCA2 (P less than 0.05) and calsequestrin-2 (P less than 0.05) protein levels. In contrast, ageing did not affect spontaneous sarcoplasmic reticulum calcium release.Ageing is associated with depression of SR calcium content, L-type calcium current, and calcium transient amplitude that may favour a progressive decline in right atrial contractile function with age.
|Nuclear p120-catenin regulates the anoikis resistance of mouse lobular breast cancer cells through Kaiso-dependent Wnt11 expression. |
van de Ven, RA; Tenhagen, M; Meuleman, W; van Riel, JJ; Schackmann, RC; Derksen, PW
Disease models & mechanisms 8 373-84 2015
E-cadherin inactivation underpins the progression of invasive lobular breast carcinoma (ILC). In ILC, p120-catenin (p120) translocates to the cytosol where it controls anchorage independence through the Rho-Rock signaling pathway, a key mechanism driving tumor growth and metastasis. We now demonstrate that anchorage-independent ILC cells show an increase in nuclear p120, which results in relief of transcriptional repression by Kaiso. To identify the Kaiso target genes that control anchorage independence we performed genome-wide mRNA profiling on anoikis-resistant mouse ILC cells, and identified 29 candidate target genes, including the established Kaiso target Wnt11. Our data indicate that anchorage-independent upregulation of Wnt11 in ILC cells is controlled by nuclear p120 through inhibition of Kaiso-mediated transcriptional repression. Finally, we show that Wnt11 promotes activation of RhoA, which causes ILC anoikis resistance. Our findings thereby establish a mechanistic link between E-cadherin loss and subsequent control of Rho-driven anoikis resistance through p120- and Kaiso-dependent expression of Wnt11.
|Calpain-Mediated Degradation of Drebrin by Excitotoxicity In vitro and In vivo. |
Chimura, T; Launey, T; Yoshida, N
PloS one 10 e0125119 2015
The level of drebrin, an evolutionarily conserved f-actin-binding protein that regulates synaptic structure and function, is reduced in the brains of patients with chronic neurodegenerative diseases such as Alzheimer's disease (AD) and Down's syndrome (DS). It was suggested that excitotoxic neuronal death caused by overactivation of NMDA-type glutamate receptors (NMDARs) occurs in AD and DS; however, the relationship between excitotoxicity and drebrin loss is unknown. Here, we show that drebrin is a novel target of calpain-mediated proteolysis under excitotoxic conditions induced by the overactivation of NMDARs. In cultured rodent neurons, degradation of drebrin was confirmed by the detection of proteolytic fragments, as well as a reduction in the amount of full-length drebrin. Notably, the NMDA-induced degradation of drebrin in mature neurons occurred concomitantly with a loss of f-actin. Furthermore, pharmacological inhibition of f-actin loss facilitated the drebrin degradation, suggesting a functional linkage between f-actin and drebrin degradation. Biochemical analyses using purified drebrin and calpain revealed that calpain degraded drebrin directly in vitro. Furthermore, cerebral ischemia also induced the degradation of drebrin in vivo. These findings suggest that calpain-mediated degradation of drebrin is a fundamental pathology of neurodegenerative diseases mediated by excitotoxicity, regardless of whether they are acute or chronic. Drebrin regulates the synaptic clustering of NMDARs; therefore, degradation of drebrin under excitotoxic conditions may modulate NMDAR-mediated signal transductions, including pro-survival signaling. Overall, the results presented here provide novel insights into the molecular basis of cellular responses to excitotoxicity in vitro and in vivo.
|Dendritic Cell-Mediated Phagocytosis but Not Immune Activation Is Enhanced by Plasmin. |
Borg, RJ; Samson, AL; Au, AE; Scholzen, A; Fuchsberger, M; Kong, YY; Freeman, R; Mifsud, NA; Plebanski, M; Medcalf, RL
PloS one 10 e0131216 2015
Removal of dead cells in the absence of concomitant immune stimulation is essential for tissue homeostasis. We recently identified an injury-induced protein misfolding event that orchestrates the plasmin-dependent proteolytic degradation of necrotic cells. As impaired clearance of dead cells by the innate immune system predisposes to autoimmunity, we determined whether plasmin could influence endocytosis and immune cell stimulation by dendritic cells - a critical cell that links the innate and adaptive immune systems. We find that plasmin generated on the surface of necrotic cells enhances their phagocytic removal by human monocyte-derived dendritic cells. Plasmin also promoted phagocytosis of protease-resistant microparticles by diverse mouse dendritic cell sub-types both in vitro and in vivo. Together with an increased phagocytic capacity, plasmin-treated dendritic cells maintain an immature phenotype, exhibit reduced migration to lymph nodes, increase their expression/release of the immunosuppressive cytokine TGF-β, and lose their capacity to mount an allogeneic response. Collectively, our findings support a novel role for plasmin formed on dead cells and other phagocytic targets in maintaining tissue homeostasis by increasing the phagocytic function of dendritic cells while simultaneously decreasing their immunostimulatory capacity consistent with producing an immunosuppressive state.
|Further Insights into the Allan-Herndon-Dudley Syndrome: Clinical and Functional Characterization of a Novel MCT8 Mutation. |
Armour, CM; Kersseboom, S; Yoon, G; Visser, TJ
PloS one 10 e0139343 2015
Mutations in the thyroid hormone (TH) transporter MCT8 have been identified as the cause for Allan-Herndon-Dudley Syndrome (AHDS), characterized by severe psychomotor retardation and altered TH serum levels. Here we report a novel MCT8 mutation identified in 4 generations of one family, and its functional characterization.Proband and family members were screened for 60 genes involved in X-linked cognitive impairment and the MCT8 mutation was confirmed. Functional consequences of MCT8 mutations were studied by analysis of [125I]TH transport in fibroblasts and transiently transfected JEG3 and COS1 cells, and by subcellular localization of the transporter.The proband and a male cousin demonstrated clinical findings characteristic of AHDS. Serum analysis showed high T3, low rT3, and normal T4 and TSH levels in the proband. A MCT8 mutation (c.869Cgreater than T; p.S290F) was identified in the proband, his cousin, and several female carriers. Functional analysis of the S290F mutant showed decreased TH transport, metabolism and protein expression in the three cell types, whereas the S290A mutation had no effect. Interestingly, both uptake and efflux of T3 and T4 was impaired in fibroblasts of the proband, compared to his healthy brother. However, no effect of the S290F mutation was observed on TH efflux from COS1 and JEG3 cells. Immunocytochemistry showed plasma membrane localization of wild-type MCT8 and the S290A and S290F mutants in JEG3 cells.We describe a novel MCT8 mutation (S290F) in 4 generations of a family with Allan-Herndon-Dudley Syndrome. Functional analysis demonstrates loss-of-function of the MCT8 transporter. Furthermore, our results indicate that the function of the S290F mutant is dependent on cell context. Comparison of the S290F and S290A mutants indicates that it is not the loss of Ser but its substitution with Phe, which leads to S290F dysfunction.
|Autophagy is a critical regulator of memory CD8(+) T cell formation. |
Puleston, DJ; Zhang, H; Powell, TJ; Lipina, E; Sims, S; Panse, I; Watson, AS; Cerundolo, V; Townsend, AR; Klenerman, P; Simon, AK
eLife 3 2014
During infection, CD8(+) T cells initially expand then contract, leaving a small memory pool providing long lasting immunity. While it has been described that CD8(+) T cell memory formation becomes defective in old age, the cellular mechanism is largely unknown. Autophagy is a major cellular lysosomal degradation pathway of bulk material, and levels are known to fall with age. In this study, we describe a novel role for autophagy in CD8(+) T cell memory formation. Mice lacking the autophagy gene Atg7 in T cells failed to establish CD8(+) T cell memory to influenza and MCMV infection. Interestingly, autophagy levels were diminished in CD8(+) T cells from aged mice. We could rejuvenate CD8(+) T cell responses in elderly mice in an autophagy dependent manner using the compound spermidine. This study reveals a cell intrinsic explanation for poor CD8(+) T cell memory in the elderly and potentially offers novel immune modulators to improve aged immunity.
|Partial restoration of cardiac function with ΔPDZ nNOS in aged mdx model of Duchenne cardiomyopathy. |
Lai, Yi, et al.
Hum. Mol. Genet., (2014) 2014
Transgenic gene deletion/over-expression studies have established the cardioprotective role of neuronal nitric oxide synthase (nNOS). However, it remains unclear whether nNOS-mediated heart protection can be translated to gene therapy. In this study, we generated an adeno-associated virus (AAV) nNOS vector and tested its therapeutic efficacy in the aged mdx model of Duchenne cardiomyopathy. A PDZ domain-deleted nNOS gene (ΔPDZ nNOS) was packaged into tyrosine mutant AAV-9 and delivered to the heart of ∼14-month-old female mdx mice, a phenotypic model of Duchenne cardiomyopathy. Seven months later, we observed robust nNOS expression in the myocardium. Supra-physiological ΔPDZ nNOS expression significantly reduced myocardial fibrosis, inflammation and apoptosis. Importantly, electrocardiography and left ventricular hemodynamics were significantly improved in treated mice. Additional studies revealed increased phosphorylation of phospholamban and p70S6K. Collectively, we have demonstrated the therapeutic efficacy of the AAV ΔPDZ nNOS vector in a symptomatic Duchenne cardiomyopathy model. Our results suggest that the cardioprotective role of ΔPDZ nNOS is likely through reduced apoptosis, enhanced phospholamban phosphorylation and improved Akt/mTOR/p70S6K signaling. Our study has opened the door to treat Duchenne cardiomyopathy with ΔPDZ nNOS gene transfer.
|Effect of pirfenidone on proliferation, TGF-β-induced myofibroblast differentiation and fibrogenic activity of primary human lung fibroblasts. |
Conte, Enrico, et al.
Eur J Pharm Sci, 58C: 13-19 (2014) 2014
Pirfenidone is an orally active small molecule that has been shown to inhibit the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis. Although pirfenidone exhibits well documented antifibrotic and antiinflammatory activities, in vitro and in vivo, its molecular targets and mechanisms of action have not been elucidated. In this study, we investigated the effects of pirfenidone on proliferation, TGF-β-induced differentiation and fibrogenic activity of primary human lung fibroblasts (HLFs). Pirfenidone reduced fibroblast proliferation and attenuated TGF-β-induced α-smooth muscle actin (SMA) and pro-collagen (Col)-I mRNA and protein levels. Importantly, pirfenidone inhibited TGF-β-induced phosphorylation of Smad3, p38, and Akt, key factors in the TGF-β pathway. Together, these results demonstrate that pirfenidone modulates HLF proliferation and TGF-β-mediated differentiation into myofibroblasts by attenuating key TGF-β-induced signaling pathways.
|A majority of human melanoma cell lines exhibits an S phase-specific defect in excision of UV-induced DNA photoproducts. |
Bélanger, F; Rajotte, V; Drobetsky, EA
PloS one 9 e85294 2014
It is well established that efficient removal of highly-promutagenic UV-induced dipyrimidine photoproducts via nucleotide excision repair (NER) is required for protection against sunlight-associated malignant melanoma. Nonetheless, the extent to which reduced NER capacity might contribute to individual melanoma susceptibility in the general population remains unclear. Here we show that among a panel of 14 human melanoma strains, 11 exhibit significant inhibition of DNA photoproduct removal during S phase relative to G0/G1 or G2/M. Evidence is presented that this cell cycle-specific NER defect correlates with enhanced apoptosis and reduced clonogenic survival following UV irradiation. In addition, melanoma strains deficient in S phase-specific DNA photoproduct removal manifest significantly lower levels of phosphorylated histone H2AX at 1 h post-UV, suggesting diminished activation of ataxia telangiectasia and Rad 3-related (ATR) kinase, i.e., a primary orchestrator of the cellular response to UV-induced DNA replication stress. Consistently, in the case of DNA photoproduct excision-proficient melanoma cells, siRNA-mediated depletion of ATR (but not of its immediate downstream effector kinase Chk1) engenders deficient NER specifically during S. On the other hand simultaneous siRNA-mediated depletion of ataxia telangiectasia mutated kinase (ATM) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) exerts no significant effect on either phosphorylation of H2AX at 1 h post-UV or the efficiency of DNA photoproduct removal. Our data suggest that defective NER exclusively during S phase, possibly associated with decreased ATR signaling, may constitute an heretofore unrecognized determinant in melanoma pathogenesis.
|Lys11- and Lys48-linked ubiquitin chains interact with p97 during endoplasmic-reticulum-associated degradation. |
Locke, M; Toth, JI; Petroski, MD
The Biochemical journal 459 205-16 2014
The ATPase associated with various cellular activities p97 has a critical function in the cytoplasmic degradation of proteins misfolded in the ER (endoplasmic reticulum) through a mechanism known as ERAD (ER-associated degradation). During this process, p97 binds polyubiquitinated ERAD substrates and couples ATP hydrolysis to their dislocation from the ER as a prerequisite to destruction by the proteasome. The ubiquitin signals important for this process are not fully understood. In the present paper we report that p97 interacts with Lys11- and Lys48-linked ubiquitin polymers, but not those containing Lys63 linkages. Disruption of p97 through siRNA-mediated depletion, dominant-negative overexpression or chemical inhibition results in the accumulation of Lys11 and Lys48 ubiquitin chains predominantly at the ER membrane, and is associated with ER stress induction. We show that a catalytically inactive deubiquitinating enzyme and p97 cofactor YOD1 enhances the accumulation of Lys11- and Lys48-linked polyubiquitin in the cytoplasm, at the ER membrane and bound to p97. In addition to general effects on p97-associated ubiquitin polymers, the ERAD substrate CD3δ is modified with both Lys11 and Lys48 ubiquitin chains prior to p97-dependent dislocation. Collectively, the results of the present study are consistent with a major role for p97 in the recognition of Lys11 and Lys48 polyubiquitinated proteins before their degradation by the proteasome.
|The (pro)renin receptor mediates constitutive PLZF-independent pro-proliferative effects which are inhibited by bafilomycin but not genistein. |
Kirsch, S; Schrezenmeier, E; Klare, S; Zaade, D; Seidel, K; Schmitz, J; Bernhard, S; Lauer, D; Slack, M; Goldin-Lang, P; Unger, T; Zollmann, FS; Funke-Kaiser, H
International journal of molecular medicine 33 795-808 2014
The (pro)renin receptor [(P)RR] is crucial for cardio-renal pathophysiology. The distinct molecular mechanisms of this receptor are still incompletely understood. The (P)RR is able to interact with different signalling proteins such as promyelocytic leukemia zinc finger protein (PLZF) and Wnt receptors. Moreover, domains of the (P)RR are essential for V-ATPase activity. V-ATPase- and Wnt-mediated effects imply constitutive, i.e., (pro)renin-independent functions of the (P)RR. Regarding ligand-dependent (P)RR signalling, the role of prorenin glycosylation is currently unknown. Therefore, the aim of this study was to analyse the contribution of constitutive (P)RR activity to its cellular effects and the relevance of prorenin glycosylation on its ligand activity. We were able to demonstrate that high glucose induces (P)RR signal transduction whereas deglycosylation of prorenin abolishes its intrinsic activity in neuronal and epithelial cells. By using siRNA against (P)RR or PLZF as well as the PLZF translocation blocker genistein and the specific V-ATPase inhibitor bafilomycin, we were able to dissect three distinct sub-pathways downstream of the (P)RR. The V-ATPase function is ligand-independently associated with strong pro-proliferative effects whereas prorenin causes moderate proliferation in vitro. In contrast, PLZF per se [i.e., in the absence of (pro)renin] does not interfere with cell number.
|Gambogenic acid kills lung cancer cells through aberrant autophagy. |
Mei, W; Dong, C; Hui, C; Bin, L; Fenggen, Y; Jingjing, S; Cheng, P; Meiling, S; Yawen, H; Xiaoshan, W; Guanghui, W; Zhiwu, C; Qinglin, L
PloS one 9 e83604 2014
Lung cancer is one of the most common types of cancer and causes 1.38 million deaths annually, as of 2008 worldwide. Identifying natural anti-lung cancer agents has become very important. Gambogenic acid (GNA) is one of the active compounds of Gamboge, a traditional medicine that was used as a drastic purgative, emetic, or vermifuge for treating tapeworm. Recently, increasing evidence has indicated that GNA exerts promising anti-tumor effects; however, the underlying mechanism remains unclear. In the present paper, we found that GNA could induce the formation of vacuoles, which was linked with autophagy in A549 and HeLa cells. Further studies revealed that GNA triggers the initiation of autophagy based on the results of MDC staining, AO staining, accumulation of LC3 II, activation of Beclin 1 and phosphorylation of P70S6K. However, degradation of p62 was disrupted and free GFP could not be released in GNA treated cells, which indicated a block in the autophagy flux. Further studies demonstrated that GNA blocks the fusion between autophagosomes and lysosomes by inhibiting acidification in lysosomes. This dysfunctional autophagy plays a pro-death role in GNA-treated cells by activating p53, Bax and cleaved caspase-3 while decreasing Bcl-2. Beclin 1 knockdown greatly decreased GNA-induced cell death and the effects on p53, Bax, cleaved caspase-3 and Bcl-2. Similar results were obtained using a xenograft model. Our findings show, for the first time, that GNA can cause aberrant autophagy to induce cell death and may suggest the potential application of GNA as a tool or viable drug in anticancer therapies.
|Gene and MicroRNA transcriptome analysis of Parkinson's related LRRK2 mouse models. |
Dorval, V; Mandemakers, W; Jolivette, F; Coudert, L; Mazroui, R; De Strooper, B; Hébert, SS
PloS one 9 e85510 2014
Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of genetic Parkinson's disease (PD). The biological function of LRRK2 and how mutations lead to disease remain poorly defined. It has been proposed that LRRK2 could function in gene transcription regulation; however, this issue remains controversial. Here, we investigated in parallel gene and microRNA (miRNA) transcriptome profiles of three different LRRK2 mouse models. Striatal tissue was isolated from adult LRRK2 knockout (KO) mice, as well as mice expressing human LRRK2 wildtype (hLRRK2-WT) or the PD-associated R1441G mutation (hLRRK2-R1441G). We identified a total of 761 genes and 24 miRNAs that were misregulated in the absence of LRRK2 when a false discovery rate of 0.2 was applied. Notably, most changes in gene expression were modest (i.e., less than 2 fold). By real-time quantitative RT-PCR, we confirmed the variations of selected genes (e.g., adra2, syt2, opalin) and miRNAs (e.g., miR-16, miR-25). Surprisingly, little or no changes in gene expression were observed in mice expressing hLRRK2-WT or hLRRK2-R1441G when compared to non-transgenic controls. Nevertheless, a number of miRNAs were misexpressed in these models. Bioinformatics analysis identified several miRNA-dependent and independent networks dysregulated in LRRK2-deficient mice, including PD-related pathways. These results suggest that brain LRRK2 plays an overall modest role in gene transcription regulation in mammals; however, these effects seem context and RNA type-dependent. Our data thus set the stage for future investigations regarding LRRK2 function in PD development.
|Modulation of mitochondrial function and autophagy mediates carnosine neuroprotection against ischemic brain damage. |
Baek, SH; Noh, AR; Kim, KA; Akram, M; Shin, YJ; Kim, ES; Yu, SW; Majid, A; Bae, ON
Stroke; a journal of cerebral circulation 45 2438-43 2014
Despite the rapidly increasing global burden of ischemic stroke, no therapeutic options for neuroprotection against stroke currently exist. Recent studies have shown that autophagy plays a key role in ischemic neuronal death, and treatments that target autophagy may represent a novel strategy in neuroprotection. We investigated whether autophagy is regulated by carnosine, an endogenous pleiotropic dipeptide that has robust neuroprotective activity against ischemic brain damage.We examined the effect of carnosine on mitochondrial dysfunction and autophagic processes in rat focal ischemia and in neuronal cultures.Autophagic pathways such as reduction of phosphorylated mammalian target of rapamycin (mTOR)/p70S6K and the conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II were enhanced in the ischemic brain. However, treatment with carnosine significantly attenuated autophagic signaling in the ischemic brain, with improvement of brain mitochondrial function and mitophagy signaling. The protective effect of carnosine against autophagy was also confirmed in primary cortical neurons.Taken together, our data suggest that the neuroprotective effect of carnosine is at least partially mediated by mitochondrial protection and attenuation of deleterious autophagic processes. Our findings shed new light on the mechanistic pathways that this exciting neuroprotective agent influences.
|MALT1 auto-proteolysis is essential for NF-κB-dependent gene transcription in activated lymphocytes. |
Baens, M; Bonsignore, L; Somers, R; Vanderheydt, C; Weeks, SD; Gunnarsson, J; Nilsson, E; Roth, RG; Thome, M; Marynen, P
PloS one 9 e103774 2014
Mucosa-associated lymphoid tissue 1 (MALT1) controls antigen receptor-mediated signalling to nuclear factor κB (NF-κB) through both its adaptor and protease function. Upon antigen stimulation, MALT1 forms a complex with BCL10 and CARMA1, which is essential for initial IκBα phosphorylation and NF-κB nuclear translocation. Parallel induction of MALT1 protease activity serves to inactivate negative regulators of NF-κB signalling, such as A20 and RELB. Here we demonstrate a key role for auto-proteolytic MALT1 cleavage in B- and T-cell receptor signalling. MALT1 cleavage occurred after Arginine 149, between the N-terminal death domain and the first immunoglobulin-like region, and did not affect its proteolytic activity. Jurkat T cells expressing an un-cleavable MALT1-R149A mutant showed unaltered initial IκBα phosphorylation and normal nuclear accumulation of NF-κB subunits. Nevertheless, MALT1 cleavage was required for optimal activation of NF-κB reporter genes and expression of the NF-κB targets IL-2 and CSF2. Transcriptome analysis confirmed that MALT1 cleavage after R149 was required to induce NF-κB transcriptional activity in Jurkat T cells. Collectively, these data demonstrate that auto-proteolytic MALT1 cleavage controls antigen receptor-induced expression of NF-κB target genes downstream of nuclear NF-κB accumulation.
|Docosahexaenoic acid reverses angiotensin II-induced RECK suppression and cardiac fibroblast migration. |
Siddesha, JM; Valente, AJ; Yoshida, T; Sakamuri, SS; Delafontaine, P; Iba, H; Noda, M; Chandrasekar, B
Cellular signalling 26 933-41 2014
The omega-3 polyunsaturated fatty acids (ω-3 fatty acids) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been reported to inhibit or delay the progression of cardiovascular diseases, including myocardial fibrosis. Recently we reported that angiotensin II (Ang II) promotes cardiac fibroblast (CF) migration by suppressing the MMP regulator reversion-inducing-cysteine-rich protein with Kazal motifs (RECK), through a mechanism dependent on AT1, ERK, and Sp1. Here we investigated the role of miR-21 in Ang II-mediated RECK suppression, and determined whether the ω-3 fatty acids reverse these effects. Ang II induced miR-21 expression in primary mouse cardiac fibroblasts (CFs) via ERK-dependent AP-1 and STAT3 activation, and while a miR-21 inhibitor reversed Ang II-induced RECK suppression, a miR-21 mimic inhibited both RECK expression and Ang II-induced CF migration. Moreover, Ang II suppressed the pro-apoptotic PTEN, and the ERK negative regulator Sprouty homologue 1 (SPRY1), but induced the metalloendopeptidase MMP2, all in a manner that was miR-21-dependent. Further, forced expression of PTEN inhibited Akt phosphorylation, Sp1 activation, and MMP2 induction. Notably, while both EPA and DHA reversed Ang II-mediated RECK suppression, DHA appeared to be more effective, and reversed Ang II-induced miR-21 expression, RECK suppression, MMP2 induction, and CF migration. These results indicate that Ang II-induced CF migration is differentially regulated by miR-21-mediated MMP induction and RECK suppression, and that DHA has the potential to upregulate RECK, and therefore may exert potential beneficial effects in cardiac fibrosis.
|Development and characterization of 3-(benzylsulfonamido)benzamides as potent and selective SIRT2 inhibitors. |
Khanfar, MA; Quinti, L; Wang, H; Choi, SH; Kazantsev, AG; Silverman, RB
European journal of medicinal chemistry 76 414-26 2014
Inhibitors of sirtuin-2 deacetylase (SIRT2) have been shown to be protective in various models of Huntington's disease (HD) by decreasing polyglutamine aggregation, a hallmark of HD pathology. The present study was directed at optimizing the potency of SIRT2 inhibitors containing the neuroprotective sulfobenzoic acid scaffold and improving their pharmacology. To achieve that goal, 176 analogues were designed, synthesized, and tested in deacetylation assays against the activities of major human sirtuins SIRT1-3. This screen yielded 15 compounds with enhanced potency for SIRT2 inhibition and 11 compounds having SIRT2 inhibition equal to reference compound AK-1. The newly synthesized compounds also demonstrated higher SIRT2 selectivity over SIRT1 and SIRT3. These candidates were subjected to a dose-response bioactivity assay, measuring an increase in α-tubulin K40 acetylation in two neuronal cell lines, which yielded five compounds bioactive in both cell lines and eight compounds bioactive in at least one of the cell lines tested. These bioactive compounds were subsequently tested in a tertiary polyglutamine aggregation assay, which identified five inhibitors. ADME properties of the bioactive SIRT2 inhibitors were assessed, which revealed a significant improvement of the pharmacological properties of the new entities, reaching closer to the goal of a clinically-viable candidate.
|Doublecortin may play a role in defining chondrocyte phenotype. |
Ge, D; Zhang, QS; Zabaleta, J; Zhang, Q; Liu, S; Reiser, B; Bunnell, BA; Braun, SE; O'Brien, MJ; Savoie, FH; You, Z
International journal of molecular sciences 15 6941-60 2014
Embryonic development of articular cartilage has not been well understood and the role of doublecortin (DCX) in determination of chondrocyte phenotype is unknown. Here, we use a DCX promoter-driven eGFP reporter mouse model to study the dynamic gene expression profiles in mouse embryonic handplates at E12.5 to E13.5 when the condensed mesenchymal cells differentiate into either endochondral chondrocytes or joint interzone cells. Illumina microarray analysis identified a variety of genes that were expressed differentially in the different regions of mouse handplate. The unique expression patterns of many genes were revealed. Cytl1 and 3110032G18RIK were highly expressed in the proximal region of E12.5 handplate and the carpal region of E13.5 handplate, whereas Olfr538, Kctd15, and Cited1 were highly expressed in the distal region of E12.5 and the metacarpal region of E13.5 handplates. There was an increasing gradient of Hrc expression in the proximal to distal direction in E13.5 handplate. Furthermore, when human DCX protein was expressed in human adipose stem cells, collagen II was decreased while aggrecan, matrilin 2, and GDF5 were increased during the 14-day pellet culture. These findings suggest that DCX may play a role in defining chondrocyte phenotype.
|LCAT deficiency does not impair amyloid metabolism in APP/PS1 mice. |
Stukas, S; Freeman, L; Lee, M; Wilkinson, A; Ossoli, A; Vaisman, B; Demosky, S; Chan, J; Hirsch-Reinshagen, V; Remaley, AT; Wellington, CL
Journal of lipid research 55 1721-1729 2014
A key step in plasma HDL maturation from discoidal to spherical particles is the esterification of cholesterol to cholesteryl ester, which is catalyzed by LCAT. HDL-like lipoproteins in cerebrospinal fluid (CSF) are also spherical, whereas nascent lipoprotein particles secreted from astrocytes are discoidal, suggesting that LCAT may play a similar role in the CNS. In plasma, apoA-I is the main LCAT activator, while in the CNS, it is believed to be apoE. apoE is directly involved in the pathological progression of Alzheimer's disease, including facilitating β-amyloid (Aβ) clearance from the brain, a function that requires its lipidation by ABCA1. However, whether apoE particle maturation by LCAT is also required for Aβ clearance is unknown. Here we characterized the impact of LCAT deficiency on CNS lipoprotein metabolism and amyloid pathology. Deletion of LCAT from APP/PS1 mice resulted in a pronounced decrease of apoA-I in plasma that was paralleled by decreased apoA-I levels in CSF and brain tissue, whereas apoE levels were unaffected. Furthermore, LCAT deficiency did not increase Aβ or amyloid in APP/PS1 LCAT(-/-) mice. Finally, LCAT expression and plasma activity were unaffected by age or the onset of Alzheimer's-like pathology in APP/PS1 mice. Taken together, these results suggest that apoE-containing discoidal HDLs do not require LCAT-dependent maturation to mediate efficient Aβ clearance.
|The protease Omi regulates mitochondrial biogenesis through the GSK3β/PGC-1α pathway. |
Xu, R; Hu, Q; Ma, Q; Liu, C; Wang, G
Cell death & disease 5 e1373 2014
Loss of the mitochondrial protease activity of Omi causes mitochondrial dysfunction, neurodegeneration with parkinsonian features and premature death in mnd2 (motor neuron degeneration 2) mice. However, the detailed mechanisms underlying this pathology remain largely unknown. Here, we report that Omi participates in the process of mitochondrial biogenesis, which has been linked to several neurodegenerative diseases. The mitochondrial biogenesis is deficit in mnd2 mice, evidenced by severe decreases of mitochondrial components, mitochondrial DNA and mitochondrial density. Omi cleaves glycogen synthase kinase 3β (GSK3β), a kinase promoting PPARγ coactivator-1α (PGC-1α) degradation, to regulate PGC-1α, a factor important for the mitochondrial biogenesis. In mnd2 mice, GSK3β abundance is increased and PGC-1α abundance is decreased significantly. Inhibition of GSK3β by SB216763 or overexpression of PGC-1α can restore mitochondrial biogenesis in mnd2 mice or Omi-knockdown N2a cells. Furthermore, there is a significant improvement of the movement ability of mnd2 mice after SB216763 treatment. Thus, our study identified Omi as a novel regulator of mitochondrial biogenesis, involving in Omi protease-deficient-induced neurodegeneration.
|Serotonin 5-HT3 receptor-mediated vomiting occurs via the activation of Ca2+/CaMKII-dependent ERK1/2 signaling in the least shrew (Cryptotis parva). |
Zhong, W; Hutchinson, TE; Chebolu, S; Darmani, NA
PloS one 9 e104718 2014
Stimulation of 5-HT3 receptors (5-HT3Rs) by 2-methylserotonin (2-Me-5-HT), a selective 5-HT3 receptor agonist, can induce vomiting. However, downstream signaling pathways for the induced emesis remain unknown. The 5-HT3R channel has high permeability to extracellular calcium (Ca(2+)) and upon stimulation allows increased Ca(2+) influx. We examined the contribution of Ca(2+)/calmodulin-dependent protein kinase IIα (Ca(2+)/CaMKIIα), interaction of 5-HT3R with calmodulin, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling to 2-Me-5-HT-induced emesis in the least shrew. Using fluo-4 AM dye, we found that 2-Me-5-HT augments intracellular Ca(2+) levels in brainstem slices and that the selective 5-HT3R antagonist palonosetron, can abolish the induced Ca(2+) signaling. Pre-treatment of shrews with either: i) amlodipine, an antagonist of L-type Ca(2+) channels present on the cell membrane; ii) dantrolene, an inhibitor of ryanodine receptors (RyRs) Ca2+-release channels located on the endoplasmic reticulum (ER); iii) a combination of their less-effective doses; or iv) inhibitors of CaMKII (KN93) and ERK1/2 (PD98059); dose-dependently suppressed emesis caused by 2-Me-5-HT. Administration of 2-Me-5-HT also significantly: i) enhanced the interaction of 5-HT3R with calmodulin in the brainstem as revealed by immunoprecipitation, as well as their colocalization in the area postrema (brainstem) and small intestine by immunohistochemistry; and ii) activated CaMKIIα in brainstem and in isolated enterochromaffin cells of the small intestine as shown by Western blot and immunocytochemistry. These effects were suppressed by palonosetron. 2-Me-5-HT also activated ERK1/2 in brainstem, which was abrogated by palonosetron, KN93, PD98059, amlodipine, dantrolene, or a combination of amlodipine plus dantrolene. However, blockade of ER inositol-1, 4, 5-triphosphate receptors by 2-APB, had no significant effect on the discussed behavioral and biochemical parameters. This study demonstrates that Ca(2+) mobilization via extracellular Ca(2+) influx through 5-HT3Rs/L-type Ca(2+) channels, and intracellular Ca(2+) release via RyRs on ER, initiate Ca(2+)-dependent sequential activation of CaMKIIα and ERK1/2, which contribute to the 5-HT3R-mediated, 2-Me-5-HT-evoked emesis.
|Eccentric contractions disrupt FKBP12 content in mouse skeletal muscle. |
Baumann, CW; Rogers, RG; Gahlot, N; Ingalls, CP
Physiological reports 2 2014
Strength deficits associated with eccentric contraction-induced muscle injury stem, in part, from impaired voltage-gated sarcoplasmic reticulum (SR) Ca(2+) release. FKBP12 is a 12-kD immunophilin known to bind to the SR Ca(2+) release channel (ryanodine receptor, RyR1) and plays an important role in excitation-contraction coupling. To assess the effects of eccentric contractions on FKBP12 content, we measured anterior crural muscle (tibialis anterior [TA], extensor digitorum longus [EDL], extensor hallucis longus muscles) strength and FKBP12 content in pellet and supernatant fractions after centrifugation via immunoblotting from mice before and after a single bout of either 150 eccentric or concentric contractions. There were no changes in peak isometric torque or FKBP12 content in TA muscles after concentric contractions. However, FKBP12 content was reduced in the pelleted fraction immediately after eccentric contractions, and increased in the soluble protein fraction 3 day after injury induction. FKBP12 content was correlated (P = 0.025; R(2) = 0.38) to strength deficits immediately after injury induction. In summary, eccentric contraction-induced muscle injury is associated with significant alterations in FKBP12 content after injury, and is correlated with changes in peak isometric torque.
|Gene therapy prolongs survival and restores function in murine and canine models of myotubular myopathy. |
Childers, MK; Joubert, R; Poulard, K; Moal, C; Grange, RW; Doering, JA; Lawlor, MW; Rider, BE; Jamet, T; Danièle, N; Martin, S; Rivière, C; Soker, T; Hammer, C; Van Wittenberghe, L; Lockard, M; Guan, X; Goddard, M; Mitchell, E; Barber, J; Williams, JK; Mack, DL; Furth, ME; Vignaud, A; Masurier, C; Mavilio, F; Moullier, P; Beggs, AH; Buj-Bello, A
Science translational medicine 6 220ra10 2014
Loss-of-function mutations in the myotubularin gene (MTM1) cause X-linked myotubular myopathy (XLMTM), a fatal, congenital pediatric disease that affects the entire skeletal musculature. Systemic administration of a single dose of a recombinant serotype 8 adeno-associated virus (AAV8) vector expressing murine myotubularin to Mtm1-deficient knockout mice at the onset or at late stages of the disease resulted in robust improvement in motor activity and contractile force, corrected muscle pathology, and prolonged survival throughout a 6-month study. Similarly, single-dose intravascular delivery of a canine AAV8-MTM1 vector in XLMTM dogs markedly improved severe muscle weakness and respiratory impairment, and prolonged life span to more than 1 year in the absence of toxicity or a humoral or cell-mediated immune response. These results demonstrate the therapeutic efficacy of AAV-mediated gene therapy for myotubular myopathy in small- and large-animal models, and provide proof of concept for future clinical trials in XLMTM patients.
|Alleviation of behavioral hypersensitivity in mouse models of inflammatory pain with two structurally different casein kinase 1 (CK1) inhibitors. |
Kurihara, T; Sakurai, E; Toyomoto, M; Kii, I; Kawamoto, D; Asada, T; Tanabe, T; Yoshimura, M; Hagiwara, M; Miyata, A
Molecular pain 10 17 2014
The phylogenetically highly conserved CK1 protein kinases consisting of at least seven isoforms form a distinct family within the eukaryotic protein kinases. CK1 family members play crucial roles in a wide range of signaling activities. However, the functional role of CK1 in somatosensory pain signaling has not yet been fully understood. The aim of this study was to clarify the role of CK1 in the regulation of inflammatory pain in mouse carrageenan and complete Freund's adjuvant (CFA) models.We have used two structurally different CK1 inhibitors, TG003 and IC261. TG003, which was originally identified as a cdc2-like kinase inhibitor, had potent inhibitory effects on CK1 isoforms in vitro and in cultured cells. Intrathecal injection of either TG003 (1-100 pmol) or IC261 (0.1-1 nmol) dose-dependently decreased mechanical allodynia and thermal hyperalgesia induced by carrageenan or CFA. Bath-application of either TG003 (1 μM) or IC261 (1 μM) had only marginal effects on spontaneous excitatory postsynaptic currents (sEPSCs) recorded in the substantia gelatinosa neurons of control mice. However, both compounds decreased the frequency of sEPSCs in both inflammatory pain models.These results suggest that CK1 plays an important pathophysiological role in spinal inflammatory pain transmission, and that inhibition of the CK1 activity may provide a novel strategy for the treatment of inflammatory pain.
|Mutations in TJP2 cause progressive cholestatic liver disease. |
Sambrotta, M; Strautnieks, S; Papouli, E; Rushton, P; Clark, BE; Parry, DA; Logan, CV; Newbury, LJ; Kamath, BM; Ling, S; Grammatikopoulos, T; Wagner, BE; Magee, JC; Sokol, RJ; Mieli-Vergani, G; , ; Smith, JD; Johnson, CA; McClean, P; Simpson, MA; Knisely, AS; Bull, LN; Thompson, RJ
Nature genetics 46 326-8 2014
Elucidating genetic causes of cholestasis has proved to be important in understanding the physiology and pathophysiology of the liver. Here we show that protein-truncating mutations in the tight junction protein 2 gene (TJP2) cause failure of protein localization and disruption of tight-junction structure, leading to severe cholestatic liver disease. These findings contrast with those in the embryonic-lethal knockout mouse, highlighting differences in redundancy in junctional complexes between organs and species.
|KLHL40 deficiency destabilizes thin filament proteins and promotes nemaline myopathy. |
Garg, A; O'Rourke, J; Long, C; Doering, J; Ravenscroft, G; Bezprozvannaya, S; Nelson, BR; Beetz, N; Li, L; Chen, S; Laing, NG; Grange, RW; Bassel-Duby, R; Olson, EN
The Journal of clinical investigation 124 3529-39 2014
Nemaline myopathy (NM) is a congenital myopathy that can result in lethal muscle dysfunction and is thought to be a disease of the sarcomere thin filament. Recently, several proteins of unknown function have been implicated in NM, but the mechanistic basis of their contribution to disease remains unresolved. Here, we demonstrated that loss of a muscle-specific protein, kelch-like family member 40 (KLHL40), results in a nemaline-like myopathy in mice that closely phenocopies muscle abnormalities observed in KLHL40-deficient patients. We determined that KLHL40 localizes to the sarcomere I band and A band and binds to nebulin (NEB), a protein frequently implicated in NM, as well as a putative thin filament protein, leiomodin 3 (LMOD3). KLHL40 belongs to the BTB-BACK-kelch (BBK) family of proteins, some of which have been shown to promote degradation of their substrates. In contrast, we found that KLHL40 promotes stability of NEB and LMOD3 and blocks LMOD3 ubiquitination. Accordingly, NEB and LMOD3 were reduced in skeletal muscle of both Klhl40-/- mice and KLHL40-deficient patients. Loss of sarcomere thin filament proteins is a frequent cause of NM; therefore, our data that KLHL40 stabilizes NEB and LMOD3 provide a potential basis for the development of NM in KLHL40-deficient patients.
|Genetic deletion of SEPT7 reveals a cell type-specific role of septins in microtubule destabilization for the completion of cytokinesis. |
Menon, MB; Sawada, A; Chaturvedi, A; Mishra, P; Schuster-Gossler, K; Galla, M; Schambach, A; Gossler, A; Förster, R; Heuser, M; Kotlyarov, A; Kinoshita, M; Gaestel, M
PLoS genetics 10 e1004558 2014
Cytokinesis terminates mitosis, resulting in separation of the two sister cells. Septins, a conserved family of GTP-binding cytoskeletal proteins, are an absolute requirement for cytokinesis in budding yeast. We demonstrate that septin-dependence of mammalian cytokinesis differs greatly between cell types: genetic loss of the pivotal septin subunit SEPT7 in vivo reveals that septins are indispensable for cytokinesis in fibroblasts, but expendable in cells of the hematopoietic system. SEPT7-deficient mouse embryos fail to gastrulate, and septin-deficient fibroblasts exhibit pleiotropic defects in the major cytokinetic machinery, including hyperacetylation/stabilization of microtubules and stalled midbody abscission, leading to constitutive multinucleation. We identified the microtubule depolymerizing protein stathmin as a key molecule aiding in septin-independent cytokinesis, demonstrated that stathmin supplementation is sufficient to override cytokinesis failure in SEPT7-null fibroblasts, and that knockdown of stathmin makes proliferation of a hematopoietic cell line sensitive to the septin inhibitor forchlorfenuron. Identification of septin-independent cytokinesis in the hematopoietic system could serve as a key to identify solid tumor-specific molecular targets for inhibition of cell proliferation.
|In vitro membrane reconstitution of the T-cell receptor proximal signaling network. |
Hui, E; Vale, RD
Nature structural & molecular biology 21 133-42 2014
T-cell receptor (TCR) phosphorylation is controlled by a complex network that includes Lck, a Src family kinase (SFK), the tyrosine phosphatase CD45 and the Lck-inhibitory kinase Csk. How these competing phosphorylation and dephosphorylation reactions are modulated to produce T-cell triggering is not fully understood. Here we reconstituted this signaling network using purified enzymes on liposomes, recapitulating the membrane environment in which they normally interact. We demonstrate that Lck's enzymatic activity can be regulated over an ~10-fold range by controlling its phosphorylation state. By varying kinase and phosphatase concentrations, we constructed phase diagrams that reveal ultrasensitivity in the transition from the quiescent to the phosphorylated state and demonstrate that co-clustering TCR and Lck or detaching Csk from the membrane can trigger TCR phosphorylation. Our results provide insight into the mechanism of TCR signaling as well as other signaling pathways involving SFKs.
|CNS expression of murine fragile X protein (FMRP) as a function of CGG-repeat size. |
Ludwig, AL; Espinal, GM; Pretto, DI; Jamal, AL; Arque, G; Tassone, F; Berman, RF; Hagerman, PJ
Human molecular genetics 23 3228-38 2014
Large expansions of a CGG-repeat element (greater than 200 repeats; full mutation) in the fragile X mental retardation 1 (FMR1) gene cause fragile X syndrome (FXS), the leading single-gene form of intellectual disability and of autism spectrum disorder. Smaller expansions (55-200 CGG repeats; premutation) result in the neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS). Whereas FXS is caused by gene silencing and insufficient FMR1 protein (FMRP), FXTAS is thought to be caused by 'toxicity' of expanded-CGG-repeat mRNA. However, as FMRP expression levels decrease with increasing CGG-repeat length, lowered protein may contribute to premutation-associated clinical involvement. To address this issue, we measured brain Fmr1 mRNA and FMRP levels as a function of CGG-repeat length in a congenic (CGG-repeat knock-in) mouse model using 57 wild-type and 97 expanded-CGG-repeat mice carrying up to ~250 CGG repeats. While Fmr1 message levels increased with repeat length, FMRP levels trended downward over the same range, subject to significant inter-subject variation. Human comparisons of protein levels in the frontal cortex of 7 normal and 17 FXTAS individuals revealed that the mild FMRP decrease in mice mirrored the more limited data for FMRP expression in the human samples. In addition, FMRP expression levels varied in a subset of mice across the cerebellum, frontal cortex, and hippocampus, as well as at different ages. These results provide a foundation for understanding both the CGG-repeat-dependence of FMRP expression and for interpreting clinical phenotypes in premutation carriers in terms of the balance between elevated mRNA and lowered FMRP expression levels.
|FAK inhibition abrogates the malignant phenotype in aggressive pediatric renal tumors. |
Megison, ML; Gillory, LA; Stewart, JE; Nabers, HC; Mrozcek-Musulman, E; Beierle, EA
Molecular cancer research : MCR 12 514-26 2014
Despite the tremendous advances in the treatment of childhood kidney tumors, there remain subsets of pediatric renal tumors that continue to pose a therapeutic challenge, mainly malignant rhabdoid kidney tumors and nonosseous renal Ewing sarcoma. Children with advanced, metastatic, or relapsed disease have a poor disease-free survival rate. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumor development and progression. FAK has been found in other pediatric solid tumors and in adult renal cellular carcinoma, leading to the hypothesis that FAK contributes to pediatric kidney tumors and would affect cellular survival. In the current study, FAK was present and phosphorylated in pediatric kidney tumor specimens. Moreover, the effects of FAK inhibition upon G401 and SK-NEP-1 cell lines were examined using a number of parallel approaches to block FAK, including RNA interference and small-molecule FAK inhibitors. FAK inhibition resulted in decreased cellular survival, invasion and migration, and increased apoptosis. Furthermore, small-molecule inhibition of FAK led to decreased SK-NEP-1 xenograft growth in vivo. These data deepen the knowledge of the tumorigenic process in pediatric renal tumors, and provide desperately needed therapeutic strategies and targets for these rare, but difficult to treat, malignancies.This study provides a fundamental understanding of tumorigenesis in difficult to treat renal tumors and provides an impetus for new avenues of research and potential for novel, targeted therapies.
|Specificity of anti-tau antibodies when analyzing mice models of Alzheimer's disease: problems and solutions. |
Petry, FR; Pelletier, J; Bretteville, A; Morin, F; Calon, F; Hébert, SS; Whittington, RA; Planel, E
PloS one 9 e94251 2014
Aggregates of hyperphosphorylated tau protein are found in a group of diseases called tauopathies, which includes Alzheimer's disease. The causes and consequences of tau hyperphosphorylation are routinely investigated in laboratory animals. Mice are the models of choice as they are easily amenable to transgenic technology; consequently, their tau phosphorylation levels are frequently monitored by Western blotting using a panel of monoclonal/polyclonal anti-tau antibodies. Given that mouse secondary antibodies can recognize endogenous mouse immunoglobulins (Igs) and the possible lack of specificity with some polyclonal antibodies, non-specific signals are commonly observed. Here, we characterized the profiles of commonly used anti-tau antibodies in four different mouse models: non-transgenic mice, tau knock-out (TKO) mice, 3xTg-AD mice, and hypothermic mice, the latter a positive control for tau hyperphosphorylation. We identified 3 tau monoclonal antibody categories: type 1, characterized by high non-specificity (AT8, AT180, MC1, MC6, TG-3), type 2, demonstrating low non-specificity (AT270, CP13, CP27, Tau12, TG5), and type 3, with no non-specific signal (DA9, PHF-1, Tau1, Tau46). For polyclonal anti-tau antibodies, some displayed non-specificity (pS262, pS409) while others did not (pS199, pT205, pS396, pS404, pS422, A0024). With monoclonal antibodies, most of the interfering signal was due to endogenous Igs and could be eliminated by different techniques: i) using secondary antibodies designed to bind only non-denatured Igs, ii) preparation of a heat-stable fraction, iii) clearing Igs from the homogenates, and iv) using secondary antibodies that only bind the light chain of Igs. All of these techniques removed the non-specific signal; however, the first and the last methods were easier and more reliable. Overall, our study demonstrates a high risk of artefactual signal when performing Western blotting with routinely used anti-tau antibodies, and proposes several solutions to avoid non-specific results. We strongly recommend the use of negative (i.e., TKO) and positive (i.e., hypothermic) controls in all experiments.
|Deletion of the EphA2 receptor exacerbates myocardial injury and the progression of ischemic cardiomyopathy. |
O'Neal, WT; Griffin, WF; Kent, SD; Faiz, F; Hodges, J; Vuncannon, J; Virag, JA
Frontiers in physiology 5 132 2014
EphrinA1-EphA-receptor signaling is protective during myocardial infarction (MI). The EphA2-receptor (EphA2-R) potentially mediates cardiomyocyte survival. To determine the role of the EphA2-R in acute non-reperfused myocardial injury in vivo, infarct size, inflammatory cell density, NF-κB, p-AKT/Akt, and MMP-2 protein levels, and changes in ephrinA1/EphA2-R gene expression profile were assessed 4 days post-MI in B6129 wild-type (WT) and EphA2-R-mutant (EphA2-R-M) mice lacking a functional EphA2-R. Fibrosis, capillary density, morphometry of left ventricular chamber and infarct dimensions, and cardiac function also were measured 4 weeks post-MI to determine the extent of ventricular remodeling. EphA2-R-M infarct size and area of residual necrosis were 31.7% and 113% greater than WT hearts, respectively. Neutrophil and macrophage infiltration were increased by 46% and 84% in EphA2-R-M hearts compared with WT, respectively. NF-κB protein expression was 1.9-fold greater in EphA2-R-M hearts at baseline and 56% less NF-κB after infarction compared with WT. EphA6 gene expression was 2.5-fold higher at baseline and increased 9.8-fold 4 days post-MI in EphA2-R-M hearts compared with WT. EphrinA1 gene expression in EphA2-R-M hearts was unchanged at baseline and decreased by 42% 4 days post-MI compared with WT hearts. EphA2-R-M hearts had 66.7% less expression of total Akt protein and 59% less p-Akt protein than WT hearts post-MI. EphA2-R-M hearts 4 weeks post-MI had increased chamber dilation and interstitial fibrosis and decreased MMP-2 expression and capillary density compared with WT. In conclusion, the EphA2-R is necessary to appropriately modulate the inflammatory response and severity of early injury during acute MI, thereby influencing the progression of ischemic cardiomyopathy.
|Loss of insulin receptor in osteoprogenitor cells impairs structural strength of bone. |
Thrailkill, K; Bunn, RC; Lumpkin, C; Wahl, E; Cockrell, G; Morris, L; Kahn, CR; Fowlkes, J; Nyman, JS
Journal of diabetes research 2014 703589 2014
Type 1 diabetes mellitus (T1D) is associated with decreased bone mineral density, a deficit in bone structure, and subsequently an increased risk of fragility fracture. These clinical observations, paralleled by animal models of T1D, suggest that the insulinopenia of T1D has a deleterious effect on bone. To further examine the action of insulin signaling on bone development, we generated mice with an osteoprogenitor-selective (osterix-Cre) ablation of the insulin receptor (IR), designated OIRKO. OIRKO mice exhibited an 80% decrease in IR in osteoblasts. Prenatal elimination of IR did not affect fetal survival or gross morphology. However, loss of IR in mouse osteoblasts resulted in a postnatal growth-constricted phenotype. By 10-12 weeks of age, femurs of OIRKO mice were more slender, with a thinner diaphyseal cortex and, consequently, a decrease in whole bone strength when subjected to bending. In male mice alone, decreased metaphyseal trabecular bone, with thinner and more rodlike trabeculae, was also observed. OIRKO mice did not, however, exhibit abnormal glucose tolerance. The skeletal phenotype of the OIRKO mouse appeared more severe than that of previously reported bone-specific IR knockdown models, and confirms that insulin receptor expression in osteoblasts is critically important for proper bone development and maintenance of structural integrity.
|Diverse matrix metalloproteinase functions regulate cancer amoeboid migration. |
Orgaz, JL; Pandya, P; Dalmeida, R; Karagiannis, P; Sanchez-Laorden, B; Viros, A; Albrengues, J; Nestle, FO; Ridley, AJ; Gaggioli, C; Marais, R; Karagiannis, SN; Sanz-Moreno, V
Nature communications 5 4255 2014
Rounded-amoeboid cancer cells use actomyosin contractility driven by Rho-ROCK and JAK-STAT3 to migrate efficiently. It has been suggested that rounded-amoeboid cancer cells do not require matrix metalloproteinases (MMPs) to invade. Here we compare MMP levels in rounded-amoeboid and elongated-mesenchymal melanoma cells. Surprisingly, we find that rounded-amoeboid melanoma cells secrete higher levels of several MMPs, including collagenase MMP-13 and gelatinase MMP-9. As a result, rounded-amoeboid melanoma cells degrade collagen I more efficiently than elongated-mesenchymal cells. Furthermore, using a non-catalytic mechanism, MMP-9 promotes rounded-amoeboid 3D migration through regulation of actomyosin contractility via CD44 receptor. MMP-9 is upregulated in a panel of rounded-amoeboid compared with elongated-mesenchymal melanoma cell lines and its levels are controlled by ROCK-JAK-STAT3 signalling. MMP-9 expression increases during melanoma progression and it is particularly prominent in the invasive fronts of lesions, correlating with cell roundness. Therefore, rounded-amoeboid cells use both catalytic and non-catalytic activities of MMPs for invasion.
|In utero exposure to valproic acid changes sleep in juvenile rats: a model for sleep disturbances in autism. |
Cusmano, DM; Mong, JA
Sleep 37 1489-99 2014
To determine whether sleep disturbances are found in the valproic acid model of autism spectrum disorders (ASD).Comparative study for sleep behavior, sleep architecture, electroencephalogram (EEG) spectral analysis, and glutamic acid decarboxylase (GAD) 65/67 protein expression in juvenile rats exposed to valproic acid (VPA), sodium salt, or saline in utero.N/A.Juvenile (postnatal day 32) male and female Sprague-Dawley rats.In utero exposure to either saline or 400 mg/kg VPA administered intraperitoneally to the dams on gestational day 12.5. On postnatal days 22-24, all rats were implanted with transmitters to record EEG and electromyogram (EMG) activity.During the light phase, when nocturnal animals are typically quiescent, the VPA-exposed animals spent significantly more time in wake (∼35 min) and significantly less time in non-rapid eye movement (NREM) sleep (∼26 min) compared to the saline controls. Furthermore, spectral analysis of the EEG revelled that VPA-exposed animals exhibited increased high-frequency activity during wake and rapid eye movement (REM) sleep and reduced theta power across all vigilance states. Interestingly, the gamma-aminobutyric acid (GABA)-ergic system, which modulates the induction and maintenance of sleep states, was also disrupted, with reduced levels of both GAD 65 and GAD67 in the cortical tissue of VPA-exposed animals compared to saline controls.To date, the current animal models of ASD have been underutilized in the investigation of associated sleep disturbances. The VPA animal model recapitulates aspects of sleep disruptions reported clinically, providing a tool to investigate cellular and molecular dysregulation contributing to sleep disruptions in ASD.
|ROCK inhibitor is not required for embryoid body formation from singularized human embryonic stem cells. |
Pettinato, G; Vanden Berg-Foels, WS; Zhang, N; Wen, X
PloS one 9 e100742 2014
We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications.
|Proteomic identification of S-nitrosylated proteins in the parasite Entamoeba histolytica by resin-assisted capture: insights into the regulation of the Gal/GalNAc lectin by nitric oxide. |
Hertz, R; Ben Lulu, S; Shahi, P; Trebicz-Geffen, M; Benhar, M; Ankri, S
PloS one 9 e91518 2014
Entamoeba histolytica is a gastrointestinal protozoan parasite that causes amebiasis, a disease which has a worldwide distribution with substantial morbidity and mortality. Nitrosative stress, which is generated by innate immune cells, is one of the various environmental challenges that E. histolytica encounters during its life cycle. Although the effects of nitric oxide (NO) on the regulation of gene expression in this parasite have been previously investigated, our knowledge on S-nitrosylated proteins in E.histolytica is lacking. In order to fill this knowledge gap, we performed a large-scale detection of S-nitrosylated (SNO) proteins in E.histolytica trophozoites that were treated with the NO donor, S-nitrosocysteine by resin-assisted capture (RAC). We found that proteins involved in glycolysis, gluconeogenesis, translation, protein transport, and adherence to target cells such as the heavy subunit of Gal/GalNac lectin are among the S-nitrosylated proteins that were enriched by SNO-RAC. We also found that the S-nitrosylated cysteine residues in the carbohydrate recognition domain (CRD) of Gal/GalNAc lectin impairs its function and contributes to the inhibition of E.histolytica adherence to host cells. Collectively, these results advance our understanding of the mechanism of reduced E.histolytica adherence to mammalian cells by NO and emphasize the importance of NO as a regulator of key physiological functions in E.histolytica.
|NLRX1 prevents mitochondrial induced apoptosis and enhances macrophage antiviral immunity by interacting with influenza virus PB1-F2 protein. |
Jaworska, J; Coulombe, F; Downey, J; Tzelepis, F; Shalaby, K; Tattoli, I; Berube, J; Rousseau, S; Martin, JG; Girardin, SE; McCullers, JA; Divangahi, M
Proceedings of the National Academy of Sciences of the United States of America 111 E2110-9 2014
To subvert host immunity, influenza A virus (IAV) induces early apoptosis in innate immune cells by disrupting mitochondria membrane potential via its polymerase basic protein 1-frame 2 (PB1-F2) accessory protein. Whether immune cells have mechanisms to counteract PB1-F2-mediated apoptosis is currently unknown. Herein, we define that the host mitochondrial protein nucleotide-binding oligomerization domain-like receptor (NLR)X1 binds to viral protein PB1-F2, preventing IAV-induced macrophage apoptosis and promoting both macrophage survival and type I IFN signaling. We initially observed that Nlrx1-deficient mice infected with IAV exhibited increased pulmonary viral replication, as well as enhanced inflammatory-associated pulmonary dysfunction and morbidity. Analysis of the lungs of IAV-infected mice revealed markedly enhanced leukocyte recruitment but impaired production of type I IFN in Nlrx1(-/-) mice. Impaired type I IFN production and enhanced viral replication was recapitulated in Nlrx1(-/-) macrophages and was associated with increased mitochondrial mediated apoptosis. Through gain- and loss-of-function strategies for protein interaction, we identified that NLRX1 directly bound PB1-F2 in the mitochondria of macrophages. Using a recombinant virus lacking PB1-F2, we confirmed that deletion of PB1-F2 abrogated NLRX1-dependent macrophage type I IFN production and apoptosis. Thus, our results demonstrate that NLRX1 acts as a mitochondrial sentinel protecting macrophages from PB1-F2-induced apoptosis and preserving their antiviral function. We further propose that NLRX1 is critical for macrophage immunity against IAV infection by sensing the extent of viral replication and maintaining a protective balance between antiviral immunity and excessive inflammation within the lungs.
|Evidence for a novel antioxidant function and isoform-specific regulation of the human p66Shc gene. |
Miyazawa, M; Tsuji, Y
Molecular biology of the cell 25 2116-27 2014
The mammalian Shc family, composed of p46, p52, and p66 isoforms, serves as an adaptor protein in cell growth and stress response. p66Shc was shown to be a negative lifespan regulator by acting as a prooxidant protein in mitochondria; however, the regulatory mechanisms of p66Shc expression and function are incompletely understood. This study provides evidence for new features of p66Shc serving as an antioxidant and critical protein in cell differentiation. Unique among the Shc family, transcription of p66Shc is activated through the antioxidant response element (ARE)-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in K562 human erythroleukemia and other cell types after treatment with hemin, an iron-containing porphyrin. Phosphorylated p66Shc at Ser-36, previously reported to be prone to mitochondrial localization, is increased by hemin treatment, but p66Shc remains exclusively in the cytoplasm. p66Shc knockdown inhibits hemin-induced erythroid differentiation, in which reactive oxygen species production and apoptosis are significantly enhanced in conjunction with suppression of other ARE-dependent antioxidant genes. Conversely, p66Shc overexpression is sufficient for inducing erythroid differentiation. Collectively these results demonstrate the isoform-specific regulation of the Shc gene by the Nrf2-ARE pathway and a new antioxidant role of p66Shc in the cytoplasm. Thus p66Shc is a bifunctional protein involved in cellular oxidative stress response and differentiation.
|Formation of nuclear bodies by the lncRNA Gomafu-associating proteins Celf3 and SF1. |
Ishizuka, A; Hasegawa, Y; Ishida, K; Yanaka, K; Nakagawa, S
Genes to cells : devoted to molecular & cellular mechanisms 19 704-21 2014
Gomafu/MIAT/Rncr2 is a long noncoding RNA that has been proposed to control retinal cell specification, stem cell differentiation and alternative splicing of schizophrenia-related genes. However, how Gomafu controls these biological processes at the molecular level has remained largely unknown. In this study, we identified the RNA-binding protein Celf3 as a novel Gomafu-associating protein. Knockdown of Celf3 led to the down-regulation of Gomafu, and cross-link RNA precipitation analysis confirmed specific binding between Celf3 and Gomafu. In the neuroblastoma cell line Neuro2A, Celf3 formed novel nuclear bodies (named CS bodies) that colocalized with SF1, another Gomafu-binding protein. Gomafu, however, was not enriched in the CS bodies; instead, it formed distinct nuclear bodies in separate regions in the nucleus. These observations suggest that Gomafu indirectly modulates the function of the splicing factors SF1 and Celf3 by sequestering these proteins into separate nuclear bodies.
|Relationship of MMP-14 and TIMP-3 expression with macrophage activation and human atherosclerotic plaque vulnerability. |
Johnson, JL; Jenkins, NP; Huang, WC; Di Gregoli, K; Sala-Newby, GB; Scholtes, VP; Moll, FL; Pasterkamp, G; Newby, AC
Mediators of inflammation 2014 276457 2014
Matrix metalloproteinase-14 (MMP-14) promotes vulnerable plaque morphology in mice, whereas tissue inhibitor of metalloproteinases-3 (TIMP-3) overexpression is protective. MMP-14(hi) TIMP-3(lo) rabbit foam cells are more invasive and more prone to apoptosis than MMP-14(lo) TIMP-3(hi) cells. We investigated the implications of these findings for human atherosclerosis. In vitro generated macrophages and foam-cell macrophages, together with atherosclerotic plaques characterised as unstable or stable, were examined for expression of MMP-14, TIMP-3, and inflammatory markers. Proinflammatory stimuli increased MMP-14 and decreased TIMP-3 mRNA and protein expression in human macrophages. However, conversion to foam-cells with oxidized LDL increased MMP-14 and decreased TIMP-3 protein, independently of inflammatory mediators and partly through posttranscriptional mechanisms. Within atherosclerotic plaques, MMP-14 was prominent in foam-cells with either pro- or anti-inflammatory macrophage markers, whereas TIMP-3 was present in less foamy macrophages and colocalised with CD206. MMP-14 positive macrophages were more abundant whereas TIMP-3 positive macrophages were less abundant in plaques histologically designated as rupture prone. We conclude that foam-cells characterised by high MMP-14 and low TIMP-3 expression are prevalent in rupture-prone atherosclerotic plaques, independent of pro- or anti-inflammatory activation. Therefore reducing MMP-14 activity and increasing that of TIMP-3 could be valid therapeutic approaches to reduce plaque rupture and myocardial infarction.
|Western Blotting, Immunohistochemistry||Human||25301980|
|Collapsin response mediator protein 5 (CRMP5) induces mitophagy, thereby regulating mitochondrion numbers in dendrites. |
Brot, S; Auger, C; Bentata, R; Rogemond, V; Ménigoz, S; Chounlamountri, N; Girard-Egrot, A; Honnorat, J; Moradi-Améli, M
The Journal of biological chemistry 289 2261-76 2014
Degradation of damaged mitochondria by mitophagy is an essential process to ensure cell homeostasis. Because neurons, which have a high energy demand, are particularly dependent on the mitochondrial dynamics, mitophagy represents a key mechanism to ensure correct neuronal function. Collapsin response mediator proteins 5 (CRMP5) belongs to a family of cytosolic proteins involved in axon guidance and neurite outgrowth signaling during neural development. CRMP5, which is highly expressed during brain development, plays an important role in the regulation of neuronal polarity by inhibiting dendrite outgrowth at early developmental stages. Here, we demonstrated that CRMP5 was present in vivo in brain mitochondria and is targeted to the inner mitochondrial membrane. The mitochondrial localization of CRMP5 induced mitophagy. CRMP5 overexpression triggered a drastic change in mitochondrial morphology, increased the number of lysosomes and double membrane vesicles termed autophagosomes, and enhanced the occurrence of microtubule-associated protein 1 light chain 3 (LC3) at the mitochondrial level. Moreover, the lipidated form of LC3, LC3-II, which triggers autophagy by insertion into autophagosomes, enhanced mitophagy initiation. Lysosomal marker translocates at the mitochondrial level, suggesting autophagosome-lysosome fusion, and induced the reduction of mitochondrial content via lysosomal degradation. We show that during early developmental stages the strong expression of endogenous CRMP5, which inhibits dendrite growth, correlated with a decrease of mitochondrial content. In contrast, the knockdown or a decrease of CRMP5 expression at later stages enhanced mitochondrion numbers in cultured neurons, suggesting that CRMP5 modulated these numbers. Our study elucidates a novel regulatory mechanism that utilizes CRMP5-induced mitophagy to orchestrate proper dendrite outgrowth and neuronal function.
|SERPINB3 protects from oxidative damage by chemotherapeutics through inhibition of mitochondrial respiratory complex I. |
Ciscato, F; Sciacovelli, M; Villano, G; Turato, C; Bernardi, P; Rasola, A; Pontisso, P
Oncotarget 5 2418-27 2014
SERPINB3 (SB3) is a serine protease inhibitor overexpressed in several malignancies of epithelial origin, including primary liver cancer, where it inhibits apoptosis through poorly defined mechanisms. In the present study we analyze the effect of SB3 on hepatoma cell death elicited by a panel of chemotherapeutic agents. We report that SB3 shields cells from the toxicity of drugs with a pro-oxidant action such as doxorubicin, cisplatin and EM20-25. The rapid rise in ROS levels prompted by these compounds causes opening of the mitochondrial permeability transition pore (PTP), irreversibly committing cells to death. We find that a fraction of SB3 locates in mitochondrial inner compartments, and that this mitochondrial fraction increases under conditions of oxidative stress. Mitochondrial SB3 inhibits ROS generation and the ensuing PTP induction and cell death through an inhibitory interaction with respiratory Complex I. These findings identify a novel mechanism of action of SB3 that contributes to tumor cell resistance to anti-neoplastic drugs.
|Human osteoarthritic cartilage shows reduced in vivo expression of IL-4, a chondroprotective cytokine that differentially modulates IL-1β-stimulated production of chemokines and matrix-degrading enzymes in vitro. |
Assirelli, E; Pulsatelli, L; Dolzani, P; Platano, D; Olivotto, E; Filardo, G; Trisolino, G; Facchini, A; Borzì, RM; Meliconi, R
PloS one 9 e96925 2014
In osteoarthritis (OA), an inflammatory environment is responsible for the imbalance between the anabolic and catabolic activity of chondrocytes and, thus, for articular cartilage derangement. This study was aimed at providing further insight into the impairment of the anabolic cytokine IL-4 and its receptors in human OA cartilage, as well as the potential ability of IL-4 to antagonize the catabolic phenotype induced by IL-1β.The in vivo expression of IL-4 and IL-4 receptor subunits (IL-4R, IL-2Rγ, IL-13Rα1) was investigated on full thickness OA or normal knee cartilage. IL-4 expression was found to be significantly lower in OA, both in terms of the percentage of positive cells and the amount of signal per cell. IL-4 receptor type I and II were mostly expressed in mid-deep cartilage layers. No significant difference for each IL-4 receptor subunit was noted. IL-4 anti-inflammatory and anti-catabolic activity was assessed in vitro in the presence of IL-1β and/or IL-4 for 24 hours using differentiated high density primary OA chondrocyte also exhibiting the three IL-4 R subunits found in vivo. Chemokines, extracellular matrix degrading enzymes and their inhibitors were evaluated at mRNA (real time PCR) and protein (ELISA or western blot) levels. IL-4 did not affect IL-1β-induced mRNA expression of GRO-α/CXCL1, IL-8/CXCL8, ADAMTS-5, TIMP-1 or TIMP-3. Conversely, IL-4 significantly inhibited RANTES/CCL5, MIP-1α/CCL3, MIP-1β/CCL4, MMP-13 and ADAMTS-4. These results were confirmed at protein level for RANTES/CCL5 and MMP-13.Our results indicate for the first time that OA cartilage has a significantly lower expression of IL-4. Furthermore, we found differences in the spectrum of biological effects of IL-4. The findings that IL-4 has the ability to hamper the IL-1β-induced release of both MMP-13 and CCL5/RANTES, both markers of OA chondrocytes, strongly indicates IL-4 as a pivotal anabolic cytokine in cartilage whose impairment impacts on OA pathogenesis.
|Mammary gland specific knockdown of the physiological surge in Cx26 during lactation retains normal mammary gland development and function. |
Stewart, MK; Plante, I; Bechberger, JF; Naus, CC; Laird, DW
PloS one 9 e101546 2014
Connexin26 (Cx26) is the major Cx protein expressed in the human mammary gland and is up-regulated during pregnancy while remaining elevated throughout lactation. It is currently unknown if patients with loss-of-function Cx26 mutations that result in hearing loss and skin diseases have a greater susceptibility to impaired breast development. To investigate if Cx26 plays a critical role in mammary gland development and differentiation, a novel Cx26 conditional knockout mouse model was generated by crossing Cx26fl/fl mice with mice expressing Cre under the β-Lactoglobulin promoter. Conditional knockdown of Cx26 from the mammary gland resulted in a dramatic reduction in detectable gap junction plaques confirmed by a significant ∼65-70% reduction in Cx26 mRNA and protein throughout parturition and lactation. Interestingly, this reduction was accompanied by a decrease in mammary gland Cx30 gap junction plaques at parturition, while no change was observed for Cx32 or Cx43. Whole mount, histological and immunofluorescent assessment of breast tissue revealed comparatively normal lobuloalveolar development following pregnancy in the conditionally knockdown mice compared to control mice. In addition, glands from genetically-modified mice were capable of producing milk proteins that were evident in the lumen of alveoli and ducts at similar levels as controls, suggesting normal gland function. Together, our results suggest that low levels of Cx26 expression throughout pregnancy and lactation, and not the physiological surge in Cx26, is sufficient for normal gland development and function.
|Growth arrest and forced differentiation of human primary glioblastoma multiforme by a novel small molecule. |
Kang, TW; Choi, SW; Yang, SR; Shin, TH; Kim, HS; Yu, KR; Hong, IS; Ro, S; Cho, JM; Kang, KS
Scientific reports 4 5546 2014
Glioblastoma multiforme is the most common malignant brain tumor in adults, with an average survival of less than one year due to its resistance to therapy. Recent studies reported that GBM initiates from CD133-expressing cancer stem cells (CSC). However, the efficacy of CSC targeting is limited. A newly developed approach in cancer treatment is the forced differentiation of cancer cells. Here, we show that the treatment of the novel small molecule, CG500354, into CD133-expressing human primary GBM cells induces growth arrest by cell cycle regulators, p53, p21, p27 and phase-specific cyclins, and neural differentiation, as confirmed by neural progenitor/precursor markers, nestin, GFAP and Tuj1. When GBM-derived cells caused the tumors in NOD/SCID mice, CG500354 induced GBM-derived cells differentiation into Tuj1 and GFAP expressing cells. We next demonstrated that CG500354 plays a tumor-suppressive role via cAMP/CREB signaling pathway. CG500354 increases not only the extracellular cAMP level but also the protein level of PKA and CREB. Additionally, both mimetic substances, Forskolin and Rolipram, revealed comparable results with CG500354. Our findings indicate that induction of growth arrest and neural differentiation via cAMP/CREB signaling pathway by CG500354 treatment suggests the novel targeting of PDE4D in the development of new drugs for brain tumor therapy.
|WIPI-dependent autophagy during neutrophil differentiation of NB4 acute promyelocytic leukemia cells. |
Brigger, D; Proikas-Cezanne, T; Tschan, MP
Cell death & disease 5 e1315 2014
Members of the WD-repeat protein interacting with phosphoinositides (WIPI) family are phosphatidylinositol 3-phosphate (PI3P) effectors that are essential for the formation of autophagosomes. Autophagosomes, unique double-membraned organelles, are characteristic for autophagy, a bulk degradation mechanism with cytoprotective and homeostatic function. Both, WIPI-1 and WIPI-2 are aberrantly expressed in several solid tumors, linking these genes to carcinogenesis. We now found that the expression of WIPI-1 was significantly reduced in a large cohort of 98 primary acute myeloid leukemia (AML) patient samples (complex karyotypes; t(8;21); t(15,17); inv(16)). In contrast, the expression of WIPI-2 was only reduced in acute promyelocytic leukemia (APL), a distinct subtype of AML (t(15,17)). As AML cells are blocked in their differentiation, we tested if the expression levels of WIPI-1 and WIPI-2 increase during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of APL. According to the higher WIPI-1 expression in granulocytes compared with immature blast cells, WIPI-1 but not WIPI-2 expression was significantly induced during neutrophil differentiation of NB4 APL cells. Interestingly, the induction of WIPI-1 expression was dependent on the transcription factor PU.1, a master regulator of myelopoiesis, supporting our notion that WIPI-1 expression is reduced in AML patients lacking proper PU-1 activity. Further, knocking down WIPI-1 in NB4 cells markedly attenuated the autophagic flux and significantly reduced neutrophil differentiation. This result was also achieved by knocking down WIPI-2, suggesting that both WIPI-1 and WIPI-2 are functionally required and not redundant in mediating the PI3P signal at the onset of autophagy in NB4 cells. In line with these data, downregulation of PI3KC3 (hVPS34), which generates PI3P upstream of WIPIs, also inhibited neutrophil differentiation. In conclusion, we demonstrate that both WIPI-1 and WIPI-2 are required for the PI3P-dependent autophagic activity during neutrophil differentiation, and that PU.1-dependent WIPI-1 expression is significantly repressed in primary AML patient samples and that the induction of autophagic flux is associated with neutrophil differentiation of APL cells.
|Krüppel-like factor 4 mediates lysophosphatidic acid-stimulated migration and proliferation of PC3M prostate cancer cells. |
Shin, SH; Kwon, YW; Heo, SC; Jeong, GO; Kim, BR; Seo, EJ; Kim, JH
Experimental & molecular medicine 46 e104 2014
Prostate cancer is the most frequently diagnosed malignancy and the second leading cause of cancer mortality among men in the United States. Accumulating evidence suggests that lysophosphatidic acid (LPA) serves as an autocrine/paracrine mediator to affect initiation, progression and metastasis of prostate cancer. In the current study, we demonstrate that LPA stimulates migration and proliferation of highly metastatic human prostate cancer, PC-3M-luc-C6 cells. LPA-induced migration of PC-3M-luc-C6 cells was abrogated by pretreatment of PC-3M-luc-C6 cells with the LPA receptor 1/3 inhibitor Ki16425 or small interfering RNA (siRNA)-mediated silencing of endogenous LPA receptor 1, implicating a key role of the LPA-LPA receptor 1 signaling axis in migration of PC-3M-luc-C6 cells. In addition, LPA treatment resulted in augmented expression levels of Krüppel-like factor 4 (KLF4), and siRNA or short-hairpin RNA (shRNA)-mediated silencing of KLF4 expression resulted in the abolishment of LPA-stimulated migration and proliferation of PC-3M-luc-C6 cells. shRNA-mediated silencing of KLF4 expression resulted in the inhibition of in vivo growth of PC-3M-luc-C6 cells in a xenograft transplantation animal model. Taken together, these results suggest a key role of LPA-induced KLF4 expression in cell migration and proliferation of prostate cancer cells in vitro and in vivo.
|Congenital heart disease protein 5 associates with CASZ1 to maintain myocardial tissue integrity. |
Sojka, S; Amin, NM; Gibbs, D; Christine, KS; Charpentier, MS; Conlon, FL
Development (Cambridge, England) 141 3040-9 2014
The identification and characterization of the cellular and molecular pathways involved in the differentiation and morphogenesis of specific cell types of the developing heart are crucial to understanding the process of cardiac development and the pathology associated with human congenital heart disease. Here, we show that the cardiac transcription factor CASTOR (CASZ1) directly interacts with congenital heart disease 5 protein (CHD5), which is also known as tryptophan-rich basic protein (WRB), a gene located on chromosome 21 in the proposed region responsible for congenital heart disease in individuals with Down's syndrome. We demonstrate that loss of CHD5 in Xenopus leads to compromised myocardial integrity, improper deposition of basement membrane, and a resultant failure of hearts to undergo cell movements associated with cardiac formation. We further report that CHD5 is essential for CASZ1 function and that the CHD5-CASZ1 interaction is necessary for cardiac morphogenesis. Collectively, these results establish a role for CHD5 and CASZ1 in the early stages of vertebrate cardiac development.
|MHC class I limits hippocampal synapse density by inhibiting neuronal insulin receptor signaling. |
Dixon-Salazar, TJ; Fourgeaud, L; Tyler, CM; Poole, JR; Park, JJ; Boulanger, LM
The Journal of neuroscience : the official journal of the Society for Neuroscience 34 11844-56 2014
Proteins of the major histocompatibility complex class I (MHCI) negatively regulate synapse density in the developing vertebrate brain (Glynn et al., 2011; Elmer et al., 2013; Lee et al., 2014), but the underlying mechanisms remain largely unknown. Here we identify a novel MHCI signaling pathway that involves the inhibition of a known synapse-promoting factor, the insulin receptor. Dominant-negative insulin receptor constructs decrease synapse density in the developing Xenopus visual system (Chiu et al., 2008), and insulin receptor activation increases dendritic spine density in mouse hippocampal neurons in vitro (Lee et al., 2011). We find that genetically reducing cell surface MHCI levels increases synapse density selectively in regions of the hippocampus where insulin receptors are expressed, and occludes the neuronal insulin response by de-repressing insulin receptor signaling. Pharmacologically inhibiting insulin receptor signaling in MHCI-deficient animals rescues synapse density, identifying insulin receptor signaling as a critical mediator of the tonic inhibitory effects of endogenous MHCI on synapse number. Insulin receptors co-immunoprecipitate MHCI from hippocampal lysates, and MHCI unmasks a cytoplasmic epitope of the insulin receptor that mediates downstream signaling. These results identify an important role for an MHCI-insulin receptor signaling pathway in circuit patterning in the developing brain, and suggest that changes in MHCI expression could unexpectedly regulate neuronal insulin sensitivity in the aging and diseased brain.
|EphA2-receptor deficiency exacerbates myocardial infarction and reduces survival in hyperglycemic mice. |
DuSablon, A; Kent, S; Coburn, A; Virag, J
Cardiovascular diabetology 13 114 2014
We have previously shown that EphrinA1/EphA expression profile changes in response to myocardial infarction (MI), exogenous EphrinA1-Fc administration following MI positively influences wound healing, and that deletion of the EphA2 Receptor (EphA2-R) exacerbates injury and remodeling. To determine whether or not ephrinA1-Fc would be of therapeutic value in the hyperglycemic infarcted heart, it is critical to evaluate how ephrinA1/EphA signaling changes in the hyperglycemic myocardium in response to MI.Streptozotocin (STZ)-induced hyperglycemia in wild type (WT) and EphA2-receptor mutant (EphA2-R-M) mice was initiated by an intraperitoneal injection of STZ (150 mg/kg) 10 days before surgery. MI was induced by permanent ligation of the left anterior descending coronary artery and analyses were performed at 4 days post-MI. ANOVAs with Student-Newman Keuls multiple comparison post-hoc analysis illustrated which groups were significantly different, with significance of at least p less than 0.05.Both WT and EphA2-R-M mice responded adversely to STZ, but only hyperglycemic EphA2-R-M mice had lower ejection fraction (EF) and fractional shortening (FS). At 4 days post-MI, we observed greater post-MI mortality in EphA2-R-M mice compared with WT and this was greater still in the EphA2-R-M hyperglycemic mice. Although infarct size was greater in hyperglycemic WT mice vs normoglycemic mice, there was no difference between hyperglycemic EphA2-R-M mice and normoglycemic EphA2-R-M mice. The hypertrophic response that normally occurs in viable myocardium remote to the infarct was noticeably absent in epicardial cardiomyocytes and cardiac dysfunction worsened in hyperglycemic EphA2-R-M hearts post-MI. The characteristic interstitial fibrotic response in the compensating myocardium remote to the infarct also did not occur in hyperglycemic EphA2-R-M mouse hearts to the same extent as that observed in the hyperglycemic WT mouse hearts. Differences in neutrophil and pan-leukocyte infiltration and serum cytokines implicate EphA2-R in modulation of injury and the differences in ephrinA1 and EphA6-R expression in governing this are discussed.We conclude that EphA2-mutant mice are more prone to hyperglycemia-induced increased injury, decreased survival, and worsened LV remodeling due to impaired wound healing.
|Mmu-miR-615-3p regulates lipoapoptosis by inhibiting C/EBP homologous protein. |
Miyamoto, Y; Mauer, AS; Kumar, S; Mott, JL; Malhi, H
PloS one 9 e109637 2014
Lipoapoptosis occurring due to an excess of saturated free fatty acids such as palmitate is a key pathogenic event in the initiation of nonalcoholic fatty liver disease. Palmitate loading of cells activates the endoplasmic reticulum stress response, including induction of the proapoptotic transcription factor C/EBP homologous protein (CHOP). Furthermore, the loss of microRNAs is implicated in regulating apoptosis under conditions of endoplasmic reticulum (ER) stress. The aim of this study was to identify specific microRNAs regulating CHOP expression during palmitate-induced ER stress. Five microRNAs were repressed under palmitate-induced endoplasmic reticulum stress conditions in hepatocyte cell lines (miR-92b-3p, miR-328-3p, miR-484, miR-574-5p, and miR-615-3p). We identified miR-615-3p as a candidate microRNA which was repressed by palmitate treatment and regulated CHOP protein expression, by RNA sequencing and in silico analyses, respectively. There is a single miR-615-3p binding site in the 3'untranslated region (UTR) of the Chop transcript. We characterized this as a functional binding site using a reporter gene-based assay. Augmentation of miR-615-3p levels, using a precursor molecule, repressed CHOP expression; and under these conditions palmitate- or tunicamycin-induced cell death were significantly reduced. Our results suggest that palmitate-induced apoptosis requires maximal expression of CHOP which is achieved via the downregulation of its repressive microRNA, miR-615-3p. We speculate that enhancement of miR-615-3p levels may be of therapeutic benefit by inhibiting palmitate-induced hepatocyte lipoapoptosis.
|Ttyh1 protein is expressed in glia in vitro and shows elevated expression in activated astrocytes following status epilepticus. |
Wiernasz, E; Kaliszewska, A; Brutkowski, W; Bednarczyk, J; Gorniak, M; Kaza, B; Lukasiuk, K
Neurochemical research 39 2516-26 2014
In a previous study, we showed that Ttyh1 protein is expressed in neurons in vitro and in vivo in the form of punctuate structures, which are localized to neuropil and neuronal somata. Herein, we provide the first description of Ttyh1 protein expression in astrocytes, oligodendrocytes and microglia in vitro. Moreover, using double immunofluorescence, we show Ttyh1 protein expression in activated astrocytes in the hippocampus following amygdala stimulation-induced status epilepticus. We demonstrate that in migrating astrocytes in in vitro wound model Ttyh1 concentrates at the edges of extending processes. These data suggest that Ttyh1 not only participates in shaping neuronal morphology, as previously described, but may also play a role in the function of activated glia in brain pathology. To localize Ttyh1 expression in the cellular compartments of neurons and astrocytes, we performed in vitro double immunofluorescent staining using markers for the following subcellular structures: endoplasmic reticulum (GRP78), Golgi apparatus (GM130), clathrin-coated vehicles (clathrin), early endosomes (Rab5 and APPL2), recycling endosomes (Rab11), trans-Golgi network (TGN46), endoplasmic reticulum membrane (calnexin), late endosomes and lysosomes (LAMP1) and synaptic vesicles (synaptoporin and synaptotagmin 1). We found that Ttyh1 is present in the endoplasmic reticulum, Golgi apparatus and clathrin-coated vesicles (clathrin) in both neurons and astrocytes and also in late endosomes or lysosomes in astrocytes. The presence of Ttyh1 was negligible in early endosomes, recycling endosomes, trans-Golgi network, endoplasmic reticulum membrane and synaptic vesicles.
|Chemically induced mouse liver tumors are resistant to treatment with atorvastatin. |
Braeuning, A; Bucher, P; Hofmann, U; Buchmann, A; Schwarz, M
BMC cancer 14 766 2014
Atorvastatin is a potent inhibitor of the mevalonate pathway and widely used as a hypolipidemic drug. Some epidemiological studies and animal experiments indicate that the long-term use of atorvastatin and structurally related drugs might be associated with a reduced risk of developing hepatocellular carcinoma (HCC), the most common hepatocellular malignancy in humans. However, the potential of atorvastatin to inhibit HCC formation is controversially discussed.Hepatocellular tumors were chemically induced by treatment of C3H/He mice with 10 μg/g body weight N-nitrosodiethylamine and the ability of atorvastatin to interfere with tumor formation was investigated by treatment of mice with 0.1% atorvastatin in the diet for 6 months. Tumor size and tumor multiplicity were analyzed, as were tissue levels of cholesterol and atorvastatin.Atorvastatin treatment efficiently reduced serum cholesterol levels. However, the growth of tumors driven by activated MAPK (mitogen-activated protein kinase) signaling was not attenuated by the presence of the drug, as evidenced by a lack of reduction of tumor volume or tumor multiplicity by atorvastatin. Levels of the atorvastatin uptake transporters Oatp1a4 and Oatp1b2 were down-regulated at the mRNA and protein levels in chemically induced mouse liver tumors, but without striking effects on atorvastatin concentrations in the tumor tissue.In summary, the present data provide substantial evidence that atorvastatin does not beneficially influence tumor growth in mouse liver and thereby challenge the hypothesis that statin use might protect against hepatocellular cancer.
|An antiapoptotic role for telomerase RNA in human immune cells independent of telomere integrity or telomerase enzymatic activity. |
Gazzaniga, FS; Blackburn, EH
Blood 124 3675-84 2014
Telomerase is a ribonucleoprotein complex that adds telomeric DNA to the ends of linear chromosomes. It contains two core canonical components: the essential RNA component, hTR, which provides the template for DNA synthesis, and the reverse transcriptase protein component, hTERT. Low telomerase activity in circulating peripheral blood mononuclear cells has been associated with a variety of diseases. It is unknown, however, whether telomerase, in addition to its long-term requirement for telomere maintenance, is also necessary for short-term immune cell proliferation and survival. We report that overexpression of enzymatically inactive hTR mutants protected against dexamethasone-induced apoptosis in stimulated CD4 T cells. Furthermore, hTR knockdown reproducibly induced apoptosis in the absence of any detectable telomere shortening or DNA damage response. In contrast, hTERT knockdown did not induce apoptosis. Strikingly, overexpression of hTERT protein caused apoptosis that was rescued by overexpression of enzymatically inactive hTR mutants. Hence, we propose that hTR can function as a noncoding RNA that protects from apoptosis independent of its function in telomerase enzymatic activity and long-term telomere maintenance in normal human immune cells. These results imply that genetic or environmental factors that alter hTR levels can directly affect immune cell function to influence health and disease.
|Loss of Dlg-1 in the mouse lens impairs fibroblast growth factor receptor signaling. |
Lee, S; Griep, AE
PloS one 9 e97470 2014
Coordination of cell proliferation, differentiation and survival is essential for normal development and maintenance of tissues in the adult organism. Growth factor receptor tyrosine kinase signaling pathways and planar cell polarity pathways are two regulators of many developmental processes. We have previously shown through analysis of mice conditionally null in the lens for the planar cell polarity gene (PCP), Dlg-1, that Dlg-1 is required for fiber differentiation. Herein, we asked if Dlg-1 is a regulator of the Fibroblast growth factor receptor (Fgfr) signaling pathway, which is known to be required for fiber cell differentiation. Western blot analysis of whole fiber cell extracts from control and Dlg-1 deficient lenses showed that levels of the Fgfr signaling intermediates pErk, pAkt, and pFrs2α, the Fgfr target, Erm, and the fiber cell specific protein, Mip26, were reduced in the Dlg-1 deficient fiber cells. The levels of Fgfr2 were decreased in Dlg-1 deficient lenses compared to controls. Conversely, levels of Fgfr1 in Dlg-1 deficient lenses were increased compared to controls. The changes in Fgfr levels were found to be specifically in the triton insoluble, cytoskeletal associated fraction of Dlg-1 deficient lenses. Immunofluorescent staining of lenses from E13.5 embryos showed that expression levels of pErk were reduced in the transition zone, a region of the lens that exhibits PCP, in the Dlg-1 deficient lenses as compared to controls. In control lenses, immunofluorescent staining for Fgfr2 was observed in the epithelium, transition zone and fibers. By E13.5, the intensity of staining for Fgfr2 was reduced in these regions of the Dlg-1 deficient lenses. Thus, loss of Dlg-1 in the lens impairs Fgfr signaling and leads to altered levels of Fgfrs, suggesting that Dlg-1 is a modulator of Fgfr signaling pathway at the level of the receptors and that Dlg-1 regulates fiber cell differentiation through its role in PCP.
|Regulation of human CYP2C9 expression by electrophilic stress involves activator protein 1 activation and DNA looping. |
Makia, NL; Surapureddi, S; Monostory, K; Prough, RA; Goldstein, JA
Molecular pharmacology 86 125-37 2014
Cytochrome P450 (CYP)2C9 and CYP2C19 are important human enzymes that metabolize therapeutic drugs, environmental chemicals, and physiologically important endogenous compounds. Initial studies using primary human hepatocytes showed induction of both the CYP2C9 and CYP2C19 genes by tert-butylhydroquinone (tBHQ). As a pro-oxidant, tBHQ regulates the expression of cytoprotective genes by activation of redox-sensing transcription factors, such as the nuclear factor E2-related factor 2 (Nrf2) and members of the activator protein 1 (AP-1) family of proteins. The promoter region of CYP2C9 contains two putative AP-1 sites (TGAGTCA) at positions -2201 and -1930, which are also highly conserved in CYP2C19. The CYP2C9 promoter is activated by ectopic expression of cFos and JunD, whereas Nrf2 had no effect. Using specific kinase inhibitors for mitogen-activated protein kinase, we showed that extracellular signal-regulated kinase and Jun N-terminal kinase are essential for tBHQ-induced expression of CYP2C9. Electrophoretic mobility shift assays demonstrate that cFos distinctly interacts with the distal AP-1 site and JunD with the proximal site. Because cFos regulates target genes as heterodimers with Jun proteins, we hypothesized that DNA looping might be required to bring the distal and proximal AP-1 sites together to activate the CYP2C9 promoter. Chromosome conformation capture analyses confirmed the formation of a DNA loop in the CYP2C9 promoter, possibly allowing interaction between cFos at the distal site and JunD at the proximal site to activate CYP2C9 transcription in response to electrophiles. These results indicate that oxidative stress generated by exposure to electrophilic xenobiotics and metabolites induces the expression of CYP2C9 and CYP2C19 in human hepatocytes.
|Arachidonic acid-dependent gene regulation during preadipocyte differentiation controls adipocyte potential. |
Nikolopoulou, E; Papacleovoulou, G; Jean-Alphonse, F; Grimaldi, G; Parker, MG; Hanyaloglu, AC; Christian, M
Journal of lipid research 55 2479-90 2014
Arachidonic acid (AA) is a major PUFA that has been implicated in the regulation of adipogenesis. We examined the effect of a short exposure to AA at different stages of 3T3-L1 adipocyte differentiation. AA caused the upregulation of fatty acid binding protein 4 (FABP4/aP2) following 24 h of differentiation. This was mediated by the prostaglandin F(2α) (PGF(2α)), as inhibition of cyclooxygenases or PGF(2α) receptor signaling counteracted the AA-mediated aP2 induction. In addition, calcium, protein kinase C, and ERK are all key elements of the pathway through which AA induces the expression of aP2. We also show that treatment with AA during the first 24 h of differentiation upregulates the expression of the transcription factor Fos-related antigen 1 (Fra-1) via the same pathway. Finally, treatment with AA for 24 h at the beginning of the adipocyte differentiation is sufficient to inhibit the late stages of adipogenesis through a Fra-1-dependent pathway, as Fra-1 knockdown rescued adipogenesis. Our data show that AA is able to program the differentiation potential of preadipocytes by regulating gene expression at the early stages of adipogenesis.
|Cell migration is regulated by AGE-RAGE interaction in human oral cancer cells in vitro. |
Ko, SY; Ko, HA; Shieh, TM; Chang, WC; Chen, HI; Chang, SS; Lin, IH
PloS one 9 e110542 2014
Advanced glycation end products (AGEs) are produced in an irreversible non-enzymatic reaction of carbohydrates and proteins. Patients with diabetes mellitus (DM) are known to have elevated AGE levels, which is viewed as a risk factor of diabetes-related complications. In a clinical setting, it has been shown that patients with oral cancer in conjunction with DM have a higher likelihood of cancer metastasis and lower cancer survival rates. AGE-RAGE (a receptor of AGEs) is also correlated with metastasis and angiogenesis. Recent studies have suggested that the malignancy of cancer may be enhanced by glyceraldehyde-derived AGEs; however, the underlying mechanism remains unclear. This study examined the apparently close correlation between AGE-RAGE and the malignancy of SAS oral cancer cell line. In this study, AGEs increased ERK phosphorylation, enhanced cell migration, and promoted the expression of RAGE, MMP2, and MMP9. Using PD98059, RAGE antibody, and RAGE RNAi to block RAGE pathway resulted in the inhibition of ERK phosphorylation. Cell migration, MMP2 and MMP9 expression were also reduced by this treatment. Our findings demonstrate the importance of AGE-RAGE with regard to the malignancy of oral cancer, and help to explain the poor prognosis of DM subjects with oral cancer.
|Preclinical evaluation of engineered oncolytic herpes simplex virus for the treatment of pediatric solid tumors. |
Megison, ML; Gillory, LA; Stewart, JE; Nabers, HC; Mroczek-Musulman, E; Waters, AM; Coleman, JM; Kelly, V; Markert, JM; Gillespie, GY; Friedman, GK; Beierle, EA
PloS one 9 e86843 2014
Recently, investigators showed that mice with syngeneic murine gliomas that were treated with a neuroattenuated oncolytic herpes simplex virus-1 (oHSV), M002, had a significant increase in survival. M002 has deletions in both copies of the γ134.5 gene, enabling replication in tumor cells but precluding infection of normal cells. Previous studies have shown antitumor effects of other oHSV against a number of adult tumors including hepatocellular carcinoma and renal cell carcinoma. The purpose of the current study was to investigate the oncolytic potential of M002 against difficult to treat pediatric liver and kidney tumors. We showed that the oHSV, M002, infected, replicated, and decreased cell survival in hepatoblastoma, malignant rhabdoid kidney tumor, and renal sarcoma cell lines. In addition, we showed that in murine xenografts, treatment with M002 significantly increased survival and decreased tumor growth. Finally, these studies showed that the primary entry protein for oHSV, CD111 (nectin-1) was present in human hepatoblastoma and malignant rhabdoid kidney tumor specimens. We concluded that M002 effectively targeted these rare aggressive tumor types and that M002 may have potential for use in children with unresponsive or relapsed pediatric solid tumors.
|Arterial dysfunction but maintained systemic blood pressure in cavin-1-deficient mice. |
Swärd, K; Albinsson, S; Rippe, C
PloS one 9 e92428 2014
Caveolae are omega-shaped plasma membrane micro-domains that are abundant in cells of the vascular system. Formation of caveolae depends on caveolin-1 and cavin-1 and lack of either protein leads to loss of caveolae. Mice with caveolin-1 deficiency have dysfunctional blood vessels, but whether absence of cavin-1 similarly leads to vascular dysfunction is not known. Here we addressed this hypothesis using small mesenteric arteries from cavin-1-deficient mice. Cavin-1-reporter staining was intense in mesenteric arteries, brain arterioles and elsewhere in the vascular system, with positive staining of both endothelial and smooth muscle cells. Arterial expression of cavin-1, -2 and -3 was reduced in knockout (KO) arteries as was expression of caveolin-1, -2 and -3. Caveolae were absent in the endothelial and smooth muscle layers of small mesenteric arteries as determined by electron microscopy. Arginase, a negative regulator of nitric oxide production, was elevated in cavin-1 deficient arteries as was contraction in response to the α1-adrenergic agonist cirazoline. Detailed assessment of vascular dimensions revealed increased media thickness and reduced distensibility, arguing that enhanced contraction was due to increased muscle mass. Contrasting with increased α1-adrenergic contraction, myogenic tone was essentially absent and this appeared to be due in part to increased nitric oxide production. Vasomotion was less frequent in the knock-out vessels. In keeping with the opposing influences on arterial resistance of increased agonist-induced contractility and reduced myogenic tone, arterial blood pressure was unchanged in vivo. We conclude that deficiency of cavin-1 affects the function of small arteries, but that opposing influences on arterial resistance balance each other such that systemic blood pressure in unstressed mice is well maintained.
|Sox9 mediates Notch1-induced mesenchymal features in lung adenocarcinoma. |
Capaccione, KM; Hong, X; Morgan, KM; Liu, W; Bishop, JM; Liu, L; Markert, E; Deen, M; Minerowicz, C; Bertino, JR; Allen, T; Pine, SR
Oncotarget 5 3636-50 2014
Sox9 has gained increasing importance both functionally and as a prognostic factor in cancer. We demonstrate a functional role for Sox9 in inducing a mesenchymal phenotype in lung ADC. We show that Sox9 mRNA and protein are overexpressed in lung ADC, particularly those with KRAS mutations. Sox9 expression correlated with the Notch target gene Hes1, and numerous other Notch pathway components. We observed that Sox9 is a potent inducer of lung cancer cell motility and invasion, and a negative regulator of E-cadherin, a key protein that is lost during epithelial-mesenchymal transition (EMT). Moreover, we show that Notch1 signaling directly regulates Sox9 expression through a SOX9 promoter binding site, independently of the TGF-β pathway, and that Sox9 participates in Notch-1 induced cell motility, cell invasion, and loss of E-cadherin expression. Together, the results identify a new functional role for a Notch1-Sox9 signaling axis in lung ADC that may explain the correlation of Sox9 with tumor progression, higher tumor grade, and poor lung cancer survival. In addition to Notch and TGF-β, Sox9 also acts downstream of NF-κB, BMP, EGFR, and Wnt/β-catenin signaling. Thus, Sox9 could potentially act as a hub to mediate cross-talk among key oncogenic pathways in lung ADC. Targeting Sox9 expression or transcriptional activity could potentially reduce resistance to targeted therapy for lung ADC caused by pathway redundancy.
|Sequence of events in measles virus replication: role of phosphoprotein-nucleocapsid interactions. |
Brunel, J; Chopy, D; Dosnon, M; Bloyet, LM; Devaux, P; Urzua, E; Cattaneo, R; Longhi, S; Gerlier, D
Journal of virology 88 10851-63 2014
The genome of nonsegmented negative-strand RNA viruses is tightly embedded within a nucleocapsid made of a nucleoprotein (N) homopolymer. To ensure processive RNA synthesis, the viral polymerase L in complex with its cofactor phosphoprotein (P) binds the nucleocapsid that constitutes the functional template. Measles virus P and N interact through two binding sites. While binding of the P amino terminus with the core of N (NCORE) prevents illegitimate encapsidation of cellular RNA, the interaction between their C-terminal domains, P(XD) and N(TAIL) is required for viral RNA synthesis. To investigate the binding dynamics between the two latter domains, the P(XD) F497 residue that makes multiple hydrophobic intramolecular interactions was mutated. Using a quantitative mammalian protein complementation assay and recombinant viruses, we found that an increase in P(XD)-to-N(TAIL) binding strength is associated with a slower transcript accumulation rate and that abolishing the interaction renders the polymerase nonfunctional. The use of a newly developed system allowing conditional expression of wild-type or mutated P genes, revealed that the loss of the P(XD)-N(TAIL) interaction results in reduced transcription by preformed transcriptases, suggesting reduced engagement on the genomic template. These intracellular data indicate that the viral polymerase entry into and progression along its genomic template relies on a protein-protein interaction that serves as a tightly controlled dynamic anchor.Mononegavirales have a unique machinery to replicate RNA. Processivity of their polymerase is only achieved when the genome template is entirely embedded into a helical homopolymer of nucleoproteins that constitutes the nucleocapsid. The polymerase binds to the nucleocapsid template through the phosphoprotein. How the polymerase complex enters and travels along the nucleocapsid template to ensure uninterrupted synthesis of up to ∼ 6,700-nucleotide messenger RNAs from six to ten consecutive genes is unknown. Using a quantitative protein complementation assay and a biGene-biSilencing system allowing conditional expression of two P genes copies, the role of the P-to-N interaction in polymerase function was further characterized. We report here a dynamic protein anchoring mechanism that differs from all other known polymerases that rely only onto a sustained and direct binding to their nucleic acid template.
|The inhibition of N-glycosylation of glycoprotein 130 molecule abolishes STAT3 activation by IL-6 family cytokines in cultured cardiac myocytes. |
Matsuo, R; Morihara, H; Mohri, T; Murasawa, S; Takewaki, K; Nakayama, H; Maeda, M; Fujio, Y
PloS one 9 e111097 2014
Interleukin-6 (IL-6) family cytokines play important roles in cardioprotection against pathological stresses. IL-6 cytokines bind to their specific receptors and activate glycoprotein 130 (gp130), a common receptor, followed by further activation of STAT3 and extracellular signal-regulated kinase (ERK)1/2 through janus kinases (JAKs); however the importance of glycosylation of gp130 remains to be elucidated in cardiac myocytes. In this study, we examined the biological significance of gp130 glycosylation using tunicamycin (Tm), an inhibitor of enzyme involved in N-linked glycosylation. In cardiomyocytes, the treatment with Tm completely replaced the glycosylated form of gp130 with its unglycosylated one. Tm treatment inhibited leukemia inhibitory factor (LIF)-mediated activation of STAT3 and ERK1/2. Similarly, IL-11 failed to activate STAT3 and ERK1/2 in the presence of Tm. Interestingly, Tm inhibited the activation of JAKs 1 and 2, without influencing the expression of suppressor of cytokine signalings (SOCSs) and protein-tyrosine phosphatase 1B (PTP1B), which are endogenous inhibitors of JAKs. To exclude the possibility that Tm blocks LIF and IL-11 signals by inhibiting the glycosylation of their specific receptors, we investigated whether the stimulation with IL-6 plus soluble IL-6 receptor (sIL-6R) could transduce their signals in Tm-treated cardiomyocytes and found that this stimulation was unable to activate the downstream signals. Collectively, these findings indicate that glycosylation of gp130 is essential for signal transduction of IL-6 family cytokines in cardiomyocytes.
|Genes and gene networks involved in sodium fluoride-elicited cell death accompanying endoplasmic reticulum stress in oral epithelial cells. |
Tabuchi, Y; Yunoki, T; Hoshi, N; Suzuki, N; Kondo, T
International journal of molecular sciences 15 8959-78 2014
Here, to understand the molecular mechanisms underlying cell death induced by sodium fluoride (NaF), we analyzed gene expression patterns in rat oral epithelial ROE2 cells exposed to NaF using global-scale microarrays and bioinformatics tools. A relatively high concentration of NaF (2 mM) induced cell death concomitant with decreases in mitochondrial membrane potential, chromatin condensation and caspase-3 activation. Using 980 probe sets, we identified 432 up-regulated and 548 down-regulated genes, that were differentially expressed by greater than 2.5-fold in the cells treated with 2 mM of NaF and categorized them into 4 groups by K-means clustering. Ingenuity® pathway analysis revealed several gene networks from gene clusters. The gene networks Up-I and Up-II included many up-regulated genes that were mainly associated with the biological function of induction or prevention of cell death, respectively, such as Atf3, Ddit3 and Fos (for Up-I) and Atf4 and Hspa5 (for Up-II). Interestingly, knockdown of Ddit3 and Hspa5 significantly increased and decreased the number of viable cells, respectively. Moreover, several endoplasmic reticulum (ER) stress-related genes including, Ddit3, Atf4 and Hapa5, were observed in these gene networks. These findings will provide further insight into the molecular mechanisms of NaF-induced cell death accompanying ER stress in oral epithelial cells.
|Diversification of TAM receptor tyrosine kinase function. |
Zagórska, A; Través, PG; Lew, ED; Dransfield, I; Lemke, G
Nature immunology 15 920-8 2014
The clearance of apoptotic cells is critical for both tissue homeostasis and the resolution of inflammation. We found that the TAM receptor tyrosine kinases Axl and Mer had distinct roles as phagocytic receptors in these two settings, in which they exhibited divergent expression, regulation and activity. Mer acted as a tolerogenic receptor in resting macrophages and during immunosuppression. In contrast, Axl was an inflammatory response receptor whose expression was induced by proinflammatory stimuli. Axl and Mer differed in their ligand specificities, ligand-receptor complex formation in tissues, and receptor shedding upon activation. These differences notwithstanding, phagocytosis by either protein was strictly dependent on receptor activation triggered by bridging of TAM receptor-ligand complexes to the 'eat-me' signal phosphatidylserine on the surface of apoptotic cells.
|SPOP suppresses tumorigenesis by regulating Hedgehog/Gli2 signaling pathway in gastric cancer. |
Zeng, C; Wang, Y; Lu, Q; Chen, J; Zhang, J; Liu, T; Lv, N; Luo, S
Journal of experimental & clinical cancer research : CR 33 75 2014
Recent evidence suggests that aberrant activation of Hedgehog (Hh) signaling by Gli transcription factors is characteristic of a variety of aggressive human carcinomas including gastric cancer. Speckle-type POZ protein, SPOP, is an E3 ubiquitin ligase adaptor, and it is found to inhibit oncogenic signaling. However, the molecular mechanisms are largely unknown.In this study, we characterized the expression of SPOP in 88 pairs of gastric cancer tissues and adjacent tissues by immunohistochemical staining and Western blotting. The relationship between SPOP expression and clinical pathologic factors was analyzed. Transfected gastric cancer cell lines were used in cell viability, wound healing and colony formation assays. The interaction of SPOP with Gli2 and other related apoptotic proteins was assessed by immunoprecipitation, Western blotting, real-time PCR and dual luciferase reporter assays. Intracellular interaction of SPOP and Gli2 was visualized by immunofluorescent staining in gastric cancer cells.Immunohistochemical staining of SPOP can be detected in gastric cancer tissues but much less than adjacent gastric tissues (P less than 0.01). High SPOP expression is negatively correlated with lymph node metastasis, poor histological differentiation, and tumor malignancy according to TNM staging. In vitro experiments revealed that over-expression of SPOP prevented tumor cells from proliferation, migration and colony formation in gastric cancer cell lines. Likewise, repression of SPOP promoted cell viability, migration, proliferation, and attenuated apoptosis. Mechanistic studies revealed that increasing SPOP accelerated Gli2 degradation but regardless of Gli2 synthesis. Furthermore, cytoplasmic Gli2 decreased markedly along with the abundant expression of SPOP in MKN45 cells.Our findings indicate that SPOP plays critical roles in suppressing gastric tumorigenesis through inhibiting Hh/Gli2 signaling pathway. It may provide an alternative strategy for developing therapeutic agents of gastric cancer in future.
|The microRNA expression signature of bladder cancer by deep sequencing: the functional significance of the miR-195/497 cluster. |
Itesako, T; Seki, N; Yoshino, H; Chiyomaru, T; Yamasaki, T; Hidaka, H; Yonezawa, T; Nohata, N; Kinoshita, T; Nakagawa, M; Enokida, H
PloS one 9 e84311 2014
Current genome-wide microRNA (miRNA) expression signature analysis using deep sequencing technologies can drive the discovery of novel cancer pathways regulated by oncogenic and/or tumor suppressive miRNAs. We determined the genome-wide miRNA expression signature in bladder cancer (BC) by deep sequencing technology. A total of ten small RNA libraries were sequenced (five BCs and five samples of histologically normal bladder epithelia (NBE)), and 13,190,619 to 18,559,060 clean small RNA reads were obtained. A total of 933 known miRNAs and 17 new miRNA candidates were detected in this analysis. Among the known miRNAs, a total of 60 miRNAs were significantly downregulated in BC compared with NBE. We also found that several miRNAs, such as miR-1/133a, miR-206/133b, let-7c/miR-99a, miR-143/145 and miR-195/497, were located close together at five distinct loci and constituted clustered miRNAs. Among these clustered miRNAs, we focused on the miR-195/497 cluster because this clustered miRNA had not been analyzed in BC. Transfection of mature miR-195 or miR-497 in two BC cell lines (BOY and T24) significantly inhibited cancer cell proliferation, migration and invasion, suggesting that the miR-195/497 cluster functioned as tumor suppressors in BC. Regarding the genes targeted by the miR-195/497 cluster, the TargetScan algorithm showed that 6,730 genes were putative miR-195/497 targets, and 113 significantly enriched signaling pathways were identified in this analysis. The "Pathways in cancer" category was the most enriched, involving 104 candidate target genes. Gene expression data revealed that 27 of 104 candidate target genes were actually upregulated in BC clinical specimens. Luciferase reporter assays and Western blotting demonstrated that BIRC5 and WNT7A were directly targeted by miR-195/497. In conclusion, aberrant expression of clustered miRNAs was identified by deep sequencing, and downregulation of miR-195/497 contributed to BC progression and metastasis. Tumor suppressive miRNA-mediated cancer pathways provide new insights into the potential mechanisms of BC oncogenesis.
|Development of oral epithelial cell line ROE2 with differentiation potential from transgenic rats harboring temperature-sensitive simian virus40 large T-antigen gene. |
Tabuchi, Y; Wada, S; Ikegame, M; Kariya, A; Furusawa, Y; Hoshi, N; Yunoki, T; Suzuki, N; Takasaki, I; Kondo, T; Suzuki, Y
Experimental animals / Japanese Association for Laboratory Animal Science 63 31-44 2014
We have developed an immortalized oral epithelial cell line, ROE2, from fetal transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene. The cells grew continuously at either a permissive temperature of 33°C or an intermediate temperature of 37°C. At the nonpermissive temperature of 39°C, on the other hand, growth decreased significantly, and the Sub-G1 phase of the cell cycle increased, indicating that the cells undergo apoptosis at a nonpermissive temperature. Histological and immunocytochemical analyses revealed that ROE2 cells at 37°C had a stratified epithelial-like morphology and expressed cytokeratins Krt4 and Krt13, marker proteins for oral nonkeratinized epithelial cells. Global-scale comprehensive microarray analysis, coupled with bioinformatics tools, demonstrated a significant gene network that was obtained from the upregulated genes. The gene network contained 16 genes, including Cdkn1a, Fos, Krt13, and Prdm1, and was associated mainly with the biological process of skin development in the category of biological functions, organ development. These four genes were validated by quantitative real-time polymerase chain reaction, and the results were nearly consistent with the microarray data. It is therefore anticipated that this cell line will be useful as an in vitro model for studies such as physiological functions, as well as for gene expression in oral epithelial cells.
|Adenosine triphosphate concentrations are higher in the brain of APOE3- compared to APOE4-targeted replacement mice and can be modulated by curcumin. |
Chin, D; Hagl, S; Hoehn, A; Huebbe, P; Pallauf, K; Grune, T; Frank, J; Eckert, GP; Rimbach, G
Genes & nutrition 9 397 2014
Curcumin from Curcuma longa may exert putative neuroprotective properties in the brain. Impaired mitochondrial function is implicated in Alzheimer's disease and the presence of the apolipoprotein (APO) E4 genotype, which is a risk factor for late-onset Alzheimer's disease, may aggravate mitochondrial malfunction. Here, we report that in the brain of 16-month-old APOE4-targeted replacement mice, adenosine triphosphate (ATP) concentrations were significantly lower than in APOE3 mice. A 3-month dietary supplementation of 0.2 % curcumin numerically increased ATP concentrations in APOE3 and significantly in APOE4 mice compared to the respective controls. Curcumin significantly induced the transcription of peroxisome proliferator-activated receptor (PPAR) γ and mitochondrial transcription factor A (TFAM) in APOE3, but not in APOE4 mice. Moreover, PPARγ coactivator (PGC)-1α and guanine-adenine repeat binding protein α (GABPa) mRNA was only increased in APOE3 mice. Consistent with these observations, protein expression of mitochondrial respiratory complexes, especially of complex IV, also appeared to be increased in APOE3 mice. In conclusion, we provide evidence that curcumin affects mitochondrial function and gene and protein expression in the murine brain despite its low bioavailability and carriers of the Alzheimer's disease-risk genotype APOE4 may be less responsive to dietary curcumin than APOE3 carriers.
|Gasdermin-B promotes invasion and metastasis in breast cancer cells. |
Hergueta-Redondo, M; Sarrió, D; Molina-Crespo, Á; Megias, D; Mota, A; Rojo-Sebastian, A; García-Sanz, P; Morales, S; Abril, S; Cano, A; Peinado, H; Moreno-Bueno, G
PloS one 9 e90099 2014
Gasdermin B (GSDMB) belongs to the Gasdermin protein family that comprises four members (GSDMA-D). Gasdermin B expression has been detected in some tumor types such as hepatocarcinomas, gastric and cervix cancers; and its over-expression has been related to tumor progression. At least four splicing isoforms of GSDMB have been identified, which may play differential roles in cancer. However, the implication of GSDMB in carcinogenesis and tumor progression is not well understood. Here, we uncover for the first time the functional implication of GSDMB in breast cancer. Our data shows that high levels of GSDMB expression is correlated with reduced survival and increased metastasis in breast cancer patients included in an expression dataset (greater than 1,000 cases). We demonstrate that GSDMB is upregulated in breast carcinomas compared to normal breast tissue, being the isoform 2 (GSDMB-2) the most differentially expressed. In order to evaluate the functional role of GSDMB in breast cancer two GSDMB isoforms were studied (GSDMB-1 and GSDMB-2). The overexpression of both isoforms in the MCF7 breast carcinoma cell line promotes cell motility and invasion, while its silencing in HCC1954 breast carcinoma cells decreases the migratory and invasive phenotype. Importantly, we demonstrate that both isoforms have a differential role on the activation of Rac-1 and Cdc-42 Rho-GTPases. Moreover, our data support that GSMDB-2 induces a pro-tumorigenic and pro-metastatic behavior in mouse xenograft models as compared to GSDMB-1. Finally, we observed that although both GSDMB isoforms interact in vitro with the chaperone Hsp90, only the GSDMB-2 isoform relies on this chaperone for its stability. Taken together, our results provide for the first time evidences that GSDMB-2 induces invasion, tumor progression and metastasis in MCF7 cells and that GSDMB can be considered as a new potential prognostic marker in breast cancer.
|A gemcitabine sensitivity screen identifies a role for NEK9 in the replication stress response. |
Smith, SC; Petrova, AV; Madden, MZ; Wang, H; Pan, Y; Warren, MD; Hardy, CW; Liang, D; Liu, EA; Robinson, MH; Rudra, S; Wang, J; Ehdaivand, S; Torres, MA; Wang, Y; Yu, DS
Nucleic acids research 42 11517-27 2014
The Replication Stress Response (RSR) is a signaling network that recognizes challenges to DNA replication and coordinates diverse DNA repair and cell-cycle checkpoint pathways. Gemcitabine is a nucleoside analogue that causes cytotoxicity by inducing DNA replication blocks. Using a synthetic lethal screen of a RNAi library of nuclear enzymes to identify genes that when silenced cause gemcitabine sensitization or resistance in human triple-negative breast cancer cells, we identified NIMA (never in mitosis gene A)-related kinase 9 (NEK9) as a key component of the RSR. NEK9 depletion in cells leads to replication stress hypersensitivity, spontaneous accumulation of DNA damage and RPA70 foci, and an impairment in recovery from replication arrest. NEK9 protein levels also increase in response to replication stress. NEK9 complexes with CHK1, and moreover, NEK9 depletion impairs CHK1 autophosphorylation and kinase activity in response to replication stress. Thus, NEK9 is a critical component of the RSR that promotes CHK1 activity, maintaining genome integrity following challenges to DNA replication.
|Nuclear translocation uncovers the amyloid peptide Aβ42 as a regulator of gene transcription. |
Barucker, C; Harmeier, A; Weiske, J; Fauler, B; Albring, KF; Prokop, S; Hildebrand, P; Lurz, R; Heppner, FL; Huber, O; Multhaup, G
The Journal of biological chemistry 289 20182-91 2014
Although soluble species of the amyloid-β peptide Aβ42 correlate with disease symptoms in Alzheimer disease, little is known about the biological activities of amyloid-β (Aβ). Here, we show that Aβ peptides varying in lengths from 38 to 43 amino acids are internalized by cultured neuroblastoma cells and can be found in the nucleus. By three independent methods, we demonstrate direct detection of nuclear Aβ42 as follows: (i) biochemical analysis of nuclear fractions; (ii) detection of biotin-labeled Aβ in living cells by confocal laser scanning microscopy; and (iii) transmission electron microscopy of Aβ in cultured cells, as well as brain tissue of wild-type and transgenic APPPS1 mice (overexpression of amyloid precursor protein and presenilin 1 with Swedish and L166P mutations, respectively). Also, this study details a novel role for Aβ42 in nuclear signaling, distinct from the amyloid precursor protein intracellular domain. Chromatin immunoprecipitation showed that Aβ42 specifically interacts as a repressor of gene transcription with LRP1 and KAI1 promoters. By quantitative RT-PCR, we confirmed that mRNA levels of the examined candidate genes were exclusively decreased by the potentially neurotoxic Aβ42 wild-type peptide. Shorter peptides (Aβ38 or Aβ40) and other longer peptides (nontoxic Aβ42 G33A substitution or Aβ43) did not affect mRNA levels. Overall, our data indicate that the nuclear translocation of Aβ42 impacts gene regulation, and deleterious effects of Aβ42 in Alzheimer disease pathogenesis may be influenced by altering the expression profiles of disease-modifying genes.
|Alternatively spliced tissue factor is not sufficient for embryonic development. |
Sluka, SH; Akhmedov, A; Vogel, J; Unruh, D; Bogdanov, VY; Camici, GG; Lüscher, TF; Ruf, W; Tanner, FC
PloS one 9 e97793 2014
Tissue factor (TF) triggers blood coagulation and is translated from two mRNA splice isoforms, encoding membrane-anchored full-length TF (flTF) and soluble alternatively-spliced TF (asTF). The complete knockout of TF in mice causes embryonic lethality associated with failure of the yolk sac vasculature. Although asTF plays roles in postnatal angiogenesis, it is unknown whether it activates coagulation sufficiently or makes previously unrecognized contributions to sustaining integrity of embryonic yolk sac vessels. Using gene knock-in into the mouse TF locus, homozygous asTF knock-in (asTFKI) mice, which express murine asTF in the absence of flTF, exhibited embryonic lethality between day 9.5 and 10.5. Day 9.5 homozygous asTFKI embryos expressed asTF protein, but no procoagulant activity was detectable in a plasma clotting assay. Although the α-smooth-muscle-actin positive mesodermal layer as well as blood islands developed similarly in day 8.5 wild-type or homozygous asTFKI embryos, erythrocytes were progressively lost from disintegrating yolk sac vessels of asTFKI embryos by day 10.5. These data show that in the absence of flTF, asTF expressed during embryonic development has no measurable procoagulant activity, does not support embryonic vessel stability by non-coagulant mechanisms, and fails to maintain a functional vasculature and embryonic survival.
|Interleukin-17 Indirectly Promotes M2 Macrophage Differentiation through Stimulation of COX-2/PGE2 Pathway in the Cancer Cells. |
Li, Q; Liu, L; Zhang, Q; Liu, S; Ge, D; You, Z
Cancer research and treatment : official journal of Korean Cancer Association 46 297-306 2014
Interleukin-17 (IL-17) is a proinflammatory cytokine that plays important roles in inflammation, autoimmunity, and cancer. The purpose of this study was to determine if IL-17 indirectly regulates macrophage differentiation through up-regulation of cyclooxygenase-2 (COX-2) expression in the cancer cell lines.Human cervical cancer HeLa, human lung cancer A549, and mouse prostate cancer Myc-CaP/CR cell lines were treated with recombinant IL-17; Western blot analysis, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction analysis were utilized to examine the cellular responses.IL-17 up-regulated expression of COX-2 mRNA and protein in HeLa, A549, and Myc- CaP/CR cell lines. IL-17's effects were mediated through nuclear factor-κB and ERK1/2 signaling pathways as the inhibitors of these pathways could inhibit IL-17- induced COX-2 expression. The conditional medium obtained from the cancer cells contained prostaglandin E2, the levels of which were increased by IL-17 treatment. When treated with the conditional medium, particularly with the IL-17-induced conditional medium, mouse RAW264.7 macrophages and human THP-1 monocytes expressed higher levels of IL-10 (a marker of M2 macrophages) than inducible nitric oxide synthase or tumor necrosis factor α (markers of M1 macrophages). In contrast, when RAW264.7 and THP-1 cells were treated directly with IL-17, expression of these marker genes was not markedly changed.The results of this study suggest that IL-17 indirectly promotes M2 macrophage differentiation through stimulation of the COX-2/PGE2 pathway in the cancer cells, thus IL-17 plays an indirect role in regulating the tumor immune microenvironment.
|Modulation of network activity and induction of homeostatic synaptic plasticity by enzymatic removal of heparan sulfates. |
Korotchenko, S; Cingolani, LA; Kuznetsova, T; Bologna, LL; Chiappalone, M; Dityatev, A
Philosophical transactions of the Royal Society of London. Series B, Biological sciences 369 20140134 2014
Heparan sulfates (HSs) are complex and highly active molecules that are required for synaptogenesis and long-term potentiation. A deficit in HSs leads to autistic phenotype in mice. Here, we investigated the long-term effect of heparinase I, which digests highly sulfated HSs, on the spontaneous bioelectrical activity of neuronal networks in developing primary hippocampal cultures. We found that chronic heparinase treatment led to a significant reduction of the mean firing rate of neurons, particularly during the period of maximal neuronal activity. Furthermore, firing pattern in heparinase-treated cultures often appeared as epileptiform bursts, with long periods of inactivity between them. These changes in network activity were accompanied by an increase in the frequency and amplitude of miniature postsynaptic excitatory currents, which could be described by a linear up-scaling of current amplitudes. Biochemically, we observed an upregulation in the expression of the glutamate receptor subunit GluA1, but not GluA2, and a strong increase in autophosphorylation of α and β Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), without changes in the levels of kinase expression. These data suggest that a deficit in HSs triggers homeostatic synaptic plasticity and drastically affects functional maturation of neural network.
|Myogenic bladder defects in mouse models of human oculodentodigital dysplasia. |
Huang, T; Shao, Q; Barr, K; Simek, J; Fishman, GI; Laird, DW
The Biochemical journal 457 441-9 2014
To date, over 65 mutations in the gene encoding Cx43 (connexin43) have been linked to the autosomal-dominant disease ODDD (oculodentodigital dysplasia). A subset of these patients experience bladder incontinence which could be due to underlying neurogenic deterioration or aberrant myogenic regulation. BSMCs (bladder smooth muscle cells) from wild-type and two Cx43 mutant lines (Cx43(G60S) and Cx43(I130T)) that mimic ODDD exhibit a significant reduction in total Cx43. Dye transfer studies revealed that the G60S mutant was a potent dominant-negative inhibitor of co-expressed Cx43, a property not equally shared by the I130T mutant. BSMCs from both mutant mouse strains were defective in their ability to contract, which is indicative of phenotype changes due to harbouring the Cx43 mutants. Upon stretching, Cx43 levels were significantly elevated in controls and mutants containing BSMCs, but the non-muscle myosin heavy chain A levels were only reduced in cells from control mice. Although the Cx43(G60S) mutant mice showed no difference in voided urine volume or frequency, the Cx43(I130T) mice voided less frequently. Thus, similar to the diversity of morbidities seen in ODDD patients, genetically modified mice also display mutation-specific changes in bladder function. Furthermore, although mutant mice have compromised smooth muscle contraction and response to stretch, overriding bladder defects in Cx43(I130T) mice are likely to be complemented by neurogenic changes.
|Insulin like growth factor 2 regulation of aryl hydrocarbon receptor in MCF-7 breast cancer cells. |
Tomblin, JK; Salisbury, TB
Biochemical and biophysical research communications 443 1092-6 2014
Insulin like growth factor (IGF)-1 and IGF-2 stimulate normal growth, development and breast cancer cell proliferation. Cyclin D1 (CCND1) promotes cell cycle by inhibiting retinoblastoma protein (RB1). The aryl hydrocarbon receptor (AHR) is a major xenobiotic receptor that also regulates cell cycle. The purpose of this study was to investigate whether IGF-2 promotes MCF-7 breast cancer proliferation by inducing AHR. Western blot and quantitative real time PCR (Q-PCR) analysis revealed that IGF-2 induced an approximately 2-fold increase (Pless than .001) in the expression of AHR and CCND1. Chromatin immunoprecipitation (ChIP), followed by Q-PCR indicated that IGF-2 promoted (Pless than .001) a 7-fold increase in AHR binding on the CCND1 promoter. AHR knockdown significantly (Pless than .001) inhibited IGF-2 stimulated increases in CCND1 mRNA and protein. AHR knockdown cells were less (Pless than .001) responsive to the proliferative effects of IGF-2 than control cells. Collectively, our findings have revealed a new regulatory mechanism by which IGF-2 induction of AHR promotes the expression of CCND1 and the proliferation of MCF-7 cells. This previously uncharacterized pathway could be important for the proliferation of IGF responsive cancer cells that also express AHR.
|Down-regulation of Gli transcription factor leads to the inhibition of migration and invasion of ovarian cancer cells via integrin β4-mediated FAK signaling. |
Chen, Q; Xu, R; Zeng, C; Lu, Q; Huang, D; Shi, C; Zhang, W; Deng, L; Yan, R; Rao, H; Gao, G; Luo, S
PloS one 9 e88386 2014
Recent evidence suggests that aberrant activation of Hedgehog (Hh) signaling by Gli transcription factors is characteristic of a variety of aggressive human carcinomas including ovarian cancer. Therefore, chemotherapeutic agents that inhibit activation of Gli transcription factors have emerged as promising novel therapeutic drugs for ovarian cancer.In this study, we show that activation of Hh signaling promoted cellular migration and invasion, whereas blockade of Hh signaling with GANT61 suppressed cellular migration and invasion in ovarian cancer cells. After treatment with GANT61, cDNA microarray analyses revealed changes in many genes such as Integrin β4 subunit (ITGB4), focal adhesion kinase (FAK), etc. Furthermore, ITGB4 expression was up-regulated by Sonic Hedgehog (Shh) ligand and down-regulated by Hh signaling inhibitor. The Shh-mediated ovarian cell migration and invasion was blocked by neutralizing antibodies to ITGB4. In addition, phosphorylations of FAK were increased by Shh and decreased by Hh signaling inhibitor. Inhibition of Gli1 expression using siRNA mimicked the effects of GANT61 treatment, supporting the specificity of GANT61. Further investigations showed that activation of FAK was required for Shh-mediated cell migration and invasion. Finally, we found that down-regulation of Gli reduced the expression of ITGB4 and the phosphorylated FAK, resulting in the inhibition of tumor growth in vivo.The Hh signaling pathway induces cell migration and invasion through ITGB4-mediated activation of FAK in ovarian cancer. Our findings suggest that the diminishment of crosstalk between phosphorylated FAK and ITGB4 due to the down-regulation of Gli family transcription factors might play a pivotal role for inhibiting ovarian cancer progression.
|Lack of synergistic effect of resveratrol and sigma-1 receptor agonist (PRE-084) in SOD1G⁹³A ALS mice: overlapping effects or limited therapeutic opportunity? |
Mancuso, R; Del Valle, J; Morell, M; Pallás, M; Osta, R; Navarro, X
Orphanet journal of rare diseases 9 78 2014
Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease characterized by the loss of motoneurons (MNs) in the spinal cord, brainstem and motor cortex, causing progressive paralysis and death. Nowadays, there is no effective therapy and most patients die 2-5 years after diagnosis. Sigma-1R is a transmembrane protein highly expressed in the CNS and specially enriched in MNs. Mutations on the Sigma-1R leading to frontotemporal lobar degeneration-ALS were recently described in human patients. We previously reported the therapeutic role of the selective sigma-1R agonist 2-(4-morpholi-nethyl)1-phenylcyclohexanecarboxylate (PRE-084) in SOD1G93A ALS mice, that promoted spinal MN preservation and extended animal survival by controlling NMDA receptor calcium influx. Resveratrol (RSV, trans-3,4',5-trihydroxystilbene) is a natural polyphenol with promising neuroprotective effects. We recently found that RSV administration to SOD1G93A mice preserves spinal MN function and increases mice survival. These beneficial effects were associated to activation of Sirtuin 1 (Sirt1) and AMP-activated protein kinase (AMPK) pathways, leading to the modulation of autophagy and an increase of mitochondrial biogenesis. The main goal of this work was to assess the effect of combined RSV and PRE-084 administration in SOD1G93A ALS mice.We determined the locomotor performance of the animals by rotarod test and evaluated spinal motoneuron function using electrophysiological tests.RSV plus PRE-084 treatment from 8 weeks of age significantly improved locomotor performance and spinal MN function, accompanied by a significant reduction of MN degeneration and an extension of mice lifespan. In agreement with our previous findings, there was an induction of PKC-specific phosphorylation of the NMDA-NR1 subunit and an increased expression and activation of Sirt1 and AMPK in the ventral spinal cord of treated SOD1G93A animals.Although combined PRE and RSV treatment significantly ameliorated SOD1G93A mice, it did not show a synergistic effect compared to RSV-only and PRE-084-only treated groups.
|Cotranscriptional histone H2B monoubiquitylation is tightly coupled with RNA polymerase II elongation rate. |
Fuchs, G; Hollander, D; Voichek, Y; Ast, G; Oren, M
Genome research 24 1572-83 2014
Various histone modifications decorate nucleosomes within transcribed genes. Among these, monoubiquitylation of histone H2B (H2Bub1) and methylation of histone H3 on lysines 36 (H3K36me2/3) and 79 (H3K79me2/3) correlate positively with gene expression. By measuring the progression of the transcriptional machinery along genes within live cells, we now report that H2B monoubiquitylation occurs cotranscriptionally and accurately reflects the advance of RNA polymerase II (Pol II). In contrast, H3K36me3 and H3K79me2 are less dynamic and represent Pol II movement less faithfully. High-resolution ChIP-seq reveals that H2Bub1 levels are selectively reduced at exons and decrease in an exon-dependent stepwise manner toward the 3' end of genes. Exonic depletion of H2Bub1 in gene bodies is highly correlated with Pol II pausing at exons, suggesting elongation rate changes associated with intron-exon structure. In support of this notion, H2Bub1 levels were found to be significantly correlated with transcription elongation rates measured in various cell lines. Overall, our data shed light on the organization of H2Bub1 within transcribed genes and single out H2Bub1 as a reliable marker for ongoing transcription elongation.
|p53 Down regulates PDGF-induced formation of circular dorsal ruffles in rat aortic smooth muscle cells. |
Payne, LJ; Eves, RL; Jia, L; Mak, AS
PloS one 9 e108257 2014
The tumor suppressor, p53, negatively regulates cell migration and invasion in addition to its role in apoptosis, cell cycle regulation and senescence. Here, we study the roles of p53 in PDGF-induced circular dorsal ruffle (CDR) formation in rat aortic smooth muscle (RASM) cells. In primary and immortalized RASM cells, up-regulation of p53 expression or increase in activity with doxorubicin inhibits CDR formation. In contrast, shRNA-knockdown of p53 or inhibition of its activity with pifithrin α promotes CDR formation. p53 acts by up-regulating PTEN expression, which antagonizes Rac and Cdc42 activation. Both lipid and protein phosphatase activities of PTEN are required for maximal suppression of CDR, but the lipid activity clearly plays the dominant role. N-WASP, the downstream effector of Cdc42, is the major positive contributor to CDR formation in RASM, and is an indirect target of p53. The Rac effector, WAVE2, appears to also play a minor role, while WAVE1 has no significant effect in CDR formation. In sum, we propose that p53 suppresses PDGF-induced CDR formation in RASM cells by upregulating PTEN leading mainly to the inhibition of the Cdc42-N-WASP pathway.
|Pharmacological targeting of the β-amyloid precursor protein intracellular domain. |
Branca, C; Sarnico, I; Ruotolo, R; Lanzillotta, A; Viscomi, AR; Benarese, M; Porrini, V; Lorenzini, L; Calzà, L; Imbimbo, BP; Ottonello, S; Pizzi, M
Scientific reports 4 4618 2014
Amyloid precursor protein (APP) intracellular domain (AICD) is a product of APP processing with transcriptional modulation activity, whose overexpression causes various Alzheimer's disease (AD)-related dysfunctions. Here we report that 1-(3',4'-dichloro-2-fluoro[1,1'-biphenyl]-4-yl)-cyclopropanecarboxylic acid) (CHF5074), a compound that favorably affects neurodegeneration, neuroinflammation and memory deficit in transgenic mouse models of AD, interacts with the AICD and impairs its nuclear activity. In neuroglioma-APPswe cells, CHF5074 shifted APP cleavage from Aβ42 to the less toxic Aβ38 peptide without affecting APP-C-terminal fragment, nor APP levels. As revealed by photoaffinity labeling, CHF5074 does not interact with γ-secretase, but binds to the AICD and lowers its nuclear translocation. In vivo treatment with CHF5074 reduced AICD occupancy as well as histone H3 acetylation levels and transcriptional output of the AICD-target gene KAI1. The data provide new mechanistic insights on this compound, which is under clinical investigation for AD treatment/prevention, as well as on the contribution of the AICD to AD pathology.
|UVB induces a genome-wide acting negative regulatory mechanism that operates at the level of transcription initiation in human cells. |
Gyenis, A; Umlauf, D; Ujfaludi, Z; Boros, I; Ye, T; Tora, L
PLoS genetics 10 e1004483 2014
Faithful transcription of DNA is constantly threatened by different endogenous and environmental genotoxic effects. Transcription coupled repair (TCR) has been described to stop transcription and quickly remove DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. This repair mechanism has been well characterized in the past using individual target genes. Moreover, numerous efforts investigated the fate of blocked RNA polymerase II (Pol II) during DNA repair mechanisms and suggested that stopped Pol II complexes can either backtrack, be removed and degraded or bypass the lesions to allow TCR. We investigated the effect of a non-lethal dose of UVB on global DNA-bound Pol II distribution in human cells. We found that the used UVB dose did not induce Pol II degradation however surprisingly at about 93% of the promoters of all expressed genes Pol II occupancy was seriously reduced 2-4 hours following UVB irradiation. The presence of Pol II at these cleared promoters was restored 5-6 hours after irradiation, indicating that the negative regulation is very dynamic. We also identified a small set of genes (including several p53 regulated genes), where the UVB-induced Pol II clearing did not operate. Interestingly, at promoters, where Pol II promoter clearance occurs, TFIIH, but not TBP, follows the behavior of Pol II, suggesting that at these genes upon UVB treatment TFIIH is sequestered for DNA repair by the TCR machinery. In agreement, in cells where the TCR factor, the Cockayne Syndrome B protein, was depleted UVB did not induce Pol II and TFIIH clearance at promoters. Thus, our study reveals a UVB induced negative regulatory mechanism that targets Pol II transcription initiation on the large majority of transcribed gene promoters, and a small subset of genes, where Pol II escapes this negative regulation.
|Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy. |
Yuen, M; Sandaradura, SA; Dowling, JJ; Kostyukova, AS; Moroz, N; Quinlan, KG; Lehtokari, VL; Ravenscroft, G; Todd, EJ; Ceyhan-Birsoy, O; Gokhin, DS; Maluenda, J; Lek, M; Nolent, F; Pappas, CT; Novak, SM; D'Amico, A; Malfatti, E; Thomas, BP; Gabriel, SB; Gupta, N; Daly, MJ; Ilkovski, B; Houweling, PJ; Davidson, AE; Swanson, LC; Brownstein, CA; Gupta, VA; Medne, L; Shannon, P; Martin, N; Bick, DP; Flisberg, A; Holmberg, E; Van den Bergh, P; Lapunzina, P; Waddell, LB; Sloboda, DD; Bertini, E; Chitayat, D; Telfer, WR; Laquerrière, A; Gregorio, CC; Ottenheijm, CA; Bönnemann, CG; Pelin, K; Beggs, AH; Hayashi, YK; Romero, NB; Laing, NG; Nishino, I; Wallgren-Pettersson, C; Melki, J; Fowler, VM; MacArthur, DG; North, KN; Clarke, NF
The Journal of clinical investigation 124 4693-708 2014
Nemaline myopathy (NM) is a genetic muscle disorder characterized by muscle dysfunction and electron-dense protein accumulations (nemaline bodies) in myofibers. Pathogenic mutations have been described in 9 genes to date, but the genetic basis remains unknown in many cases. Here, using an approach that combined whole-exome sequencing (WES) and Sanger sequencing, we identified homozygous or compound heterozygous variants in LMOD3 in 21 patients from 14 families with severe, usually lethal, NM. LMOD3 encodes leiomodin-3 (LMOD3), a 65-kDa protein expressed in skeletal and cardiac muscle. LMOD3 was expressed from early stages of muscle differentiation; localized to actin thin filaments, with enrichment near the pointed ends; and had strong actin filament-nucleating activity. Loss of LMOD3 in patient muscle resulted in shortening and disorganization of thin filaments. Knockdown of lmod3 in zebrafish replicated NM-associated functional and pathological phenotypes. Together, these findings indicate that mutations in the gene encoding LMOD3 underlie congenital myopathy and demonstrate that LMOD3 is essential for the organization of sarcomeric thin filaments in skeletal muscle.
|Reducing dynamin 2 expression rescues X-linked centronuclear myopathy. |
Cowling, BS; Chevremont, T; Prokic, I; Kretz, C; Ferry, A; Coirault, C; Koutsopoulos, O; Laugel, V; Romero, NB; Laporte, J
The Journal of clinical investigation 124 1350-63 2014
Centronuclear myopathies (CNM) are congenital disorders associated with muscle weakness and abnormally located nuclei in skeletal muscle. An autosomal dominant form of CNM results from mutations in the gene encoding dynamin 2 (DNM2), and loss-of-function mutations in the gene encoding myotubularin (MTM1) result in X-linked CNM (XLCNM, also called myotubular myopathy), which promotes severe neonatal hypotonia and early death. Currently, no effective treatments exist for XLCNM. Here, we found increased DNM2 levels in XLCNM patients and a mouse model of XLCNM (Mtm1(-/y)). Generation of Mtm1(-/y) mice that were heterozygous for Dnm2 revealed that reduction of DNM2 in XLCNM mice restored life span, whole-body strength, and diaphragm function and increased muscle strength. Additionally, classic CNM-associated histological features, including fiber atrophy and nuclei mispositioning, were absent or reduced. Ultrastructural analysis revealed improvement of sarcomere organization and triad structures. Skeletal muscle-specific decrease of Dnm2 during embryogenesis or in young mice after disease onset revealed that the rescue associated with downregulation of Dnm2 is cell autonomous and is able to stop and potentially revert XLCNM progression. These data indicate that MTM1 and DNM2 regulate muscle organization and force through a common pathway. Furthermore, despite DNM2 being a key mechanoenzyme, its reduction is beneficial for XLCNM and represents a potential therapeutic approach for patients.
|An antagonistic interaction between PlexinB2 and Rnd3 controls RhoA activity and cortical neuron migration. |
Azzarelli, R; Pacary, E; Garg, R; Garcez, P; van den Berg, D; Riou, P; Ridley, AJ; Friedel, RH; Parsons, M; Guillemot, F
Nature communications 5 3405 2014
A transcriptional programme initiated by the proneural factors Neurog2 and Ascl1 controls successive steps of neurogenesis in the embryonic cerebral cortex. Previous work has shown that proneural factors also confer a migratory behaviour to cortical neurons by inducing the expression of the small GTP-binding proteins such as Rnd2 and Rnd3. However, the directionality of radial migration suggests that migrating neurons also respond to extracellular signal-regulated pathways. Here we show that the Plexin B2 receptor interacts physically and functionally with Rnd3 and stimulates RhoA activity in migrating cortical neurons. Plexin B2 competes with p190RhoGAP for binding to Rnd3, thus blocking the Rnd3-mediated inhibition of RhoA and also recruits RhoGEFs to directly stimulate RhoA activity. Thus, an interaction between the cell-extrinsic Plexin signalling pathway and the cell-intrinsic Ascl1-Rnd3 pathway determines the level of RhoA activity appropriate for cortical neuron migration.
|The HIV-1 Tat protein modulates CD4 expression in human T cells through the induction of miR-222. |
Orecchini, E; Doria, M; Michienzi, A; Giuliani, E; Vassena, L; Ciafrè, SA; Farace, MG; Galardi, S
RNA biology 11 334-8 2014
Several cellular microRNAs show substantial changes in expression during HIV-1 infection and their active role in the viral life cycle is progressively emerging. In the present study, we found that HIV-1 infection of Jurkat T cells significantly induces the expression of miR-222. We show that this induction depends on HIV-1 Tat protein, which is able to increase the transcriptional activity of NFkB on miR-222 promoter. Moreover, we demonstrate that miR-222 directly targets CD4, a key receptor for HIV-1, thus reducing its expression. We propose that Tat, by inducing miR-222 expression, complements the CD4 downregulation activity exerted by other viral proteins (i.e., Nef, Vpu, and Env), and we suggest that this represents a novel mechanism through which HIV-1 efficiently represses CD4 expression in infected cells.
|NOTCH1 Can Initiate NF-κB Activation via Cytosolic Interactions with Components of the T Cell Signalosome. |
Shin, HM; Tilahun, ME; Cho, OH; Chandiran, K; Kuksin, CA; Keerthivasan, S; Fauq, AH; Golde, TE; Miele, L; Thome, M; Osborne, BA; Minter, LM
Frontiers in immunology 5 249 2014
T cell stimulation requires the input and integration of external signals. Signaling through the T cell receptor (TCR) is known to induce formation of the membrane-tethered CBM complex, comprising CARMA1, BCL10, and MALT1, which is required for TCR-mediated NF-κB activation. TCR signaling has been shown to activate NOTCH proteins, transmembrane receptors also implicated in NF-κB activation. However, the link between TCR-mediated NOTCH signaling and early events leading to induction of NF-κB activity remains unclear. In this report, we demonstrate a novel cytosolic function for NOTCH1 and show that it is essential to CBM complex formation. Using a model of skin allograft rejection, we show in vivo that NOTCH1 acts in the same functional pathway as PKCθ, a T cell-specific kinase important for CBM assembly and classical NF-κB activation. We further demonstrate in vitro NOTCH1 associates physically with PKCθ and CARMA1 in the cytosol. Unexpectedly, when NOTCH1 expression was abrogated using RNAi approaches, interactions between CARMA1, BCL10, and MALT1 were lost. This failure in CBM assembly reduced inhibitor of kappa B alpha phosphorylation and diminished NF-κB-DNA binding. Finally, using a luciferase gene reporter assay, we show the intracellular domain of NOTCH1 can initiate robust NF-κB activity in stimulated T cells, even when NOTCH1 is excluded from the nucleus through modifications that restrict it to the cytoplasm or hold it tethered to the membrane. Collectively, these observations provide evidence that NOTCH1 may facilitate early events during T cell activation by nucleating the CBM complex and initiating NF-κB signaling.
|Progranulin protects against amyloid β deposition and toxicity in Alzheimer's disease mouse models. |
Minami, SS; Min, SW; Krabbe, G; Wang, C; Zhou, Y; Asgarov, R; Li, Y; Martens, LH; Elia, LP; Ward, ME; Mucke, L; Farese, RV; Gan, L
Nature medicine 20 1157-64 2014
Haploinsufficiency of the progranulin (PGRN) gene (GRN) causes familial frontotemporal lobar degeneration (FTLD) and modulates an innate immune response in humans and in mouse models. GRN polymorphism may be linked to late-onset Alzheimer's disease (AD). However, the role of PGRN in AD pathogenesis is unknown. Here we show that PGRN inhibits amyloid β (Aβ) deposition. Selectively reducing microglial expression of PGRN in AD mouse models impaired phagocytosis, increased plaque load threefold and exacerbated cognitive deficits. Lentivirus-mediated PGRN overexpression lowered plaque load in AD mice with aggressive amyloid plaque pathology. Aβ plaque load correlated negatively with levels of hippocampal PGRN, showing the dose-dependent inhibitory effects of PGRN on plaque deposition. PGRN also protected against Aβ toxicity. Lentivirus-mediated PGRN overexpression prevented spatial memory deficits and hippocampal neuronal loss in AD mice. The protective effects of PGRN against Aβ deposition and toxicity have important therapeutic implications. We propose enhancing PGRN as a potential treatment for PGRN-deficient FTLD and AD.
|Differential TAM receptor-ligand-phospholipid interactions delimit differential TAM bioactivities. |
Lew, ED; Oh, J; Burrola, PG; Lax, I; Zagórska, A; Través, PG; Schlessinger, J; Lemke, G
eLife 3 2014
The TAM receptor tyrosine kinases Tyro3, Axl, and Mer regulate key features of cellular physiology, yet the differential activities of the TAM ligands Gas6 and Protein S are poorly understood. We have used biochemical and genetic analyses to delineate the rules for TAM receptor-ligand engagement and find that the TAMs segregate into two groups based on ligand specificity, regulation by phosphatidylserine, and function. Tyro3 and Mer are activated by both ligands but only Gas6 activates Axl. Optimal TAM signaling requires coincident TAM ligand engagement of both its receptor and the phospholipid phosphatidylserine (PtdSer): Gas6 lacking its PtdSer-binding 'Gla domain' is significantly weakened as a Tyro3/Mer agonist and is inert as an Axl agonist, even though it binds to Axl with wild-type affinity. In two settings of TAM-dependent homeostatic phagocytosis, Mer plays a predominant role while Axl is dispensable, and activation of Mer by Protein S is sufficient to drive phagocytosis.
|G9a influences neuronal subtype specification in striatum. |
Maze, I; Chaudhury, D; Dietz, DM; Von Schimmelmann, M; Kennedy, PJ; Lobo, MK; Sillivan, SE; Miller, ML; Bagot, RC; Sun, H; Turecki, G; Neve, RL; Hurd, YL; Shen, L; Han, MH; Schaefer, A; Nestler, EJ
Nature neuroscience 17 533-9 2014
Cocaine-mediated repression of the histone methyltransferase (HMT) G9a has recently been implicated in transcriptional, morphological and behavioral responses to chronic cocaine administration. Here, using a ribosomal affinity purification approach, we found that G9a repression by cocaine occurred in both Drd1-expressing (striatonigral) and Drd2-expressing (striatopallidal) medium spiny neurons. Conditional knockout and overexpression of G9a within these distinct cell types, however, revealed divergent behavioral phenotypes in response to repeated cocaine treatment. Our studies further indicated that such developmental deletion of G9a selectively in Drd2 neurons resulted in the unsilencing of transcriptional programs normally specific to striatonigral neurons and in the acquisition of Drd1-associated projection and electrophysiological properties. This partial striatopallidal to striatonigral 'switching' phenotype in mice indicates a new role for G9a in contributing to neuronal subtype identity and suggests a critical function for cell type-specific histone methylation patterns in the regulation of behavioral responses to environmental stimuli.
|Increased macroH2A1.1 expression correlates with poor survival of triple-negative breast cancer patients. |
Lavigne, AC; Castells, M; Mermet, J; Kocanova, S; Dalvai, M; Bystricky, K
PloS one 9 e98930 2014
Epithelial-Mesenchymal Transition (EMT) features appear to be key events in development and progression of breast cancer. Epigenetic modifications contribute to the establishment and maintenance of cancer subclasses, as well as to the EMT process. Whether histone variants contribute to these transformations is not known. We investigated the relative expression levels of histone macroH2A1 splice variants and correlated it with breast cancer status/prognosis/types.To detect differential expression of macroH2A1 variant mRNAs in breast cancer cells and tumor samples, we used the following databases: GEO, EMBL-EBI and publisher databases (may-august 2012). We extracted macroH2A1.1/macroH2A1 mRNA ratios and performed correlation studies on intrinsic molecular subclasses of breast cancer and on molecular characteristics of EMT. Associations between molecular and survival data were determined.We found increased macroH2A1.1/macroH2A1 mRNA ratios to be associated with the claudin-low intrinsic subtype in breast cancer cell lines. At the molecular level this association translates into a positive correlation between macroH2A1 ratios and molecular characteristics of the EMT process. Moreover, untreated Triple Negative Breast Cancers presenting a high macroH2A1.1 mRNA ratio exhibit a poor outcome.These results provide first evidence that macroH2A1.1 could be exploited as an actor in the maintenance of a transient cellular state in EMT progress towards metastatic development of breast tumors.
|Cellular stress amplifies TLR3/4-induced CXCL1/2 gene transcription in mononuclear phagocytes via RIPK1. |
Zhao, C; Pavicic, PG; Datta, S; Sun, D; Novotny, M; Hamilton, TA
Journal of immunology (Baltimore, Md. : 1950) 193 879-88 2014
The impact of environmental stressors on the magnitude of specific chemokine gene expression was examined in mouse bone marrow-derived macrophages stimulated through various TLRs. Levels of TLR-stimulated CXCL1 and CXCL2 but not CXCL10 or CCL5 mRNAs were selectively enhanced (greater than 10-fold) in stressed macrophages. The amplification was also manifested for other proinflammatory cytokines, including TNF-α, IL-1α, and IL-6. Responses through TLR3 and TLR4 exhibited the greatest sensitivity, reflecting a requirement for Toll/IL-IR domain-containing adaptor-inducing IFN-β (TRIF), the adaptor protein selectively associated with these TLRs. IFN regulatory factor 3, a transcription factor that is downstream of TLR4/TRIF signaling, was not required for sensitivity to stress-induced chemokine amplification. c/EBP homologous protein and X box binding protein 1 have been reported to enhance inflammatory cytokine responses but are not required for amplification of TLR3/4-induced CXCL1 expression. Rather, receptor-interacting protein kinase 1, a kinase also linked with TLR3/4/TRIF signaling, is required and involves a stress-dependent increase in its abundance and ubiquitination. Whereas NF-κB activation is necessary for TLR-induced chemokine gene transcription, this factor does not appear to be the primary mechanistic target of environmental stress. The application of stress also enhanced chemokine expression in macrophages infiltrating the peritoneal cavity but was not observed in the resident peritoneal cells or in the liver. These findings identify novel mechanisms for modulating the magnitude and duration of selective TLR-induced chemokine and cytokine gene expression and further establish the importance of cell stress pathways in coordinating the outcomes of cellular and tissue injury.
|Angiotensin II induces membrane trafficking of natively expressed transient receptor potential vanilloid type 4 channels in hypothalamic 4B cells. |
Saxena, A; Bachelor, M; Park, YH; Carreno, FR; Nedungadi, TP; Cunningham, JT
American journal of physiology. Regulatory, integrative and comparative physiology 307 R945-55 2014
Transient receptor potential vanilloid family type 4 (TRPV4) channels are expressed in central neuroendocrine neurons and have been shown to be polymodal in other systems. We previously reported that in the rodent, a model of dilutional hyponatremia associated with hepatic cirrhosis, TRPV4 expression is increased in lipid rafts from the hypothalamus and that this effect may be angiotensin dependent. In this study, we utilized the immortalized neuroendocrine rat hypothalamic 4B cell line to more directly test the effects of angiotensin II (ANG II) on TRPV4 expression and function. Our results demonstrate the expression of corticotropin-releasing factor (CRF) transcripts, for sex-determining region Y (SRY) (male genotype), arginine vasopressin (AVP), TRPV4, and ANG II type 1a and 1b receptor in 4B cells. After a 1-h incubation in ANG II (100 nM), 4B cells showed increased TRPV4 abundance in the plasma membrane fraction, and this effect was prevented by the ANG II type 1 receptor antagonist losartan (1 μM) and by a Src kinase inhibitor PP2 (10 μM). Ratiometric calcium imaging experiments demonstrated that ANG II incubation potentiated TRPV4 agonist (GSK 1016790A, 20 nM)-induced calcium influx (control 18.4 ± 2.8% n = 5 and ANG II 80.5 ± 2.4% n = 5). This ANG II-induced increase in calcium influx was also blocked by 1 μM losartan and 10 μM PP2 (losartan 26.4 ± 3.8% n = 5 and PP2 19.7 ± 3.9% n = 5). Our data suggests that ANG II can increase TRPV4 channel membrane expression in 4B cells through its action on AT1R involving a Src kinase pathway.
|Forkhead transcription factor FOXO3a levels are increased in Huntington disease because of overactivated positive autofeedback loop. |
Kannike, K; Sepp, M; Zuccato, C; Cattaneo, E; Timmusk, T
The Journal of biological chemistry 289 32845-57 2014
Huntington disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by an increased number of CAG repeats in the HTT gene coding for huntingtin. Decreased neurotrophic support and increased mitochondrial and excitotoxic stress have been reported in HD striatal and cortical neurons. The members of the class O forkhead (FOXO) transcription factor family, including FOXO3a, act as sensor proteins that are activated upon decreased survival signals and/or increased cellular stress. Using immunocytochemical screening in mouse striatal Hdh(7/7) (wild type), Hdh(7/109) (heterozygous for HD mutation), and Hdh(109/109) (homozygous for HD mutation) cells, we identified FOXO3a as a differentially regulated transcription factor in HD. We report increased nuclear FOXO3a levels in mutant Hdh cells. Additionally, we show that treatment with mitochondrial toxin 3-nitropropionic acid results in enhanced nuclear localization of FOXO3a in wild type Hdh(7/7) cells and in rat primary cortical neurons. Furthermore, mRNA levels of Foxo3a are increased in mutant Hdh cells compared with wild type cells and in 3-nitropropionic acid-treated primary neurons compared with untreated neurons. A similar increase was observed in the cortex of R6/2 mice and HD patient post-mortem caudate tissue compared with controls. Using chromatin immunoprecipitation and reporter assays, we demonstrate that FOXO3a regulates its own transcription by binding to the conserved response element in Foxo3a promoter. Altogether, the findings of this study suggest that FOXO3a levels are increased in HD cells as a result of overactive positive feedback loop.
|Acetylsalicylic acid inhibits IL-18-induced cardiac fibroblast migration through the induction of RECK. |
Siddesha, JM; Valente, AJ; Sakamuri, SS; Gardner, JD; Delafontaine, P; Noda, M; Chandrasekar, B
Journal of cellular physiology 229 845-55 2014
The pathogenesis of cardiac fibrosis and adverse remodeling is thought to involve the ROS-dependent induction of inflammatory cytokines and matrix metalloproteinases (MMPs), and the activation and migration of cardiac fibroblasts (CF). Here we investigated the role of RECK (reversion-inducing-cysteine-rich protein with Kazal motifs), a unique membrane-anchored MMP regulator, on IL-18-induced CF migration, and the effect of acetylsalicylic acid (ASA) on this response. In a Matrigel invasion assay, IL-18-induced migration of primary mouse CF was dependent on both IKK/NF-κB- and JNK/AP-1-mediated MMP9 induction and Sp1-mediated RECK suppression, mechanisms that required Nox4-dependent H(2)O(2) generation. Notably, forced expression of RECK attenuated IL-18-induced MMP9 activation and CF migration. Further, therapeutic concentrations of ASA inhibited IL-18-induced H(2)O(2) generation, MMP9 activation, RECK suppression, and CF migration. The salicylic acid moiety of ASA similarly attenuated IL-18-induced CF migration. Thus, ASA may exert potential beneficial effect in cardiac fibrosis through multiple protective mechanisms.
|The sequence-specific transcription factor c-Jun targets Cockayne syndrome protein B to regulate transcription and chromatin structure. |
Lake, RJ; Boetefuer, EL; Tsai, PF; Jeong, J; Choi, I; Won, KJ; Fan, HY
PLoS genetics 10 e1004284 2014
Cockayne syndrome is an inherited premature aging disease associated with numerous developmental and neurological defects, and mutations in the gene encoding the CSB protein account for the majority of Cockayne syndrome cases. Accumulating evidence suggests that CSB functions in transcription regulation, in addition to its roles in DNA repair, and those defects in this transcriptional activity might contribute to the clinical features of Cockayne syndrome. Transcription profiling studies have so far uncovered CSB-dependent effects on gene expression; however, the direct targets of CSB's transcriptional activity remain largely unknown. In this paper, we report the first comprehensive analysis of CSB genomic occupancy during replicative cell growth. We found that CSB occupancy sites display a high correlation to regions with epigenetic features of promoters and enhancers. Furthermore, we found that CSB occupancy is enriched at sites containing the TPA-response element. Consistent with this binding site preference, we show that CSB and the transcription factor c-Jun can be found in the same protein-DNA complex, suggesting that c-Jun can target CSB to specific genomic regions. In support of this notion, we observed decreased CSB occupancy of TPA-response elements when c-Jun levels were diminished. By modulating CSB abundance, we found that CSB can influence the expression of nearby genes and impact nucleosome positioning in the vicinity of its binding site. These results indicate that CSB can be targeted to specific genomic loci by sequence-specific transcription factors to regulate transcription and local chromatin structure. Additionally, comparison of CSB occupancy sites with the MSigDB Pathways database suggests that CSB might function in peroxisome proliferation, EGF receptor transactivation, G protein signaling and NF-κB activation, shedding new light on the possible causes and mechanisms of Cockayne syndrome.
|Prognostic impact of PHIP copy number in melanoma: linkage to ulceration. |
Bezrookove, V; De Semir, D; Nosrati, M; Tong, S; Wu, C; Thummala, S; Dar, AA; Leong, SP; Cleaver, JE; Sagebiel, RW; Miller, JR; Kashani-Sabet, M
The Journal of investigative dermatology 134 783-90 2014
Ulceration is an important prognostic factor in melanoma whose biologic basis is poorly understood. Here we assessed the prognostic impact of pleckstrin homology domain-interacting protein (PHIP) copy number and its relationship to ulceration. PHIP copy number was determined using fluorescence in situ hybridization (FISH) in a tissue microarray cohort of 238 melanomas. Elevated PHIP copy number was associated with significantly reduced distant metastasis-free survival (DMFS; P=0.01) and disease-specific survival (DSS; P=0.009) by Kaplan-Meier analyses. PHIP FISH scores were independently predictive of DMFS (P=0.03) and DSS (P=0.03). Increased PHIP copy number was an independent predictor of ulceration status (P=0.04). The combined impact of increased PHIP copy number and tumor vascularity on ulceration status was highly significant (Pless than 0.0001). Stable suppression of PHIP in human melanoma cells resulted in significantly reduced glycolytic activity in vitro, with lower expression of lactate dehydrogenase 5, hypoxia-inducible factor 1 alpha subunit, and vascular endothelial growth factor, and was accompanied by reduced microvessel density in vivo. These results provide further support for PHIP as a molecular prognostic marker of melanoma, and reveal a significant linkage between PHIP levels and ulceration. Moreover, they suggest that ulceration may be driven by increased glycolysis and angiogenesis.
|Active and passive immunization strategies based on the SDPM1 peptide demonstrate pre-clinical efficacy in the APPswePSEN1dE9 mouse model for Alzheimer's disease. |
Camboni, M; Wang, CM; Miranda, C; Yoon, JH; Xu, R; Zygmunt, D; Kaspar, BK; Martin, PT
Neurobiology of disease 62 31-43 2014
Recent clinical and pre-clinical studies suggest that both active and passive immunization strategies targeting Aβ amyloid may have clinical benefit in Alzheimer's disease. Here, we demonstrate that vaccination of APPswePSEN1dE9 mice with SDPM1, an engineered non-native Aβ amyloid-specific binding peptide, lowers brain Aβ amyloid plaque burden and brain Aβ1-40 and Aβ1-42 peptide levels, improves cognitive learning and memory in Morris water maze tests and increases the expression of synaptic brain proteins. This was the case in young mice immunized prior to development of significant brain amyloid burden, and in older mice, where brain amyloid was already present. Active immunization was optimized using ALUM as an adjuvant to stimulate production of anti-SDPM1 and anti-Aβ amyloid antibodies. Intracerebral injection of P4D6, an SDPM1 peptide-mimotope antibody, also lowered brain amyloid plaque burden in APPswePSEN1dE9 mice. Additionally, P4D6 inhibited Aβ amyloid-mediated toxicity in cultured neuronal cells. The protein sequence of the variable domain within the P4D6 heavy chain was found to mimic a multimer of the SDPM1 peptide motif. These data demonstrate the efficacy of active and passive vaccine strategies to target Aβ amyloid oligomers using an engineered peptide-mimotope strategy.
|Gastritis promotes an activated bone marrow-derived mesenchymal stem cell with a phenotype reminiscent of a cancer-promoting cell. |
Donnelly, JM; Engevik, AC; Engevik, M; Schumacher, MA; Xiao, C; Yang, L; Worrell, RT; Zavros, Y
Digestive diseases and sciences 59 569-82 2014
Bone marrow-derived mesenchymal stem cells (BM-MSCs) promote gastric cancer in response to gastritis. In culture, BM-MSCs are prone to mutation with continued passage but it is unknown whether a similar process occurs in vivo in response to gastritis.The purpose of this study was to identify the role of chronic gastritis in the transformation of BM-MSCs leading to an activated cancer-promoting phenotype.Age matched C57BL/6 (BL/6) and gastrin deficient (GKO) mice were used for isolation of stomach, serum and mesenchymal stem cells (MSCs) at 3 and 6 months of age. MSC activation was assessed by growth curve analysis, fluorescence-activated cell sorting and xenograft assays. To allow for the isolation of bone marrow-derived stromal cells and assay in response to chronic gastritis, IRG/Vav-1(Cre) mice that expressed both enhanced green fluorescent protein-expressing hematopoietic cells and red fluorescent protein-expressing stromal cells were generated. In a parabiosis experiment, IRG/Vav-1(Cre) mice were paired to either an uninfected Vav-1(Cre) littermate or a BL/6 mouse inoculated with Helicobacter pylori.GKO mice displayed severe atrophic gastritis accompanied by elevated gastric tissue and circulating transforming growth factor beta (TGFβ) by 3 months of age. Compared to BM-MSCs isolated from uninflamed BL/6 mice, BM-MSCs isolated from GKO mice displayed an increased proliferative rate and elevated phosphorylated-Smad3 suggesting active TGFβ signaling. In xenograft assays, mice injected with BM-MSCs from 6-month-old GKO animals displayed tumor growth. RFP+ stromal cells were rapidly recruited to the gastric mucosa of H. pylori parabionts and exhibited changes in gene expression.Gastritis promotes the in vivo activation of BM-MSCs to a phenotype reminiscent of a cancer-promoting cell.
|An investigation of the effects of the core protein telomerase reverse transcriptase on Wnt signaling in breast cancer cells. |
Listerman, I; Gazzaniga, FS; Blackburn, EH
Molecular and cellular biology 34 280-9 2014
Telomerase canonically maintains telomeres, but recent reports have suggested that the core protein mammalian telomerase reverse transcriptase (TERT) component, together with the chromatin remodeling factor BRG1 and β-catenin, may also bind to and promote expression of Wnt target genes. However, this proposed noncanonical role of TERT in Wnt signaling has been controversial. Here, we investigated the effects of human TERT (hTERT) on Wnt signaling in human breast cancer lines and HeLa cells. We failed to find evidence for physical association of hTERT with BRG1 or β-catenin; instead, we present evidence that anti-FLAG antibody cross-reactivity properties may explain the previously reported interaction of hTERT with β-catenin. Furthermore, altering hTERT levels in four different breast cancer cell lines caused minimal and discordant effects on Wnt target and Wnt pathway gene expression. Although hTERT's role in Wnt signaling was addressed only indirectly, no significant representation of Wnt target genes was detected in chromatin immunoprecipitation-sequencing (ChIP-seq) and chromatin isolation by RNA purification and sequencing (ChIRP-seq) loci cooccupied in HeLa S3 cells by both BRG1 and hTR. In summary, our evidence fails to support the idea of a biologically consistent hTERT interaction with the Wnt pathway in human breast cancer cells, and any detectable influence of hTERT depended on cell type and experimental system.
|Congenital brain serotonin deficiency leads to reduced ethanol sensitivity and increased ethanol consumption in mice. |
Sachs, BD; Salahi, AA; Caron, MG
Neuropharmacology 77 177-84 2014
Serotonergic dysfunction has been hypothesized to play an important role in the pathophysiology of alcoholism. However, whether congenital serotonin (5-HT) deficiency leads to increased alcohol consumption or affects ethanol-related behaviors has not been established. Here, we use a transgenic mouse line that expresses a hypofunctional variant of the 5-HT synthesis enzyme, tryptophan hydroxylase 2, to examine the impact of 5-HT deficiency on responses to alcohol. We demonstrate that these 5-HT-deficient transgenic animals (Tph2KI mice) recover their righting reflex more rapidly than wild-type controls following a high dose of ethanol and exhibit blunted locomotor retardation in response to repeated ethanol administration. In addition, compared to WT controls, 5-HT-deficient animals consume significantly more ethanol and exhibit increased preference for ethanol in two-bottle choice tests. Our data also suggest that 5-HT plays a critical role in mediating the effects of ethanol on Akt/GSK3β signaling in the nucleus accumbens. Overall, our results corroborate previous theories regarding the importance of brain 5-HT levels in mediating responsiveness to alcohol and demonstrate, for the first time, that congenital 5-HT deficiency leads to increased ethanol consumption and decreased sensitivity to the sedative-like effects of ethanol, perhaps in part through modulating Akt/GSK3β signaling.
|Cell-type specific increases in female hamster nucleus accumbens spine density following female sexual experience. |
Staffend, NA; Hedges, VL; Chemel, BR; Watts, VJ; Meisel, RL
Brain structure & function 219 2071-81 2014
Female sexual behavior is an established model of a naturally motivated behavior which is regulated by activity within the mesolimbic dopamine system. Repeated activation of the mesolimbic circuit by female sexual behavior elevates dopamine release and produces persistent postsynaptic alterations to dopamine D1 receptor signaling within the nucleus accumbens. Here we demonstrate that sexual experience in female Syrian hamsters significantly increases spine density and alters morphology selectively in D1 receptor-expressing medium spiny neurons within the nucleus accumbens core, with no corresponding change in dopamine receptor binding or protein expression. Our findings demonstrate that previous life experience with a naturally motivated behavior has the capacity to induce persistent structural alterations to the mesolimbic circuit that can increase reproductive success and are analogous to the persistent structural changes following repeated exposure to many drugs of abuse.
|Distribution of language-related Cntnap2 protein in neural circuits critical for vocal learning. |
Condro, MC; White, SA
The Journal of comparative neurology 522 169-85 2014
Variants of the contactin associated protein-like 2 (Cntnap2) gene are risk factors for language-related disorders including autism spectrum disorder, specific language impairment, and stuttering. Songbirds are useful models for study of human speech disorders due to their shared capacity for vocal learning, which relies on similar cortico-basal ganglia circuitry and genetic factors. Here we investigate Cntnap2 protein expression in the brain of the zebra finch, a songbird species in which males, but not females, learn their courtship songs. We hypothesize that Cntnap2 has overlapping functions in vocal learning species, and expect to find protein expression in song-related areas of the zebra finch brain. We further expect that the distribution of this membrane-bound protein may not completely mirror its mRNA distribution due to the distinct subcellular localization of the two molecular species. We find that Cntnap2 protein is enriched in several song control regions relative to surrounding tissues, particularly within the adult male, but not female, robust nucleus of the arcopallium (RA), a cortical song control region analogous to human layer 5 primary motor cortex. The onset of this sexually dimorphic expression coincides with the onset of sensorimotor learning in developing males. Enrichment in male RA appears due to expression in projection neurons within the nucleus, as well as to additional expression in nerve terminals of cortical projections to RA from the lateral magnocellular nucleus of the nidopallium. Cntnap2 protein expression in zebra finch brain supports the hypothesis that this molecule affects neural connectivity critical for vocal learning across taxonomic classes.
|miR-661 downregulates both Mdm2 and Mdm4 to activate p53. |
Hoffman, Y; Bublik, DR; Pilpel, Y; Oren, M
Cell death and differentiation 21 302-9 2014
The p53 pathway is pivotal in tumor suppression. Cellular p53 activity is subject to tight regulation, in which the two related proteins Mdm2 and Mdm4 have major roles. The delicate interplay between the levels of Mdm2, Mdm4 and p53 is crucial for maintaining proper cellular homeostasis. microRNAs (miRNAs) are short non-coding RNAs that downregulate the level and translatability of specific target mRNAs. We report that miR-661, a primate-specific miRNA, can target both Mdm2 and Mdm4 mRNA in a cell type-dependent manner. miR-661 interacts with Mdm2 and Mdm4 RNA within living cells. The inhibitory effect of miR-661 is more prevalent on Mdm2 than on Mdm4. Interestingly, the predicted miR-661 targets in both mRNAs reside mainly within Alu elements, suggesting a primate-specific mechanism for regulatory diversification during evolution. Downregulation of Mdm2 and Mdm4 by miR-661 augments p53 activity and inhibits cell cycle progression in p53-proficient cells. Correspondingly, low miR-661 expression correlates with bad outcome in breast cancers that typically express wild-type p53. In contrast, the miR-661 locus tends to be amplified in tumors harboring p53 mutations, and miR-661 promotes migration of cells derived from such tumors. Thus, miR-661 may either suppress or promote cancer aggressiveness, depending on p53 status.
|Vascular delivery of rAAVrh74.MCK.GALGT2 to the gastrocnemius muscle of the rhesus macaque stimulates the expression of dystrophin and laminin α2 surrogates. |
Chicoine, LG; Rodino-Klapac, LR; Shao, G; Xu, R; Bremer, WG; Camboni, M; Golden, B; Montgomery, CL; Shontz, K; Heller, KN; Griffin, DA; Lewis, S; Coley, BD; Walker, CM; Clark, KR; Sahenk, Z; Mendell, JR; Martin, PT
Molecular therapy : the journal of the American Society of Gene Therapy 22 713-24 2014
Overexpression of GALGT2 in skeletal muscle can stimulate the glycosylation of α dystroglycan and the upregulation of normally synaptic dystroglycan-binding proteins, some of which are dystrophin and laminin α2 surrogates known to be therapeutic for several forms of muscular dystrophy. This article describes the vascular delivery of GALGT2 gene therapy in a large animal model, the rhesus macaque. Recombinant adeno-associated virus, rhesus serotype 74 (rAAVrh74), was used to deliver GALGT2 via the femoral artery to the gastrocnemius muscle using an isolated focal limb perfusion method. GALGT2 expression averaged 44 ± 4% of myofibers after treatment in macaques with low preexisting anti-rAAVrh74 serum antibodies, and expression was reduced to 9 ± 4% of myofibers in macaques with high preexisting rAAVrh74 immunity (P less than 0.001; n = 12 per group). This was the case regardless of the addition of immunosuppressants, including prednisolone, tacrolimus, and mycophenolate mofetil. GALGT2-treated macaque muscles showed increased glycosylation of α dystroglycan and increased expression of dystrophin and laminin α2 surrogate proteins, including utrophin, plectin1, agrin, and laminin α5. These experiments demonstrate successful transduction of rhesus macaque muscle with rAAVrh74.MCK.GALGT2 after vascular delivery and induction of molecular changes thought to be therapeutic in several forms of muscular dystrophy.
|Differential postpartum sensitivity to the anxiety-modulating effects of offspring contact is associated with innate anxiety and brainstem levels of dopamine beta-hydroxylase in female laboratory rats. |
Ragan, CM; Lonstein, JS
Neuroscience 256 433-44 2014
In female mammals, the postpartum period involves dramatic shifts in many socioemotional behaviors. This includes a suppression of anxiety-related behaviors that requires recent physical contact with offspring. Factors contributing to differences among females in their susceptibility to the anxiety-modulating effect of offspring contact are unknown, but could include their innate anxiety and brain monoaminergic activity. Anxiety behavior was assessed in a large group of nulliparous female rats and the least-anxious and most-anxious tertiles were mated. Anxiety was assessed again postpartum after females were permitted or prevented from contacting their offspring 4 h before testing. Levels of dopamine β-hydroxylase (DBH, norepinephrine synthesizing enzyme) and tryptophan hydroxylase-2 (TPH2, serotonin synthesizing enzyme) were measured in the brainstem and dorsal raphe, respectively. It was found that anxiety-related behavior in the two groups did not differ when dams were permitted contact with offspring before testing. Removal of the offspring before testing, however, differentially affected anxiety based on dams' innate anxiety. Specifically, dams reverted back to their pre-mating levels of anxiety such that offspring removal slightly increased anxiety in the most-anxious females but greatly lowered anxiety in the least-anxious females. This reduction in anxiety in the least-anxious females after litter removal was associated with lower brainstem DBH. There was no relationship between females' anxiety and dorsal raphe TPH2. Thus, a primary effect of recent contact with offspring on anxiety-related behavior in postpartum rats is to shift females away from their innate anxiety to a more moderate level of responding. This effect is particularly true for females with the lowest anxiety, may be mediated by central noradrenergic systems, and has implications for their ability to attend to their offspring.
|Inhibition of succinate dehydrogenase by the mitochondrial chaperone TRAP1 has anti-oxidant and anti-apoptotic effects on tumor cells. |
Guzzo, G; Sciacovelli, M; Bernardi, P; Rasola, A
Oncotarget 5 11897-908 2014
TRAP1 is a mitochondrial chaperone highly expressed in many tumor types; it inhibits respiratory complex II, down-modulating its succinate dehydrogenase (SDH) enzymatic activity. SDH inhibition in turn leads to a pseudohypoxic state caused by succinate-dependent HIF1α stabilization and promotes neoplastic growth. Here we report that TRAP1 inhibition of SDH also shields cells from oxidative insults and from the ensuing lethal opening of the mitochondrial permeability transition pore. This anti-oxidant activity of TRAP1 protects tumor cells from death in conditions of nutrient paucity that mimic those encountered in the neoplasm during the process of malignant accrual, and it is required for in vitro tumorigenic growth. Our findings demonstrate that SDH inhibition by TRAP1 is oncogenic not only by inducing pseudohypoxia, but also by protecting tumor cells from oxidative stress.
|Intravenously injected human apolipoprotein A-I rapidly enters the central nervous system via the choroid plexus. |
Stukas, S; Robert, J; Lee, M; Kulic, I; Carr, M; Tourigny, K; Fan, J; Namjoshi, D; Lemke, K; DeValle, N; Chan, J; Wilson, T; Wilkinson, A; Chapanian, R; Kizhakkedathu, JN; Cirrito, JR; Oda, MN; Wellington, CL
Journal of the American Heart Association 3 e001156 2014
Brain lipoprotein metabolism is dependent on lipoprotein particles that resemble plasma high-density lipoproteins but that contain apolipoprotein (apo) E rather than apoA-I as their primary protein component. Astrocytes and microglia secrete apoE but not apoA-I; however, apoA-I is detectable in both cerebrospinal fluid and brain tissue lysates. The route by which plasma apoA-I enters the central nervous system is unknown.Steady-state levels of murine apoA-I in cerebrospinal fluid and interstitial fluid are 0.664 and 0.120 μg/mL, respectively, whereas brain tissue apoA-I is ≈10% to 15% of its levels in liver. Recombinant, fluorescently tagged human apoA-I injected intravenously into mice localizes to the choroid plexus within 30 minutes and accumulates in a saturable, dose-dependent manner in the brain. Recombinant, fluorescently tagged human apoA-I accumulates in the brain for 2 hours, after which it is eliminated with a half-life of 10.3 hours. In vitro, human apoA-I is specifically bound, internalized, and transported across confluent monolayers of primary human choroid plexus epithelial cells and brain microvascular endothelial cells.Following intravenous injection, recombinant human apoA-I rapidly localizes predominantly to the choroid plexus. Because apoA-I mRNA is undetectable in murine brain, our results suggest that plasma apoA-I, which is secreted from the liver and intestine, gains access to the central nervous system primarily by crossing the blood-cerebrospinal fluid barrier via specific cellular mediated transport, although transport across the blood-brain barrier may also contribute to a lesser extent.
|Specific deficiency of Plzf paralog, Zbtb20, in Sertoli cells does not affect spermatogenesis and fertility in mice. |
Jiang, X; Zhang, H; Yin, S; Zhang, Y; Yang, W; Zheng, W; Wang, L; Wang, Z; Bukhari, I; Cooke, HJ; Iqbal, F; Shi, Q
Scientific reports 4 7062 2014
Ztbt20 is a POK family transcription factor and primarily functions through its conserved C2H2 Krüppel type zinc finger and BTB/POZ domains. The present study was designed to define the function of the Zbtb20, in vivo, during mouse spermatogenesis. Immunohistochemical studies revealed that ZBTB20 protein was localized specifically in the nuclei of Sertoli cells in seminiferous tubules. To investigate its role during spermatogenesis, we crossed Amh-Cre transgenic mice with Zbtb20 floxp mice to generate conditionally knockout mice (cKO) in which Zbtb20 was specifically deleted in Sertoli cells. The cKO mice were fertile and did not show any detectable abnormalities in spermatogenesis. Taken together, though specific deletion of transcription factor Zbtb20 in Sertoli cells has no apparent influence on spermatogenesis, its specific localization in Sertoli cells makes Zbtb20 a useful marker for the identification of Sertoli cells in seminiferous tubules.
|Loss of compensatory pro-survival and anti-apoptotic modulator, IKKε, sensitizes ovarian cancer cells to CHEK1 loss through an increased level of p21. |
Kim, MK; Min, DJ; Wright, G; Goldlust, I; Annunziata, CM
Oncotarget 5 12788-802 2014
Ovarian cancer (OC) is extremely heterogeneous, implying that therapeutic strategies should be specifically designed based on molecular characteristics of an individual's tumor. Previously, we showed that IKKε promotes invasion and metastasis in a subset of OCs. Here, we identified CHEK1 as an IKKε-dependent lethal gene from shRNA kinome library screen. In subsequent pharmacological intervention studies, the co-inhibition of IKKε and CHEK1 was more effective in killing OC cells than single treatment. At the molecular level, co-inhibition dramatically decreased pro-survival proteins, but increased proteins involved in DNA damage and apoptosis. IKKε-knockdown increased p21 levels, while overexpression of wild-type IKKε, but not a kinase dead IKKε mutant decreased p21 levels. We further demonstrated that the depletion of p21 rendered OC cells more resistant to cell death induced by co-inhibition of IKKε and CHEK1. In conclusion, we revealed a novel interplay between IKKε, CHEK1 and p21 signaling in survival of OC. Our study provides a rationale for the clinical development of specific IKKε inhibitor and for usage of IKKε as an exploratory marker for resistance to CHEK1 inhibitors in the clinic. The interplay provides one potential explanation as to why very few clinical responses were achieved in patients treated with single-agent CHEK1 inhibitors.
|Biallelic truncating mutations in FMN2, encoding the actin-regulatory protein Formin 2, cause nonsyndromic autosomal-recessive intellectual disability. |
Law, R; Dixon-Salazar, T; Jerber, J; Cai, N; Abbasi, AA; Zaki, MS; Mittal, K; Gabriel, SB; Rafiq, MA; Khan, V; Nguyen, M; Ali, G; Copeland, B; Scott, E; Vasli, N; Mikhailov, A; Khan, MN; Andrade, DM; Ayaz, M; Ansar, M; Ayub, M; Vincent, JB; Gleeson, JG
American journal of human genetics 95 721-8 2014
Dendritic spines represent the major site of neuronal activity in the brain; they serve as the receiving point for neurotransmitters and undergo rapid activity-dependent morphological changes that correlate with learning and memory. Using a combination of homozygosity mapping and next-generation sequencing in two consanguineous families affected by nonsyndromic autosomal-recessive intellectual disability, we identified truncating mutations in formin 2 (FMN2), encoding a protein that belongs to the formin family of actin cytoskeleton nucleation factors and is highly expressed in the maturing brain. We found that FMN2 localizes to punctae along dendrites and that germline inactivation of mouse Fmn2 resulted in animals with decreased spine density; such mice were previously demonstrated to have a conditioned fear-learning defect. Furthermore, patient neural cells derived from induced pluripotent stem cells showed correlated decreased synaptic density. Thus, FMN2 mutations link intellectual disability either directly or indirectly to the regulation of actin-mediated synaptic spine density.
|miR-214-mediated downregulation of RNF8 induces chromosomal instability in ovarian cancer cells. |
Wang, Z; Yin, H; Zhang, Y; Feng, Y; Yan, Z; Jiang, X; Bukhari, I; Iqbal, F; Cooke, HJ; Shi, Q
Cell cycle (Georgetown, Tex.) 13 3519-28 2014
Defective DNA damage response (DDR) is frequently associated with carcinogenesis. Abrogation of DDR leads to chromosomal instability, a most common characteristic of tumors. However, the molecular mechanisms underlying regulation of DDR are still elusive. The ubiquitin ligase RNF8 mediates the ubiquitination of γH2AX and recruits 53BP1 and BRCA1 to DNA damage sites which promotes DDR and inhibits chromosomal instability. Though RNF8 is a key player involved in DDR, regulation of its expression is still poorly understood. Here, we show that miR-214 could abrogate DDR by repressing RNF8 expression through direct binding to 3'-untranslated region (3' UTR) of RNF8 mRNA in human ovarian cancer cells. Antagonizing miR-214 by expressing its inhibitors in A2780 cells significantly increased RNF8 expression and thus promoted DNA damage repair. Consistent with the role of miR-214 in regulating RNF8 expression, the impaired DNA repair induced by miR-214 overexpression can be rescued by overexpressing RNF8 mRNA lacking the 3' UTR. Together, our results indicate that down-regulation of RNF8 mediated by miR-214 impedes DNA damage response to induce chromosomal instability in ovarian cancers, which may facilitate the understanding of mechanisms underlying chromosomal instability.
|Formation of well-defined embryoid bodies from dissociated human induced pluripotent stem cells using microfabricated cell-repellent microwell arrays. |
Pettinato, G; Wen, X; Zhang, N
Scientific reports 4 7402 2014
A simple, scalable, and reproducible technology that allows direct formation of large numbers of homogeneous and synchronized embryoid bodies (EBs) of defined sizes from dissociated human induced pluripotent stem cells (hiPSCs) was developed. Non-cell-adhesive hydrogels were used to create round-bottom microwells to host dissociated hiPSCs. No Rho-associated kinase inhibitor (ROCK-i), or centrifugation was needed and the side effects of ROCK-i can be avoided. The key requirement for the successful EB formation in addition to the non-cell-adhesive round-bottom microwells is the input cell density per microwell. Too few or too many cells loaded into the microwells will compromise the EB formation process. In parallel, we have tested our microwell-based system for homogeneous hEB formation from dissociated human embryonic stem cells (hESCs). Successful production of homogeneous hEBs from dissociated hESCs in the absence of ROCK-i and centrifugation was achieved within an optimal range of input cell density per microwell. Both the hiPSC- and hESC-derived hEBs expressed key proteins characteristic of all the three developmental germ layers, confirming their EB identity. This novel EB production technology may represent a versatile platform for the production of homogeneous EBs from dissociated human pluripotent stem cells (hPSCs).
|Neuroprotective and anti-inflammatory roles of the phosphatase and tensin homolog deleted on chromosome Ten (PTEN) Inhibition in a Mouse Model of Temporal Lobe Epilepsy. |
Grande, V; Manassero, G; Vercelli, A
PloS one 9 e114554 2014
Excitotoxic damage represents the major mechanism leading to cell death in many human neurodegenerative diseases such as ischemia, trauma and epilepsy. Caused by an excess of glutamate that acts on metabotropic and ionotropic excitatory receptors, excitotoxicity activates several death signaling pathways leading to an extensive neuronal loss and a consequent strong activation of astrogliosis. Currently, the search for a neuroprotective strategy is aimed to identify the level in the signaling pathways to block excitotoxicity avoiding the loss of important physiological functions and side effects. To this aim, PTEN can be considered an ideal candidate: downstream the excitatory receptors activated in excitotoxicity (whose inhibition was shown to be not clinically viable), it is involved in neuronal damage and in the first stage of the reactive astrogliosis in vivo. In this study, we demonstrated the involvement of PTEN in excitotoxicity through its pharmacological inhibition by dipotassium bisperoxo (picolinato) oxovanadate [bpv(pic)] in a model of temporal lobe epilepsy, obtained by intraperitoneal injection of kainate in 2-month-old C57BL/6J male mice. We have demonstrated that inhibition of PTEN by bpv(pic) rescues neuronal death and decreases the reactive astrogliosis in the CA3 area of the hippocampus caused by systemic administration of kainate. Moreover, the neurotoxin administration increases significantly the scanty presence of mitochondrial PTEN that is significantly decreased by the administration of the inhibitor 6 hr after the injection of kainate, suggesting a role of PTEN in mitochondrial apoptosis. Taken together, our results confirm the key role played by PTEN in the excitotoxic damage and the strong anti-inflammatory and neuroprotective potential of its inhibition.
|Constitutive and ligand-induced EGFR signalling triggers distinct and mutually exclusive downstream signalling networks. |
Chakraborty, S; Li, L; Puliyappadamba, VT; Guo, G; Hatanpaa, KJ; Mickey, B; Souza, RF; Vo, P; Herz, J; Chen, MR; Boothman, DA; Pandita, TK; Wang, DH; Sen, GC; Habib, AA
Nature communications 5 5811 2014
Epidermal growth factor receptor (EGFR) overexpression plays an important oncogenic role in cancer. Regular EGFR protein levels are increased in cancer cells and the receptor then becomes constitutively active. However, downstream signals generated by constitutively activated EGFR are unknown. Here we report that the overexpressed EGFR oscillates between two distinct and mutually exclusive modes of signalling. Constitutive or non-canonical EGFR signalling activates the transcription factor IRF3 leading to expression of IFI27, IFIT1 and TRAIL. Ligand-mediated activation of EGFR switches off IRF3-dependent transcription, activates canonical extracellular signal-regulated kinase (ERK) and Akt signals, and confers sensitivity to chemotherapy and virus-induced cell death. Mechanistically, the distinct downstream signals result from a switch of EGFR-associated proteins. EGFR constitutively complexes with IRF3 and TBK1 leading to TBK1 and IRF3 phosphorylation. Addition of epidermal growth factor dissociates TBK1, IRF3 and EGFR leading to a loss of IRF3 activity, Shc-EGFR association and ERK activation. Finally, we provide evidence for non-canonical EGFR signalling in glioblastoma.
|Control of lung vascular permeability and endotoxin-induced pulmonary oedema by changes in extracellular matrix mechanics. |
Mammoto, Akiko, et al.
Nat Commun, 4: 1759 (2013) 2013
Increased vascular permeability contributes to many diseases, including acute respiratory distress syndrome, cancer and inflammation. Most past work on vascular barrier function has focused on soluble regulators, such as tumour-necrosis factor-α. Here we show that lung vascular permeability is controlled mechanically by changes in extracellular matrix structure. Our studies reveal that pulmonary vascular leakage can be increased by altering extracellular matrix compliance in vitro and by manipulating lysyl oxidase-mediated collagen crosslinking in vivo. Either decreasing or increasing extracellular matrix stiffness relative to normal levels disrupts junctional integrity and increases vascular leakage. Importantly, endotoxin-induced increases of vascular permeability are accompanied by concomitant increases in extracellular matrix rigidity and lysyl oxidase activity, which can be prevented by inhibiting lysyl oxidase activity. The identification of lysyl oxidase and the extracellular matrix as critical regulators of lung vascular leakage might lead to the development of new therapeutic approaches for the treatment of pulmonary oedema and other diseases caused by abnormal vascular permeability.
|IL-22 contributes to TGF-β1-mediated epithelial-mesenchymal transition in asthmatic bronchial epithelial cells. |
Johnson, JR; Nishioka, M; Chakir, J; Risse, PA; Almaghlouth, I; Bazarbashi, AN; Plante, S; Martin, JG; Eidelman, D; Hamid, Q
Respiratory research 14 118 2013
Allergic asthma is characterized by airway inflammation in response to antigen exposure, leading to airway remodeling and lung dysfunction. Epithelial-mesenchymal transition (EMT) may play a role in airway remodeling through the acquisition of a mesenchymal phenotype in airway epithelial cells. TGF-β1 is known to promote EMT; however, other cytokines expressed in severe asthma with extensive remodeling, such as IL-22, may also contribute to this process. In this study, we evaluated the contribution of IL-22 to EMT in primary bronchial epithelial cells from healthy and asthmatic subjects.Primary bronchial epithelial cells were isolated from healthy subjects, mild asthmatics and severe asthmatics (n=5 patients per group). The mRNA and protein expression of epithelial and mesenchymal cell markers and EMT-associated transcription factors was evaluated following stimulation with TGF-β1, IL-22 and TGF-β1+IL-22.Primary bronchial epithelial cells stimulated with TGF-β1 underwent EMT, demonstrated by decreased expression of epithelial markers (E-cadherin and MUC5AC) and increased expression of mesenchymal markers (N-cadherin and vimentin) and EMT-associated transcription factors. IL-22 alone had no effect on epithelial or mesenchymal gene expression. However, IL-22+TGF-β1 promoted the expression of some EMT transcription factors (Snail1 and Zeb1) and led to a more profound cadherin shift, but only in cells obtained from severe asthmatics.The impact of IL-22 on airway epithelial cells depends on the cytokine milieu and the clinical phenotype of the patient. Further studies are required to determine the molecular mechanism of IL-22 and TGF-β1 cooperativity in driving EMT in primary human bronchial epithelial cells.
|Elevated pulmonary arterial pressure and altered expression of Ddah1 and Arg1 in mice lacking cavin-1/PTRF. |
Swärd, K; Sadegh, MK; Mori, M; Erjefält, JS; Rippe, C
Physiological reports 1 e00008 2013
Caveolae are invaginations in the plasma membrane that depend on caveolins and cavins for maturation. Here, we investigated the pulmonary phenotype in mice lacking cavin-1. Bright field and electron-microscopy showed that the cavin-1-deficient mice lacked caveolae in the lung, had an increased lung tissue density, and exhibited hypertrophic remodeling of pulmonary arteries. The right ventricle of the heart moreover had an increased mass and the right ventricular pressure was elevated. A microarray analysis revealed upregulation of Arg1 and downregulation of Ddah1, molecules whose altered expression has previously been associated with pulmonary arterial hypertension. Taken together, this work demonstrates vascular remodeling and increased pulmonary blood pressure in cavin-1 deficient mice and associates this phenotype with altered expression of Arg1 and Ddah1.
|Mir-29 repression in bladder outlet obstruction contributes to matrix remodeling and altered stiffness. |
Ekman, M; Bhattachariya, A; Dahan, D; Uvelius, B; Albinsson, S; Swärd, K
PloS one 8 e82308 2013
Recent work has uncovered a role of the microRNA (miRNA) miR-29 in remodeling of the extracellular matrix. Partial bladder outlet obstruction is a prevalent condition in older men with prostate enlargement that leads to matrix synthesis in the lower urinary tract and increases bladder stiffness. Here we tested the hypothesis that miR-29 is repressed in the bladder in outlet obstruction and that this has an impact on protein synthesis and matrix remodeling leading to increased bladder stiffness. c-Myc, NF-κB and SMAD3, all of which repress miR-29, were activated in the rat detrusor following partial bladder outlet obstruction but at different times. c-Myc and NF-κB activation occurred early after obstruction, and SMAD3 phosphorylation increased later, with a significant elevation at 6 weeks. c-Myc, NF-κB and SMAD3 activation, respectively, correlated with repression of miR-29b and miR-29c at 10 days of obstruction and with repression of miR-29c at 6 weeks. An mRNA microarray analysis showed that the reduction of miR-29 following outlet obstruction was associated with increased levels of miR-29 target mRNAs, including mRNAs for tropoelastin, the matricellular protein Sparc and collagen IV. Outlet obstruction increased protein levels of eight out of eight examined miR-29 targets, including tropoelastin and Sparc. Transfection of human bladder smooth muscle cells with antimiR-29c and miR-29c mimic caused reciprocal changes in target protein levels in vitro. Tamoxifen inducible and smooth muscle-specific deletion of Dicer in mice reduced miR-29 expression and increased tropoelastin and the thickness of the basal lamina surrounding smooth muscle cells in the bladder. It also increased detrusor stiffness independent of outlet obstruction. Taken together, our study supports a model where the combined repressive influences of c-Myc, NF-κB and SMAD3 reduce miR-29 in bladder outlet obstruction, and where the resulting drop in miR-29 contributes to matrix remodeling and altered passive mechanical properties of the detrusor.
|Secreted hCLCA1 is a signaling molecule that activates airway macrophages. |
Ching, JC; Lobanova, L; Loewen, ME
PloS one 8 e83130 2013
The CLCA gene family produces both secreted and membrane-associated proteins that modulate ion-channel function, drive mucus production and have a poorly understood pleiotropic effect on airway inflammation. The primary up-regulated human CLCA ortholog in airway inflammation is hCLCA1. Here we show that this protein can activate airway macrophages, inducing them to express cytokines and to undertake a pivotal role in airway inflammation. In a U-937 airway macrophage-monocyte cell line, conditioned media from HEK 293 cells heterologously expressing hCLCA1 (with or without fetal bovine serum) increased the levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α and IL-8). This effect was independent of the metalloprotease domain of hCLCA1. Primary porcine alveolar macrophages were similarly activated, demonstrating the effect was not cell line dependent. Similarly, immuno-purified hCLCA1 at physiologically relevant concentration of ~100 pg/mL was able to activate macrophages and induce pro-inflammatory response. This cytokine response increased with higher concentration of immuno-purified hCLCA1. These findings demonstrate the ability of hCLCA1 to function as a signaling molecule and activate macrophages, central regulators of airway inflammation.
|Aggravation of nonalcoholic steatohepatitis by moderate alcohol consumption is associated with decreased SIRT1 activity in rats. |
Nascimento, AF; Ip, BC; Luvizotto, RA; Seitz, HK; Wang, XD
Hepatobiliary surgery and nutrition 2 252-9 2013
Chronic alcohol intake decreases adiponectin and sirtuin 1 (SIRT1) expressions, both of which have been implicated in various biological processes including inflammation, apoptosis and metabolism. We have previously shown that moderate consumption of alcohol aggravates liver inflammation and apoptosis in rats with pre-existing nonalcoholic steatohepatitis (NASH). This study investigated whether moderate alcohol intake alters SIRT1 activity, adiponectin/Adiponectin receptor (AdipoR)-related signaling and lipid metabolism in a pre-existing NASH status. Sprague-Dawley rats were fed with a high-fat diet (71% energy from fat) for 6 weeks to induce NASH then subsequently divided into 2 sub-groups: fed either a modified high-fat diet (HFD, 55% energy from fat) or a modified high-fat alcoholic diet (HFA, 55% energy from fat and 16% energy from ethanol) for an additional 4 weeks. We observed in comparison to HFD group, HFA increased hepatic nuclear SIRT1 protein but decreased its deacetylase activity. SREBP-1c protein expression and FAS mRNA levels were significantly upregulated, while DGAT1/2 and CPT-I mRNA levels were downregulated in the livers of HFA compared to HFD. Although hepatic AdipoR1 decreased, HFA did not alter AdipoR2 and their downstream signaling. There were no significant changes in plasma adiponectin and free fatty acids (FFA), as well as adiponectin expression in adipose tissue between the two groups. The present study indicates that suppression in SIRT1 deacetylase activity contributes to alcohol-exacerbated hepatic inflammation and apoptosis in rats with pre-existing NASH. In addition, moderate alcohol intake did not modulate adiponectin/AdipoR signaling axis in this model.
|Alternative splicing of the RAGE cytoplasmic domain regulates cell signaling and function. |
Jules, J; Maiguel, D; Hudson, BI
PloS one 8 e78267 2013
The Receptor for Advanced Glycation End-products (RAGE) is a multi-ligand receptor present on most cell types. Upregulation of RAGE is seen in a number of pathological states including, inflammatory and vascular disease, dementia, diabetes and various cancers. We previously demonstrated that alternative splicing of the RAGE gene is an important mechanism which regulates RAGE signaling through the production of soluble ligand decoy isoforms. However, no studies have identified any alternative splice variants within the intracellular region of RAGE, a region critical for RAGE signaling. Herein, we have cloned and characterized a novel splice variant of RAGE that has a truncated intracellular domain (RAGEΔICD). RAGEΔICD is prevalent in both human and mouse tissues including lung, brain, heart and kidney. Expression of RAGEΔICD in C6 glioma cells impaired RAGE-ligand induced signaling through various MAP kinase pathways including ERK1/2, p38 and SAPK/JNK. Moreover, RAGEΔICD significantly affected tumor cell properties through altering cell migration, invasion, adhesion and viability in C6 glioma cells. Furthermore, C6 glioma cells expressing RAGEΔICD exhibited drastic inhibition on tumorigenesis in soft agar assays. Taken together, these data indicate that RAGEΔICD represents a novel endogenous mechanism to regulate RAGE signaling. Significantly, RAGEΔICD could play an important role in RAGE related disease states through down regulation of RAGE signaling.
|c-MET regulates myoblast motility and myocyte fusion during adult skeletal muscle regeneration. |
Webster, MT; Fan, CM
PloS one 8 e81757 2013
Adult muscle stem cells, satellite cells (SCs), endow skeletal muscle with tremendous regenerative capacity. Upon injury, SCs activate, proliferate, and migrate as myoblasts to the injury site where they become myocytes that fuse to form new muscle. How migration is regulated, though, remains largely unknown. Additionally, how migration and fusion, which both require dynamic rearrangement of the cytoskeleton, might be related is not well understood. c-MET, a receptor tyrosine kinase, is required for myogenic precursor cell migration into the limb for muscle development during embryogenesis. Using a genetic system to eliminate c-MET function specifically in adult mouse SCs, we found that c-MET was required for muscle regeneration in response to acute muscle injury. c-MET mutant myoblasts were defective in lamellipodia formation, had shorter ranges of migration, and migrated slower compared to control myoblasts. Surprisingly, c-MET was also required for efficient myocyte fusion, implicating c-MET in dual functions of regulating myoblast migration and myocyte fusion.
|Rsk2 Knockdown in PC12 Cells Results in Sp1 Dependent Increased Expression of the Gria2 Gene, Encoding the AMPA Receptor Subunit GluR2. |
Mehmood, T; Schneider, A; Pannetier, S; Hanauer, A
International journal of molecular sciences 14 3358-75 2013
The RSK2 protein is a member of the RSK serine-threonine protein kinase family and is encoded by the X-linked rps6ka3 gene in human. Highly heterogeneous loss-of-function mutations affecting this gene are responsible for a severe syndromic form of cognitive impairment, Coffin-Lowry syndrome. RSK2, which is highly conserved in mammals, acts at the distal end of the Ras-ERK signaling pathway and is activated in response to growth factors and neurotransmitters. RSK2 is highly expressed in the hippocampus, and Rsk2-KO mice display spatial learning and memory impairment. We recently showed that ERK1/2 activity is abnormally increased in the hippocampus of Rsk2-KO mice as well as the expression of the AMPA receptor subunit GluR2. The mechanism via which RSK2 deficiency affects the expression of GluR2 in neural cells was unknown. To address this issue we constitutively suppressed the expression of RSK2 in PC12 cells via vector-based shRNA in the present study. We show that Rsk2 silencing leads also to an elevation of ERK1/2 phosphorylation as well as of GluR2 expression and that the increased level of GluR2 expression results from the increased ERK1/2 activity on the transcription factor Sp1. Our results provide evidence that RSK2 modulates ERK1/2 activity on Sp1, which regulates GluR2 expression through transcriptional activation.
|Visceral endoderm expression of Yin-Yang1 (YY1) is required for VEGFA maintenance and yolk sac development. |
Rhee, S; Guerrero-Zayas, MI; Wallingford, MC; Ortiz-Pineda, P; Mager, J; Tremblay, KD
PloS one 8 e58828 2013
Mouse embryos lacking the polycomb group gene member Yin-Yang1 (YY1) die during the peri-implantation stage. To assess the post-gastrulation role of YY1, a conditional knock-out (cKO) strategy was used to delete YY1 from the visceral endoderm of the yolk sac and the definitive endoderm of the embryo. cKO embryos display profound yolk sac defects at 9.5 days post coitum (dpc), including disrupted angiogenesis in mesoderm derivatives and altered epithelial characteristics in the visceral endoderm. Significant changes in both cell death and proliferation were confined to the YY1-expressing yolk sac mesoderm indicating that loss of YY1 in the visceral endoderm causes defects in the adjacent yolk sac mesoderm. Production of Vascular Endothelial Growth Factor A (VEGFA) by the visceral endoderm is essential for normal growth and development of the yolk sac vasculature. Reduced levels of VEGFA are observed in the cKO yolk sac, suggesting a cause for the angiogenesis defects. Ex vivo culture with exogenous VEGF not only rescued angiogenesis and apoptosis in the cKO yolk sac mesoderm, but also restored the epithelial defects observed in the cKO visceral endoderm. Intriguingly, blocking the activity of the mesoderm-localized VEGF receptor, FLK1, recapitulates both the mesoderm and visceral endoderm defects observed in the cKO yolk sac. Taken together, these results demonstrate that YY1 is responsible for maintaining VEGF in the developing visceral endoderm and that a VEGF-responsive paracrine signal, originating in the yolk sac mesoderm, is required to promote normal visceral endoderm development.
|Early versus late-phase consolidation of opiate reward memories requires distinct molecular and temporal mechanisms in the amygdala-prefrontal cortical pathway. |
Gholizadeh, S; Sun, N; De Jaeger, X; Bechard, M; Coolen, L; Laviolette, SR
PloS one 8 e63612 2013
The consolidation of newly acquired memories involves the temporal transition from a recent, less stable trace to a more permanent consolidated form. Opiates possess potent rewarding effects and produce powerful associative memories. The activation of these memories is associated with opiate abuse relapse phenomena and the persistence of compulsive opiate dependence. However, the neuronal, molecular and temporal mechanisms by which associative opiate reward memories are consolidated are not currently understood. We report that the consolidation of associative opiate reward memories involves a temporal and molecular switch between the basolateral nucleus of the amygdala (BLA) (early consolidation phase) to the medial prefrontal cortex (mPFC) (late consolidation phase). We demonstrate at the molecular, behavioral and neuronal levels that the consolidation of a recently acquired opiate reward memory involves an extracellular signal-related kinase (ERK)-dependent phosphorylation process within the BLA. In contrast, later-stage consolidation of a newly acquired memory is dependent upon a calcium-calmodulin-dependent (CaMKII), ERK-independent, mechanism in the mPFC, over a 12 hr temporal gradient. In addition, using in vivo multi-unit neuronal recordings in the mPFC, we report that protein synthesis within the BLA modulates the consolidation of opiate-reward memory in neuronal mPFC sub-populations, via the same temporal dynamic.
|Active Component of Antrodia cinnamomea Mycelia Targeting Head and Neck Cancer Initiating Cells through Exaggerated Autophagic Cell Death. |
Chang, CW; Chen, CC; Wu, MJ; Chen, YS; Chen, CC; Sheu, SJ; Lin, TW; Chou, SH; Lin, SC; Liu, CJ; Lee, TC; Huang, CY; Lo, JF
Evidence-based complementary and alternative medicine : eCAM 2013 946451 2013
Head and neck squamous cell carcinoma (HNSCC) is a highly lethal cancer. Previously, we identify head and neck cancer initiating cells (HN-CICs), which are highly tumorigenic and resistant to conventional therapy. Therefore, development of drug candidates that effectively target HN-CICs would benefit future head and neck cancer therapy. In this study, we first successfully screened for an active component, named YMGKI-1, from natural products of Antrodia cinnamomea Mycelia (ACM), which can target the stemness properties of HNSCC. Treatment of YMGKI-1 significantly downregulated the aldehyde dehydrogenase (ALDH) activity, one of the characteristics of CIC in HNSCC cells. Additionally, the tumorigenic properties of HNSCC cells were attenuated by YMGKI-1 treatment in vivo. Further, the stemness properties of HN-CICs, which are responsible for the malignancy of HNSCC, were also diminished by YMGKI-1 treatment. Strikingly, YMGKI-1 also effectively suppressed the cell viability of HN-CICs but not normal stem cells. Finally, YMGKI-1 induces the cell death of HN-CICs by dysregulating the exaggerated autophagic signaling pathways. Together, our results indicate that YMGKI-1 successfully lessens stemness properties and tumorigenicity of HN-CICs. These findings provide a new drug candidate from purified components of ACM as an alternative therapy for head and neck cancer in the future.
|The role of phospholipase D in modulating the MTOR signaling pathway in polycystic kidney disease. |
Liu, Y; Käch, A; Ziegler, U; Ong, AC; Wallace, DP; Arcaro, A; Serra, AL
PloS one 8 e73173 2013
The mammalian target of rapamycin (mTOR) signaling pathway is aberrantly activated in polycystic kidney disease (PKD). Emerging evidence suggests that phospholipase D (PLD) and its product phosphatidic acid (PA) regulate mTOR activity. In this study, we assessed in vitro the regulatory function of PLD and PA on the mTOR signaling pathway in PKD. We found that the basal level of PLD activity was elevated in PKD cells. Targeting PLD by small molecule inhibitors reduced cell proliferation and blocked mTOR signaling, whereas exogenous PA stimulated mTOR signaling and abolished the inhibitory effect of PLD on PKD cell proliferation. We also show that blocking PLD activity enhanced the sensitivity of PKD cells to rapamycin and that combining PLD inhibitors and rapamycin synergistically inhibited PKD cell proliferation. Furthermore, we demonstrate that targeting mTOR did not induce autophagy, whereas targeting PLD induced autophagosome formation. Taken together, our findings suggest that deregulated mTOR pathway activation is mediated partly by increased PLD signaling in PKD cells. Targeting PLD isoforms with pharmacological inhibitors may represent a new therapeutic strategy in PKD.
|Aryl hydrocarbon receptor ligands inhibit igf-ii and adipokine stimulated breast cancer cell proliferation. |
Salisbury, TB; Morris, GZ; Tomblin, JK; Chaudhry, AR; Cook, CR; Santanam, N
ISRN endocrinology 2013 104850 2013
Obesity increases human cancer risk and the risk for cancer recurrence. Adipocytes secrete paracrine factors termed adipokines that stimulate signaling in cancer cells that induce proliferation. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that plays roles in tumorigenesis, is regulated by exogenous lipophilic chemicals, and has been explored as a therapeutic target for cancer therapy. Whether exogenous AHR ligands modulate adipokine stimulated breast cancer cell proliferation has not been investigated. We provide evidence that adipocytes secrete insulin-like growth factor 2 (IGF-2) at levels that stimulate the proliferation of human estrogen receptor (ER) positive breast cancer cells. Using highly specific AHR ligands and AHR short interfering RNA (AHR-siRNA), we show that specific ligand-activated AHR inhibits adipocyte secretome and IGF-2-stimulated breast cancer cell proliferation. We also report that a highly specific AHR agonist significantly (P less than 0.05) inhibits the expression of E2F1, CCND1 (known as Cyclin D1), MYB, SRC, JAK2, and JUND in breast cancer cells. Collectively, these data suggest that drugs that target the AHR may be useful for treating cancer in human obesity.
|Stretch-sensitive down-regulation of the miR-144/451 cluster in vascular smooth muscle and its role in AMP-activated protein kinase signaling. |
Turczyńska, KM; Bhattachariya, A; Säll, J; Göransson, O; Swärd, K; Hellstrand, P; Albinsson, S
PloS one 8 e65135 2013
Vascular smooth muscle cells are constantly exposed to mechanical force by the blood pressure, which is thought to regulate smooth muscle growth, differentiation and contractile function. We have previously shown that the expression of microRNAs (miRNAs), small non-coding RNAs, is essential for regulation of smooth muscle phenotype including stretch-dependent contractile differentiation. In this study, we have investigated the effect of mechanical stretch on miRNA expression and the role of stretch-sensitive miRNAs for intracellular signaling in smooth muscle. MiRNA array analysis, comparing miRNA levels in stretched versus non-stretched portal veins, revealed a dramatic decrease in the miR-144/451 cluster level. Because this miRNA cluster is predicted to target AMPK pathway components, we next examined activation of this pathway. Diminished miR-144/451 expression was inversely correlated with increased phosphorylation of AMPKα at Thr172 in stretched portal vein. Similar to the effect of stretch, contractile differentiation could be induced in non-stretched portal veins by the AMPK activator, AICAR. Transfection with miR-144/451 mimics reduced the protein expression level of mediators in the AMPK pathway including MO25α, AMPK and ACC. This effect also decreased AICAR-induced activation of the AMPK signaling pathway. In conclusion, our results suggest that stretch-induced activation of AMPK in vascular smooth muscle is in part regulated by reduced levels of miR-144/451 and that this effect may play a role in promoting contractile differentiation of smooth muscle cells.
|Substratum compliance modulates corneal fibroblast to myofibroblast transformation. |
Dreier, B; Thomasy, SM; Mendonsa, R; Raghunathan, VK; Russell, P; Murphy, CJ
Investigative ophthalmology & visual science 54 5901-7 2013
The transformation of fibroblasts to myofibroblasts is critical to corneal wound healing, stromal haze formation, and scarring. It has recently been demonstrated that the provision of biomimetic substratum topographic cues inhibits the progression toward the myofibroblast phenotype under the influence of transforming growth factor β1 (TGF-β1). The objective of this study was to determine the effect of another fundamental biophysical cue, substrate compliance, on TGF-β1-induced myofibroblast transformation of primary corneal cells isolated from human and rabbit corneas.Human and rabbit corneal fibroblasts were cultured on surfaces of varying substrate compliance (4-71 kPa) and tissue culture plastic (TCP) (greater than 1 gigapascal [GPa]). Cells were cultured in media containing TGF-β1 at concentrations of 0, 1, or 10 ng/mL for 72 hours. RNA and protein were collected from cells cultured on polyacrylamide gels and TCP and were analyzed for the expression of α-smooth muscle actin (α-SMA), a key marker of myofibroblast transformation, using quantitative PCR, immunocytochemistry, and Western blot.Cells grown on more compliant substrates demonstrated significantly reduced amounts of α-SMA mRNA compared with TCP. Immunocytochemistry and Western blot analysis determining the presence of α-SMA corroborated this finding, thus confirming a reduced transformation to the myofibroblast phenotype on more compliant substrates compared with cells on TCP in the presence of TGF-β1.These data indicate that substrate compliance modulates TGF-β1-induced expression of α-SMA and thus influences myofibroblast transformation in the corneal stroma. This provides further evidence that biomimetic biophysical cues inhibit myofibroblast transformation and participate in stabilizing the native cellular phenotype.
|Differential regulation of cell functions by CSD peptide subdomains. |
Reese, C; Dyer, S; Perry, B; Bonner, M; Oates, J; Hofbauer, A; Sessa, W; Bernatchez, P; Visconti, RP; Zhang, J; Hatfield, CM; Silver, RM; Hoffman, S; Tourkina, E
Respiratory research 14 90 2013
In fibrotic lung diseases, expression of caveolin-1 is decreased in fibroblasts and monocytes. The effects of this deficiency are reversed by treating cells or animals with the caveolin-1 scaffolding domain peptide (CSD, amino acids 82-101 of caveolin-1) which compensates for the lack of caveolin-1. Here we compare the function of CSD subdomains (Cav-A, Cav-B, Cav-C, Cav-AB, and Cav-BC) and mutated versions of CSD (F92A and T90A/T91A/F92A).Migration toward the chemokine CXCL12 and the associated expression of F-actin, CXCR4, and pSmad 2/3 were studied in monocytes from healthy donors and SSc patients. Fibrocyte differentiation was studied using PBMC from healthy donors and SSc patients. Collagen I secretion and signaling were studied in fibroblasts derived from the lung tissue of healthy subjects and SSc patients.Cav-BC and CSD at concentrations as low as 0.01 μM inhibited the hypermigration of SSc monocytes and TGFβ-activated Normal monocytes and the differentiation into fibrocytes of SSc and Normal monocytes. While CSD also inhibited the migration of poorly migrating Normal monocytes, Cav-A (and other subdomains to a lesser extent) promoted the migration of Normal monocytes while inhibiting the hypermigration of TGFβ-activated Normal monocytes. The effects of versions of CSD on migration may be mediated in part via their effects on CXCR4, F-actin, and pSmad 2/3 expression. Cav-BC was as effective as CSD in inhibiting fibroblast collagen I and ASMA expression and MEK/ERK signaling. Cav-C and Cav-AB also inhibited collagen I expression, but in many cases did not affect ASMA or MEK/ERK. Cav-A increased collagen I expression in scleroderma lung fibroblasts. Full effects on fibroblasts of versions of CSD required 5 μM peptide.Cav-BC retains most of the anti-fibrotic functions of CSD; Cav-A exhibits certain pro-fibrotic functions. Results obtained with subdomains and mutated versions of CSD further suggest that the critical functional residues in CSD depend on the cell type and readout being studied. Monocytes may be more sensitive to versions of CSD than fibroblasts and endothelial cells because the baseline level of caveolin-1 in monocytes is much lower than in these other cell types.
|Major differences between tumor and normal human cell fates after exposure to chemotherapeutic monofunctional alkylator. |
Gupte, M; Tuck, AN; Sharma, VP; Williams, KJ
PloS one 8 e74071 2013
The major dilemma of cancer chemotherapy has always been a double-edged sword, producing resistance in tumor cells and life-threatening destruction of nontumorigenic tissue. Glioblastoma is the most common form of primary brain tumor, with median survival at 14 months after surgery, radiation and temozolomide (monofunctional alkylator) therapy. Treatment failure is most often due to temozolomide-resistant tumor growth. The underlying basis for development of tumor cell resistance to temozolomide instead of death is not understood. Our current results demonstrate that both cervical carcinoma (HeLa MR) and glioblastoma (U251) tumor cells exposed to an equivalent chemotherapeutic concentration of a monofunctional alkylator undergo multiple cell cycles, maintenance of metabolic activity, and a prolonged time to death that involves accumulation of Apoptosis Inducing Factor (AIF) within the nucleus. A minority of the tumor cell population undergoes senescence, with minimal caspase cleavage. Surviving tumor cells are comprised of a very small subpopulation of individual cells that eventually resume proliferation, out of which resistant cells emerge. In contrast, normal human cells (MCF12A) exposed to a monofunctional alkylator undergo an immediate decrease in metabolic activity and subsequent senescence. A minority of the normal cell population undergoes cell death by the caspase cleavage pathway. All cytotoxic events occur within the first cell cycle in nontumorigenic cells. In summation, we have demonstrated that two different highly malignant tumor cell lines slowly undergo very altered cellular and temporal responses to chemotherapeutic monofunctional alkylation, as compared to rapid responses of normal cells. In the clinic, this produces resistance and growth of tumor cells, cytotoxicity of normal cells, and death of the patient.
|POU-III transcription factors (Brn1, Brn2, and Oct6) influence neurogenesis, molecular identity, and migratory destination of upper-layer cells of the cerebral cortex. |
Dominguez, MH; Ayoub, AE; Rakic, P
Cerebral cortex (New York, N.Y. : 1991) 23 2632-43 2013
The upper layers (II-IV) are the most prominent distinguishing feature of mammalian neocortex compared with avian or reptilian dorsal cortex, and are vastly expanded in primates. Although the time-dependent embryonic generation of upper-layer cells is genetically instructed within their parental progenitors, mechanisms governing cell-intrinsic fate transitions remain obscure. POU-homeodomain transcription factors Pou3f3 and Pou3f2 (Brn1 and Brn2) are known to label postmitotic upper-layer cells, and are redundantly required for their production. We find that the onset of Pou3f3/2 expression actually occurs in ventricular zone (VZ) progenitors, and that Pou3f3/2 subsequently label neural progeny switching from deep-layer Ctip2(+) identity to Satb2(+) upper-layer fate as they migrate to proper superficial positions. By using an Engrailed dominant-negative repressor, we show that sustained neurogenesis after the deep- to upper-layer transition requires the proneual action of Pou3fs in VZ progenitors. Conversely, single-gene overexpression of any Pou3f in early neural progenitors is sufficient to specify the precocious birth of Satb2(+) daughter neurons that extend axons to the contralateral hemisphere, as well as exhibit robust pia-directed migration that is characteristic of upper-layer cells. Finally, we demonstrate that Pou3fs influence multiple stages of neurogenesis by suppressing Notch effector Hes5, and promoting the expression of proneural transcription factors Tbr2 and Tbr1.
|A mutation in the start codon of γ-crystallin D leads to nuclear cataracts in the Dahl SS/Jr-Ctr strain. |
Johnson, AC; Lee, JW; Harmon, AC; Morris, Z; Wang, X; Fratkin, J; Rapp, JP; Gomez-Sanchez, E; Garrett, MR
Mammalian genome : official journal of the International Mammalian Genome Society 24 95-104 2013
Cataracts are a major cause of blindness. The most common forms of cataracts are age- and UV-related and develop mostly in the elderly, while congenital cataracts appear at birth or in early childhood. The Dahl salt-sensitive (SS/Jr) rat is an extensively used model of salt-sensitive hypertension that exhibits concomitant renal disease. In the mid-1980s, cataracts appeared in a few animals in the Dahl S colony, presumably the result of a spontaneous mutation. The mutation was fixed and bred to establish the SS/Jr-Ctr substrain. The SS/Jr-Ctr substrain has been used exclusively by a single investigator to study the role of steroids and hypertension. Using a classical positional cloning approach, we localized the cataract gene with high resolution to a less than 1-Mbp region on chromosome 9 using an F1(SS/Jr-Ctr × SHR) × SHR backcross population. The 1-Mbp region contained only 13 genes, including 4 genes from the γ-crystallins (Cryg) gene family, which are known to play a role in cataract formation. All of the γ-crystallins were sequenced and a novel point mutation in the start codon (ATG → GTG) of the Crygd gene was identified. This led to the complete absence of the CRYGD protein in the eyes of the SS/Jr-Ctr strain. In summary, the identification of the genetic cause in this novel cataract model may provide an opportunity to better understand the development of cataracts, particularly in the context of hypertension.
|miR-205 negatively regulates the androgen receptor and is associated with adverse outcome of prostate cancer patients. |
Hagman, Z; Haflidadóttir, BS; Ceder, JA; Larne, O; Bjartell, A; Lilja, H; Edsjö, A; Ceder, Y
British journal of cancer 108 1668-76 2013
The microRNA-205 (miR-205) has been shown to be deregulated in prostate cancer (PCa). Here we continue to investigate the prognostic and therapeutic potential of this microRNA.The expression of miR-205 is measured by qRT-PCR and in situ hybridisation in a well-documented PCa cohort. An AGO2-based RIP-Chip assay is used to identify targets that are verified with western blots, luciferase reporter assay, ELISA and immunohistochemistry.The expression of miR-205 is inversely correlated to the occurrence of metastases and shortened overall survival, and is lower in castration-resistant PCa patients. The miR-205 expression is mainly localised to the basal cells of benign prostate tissues. Genes regulated by miR-205 are enriched in, for example, the MAPK/ERK, Toll-like receptor and IL-6 signaling pathways. We demonstrate binding of miR-205 to the 3'UTR of androgen receptor (AR) and decrease of both AR transcript and protein levels. This finding was corroborated in the patient cohort were miR-205 expression inversely correlated to AR immunostaining in malignant prostate cells and to serum levels of prostate-specific antigen, an androgen-regulated protein.Taken together, these findings imply that miR-205 might have therapeutic potential, especially for the castration resistant and currently untreatable form of PCa.
|Rapid assessment of RNAi-mediated protein depletion by selected reaction monitoring mass spectrometry. |
Glukhova, VA; Tomazela, DM; Findlay, GD; Monnat, RJ; MacCoss, MJ
Journal of proteome research 12 3246-54 2013
We describe the use of a targeted proteomics approach, selected reaction monitoring (SRM) mass spectrometry, to detect and assess RNAi-mediated depletion or "knockdown" of specific proteins from human cells and from Drosophila flies. This label-free approach does not require any specific reagents to confirm the depletion of RNAi target protein(s) in unfractionated cell or whole organism extracts. The protocol described here is general, can be developed rapidly, and can be multiplexed to detect and measure multiple proteins at once. Furthermore, the methodology can be extended to any tandem mass spectrometer, making it widely accessible. This methodology will be applicable to a wide range of basic science and clinical questions where RNAi-mediated protein depletion needs to be verified, or where differences in relative abundance of target proteins need to be rapidly assessed between samples.
|Willow bark extract increases antioxidant enzymes and reduces oxidative stress through activation of Nrf2 in vascular endothelial cells and Caenorhabditis elegans. |
Ishikado, A; Sono, Y; Matsumoto, M; Robida-Stubbs, S; Okuno, A; Goto, M; King, GL; Blackwell, TK; Makino, T
Free radical biology & medicine 65 1506-15 2013
Willow bark extract (WBE) is listed in the European Pharmacopoeia and has been traditionally used for treating fever, pain, and inflammation. Recent studies have demonstrated its clinical usefulness. This study investigated the antioxidative effects of WBE in human umbilical vein endothelial cells (HUVECs) and Caenorhabditis elegans. WBE prevented oxidative-stress-induced cytotoxicity of HUVECs and death of C. elegans. WBE dose-dependently increased mRNA and protein expression levels of the nuclear factor erythroid 2-related factor 2 (Nrf2) target genes heme oxygenase-1, γ-glutamylcysteine ligase modifier and catalytic subunits, and p62 and intracellular glutathione (GSH) in HUVECs. In the nematode C. elegans, WBE increased the expression of the gcs-1::green fluorescent protein reporter, a well-characterized target of the Nrf2 ortholog SKN-1, in a manner that was SKN-1-dependent. WBE increased intranuclear expression and DNA binding of Nrf2 and the activity of an antioxidant response element (ARE) reporter plasmid in HUVECs. WBE-induced expression of Nrf2-regulated genes and increased GSH levels in HUVECs were reduced by Nrf2 and p38 small interfering (si) RNAs and by the p38-specific inhibitor SB203580. Nrf2 siRNA reduced the cytoprotective effect of WBE against oxidative stress in HUVECs. Salicin, a major anti-inflammatory ingredient of WBE, failed to activate ARE-luciferase activity, whereas a salicin-free WBE fraction showed intensive activity. WBE induced antioxidant enzymes and prevented oxidative stress through activation of Nrf2 independent of salicin, providing a new potential explanation for the clinical usefulness of WBE.
|p53 is required for brown adipogenic differentiation and has a protective role against diet-induced obesity. |
Molchadsky, A; Ezra, O; Amendola, PG; Krantz, D; Kogan-Sakin, I; Buganim, Y; Rivlin, N; Goldfinger, N; Folgiero, V; Falcioni, R; Sarig, R; Rotter, V
Cell death and differentiation 20 774-83 2013
Proper regulation of white and brown adipogenic differentiation is important for maintaining an organism's metabolic profile in a homeostatic state. The recent observations showing that the p53 tumor suppressor plays a role in metabolism raise the question of whether it is involved in the regulation of white and brown adipocyte differentiation. By using several in vitro models, representing various stages of white adipocyte differentiation, we found that p53 exerts a suppressive effect on white adipocyte differentiation in both mouse and human cells. Moreover, our in vivo analysis indicated that p53 is implicated in protection against diet-induced obesity. In striking contrast, our data shows that p53 exerts a positive regulatory effect on brown adipocyte differentiation. Abrogation of p53 function in skeletal muscle committed cells reduced their capacity to differentiate into brown adipocytes and histological analysis of brown adipose tissue revealed an impaired morphology in both embryonic and adult p53-null mice. Thus, depending on the specific adipogenic differentiation program, p53 may exert a positive or a negative effect. This cell type dependent regulation reflects an additional modality of p53 in maintaining a homeostatic state, not only in the cell, but also in the organism at large.
|Prefoldin protects neuronal cells from polyglutamine toxicity by preventing aggregation formation. |
Tashiro, E; Zako, T; Muto, H; Itoo, Y; Sörgjerd, K; Terada, N; Abe, A; Miyazawa, M; Kitamura, A; Kitaura, H; Kubota, H; Maeda, M; Momoi, T; Iguchi-Ariga, SM; Kinjo, M; Ariga, H
The Journal of biological chemistry 288 19958-72 2013
Huntington disease is caused by cell death after the expansion of polyglutamine (polyQ) tracts longer than ∼40 repeats encoded by exon 1 of the huntingtin (HTT) gene. Prefoldin is a molecular chaperone composed of six subunits, PFD1-6, and prevents misfolding of newly synthesized nascent polypeptides. In this study, we found that knockdown of PFD2 and PFD5 disrupted prefoldin formation in HTT-expressing cells, resulting in accumulation of aggregates of a pathogenic form of HTT and in induction of cell death. Dead cells, however, did not contain inclusions of HTT, and analysis by a fluorescence correlation spectroscopy indicated that knockdown of PFD2 and PFD5 also increased the size of soluble oligomers of pathogenic HTT in cells. In vitro single molecule observation demonstrated that prefoldin suppressed HTT aggregation at the small oligomer (dimer to tetramer) stage. These results indicate that prefoldin inhibits elongation of large oligomers of pathogenic Htt, thereby inhibiting subsequent inclusion formation, and suggest that soluble oligomers of polyQ-expanded HTT are more toxic than are inclusion to cells.
|Sox2 transcriptionally regulates PQBP1, an intellectual disability-microcephaly causative gene, in neural stem progenitor cells. |
Li, C; Ito, H; Fujita, K; Shiwaku, H; Qi, Y; Tagawa, K; Tamura, T; Okazawa, H
PloS one 8 e68627 2013
PQBP1 is a nuclear-cytoplasmic shuttling protein that is engaged in RNA metabolism and transcription. In mouse embryonic brain, our previous in situ hybridization study revealed that PQBP1 mRNA was dominantly expressed in the periventricular zone region where neural stem progenitor cells (NSPCs) are located. Because the expression patterns in NSPCs are related to the symptoms of intellectual disability and microcephaly in PQBP1 gene-mutated patients, we investigated the transcriptional regulation of PQBP1 by NSPC-specific transcription factors. We selected 132 genome sequences that matched the consensus sequence for the binding of Sox2 and POU transcription factors upstream and downstream of the mouse PQBP1 gene. We then screened the binding affinity of these sequences to Sox2-Pax6 or Sox2-Brn2 with gel mobility shift assays and found 18 genome sequences that interacted with the NSPC-specific transcription factors. Some of these sequences had cis-regulatory activities in Luciferase assays and in utero electroporation into NSPCs. Furthermore we found decreased levels of expression of PQBP1 protein in NSPCs of heterozygous Sox2-knockout mice in vivo by immunohistochemistry and western blot analysis. Collectively, these results indicated that Sox2 regulated the transcription of PQBP1 in NSPCs.
|BMCC1 is an AP-2 associated endosomal protein in prostate cancer cells. |
Harris, JL; Richards, RS; Chow, CW; Lee, S; Kim, M; Buck, M; Teng, L; Clarke, R; Gardiner, RA; Lavin, MF
PloS one 8 e73880 2013
The prostate cancer antigen gene 3 (PCA3) is embedded in an intron of a second gene BMCC1 (Bcl2-/adenovirus E1B nineteen kDa-interacting protein 2 (BNIP-2) and Cdc42GAP homology BCH motif-containing molecule at the carboxyl terminal region 1) which is also upregulated in prostate cancer. BMCC1 was initially annotated as two genes (C9orf65/PRUNE and BNIPXL) on either side of PCA3 but our data suggest that it represents a single gene coding for a high molecular weight protein. Here we demonstrate for the first time the expression of a greater than 300 kDa BMCC1 protein (BMCC1-1) in prostate cancer and melanoma cell lines. This protein was found exclusively in the microsomal fraction and localised to cytoplasmic vesicles. We also observed expression of BMCC1 protein in prostate cancer sections using immunohistology. GST pull down, immunoprecipitation and mass spectrometry protein interaction studies identified multiple members of the Adaptor Related Complex 2 (AP-2) as BMCC1 interactors. Consistent with a role for BMCC1 as an AP-2 interacting endosomal protein, BMCC1 co-localised with β-adaptin at the perinuclear region of the cell. BMCC1 also showed partial co-localisation with the early endosome small GTP-ase Rab-5 as well as strong co-localisation with internalised pulse-chase labelled transferrin (Tf), providing evidence that BMCC1 is localised to functional endocytic vesicles. BMCC1 knockdown did not affect Tf uptake and AP-2 knockdown did not disperse BMCC1 vesicular distribution, excluding an essential role for BMCC1 in canonical AP-2 mediated endocytic uptake. Instead, we posit a novel role for BMCC1 in post-endocytic trafficking. This study provides fundamental characterisation of the BMCC1 complex in prostate cancer cells and for the first time implicates it in vesicle trafficking.
|Ero1-α and PDIs constitute a hierarchical electron transfer network of endoplasmic reticulum oxidoreductases. |
Araki, K; Iemura, S; Kamiya, Y; Ron, D; Kato, K; Natsume, T; Nagata, K
The Journal of cell biology 202 861-74 2013
Ero1-α and endoplasmic reticulum (ER) oxidoreductases of the protein disulfide isomerase (PDI) family promote the efficient introduction of disulfide bonds into nascent polypeptides in the ER. However, the hierarchy of electron transfer among these oxidoreductases is poorly understood. In this paper, Ero1-α-associated oxidoreductases were identified by proteomic analysis and further confirmed by surface plasmon resonance. Ero1-α and PDI were found to constitute a regulatory hub, whereby PDI induced conformational flexibility in an Ero1-α shuttle cysteine (Cys99) facilitated intramolecular electron transfer to the active site. In isolation, Ero1-α also oxidized ERp46, ERp57, and P5; however, kinetic measurements and redox equilibrium analysis revealed that PDI preferentially oxidized other oxidoreductases. PDI accepted electrons from the other oxidoreductases via its a' domain, bypassing the a domain, which serves as the electron acceptor from reduced glutathione. These observations provide an integrated picture of the hierarchy of cooperative redox interactions among ER oxidoreductases in mammalian cells.
|Acetylation of p53 stimulates miRNA processing and determines cell survival following genotoxic stress. |
Chang, J; Davis-Dusenbery, BN; Kashima, R; Jiang, X; Marathe, N; Sessa, R; Louie, J; Gu, W; Lagna, G; Hata, A
The EMBO journal 32 3192-205 2013
It is widely accepted that different forms of stress activate a common target, p53, yet different outcomes are triggered in a stress-specific manner. For example, activation of p53 by genotoxic agents, such as camptothecin (CPT), triggers apoptosis, while non-genotoxic activation of p53 by Nutlin-3 (Nut3) leads to cell-cycle arrest without significant apoptosis. Such stimulus-specific responses are attributed to differential transcriptional activation of various promoters by p53. In this study, we demonstrate that CPT, but not Nut3, induces miR-203, which downregulates anti-apoptotic bcl-w and promotes cell death in a p53-dependent manner. We find that acetylation of K120 in the DNA-binding domain of p53 augments its association with the Drosha microprocessor and promotes nuclear primary miRNA processing. Knockdown of human orthologue of Males absent On the First (hMOF), the acetyltransferase that targets K120 in p53, abolishes induction of miR-203 and cell death mediated by CPT. Thus, this study reveals that p53 acetylation at K120 plays a critical role in the regulation of the Drosha microprocessor and that post-transcriptional regulation of gene expression by p53 via miRNAs plays a role in determining stress-specific cellular outcomes.
|Sunitinib enhances neuronal survival in vitro via NF-κB-mediated signaling and expression of cyclooxygenase-2 and inducible nitric oxide synthase. |
Sanchez, A; Tripathy, D; Yin, X; Luo, J; Martinez, JM; Grammas, P
Journal of neuroinflammation 10 93 2013
Angiogenesis is tightly linked to inflammation and cancer. Regulation of angiogenesis is mediated primarily through activation of receptor tyrosine kinases, thus kinase inhibitors represent a new paradigm in anti-cancer therapy. However, these inhibitors have broad effects on inflammatory processes and multiple cell types. Sunitinib is a multitarget receptor tyrosine kinase inhibitor, which has shown promise for the treatment of glioblastoma, a highly vascularized tumor. However, there is little information as to the direct effects of sunitinib on brain-derived neurons. The objective of this study is to explore the effects of sunitinib on neuronal survival as well as on the expression of inflammatory protein mediators in primary cerebral neuronal cultures.Primary cortical neurons were exposed to various doses of sunitinib. The drug-treated cultures were assessed for survival by MTT assay and cell death by lactate dehydrogenase release. The ability of sunitinib to affect NF-κB, COX2 and NOS2 expression was determined by western blot. The NF-κB inhibitors dicoumarol, SN50 and BAY11-7085 were employed to assess the role of NF-κB in sunitinib-mediated effects on neuronal survival as well as COX2 and NOS2 expression.Treatment of neuronal cultures with sunitinib caused a dose-dependent increase in cell survival and decrease in neuronal cell death. Exposure of neurons to sunitinib also induced an increase in the expression of NF-κB, COX2 and NOS2. Inhibiting NF-κB blunted the increase in cell survival and decrease in cell death evoked by sunitinib. Treatment of cell cultures with both sunitinib and NF-κB inhibitors mitigated the increase in COX2 and NOS2 caused by sunitinib.Sunitinib increases neuronal survival and this neurotrophic effect is mediated by NF-κB. Also, the inflammatory proteins COX2 and NOS2 are upregulated by sunitinib in an NF-κB-dependent manner. These data are in agreement with a growing literature suggesting beneficial effects for inflammatory mediators such as NF-κB, COX2 and NOS2 in neurons. Further work is needed to fully explore the effects of sunitinib in the brain and its possible use as a treatment for glioblastoma. Finally, sunitinib may be useful for the treatment of a range of central nervous system diseases where neuronal injury is prominent.
|The human immunodeficiency virus type 1 Vpr protein upregulates PVR via activation of the ATR-mediated DNA damage response pathway. |
Vassena, L; Giuliani, E; Matusali, G; Cohen, ÉA; Doria, M
The Journal of general virology 94 2664-9 2013
Viral infection may induce the cell-surface expression of PVR (CD155) that, upon recognition by its cognate activating DNAM-1 receptor present on cytotoxic lymphocytes, may promote antiviral immune responses. Here we show that expression of the human immunodeficiency virus type 1 (HIV-1) Vpr protein in Jurkat T cells increases cell-surface and total PVR levels. Analysis of mutated Vpr variants indicated that Vpr uses the same protein surfaces, and hence probably the same mechanisms, to upregulate PVR and arrest the cell cycle in the G2 phase. Moreover, we found that PVR upregulation by Vpr relied on the ability of the protein to activate the ATR kinase that triggers the DNA damage response pathway and G2 arrest. Finally, we showed that Vpr contributes to PVR up-modulation in HIV-infected CD4(+) T lymphocytes and inhibits the PVR downregulating activity of the viral Nef protein.
|A novel transgenic mouse model of growth plate dysplasia reveals that decreased chondrocyte proliferation due to chronic ER stress is a key factor in reduced bone growth. |
Gualeni, B; Rajpar, MH; Kellogg, A; Bell, PA; Arvan, P; Boot-Handford, RP; Briggs, MD
Disease models & mechanisms 6 1414-25 2013
Disease mechanisms leading to different forms of chondrodysplasia include extracellular matrix (ECM) alterations and intracellular stress resulting in abnormal changes to chondrocyte proliferation and survival. Delineating the relative contribution of these two disease mechanisms is a major challenge in understanding disease pathophysiology in genetic skeletal diseases and a prerequisite for developing effective therapies. To determine the influence of intracellular stress and changes in chondrocyte phenotype to the development of chondrodysplasia, we targeted the expression of the G2320R mutant form of thyroglobulin to the endoplasmic reticulum (ER) of resting and proliferating chondrocytes. Previous studies on this mutant protein have shown that it induces intracellular aggregates and causes cell stress and death in the thyroid gland. The expression and retention of this exogenous mutant protein in resting and proliferating chondrocytes resulted in a chronic cell stress response, growth plate dysplasia and reduced bone growth, without inducing any alterations to the architecture and organization of the cartilage ECM. More significantly, the decreased bone growth seemed to be the direct result of reduced chondrocyte proliferation in the proliferative zone of growth plates in transgenic mice, without transcriptional activation of a classical unfolded protein response (UPR) or apoptosis. Overall, these data show that mutant protein retention in the ER of resting and proliferative zone chondrocytes is sufficient to cause disrupted bone growth. The specific disease pathways triggered by mutant protein retention do not necessarily involve a prototypic UPR, but all pathways impact upon chondrocyte proliferation in the cartilage growth plate.
|Knockdown of tyrosine hydroxylase in the nucleus of the solitary tract reduces elevated blood pressure during chronic intermittent hypoxia. |
Bathina, CS; Rajulapati, A; Franzke, M; Yamamoto, K; Cunningham, JT; Mifflin, S
American journal of physiology. Regulatory, integrative and comparative physiology 305 R1031-9 2013
Noradrenergic A2 neurons in nucleus tractus solitarius (NTS) respond to stressors such as hypoxia. We hypothesize that tyrosine hydroxylase (TH) knockdown in NTS reduces cardiovascular responses to chronic intermittent hypoxia (CIH), a model of the arterial hypoxemia observed during sleep apnea in humans. Adult male Sprague-Dawley rats were implanted with radiotelemetry transmitters and adeno-associated viral constructs with green fluorescent protein (GFP) reporter having either short hairpin RNA (shRNA) for TH or scrambled virus (scRNA) were injected into caudal NTS. Virus-injected rats were exposed to 7 days of CIH (alternating periods of 10% O2 and of 21% O2 from 8 AM to 4 PM; from 4 PM to 8 AM rats were exposed to 21% O2). CIH increased mean arterial pressure (MAP) and heart rate (HR) during the day in both the scRNA (n = 14, P less than 0.001 MAP and HR) and shRNA (n = 13, P less than 0.001 MAP and HR) groups. During the night, MAP and HR remained elevated in the scRNA rats (P less than 0.001 MAP and HR) but not in the shRNA group. TH immunoreactivity and protein were reduced in the shRNA group. FosB/ΔFosB immunoreactivity was decreased in paraventricular nucleus (PVN) of shRNA group (P less than 0.001). However, the shRNA group did not show any change in the FosB/ΔFosB immunoreactivity in the rostral ventrolateral medulla. Exposure to CIH increased MAP which persisted beyond the period of exposure to CIH. Knockdown of TH in the NTS reduced this CIH-induced persistent increase in MAP and reduced the transcriptional activation of PVN. This indicates that NTS A2 neurons play a role in the cardiovascular responses to CIH.
|Synthesis of mitochondrial DNA precursors during myogenesis, an analysis in purified C2C12 myotubes. |
Frangini, M; Franzolin, E; Chemello, F; Laveder, P; Romualdi, C; Bianchi, V; Rampazzo, C
The Journal of biological chemistry 288 5624-35 2013
During myogenesis, myoblasts fuse into multinucleated myotubes that acquire the contractile fibrils and accessory structures typical of striated skeletal muscle fibers. To support the high energy requirements of muscle contraction, myogenesis entails an increase in mitochondrial (mt) mass with stimulation of mtDNA synthesis and consumption of DNA precursors (dNTPs). Myotubes are quiescent cells and as such down-regulate dNTP production despite a high demand for dNTPs. Although myogenesis has been studied extensively, changes in dNTP metabolism have not been examined specifically. In differentiating cultures of C2C12 myoblasts and purified myotubes, we analyzed expression and activities of enzymes of dNTP biosynthesis, dNTP pools, and the expansion of mtDNA. Myotubes exibited pronounced post-mitotic modifications of dNTP synthesis with a particularly marked down-regulation of de novo thymidylate synthesis. Expression profiling revealed the same pattern of enzyme down-regulation in adult murine muscles. The mtDNA increased steadily after myoblast fusion, turning over rapidly, as revealed after treatment with ethidium bromide. We individually down-regulated p53R2 ribonucleotide reductase, thymidine kinase 2, and deoxyguanosine kinase by siRNA transfection to examine how a further reduction of these synthetic enzymes impacted myotube development. Silencing of p53R2 had little effect, but silencing of either mt kinase caused 50% mtDNA depletion and an unexpected decrease of all four dNTP pools independently of the kinase specificity. We suggest that during development of myotubes the shortage of even a single dNTP may affect all four pools through dysregulation of ribonucleotide reduction and/or dissipation of the non-limiting dNTPs during unproductive elongation of new DNA chains.
|γ-secretase binding sites in aged and Alzheimer's disease human cerebrum: the choroid plexus as a putative origin of CSF Aβ. |
Liu, F; Xue, ZQ; Deng, SH; Kun, X; Luo, XG; Patrylo, PR; Rose, GM; Cai, H; Struble, RG; Cai, Y; Yan, XX
The European journal of neuroscience 37 1714-25 2013
Deposition of β -amyloid (Aβ) peptides, cleavage products of β-amyloid precursor protein (APP) by β-secretase-1 (BACE1) and γ-secretase, is a neuropathological hallmark of Alzheimer's disease (AD). γ-Secretase inhibition is a therapeutical anti-Aβ approach, although changes in the enzyme's activity in AD brain are unclear. Cerebrospinal fluid (CSF) Aβ peptides are thought to derive from brain parenchyma and thus may serve as biomarkers for assessing cerebral amyloidosis and anti-Aβ efficacy. The present study compared active γ-secretase binding sites with Aβ deposition in aged and AD human cerebrum, and explored the possibility of Aβ production and secretion by the choroid plexus (CP). The specific binding density of [(3) H]-L-685,458, a radiolabeled high-affinity γ-secretase inhibitor, in the temporal neocortex and hippocampal formation was similar for AD and control cases with similar ages and post-mortem delays. The CP in post-mortem samples exhibited exceptionally high [(3) H]-L-685,458 binding density, with the estimated maximal binding sites (Bmax) reduced in the AD relative to control groups. Surgically resected human CP exhibited APP, BACE1 and presenilin-1 immunoreactivity, and β-site APP cleavage enzymatic activity. In primary culture, human CP cells also expressed these amyloidogenic proteins and released Aβ40 and Aβ42 into the medium. Overall, our results suggest that γ-secretase activity appears unaltered in the cerebrum in AD and is not correlated with regional amyloid plaque pathology. The CP appears to be a previously unrecognised non-neuronal contributor to CSF Aβ, probably at reduced levels in AD.
|Identification of a functional TPH1 polymorphism associated with irritable bowel syndrome bowel habit subtypes. |
Grasberger, H; Chang, L; Shih, W; Presson, AP; Sayuk, GS; Newberry, RD; Karagiannides, I; Pothoulakis, C; Mayer, E; Merchant, JL
The American journal of gastroenterology 108 1766-74 2013
Alterations in 5-hydroxytryptamine (5-HT) signaling have been implicated as a factor contributing to the altered bowel habit of irritable bowel syndrome (IBS) patients. Tryptophan hydroxylase 1 (TPH1) is the rate-limiting enzyme in enterochromaffin cell 5-HT biosynthesis. We hypothesized that genetic variants affecting TPH1 gene expression might alter intestinal 5-HT bioavailability and subsequently the propensity for distinct bowel habit subtypes in IBS. In this study, we assessed the only common TPH1 proximal promoter variant (-347C/A; rs7130929) and its association with bowel habit predominance in IBS.Electrophoretic mobility shift assays were performed to assess whether the -347C/A-allele variant affects the DNA binding of nuclear factors. Genotype distribution was determined for 422 IBS patients subtyped using the Rome III criteria and for 495 healthy controls recruited from two university medical centers. Association with bowel habit was tested using a multinomial logistic regression model controlling for race, anxiety, depression, and study site.Early growth response factor 1 (EGR-1) bound with higher affinity to a site comprising the minor A-allele of single-nucleotide polymorphism (SNP) -347C/A. TPH1 genotype frequencies did not differ between IBS patients and controls overall. The CC genotype was more prevalent in the IBS-D subtype (47%) than in the IBS-C (25%) and IBS-M (37%) subtypes (P=0.039) after adjusting for race and other covariates. Colonic biopsies from a small cohort of IBS patients from one center were tested for higher TPH1 mRNA expression in samples with CC compared with the CA genotype, but the results did not reach statistical significance.The TPH1 promoter SNP -347C/A differentially binds EGR-1 and correlates with IBS bowel habit subtypes and possibly colonic TPH1 expression consistent with its role in modulating intestinal 5-HT signaling.
|Myotubularin and PtdIns3P remodel the sarcoplasmic reticulum in muscle in vivo. |
Amoasii, L; Hnia, K; Chicanne, G; Brech, A; Cowling, BS; Müller, MM; Schwab, Y; Koebel, P; Ferry, A; Payrastre, B; Laporte, J
Journal of cell science 126 1806-19 2013
The sarcoplasmic reticulum (SR) is a specialized form of endoplasmic reticulum (ER) in skeletal muscle and is essential for calcium homeostasis. The mechanisms involved in SR remodeling and maintenance of SR subdomains are elusive. In this study, we identified myotubularin (MTM1), a phosphoinositide phosphatase mutated in X-linked centronuclear myopathy (XLCNM, or myotubular myopathy), as a key regulator of phosphatidylinositol 3-monophosphate (PtdIns3P) levels at the SR. MTM1 is predominantly located at the SR cisternae of the muscle triads, and Mtm1-deficient mouse muscles and myoblasts from XLCNM patients exhibit abnormal SR/ER networks. In vivo modulation of MTM1 enzymatic activity in skeletal muscle using ectopic expression of wild-type or a dead-phosphatase MTM1 protein leads to differential SR remodeling. Active MTM1 is associated with flat membrane stacks, whereas dead-phosphatase MTM1 mutant promotes highly curved cubic membranes originating from the SR and enriched in PtdIns3P. Overexpression of a tandem FYVE domain with high affinity for PtdIns3P alters the shape of the SR cisternae at the triad. Our findings, supported by the parallel analysis of the Mtm1-null mouse and an in vivo study, reveal a direct function of MTM1 enzymatic activity in SR remodeling and a key role for PtdIns3P in promoting SR membrane curvature in skeletal muscle. We propose that alteration in SR remodeling is a primary cause of X-linked centronuclear myopathy. The tight regulation of PtdIns3P on specific membrane subdomains may be a general mechanism to control membrane curvature.
|TORC1 suppression predicts responsiveness to RAF and MEK inhibition in BRAF-mutant melanoma. |
Corcoran, RB; Rothenberg, SM; Hata, AN; Faber, AC; Piris, A; Nazarian, RM; Brown, RD; Godfrey, JT; Winokur, D; Walsh, J; Mino-Kenudson, M; Maheswaran, S; Settleman, J; Wargo, JA; Flaherty, KT; Haber, DA; Engelman, JA
Science translational medicine 5 196ra98 2013
RAF and MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase) inhibitors are effective in treating patients with BRAF-mutant melanoma. However, most responses are partial and short-lived, and many patients fail to respond at all. We found that suppression of TORC1 activity in response to RAF or MEK inhibitors, as measured by decreased phosphorylation of ribosomal protein S6 (P-S6), effectively predicted induction of cell death by the inhibitor in BRAF-mutant melanoma cell lines. In resistant melanomas, TORC1 activity was maintained after treatment with RAF or MEK inhibitors, in some cases despite robust suppression of mitogen-activated protein kinase (MAPK) signaling. In in vivo mouse models, suppression of TORC1 after MAPK inhibition was necessary for induction of apoptosis and tumor response. Finally, in paired biopsies obtained from patients with BRAF-mutant melanoma before treatment and after initiation of RAF inhibitor therapy, P-S6 suppression predicted significantly improved progression-free survival. Such a change in P-S6 could be readily monitored in real time by serial fine-needle aspiration biopsies, making quantitation of P-S6 a valuable biomarker to guide treatment in BRAF-mutant melanoma.
|Developmental and visual input-dependent regulation of the CB1 cannabinoid receptor in the mouse visual cortex. |
Yoneda, T; Kameyama, K; Esumi, K; Daimyo, Y; Watanabe, M; Hata, Y
PloS one 8 e53082 2013
The mammalian visual system exhibits significant experience-induced plasticity in the early postnatal period. While physiological studies have revealed the contribution of the CB1 cannabinoid receptor (CB1) to developmental plasticity in the primary visual cortex (V1), it remains unknown whether the expression and localization of CB1 is regulated during development or by visual experience. To explore a possible role of the endocannabinoid system in visual cortical plasticity, we examined the expression of CB1 in the visual cortex of mice. We found intense CB1 immunoreactivity in layers II/III and VI. CB1 mainly localized at vesicular GABA transporter-positive inhibitory nerve terminals. The amount of CB1 protein increased throughout development, and the specific laminar pattern of CB1 appeared at P20 and remained until adulthood. Dark rearing from birth to P30 decreased the amount of CB1 protein in V1 and altered the synaptic localization of CB1 in the deep layer. Dark rearing until P50, however, did not influence the expression of CB1. Brief monocular deprivation for 2 days upregulated the localization of CB1 at inhibitory nerve terminals in the deep layer. Taken together, the expression and the localization of CB1 are developmentally regulated, and both parameters are influenced by visual experience.
|Advanced oxidation protein products induce cardiomyocyte death via Nox2/Rac1/superoxide-dependent TRAF3IP2/JNK signaling. |
Valente, AJ; Yoshida, T; Clark, RA; Delafontaine, P; Siebenlist, U; Chandrasekar, B
Free radical biology & medicine 60 125-35 2013
Advanced oxidation protein products (AOPPs) are formed during chronic oxidative stress as a result of reactions between plasma proteins and chlorinated oxidants. Their levels are elevated during various cardiovascular diseases. Because elevated AOPPs serve as independent risk factors for ischemic heart disease, and cardiomyocyte death is a hallmark of ischemic heart disease, we hypothesized that AOPPs will induce cardiomyocyte death. AOPP-modified mouse serum albumin (AOPP-MSA) induced significant death of neonatal mouse cardiomyocytes that was attenuated by knockdown of the receptor for advanced glycation end products, but not CD36. Notably, TRAF3-interacting protein 2 (TRAF3IP2; also known as CIKS or Act1) knockdown blunted AOPP-induced apoptosis. AOPP-MSA stimulated Nox2/Rac1-dependent superoxide generation, TRAF3IP2 expression, and TRAF3IP2-dependent JNK activation. The superoxide anion generating xanthine/xanthine oxidase system and hydrogen peroxide both induced TRAF3IP2 expression. Further, AOPP-MSA induced mitochondrial Bax translocation and release of cytochrome c into cytoplasm. Moreover, AOPP-MSA suppressed antiapoptotic Bcl-2 and Bcl-xL expression. These effects were reversed by TRAF3IP2 knockdown or forced expression of mutant JNK. Similar to its effects in neonatal cardiomyocytes, AOPP-MSA induced adult cardiomyocyte death in part via TRAF3IP2. These results demonstrate for the first time that AOPPs induce cardiomyocyte death via Nox2/Rac1/superoxide-dependent TRAF3IP2/JNK activation in vitro and suggest that AOPPs may contribute to myocardial injury in vivo. Thus TRAF3IP2 may represent a potential therapeutic target in ischemic heart disease.
|Combined 5-FU and ChoKα inhibitors as a new alternative therapy of colorectal cancer: evidence in human tumor-derived cell lines and mouse xenografts. |
de la Cueva, A; Ramírez de Molina, A; Alvarez-Ayerza, N; Ramos, MA; Cebrián, A; Del Pulgar, TG; Lacal, JC
PloS one 8 e64961 2013
Colorectal cancer (CRC) is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU) is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα), an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy.ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS) and thymidine kinase (TK1) levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action.Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors.
|Toward RNAi therapy for the polyglutamine disease Machado-Joseph disease. |
Costa, Mdo C; Luna-Cancalon, K; Fischer, S; Ashraf, NS; Ouyang, M; Dharia, RM; Martin-Fishman, L; Yang, Y; Shakkottai, VG; Davidson, BL; Rodríguez-Lebrón, E; Paulson, HL
Molecular therapy : the journal of the American Society of Gene Therapy 21 1898-908 2013
Machado-Joseph disease (MJD) is a dominantly inherited ataxia caused by a polyglutamine-coding expansion in the ATXN3 gene. Suppressing expression of the toxic gene product represents a promising approach to therapy for MJD and other polyglutamine diseases. We performed an extended therapeutic trial of RNA interference (RNAi) targeting ATXN3 in a mouse model expressing the full human disease gene and recapitulating key disease features. Adeno-associated virus (AAV) encoding a microRNA (miRNA)-like molecule, miRATXN3, was delivered bilaterally into the cerebellum of 6- to 8-week-old MJD mice, which were then followed up to end-stage disease to assess the safety and efficacy of anti-ATXN3 RNAi. Despite effective, lifelong suppression of ATXN3 in the cerebellum and the apparent safety of miRATXN3, motor impairment was not ameliorated in treated MJD mice and survival was not prolonged. These results with an otherwise effective RNAi agent suggest that targeting a large extent of the cerebellum alone may not be sufficient for effective human therapy. Artificial miRNAs or other nucleotide-based suppression strategies targeting ATXN3 more widely in the brain should be considered in future preclinical tests.
|Perinatal nicotine-induced transgenerational asthma. |
Rehan, VK; Liu, J; Sakurai, R; Torday, JS
American journal of physiology. Lung cellular and molecular physiology 305 L501-7 2013
Asthma is a major public health hazard worldwide. Its transgenerational inheritance has been inferred from epidemiological studies. More recently, using nicotine as a proxy for maternal smoking, we have demonstrated that an asthma-like phenotype can be inherited by rat offspring for up to two generations, i.e., multigenerationally, after the initial intrauterine exposure. We hypothesized that asthma transmission to offspring following perinatal nicotine exposure is not restricted up to F2 generation, but it also extends to subsequent generations. To test this hypothesis, using a well-established rat model of nicotine exposure-induced childhood asthma, we determined if perinatal nicotine exposure of F0 gestating dams would transmit asthma transgenerationally to F3 offspring. We now extend our findings to third-generation offspring, including abnormal pulmonary function, particularly as it relates to the occurrence in the upper airway exclusively in males, and to its effects on molecular functional markers (fibronectin and peroxisome proliferator-activated receptor γ), previously shown to be consistent with the asthma phenotype, herein expressed in fibroblasts isolated from the lung. These data, for the first time, demonstrate the transgenerational transmission of the asthma phenotype to F3 offspring following perinatal nicotine exposure of F0 dams.
|The tumour suppressor miR-34c targets MET in prostate cancer cells. |
Hagman, Z; Haflidadottir, BS; Ansari, M; Persson, M; Bjartell, A; Edsjö, A; Ceder, Y
British journal of cancer 109 1271-8 2013
The microRNA, miR-34c, is a well-established regulator of tumour suppression. It is downregulated in most forms of cancers and inhibits malignant growth by repressing genes involved in processes such as proliferation, anti-apoptosis, stemness, and migration. We have previously reported downregulation and tumour suppressive properties for miR-34c in prostate cancer (PCa).In this study, we set out to further characterize the mechanisms by which miR-34c deregulation contributes to PCa progression. The genes regulated by miR-34c in the PCa cell line PC3 were identified by microarray analyses and were found to be enriched in cell death, cell cycle, cellular growth, and cellular movement pathways. One of the identified targets was MET, a receptor tyrosine kinase activated by hepatocyte growth factor, that is crucial for metastatic progression.We confirmed the inhibitory effect of miR-34c on both MET transcript and protein levels. The binding of miR-34c to two binding sites in the 3'-UTR of MET was validated using luciferase reporter assays and target site blockers. The effect of this regulation on the miR-34c inhibition of the migratory phenotype was also confirmed. In addition, a significant inverse correlation between miR-34c expression levels and MET immunostaining was found in PCa patients.These findings provide a novel molecular mechanism of MET regulation in PCa and contribute to the increasing evidence that miR-34c has a key tumour suppressive role in PCa.
|The LKB1 tumor suppressor differentially affects anchorage independent growth of HPV positive cervical cancer cell lines. |
Mack, HI; Munger, K
Virology 446 9-16 2013
Infection with high-risk human papillomaviruses is causally linked to cervical carcinogenesis. However, most lesions caused by high-risk HPV infections do not progress to cancer. Host cell mutations contribute to malignant progression but the molecular nature of such mutations is unknown. Based on a previous study that reported an association between liver kinase B1 (LKB1) tumor suppressor loss and poor outcome in cervical cancer, we sought to determine the molecular basis for this observation. LKB1-negative cervical and lung cancer cells were reconstituted with wild type or kinase defective LKB1 mutants and we examined the importance of LKB1 catalytic activity in known LKB1-regulated processes including inhibition of cell proliferation and elevated resistance to energy stress. Our studies revealed marked differences in the biological activities of two kinase defective LKB1 mutants in the various cell lines. Thus, our results suggest that LKB1 may be a cell-type specific tumor suppressor.
|Loss of metabotropic glutamate receptor 2 escalates alcohol consumption. |
Zhou, Z; Karlsson, C; Liang, T; Xiong, W; Kimura, M; Tapocik, JD; Yuan, Q; Barbier, E; Feng, A; Flanigan, M; Augier, E; Enoch, MA; Hodgkinson, CA; Shen, PH; Lovinger, DM; Edenberg, HJ; Heilig, M; Goldman, D
Proceedings of the National Academy of Sciences of the United States of America 110 16963-8 2013
Identification of genes influencing complex traits is hampered by genetic heterogeneity, the modest effect size of many alleles, and the likely involvement of rare and uncommon alleles. Etiologic complexity can be simplified in model organisms. By genomic sequencing, linkage analysis, and functional validation, we identified that genetic variation of Grm2, which encodes metabotropic glutamate receptor 2 (mGluR2), alters alcohol preference in animal models. Selectively bred alcohol-preferring (P) rats are homozygous for a Grm2 stop codon (Grm2 *407) that leads to largely uncompensated loss of mGluR2. mGluR2 receptor expression was absent, synaptic glutamate transmission was impaired, and expression of genes involved in synaptic function was altered. Grm2 *407 was linked to increased alcohol consumption and preference in F2 rats generated by intercrossing inbred P and nonpreferring rats. Pharmacologic blockade of mGluR2 escalated alcohol self-administration in Wistar rats, the parental strain of P and nonpreferring rats. The causal role of mGluR2 in altered alcohol preference was further supported by elevated alcohol consumption in Grm2 (-/-) mice. Together, these data point to mGluR2 as an origin of alcohol preference and a potential therapeutic target.
|The TNF family member 4-1BBL sustains inflammation by interacting with TLR signaling components during late-phase activation. |
Ma, J; Bang, BR; Lu, J; Eun, SY; Otsuka, M; Croft, M; Tobias, P; Han, J; Takeuchi, O; Akira, S; Karin, M; Yagita, H; Kang, YJ
Science signaling 6 ra87 2013
Activation of Toll-like receptor (TLR)-dependent signaling leads to the expression of genes encoding proinflammatory factors, such as tumor necrosis factor-α (TNF-α), and this proinflammatory gene expression is sustained for the duration of the inflammatory response. TLR4-mediated inflammation, which occurs in two phases, depends on the TNF family member 4-1BB ligand (4-1BBL) to sustain TNF-α production during late-phase signaling. We showed that Toll-interleukin-1 receptor (TIR) domain-containing adaptor protein (TIRAP) and the kinase IRAK2 interacted with 4-1BBL to mediate late-phase TLR4 signaling. Expression of 4-1bbl depended on early TLR4 signaling that also induced Tnf expression, and 4-1BBL translocated to the plasma membrane, where it interacted with TLR4 to mediate late-phase signaling. TLR4-4-1BBL-mediated signaling depended on TIRAP and IRAK2, as well as a complex consisting of the E3 ubiquitin ligase TRAF6 (TNF receptor-associated factor 6), the kinase TAK1 (transforming growth factor-β-activated kinase 1), and the adaptor protein TAB1 (TAK-binding protein 1). Inhibition of this late-phase pathway reduced the extent of TNF-α production by mouse macrophages exposed to the TLR4 ligand lipopolysaccharide (LPS) and ameliorated LPS-induced sepsis in mice. Together, these data suggest that TIRAP and IRAK2 are critical for the sustained inflammatory response that is mediated by late-phase signaling by the TLR-4-1BBL complex.
|Histone H3K9 methyltransferase G9a represses PPARγ expression and adipogenesis. |
Wang, L; Xu, S; Lee, JE; Baldridge, A; Grullon, S; Peng, W; Ge, K
The EMBO journal 32 45-59 2013
PPARγ promotes adipogenesis while Wnt proteins inhibit adipogenesis. However, the mechanisms that control expression of these positive and negative master regulators of adipogenesis remain incompletely understood. By genome-wide histone methylation profiling in preadipocytes, we find that among gene loci encoding adipogenesis regulators, histone methyltransferase (HMT) G9a-mediated repressive epigenetic mark H3K9me2 is selectively enriched on the entire PPARγ locus. H3K9me2 and G9a levels decrease during adipogenesis, which correlates inversely with induction of PPARγ. Removal of H3K9me2 by G9a deletion enhances chromatin opening and binding of the early adipogenic transcription factor C/EBPβ to PPARγ promoter, which promotes PPARγ expression. Interestingly, G9a represses PPARγ expression in an HMT activity-dependent manner but facilitates Wnt10a expression independent of its enzymatic activity. Consistently, deletion of G9a or inhibiting G9a HMT activity promotes adipogenesis. Finally, deletion of G9a in mouse adipose tissues increases adipogenic gene expression and tissue weight. Thus, by inhibiting PPARγ expression and facilitating Wnt10a expression, G9a represses adipogenesis.
|DNA methyltransferase inhibitor zebularine inhibits human hepatic carcinoma cells proliferation and induces apoptosis. |
Nakamura, K; Aizawa, K; Nakabayashi, K; Kato, N; Yamauchi, J; Hata, K; Tanoue, A
PloS one 8 e54036 2013
Hepatocellular carcinoma is one of the most common cancers worldwide. During tumorigenesis, tumor suppressor and cancer-related genes are commonly silenced by aberrant DNA methylation in their promoter regions. Zebularine (1-(β-(D)-ribofuranosyl)-1,2-dihydropyrimidin-2-one) acts as an inhibitor of DNA methylation and exhibits chemical stability and minimal cytotoxicity both in vitro and in vivo. In this study, we explore the effect and possible mechanism of action of zebularine on hepatocellular carcinoma cell line HepG2. We demonstrate that zebularine exhibits antitumor activity on HepG2 cells by inhibiting cell proliferation and inducing apoptosis, however, it has little effect on DNA methylation in HepG2 cells. On the other hand, zebularine treatment downregulated CDK2 and the phosphorylation of retinoblastoma protein (Rb), and upregulated p21(WAF/CIP1) and p53. We also found that zebularine treatment upregulated the phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). These results suggest that the p44/42 MAPK pathway plays a role in zebularine-induced cell-cycle arrest by regulating the activity of p21(WAF/CIP1) and Rb. Furthermore, although the proapoptotic protein Bax levels were not affected, the antiapoptotic protein Bcl-2 level was downregulated with zebularine treatment. In addition, the data in the present study indicate that inhibition of the double-stranded RNA-dependent protein kinase (PKR) is involved in inducing apoptosis with zebularine. These results suggest a novel mechanism of zebularine-induced cell growth arrest and apoptosis via a DNA methylation-independent pathway in hepatocellular carcinoma.
|Adenosine transporter ENT1 regulates the acquisition of goal-directed behavior and ethanol drinking through A2A receptor in the dorsomedial striatum. |
Nam, HW; Hinton, DJ; Kang, NY; Kim, T; Lee, MR; Oliveros, A; Adams, C; Ruby, CL; Choi, DS
The Journal of neuroscience : the official journal of the Society for Neuroscience 33 4329-38 2013
Adenosine signaling has been implicated in the pathophysiology of many psychiatric disorders including alcoholism. Striatal adenosine A2A receptors (A2AR) play an essential role in both ethanol drinking and the shift from goal-directed action to habitual behavior. However, direct evidence for a role of striatal A2AR signaling in ethanol drinking and habit development has not been established. In the present study, we found that decreased A2AR-mediated CREB activity in the dorsomedial striatum (DMS) enhanced initial behavioral acquisition of goal-directed behaviors and the vulnerability to progress to excessive ethanol drinking during operant conditioning in mice lacking ethanol-sensitive adenosine transporter ENT1 (ENT1(-/-)). Using mice expressing β-galactosidase (lacZ) under the control of seven repeated CRE sites in both genotypes (CRE-lacZ/ENT1(+/+) mice and CRE-lacZ/ENT1(-/-) mice) and the dominant-negative form of CREB, we found that reduced CREB activity in the DMS was causally associated with decreased A2AR signaling and increased goal-directed ethanol drinking. Finally, we have demonstrated that the A2AR antagonist ZM241385 dampened protein kinase A activity-mediated signaling in the DMS and promoted excessive ethanol drinking in ENT1(+/+) mice, but not in ENT1(-/-) mice. Our results indicate that A2AR-mediated CREB signaling in the DMS is a key determinant in enhancing the development of goal-directed ethanol drinking in mice.
|Distinct signal transduction pathways downstream of the (P)RR revealed by microarray and ChIP-chip analyses. |
Zaade, D; Schmitz, J; Benke, E; Klare, S; Seidel, K; Kirsch, S; Goldin-Lang, P; Zollmann, FS; Unger, T; Funke-Kaiser, H
PloS one 8 e57674 2013
The (pro)renin receptor ((P)RR) signaling is involved in different pathophysiologies ranging from cardiorenal end-organ damage via diabetic retinopathy to tumorigenesis. We have previously shown that the transcription factor promyelocytic leukemia zinc finger (PLZF) is an adaptor protein of the (P)RR. Furthermore, recent publications suggest that major functions of the (P)RR are mediated ligand-independently by its transmembrane and intracellular part, which acts as an accessory protein of V-ATPases. The transcriptome and recruitmentome downstream of the V-ATPase function and PLZF in the context of the (P)RR are currently unknown. Therefore, we performed a set of microarray and chromatin-immunoprecipitation (ChIP)-chip experiments using siRNA against the (P)RR, stable overexpression of PLZF, the PLZF translocation inhibitor genistein and the specific V-ATPase inhibitor bafilomycin to dissect transcriptional pathways downstream of the (P)RR. We were able to identify distinct and overlapping genetic signatures as well as novel real-time PCR-validated target genes of the different molecular functions of the (P)RR. Moreover, bioinformatic analyses of our data confirm the role of (P)RŔs signal transduction pathways in cardiovascular disease and tumorigenesis.
|Proteomic profiling in Drosophila reveals potential Dube3a regulation of the actin cytoskeleton and neuronal homeostasis. |
Jensen, L; Farook, MF; Reiter, LT
PloS one 8 e61952 2013
The molecular defects associated with Angelman syndrome (AS) and 15q duplication autism are directly correlated to expression levels of the E3 ubiquitin ligase protein UBE3A. Here we used Drosophila melanogaster to screen for the targets of this ubiquitin ligase under conditions of both decreased (as in AS) or increased (as in dup(15)) levels of the fly Dube3a or human UBE3A proteins. Using liquid phase isoelectric focusing of proteins from whole fly head extracts we identified a total of 50 proteins that show changes in protein, and in some cases transcriptional levels, when Dube3a fluctuates. We analyzed head extracts from cytoplasmic, nuclear and membrane fractions for Dube3a regulated proteins. Our results indicate that Dube3a is involved in the regulation of cellular functions related to ATP synthesis/metabolism, actin cytoskeletal integrity, both catabolism and carbohydrate metabolism as well as nervous system development and function. Sixty-two percent of the proteins were greater than 50% identical to homologous human proteins and 8 have previously be shown to be ubiquitinated in the fly nervous system. Eight proteins may be regulated by Dube3a at the transcript level through the transcriptional co-activation function of Dube3a. We investigated one autism-associated protein, ATPα, and found that it can be ubiquitinated in a Dube3a dependent manner. We also found that Dube3a mutants have significantly less filamentous actin than wild type larvae consistent with the identification of actin targets regulated by Dube3a. The identification of UBE3A targets is the first step in unraveling the molecular etiology of AS and duplication 15q autism.
|Prostate cancer stem cell-targeted efficacy of a new-generation taxoid, SBT-1214 and novel polyenolic zinc-binding curcuminoid, CMC2.24. |
Botchkina, GI; Zuniga, ES; Rowehl, RH; Park, R; Bhalla, R; Bialkowska, AB; Johnson, F; Golub, LM; Zhang, Y; Ojima, I; Shroyer, KR
PloS one 8 e69884 2013
Prostate cancer is the second leading cause of cancer death among men. Multiple evidence suggests that a population of tumor-initiating, or cancer stem cells (CSCs) is responsible for cancer development and exceptional drug resistance, representing a highly important therapeutic target. The present study evaluated CSC-specific alterations induced by new-generation taxoid SBT-1214 and a novel polyenolic zinc-binding curcuminoid, CMC2.24, in prostate CSCs.The CD133(high)/CD44(high) phenotype was isolated from spontaneously immortalized patient-derived PPT2 cells and highly metastatic PC3MM2 cells. Weekly treatment of the NOD/SCID mice bearing PPT2- and PC3MM3-induced tumors with the SBT-1214 led to dramatic suppression of tumor growth. Four of six PPT2 and 3 of 6 PC3MM2 tumors have shown the absence of viable cells in residual tumors. In vitro, SBT-1214 (100 nM-1 µM; for 72 hr) induced about 60% cell death in CD133(high)/CD44(+/high) cells cultured on collagen I in stem cell medium (in contrast, the same doses of paclitaxel increased proliferation of these cells). The cytotoxic effects were increased when SBT-1214 was combined with the CMC2.24. A stem cell-specific PCR array assay revealed that this drug combination mediated massive inhibition of multiple constitutively up-regulated stem cell-related genes, including key pluripotency transcription factors. Importantly, this drug combination induced expression of p21 and p53, which were absent in CD133(high)/CD44(high) cells. Viable cells that survived this treatment regimen were no longer able to induce secondary spheroids, exhibited significant morphological abnormalities and died in 2-5 days.We report here that the SBT-1214 alone, or in combination with CMC2.24, possesses significant activity against prostate CD133(high)/CD44(+/high) tumor-initiating cells. This drug combination efficiently inhibits expression of the majority of stem cell-related genes and pluripotency transcription factors. In addition, it induces a previously absent expression of p21 and p53 ("gene wake-up"), which can potentially reverse drug resistance by increasing sensitivity to anti-cancer drugs.
|p53 counteracts reprogramming by inhibiting mesenchymal-to-epithelial transition. |
Brosh, R; Assia-Alroy, Y; Molchadsky, A; Bornstein, C; Dekel, E; Madar, S; Shetzer, Y; Rivlin, N; Goldfinger, N; Sarig, R; Rotter, V
Cell death and differentiation 20 312-20 2013
The process of somatic cell reprogramming is gaining increasing interest as reprogrammed cells are considered to hold a great therapeutic potential. However, with current technologies this process is relatively inefficient. Recent studies reported that inhibition of the p53 tumor suppressor profoundly facilitates reprogramming and attributed this effect to the ability of p53 to restrict proliferation and induce apoptosis. Given that mesenchymal-to-epithelial transition (MET) was recently shown to be necessary for reprogramming of fibroblasts, we investigated whether p53 counteracts reprogramming by affecting MET. We found that p53 restricts MET during the early phases of reprogramming and that this effect is primarily mediated by the ability of p53 to inhibit Klf4-dependent activation of epithelial genes. Moreover, transcriptome analysis revealed a large transcriptional signature enriched with epithelial genes, which is markedly induced by Klf4 exclusively in p53(-/-) cells. We also found that the expression of the epithelial marker E-Cadherin negatively correlates with p53 activity in a variety of mesenchymal cells even before the expression of reprogramming factors. Finally, we demonstrate that the inhibitory effect of p53 on MET is mediated by p21. We conclude that inhibition of the p53-p21 axis predisposes mesenchymal cells to the acquisition of epithelial characteristics and renders them more prone to reprogramming. Our study uncovers a novel mechanism by which p53 restrains reprogramming and highlights the role of p53 in regulating cell plasticity.
|Nuclear translocation of cardiac G protein-Coupled Receptor kinase 5 downstream of select Gq-activating hypertrophic ligands is a calmodulin-dependent process. |
Gold, JI; Martini, JS; Hullmann, J; Gao, E; Chuprun, JK; Lee, L; Tilley, DG; Rabinowitz, JE; Bossuyt, J; Bers, DM; Koch, WJ
PloS one 8 e57324 2013
G protein-Coupled Receptors (GPCRs) kinases (GRKs) play a crucial role in regulating cardiac hypertrophy. Recent data from our lab has shown that, following ventricular pressure overload, GRK5, a primary cardiac GRK, facilitates maladaptive myocyte growth via novel nuclear localization. In the nucleus, GRK5's newly discovered kinase activity on histone deacetylase 5 induces hypertrophic gene transcription. The mechanisms governing the nuclear targeting of GRK5 are unknown. We report here that GRK5 nuclear accumulation is dependent on Ca(2+)/calmodulin (CaM) binding to a specific site within the amino terminus of GRK5 and this interaction occurs after selective activation of hypertrophic Gq-coupled receptors. Stimulation of myocytes with phenylephrine or angiotensinII causes GRK5 to leave the sarcolemmal membrane and accumulate in the nucleus, while the endothelin-1 does not cause nuclear GRK5 localization. A mutation within the amino-terminus of GRK5 negating CaM binding attenuates GRK5 movement from the sarcolemma to the nucleus and, importantly, overexpression of this mutant does not facilitate cardiac hypertrophy and related gene transcription in vitro and in vivo. Our data reveal that CaM binding to GRK5 is a physiologically relevant event that is absolutely required for nuclear GRK5 localization downstream of hypertrophic stimuli, thus facilitating GRK5-dependent regulation of maladaptive hypertrophy.
|Mutant p53 attenuates the anti-tumorigenic activity of fibroblasts-secreted interferon beta. |
Madar, S; Harel, E; Goldstein, I; Stein, Y; Kogan-Sakin, I; Kamer, I; Solomon, H; Dekel, E; Tal, P; Goldfinger, N; Friedlander, G; Rotter, V
PloS one 8 e61353 2013
Mutations in the p53 tumor suppressor protein are highly frequent in tumors and often endow cells with tumorigenic capacities. We sought to examine a possible role for mutant p53 in the cross-talk between cancer cells and their surrounding stroma, which is a crucial factor affecting tumor outcome. Here we present a novel model which enables individual monitoring of the response of cancer cells and stromal cells (fibroblasts) to co-culturing. We found that fibroblasts elicit the interferon beta (IFNβ) pathway when in contact with cancer cells, thereby inhibiting their migration. Mutant p53 in the tumor was able to alleviate this response via SOCS1 mediated inhibition of STAT1 phosphorylation. IFNβ on the other hand, reduced mutant p53 RNA levels by restricting its RNA stabilizer, WIG1. These data underscore mutant p53 oncogenic properties in the context of the tumor microenvironment and suggest that mutant p53 positive cancer patients might benefit from IFNβ treatment.
|Maternal cholestasis during pregnancy programs metabolic disease in offspring. |
Papacleovoulou, G; Abu-Hayyeh, S; Nikolopoulou, E; Briz, O; Owen, BM; Nikolova, V; Ovadia, C; Huang, X; Vaarasmaki, M; Baumann, M; Jansen, E; Albrecht, C; Jarvelin, MR; Marin, JJ; Knisely, AS; Williamson, C
The Journal of clinical investigation 123 3172-81 2013
The intrauterine environment is a major contributor to increased rates of metabolic disease in adults. Intrahepatic cholestasis of pregnancy (ICP) is a liver disease of pregnancy that affects 0.5%-2% of pregnant women and is characterized by increased bile acid levels in the maternal serum. The influence of ICP on the metabolic health of offspring is unknown. We analyzed the Northern Finland birth cohort 1985-1986 database and found that 16-year-old children of mothers with ICP had altered lipid profiles. Males had increased BMI, and females exhibited increased waist and hip girth compared with the offspring of uncomplicated pregnancies. We further investigated the effect of maternal cholestasis on the metabolism of adult offspring in the mouse. Females from cholestatic mothers developed a severe obese, diabetic phenotype with hepatosteatosis following a Western diet, whereas matched mice not exposed to cholestasis in utero did not. Female littermates were susceptible to metabolic disease before dietary challenge. Human and mouse studies showed an accumulation of lipids in the fetoplacental unit and increased transplacental cholesterol transport in cholestatic pregnancy. We believe this is the first report showing that cholestatic pregnancy in the absence of altered maternal BMI or diabetes can program metabolic disease in the offspring.
|4-Hydroxy hexenal derived from docosahexaenoic acid protects endothelial cells via Nrf2 activation. |
Ishikado, A; Morino, K; Nishio, Y; Nakagawa, F; Mukose, A; Sono, Y; Yoshioka, N; Kondo, K; Sekine, O; Yoshizaki, T; Ugi, S; Uzu, T; Kawai, H; Makino, T; Okamura, T; Yamamoto, M; Kashiwagi, A; Maegawa, H
PloS one 8 e69415 2013
Recent studies have proposed that n-3 polyunsaturated fatty acids (n-3 PUFAs) have direct antioxidant and anti-inflammatory effects in vascular tissue, explaining their cardioprotective effects. However, the molecular mechanisms are not yet fully understood. We tested whether n-3 PUFAs showed antioxidant activity through the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a master transcriptional factor for antioxidant genes. C57BL/6 or Nrf2(-/-) mice were fed a fish-oil diet for 3 weeks. Fish-oil diet significantly increased the expression of heme oxygenase-1 (HO-1), and endothelium-dependent vasodilation in the aorta of C57BL/6 mice, but not in the Nrf2(-/-) mice. Furthermore, we observed that 4-hydroxy hexenal (4-HHE), an end-product of n-3 PUFA peroxidation, was significantly increased in the aorta of C57BL/6 mice, accompanied by intra-aortic predominant increase in docosahexaenoic acid (DHA) rather than that in eicosapentaenoic acid (EPA). Human umbilical vein endothelial cells were incubated with DHA or EPA. We found that DHA, but not EPA, markedly increased intracellular 4-HHE, and nuclear expression and DNA binding of Nrf2. Both DHA and 4-HHE also increased the expressions of Nrf2 target genes including HO-1, and the siRNA of Nrf2 abolished these effects. Furthermore, DHA prevented oxidant-induced cellular damage or reactive oxygen species production, and these effects were disappeared by an HO-1 inhibitor or the siRNA of Nrf2. Thus, we found protective effects of DHA through Nrf2 activation in vascular tissue, accompanied by intra-vascular increases in 4-HHE, which may explain the mechanism of the cardioprotective effects of DHA.
|Effects of mild cold shock (25°C) followed by warming up at 37°C on the cellular stress response. |
Neutelings, T; Lambert, CA; Nusgens, BV; Colige, AC
PloS one 8 e69687 2013
Temperature variations in cells, tissues and organs may occur in a number of circumstances. We report here that reducing temperature of cells in culture to 25°C for 5 days followed by a rewarming to 37°C affects cell biology and induces a cellular stress response. Cell proliferation was almost arrested during mild hypothermia and not restored upon returning to 37°C. The expression of cold shock genes, CIRBP and RBM3, was increased at 25°C and returned to basal level upon rewarming while that of heat shock protein HSP70 was inversely regulated. An activation of pro-apoptotic pathways was evidenced by FACS analysis and increased Bax/Bcl2 and BclX(S/L) ratios. Concomitant increased expression of the autophagosome-associated protein LC3II and AKT phosphorylation suggested a simultaneous activation of autophagy and pro-survival pathways. However, a large proportion of cells were dying 24 hours after rewarming. The occurrence of DNA damage was evidenced by the increased phosphorylation of p53 and H2AX, a hallmark of DNA breaks. The latter process, as well as apoptosis, was strongly reduced by the radical oxygen species (ROS) scavenger, N-acetylcysteine, indicating a causal relationship between ROS, DNA damage and cell death during mild cold shock and rewarming. These data bring new insights into the potential deleterious effects of mild hypothermia and rewarming used in various research and therapeutical fields.
|Up-regulation of SOX9 in sertoli cells from testiculopathic patients accounts for increasing anti-mullerian hormone expression via impaired androgen receptor signaling. |
Lan, KC; Chen, YT; Chang, C; Chang, YC; Lin, HJ; Huang, KE; Kang, HY
PloS one 8 e76303 2013
Testosterone provokes Sertoli cell maturation and represses AMH production. In adult patients with Sertoli-cells-only syndrome (SCOS) and androgen insensitivity syndrome (AIS), high level of AMH expression is detected in Sertoli cells due to defect of androgen/AR signaling.We postulated that up-regulation of SOX9 due to impairment of androgen/AR signaling in Sertoli cells might explain why high level of anti-Mullerian hormone (AMH) expression occur in these testiculopathic patients.Biological research of testicular specimens from men with azoospermia or mouse. The serum hormone levels were studied in 23 men with obstructive azoospermia, 33 men with SCOS azoospermia and 21 volunteers with normal seminograms during a period of 4 years. Immunohistochemical staining and reverse-transcription PCR were used to examine the relationships among AR, SOX9 and AMH expression in adult human and mouse testes. The ability of AR to repress the expression of SOX9 and AMH was evaluated in vitro in TM4 Sertoli cells and C3H10T1/2 cells.SCOS specimens showed up-regulation of SOX9 and AMH proteins but down-regulation of AR proteins in Sertoli cells. The mRNA levels of AR were significantly lower and the SOX9, AMH mRNA levels higher in all SCOS patients compared to controls (Pless than 0.05). The testosterone levels in the SCOS patients were within the normal range, but most were below the median of the controls. Furthermore, our in vitro cell line experiments demonstrated that androgen/AR signaling suppressed the gene and protein levels of AMH via repression of SOX9.Our data show that the functional androgen/AR signaling to repress SOX9 and AMH expression is essential for Sertoli cell maturation. Impairment of androgen/AR signaling promotes SOX9-mediated AMH production, accounts for impairments of Sertoli cells in SCOS azoospermic patients.
|Molecular architecture of the chick vestibular hair bundle. |
Shin, JB; Krey, JF; Hassan, A; Metlagel, Z; Tauscher, AN; Pagana, JM; Sherman, NE; Jeffery, ED; Spinelli, KJ; Zhao, H; Wilmarth, PA; Choi, D; David, LL; Auer, M; Barr-Gillespie, PG
Nature neuroscience 16 365-74 2013
Hair bundles of the inner ear have a specialized structure and protein composition that underlies their sensitivity to mechanical stimulation. Using mass spectrometry, we identified and quantified greater than 1,100 proteins, present from a few to 400,000 copies per stereocilium, from purified chick bundles; 336 of these were significantly enriched in bundles. Bundle proteins that we detected have been shown to regulate cytoskeleton structure and dynamics, energy metabolism, phospholipid synthesis and cell signaling. Three-dimensional imaging using electron tomography allowed us to count the number of actin-actin cross-linkers and actin-membrane connectors; these values compared well to those obtained from mass spectrometry. Network analysis revealed several hub proteins, including RDX (radixin) and SLC9A3R2 (NHERF2), which interact with many bundle proteins and may perform functions essential for bundle structure and function. The quantitative mass spectrometry of bundle proteins reported here establishes a framework for future characterization of dynamic processes that shape bundle structure and function.
|Nuclear expression of β-catenin promotes RB stability and resistance to TNF-induced apoptosis in colon cancer cells. |
Han, J; Soletti, RC; Sadarangani, A; Sridevi, P; Ramirez, ME; Eckmann, L; Borges, HL; Wang, JY
Molecular cancer research : MCR 11 207-18 2013
Tumor necrosis factor (TNF)-α promotes tumor development under chronic inflammation. Because TNF also activates caspase-8, selective inhibition of TNF-induced extrinsic apoptosis would be required for inflammation-associated tumor growth. In a mouse model of inflammation-associated colon carcinogenesis, we found nuclear expression of β-catenin in tumors of wild-type, but not mutant, mice that were made resistant to TNF-induced apoptosis by a germline mutation blocking caspase cleavage of the retinoblastoma (RB) protein, despite similar frequencies of β-catenin exon-3 mutations in these two genetic backgrounds. TNF-induced apoptosis was also attenuated in human colon cancer cell lines with genetically activated β-catenin. However, we found that HCT116 cells, which contain an activated allele of β-catenin but do not express nuclear β-catenin, were sensitive to TNF-induced apoptosis. In HCT116 cells, TNF stimulated efficient RB cleavage that preceded chromatin condensation. In contrast, TNF did not induce RB cleavage in colon cancer cells expressing nuclear β-catenin and these cells could be sensitized to basal and/or TNF-induced apoptosis by the knockdown of β-catenin or RB. In the apoptosis-resistant colon cancer cells, knockdown of β-catenin led to a reduction in the RB protein without affecting RB mRNA. Furthermore, ectopic expression of the caspase-resistant, but not the wild-type, RB re-established resistance to TNF-induced caspase activation in colon cancer cells without β-catenin. Together, these results suggest that nuclear β-catenin-dependent RB stabilization suppresses TNF-induced apoptosis in caspase-8-positive colon cancer cells.
|RIF1 counteracts BRCA1-mediated end resection during DNA repair. |
Feng, L; Fong, KW; Wang, J; Wang, W; Chen, J
The Journal of biological chemistry 288 11135-43 2013
BRCA1 promotes homologous recombination repair and antagonizes 53BP1-dependent nonhomologous end joining (NHEJ) pathway. However, the molecular basis of the competition between BRCA1 and 53BP1 pathways remains elusive. Here we report that RIF1 protein translocates to damage sites via ATM-dependent 53BP1 phosphorylation. Strikingly, loss of RIF1 rescues initial DNA end resection and checkpoint activation in BRCA1-depleted cells. Interestingly RIF1 accumulation at damage sites is antagonized by BRCA1 in S and G2 phases. Conversely, the translocation of BRCA1 to damage sites is inhibited by RIF1 in G1 phase. However, loss of RIF1 differs from that of 53BP1 deficiency, as it cannot fully rescue RAD51 foci formation, homologous recombination defect, and radio-hypersensitivity in BRCA1-deficient cells. This is likely because RIF1, but not 53BP1, also regulates the foci formation and chromatin loading of BLM (the Bloom syndrome helicase). Thus, RIF1 not only acts downstream of 53BP1 and counteracts BRCA1-mediated end resection but also has a secondary role in promoting BLM function in DNA repair.
|p62/SQSTM1 differentially removes the toxic mutant androgen receptor via autophagy and inclusion formation in a spinal and bulbar muscular atrophy mouse model. |
Doi, H; Adachi, H; Katsuno, M; Minamiyama, M; Matsumoto, S; Kondo, N; Miyazaki, Y; Iida, M; Tohnai, G; Qiang, Q; Tanaka, F; Yanagawa, T; Warabi, E; Ishii, T; Sobue, G
The Journal of neuroscience : the official journal of the Society for Neuroscience 33 7710-27 2013
Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders that are caused by the expansion of trinucleotide CAG repeats in the causative genes. Spinal and bulbar muscular atrophy (SBMA) is an inherited motor neuron disease that is caused by the expansion of a polyQ tract within the androgen receptor (AR). p62 is a ubiquitin- and light-chain 3-binding protein that is known to regulate the degradation of targeted proteins via autophagy and inclusion formation. In this study, we examined the effects of p62 depletion and overexpression on cultured cells and in a transgenic mouse model that overexpressed the mutant AR. Here, we demonstrate that depletion of p62 significantly exacerbated motor phenotypes and the neuropathological outcome, whereas overexpression of p62 protected against mutant AR toxicity in SBMA mice. Depletion of p62 significantly increased the levels of monomeric mutant AR and mutant AR protein complexes in an SBMA mouse model via the impairment of autophagic degradation. In addition, p62 overexpression improved SBMA mouse phenotypes by inducing cytoprotective inclusion formation. Our results demonstrate that p62 provides two different therapeutic targets in SBMA pathogenesis: (1) autophagy-dependent degradation and (2) benevolent inclusion formation of the mutant AR.
|TIP48/Reptin and H2A.Z requirement for initiating chromatin remodeling in estrogen-activated transcription. |
Dalvai, M; Fleury, L; Bellucci, L; Kocanova, S; Bystricky, K
PLoS genetics 9 e1003387 2013
Histone variants, including histone H2A.Z, are incorporated into specific genomic sites and participate in transcription regulation. The role of H2A.Z at these sites remains poorly characterized. Our study investigates changes in the chromatin environment at the Cyclin D1 gene (CCND1) during transcriptional initiation in response to estradiol in estrogen receptor positive mammary tumour cells. We show that H2A.Z is present at the transcription start-site and downstream enhancer sequences of CCND1 when the gene is poorly transcribed. Stimulation of CCND1 expression required release of H2A.Z concomitantly from both these DNA elements. The AAA+ family members TIP48/reptin and the histone variant H2A.Z are required to remodel the chromatin environment at CCND1 as a prerequisite for binding of the estrogen receptor (ERα) in the presence of hormone. TIP48 promotes acetylation and exchange of H2A.Z, which triggers a dissociation of the CCND1 3' enhancer from the promoter, thereby releasing a repressive intragenic loop. This release then enables the estrogen receptor to bind to the CCND1 promoter. Our findings provide new insight into the priming of chromatin required for transcription factor access to their target sequence. Dynamic release of gene loops could be a rapid means to remodel chromatin and to stimulate transcription in response to hormones.
|Colon cancer cells adopt an invasive phenotype without mesenchymal transition in 3-D but not 2-D culture upon combined stimulation with EGF and crypt growth factors. |
Ludwig, K; Tse, ES; Wang, JY
BMC cancer 13 221 2013
The intestinal crypt homeostasis is maintained by a combination of growth factors including Wnt, R-Spondin1, Noggin and the epidermal growth factor (EGF). In human colorectal cancer, the Wnt pathway is constitutively activated through genetic and epigenetic alterations in as many as 11 genes encoding components of this crypt stem-cell maintenance mechanism. Although the proliferation of colon cancer cells does not require Wnt, it is possible that colon cancer cells can still respond to the crypt growth factors in the colonic microenvironment. A number of studies have shown that epithelial cells behave differently in 3-D versus 2-D cultures. Because the 3-D conditions more closely mimic the in vivo environment, we examined the effects of Wnt and other crypt growth factors on colon cancer cell growth in 3-D culture.Colon cancer cells were grown in 3-D matrigel supplemented with different combinations of crypt growth factors and colonies were examined for morphology and pathways.When colon cancer cells were cultured in 3-D with EGF, they grew as round spheroid colonies. However, colon cancer cells also grew as flat, disc-like colonies when cultured with EGF plus Wnt, R-Spondin1 and Noggin. Disc colonies were found to have comparable levels of E-cadherin as the spheroid colonies, but showed decreased E-cadherin at the cell-matrix contact sites. Disc colonies also elaborated F-actin rich protrusions (FRP) at the cell-matrix edge, reminiscent of an invasive phenotype but without the expression of vimentin. These E-cadherin and F-actin alterations were not induced by the four growth factors in 2-D culture. Formation of the disc colonies was inhibited by the knockdown of β-catenin and by protein kinase inhibitors such as gefitinib, imatinib and MK-2206. Furthermore, withdrawal of the crypt growth factors was able to revert the disc colonies to spheroid growth, showing that the invasive phenotype was reversible dependent on the availability of growth factors.These findings show that colon cancer cells remain responsive to the growth factors in the crypt microenvironment and can be induced to undergo morphological transformation in the more physiologically relevant 3-D culture.
|Enhanced inhibition of urinary bladder cancer growth and muscle invasion by allyl isothiocyanate and celecoxib in combination. |
Bhattacharya, A; Li, Y; Shi, Y; Zhang, Y
Carcinogenesis 34 2593-9 2013
Allyl isothiocyanate (AITC) occurs in cruciferous vegetables that are commonly consumed by humans and has been shown to inhibit urinary bladder cancer growth and progression in previous preclinical studies. However, AITC does not significantly modulate cyclooxygenase-2 (Cox-2), whose oncogenic activity has been well documented in bladder cancer and other cancers. Celecoxib is a selective Cox-2 inhibitor and has been widely used for treatment of several diseases. Celecoxib has also been evaluated in bladder cancer patients, but its efficacy against bladder cancer as a single agent remains unclear. In a syngeneic rat model of orthotopic bladder cancer, treatment of the animals with the combination of AITC and celecoxib at low dose levels (AITC at 1 mg/kg and celecoxib at 10 mg/kg) led to increased or perhaps synergistic inhibition of bladder cancer growth and muscle invasion, compared with each agent used alone. The combination regime was also more effective than each single agent in inhibiting microvessel formation and stimulating microvessel maturation in the tumor tissues. The anticancer efficacy of the combination regime was associated with depletion of prostaglandin E2, a key downstream signaling molecule of Cox-2, caspase activation and downregulation of vascular endothelial growth factor in the tumor tissues. These data show that AITC and celecoxib complement each other for inhibition of bladder cancer and provide a novel combination approach for potential use for prevention or treatment of human bladder cancer.
|Prolidase directly binds and activates epidermal growth factor receptor and stimulates downstream signaling. |
Yang, L; Li, Y; Ding, Y; Choi, KS; Kazim, AL; Zhang, Y
The Journal of biological chemistry 288 2365-75 2013
Prolidase, also known as Xaa-Pro dipeptidase or peptidase D (PEPD), is a ubiquitously expressed cytosolic enzyme that hydrolyzes dipeptides with proline or hydroxyproline at the carboxyl terminus. In this article, however, we demonstrate that PEPD directly binds to and activates epidermal growth factor receptor (EGFR), leading to stimulation of signaling proteins downstream of EGFR, and that such activity is neither cell-specific nor dependent on the enzymatic activity of PEPD. In line with the pro-survival and pro-proliferation activities of EGFR, PEPD stimulates DNA synthesis. We further show that PEPD activates EGFR only when it is present in the extracellular space, but that PEPD is released from injured cells and tissues and that such release appears to result in EGFR activation. PEPD differs from all known EGFR ligands in that it does not possess an epidermal growth factor (EGF) motif and is not synthesized as a transmembrane precursor, but PEPD binding to EGFR can be blocked by EGF. In conclusion, PEPD is a ligand of EGFR and presents a novel mechanism of EGFR activation.
|The C-terminal module IV of connective tissue growth factor is a novel immune modulator of the Th17 response. |
Rodrigues-Díez, R; Rodrigues-Díez, RR; Rayego-Mateos, S; Suarez-Alvarez, B; Lavoz, C; Stark Aroeira, L; Sánchez-López, E; Orejudo, M; Alique, M; Lopez-Larrea, C; Ortiz, A; Egido, J; Ruiz-Ortega, M
Laboratory investigation; a journal of technical methods and pathology 93 812-24 2013
Connective tissue growth factor (CTGF/CCN2) is a matricellular protein susceptible to proteolytic degradation. CCN2 levels have been suggested as a potential risk biomarker in several chronic diseases. In body fluids, CCN2 full-length and its degradation fragments can be found; however, their in vivo effects are far from being elucidated. CCN2 was described as a profibrotic mediator, but this concept is changing to a proinflammatory cytokine. In vitro, CCN2 full-length and its C-terminal module IV (CCN2(IV)) exert proinflammatory properties. Emerging evidence suggest that Th17 cells, and its effector cytokine IL-17A, participate in chronic inflammatory diseases. Our aim was to explore whether CCN2(IV) could regulate the Th17 response. In vitro, stimulation of human naive CD4+ T lymphocytes with CCN2(IV) resulted in differentiation to Th17 phenotype. The in vivo effects of CCN2(IV) were studied in C57BL/6 mice. Intraperitoneal administration of recombinant CCN2(IV) did not change serum IL-17A levels, but caused an activation of the Th17 response in the kidney, characterized by interstitial infiltration of Th17 (IL17A+/CD4+) cells and upregulation of proinflammatory mediators. In CCN2(IV)-injected mice, elevated renal levels of Th17-related factors (IL-17A, IL-6, STAT3 and RORγt) were found, whereas Th1/Th2 cytokines or Treg-related factors (TGF-β and Foxp-3) were not modified. Treatment with an anti-IL-17A neutralizing antibody diminished CCN2(IV)-induced renal inflammation. Our findings unveil that the C-terminal module of CCN2 induces the Th17 differentiation of human Th17 cells and causes a renal Th17 inflammatory response. Furthermore, these data bear out that IL-17A targeting is a promising tool for chronic inflammatory diseases, including renal pathologies.
|Loss of ARF sensitizes transgenic BRAFV600E mice to UV-induced melanoma via suppression of XPC. |
Luo, C; Sheng, J; Hu, MG; Haluska, FG; Cui, R; Xu, Z; Tsichlis, PN; Hu, GF; Hinds, PW
Cancer research 73 4337-48 2013
Both genetic mutations and UV irradiation (UVR) can predispose individuals to melanoma. Although BRAF(V600E) is the most prevalent oncogene in melanoma, the BRAF(V600E) mutant is not sufficient to induce tumors in vivo. Mutation at the CDKN2A locus is another melanoma-predisposing event that can disrupt the function of both p16(INK4a) and ARF. Numerous studies have focused on the role of p16(INK4a) in melanoma, but the involvement of ARF, a well-known p53 activator, is still controversial. Using a transgenic BRAF(V600E) mouse model previously generated in our laboratory, we report that loss of ARF is able to enhance spontaneous melanoma formation and cause profound sensitivity to neonatal UVB exposure. Mechanistically, BRAF(V600E) and ARF deletion synergize to inhibit nucleotide excision repair by epigenetically repressing XPC and inhibiting the E2F4/DP1 complex. We suggest that the deletion of ARF promotes melanomagenesis not by abrogating p53 activation but by acting in concert with BRAF(V600E) to increase the load of DNA damage caused by UVR.
|Upregulation of miR-96 enhances cellular proliferation of prostate cancer cells through FOXO1. |
Haflidadóttir, BS; Larne, O; Martin, M; Persson, M; Edsjö, A; Bjartell, A; Ceder, Y
PloS one 8 e72400 2013
Aberrant expression of miR-96 in prostate cancer has previously been reported. However, the role and mechanism of action of miR-96 in prostate cancer has not been determined. In this study, the diagnostic and prognostic properties of miR-96 expression levels were investigated by qRT-PCR in two well documented prostate cancer cohorts. The miR-96 expression was found to be significantly higher in prostate cancer patients and correlate with WHO grade, and decreased overall survival time; patients with low levels of miR-96 lived 1.5 years longer than patients with high miR-96 levels. The therapeutic potential was further investigated in vitro, showing that ectopic levels of miR-96 enhances growth and cellular proliferation in prostate cancer cells, implying that miR-96 has oncogenic properties in this setting. We demonstrate that miR-96 expression decreases the transcript and protein levels of FOXO1 by binding to one of two predicted binding sites in the FOXO1 3'UTR sequence. Blocking this binding site completely inhibited the growth enhancement conveyed by miR-96. This finding was corroborated in a large external prostate cancer patient cohort where miR-96 expression inversely correlated to FOXO1 expression. Taken together these findings indicate that miR-96 plays a key role in prostate cancer cellular proliferation and can enhance prostate cancer progression. This knowledge might be utilized for the development of novel therapeutic tools for prostate cancer.
|Retroviral DNA methylation and epigenetic repression are mediated by the antiviral host protein Daxx. |
Shalginskikh, N; Poleshko, A; Skalka, AM; Katz, RA
Journal of virology 87 2137-50 2013
Integrated retroviral DNA is subject to epigenetic transcriptional silencing at different frequencies. This process is mediated by repressive DNA methylation and histone modifications on viral chromatin. However, the detailed mechanisms by which retroviral silencing is initiated and maintained are not well understood. Using a model system in which avian sarcoma virus (ASV) DNA is epigenetically repressed in mammalian cells, we previously found that a cellular scaffolding protein, Daxx, acts as an antiretroviral factor that promotes epigenetic repression through recruitment of histone deacetylases (HDACs). Here we show that human Daxx protein levels are increased in response to retroviral infection and that Daxx acts at the time of infection to initiate epigenetic repression. Consistent with a rapid and active antiviral epigenetic response, we found that repressive histone marks and long terminal repeat (LTR) DNA methylation could be detected within 12 h to 3 days postinfection, respectively. Daxx was also found to be required for long-term ASV silencing maintenance and full viral DNA methylation, and it was physically associated with both viral DNA and DNA methyltransferases (DNMTs). These findings support a model in which incoming retroviral protein-DNA complexes are detected by Daxx, and the integrated provirus is rapidly chromatinized and repressed by DNA methylation and histone modification as part of an antiviral response. These results uncover a possible direct and active antiviral mechanism by which DNMTs can be recruited to retroviral DNA.
|Activation of p21 by HDAC inhibitors requires acetylation of H2A.Z. |
Bellucci, L; Dalvai, M; Kocanova, S; Moutahir, F; Bystricky, K
PloS one 8 e54102 2013
Differential positioning of the histone variant H2A.Z in a p53 dependent manner was shown to regulate p21 transcription. Whether H2A.Z is involved in p21 activity in the absence of p53 is not known. The p21 gene is repressed in estrogen receptor (ER) negative cell lines that are p53-/- and hormone independent for their growth. Here we demonstrate that class I and II pan Histone deacetylase inhibitors (HDACi) induce p21 transcription and reduce cell proliferation of MDA-MB231, an ERα-negative mammary tumor cell line, in a H2A.Z dependent manner. H2A.Z is associated with the transcription start site (TSS) of the repressed p21 gene. Depleting H2A.Z did not lead to transcription of p21 but annihilated the stimulating effect of HDACi on this gene. Acetylation of H2A.Z but not of H3K9 at the p21 promoter correlated with p21 activation. We further show that HDACi treatment reduced the presence of the p400 chromatin remodeler at the p21 TSS. We propose a model in which association of p400 negatively affects p21 transcription by interfering with acetylation of H2A.Z.
|M98K-OPTN induces transferrin receptor degradation and RAB12-mediated autophagic death in retinal ganglion cells. |
Sirohi, K; Chalasani, ML; Sudhakar, C; Kumari, A; Radha, V; Swarup, G
Autophagy 9 510-27 2013
Mutations in the autophagy receptor OPTN/optineurin are associated with the pathogenesis of glaucoma and amyotrophic lateral sclerosis, but the underlying molecular basis is poorly understood. The OPTN variant, M98K has been described as a risk factor for normal tension glaucoma in some ethnic groups. Here, we examined the consequence of the M98K mutation in affecting cellular functions of OPTN. Overexpression of M98K-OPTN induced death of retinal ganglion cells (RGC-5 cell line), but not of other neuronal and non-neuronal cells. Enhanced levels of the autophagy marker, LC3-II, a post-translationally modified form of LC3, in M98K-OPTN-expressing cells and the inability of an LC3-binding-defective M98K variant of OPTN to induce cell death, suggested that autophagy contributes to cell death. Knockdown of Atg5 reduced M98K-induced death of RGC-5 cells, further supporting the involvement of autophagy. Overexpression of M98K-OPTN enhanced autophagosome formation and potentiated the delivery of transferrin receptor to autophagosomes for degradation resulting in reduced cellular transferrin receptor levels. Coexpression of transferrin receptor or supplementation of media with an iron donor reduced M98K-induced cell death. OPTN complexes with RAB12, a GTPase involved in vesicle trafficking, and M98K variant shows enhanced colocalization with RAB12. Knockdown of Rab12 increased transferrin receptor level and reduced M98K-induced cell death. RAB12 is present in autophagosomes and knockdown of Rab12 resulted in reduced formation of autolysosomes during starvation-induced autophagy, implicating a role for RAB12 in autophagy. These results also show that transferrin receptor degradation and autophagy play a crucial role in RGC-5 cell death induced by M98K variant of OPTN.
|Transcriptional regulation by the Wilms tumor protein, Wt1, suggests a role of the metalloproteinase Adamts16 in murine genitourinary development. |
Jacobi, CL; Rudigier, LJ; Scholz, H; Kirschner, KM
The Journal of biological chemistry 288 18811-24 2013
ADAMTS16 (a disintegrin and metalloproteinase with thrombospondin motifs) is a secreted mammalian metalloproteinase with unknown function. We report here that murine Adamts16 is co-expressed with the Wilms tumor protein, Wt1, in the developing glomeruli of embryonic kidneys. Adamts16 mRNA levels were significantly reduced upon transfection of embryonic murine kidney explants with Wt1 antisense vivo-morpholinos. Antisense knockdown of Adamts16 inhibited branching morphogenesis in kidney organ cultures. Adamts16 was detected by in situ mRNA hybridization and/or immunohistochemistry also in embryonic gonads and in spermatids and granulosa cells of adult testes and ovaries, respectively. Silencing of Wt1 by transfection with antisense vivo-morpholinos significantly increased Adamts16 mRNA in cultured embryonic XY gonads (11.5 and 12.5 days postconception), and reduced Adamts16 transcripts in XX gonads (12.5 and 13.5 days postconception). Three predicted Wt1 consensus motifs could be identified in the promoter and the 5'-untranslated region of the murine Adamts16 gene. Binding of Wt1 protein to these elements was verified by EMSA and ChIP. A firefly luciferase reporter gene under control of the Adamts16 promoter was activated ∼8-fold by transient co-transfection of human granulosa cells with a Wt1 expression construct. Gradual shortening of the 5'-flanking sequence successively reduced and eventually abrogated Adamts16 promoter activation by Wt1. These findings demonstrate that Wt1 differentially regulates the Adamts16 gene in XX and XY embryonic gonads. It is suggested that Adamts16 acts immediately downstream of Wt1 during murine urogenital development. We propose that Adamts16 is involved in branching morphogenesis of the kidneys in mice.
|The neural cell adhesion molecule promotes maturation of the presynaptic endocytotic machinery by switching synaptic vesicle recycling from adaptor protein 3 (AP-3)- to AP-2-dependent mechanisms. |
Shetty, A; Sytnyk, V; Leshchyns'ka, I; Puchkov, D; Haucke, V; Schachner, M
The Journal of neuroscience : the official journal of the Society for Neuroscience 33 16828-45 2013
Newly formed synapses undergo maturation during ontogenetic development via mechanisms that remain poorly understood. We show that maturation of the presynaptic endocytotic machinery in CNS neurons requires substitution of the adaptor protein 3 (AP-3) with AP-2 at the presynaptic plasma membrane. In mature synapses, AP-2 associates with the intracellular domain of the neural cell adhesion molecule (NCAM). NCAM promotes binding of AP-2 over binding of AP-3 to presynaptic membranes, thus favoring the substitution of AP-3 for AP-2 during formation of mature synapses. The presynaptic endocytotic machinery remains immature in adult NCAM-deficient (NCAM-/-) mice accumulating AP-3 instead of AP-2 and its partner protein AP180 in synaptic membranes and vesicles. NCAM deficiency or disruption of the NCAM/AP-2 complex in wild-type (NCAM+/+) neurons by overexpression of AP-2 binding-defective mutant NCAM interferes with efficient retrieval of the synaptic vesicle v-SNARE synaptobrevin 2. Abnormalities in synaptic vesicle endocytosis and recycling may thus contribute to neurological disorders associated with mutations in NCAM.
|Recessive MYL2 mutations cause infantile type I muscle fibre disease and cardiomyopathy. |
Weterman, MA; Barth, PG; van Spaendonck-Zwarts, KY; Aronica, E; Poll-The, BT; Brouwer, OF; van Tintelen, JP; Qahar, Z; Bradley, EJ; de Wissel, M; Salviati, L; Angelini, C; van den Heuvel, L; Thomasse, YE; Backx, AP; Nürnberg, G; Nürnberg, P; Baas, F
Brain : a journal of neurology 136 282-93 2013
A cardioskeletal myopathy with onset and death in infancy, morphological features of muscle type I hypotrophy with myofibrillar disorganization and dilated cardiomyopathy was previously reported in three Dutch families. Here we report the genetic cause of this disorder. Multipoint parametric linkage analysis of six Dutch patients identified a homozygous region of 2.1 Mb on chromosome 12, which was shared between all Dutch patients, with a log of odds score of 10.82. Sequence analysis of the entire linkage region resulted in the identification of a homozygous mutation in the last acceptor splice site of the myosin regulatory light chain 2 gene (MYL2) as the genetic cause. MYL2 encodes a myosin regulatory light chain (MLC-2V). The myosin regulatory light chains bind, together with the essential light chains, to the flexible neck region of the myosin heavy chain in the hexameric myosin complex and have a structural and regulatory role in muscle contraction. The MYL2 mutation results in use of a cryptic splice site upstream of the last exon causing a frameshift and replacement of the last 32 codons by 20 different codons. Whole exome sequencing of an Italian patient with similar clinical features showed compound heterozygosity for two other mutations affecting the same exon of MYL2, also resulting in mutant proteins with altered C-terminal tails. As a consequence of these mutations, the second EF-hand domain is disrupted. EF-hands, assumed to function as calcium sensors, can undergo a conformational change upon binding of calcium that is critical for interactions with downstream targets. Immunohistochemical staining of skeletal muscle tissue of the Dutch patients showed a diffuse and weak expression of the mutant protein without clear fibre specificity, while normal protein was absent. Heterozygous missense mutations in MYL2 are known to cause dominant hypertrophic cardiomyopathy; however, none of the parents showed signs of cardiomyopathy. In conclusion, the mutations in the last exon of MYL2 are responsible for a novel autosomal recessive lethal myosinopathy due to defects changing the C-terminal tail of the ventricular form of the myosin regulatory light chain. We propose 'light chain myopathy' as a name for this MYL2-associated myopathy.
|The effects of brain serotonin deficiency on behavioural disinhibition and anxiety-like behaviour following mild early life stress. |
Sachs, BD; Rodriguiz, RM; Siesser, WB; Kenan, A; Royer, EL; Jacobsen, JP; Wetsel, WC; Caron, MG
The international journal of neuropsychopharmacology / official scientific journal of the Collegium Internationale Neuropsychopharmacologicum (CINP) 16 2081-94 2013
Aberrant serotonin (5-HT) signalling and exposure to early life stress have both been suggested to play a role in anxiety- and impulsivity-related behaviours. However, whether congenital 5-HT deficiency × early life stress interactions influence the development of anxiety- or impulsivity-like behaviour has not been established. Here, we examined the effects of early life maternal separation (MS) stress on anxiety-like behaviour and behavioural disinhibition, a type of impulsivity-like behaviour, in wild-type (WT) and tryptophan hydroxylase 2 (Tph2) knock-in (Tph2KI) mice, which exhibit ~60-80% reductions in the levels of brain 5-HT due to a R439H mutation in Tph2. We also investigated the effects of 5-HT deficiency and early life stress on adult hippocampal neurogenesis, plasma corticosterone levels and several signal transduction pathways in the amygdala. We demonstrate that MS slightly increases anxiety-like behaviour in WT mice and induces behavioural disinhibition in Tph2KI animals. We also demonstrate that MS leads to a slight decrease in cell proliferation within the hippocampus and potentiates corticosterone responses to acute stress, but these effects are not affected by brain 5-HT deficiency. However, we show that 5-HT deficiency leads to significant alterations in SGK-1 and GSK3β signalling and NMDA receptor expression in the amygdala in response to MS. Together, these findings support a potential role for 5-HT-dependent signalling in the amygdala in regulating the long-term effects of early life stress on anxiety-like behaviour and behavioural disinhibition.
|Constitutively-active androgen receptor variants function independently of the HSP90 chaperone but do not confer resistance to HSP90 inhibitors. |
Gillis, JL; Selth, LA; Centenera, MM; Townley, SL; Sun, S; Plymate, SR; Tilley, WD; Butler, LM
Oncotarget 4 691-704 2013
The development of lethal, castration resistant prostate cancer is associated with adaptive changes to the androgen receptor (AR), including the emergence of mutant receptors and truncated, constitutively active AR variants. AR relies on the molecular chaperone HSP90 for its function in both normal and malignant prostate cells, but the requirement for HSP90 in environments with aberrant AR expression is largely unknown. Here, we investigated the efficacy of three HSP90 inhibitors, 17-AAG, HSP990 and AUY922, against clinically-relevant AR missense mutants and truncated variants. HSP90 inhibition effectively suppressed the signaling of wild-type AR and all AR missense mutants tested. By contrast, two truncated AR variants, AR-V7 and ARv567es, exhibited marked resistance to HSP90 inhibitors. Supporting this observation, nuclear localization of the truncated AR variants was not affected by HSP90 inhibition and AR variant:HSP90 complexes could not be detected in prostate cancer cells. Interestingly, HSP90 inhibition resulted in accumulation of AR-V7 and ARv567es in both cell lines and human tumor explants. Despite the apparent independence of AR variants from HSP90 and their treatment-associated induction, the growth of cell lines with endogenous or enforced expression of AR-V7 or ARv567es remained highly sensitive to AUY922. This study demonstrates that functional AR variant signaling does not confer resistance to HSP90 inhibition, yields insight into the interaction between AR and HSP90 and provides further impetus for the clinical application of HSP90 inhibitors in advanced prostate cancer.
|The histone methyltransferase MMSET/WHSC1 activates TWIST1 to promote an epithelial-mesenchymal transition and invasive properties of prostate cancer. |
Ezponda, T; Popovic, R; Shah, MY; Martinez-Garcia, E; Zheng, Y; Min, DJ; Will, C; Neri, A; Kelleher, NL; Yu, J; Licht, JD
Oncogene 32 2882-90 2013
Epigenetic deregulation of gene expression has a role in the initiation and progression of prostate cancer (PCa). The histone methyltransferase MMSET/WHSC1 (Multiple Myeloma SET domain) is overexpressed in a number of metastatic tumors, but its mechanism of action has not been defined. In this work, we found that PCa cell lines expressed significantly higher levels of MMSET compared with immortalized, non-transformed prostate cells. Knockdown experiments showed that, in metastatic PCa cell lines, dimethylation of lysine 36 and trimethylation of lysine 27 on histone H3 (H3K36me2 and H3K27me3, respectively) depended on MMSET expression, whereas depletion of MMSET in benign prostatic cells did not affect chromatin modifications. Knockdown of MMSET in DU145 and PC-3 tumor cells decreased cell proliferation, colony formation in soft agar and strikingly diminished cell migration and invasion. Conversely, overexpression of MMSET in immortalized, non-transformed RWPE-1 cells promoted cell migration and invasion, accompanied by an epithelial-mesenchymal transition (EMT). Among a panel of EMT-promoting genes analyzed, TWIST1 expression was strongly activated in response to MMSET. Chromatin immunoprecipitation analysis demonstrated that MMSET binds to the TWIST1 locus and leads to an increase in H3K36me2, suggesting a direct role of MMSET in the regulation of this gene. Depletion of TWIST1 in MMSET-overexpressing RWPE-1 cells blocked cell invasion and EMT, indicating that TWIST1 was a critical target of MMSET, responsible for the acquisition of an invasive phenotype. Collectively, these data suggest that MMSET has a role in PCa pathogenesis and progression through epigenetic regulation of metastasis-related genes.
|The transcription factor Runx2 is under circadian control in the suprachiasmatic nucleus and functions in the control of rhythmic behavior. |
Reale, ME; Webb, IC; Wang, X; Baltazar, RM; Coolen, LM; Lehman, MN
PloS one 8 e54317 2013
Runx2, a member of the family of runt-related transcription factors, is rhythmically expressed in bone and may be involved in circadian rhythms in bone homeostasis and osteogenesis. Runx2 is also expressed in the brain, but its function is unknown. We tested the hypothesis that in the brain, Runx2 may interact with clock-controlled genes to regulate circadian rhythms in behavior. First, we demonstrated diurnal and circadian rhythms in the expression of Runx2 in the mouse brain. Expression of Runx2 mRNA and protein mirrored that of the core clock genes, Period1 and Period2, in the suprachiasmatic nucleus (SCN), the paraventricular nucleus and the olfactory bulb. The rhythm of Runx2 expression was eliminated in the SCN of Bmal1(-/-) mice. Moreover, by crossbreeding mPer2(Luc) mice with Runx2(+/-) mice and recording bioluminescence rhythms, a significant lengthening of the period of rhythms was detected in cultured SCN of Runx2(-/-) animals compared to either Runx2(+/-) or Runx2(+/+) mice. Behavioral analyses of Runx2 mutant mice revealed that Runx2(+/-) animals displayed a significantly lengthened free-running period of running wheel activity compared to Runx2(+/+) littermates. Taken together, these findings provide evidence for clock gene-mediated rhythmic expression of Runx2, and its functional role in regulating circadian period at the level of the SCN and behavior.
|Alcohol administration blocks stress-induced impairments in memory and anxiety, and alters hippocampal neurotransmitter receptor expression in male rats. |
Gomez, JL; Lewis, MJ; Sebastian, V; Serrano, P; Luine, VN
Hormones and behavior 63 659-66 2013
Chronic exposure to stress has many deleterious effects on behavior, which can often lead to self-medication with anxiolytics, antidepressants, or alcohol. We determined the effects of alcohol administration following a stressor on established behavioral, physiological, and neural responses to stress. Male Sprague-Dawley rats received: No alcohol/No stress (CON), Alcohol alone (ALC), Stress alone (STR), or Stress plus Alcohol (STR+ALC). For seven consecutive days, two cohorts received an oral dose of 2.0 g/kg of either 20% ethanol or saline. In Cohort 1, behavioral testing began after the final treatment (day-8). Memory was tested using the object recognition (OR) and Y-maze, anxiety on the plus maze, and depression on the forced swim task. Memory on OR and Y-maze tasks was impaired in the ALC and STR groups. This deficit was reversed in the STR+ALC group, which performed not differently from the CON group. Stress alone was associated with increased anxiety, which was alleviated with alcohol treatment. No treatment effects were found in the forced swim task. In Cohort 2, hippocampal GABAα4 was upregulated in the STR+ALC group and GluN2B was upregulated in the ALC and STR+ALC groups. The STR+ALC group in Cohort 1 showed enhanced corticosterone levels after forced swim. The STR+ALC group in Cohort 2 showed increased corticosterone levels on day-1 of treatment and a habituation by day-7. In conclusion, this study found a reversal of stress-induced deficits in cognition and anxiety when alcohol was given post-stress, and changes in neurotransmitter receptor expression may contribute to these behavioral effects.
|Hypertonic stress promotes autophagy and microtubule-dependent autophagosomal clusters. |
Nunes, P; Ernandez, T; Roth, I; Qiao, X; Strebel, D; Bouley, R; Charollais, A; Ramadori, P; Foti, M; Meda, P; Féraille, E; Brown, D; Hasler, U
Autophagy 9 550-67 2013
Osmotic homeostasis is fundamental for most cells, which face recurrent alterations of environmental osmolality that challenge cell viability. Protein damage is a consequence of hypertonic stress, but whether autophagy contributes to the osmoprotective response is unknown. Here, we investigated the possible implications of autophagy and microtubule organization on the response to hypertonic stress. We show that hypertonicity rapidly induced long-lived protein degradation, LC3-II generation and Ptdlns3K-dependent formation of LC3- and ATG12-positive puncta. Lysosomotropic agents chloroquine and bafilomycin A 1, but not nutrient deprivation or rapamycin treatment, further increased LC3-II generation, as well as ATG12-positive puncta, indicating that hypertonic stress increases autophagic flux. Autophagy induction upon hypertonic stress enhanced cell survival since cell death was increased by ATG12 siRNA-mediated knockdown and reduced by rapamycin. We additionally showed that hypertonicity induces fast reorganization of microtubule networks, which is associated with strong reorganization of microtubules at centrosomes and fragmentation of Golgi ribbons. Microtubule remodeling was associated with pericentrosomal clustering of ATG12-positive autolysosomes that colocalized with SQSTM1/p62 and ubiquitin, indicating that autophagy induced by hypertonic stress is at least partly selective. Efficient autophagy by hypertonic stress required microtubule remodeling and was DYNC/dynein-dependent as autophagosome clustering was enhanced by paclitaxel-induced microtubule stabilization and was reduced by nocodazole-induced tubulin depolymerization as well as chemical (EHNA) or genetic [DCTN2/dynactin 2 (p50) overexpression] interference of DYNC activity. The data document a general and hitherto overlooked mechanism, where autophagy and microtubule remodeling play prominent roles in the osmoprotective response.
|The shift from low to high non-structural protein 1 expression in rotavirus-infected MA-104 cells. |
Martínez-Álvarez, L; Piña-Vázquez, C; Zarco, W; Padilla-Noriega, L
Memórias do Instituto Oswaldo Cruz 108 421-8 2013
A hallmark of group/species A rotavirus (RVA) replication in MA-104 cells is the logarithmic increase in viral mRNAs that occurs four-12 h post-infection. Viral protein synthesis typically lags closely behind mRNA synthesis but continues after mRNA levels plateau. However, RVA non-structural protein 1 (NSP1) is present at very low levels throughout viral replication despite showing robust protein synthesis. NSP1 has the contrasting properties of being susceptible to proteasomal degradation, but being stabilised against proteasomal degradation by viral proteins and/or viral mRNAs. We aimed to determine the kinetics of the accumulation and intracellular distribution of NSP1 in MA-104 cells infected with rhesus rotavirus (RRV). NSP1 preferentially localises to the perinuclear region of the cytoplasm of infected cells, forming abundant granules that are heterogeneous in size. Late in infection, large NSP1 granules predominate, coincident with a shift from low to high NSP1 expression levels. Our results indicate that rotavirus NSP1 is a late viral protein in MA-104 cells infected with RRV, presumably as a result of altered protein turnover.
|Phosphorylation regulates NCC stability and transporter activity in vivo. |
Yang, SS; Fang, YW; Tseng, MH; Chu, PY; Yu, IS; Wu, HC; Lin, SW; Chau, T; Uchida, S; Sasaki, S; Lin, YF; Sytwu, HK; Lin, SH
Journal of the American Society of Nephrology : JASN 24 1587-97 2013
A T60M mutation in the thiazide-sensitive sodium chloride cotransporter (NCC) is common in patients with Gitelman's syndrome (GS). This mutation prevents Ste20-related proline and alanine-rich kinase (SPAK)/oxidative stress responsive kinase-1 (OSR1)-mediated phosphorylation of NCC and alters NCC transporter activity in vitro. Here, we examined the physiologic effects of NCC phosphorylation in vivo using a novel Ncc T58M (human T60M) knock-in mouse model. Ncc(T58M/T58M) mice exhibited typical features of GS with a blunted response to thiazide diuretics. Despite expressing normal levels of Ncc mRNA, these mice had lower levels of total Ncc and p-Ncc protein that did not change with a low-salt diet that increased p-Spak. In contrast to wild-type Ncc, which localized to the apical membrane of distal convoluted tubule cells, T58M Ncc localized primarily to the cytosolic region and caused an increase in late distal convoluted tubule volume. In MDCK cells, exogenous expression of phosphorylation-defective NCC mutants reduced total protein expression levels and membrane stability. Furthermore, our analysis found diminished total urine NCC excretion in a cohort of GS patients with homozygous NCC T60M mutations. When Wnk4(D561A/+) mice, a model of pseudohypoaldosteronism type II expressing an activated Spak/Osr1-Ncc, were crossed with Ncc(T58M/T58M) mice, total Ncc and p-Ncc protein levels decreased and the GS phenotype persisted over the hypertensive phenotype. Overall, these data suggest that SPAK-mediated phosphorylation of NCC at T60 regulates NCC stability and function, and defective phosphorylation at this residue corrects the phenotype of pseudohypoaldosteronism type II.
|Epigenetic inheritance of a cocaine-resistance phenotype. |
Vassoler, FM; White, SL; Schmidt, HD; Sadri-Vakili, G; Pierce, RC
Nature neuroscience 16 42-7 2013
We delineated a heritable phenotype resulting from the self-administration of cocaine in rats. We observed delayed acquisition and reduced maintenance of cocaine self-administration in male, but not female, offspring of sires that self-administered cocaine. Brain-derived neurotrophic factor (Bdnf) mRNA and BDNF protein were increased in the medial prefrontal cortex (mPFC), and there was an increased association of acetylated histone H3 with Bdnf promoters in only the male offspring of cocaine-experienced sires. Administration of a BDNF receptor antagonist (the TrkB receptor antagonist ANA-12) reversed the diminished cocaine self-administration in male cocaine-sired rats. In addition, the association of acetylated histone H3 with Bdnf promoters was increased in the sperm of sires that self-administered cocaine. Collectively, these findings indicate that voluntary paternal ingestion of cocaine results in epigenetic reprogramming of the germline, having profound effects on mPFC gene expression and resistance to cocaine reinforcement in male offspring.
|Peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1)- and estrogen-related receptor (ERR)-induced regulator in muscle 1 (Perm1) is a tissue-specific regulator of oxidative capacity in skeletal muscle cells. |
Cho, Y; Hazen, BC; Russell, AP; Kralli, A
The Journal of biological chemistry 288 25207-18 2013
Mitochondrial oxidative metabolism and energy transduction pathways are critical for skeletal and cardiac muscle function. The expression of genes important for mitochondrial biogenesis and oxidative metabolism are under the control of members of the peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1) family of transcriptional coactivators and the estrogen-related receptor (ERR) subfamily of nuclear receptors. Perturbations in PGC-1 and/or ERR activities have been associated with alterations in capacity for endurance exercise, rates of muscle atrophy, and cardiac function. The mechanism(s) by which PGC-1 and ERR proteins regulate muscle-specific transcriptional programs is not fully understood. We show here that PGC-1α and ERRs induce the expression of a so far uncharacterized muscle-specific protein, PGC-1- and ERR-induced regulator in muscle 1 (Perm1), which regulates the expression of selective PGC-1/ERR target genes. Perm1 is required for the basal as well as PGC-1α-enhanced expression of genes with roles in glucose and lipid metabolism, energy transfer, and contractile function. Silencing of Perm1 in cultured myotubes compromises respiratory capacity and diminishes PGC-1α-induced mitochondrial biogenesis. Our findings support a role for Perm1 acting downstream of PGC-1α and ERRs to regulate muscle-specific pathways important for energy metabolism and contractile function. Elucidating the function of Perm1 may enable novel approaches for the treatment of disorders with compromised skeletal muscle bioenergetics, such as mitochondrial myopathies and age-related/disease-associated muscle atrophies.
|Proplatelet generation in the mouse requires PKCε-dependent RhoA inhibition. |
Gobbi, G; Mirandola, P; Carubbi, C; Masselli, E; Sykes, SM; Ferraro, F; Nouvenne, A; Thon, JN; Italiano, JE; Vitale, M
Blood 122 1305-11 2013
During thrombopoiesis, megakaroycytes undergo extensive cytoskeletal remodeling to form proplatelet extensions that eventually produce mature platelets. Proplatelet formation is a tightly orchestrated process that depends on dynamic regulation of both tubulin reorganization and Rho-associated, coiled-coil containing protein kinase/RhoA activity. A disruption in tubulin dynamics or RhoA activity impairs proplatelet formation and alters platelet morphology. We previously observed that protein kinase Cepsilon (PKCε), a member of the protein kinase C family of serine/threonine-kinases, expression varies during human megakaryocyte differentiation and modulates megakaryocyte maturation and platelet release. Here we used an in vitro model of murine platelet production to investigate a potential role for PKCε in proplatelet formation. By immunofluorescence we observed that PKCε colocalizes with α/β-tubulin in specific areas of the marginal tubular-coil in proplatelets. Moreover, we found that PKCε expression escalates during megakarocyte differentiation and remains elevated in proplatelets, whereas the active form of RhoA is substantially downregulated in proplatelets. PKCε inhibition resulted in lower proplatelet numbers and larger diameter platelets in culture as well as persistent RhoA activation. Finally, we demonstrate that pharmacological inhibition of RhoA is capable of reversing the proplatelet defects mediated by PKCε inhibition. Collectively, these data indicate that by regulating RhoA activity, PKCε is a critical mediator of mouse proplatelet formation in vitro.
|Regulation of transcription by the MLL2 complex and MLL complex-associated AKAP95. |
Jiang, H; Lu, X; Shimada, M; Dou, Y; Tang, Z; Roeder, RG
Nature structural & molecular biology 20 1156-63 2013
Although histone H3 Lys4 (H3K4) methylation is widely associated with gene activation, direct evidence for its causal role in transcription, through specific MLL family members, is scarce. Here we have purified a human MLL2 (Kmt2b) complex that is highly active in H3K4 methylation and chromatin transcription in a cell-free system. This effect requires S-adenosyl methionine and intact H3K4, thus establishing a direct and causal role for MLL2-mediated H3K4 methylation in transcription. We also show that human AKAP95, a chromatin-associated protein, physically and functionally associates with MLL complexes and directly enhances their methyltransferase activity. Ectopic AKAP95 stimulates expression of a chromosomal reporter gene in synergy with MLL1 or MLL2, whereas AKAP95 depletion impairs retinoic acid-mediated gene induction in embryonic stem cells. These results demonstrate an important role for AKAP95 in regulating histone methylation and gene expression, particularly during cell-fate transitions.
|Differential expression of molecular markers of synaptic plasticity in the hippocampus, prefrontal cortex, and amygdala in response to spatial learning, predator exposure, and stress-induced amnesia. |
Phillip R Zoladz,Collin R Park,Joshua D Halonen,Samina Salim,Karem H Alzoubi,Marisa Srivareerat,Monika Fleshner,Karim A Alkadhi,David M Diamond
Hippocampus 22 2012
We have studied the effects of spatial learning and predator stress-induced amnesia on the expression of calcium/calmodulin-dependent protein kinase II (CaMKII), brain-derived neurotrophic factor (BDNF) and calcineurin in the hippocampus, basolateral amygdala (BLA), and medial prefrontal cortex (mPFC). Adult male rats were given a single training session in the radial-arm water maze (RAWM) composed of 12 trials followed by a 30-min delay period, during which rats were either returned to their home cages or given inescapable exposure to a cat. Immediately following the 30-min delay period, the rats were given a single test trial in the RAWM to assess their memory for the hidden platform location. Under control (no stress) conditions, rats exhibited intact spatial memory and an increase in phosphorylated CaMKII (p-CaMKII), total CaMKII, and BDNF in dorsal CA1. Under stress conditions, rats exhibited impaired spatial memory and a suppression of all measured markers of molecular plasticity in dorsal CA1. The molecular profiles observed in the BLA, mPFC, and ventral CA1 were markedly different from those observed in dorsal CA1. Stress exposure increased p-CaMKII in the BLA, decreased p-CaMKII in the mPFC, and had no effect on any of the markers of molecular plasticity in ventral CA1. These findings provide novel observations regarding rapidly induced changes in the expression of molecular plasticity in response to spatial learning, predator exposure, and stress-induced amnesia in brainregions involved in different aspects of memory processing.
|Elevated cyclin g2 expression intersects with DNA damage checkpoint signaling and is required for a potent g2/m checkpoint arrest response to Doxorubicin. |
Maike Zimmermann,Aruni S Arachchige-Don,Michaela S Donaldson,Robert F Dallapiazza,Colleen E Cowan,Mary C Horne
The Journal of biological chemistry 287 2012
To maintain genomic integrity DNA damage response (DDR), signaling pathways have evolved that restrict cellular replication and allow time for DNA repair. CCNG2 encodes an unconventional cyclin homolog, cyclin G2 (CycG2), linked to growth inhibition. Its expression is repressed by mitogens but up-regulated during cell cycle arrest responses to anti-proliferative signals. Here we investigate the potential link between elevated CycG2 expression and DDR signaling pathways. Expanding our previous finding that CycG2 overexpression induces a p53-dependent G(1)/S phase cell cycle arrest in HCT116 cells, we now demonstrate that this arrest response also requires the DDR checkpoint protein kinase Chk2. In accord with this finding we establish that ectopic CycG2 expression increases phosphorylation of Chk2 on threonine 68. We show that DNA double strand break-inducing chemotherapeutics stimulate CycG2 expression and correlate its up-regulation with checkpoint-induced cell cycle arrest and phospho-modification of proteins in the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) signaling pathways. Using pharmacological inhibitors and ATM-deficient cell lines, we delineate the DDR kinase pathway promoting CycG2 up-regulation in response to doxorubicin. Importantly, RNAi-mediated blunting of CycG2 attenuates doxorubicin-induced cell cycle checkpoint responses in multiple cell lines. Employing stable clones, we test the effect that CycG2 depletion has on DDR proteins and signals that enforce cell cycle checkpoint arrest. Our results suggest that CycG2 contributes to DNA damage-induced G(2)/M checkpoint by enforcing checkpoint inhibition of CycB1-Cdc2 complexes.
|Age-related decrease in cerebrovascular-derived neuroprotective proteins: effect of acetaminophen. |
Tripathy, D; Sanchez, A; Yin, X; Martinez, J; Grammas, P
Microvascular research 84 278-85 2012
As the population ages, the need for effective methods to maintain brain function in older adults is increasingly pressing. Vascular disease and neurodegenerative disorders commonly co-occur in older persons. Cerebrovascular products contribute to the neuronal milieu and have important consequences for neuronal viability. In this regard vascular derived neuroprotective proteins, Such as vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), and pituitary adenylate cyclase activating peptide (PACAP) are important for maintaining neuronal viability, especially in the face of injury and disease. The objective of this study is to measure and compare levels of VEGF, PEDF and PACAP released from isolated brain microvessels of Fischer 344 rats at 6, 12, 18, and 24 months of age. Addition of acetaminophen to isolated brain microvessels is employed to determine whether this drug affects vascular expression of these neuroprotective proteins. Experiments on cultured brain endothelial cells are performed to explore the mechanisms/mediators that regulate the effect of acetaminophen on endothelial cells. The data indicate cerebrovascular expression of VEGF, PEDF and PACAP significantly decreases with age. The age-associated decrease in VEGF and PEDF is ameliorated by addition of acetaminophen to isolated brain microvessels. Also, release of VEGF, PEDF, and PACAP from cultured brain endothelial cells decreases with exposure to the oxidant stressor menadione. Acetaminophen treatment upregulates VEGF, PEDF and PACAP in brain endothelial cells exposed to oxidative stress. The effect of acetaminophen on cultured endothelial cells is in part inhibited by the selective thrombin inhibitor hirudin. The results of this study suggest that acetaminophen may be a useful agent for preserving cerebrovascular function. If a low dose of acetaminophen can counteract the decrease in vascular-derived neurotrophic factors evoked by age and oxidative stress, this drug might be useful for improving brain function in the elderly.
|Gene network and pathway analysis of mice with conditional ablation of Dicer in post-mitotic neurons. |
Dorval, V; Smith, PY; Delay, C; Calvo, E; Planel, E; Zommer, N; Buée, L; Hébert, SS
PloS one 7 e44060 2012
The small non-protein-coding microRNAs (miRNAs) have emerged as critical regulators of neuronal differentiation, identity and survival. To date, however, little is known about the genes and molecular networks regulated by neuronal miRNAs in vivo, particularly in the adult mammalian brain.We analyzed whole genome microarrays from mice lacking Dicer, the enzyme responsible for miRNA production, specifically in postnatal forebrain neurons. A total of 755 mRNA transcripts were significantly (Pless than 0.05, FDRless than 0.25) misregulated in the conditional Dicer knockout mice. Ten genes, including Tnrc6c, Dnmt3a, and Limk1, were validated by real time quantitative RT-PCR. Upregulated transcripts were enriched in nonneuronal genes, which is consistent with previous studies in vitro. Microarray data mining showed that upregulated genes were enriched in biological processes related to gene expression regulation, while downregulated genes were associated with neuronal functions. Molecular pathways associated with neurological disorders, cellular organization and cellular maintenance were altered in the Dicer mutant mice. Numerous miRNA target sites were enriched in the 3'untranslated region (3'UTR) of upregulated genes, the most significant corresponding to the miR-124 seed sequence. Interestingly, our results suggest that, in addition to miR-124, a large fraction of the neuronal miRNome participates, by order of abundance, in coordinated gene expression regulation and neuronal maintenance.Taken together, these results provide new clues into the role of specific miRNA pathways in the regulation of brain identity and maintenance in adult mice.
|Effects of HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) on NEU/HER2 overexpressing mammary tumours in MMTV-NEU-NT mice monitored by Magnetic Resonance Spectroscopy. |
Rodrigues, LM; Chung, YL; Al Saffar, NM; Sharp, SY; Jackson, LE; Banerji, U; Stubbs, M; Leach, MO; Griffiths, JR; Workman, P
BMC research notes 5 250 2012
The importance of ERBB2/NEU/HER2 in the response of breast tumours to the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG; tanespimycin) has been demonstrated in the clinic. ERBB2 is an oncoprotein client that is highly dependent on HSP90. This and other oncogenic client proteins (e.g. B-RAF, C-RAF, ALK and CDK4) are depleted by 17-AAG in both animal tumours and patients. Here we investigate by Magnetic Resonance Spectroscopy (MRS) the metabolic response of 17-AAG in spontaneous, NEU/HER2 driven mammary tumours in transgenic MMTV-NEU-NT mice and in cells isolated and cultured from these tumours.Mammary tumours were monitored by 31P MRS in vivo and in tumour extracts, comparing control and 17-AAG treated mice. A cell line derived from NEU/HER2 mammary tumours was also cultured and the effect of 17-AAG was measured by 31P MRS in cell extracts. Molecular biomarkers were assessed by immunoblotting in extracts from cells and tumours. For comparison of tumour volume, metabolite concentrations and Western blot band intensities, two-tailed unpaired t-tests were used.The NEU/HER2 mammary tumours were very sensitive to 17-AAG and responded in a dose-dependent manner to 3 daily doses of 20, 40 and 80mg/kg of 17-AAG, all of which caused significant regression. At the higher doses, 31P MRS of tumour extracts showed significant decreases in phosphocholine (PC) and phosphoethanolamine (PE) whereas no significant changes were seen at the 20mg/kg dose. Extracts of isolated cells cultured from the mammary carcinomas showed a significant decrease in viable cell number and total PME after 17-AAG treatment. Western blots confirmed the expected action of 17-AAG in inducing HSP72 and significantly depleting HSP90 client proteins, including NEU/HER2 both in tumours and in isolated cells.The data demonstrate the high degree of sensitivity of this clinically relevant NEU/HER2-driven tumour model to HSP90 inhibition by 17-AAG, consistent with the clinical data, and suggest that the metabolic signature of choline phospholipids obtained by MRS could be useful both as a preclinical and clinical tool for investigating surrogate markers of response to treatment.
|The G60S Cx43 mutant enhances keratinocyte proliferation and differentiation. |
Jared M Churko,John J Kelly,Andrew Macdonald,Jack Lee,Jacinda Sampson,Donglin Bai,Dale W Laird
Experimental dermatology 21 2012
Transient knock-down of the gap junction protein Cx43 by antisense and siRNA, or gap junction block with mimetic peptides, have been shown to enhance epidermal wound healing. However, patients with oculodentodigital dysplasia (ODDD) express mutant Cx43 that leads to a chronic reduction in gap junctional intercellular communication. To determine whether mutant Cx43 in keratinocytes would impact upon the wound healing process, we localized Cx43 in human and mouse skin tissue expressing mutant Cx43 and assessed the ability of primary keratinocytes derived from a mouse model of ODDD to proliferate, migrate and differentiate. In the epidermis from an ODDD patient and in the epidermis of mice expressing the G60S mutant or in keratinocytes obtained from mutant mice, Cx43 was frequently found within intracellular compartments and rarely localized to punctate sites of cell-cell apposition. Primary keratinocytes derived from G60S mutant mice proliferated faster but migrated similarly to keratinocytes derived from wild-type control mice. Keratinocytes derived from mutant mice expressed abundant Cx43 and higher levels of involucrin and loricrin under low calcium conditions. However, after calcium-induced differentiation, similar levels of Cx43, involucrin and loricrin were observed. Thus, we conclude that during wound healing, mutant Cx43 may enhance keratinocyte proliferation and promote early differentiation of keratinocytes.
|Pax6 is a key component of regulated glucagon secretion. |
Yvan Gosmain,Claire Cheyssac,Mounia Heddad Masson,Audrey Guérardel,Caroline Poisson,Jacques Philippe
Endocrinology 153 2012
The Pax6 transcription factor is crucial for pancreatic α-cells. Indeed, Pax6-deficient mouse models are characterized by markedly altered α-cell differentiation. Our objective was to investigate the role of Pax6 in glucagon secretion process. We used a Pax6-deficient model in rat primary enriched-α cells with specific small interfering RNA leading to a 70% knockdown of Pax6 expression. We first showed that Pax6 knockdown decreases glucagon biosynthesis as well as glucagon release. Through physiological assays, we demonstrated that the decrease of Pax6 affects specifically acute glucagon secretion in primary α-cell in response to glucose, palmitate, and glucose-dependent insulinotropic peptide (GIP) but not the response to arginine and epinephrine. We identified in Pax6 knockdown model that genes involved in glucagon secretion such as the glucokinase (GCK), G protein-coupled receptor (GPR40), and GIP receptor (GIPR) as well as the corresponding proteins were significantly decreased whereas the insulin receptor (IR) Kir6.2/Sur1, and glucose transporter 1 genes were not affected. We demonstrated that Pax6 directly binds and activates specific elements on the promoter region of the GPR40, GCK, and GIPR genes. Finally, through site-directed mutagenesis experiments, we showed that disruption of Pax6 binding on the GCK, GPR40, and GIPR gene promoters led to specific decreases of their activities in the αTC1.9 glucagon-producing cell line. Hence our results indicate that Pax6 acts on the regulation of glucagon secretion at least through the transcriptional control of GCK, GPR40, and GIPR. We propose that Pax6 is not only critical for glucagon biosynthesis but also for glucagon secretion particularly in response to nutrients.
|Impairments to the GH-IGF-I axis in hSOD1G93A mice give insight into possible mechanisms of GH dysregulation in patients with amyotrophic lateral sclerosis. |
Steyn, FJ; Ngo, ST; Lee, JD; Leong, JW; Buckley, AJ; Veldhuis, JD; McCombe, PA; Chen, C; Bellingham, MC
Endocrinology 153 3735-46 2012
GH deficiency has been found in subjects with amyotrophic lateral sclerosis (ALS). Disrupted endocrine function could contribute to the progressive muscle loss and hypermetabolism seen in ALS. It is not possible to study all the elements of the GH-IGF-I axis in ALS patients. Consequently, it remains unclear whether dysfunctional GH secretion contributes to disease pathogenesis and why GH and IGF-I directed treatment strategies are ineffective in human ALS. The hSOD1(G93A) transgenic mouse model is useful for the detailed investigation of the pathogenesis of ALS. We report that symptomatic male hSOD1(G93A) transgenic mice exhibit a deficiency in GH secretion similar to that seen in human ALS. Further characterization of the GH-IGF-I axis in hSOD1(G93A) mice reveals central and peripheral abnormalities that are not found in wild-type age-matched controls. Specifically, we observe aberrant endogenous pulsatile GH secretion, reduced pituitary GH content, and decreased circulating levels of IGF-I, indicating global GH deficiency in hSOD1(G93A) mice. Furthermore, a reduction in the expression of the IGF-I receptor α-subunit in skeletal muscle and lumbar spinal cords of hSOD1(G93A) mice suggests impaired IGF-I signaling within these tissues. This is the first account of disrupted GH secretion in a transgenic mouse model of ALS. These observations are essential for the development of effective GH and IGF-I targeted therapies in ALS.
|Inhibition of damage-regulated autophagy modulator-1 (DRAM-1) impairs neutrophil differentiation of NB4 APL cells. |
Magali Humbert,Chantal Mueller,Martin F Fey,Mario P Tschan
Leukemia research 36 2012
The damage-regulator autophagy modulator 1 (DRAM-1) is a lysosomal protein that positively regulates autophagy in a p53-dependent manner. We aimed at analyzing the role of DRAM-1 in granulocytic differentiation of APL cells. We observed a significant increase of DRAM-1 expression during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of NB4 APL cells but not in ATRA-resistant NB4-R2 cells. Next, knocking down DRAM-1 in NB4 APL cells was sufficient to impair neutrophil differentiation. Given that DRAM-1 is a transcriptional target of p53, we tested if DRAM-1 is regulated by the p53 relative p73. Indeed, inhibiting p73 prevented neutrophil differentiation and DRAM-1 induction of NB4 cells. In conclusion, we show for the first time that p73-regulated DRAM-1 is functionally involved in neutrophil differentiation of APL cells.
|PU.1 is linking the glycolytic enzyme HK3 in neutrophil differentiation and survival of APL cells. |
Federzoni, EA; Valk, PJ; Torbett, BE; Haferlach, T; Löwenberg, B; Fey, MF; Tschan, MP
Blood 119 4963-70 2012
The transcription factor PU.1 is a master regulator of myeloid differentiation and function. On the other hand, only scarce information is available on PU.1-regulated genes involved in cell survival. We now identified the glycolytic enzyme hexokinase 3 (HK3), a gene with cytoprotective functions, as transcriptional target of PU.1. Interestingly, HK3 expression is highly associated with the myeloid lineage and was significantly decreased in acute myeloid leukemia patients compared with normal granulocytes. Moreover, HK3 expression was significantly lower in acute promyelocytic leukemia (APL) compared with non-APL patient samples. In line with the observations in primary APL patient samples, we observed significantly higher HK3 expression during neutrophil differentiation of APL cell lines. Moreover, knocking down PU.1 impaired HK3 induction during neutrophil differentiation. In vivo binding of PU.1 and PML-RARA to the HK3 promoter was found, and PML-RARA attenuated PU.1 activation of the HK3 promoter. Next, inhibiting HK3 in APL cell lines resulted in significantly reduced neutrophil differentiation and viability compared with control cells. Our findings strongly suggest that HK3 is: (1) directly activated by PU.1, (2) repressed by PML-RARA, and (3) functionally involved in neutrophil differentiation and cell viability of APL cells.
|Impaired contractility and detrusor hypertrophy in cavin-1-deficient mice. |
Mardjaneh Sadegh Karbalaei,Catarina Rippe,Sebastian Albinsson,Mari Ekman,Alva Mansten,Bengt Uvelius,Karl Swärd,Karl Sw
European journal of pharmacology 689 2012
Caveolae are membrane invaginations present in a variety of cell types. Formation of caveolae depends on caveolins and on the more recently discovered family of proteins known as the cavins. Genetic ablation of caveolin-1 was previously shown to give rise to a number of urogenital alterations, but the effects of cavin-1 deletion on urogenital function remain unknown. Here we characterised detrusor contractility and structure in cavin-1-deficient mice. Electron microscopy demonstrated essentially complete lack of caveolae in the knock-out detrusor, and immunoblotting disclosed reduced levels of cavin-3 and of all caveolin proteins. Bladder weight was increased in male knock-out mice, and length-tension relationships demonstrated a reduction in depolarisation-induced contraction. Contractility in response to muscarinic receptor activation was similarly reduced. Despite these functional changes, micturition patterns were similar in conscious and freely moving animals and diuresis was unchanged. Our breeding additionally disclosed that the number of knock-out mice generated in heterozygous crosses was lower than expected, suggesting embryonic/perinatal lethality. In conclusion, this is the first study to show that cavin-1 is critical for detrusor caveolae and for the overall contractility and structure of the urinary bladder.
|Tetraploid cells from cytokinesis failure induce aneuploidy and spontaneous transformation of mouse ovarian surface epithelial cells. |
Lv, L; Zhang, T; Yi, Q; Huang, Y; Wang, Z; Hou, H; Zhang, H; Zheng, W; Hao, Q; Guo, Z; Cooke, HJ; Shi, Q
Cell cycle (Georgetown, Tex.) 11 2864-75 2012
Most ovarian cancers originate from the ovarian surface epithelium and are characterized by aneuploid karyotypes. Aneuploidy, a consequence of chromosome instability, is an early event during the development of ovarian cancers. However, how aneuploid cells are evolved from normal diploid cells in ovarian cancers remains unknown. In the present study, cytogenetic analyses of a mouse syngeneic ovarian cancer model revealed that diploid mouse ovarian surface epithelial cells (MOSECs) experienced an intermediate tetraploid cell stage, before evolving to aneuploid (mainly near-tetraploid) cells. Using long-term live-cell imaging followed by fluorescence in situ hybridization (FISH), we demonstrated that tetraploid cells originally arose from cytokinesis failure of bipolar mitosis in diploid cells, and gave rise to aneuploid cells through chromosome mis-segregation during both bipolar and multipolar mitoses. Injection of the late passage aneuploid MOSECs resulted in tumor formation in C57BL/6 mice. Therefore, we reveal a pathway for the evolution of diploid to aneuploid MOSECs and elucidate a mechanism for the development of near-tetraploid ovarian cancer cells.
|Release of the glucose-regulated protein 94 by baby hamster kidney cells. |
Yulia Evdokimovskaya,Yuri Skarga,Veronika Vrublevskaya,Oleg Morenkov
Cell biochemistry and function 30 2012
Glucose-regulated protein 94 (grp94) is a major component of the endoplasmic reticulum (ER) lumen of eukaryotic cells. We showed that grp94 is released from baby hamster kidney (BHK-21) cells into a serum-free medium. The exit of grp94 into the medium was not related to the protein discharge due to cell death and was independent of de novo protein synthesis. The treatment of cells with brefeldin A and monensin, the inhibitors of the classical pathway of protein secretion, did not decrease the extracellular level of grp94, indicating that the discharge of grp94 from cells does not occur through the ER/Golgi-dependent pathway. Exosomes, membrane vesicles secreted by several cell types, were not involved in the release of grp94 from cells. Methyl-β-cyclodextrin, a substance that disrupts the lipid raft organization, considerably reduced the extracellular level of grp94, indicating that lipid rafts are involved in the liberation of grp94 from BHK-21 cells. The results suggest that BHK-21 cells release grp94 into the serum-free medium via the nonclassical secretory pathway in which lipid rafts play an important role. Copyright
|Resveratrol helps recovery from fatty liver and protects against hepatocellular carcinoma induced by hepatitis B virus X protein in a mouse model. |
Lin, HC; Chen, YF; Hsu, WH; Yang, CW; Kao, CH; Tsai, TF
Cancer prevention research (Philadelphia, Pa.) 5 952-62 2012
Resveratrol is a natural polyphenol that has beneficial effects across species and various disease models. Here, we investigate whether resveratrol is effective against hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) using HBV X protein (HBx) transgenic mice. We found that resveratrol (30 mg/kg/d) has a therapeutic effect on HBx-induced fatty liver and the early stages of liver damage. Resveratrol decreased intracellular reactive oxygen species and transiently stimulated hepatocyte proliferation. Interestingly, resveratrol inhibited LXRα and downregulated the expression of the lipogenic genes, Srebp1-c and PPARγ. The decrease in Srebp1-c seems to further downregulate the expression of its target genes, Acc and Fas. In addition, resveratrol stimulated the activity of Ampk and SirT1. Thus, resveratrol has a pleiotropic effect on HBx transgenic mice in terms of the downregulation of lipogenesis, the promotion of transient liver regeneration, and the stimulation of antioxidant activity. Furthermore, at the later precancerous stages, resveratrol delayed HBx-mediated hepatocarcinogenesis and reduced HCC incidence from 80% to 15%, a 5.3-fold reduction. Resveratrol should be considered as a potential chemopreventive agent for HBV-associated HCC.
|Regulation of the tumor suppressor PTEN by SUMO. |
González-Santamaría, J; Campagna, M; Ortega-Molina, A; Marcos-Villar, L; de la Cruz-Herrera, CF; González, D; Gallego, P; Lopitz-Otsoa, F; Esteban, M; Rodríguez, MS; Serrano, M; Rivas, C
Cell death & disease 3 e393 2012
The crucial function of the PTEN tumor suppressor in multiple cellular processes suggests that its activity must be tightly controlled. Both, membrane association and a variety of post-translational modifications, such as acetylation, phosphorylation, and mono- and polyubiquitination, have been reported to regulate PTEN activity. Here, we demonstrated that PTEN is also post-translationally modified by the small ubiquitin-like proteins, small ubiquitin-related modifier 1 (SUMO1) and SUMO2. We identified lysine residue 266 and the major monoubiquitination site 289, both located within the C2 domain required for PTEN membrane association, as SUMO acceptors in PTEN. We demonstrated the existence of a crosstalk between PTEN SUMOylation and ubiquitination, with PTEN-SUMO1 showing a reduced capacity to form covalent interactions with monoubiquitin and accumulation of PTEN-SUMO2 conjugates after inhibition of the proteasome. Moreover, we found that virus infection induces PTEN SUMOylation and favors PTEN localization at the cell membrane. Finally, we demonstrated that SUMOylation contributes to the control of virus infection by PTEN.
|The p38/MK2-driven exchange between tristetraprolin and HuR regulates AU-rich element-dependent translation. |
Tiedje, C; Ronkina, N; Tehrani, M; Dhamija, S; Laass, K; Holtmann, H; Kotlyarov, A; Gaestel, M
PLoS genetics 8 e1002977 2012
TNF expression of macrophages is under stringent translational control that depends on the p38 MAPK/MK2 pathway and the AU-rich element (ARE) in the TNF mRNA. Here, we elucidate the molecular mechanism of phosphorylation-regulated translation of TNF. We demonstrate that translation of the TNF-precursor at the ER requires expression of the ARE-binding and -stabilizing factor human antigen R (HuR) together with either activity of the p38 MAPK/MK2 pathway or the absence of the ARE-binding and -destabilizing factor tristetraprolin (TTP). We show that phosphorylation of TTP by MK2 decreases its affinity to the ARE, inhibits its ability to replace HuR, and permits HuR-mediated initiation of translation of TNF mRNA. Since translation of TTP's own mRNA is also regulated by this mechanism, an intrinsic feedback control of the inflammatory response is ensured. The phosphorylation-regulated TTP/HuR exchange at target mRNAs provides a reversible switch between unstable/non-translatable and stable/efficiently translated mRNAs.
|Exons 5-15 of kazrin are dispensable for murine epidermal morphogenesis and homeostasis. |
Chhatriwala, MK; Cipolat, S; Sevilla, LM; Nachat, R; Watt, FM
The Journal of investigative dermatology 132 1977-87 2012
Kazrin binds to periplakin and ARVCF catenin, and regulates adhesion and differentiation of cultured human keratinocytes. To explore kazrin function in vivo, we generated a kazrin gene-trap mouse in which only exons 1-4 were expressed, fused to β-galactosidase. On transient transfection, the protein encoded by exons 1-4 did not enter the nucleus, but did cause keratinocyte shape changes. The mice had no obvious defects in skin development or homeostasis, and periplakin and desmoplakin localization was normal. Expression of the kazrin-β-galactosidase fusion protein faithfully reported endogenous kazrin expression. Kazrin was not expressed in embryonic epidermis and was first detected at postnatal day 1. In adult mice, epidermal kazrin expression was less widespread than in humans and Xenopus, being confined to the bulb of anagen hair follicles, the infundibulum, and parakeratotic tail epidermis. In anagen bulbs, kazrin was expressed by a band of cells with elongated morphology and low desmoplakin levels, suggesting a role in morphogenetic cell movements. We conclude that exons 5-15 of kazrin, encoding the nuclear localization signal and C-terminal domain, are not required for epidermal development and function. The previously reported role of kazrin in regulating cell shape appears to reside within the N-terminal coiled-coil domain encoded by exons 1-4.
|Molecular Characteristics of Clostridium perfringens TpeL Toxin and Consequences of Mono-O-GlcNAcylation of Ras in Living Cells. |
Gregor Guttenberg,Sven Hornei,Thomas Jank,Carsten Schwan,Wei Lü,Oliver Einsle,Panagiotis Papatheodorou,Klaus Aktories
The Journal of biological chemistry 287 2012
TpeL is a member of the family of clostridial glucosylating toxins produced by Clostridium perfringens type A, B, and C strains. In contrast to other members of this toxin family, it lacks a C-terminal polypeptide repeat domain, which is suggested to be involved in target cell binding. It was shown that the glucosyltransferase domain of TpeL modifies Ras in vitro by mono-O-glucosylation or mono-O-GlcNAcylation (Nagahama, M., Ohkubo, A., Oda, M., Kobayashi, K., Amimoto, K., Miyamoto, K., and Sakurai, J. (2011) Infect. Immun. 79, 905-910). Here we show that TpeL preferably utilizes UDP-N-acetylglucosamine (UDP-GlcNAc) as a sugar donor. Change of alanine 383 of TpeL to isoleucine turns the sugar donor preference from UDP-GlcNAc to UDP-glucose. In contrast to previous studies, we show that Rac is a poor substrate in vitro and in vivo and requires 1-2 magnitudes higher toxin concentrations for modification by TpeL. The toxin is autoproteolytically processed in the presence of inositol hexakisphosphate (InsP(6)) by an intrinsic cysteine protease domain, located next to the glucosyltransferase domain. A C-terminally extended TpeL full-length variant (TpeL1-1779) induces apoptosis in HeLa cells (most likely by mono-O-GlcNAcylation of Ras), and inhibits Ras signaling including Ras-Raf interaction and ERK activation. In addition, TpeL blocks Ras signaling in rat pheochromocytoma PC12 cells. TpeL is a glucosylating toxin, which modifies Ras and induces apoptosis in target cells without having a typical C-terminal polypeptide repeat domain.
|TGF-β-SMAD3 signaling mediates hepatic bile acid and phospholipid metabolism following lithocholic acid-induced liver injury. |
Tsutomu Matsubara,Naoki Tanaka,Misako Sato,Dong Wook Kang,Kristopher W Krausz,Kathleen C Flanders,Kazuo Ikeda,Hans Luecke,Lalage M Wakefield,Frank J Gonzalez
Journal of lipid research 53 2012
Transforming growth factor-β (TGFβ) is activated as a result of liver injury, such as cholestasis. However, its influence on endogenous metabolism is not known. This study demonstrated that TGFβ regulates hepatic phospholipid and bile acid homeostasis through MAD homolog 3 (SMAD3) activation as revealed by lithocholic acid-induced experimental intrahepatic cholestasis. Lithocholic acid (LCA) induced expression of TGFB1 and the receptors TGFBR1 and TGFBR2 in the liver. In addition, immunohistochemistry revealed higher TGFβ expression around the portal vein after LCA exposure and diminished SMAD3 phosphorylation in hepatocytes from Smad3-null mice. Serum metabolomics indicated increased bile acids and decreased lysophosphatidylcholine (LPC) after LCA exposure. Interestingly, in Smad3-null mice, the metabolic alteration was attenuated. LCA-induced lysophosphatidylcholine acyltransferase 4 (LPCAT4) and organic solute transporter β (OSTβ) expression were markedly decreased in Smad3-null mice, whereas TGFβ induced LPCAT4 and OSTβ expression in primary mouse hepatocytes. In addition, introduction of SMAD3 enhanced the TGFβ-induced LPCAT4 and OSTβ expression in the human hepatocellular carcinoma cell line HepG2. In conclusion, considering that Smad3-null mice showed attenuated serum ALP activity, a diagnostic indicator of cholangiocyte injury, these results strongly support the view that TGFβ-SMAD3 signaling mediates an alteration in phospholipid and bile acid metabolism following hepatic inflammation with the biliary injury.
|Identification of hamster inducible nitric oxide synthase (iNOS) promoter sequences that influence basal and inducible iNOS expression. |
Omar A Saldarriaga,Bruno L Travi,Goutam Ghosh Choudhury,Peter C Melby
Journal of leukocyte biology 92 2012
IFN-γ/LPS-activated hamster (Mesocricetus auratus) macrophages express significantly less iNOS (NOS2) than activated mouse macrophages, which contributes to the hamster's susceptibility to intracellular pathogens. We determined a mechanism responsible for differences in iNOS promoter activity in hamsters and mice. The HtPP (1.2 kb) showed low basal and inducible promoter activity when compared with the mouse, and sequences within a 100-bp region (-233 to -133) of the mouse and hamster promoters influenced this activity. Moreover, within this 100 bp, we identified a smaller region (44 bp) in the mouse promoter, which recovered basal promoter activity when swapped into the hamster promoter. The mouse homolog (100-bp region) contained a cis-element for NF-IL-6 (-153/-142), which was absent in the hamster counterpart. EMSA and supershift assays revealed that the hamster sequence did not support the binding of NF-IL-6. Introduction of a functional NF-IL-6 binding sequence into the hamster promoter or its alteration in the mouse promoter revealed the critical importance of this transcription factor for full iNOS promoter activity. Furthermore, the binding of NF-IL-6 to the iNOS promoter (-153/-142) in vivo was increased in mouse cells but was reduced in hamster cells after IFN-γ/LPS stimulation. Differences in the activity of the iNOS promoters were evident in mouse and hamster cells, so they were not merely a result of species-specific differences in transcription factors. Thus, we have identified unique DNA sequences and a critical transcription factor, NF-IL-6, which contribute to the overall basal and inducible expression of hamster iNOS.
|Self-renewal and differentiation of reactive astrocyte-derived neural stem/progenitor cells isolated from the cortical peri-infarct area after stroke. |
Shimada, IS; LeComte, MD; Granger, JC; Quinlan, NJ; Spees, JL
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 7926-40 2012
In response to stroke, subpopulations of cortical reactive astrocytes proliferate and express proteins commonly associated with neural stem/progenitor cells such as glial fibrillary acidic protein (GFAP) and Nestin. To examine the stem cell-related properties of cortical reactive astrocytes after injury, we generated GFAP-CreER(TM);tdRFP mice to permanently label reactive astrocytes. We isolated cells from the cortical peri-infarct area 3 d after stroke, and cultured them in neural stem cell medium containing epidermal growth factor and basic fibroblast growth factor. We observed tdRFP-positive neural spheres in culture, suggestive of tdRFP-positive reactive astrocyte-derived neural stem/progenitor cells (Rad-NSCs). Cultured Rad-NSCs self-renewed and differentiated into neurons, astrocytes, and oligodendrocytes. Pharmacological inhibition and conditional knock-out mouse studies showed that Presenilin 1 and Notch 1 controlled neural sphere formation by Rad-NSCs after stroke. To examine the self-renewal and differentiation potential of Rad-NSCs in vivo, Rad-NSCs were transplanted into embryonic, neonatal, and adult mouse brains. Transplanted Rad-NSCs were observed to persist in the subventricular zone and secondary Rad-NSCs were isolated from the host brain 28 d after transplantation. In contrast with neurogenic postnatal day 4 NSCs and adult NSCs from the subventricular zone, transplanted Rad-NSCs differentiated into astrocytes and oligodendrocytes, but not neurons, demonstrating that Rad-NSCs had restricted differentiation in vivo. Our results indicate that Rad-NSCs are unlikely to be suitable for neuronal replacement in the absence of genetic or epigenetic modification.
|ASC-dependent RIP2 kinase regulates reduced PGE2 production in chronic periodontitis. |
Taxman, DJ; Lei, Y; Zhang, S; Holley-Guthrie, E; Offenbacher, S; Ting, JP
Journal of dental research 91 877-82 2012
Levels of prostaglandin E(2) (PGE(2)) and its processing enzyme, prostaglandin-endoperoxide-synthase-2/ cyclooxygenase-2 (PTGS2/COX-2), are elevated in actively progressing periodontal lesions, but suppressed in chronic disease. COX-2 expression is regulated through inflammatory signaling that converges on the mitogen-activated protein kinase (MAPK) pathway. Emerging evidence suggests a role for the inflammatory adaptor protein, ASC/Pycard, in MAPK activation. We postulated that ASC may represent a mediator of the MAPK-mediated regulatory network of PGE(2) production. Using RNAi-mediated gene slicing, we demonstrated that ASC regulates COX-2 expression and PGE(2) production in THP1 monocytic cells following infection with Porphyromonas gingivalis (Pg). Production of PGE(2) did not require the inflammasome adaptor function of ASC, but was dependent on MAPK activation. Furthermore, the MAP kinase kinase kinase CARD domain-containing protein RIPK2 was induced by Pg in an ASC-dependent manner. Reduced ASC and RIPK2 levels were revealed by orthogonal comparison of the expression of the RIPK family in ASC-deficient THP1 cells with that in chronic periodontitis patients. We show that pharmacological inhibition of RIPK2 represses PGE(2) secretion, and RNAi-mediated silencing of RIPK2 leads to diminished MAPK activation and PGE(2) secretion. These findings identify a novel ASC-RIPK2 axis in the generation of PGE(2) that is repressed in patients diagnosed with chronic adult periodontitis.
|HIV-1 infection and first line ART induced differential responses in mitochondria from blood lymphocytes and monocytes: the ANRS EP45 "Aging" study. |
Perrin, Sophie, et al.
PLoS ONE, 7: e41129 (2012) 2012
The ANRS EP45 "Aging" study investigates the cellular mechanisms involved in the accelerated aging of HIV-1 infected and treated patients. The data reported focus on mitochondria, organelles known to be involved in cell senescence.
|A gatekeeper residue for NEDD8-activating enzyme inhibition by MLN4924. |
Toth, JI; Yang, L; Dahl, R; Petroski, MD
Cell reports 1 309-16 2012
Inhibition of NEDD8-activating enzyme (NAE) has emerged as a highly promising approach to treat cancer through the adenosine sulfamate analog MLN4924. Here, we show that selective pressure results in HCT116 colorectal carcinoma cells with decreased MLN4924 sensitivity and identify a single-nucleotide transition that changes alanine 171 to threonine (A171T) of the NAE subunit UBA3. This reduces the enzyme's affinity for MLN4924 and ATP while increasing NEDD8 activation at physiological ATP concentrations. Expression of UBA3 A171T is sufficient to decrease MLN4924 sensitivity of naive HCT116 cells, indicating that it is a dominant suppressor of MLN4924-mediated cell death. Our data suggest that the on-target potency of MLN4924 selects for a point mutation in NAE that overcomes the molecule's inhibitory effects, allowing cancer cell survival.
|An evaluation of oligonucleotide-based therapeutic strategies for polyQ diseases. |
Fiszer, A; Olejniczak, M; Switonski, PM; Wroblewska, JP; Wisniewska-Kruk, J; Mykowska, A; Krzyzosiak, WJ
BMC molecular biology 13 6 2012
RNA interference (RNAi) and antisense strategies provide experimental therapeutic agents for numerous diseases, including polyglutamine (polyQ) disorders caused by CAG repeat expansion. We compared the potential of different oligonucleotide-based strategies for silencing the genes responsible for several polyQ diseases, including Huntington's disease and two spinocerebellar ataxias, type 1 and type 3. The strategies included nonallele-selective gene silencing, gene replacement, allele-selective SNP targeting and CAG repeat targeting.Using the patient-derived cell culture models of polyQ diseases, we tested various siRNAs, and antisense reagents and assessed their silencing efficiency and allele selectivity. We showed considerable allele discrimination by several SNP targeting siRNAs based on a weak G-G or G-U pairing with normal allele and strong G-C pairing with mutant allele at the site of RISC-induced cleavage. Among the CAG repeat targeting reagents the strongest allele discrimination is achieved by miRNA-like functioning reagents that bind to their targets and inhibit their translation without substantial target cleavage. Also, morpholino analog performs well in mutant and normal allele discrimination but its efficient delivery to cells at low effective concentration still remains a challenge.Using three cellular models of polyQ diseases and the same experimental setup we directly compared the performance of different oligonucleotide-based treatment strategies that are currently under development. Based on the results obtained by us and others we discussed the advantages and drawbacks of these strategies considering them from several different perspectives. The strategy aimed at nonallele-selective inhibiting of causative gene expression by targeting specific sequence of the implicated gene is the easiest to implement but relevant benefits are still uncertain. The gene replacement strategy that combines the nonallele-selective gene silencing with the expression of the exogenous normal allele is a logical extension of the former and it deserves to be explored further. Both allele-selective RNAi approaches challenge cellular RNA interference machinery to show its ability to discriminate between similar sequences differing in either single base substitutions or repeated sequence length. Although both approaches perform well in allele discrimination most of our efforts are focused on repeat targeting due to its potentially higher universality.
|Cyclic mechanical strain maintains Nanog expression through PI3K/Akt signaling in mouse embryonic stem cells. |
Rie Horiuchi,Takayuki Akimoto,Zhang Hong,Takashi Ushida
Experimental cell research 318 2012
Mechanical strain has been reported to affect the proliferation/differentiation of many cell types; however, the effects of mechanotransduction on self-renewal as well as pluripotency of embryonic stem (ES) cells remains unknown. To investigate the effects of mechanical strain on mouse ES cell fate, we examined the expression of Nanog, which is an essential regulator of self-renewal and pluripotency as well as Nanog-associated intracellular signaling during uniaxial cyclic mechanical strain. The mouse ES cell line, CCE was plated onto elastic membranes, and we applied 10% strain at 0.17Hz. The expression of Nanog was reduced during ES cell differentiation in response to the withdrawal of leukemia inhibitory factor (LIF); however, two days of cyclic mechanical strain attenuated this reduction of Nanog expression. On the other hand, the cyclic mechanical strain promoted PI3K-Akt signaling, which is reported as an upstream of Nanog transcription. The cyclic mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor wortmannin. Furthermore, cytochalasin D, an inhibitor of actin polymerization, also inhibited the mechanical strain-induced increase in phospho-Akt. These findings imply that mechanical force plays a role in regulating Nanog expression in ES cells through the actin cytoskeleton-PI3K-Akt signaling.
|MicroRNA-133a regulates DNA methylation in diabetic cardiomyocytes. |
Chavali, V; Tyagi, SC; Mishra, PK
Biochemical and biophysical research communications 425 668-72 2012
We tested the hypothesis that miR-133a regulates DNA methylation by inhibiting Dnmt-1 (maintenance) and Dnmt-3a and -3b (de novo) methyl transferases in diabetic hearts by using Ins2(+/-) Akita (diabetic) and C57BL/6J (WT), mice and HL1 cardiomyocytes. The specific role of miR-133a in DNA methylation in diabetes was assessed by two treatment groups (1) scrambled, miR-133a mimic, anti-miR-133a, and (2) 5mM glucose (CT), 25 mM glucose (HG) and HG+miR-133a mimic. The levels of miR-133a, Dnmt-1, -3a and -3b were measured by multiplex RT-PCR, qPCR and Western blotting. The results revealed that miR-133a is inhibited but Dnmt-1 and -3b are induced in Akita suggesting that attenuation of miR-133a induces both maintenance (Dnmt-1) - and de novo - methylation (Dnmt-3b) in diabetes. The up regulation of Dnmt-3a in Akita hearts elicits intricate and antagonizing interaction between Dnmt-3a and -3b. In cardiomyocytes, over expression of miR-133a inhibits but silencing of miR-133a induces Dnmt-1, -3a and -3b elucidating the involvement of miR-133a in regulation of DNA methylation. The HG treatment up regulates only Dnmt-1 and not Dnmt-3a and -3b suggesting that acute hyperglycemia triggers only maintenance methylation. The over expression of miR-133a mitigates glucose mediated induction of Dnmt-1 illustrating the role of miR-133a in regulation of DNA methylation in diabetes.
|Porphyromonas gingivalis mediates inflammasome repression in polymicrobial cultures through a novel mechanism involving reduced endocytosis. |
Taxman, DJ; Swanson, KV; Broglie, PM; Wen, H; Holley-Guthrie, E; Huang, MT; Callaway, JB; Eitas, TK; Duncan, JA; Ting, JP
The Journal of biological chemistry 287 32791-9 2012
The interleukin (IL)-1β-processing inflammasome has recently been identified as a target for pathogenic evasion of the inflammatory response by a number of bacteria and viruses. We postulated that the periodontal pathogen, Porphyromonas gingivalis may suppress the inflammasome as a mechanism for its low immunogenicity and pathogenic synergy with other, more highly immunogenic periodontal bacteria. Our results show that P. gingivalis lacks signaling capability for the activation of the inflammasome in mouse macrophages. Furthermore, P. gingivalis can suppress inflammasome activation by another periodontal bacterium, Fusobacterium nucleatum. This repression affects IL-1β processing, as well as other inflammasome-mediated processes, including IL-18 processing and cell death, in both human and mouse macrophages. F. nucleatum activates IL-1β processing through the Nlrp3 inflammasome; however, P. gingivalis repression is not mediated through reduced levels of inflammasome components. P. gingivalis can repress Nlrp3 inflammasome activation by Escherichia coli, and by danger-associated molecular patterns and pattern-associated molecular patterns that mediate activation through endocytosis. However, P. gingivalis does not suppress Nlrp3 inflammasome activation by ATP or nigericin. This suggests that P. gingivalis may preferentially suppress endocytic pathways toward inflammasome activation. To directly test whether P. gingivalis infection affects endocytosis, we assessed the uptake of fluorescent particles in the presence or absence of P. gingivalis. Our results show that P. gingivalis limits both the number of cells taking up beads and the number of beads taken up for bead-positive cells. These results provide a novel mechanism of pathogen-mediated inflammasome inhibition through the suppression of endocytosis.
|Mouse model of muscleblind-like 1 overexpression: skeletal muscle effects and therapeutic promise. |
Chamberlain, CM; Ranum, LP
Human molecular genetics 21 4645-54 2012
Myotonic dystrophy (DM) is a multisystemic disease caused by CTG or CCTG expansion mutations. There is strong evidence that DM1 CUG and DM2 CCUG expansion transcripts sequester muscleblind-like (MBNL) proteins and that loss of MBNL function causes alternative splicing abnormalities that contribute to disease. Because MBNL1 loss is thought to play an important role in disease and localized AAV delivery of MBNL1 partially rescues skeletal muscle pathology in DM mice, there is strong interest in MBNL1 overexpression as a therapeutic strategy. We developed the first transgenic MBNL1 overexpression mouse model (MBNL1-OE) to test the safety and efficacy of multisystemic MBNL1 overexpression. First, we demonstrate that MBNL1 overexpression is generally well-tolerated in skeletal muscle. Second, we show the surprising result that premature shifts in alternative splicing of MBNL1-regulated genes in multiple organ systems are compatible with life and do not cause embryonic lethality. Third, we show for the first time that early and long-term MBNL1 overexpression prevents CUG-induced myotonia, myopathy and alternative splicing abnormalities in DM1 mice. In summary, MBNL1 overexpression may be a valuable strategy for treating the skeletal muscle features of DM.
|Activity-dependent local translation of matrix metalloproteinase-9. |
Dziembowska, M; Milek, J; Janusz, A; Rejmak, E; Romanowska, E; Gorkiewicz, T; Tiron, A; Bramham, CR; Kaczmarek, L
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 14538-47 2012
Local, synaptic synthesis of new proteins in response to neuronal stimulation plays a key role in the regulation of synaptic morphogenesis. Recent studies indicate that matrix metalloproteinase-9 (MMP-9), an endopeptidase that regulates the pericellular environment through cleavage of its protein components, plays a critical role in regulation of spine morphology and synaptic plasticity. Here, we sought to determine whether MMP-9 mRNA is transported to dendrites for local translation and protein release. First, dendritic transport of MMP-9 mRNA was seen in primary hippocampal neuronal cultures treated with glutamate and in dentate gyrus granule cells in adult anesthetized rats after induction of long-term potentiation. Second, rapid, activity-dependent polyadenylation of MMP-9 mRNA; association of the mRNA with actively translating polysomes; and de novo MMP-9 protein synthesis were obtained in synaptoneurosomes isolated from rat hippocampus. Third, glutamate stimulation of cultured hippocampal neurons evoked a rapid (in minutes) increase in MMP-9 activity, as measured by cleavage of its native substrate, β-dystroglycan. This activity was reduced by the polyadenylation inhibitor, thus linking MMP-9 translation with protein function. In aggregate, our findings show that MMP-9 mRNA is transported to dendrites and locally translated and that the protein is released in an activity-dependent manner. Acting in concert with other dendritically synthesized proteins, locally secreted MMP-9 may contribute to the structural and functional plasticity of the activated synapses.
|Mouse tissues that undergo neoplastic progression after K-Ras activation are distinguished by nuclear translocation of phospho-Erk1/2 and robust tumor suppressor responses. |
Parikh, N; Shuck, RL; Nguyen, TA; Herron, A; Donehower, LA
Molecular cancer research : MCR 10 845-55 2012
Mutation of K-Ras is a frequent oncogenic event in human cancers, particularly cancers of lungs, pancreas, and colon. It remains unclear why some tissues are more susceptible to Ras-induced transformation than others. Here, we globally activated a mutant oncogenic K-Ras allele (K-Ras(G12D)) in mice and examined the tissue-specific effects of this activation on cancer pathobiology, Ras signaling, tumor suppressor, DNA damage, and inflammatory responses. Within 5 to 6 weeks of oncogenic Ras activation, mice develop oral and gastric papillomas, lung adenomas, and hematopoietic hyperproliferation and turn moribund. The oral, gastric, and lung premalignant lesions display activated extracellular signal-regulated kinases (Erk)1/2 and NF-κB signaling as well as activated tumor suppressor and DNA damage responses. Other organs such as pancreas, liver, and small intestine do not exhibit neoplastic progression within 6 weeks following K-Ras(G12D) activation and do not show a potent tumor suppressor response. Even though robust Erk1/2 signaling is activated in all the tissues examined, the pErk1/2 distribution remains largely cytoplasmic in K-Ras(G12D)-refractory tissues (pancreas, liver, and intestines) as opposed to a predominantly nuclear localization in K-Ras(G12D)-induced neoplasms of lung, oral, and gastric mucosa. The downstream targets of Ras signaling, pElk-1 and c-Myc, are elevated in K-Ras(G12D)-induced neoplastic lesions but not in K-Ras(G12D)-refractory tissues. We propose that oncogenic K-Ras-refractory tissues delay oncogenic progression by spatially limiting the efficacy of Ras/Raf/Erk1/2 signaling, whereas K-Ras-responsive tissues exhibit activated Ras/Raf/Erk1/2 signaling, rapidly form premalignant tumors, and activate potent antitumor responses that effectively prevent further malignant progression.
|Gene Silencing of the Mitochondrial Adaptor p66Shc Suppresses Vascular Hyperglycemic Memory in Diabetes. |
Paneni, Francesco, et al.
Circulation research, (2012) 2012
Rationale:Hyperglycemic memory may explain why intensive glucose control has failed to improve cardiovascular outcomes in patients with diabetes. Indeed, hyperglycemia promotes vascular dysfunction even after glucose normalization. However, the molecular mechanisms of this phenomenon remain to be elucidated.Objective:The present study investigated the role of mitochondrial adaptor p66(Shc) in this setting.Methods and Results:In human aortic endothelial cells (HAECs) exposed to high glucose and aortas of diabetic mice, activation of p66(Shc) by protein kinase C βII (PKCβII) persisted after returning to normoglycemia. Persistent p66(Shc) upregulation and mitochondrial translocation were associated with continued reactive oxygen species (ROS) production, reduced nitric oxide bioavailability, and apoptosis. We show that (Shc) gene overexpression was epigenetically regulated by promoter CpG hypomethylation and general control nonderepressible 5-induced histone 3 acetylation. Furthermore, p66(Shc)-derived ROS production maintained PKCβII upregulation and PKCβII-dependent inhibitory phosphorylation of endothelial nitric oxide synthase at Thr-495, leading to a detrimental vicious cycle despite restoration of normoglycemia. Moreover, p66(Shc) activation accounted for the persistent elevation of the advanced glycated end product precursor methylglyoxal. In vitro and in vivo gene silencing of p66(Shc), performed at the time of glucose normalization, blunted ROS production, restored endothelium-dependent vasorelaxation, and attenuated apoptosis by limiting cytochrome c release, caspase 3 activity, and cleavage of poly (ADP-ribose) polymerase.Conclusions:p66(Shc) is the key effector driving vascular hyperglycemic memory in diabetes. Our study provides molecular insights for the progression of diabetic vascular complications despite glycemic control and may help to define novel therapeutic targets.
|Regulation of nuclear factor κB (NF-κB) transcriptional activity via p65 acetylation by the chaperonin containing TCP1 (CCT). |
Pejanovic, N; Hochrainer, K; Liu, T; Aerne, BL; Soares, MP; Anrather, J
PloS one 7 e42020 2012
The NF-κB family member p65 is central to inflammation and immunity. The purpose of this study was to identify and characterize evolutionary conserved genes modulating p65 transcriptional activity. Using an RNAi screening approach, we identified chaperonin containing TCP1 subunit η (CCTη) as a regulator of Drosophila NF-κB proteins, Dorsal and Dorsal-related immunity factor (Dif). CCTη was also found to regulate NF-κB-driven transcription in mammalian cells, acting in a promoter-specific context, downstream of IκB kinase (IKK). CCTη knockdown repressed IκBα and CXCL2/MIP2 transcription during the early phase of NF-κB activation while impairing the termination of CCL5/RANTES and CXCL10/IP10 transcription. The latter effect was associated with increased DNA binding and reduced p65 acetylation, presumably by altering the activity of histone acetyltransferase CREB-binding protein (CBP). We identified p65 lysines (K) 122 and 123 as target residues mediating the CCTη-driven termination of NF-κB-dependent transcription. We propose that CCTη regulates NF-κB activity in a manner that resolves inflammation.
|Immunohistochemical detection of glypican-5 in paraffin-embedded material: an optimized method for a novel research antibody. |
Khin Thway,Joanna Selfe,Janet Shipley
Applied immunohistochemistry & molecular morphology : AIMM / official publication of the Society for Applied Immunohistochemistry 20 2012
Glypican-5 (GPC5) is a cell surface heparan sulfate proteoglycan and 1 of 6 closely related members of the glypican family in mammals. Glypicans are predominantly expressed during development in cell-specific and tissue-specific contexts, and the expression of some is linked to developmental disorders and several visceral malignancies. We have previously shown that the region of amplification at 13q31.3 in a subset of rhabdomyosarcomas contains the GPC5 locus, and by copy number and gene expression analyses, that GPC5 is consistently expressed and upregulated in amplified tumors. As the immunohistochemical profile of GPC5 is untested, our aim was to optimize a commercially available anti-human GPC5 antibody for immunohistochemical use in formalin-fixed and paraffin-embedded (FFPE) tissue. Quantitative real-time polymerase chain reaction analyses of normal tissue samples indicated that the brain and testis highly expressed GPC5. High protein expression in these tissues and a cell line constructed to overexpress GPC5 were demonstrated by Western blotting. These normal tissues and the isogenic cell line were FFPE, and immunohistochemical expression of GPC5 was assessed using different methods of antigen retrieval, detection, and primary antibody concentration. The optimum conditions for detection were by heat-induced antigen retrieval, in sodium citrate buffer at pH 6. Enzyme-mediated retrieval did not produce effective detection, producing weaker, less well-localized GPC5 expression. We demonstrate that anti-human GPC5 antibody is amenable to use in FFPE tissue and with the optimized protocol we describe shows specific cellular localization and good staining intensity with minimal background staining.
|Proteomic and protein interaction network analysis of human T lymphocytes during cell-cycle entry. |
Stephen J Orr,Daniel R Boutz,Rong Wang,Constantinos Chronis,Nicholas C Lea,Thivyan Thayaparan,Emma Hamilton,Hanna Milewicz,Eric Blanc,Ghulam J Mufti,Edward M Marcotte,N Shaun B Thomas
Molecular systems biology 8 2012
Regulating the transition of cells such as T lymphocytes from quiescence (G(0)) into an activated, proliferating state involves initiation of cellular programs resulting in entry into the cell cycle (proliferation), the growth cycle (blastogenesis, cell size) and effector (functional) activation. We show the first proteomic analysis of protein interaction networks activated during entry into the first cell cycle from G(0). We also provide proof of principle that blastogenesis and proliferation programs are separable in primary human T cells. We employed a proteomic profiling method to identify large-scale changes in chromatin/nuclear matrix-bound and unbound proteins in human T lymphocytes during the transition from G(0) into the first cell cycle and mapped them to form functionally annotated, dynamic protein interaction networks. Inhibiting the induction of two proteins involved in two of the most significantly upregulated cellular processes, ribosome biogenesis (eIF6) and hnRNA splicing (SF3B2/SF3B4), showed, respectively, that human T cells can enter the cell cycle without growing in size, or increase in size without entering the cell cycle.
|Smad2 Decelerates Re-epithelialization during Gingival Wound Healing. |
K Tomikawa,T Yamamoto,N Shiomi,M Shimoe,S Hongo,K Yamashiro,T Yamaguchi,H Maeda,S Takashiba
Journal of dental research 91 2012
During periodontal regeneration, inhibition of gingival downgrowth is necessary to promote migration of mesenchymal cells into the defects. Transforming growth factor (TGF)-β is a pleiotropic cytokine that has numerous cell functions, including regulation of epithelial growth. Recent studies have shown that Smad2, a downstream transcription factor of TGF-β, plays crucial roles in wound healing in the epithelia. Therefore, we investigated the effects of Smad2 overexpression on re-epithelialization of gingival wounds. Transgenic mice overexpressing smad2 driven by the keratin 14 promoter (k14-smad2) were confirmed to have significant Smad2 phosphorylation in gingival basal epithelia. Punch wounds were made in the palatal gingiva, and wound healing was assessed histologically for 7 days. Re-epithelialization was significantly retarded on day 2, while collagen deposition was enhanced on day 7 in k14-smad2 compared with wild-type mice. Moreover, expression of keratin 16 (K16), an indicator of keratinocyte migration, was significantly inhibited in wound-edge keratinocytes in k14-smad2. The inhibition of K16 coincided with the induction of Smad2 in the corresponding epithelia, while BrdU incorporation was unaffected. These results indicated that Smad2 has inhibitory effects in regulating keratinocyte migration during gingival wound healing. TGF-β/Smad2 signaling mediating alteration of K16 expression must be tightly regulated during periodontal regeneration.
|Imaging immune and metabolic cells of visceral adipose tissues with multimodal nonlinear optical microscopy. |
Urasaki, Y; Johlfs, MG; Fiscus, RR; Le, TT
PloS one 7 e38418 2012
Visceral adipose tissue (VAT) inflammation is recognized as a mechanism by which obesity is associated with metabolic diseases. The communication between adipose tissue macrophages (ATMs) and adipocytes is important to understanding the interaction between immunity and energy metabolism and its roles in obesity-induced diseases. Yet visualizing adipocytes and macrophages in complex tissues is challenging to standard imaging methods. Here, we describe the use of a multimodal nonlinear optical (NLO) microscope to characterize the composition of VATs of lean and obese mice including adipocytes, macrophages, and collagen fibrils in a label-free manner. We show that lipid metabolism processes such as lipid droplet formation, lipid droplet microvesiculation, and free fatty acids trafficking can be dynamically monitored in macrophages and adipocytes. With its versatility, NLO microscopy should be a powerful imaging tool to complement molecular characterization of the immunity-metabolism interface.
|Humanized c-Myc mouse. |
Lehmann, FM; Feicht, S; Helm, F; Maurberger, A; Ladinig, C; Zimber-Strobl, U; Kühn, R; Mautner, J; Gerbitz, A; Bornkamm, GW
PloS one 7 e42021 2012
A given tumor is usually dependent on the oncogene that is activated in the respective tumor entity. This phenomenon called oncogene addiction provides the rationale for attempts to target oncogene products in a therapeutic manner, be it by small molecules, by small interfering RNAs (siRNA) or by antigen-specific T cells. As the proto-oncogene product is required also for the function of normal cells, this raises the question whether there is a therapeutic window between the adverse effects of specific inhibitors or T cells to normal tissue that may limit their application, and their beneficial tumor-specific therapeutic action. To address this crucial question, suitable mouse strains need to be developed, that enable expression of the human proto-oncogene not only in tumor but also in normal cells. The aim of this work is to provide such a mouse strain for the human proto-oncogene product c-MYC.We generated C57BL/6-derived embryonic stem cells that are transgenic for a humanized c-Myc gene and established a mouse strain (hc-Myc) that expresses human c-MYC instead of the murine ortholog. These transgenic animals harbor the humanized c-Myc gene integrated into the endogenous murine c-Myc locus. Despite the lack of the endogenous murine c-Myc gene, homozygous mice show a normal phenotype indicating that human c-MYC can replace its murine ortholog.The newly established hc-Myc mouse strain provides a model system to study in detail the adverse effects of therapies that target the human c-MYC protein. To mimic the clinical situation, hc-Myc mice may be cross-bred to mice that develop tumors due to overexpression of human c-MYC. With these double transgenic mice it will be possible to study simultaneously the therapeutic efficiency and adverse side effects of MYC-specific therapies in the same mouse.
|Effects of an early experience of reward through maternal contact or its denial on laterality of protein expression in the developing rat hippocampus. |
Raftogianni, A; Stamatakis, A; Papadopoulou, A; Vougas, K; Anagnostopoulos, AK; Stylianopoulou, F; Tsangaris, GT
PloS one 7 e48337 2012
Laterality is a basic characteristic of the brain which is detectable early in life. Although early experiences affect laterality of the mature brain, there are no reports on their immediate neurochemical effects during neonatal life, which could provide evidence as to the mechanisms leading to the lateralized brain. In order to address this issue, we determined the differential protein expression profile of the left and right hippocampus of 13-day-old rat control (CTR) pups, as well as following exposure to an early experience involving either receipt (RER) or denial (DER) of the expected reward of maternal contact. Proteomic analysis was performed by 2-dimensional polyacrylamide gel electrophoresis (PAGE) followed by mass spectroscopy. The majority of proteins found to be differentially expressed either between the three experimental groups (DER, RER, CTR) or between the left and right hemisphere were cytoskeletal (34%), enzymes of energy metabolism (32%), and heat shock proteins (17%). In all three groups more proteins were up-regulated in the left compared to the right hippocampus. Tubulins were found to be most often up-regulated, always in the left hippocampus. The differential expression of β-tubulin, β-actin, dihydropyrimidinase like protein 1, glial fibrillary acidic protein (GFAP) and Heat Shock protein 70 revealed by the proteomic analysis was in general confirmed by Western blots. Exposure to the early experience affected brain asymmetry: In the RER pups the ratio of proteins up-regulated in the left hippocampus to those in the right was 1.8, while the respective ratio was 3.6 in the CTR and 3.4 in the DER. Our results could contribute to the elucidation of the cellular mechanisms mediating the effects of early experiences on the vulnerability for psychopathology, since proteins shown in our study to be differentially expressed (e.g. tubulins, dihydropyrimidinase like proteins, 14-3-3 protein, GFAP, ATP synthase, α-internexin) have also been identified in proteomic analyses of post-mortem brains from psychiatric patients.
|Classical macrophage activation up-regulates several matrix metalloproteinases through mitogen activated protein kinases and nuclear factor-κB. |
Huang, WC; Sala-Newby, GB; Susana, A; Johnson, JL; Newby, AC
PloS one 7 e42507 2012
Remodelling of the extracellular matrix (ECM) and cell surface by matrix metalloproteinases (MMPs) is an important function of monocytes and macrophages. Recent work has emphasised the diverse roles of classically and alternatively activated macrophages but the consequent regulation of MMPs and their inhibitors has not been studied comprehensively. Classical activation of macrophages derived in vitro from un-fractionated CD16(+/-) or negatively-selected CD16(-) macrophages up-regulated MMP-1, -3, -7, -10, -12, -14 and -25 and decreased TIMP-3 steady-state mRNA levels. Bacterial lipopolysaccharide, IL-1 and TNFα were more effective than interferonγ except for the effects on MMP-25, and TIMP-3. By contrast, alternative activation decreased MMP-2, -8 and -19 but increased MMP -11, -12, -25 and TIMP-3 steady-state mRNA levels. Up-regulation of MMPs during classical activation depended on mitogen activated protein kinases, phosphoinositide-3-kinase and inhibitor of κB kinase-2. Effects of interferonγ depended on janus kinase-2. Where investigated, similar effects were seen on protein concentrations and collagenase activity. Moreover, activity of MMP-1 and -10 co-localised with markers of classical activation in human atherosclerotic plaques in vivo. In conclusion, classical macrophage activation selectively up-regulates several MMPs in vitro and in vivo and down-regulates TIMP-3, whereas alternative activation up-regulates a distinct group of MMPs and TIMP-3. The signalling pathways defined here suggest targets for selective modulation of MMP activity.
|HIV protease inhibitors do not cause the accumulation of prelamin A in PBMCs from patients receiving first line therapy: the ANRS EP45 "aging" study. |
Perrin, S; Cremer, J; Faucher, O; Reynes, J; Dellamonica, P; Micallef, J; Solas, C; Lacarelle, B; Stretti, C; Kaspi, E; Robaglia-Schlupp, A; Nicolino-Brunet, C; Tamalet, CN; Tamalet, C; Lévy, N; Poizot-Martin, I; Cau, P; Roll, P
PloS one 7 e53035 2012
The ANRS EP45 "Aging" study investigates the cellular mechanisms involved in the accelerated aging of HIV-1 infected and treated patients. The present report focuses on lamin A processing, a pathway known to be altered in systemic genetic progeroid syndromes.35 HIV-1 infected patients being treated with first line antiretroviral therapy (ART, mean duration at inclusion: 2.7±1.3 years) containing boosted protease inhibitors (PI/r) (comprising lopinavir/ritonavir in 65% of patients) were recruited together with 49 seronegative age- and sex-matched control subjects (http://clinicaltrials.gov/, NCT01038999). In more than 88% of patients, the viral load was less than 40 copies/ml and the CD4+ cell count was greater than 500/mm³. Prelamin A processing in peripheral blood mononuclear cells (PBMCs) from patients and controls was analysed by western blotting at inclusion. PBMCs from patients were also investigated at 12 and 24 months after enrolment in the study. PBMCs from healthy controls were also incubated with boosted lopinavir in culture medium containing various concentrations of proteins (4 to 80 g/L).Lamin A precursor was not observed in cohort patient PBMC regardless of the PI/r used, the dose and the plasma concentration. Prelamin A was detected in PBMC incubated in culture medium containing a low protein concentration (4 g/L) but not in plasma (60-80 g/L) or in medium supplemented with BSA (40 g/L), both of which contain a high protein concentration.Prelamin A processing abnormalities were not observed in PBMCs from patients under the PI/r first line regimen. Therefore, PI/r do not appear to contribute to lamin A-related aging in PBMCs. In cultured PBMCs from healthy donors, prelamin A processing abnormalities were only observed when the protein concentration in the culture medium was low, thus increasing the amount of PI available to enter cells. ClinicalTrials.gov NCT01038999 http://clinicaltrials.gov/ct2/show/NCT01038999.
|Malat1 is not an essential component of nuclear speckles in mice. |
Nakagawa, S; Ip, JY; Shioi, G; Tripathi, V; Zong, X; Hirose, T; Prasanth, KV
RNA (New York, N.Y.) 18 1487-99 2012
Malat1 is an abundant long, noncoding RNA that localizes to nuclear bodies known as nuclear speckles, which contain a distinct set of pre-mRNA processing factors. Previous studies in cell culture have demonstrated that Malat1 interacts with pre-mRNA splicing factors, including the serine- and arginine-rich (SR) family of proteins, and regulates a variety of biological processes, including cancer cell migration, synapse formation, cell cycle progression, and responses to serum stimulation. To address the physiological function of Malat1 in a living organism, we generated Malat1-knockout (KO) mice using homologous recombination. Unexpectedly, the Malat1-KO mice were viable and fertile, showing no apparent phenotypes. Nuclear speckle markers were also correctly localized in cells that lacked Malat1. However, the cellular levels of another long, noncoding RNA--Neat1--which is an architectural component of nuclear bodies known as paraspeckles, were down-regulated in a particular set of tissues and cells lacking Malat1. We propose that Malat1 is not essential in living mice maintained under normal laboratory conditions and that its function becomes apparent only in specific cell types and under particular conditions.
|NKCC1 upregulation disrupts chloride homeostasis in the hypothalamus and increases neuronal activity-sympathetic drive in hypertension. |
Ye, ZY; Li, DP; Byun, HS; Li, L; Pan, HL
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 8560-8 2012
Hypertension is a major risk factor for coronary artery disease, stroke, and kidney failure. However, the etiology of hypertension in most patients is poorly understood. Increased sympathetic drive emanating from the hypothalamic paraventricular nucleus (PVN) plays a major role in the development of hypertension. Na(+)-K(+)-2Cl(-) cotransporter-1 (NKCC1) in the brain is critically involved in maintaining chloride homeostasis and in neuronal responses mediated by GABA(A) receptors. Here we present novel evidence that the GABA reversal potential (E(GABA)) of PVN presympathetic neurons undergoes a depolarizing shift that diminishes GABA inhibition in spontaneously hypertensive rats (SHRs). Inhibition of NKCC1, but not KCC2, normalizes E(GABA) and restores GABA inhibition of PVN neurons in SHRs. The mRNA and protein levels of NKCC1, but not KCC2, in the PVN are significantly increased in SHRs, and the NKCC1 proteins on the plasma membrane are highly glycosylated. Inhibiting NKCC1 N-glycosylation restores E(GABA) and GABAergic inhibition of PVN presympathetic neurons in SHRs. Furthermore, NKCC1 inhibition significantly reduces the sympathetic vasomotor tone and augments the sympathoinhibitory responses to GABA(A) receptor activation in the PVN in SHRs. These findings suggest that increased NKCC1 activity and glycosylation disrupt chloride homeostasis and impair synaptic inhibition in the PVN to augment the sympathetic drive in hypertension. This information greatly improves our understanding of the pathogenesis of hypertension and helps to design better treatment strategies for neurogenic hypertension.
|Novel Insights into the Molecular Mechanism of Action of DNA Hypomethylating Agents: Role of Protein Kinase C δ in Decitabine-Induced Degradation of DNA Methyltransferase 1. |
Datta, J; Ghoshal, K; Motiwala, T; Jacob, ST
Genes & cancer 3 71-81 2012
We have previously demonstrated proteasomal degradation of DNMT1 in mammalian cells following treatment with several DNA hypomethylating agents. Here, we demonstrate dose-dependent degradation of Dnmt1 in mouse embryonic stem (ES) cells expressing catalytic site mutant (cys-ser), confirming that the covalent bond formation between Dnmt1 and decitabine-incorporated DNA is not essential for this process. DNMT1o, the oocyte-specific isoform that lacks the N-terminal 118-amino acid domain, did not undergo decitabine-mediated degradation, which further proves the requirement of multiple domains including nuclear localization signal, KEN box, and BAH domains for this process. Analysis of glycerol density gradient fractions of micrococcal nuclease-digested nuclei showed that both nucleosomal and nucleoplasmic DNMT1 are degraded upon decitabine treatment. Among different inhibitors tested, the inhibitors of the proteasomal pathway and several protein kinases impeded decitabine-induced DNMT1 degradation. The maximal effect caused by inhibiting protein kinase C (PKC) persuaded us to investigate further its role in decitabine-mediated DNMT1 degradation. Blockage of the degradation process after treatment with rottlerin, an inhibitor of PKCδ, or after siRNA-mediated depletion of PKCδ, indicated that this protein kinase is involved in decitabine-mediated depletion of DNMT1. PKCδ interacted with and phosphorylated DNMT1 in vitro. Moreover, rottlerin inhibited both basal and decitabine-induced phosphorylation of DNMT1. These studies provide substantial evidence that decitabine-induced degradation of the maintenance methyltransferase DNMT1 does not require covalent bond formation with the substrate and also elucidate its underlying molecular mechanism.
|Interactions between inflammatory signals and the progesterone receptor in regulating gene expression in pregnant human uterine myocytes. |
Yun Lee,Suren R Sooranna,Vasso Terzidou,Mark Christian,Jan Brosens,Kaisa Huhtinen,Matti Poutanen,Geraint Barton,Mark R Johnson,Phillip R Bennett
Journal of cellular and molecular medicine 16 2012
The absence of a fall in circulating progesterone levels has led to the concept that human labour is associated with 'functional progesterone withdrawal' caused through changes in the expression or function of progesterone receptor (PR). At the time of labour, the human uterus is heavily infiltrated with inflammatory cells, which release cytokines to create a 'myometrial inflammation' via NF-κB activation. The negative interaction between NF-κB and PR, may represent a mechanism to account for 'functional progesterone withdrawal' at term. Conversely, PR may act to inhibit NF-κB function and so play a role in inhibition of myometrial inflammation during pregnancy. To model this inter-relationship, we have used small interfering (si) RNA-mediated knock-down of PR in human pregnant myocytes and whole genome microarray analysis to identify genes regulated through PR. We then activated myometrial inflammation using IL-1β stimulation to determine the role of PR in myometrial inflammation regulation. Through PR-knock-down, we found that PR regulates gene networks involved in myometrial quiescence and extracellular matrix integrity. Activation of myometrial inflammation was found to antagonize PR-induced gene expression, of genes normally upregulated via PR. We found that PR does not play a role in repression of pro-inflammatory gene networks induced by IL-1β and that only MMP10 was significantly regulated in opposite directions by IL-1β and PR. We conclude that progesterone acting through PR does not generally inhibit myometrial inflammation. Activation of myometrial inflammation does cause 'functional progesterone withdrawal' but only in the context of genes normally upregulated via PR.
|High throughput screening for compounds that alter muscle cell glycosylation identifies new role for N-glycans in regulating sarcolemmal protein abundance and laminin binding. |
Cabrera, PV; Pang, M; Marshall, JL; Kung, R; Nelson, SF; Stalnaker, SH; Wells, L; Crosbie-Watson, RH; Baum, LG
The Journal of biological chemistry 287 22759-70 2012
Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function.
|Behavioral phenotype and BDNF differences related to apoE isoforms and sex in young transgenic mice. |
Ingrid Reverte,Anders Bue Klein,Cecilia Ratner,José L Domingo,Maria Teresa Colomina
Experimental neurology 237 2012
Human apolipoprotein E (apoE) plays an important role in lipid transport and distribution, being involved in neurite growth and neuroprotection in the brain. In humans, the apoE4 isoform is a risk factor for developing Azheimer's disease (AD), while apoE2 seems to provide neuroprotection. However, very little information is available on apoE2 genotype. In the present study, we have characterized behavioral and learning phenotypes in young transgenic mice apoE2, apoE3 and apoE4 of both sexes. We have also determined the levels of brain-derived neurotrophic factor (BDNF) and its receptor TrkB in cortex and hippocampus of male and female mice carrying either genotype. Our results show a worse performance of apoE4 and apoE2 mice in the acquisition of a spatial task compared to apoE3 mice, and a worse retention in apoE2 mice compared to the other two genotypes. On the other hand, an increase in the exploration of an open-field, which is compatible with a hyperactive behavior, was found in apoE2 females, while a decreased activity was observed in apoE4 mice. Increased BDNF levels in the frontal cortex were observed in apoE2 mice compared to apoE3. These results underscore behavioral differences between apoE genotypes in young mice, as well as the existence of interactions between genotype and gender, providing new valuable information on the apoE2 genotype.
|Vezatin is essential for dendritic spine morphogenesis and functional synaptic maturation. |
Danglot, L; Freret, T; Le Roux, N; Narboux Nême, N; Burgo, A; Hyenne, V; Roumier, A; Contremoulins, V; Dauphin, F; Bizot, JC; Vodjdani, G; Gaspar, P; Boulouard, M; Poncer, JC; Galli, T; Simmler, MC
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 9007-22 2012
Vezatin is an integral membrane protein associated with cell-cell adhesion complex and actin cytoskeleton. It is expressed in the developing and mature mammalian brain, but its neuronal function is unknown. Here, we show that Vezatin localizes in spines in mature mouse hippocampal neurons and codistributes with PSD95, a major scaffolding protein of the excitatory postsynaptic density. Forebrain-specific conditional ablation of Vezatin induced anxiety-like behavior and impaired cued fear-conditioning memory response. Vezatin knock-down in cultured hippocampal neurons and Vezatin conditional knock-out in mice led to a significantly increased proportion of stubby spines and a reduced proportion of mature dendritic spines. PSD95 remained tethered to presynaptic terminals in Vezatin-deficient hippocampal neurons, suggesting that the reduced expression of Vezatin does not compromise the maintenance of synaptic connections. Accordingly, neither the amplitude nor the frequency of miniature EPSCs was affected in Vezatin-deficient hippocampal neurons. However, the AMPA/NMDA ratio of evoked EPSCs was reduced, suggesting impaired functional maturation of excitatory synapses. These results suggest a role of Vezatin in dendritic spine morphogenesis and functional synaptic maturation.
|Disruption of RAB40AL function leads to Martin--Probst syndrome, a rare X-linked multisystem neurodevelopmental human disorder. |
Bedoyan, JK; Schaibley, VM; Peng, W; Bai, Y; Mondal, K; Shetty, AC; Durham, M; Micucci, JA; Dhiraaj, A; Skidmore, JM; Kaplan, JB; Skinner, C; Schwartz, CE; Antonellis, A; Zwick, ME; Cavalcoli, JD; Li, JZ; Martin, DM
Journal of medical genetics 49 332-40 2012
Martin--Probst syndrome (MPS) is a rare X-linked disorder characterised by deafness, cognitive impairment, short stature and distinct craniofacial dysmorphisms, among other features. The authors sought to identify the causative mutation for MPS.Massively parallel sequencing in two affected, related male subjects with MPS identified a RAB40AL (also called RLGP) missense mutation (chrX:102,079,078-102,079,079AC→GA p.D59G; hg18). RAB40AL encodes a small Ras-like GTPase protein with one suppressor of cytokine signalling box. The p.D59G variant is located in a highly conserved region of the GTPase domain between β-2 and β-3 strands. Using RT-PCR, the authors show that RAB40AL is expressed in human fetal and adult brain and kidney, and adult lung, heart, liver and skeletal muscle. RAB40AL appears to be a primate innovation, with no orthologues found in mouse, Xenopus or zebrafish. Western analysis and fluorescence microscopy of GFP-tagged RAB40AL constructs from transiently transfected COS7 cells show that the D59G missense change renders RAB40AL unstable and disrupts its cytoplasmic localisation.This is the first study to show that mutation of RAB40AL is associated with a human disorder. Identification of RAB40AL as the gene mutated in MPS allows for further investigations into the molecular mechanism(s) of RAB40AL and its roles in diverse processes such as cognition, hearing and skeletal development.
|An aberrant cerebellar development in mice lacking matrix metalloproteinase-3. |
Inge Van Hove,Mieke Verslegers,Tom Buyens,Nathalie Delorme,Kim Lemmens,Stijn Stroobants,Ilse Gantois,Rudi D'Hooge,Lieve Moons
Molecular neurobiology 45 2012
Cell-cell and cell-matrix interactions are necessary for neuronal patterning and brain wiring during development. Matrix metalloproteinases (MMPs) are proteolytic enzymes capable of remodelling the pericellular environment and regulating signaling pathways through cleavage of a large degradome. MMPs have been suggested to affect cerebellar development, but the specific role of different MMPs in cerebellar morphogenesis remains unclear. Here, we report a role for MMP-3 in the histogenesis of the mouse cerebellar cortex. MMP-3 expression peaks during the second week of postnatal cerebellar development and is most prominently observed in Purkinje cells (PCs). In MMP-3 deficient (MMP-3(-/-)) mice, a protracted granule cell (GC) tangential migration and a delayed GC radial migration results in a thicker and persistent external granular layer, a retarded arrival of GCs in the inner granular layer, and a delayed GABAergic interneuron migration. Importantly, these neuronal migration anomalies, as well as the consequent disturbed synaptogenesis on PCs, seem to be caused by an abnormal PC dendritogenesis, which results in reduced PC dendritic trees in the adult cerebellum. Of note, these developmental and adult cerebellar defects might contribute to the aberrant motor phenotype observed in MMP-3(-/-) mice and suggest an involvement of MMP-3 in mouse cerebellar development.
|Tumor suppressive microRNA-1285 regulates novel molecular targets: aberrant expression and functional significance in renal cell carcinoma. |
Hiedo Hidaka,Naohiko Seki,Hirofumi Yoshino,Takeshi Yamasaki,Yasutoshi Yamada,Nijiro Nohata,Miki Fuse,Masayuki Nakagawa,Hideki Enokida
Oncotarget 3 2012
MicroRNAs (miRNA) are non-coding RNAs, approximately 22 nucleotides in length, which function as post-transcriptional regulators. A large body of evidence indicates that miRNAs regulate the expression of cancer-related genes involved in proliferation, migration, invasion, and metastasis. The aim of this study was to identify novel cancer networks in renal cell carcinoma (RCC) based on miRNA expression signatures obtained from RCC clinical specimens. Expression signatures revealed that 103 miRNAs were significantly downregulated (more than 0.5-fold change) in RCC specimens. Functional screening (cell proliferation assays) was performed to identify tumor suppressive activities of 20 downregulated miRNAs. Restoration of mature miRNAs in cancer cells showed that 14 miRNAs (miR-1285, miR-206, miR-1, miR-135a, miR-429, miR-200c, miR-1291, miR-133b, miR-508-3p, miR-360-3p, miR-509-5p, miR-218, miR-335, miR-1255b and miR-1285) markedly inhibited cancer cell proliferation, suggesting that these miRNAs were candidate tumor suppressive miRNAs in RCC. We focused on miR-1285 because it significantly inhibited cancer cell proliferation, invasion, and migration following its transfection. We addressed miR-1285-regulated cancer networks by using genome-wide gene expression analysis and bioinformatics. The data showed that transglutaminase 2 (TGM2) was directly regulated by miR-1285. Silencing of the target gene demonstrated significant inhibition of cell proliferation and invasion in the RCC cells. Furthermore, immunohistochemistry showed that TGM2 expression levels in RCC specimens were significantly higher than those in normal renal tissues. Downregulation of tumor suppressive miR-1285, which targets oncogenic genes including TGM2, might contribute to RCC development. Thus, miR-1285 modulates a novel molecular target and provides new insights into potential mechanisms of RCC oncogenesis.
|The involvement of integrin β1 signaling in the migration and myofibroblastic differentiation of skin fibroblasts on anisotropic collagen-containing nanofibers. |
Huang, C; Fu, X; Liu, J; Qi, Y; Li, S; Wang, H
Biomaterials 33 1791-800 2012
Utilization of nanofibrous matrices for skin wound repair holds great promise due to their morphological and dimensional similarity to native extracellular matrix (ECM). It becomes highly desired to understand how various nanofibrous matrices regulate skin cell behaviors and intracellular signaling pathways, important to tuning the functionality of tissue-engineered skin grafts and affecting the wound healing process. In this study, the phenotypic expressions of normal human dermal fibroblasts (NHDFs) on collagen-containing nanofibrous matrices with either isotropic (i.e., fibers collected randomly with no alignment) or anisotropic (i.e., fibers collected with alignment) fiber organizations were studied by immunostaining, migration assay and molecular analyses. Results showed that both nanofibrous matrices supported the attachment and growth of NHDFs similarly, while showing different cell morphology with distinct variation in focal adhesion formation and distribution. Anisotropic nanofibers significantly triggered the integrin β1 signaling pathway in NHDFs as evidenced by an increase of active integrin β1 (130 kD mature form) and phosphorylation of focal adhesion kinase (FAK) at Tyr-397. Anisotropic matrices also promoted the migration of NHDFs along the fibers, while neutralization of the integrin β1 activity abolished this promotion. Moreover, the fibroblast-to-myofibroblast differentiation was greatly enhanced for the NHDFs cultured on anisotropic nanofibrous matrices over a period of 48 h. Inhibition of cellular integrin β1 activity by neutralizing antibody eliminated this enhancement. These findings suggest the important role of integrin β1 signaling pathway in regulating the nanofiber-induced fibroblast phenotypic alteration and providing insightful understanding of the possible application of collagen-containing nanofibrous matrices for skin regeneration.
|ARNO regulates VEGF-dependent tissue responses by stabilizing endothelial VEGFR-2 surface expression. |
Mannell, HK; Pircher, J; Chaudhry, DI; Alig, SK; Koch, EG; Mettler, R; Pohl, U; Krötz, F
Cardiovascular research 93 111-9 2012
The vascular endothelial growth factor (VEGF) stimulates angiogenesis by induction of vessel permeability, proliferation, and migration of endothelial cells, an important process in ischaemic diseases. ADP-ribosylation factor (ARF) nucleotide-binding site opener (ARNO) (cytohesin-2) is a guanine exchange factor important for cellular signalling through ARF GTPases. However, a role for ARNO in VEGF-dependent endothelial processes has so far not been documented. Therefore, we investigated whether ARNO has a role in VEGF-dependent activation of endothelial cells and thus vessel permeability.ARNO expression was observed in endothelial cells in vitro by RT-PCR, western blotting, and immunofluorescence as well as ex vivo by immunohistochemical staining of mouse aorta. Treatment with the cytohesin inhibitor SecinH3 or with an ARNO siRNA prevented VEGF-dependent Akt activation, assessed by detection of phosphorylated Akt, and proliferation of endothelial cells in vitro, measured by methylthiazoletetrazolium (MTT) reduction. In addition, ARNO suppression reduced VEGF-induced permeability in vessels of the mouse (C57BL/6) cremaster muscle in vivo, as measured by extravasation of fluorescein isothiocyanate (FITC)-dextran. Moreover, ARNO knock-down accelerated ligand-induced reduction in vascular endothelial growth factor receptor-2 (VEGFR-2) surface expression, internalization, and degradation, as assessed by flow cytometry and western blotting, respectively.Our findings indicate an important and novel role for endothelial ARNO in VEGF-dependent initiation of angiogenesis by regulation of VEGFR-2 internalization in endothelial cells, resulting in the activation of the Akt pathway, vessel permeability, and ultimately endothelial proliferation. Thus, ARNO may be a new essential player in endothelial signalling and angiogenesis.
|BRCA2 protein deficiency exaggerates doxorubicin-induced cardiomyocyte apoptosis and cardiac failure. |
Singh, KK; Shukla, PC; Quan, A; Desjardins, JF; Lovren, F; Pan, Y; Garg, V; Gosal, S; Garg, A; Szmitko, PE; Schneider, MD; Parker, TG; Stanford, WL; Leong-Poi, H; Teoh, H; Al-Omran, M; Verma, S
The Journal of biological chemistry 287 6604-14 2012
The tumor suppressor breast cancer susceptibility gene 2 (BRCA2) plays an important role in the repair of DNA damage, and loss of BRCA2 predisposes carriers to breast and ovarian cancers. Doxorubicin (DOX) remains the cornerstone of chemotherapy in such individuals. However, it is often associated with cardiac failure, which once manifests carries a poor prognosis. Because BRCA2 regulates genome-wide stability and facilitates DNA damage repair, we hypothesized that loss of BRCA2 may increase susceptibility to DOX-induced cardiac failure. To this aim, we generated cardiomyocyte-specific BRCA2 knock-out (CM-BRCA2(-/-)) mice using the Cre-loxP technology and evaluated their basal and post-DOX treatment phenotypes. Although CM-BRCA2(-/-) mice exhibited no basal cardiac phenotype, DOX treatment resulted in markedly greater cardiac dysfunction and mortality in CM-BRCA2(-/-) mice compared with control mice. Apoptosis in left ventricular (LV) sections from CM-BRCA2(-/-) mice compared with that in corresponding sections from wild-type (WT) littermate controls was also significantly enhanced after DOX treatment. Microscopic examination of LV sections from DOX-treated CM-BRCA2(-/-) mice revealed a greater number of DNA double-stranded breaks and the absence of RAD51 focus formation, an essential marker of double-stranded break repair. The levels of p53 and the p53-related proapoptotic proteins p53-up-regulated modulator of apoptosis (PUMA) and Bax were significantly increased in samples from CM-BRCA2(-/-) mice. This corresponded with increased Bax to Bcl-2 ratios and elevated cytochrome c release in the LV sections of DOX-treated CM-BRCA2(-/-) mice. Taken together, these data suggest a critical and previously unrecognized role of BRCA2 as a gatekeeper of DOX-induced cardiomyocyte apoptosis and susceptibility to overt cardiac failure. Pharmacogenomic studies evaluating cardiac function in BRCA2 mutation carriers treated with doxorubicin are encouraged.
|Glomerular MYH9 expression is reduced by HIV-1. |
Hays, T; D'Agati, VD; Garellek, JA; Warren, T; Trubin, ME; Hyink, DP; He, JC; Klotman, PE
AIDS (London, England) 26 797-803 2012
The continuing disease burden of HIV-associated nephropathy (HIVAN) warrants better elucidation of its pathogenic mechanisms. Given that loss of MYH9 function causes a Mendelian renal disease, we hypothesized that renal expression of MYH9 is down-regulated by HIV-1 in HIVAN pathogenesis.Using immunofluorescence, we determined that glomerular expression of MYH9 was reduced in the kidneys of HIV-1 transgenic mice. We further determined that Myh9 expression was reduced in HIV-1 transgenic podocytes, statistically significantly at the protein level, and that MYH9 expression was significantly reduced at protein and message level in human podocytes transduced with HIV-1. In analyzing expression in human tissue, we confirmed that MYH9 is abundantly expressed in glomeruli, and podocytes specifically. Finally, we found that MYH9 expression was significantly reduced in human glomeruli in the setting of HIVAN.We conclude that the podocyte host response to HIV-1 includes down-regulation of MYH9 expression, and hypothesize that this down-regulation might play a role in the pathogenesis of HIVAN.
|Functional and molecular evidence of myelin- and neuroprotection by thyroid hormone administration in experimental allergic encephalomyelitis. |
M L Dell'Acqua,L Lorenzini,G D'Intino,S Sivilia,P Pasqualetti,V Panetta,M Paradisi,M M Filippi,C Baiguera,M Pizzi,L Giardino,P M Rossini,L Calzà
Neuropathology and applied neurobiology 38 2012
Recent data in mouse and rat demyelination models indicate that administration of thyroid hormone (TH) has a positive effect on the demyelination/remyelination balance. As axonal pathology has been recognized as an early neuropathological event in multiple sclerosis, and remyelination is considered a pre-eminent neuroprotective strategy, in this study we investigated whether TH administration improves nerve impulse propagation and protects axons.
|An essential requirement for β1 integrin in the assembly of extracellular matrix proteins within the vascular wall. |
Turlo, KA; Noel, OD; Vora, R; LaRussa, M; Fassler, R; Hall-Glenn, F; Iruela-Arispe, ML
Developmental biology 365 23-35 2012
β1 integrin has been shown to contribute to vascular smooth muscle cell differentiation, adhesion and mechanosensation in vitro. Here we showed that deletion of β1 integrin at the onset of smooth muscle differentiation resulted in interrupted aortic arch, aneurysms and failure to assemble extracellular matrix proteins. These defects result in lethality prior to birth. Our data indicates that β1 integrin is not required for the acquisition, but it is essential for the maintenance of the smooth muscle cell phenotype, as levels of critical smooth muscle proteins are gradually reduced in mutant mice. Furthermore, while deposition of extracellular matrix was not affected, its structure was disrupted. Interestingly, defects in extracellular matrix and vascular wall assembly, were restricted to the aortic arch and its branches, compromising the brachiocephalic and carotid arteries and to the exclusion of the descending aorta. Additional analysis of β1 integrin in the pharyngeal arch smooth muscle progenitors was performed using wnt1Cre. Neural crest cells deleted for β1 integrin were able to migrate to the pharyngeal arches and associate with endothelial lined arteries; but exhibited vascular remodeling defects and early lethality. This work demonstrates that β1 integrin is dispensable for migration and initiation of the smooth muscle differentiation program, however, it is essential for remodeling of the pharyngeal arch arteries and for the assembly of the vessel wall of their derivatives. It further establishes a critical role of β1 integrin in the protection against aneurysms that is particularly confined to the ascending aorta and its branches.
|Targeting human epidermal growth factor receptor 2 by a cell-penetrating peptide-affibody bioconjugate. |
Srinath Govindarajan,Jeyarajan Sivakumar,Prathyusha Garimidi,Nandini Rangaraj,Jerald M Kumar,Nalam M Rao,Vijaya Gopal
Biomaterials 33 2012
Cell-penetrating peptide (CPP)-based delivery systems represent a strategy that facilitates DNA import efficiently and non-specifically into cells. To introduce specificity, we devised an approach that combines a cell-penetrating peptide, TAT-Mu (TM) and a targeting ligand, an HER2 antibody mimetic-affibody (AF), designated as TMAF to deliver nucleic acids into the cells. In this study, we synthesized TMAF protein and its truncated versions, i.e. MAF and AF, by expressing the corresponding plasmids in Escherichia coli BL21(DE3)pLysS cells. Purified TMAF binds DNA efficiently and protects plasmid DNA from DNaseI action. Transfection of HER2+ breast cancer cell lines MDA-MB-453, SK-OV-3, SK-BR-3 and an ovarian cancer cell line with plasmid DNA pCMV?-gal, resulted in enhanced ?-galactosidase activity when compared to control MDA-MB-231 cells. Maximal activity observed in MDA-MB-453 cells at DNA:TMAF:Protamine sulphate (PS) corresponding to 1:8:2 charge ratios. Further the observed gene transfection was resistant to serum, sensitive to the presence of free AF and non-toxic. Variants of TMAF although non-toxic, were far less efficient indicating the effective role of the TAT and Mu domains. The observed DNA uptake and reporter gene activity mediated by TMAFin vitro could be linked with the cell-surface density of tyrosine kinase receptor HER2 (ErbB2) levels estimated by Western blot. Further, we confirmed the efficacy of DNA transfer by TMAF protein in xenograft mouse models using MDA-MB-453 cells. Expression of ?-galactosidase as the reporter gene, upon intratumoral injection of DNA, in complex with TMAF, lends credence to specific DNA import and distribution within the tumor tissue that was attributed to high HER2 receptor overexpression in MDA-MB-453 cells. Through delivery of anti-TF hshRNA: TMAF: PS complex, we demonstrate specific knockdown of tissue factor (TF) in MDA-MB-453 cells in vitro. Most importantly, in a xenograft mouse model, we observe significant (P<0.05) and specific reduction of tumor volume when anti-TF hshRNA: TMAF: PS complex was injected intratumorally. Collectively our data indicate that AF-based chimeric peptides with nucleic acid binding properties may provide an effective tumor specific strategy to deliver therapeutic nucleic acids.
|The transcription factor Erg controls endothelial cell quiescence by repressing activity of nuclear factor (NF)-κB p65. |
Dryden, NH; Sperone, A; Martin-Almedina, S; Hannah, RL; Birdsey, GM; Khan, ST; Layhadi, JA; Mason, JC; Haskard, DO; Göttgens, B; Randi, AM
The Journal of biological chemistry 287 12331-42 2012
The interaction of transcription factors with specific DNA sequences is critical for activation of gene expression programs. In endothelial cells (EC), the transcription factor NF-κB is important in the switch from quiescence to activation, and is tightly controlled to avoid excessive inflammation and organ damage. Here we describe a novel mechanism that controls the activation of NF-κB in EC. The transcription factor Erg, the most highly expressed ETS member in resting EC, controls quiescence by repressing proinflammatory gene expression. Focusing on intercellular adhesion molecule 1(ICAM)-1 as a model, we identify two ETS binding sites (EBS -118 and -181) within the ICAM-1 promoter required for Erg-mediated repression. We show that Erg binds to both EBS -118 and EBS -181, the latter located within the NF-κB binding site. Interestingly, inhibition of Erg expression in quiescent EC results in increased NF-κB-dependent ICAM-1 expression, indicating that Erg represses basal NF-κB activity. Erg prevents NF-κB p65 from binding to the ICAM-1 promoter, suggesting a direct mechanism of interference. Gene set enrichment analysis of transcriptome profiles of Erg and NF-κB-dependent genes, together with chromatin immunoprecipitation (ChIP) studies, reveals that this mechanism is common to other proinflammatory genes, including cIAP-2 and IL-8. These results identify a role for Erg as a gatekeeper controlling vascular inflammation, thus providing an important barrier to protect against inappropriate endothelial activation.
|Methylseleninic acid enhances paclitaxel efficacy for the treatment of triple-negative breast cancer. |
Qi, Y; Fu, X; Xiong, Z; Zhang, H; Hill, SM; Rowan, BG; Dong, Y
PloS one 7 e31539 2012
A major challenge in breast cancer therapy is the lack of an effective therapeutic option for a particularly aggressive subtype of breast cancer, triple-negative breast cancer. Here we provide the first preclinical evidence that a second-generation selenium compound, methylseleninic acid, significantly enhances the anticancer efficacy of paclitaxel in triple-negative breast cancer. Through combination-index value calculation, we demonstrated that methylseleninic acid synergistically enhanced the growth inhibitory effect of paclitaxel in triple-negative breast cancer cells. The synergism was attributable to more pronounced induction of caspase-mediated apoptosis, arrest of cell cycle progression at the G2/M checkpoint, and inhibition of cell proliferation. Treatment of SCID mice bearing MDA-MB-231 triple-negative breast cancer xenografts for four weeks with methylseleninic acid (4.5 mg/kg/day, orally) and paclitaxel (10 mg/kg/week, through intraperitoneal injection) resulted in a more pronounced inhibition of tumor growth compared with either agent alone. The attenuated tumor growth correlated with a decrease in tumor cell proliferation and an induction of apoptosis. The in vivo study also indicated the safety of using methylseleninic acid in the combination regime. Our findings thus provide strong justification for the further development of methylseleninic acid and paclitaxel combination therapy for the treatment of triple-negative breast cancer.
|Osteogenic differentiation of stem cells alters vitamin d receptor expression. |
Rene Olivares-Navarrete,Ken Sutha,Sharon L Hyzy,Daphne L Hutton,Zvi Schwartz,Todd McDevitt,Barbara D Boyan
Stem cells and development 21 2012
Pluripotent and multipotent stem cells adopt an osteoblastic phenotype when cultured in environments that enhance their osteogenic potential. Embryonic stem cells differentiated as embryoid bodies (EBs) in osteogenic medium containing β-glycerophosphate exhibit increased expression of bone markers, indicating that cells are osteoblastic. Interestingly, 1α,25-dihydroxyvitaminD3 (1,25D) enhances the osteogenic phenotype not just in EBs but also in multipotent adult mesenchymal stem cells (MSCs). 1,25D acts on osteoblasts via classical vitamin D receptors (VDR) and via a membrane 1,25D-binding protein [protein disulfide isomerase family A, member 3 (PDIA3)], which activates protein kinase C -signaling. The aims of this study were to determine whether these receptors are regulated during osteogenic differentiation of stem cells and if stem cells and differentiated progeny are responsive to 1,25D. mRNA and protein levels for VDR, PDIA3, and osteoblast-associated proteins were measured in undifferentiated cells and in cells treated with osteogenic medium. Mouse EBs expressed both VDR and PDIA3, but VDR increased as cells underwent osteogenic differentiation. Human MSCs expressed Pdia3 at constant levels throughout differentiation, but VDR increased in cells treated with osteogenic medium. These results suggest that both 1,25D signaling mechanisms are important, with PDIA3 playing a greater role during early events and VDR playing a greater role in later stages of differentiation. Understanding these coordinated events provide a powerful tool to control pluripotent and multipotent stem cell differentiation through induction medium.
|p53, a novel regulator of lipid metabolism pathways. |
Ido Goldstein,Osnat Ezra,Noa Rivlin,Alina Molchadsky,Shalom Madar,Naomi Goldfinger,Varda Rotter
Journal of hepatology 56 2012
In this study we aimed at characterizing the regulation of hepatic metabolic pathways by the p53 transcription factor.
|Role of renal DJ-1 in the pathogenesis of hypertension associated with increased reactive oxygen species production. |
Cuevas, S; Zhang, Y; Yang, Y; Escano, C; Asico, L; Jones, JE; Armando, I; Jose, PA
Hypertension 59 446-52 2012
The D(2) dopamine receptor (D(2)R) is important in the pathogenesis of essential hypertension. We have already reported that systemic deletion of the D(2)R gene in mice results in reactive oxygen species (ROS)-dependent hypertension, suggesting that the D(2)R has antioxidant effects. However, the mechanism of this effect is unknown. DJ-1 is a protein that has antioxidant properties. D(2)R and DJ-1 are expressed in the mouse kidney and colocalize and coimunoprecipitate in mouse renal proximal tubule cells. We hypothesized that D(2)Rs regulate renal ROS production in the kidney through regulation of DJ-1 expression or function. Heterozygous D(2)(+/-) mice have increased blood pressure, urinary 8-isoprostanes, and renal Nox 4 expression, but decreased renal DJ-1 expression. Silencing D(2)R expression in mouse renal proximal tubule cells increases ROS production and decreases the expression of DJ-1. Conversely, treatment of these cells with a D(2)R agonist increases DJ-1 expression and decreases Nox 4 expression and NADPH oxidase activity, effects that are partially blocked by a D(2)R antagonist. Silencing DJ-1 expression in mouse renal proximal tubule cells increases ROS production and Nox 4 expression. Selective renal DJ-1 silencing by the subcapsular infusion of DJ-1 siRNA in mice increases blood pressure, renal Nox4 expression, and NADPH oxidase activity. These results suggest that the inhibitory effects of D(2)R on renal ROS production are at least, in part, mediated by a positive regulation of DJ-1 expression/function and that DJ-1 may have a role in the prevention of hypertension associated with increased ROS production.
|Mice lacking urea transporter UT-B display depression-like behavior. |
Xin Li,Jianhua Ran,Hong Zhou,Tianluo Lei,Li Zhou,Jingyan Han,Baoxue Yang
Journal of molecular neuroscience : MN 46 2012
Urea transporter B is one of urea transporters that selectively transport urea driven by urea gradient across membrane and expressed abundantly in brain. To determine the physiological role of UT-B in brain, UT-B localization, urea concentration, tissue morphology of brain, and behavioral phenotypes were studied in UT-B heterozygous mice via UT-B null mice. UT-B mRNA was expressed in olfactory bulb, cortex, caudate nucleus, hippocampus and hypothalamus of UT-B heterozygous mice. UT-B null mice exhibited depression-like behavior, with urea accumulation, nitric oxide reduction, and selective neuronal nitric oxide synthase level increase in hippocampus. After acute urea loading, the urea level increased, NO production decreased in hippocampus from both types of mice. Moreover, urea level was higher, and NO concentration was lower consistently in UT-B null hippocampus than that in heterozygous hippocampus. In vitro, 25 mM urea inhibited NO production too. Furthermore, UT-B knockout induced a long-lasting notable decrease in regional cerebral blood flow and altered morphology, such as loss of neurons in CA3 region, swelling, and membranous myelin-like structure formation within myelinated and unmyelinated fibers in hippocampus. These results suggest that urea accumulation in the hippocampus induced by UT-B deletion can cause depression-like behavior, which possibly attribute to disturbance in NOS/NO system.
|Carbonic anhydrase XIV in the normal and hypertrophic myocardium. |
Lorena A Vargas,Bernardo V Alvarez
Journal of molecular and cellular cardiology 52 2012
Two AE3 transcripts, full-length (AE3fl) and cardiac (AE3c) are expressed in the heart. AE3 catalyzes electroneutral Cl(-)/HCO(3)(-) exchange across cardiomyocyte sarcolemma. AE proteins associate with carbonic anhydrases (CA), including CAII and CAIV, forming a HCO(3)(-) transport metabolon (BTM), increasing HCO(3)(-) fluxes and regulating cardiomyocytes pH. CAXIV, which is also expressed in the heart's sarcolemma, is a transmembrane enzyme with an extracellular catalytic domain. Herein, AE3/CAXIV physical association was examined by coimmunoprecipitation using rodent heart lysates. CAXIV immunoprecipitated with anti-AE3 antibody and both AE3fl and AE3c were reciprocally immunoprecipitated using anti-CAXIV antibody, indicating AE3fl-AE3c/CAXIV interaction in the myocardium. Coimmunoprecipitation experiments on heart lysates from a mouse with targeted disruption of the ae3 gene, failed to pull down AE3 with the CAXIV antibody. Confocal images demonstrated colocalization of CAXIV and AE3 in mouse ventricular myocytes. Functional association of AE3fl and CAXIV was examined in isolated hypertrophic rat cardiomyocytes, using fluorescence measurements of BCECF to monitor cytosolic pH. Hypertrophic cardiomyocytes of spontaneously hypertensive rats (SHR) presented elevated myocardial AE-mediated Cl(-)/HCO(3)(-) exchange activity (J(HCO3-) mM.min(-1)) compared to normal (Wistar) rats (7.5±1.3, n=4 versus 2.9±0.1, n=6, respectively). AE3fl, AE3c, CAII, CAIV, and CAIX protein expressions were similar in SHR and Wistar rat hearts. However, immunoblots revealed a twofold increase of CAXIV protein expression in the SHR myocardium compared to normal hearts (n=11). Furthermore, the CA-inhibitor, benzolamide, neutralized the stimulatory effect of extracellular CA on AE3 transport activity (3.7±1.5, n=3), normalizing AE3-dependent HCO(3)(-) fluxes in SHR. CAXIV/AE3 interaction constitutes an extracellular component of a BTM which potentiates AE3-mediated HCO(3)(-) transport in the heart. Increased CAXIV expression and consequent AE3/CAXIV complex formation would render AE3 hyperactive in the SHR heart.
|RNA editing of the Q/R site of GluA2 in different cultured cell lines that constitutively express different levels of RNA editing enzyme ADAR2. |
Takenari Yamashita,Chieko Tadami,Yoshinori Nishimoto,Takuto Hideyama,Daisuke Kimura,Takeshi Suzuki,Shin Kwak
Neuroscience research 73 2012
Adenosine deaminase acting on RNA 2 (ADAR2) catalyzes RNA editing at the glutamine/arginine (Q/R) site of GluA2, and an ADAR2 deficiency may play a role in the death of motor neurons in ALS patients. The expression level of ADAR2 mRNA is a determinant of the editing activity at the GluA2 Q/R site in human brain but not in cultured cells. Therefore, we investigated the extent of Q/R site-editing in the GluA2 mRNA and pre-mRNA as well as the ADAR2 mRNA and GluA2 mRNA and pre-mRNA levels in various cultured cell lines. The extent of the GluA2 mRNA editing was 100% except in SH-SY5Y cells, which have a much lower level of ADAR2 than the other cell lines examined. The ADAR2 activity at the GluA2 pre-mRNA Q/R site correlated with the ADAR2 mRNA level relative to the GluA2 pre-mRNA. SH-SY5Y cells expressed higher level of the GluA2 mRNA in the cytoplasm compared with other cell lines. These results suggest that the ADAR2 expression level reflects editing activity at the GluA2 Q/R site and that although the edited GluA2 pre-mRNA is readily spliced, the unedited GluA2 pre-mRNA is also spliced and transported to the cytoplasm when ADAR2 expression is low.
|The role of notch 1 activation in cardiosphere derived cell differentiation. |
Chen, L; Ashraf, M; Wang, Y; Zhou, M; Zhang, J; Qin, G; Rubinstein, J; Weintraub, NL; Tang, Y
Stem cells and development 21 2122-9 2012
Cardiosphere derived cells (CDC) are present in the human heart and include heterogeneous cell populations of cardiac progenitor cells, multipotent progenitors that play critical roles in the physiological and pathological turnover of heart tissue. Little is known about the molecular pathways that control the differentiation of CDC. In this study, we examined the role of Notch 1/J kappa-recombining binding protein (RBPJ) signaling, a critical cell-fate decision pathway, in CDC differentiation. We isolated CDC from mouse cardiospheres and analyzed the differentiation of transduced cells expressing the Notch1 intracellular domain (N1-ICD), the active form of Notch1, using a terminal differentiation marker polymerase chain reaction (PCR) array. We found that Notch1 primarily supported the differentiation of CDC into smooth muscle cells (SMC), as demonstrated by the upreguation of key SMC proteins, including smooth muscle myosin heavy chain (Myh11) and SM22α (Tagln), in N1-ICD expressing CDC. Conversely, genetic ablation of RBPJ in CDC diminished the expression of SMC differentiation markers, confirming that SMC differentiation CDC is dependent on RBPJ. Finally, in vivo experiments demonstrate enhanced numbers of smooth muscle actin-expressing implanted cells after an injection of N1-ICD-expressing CDC into ischemic myocardium (44±8/high power field (hpf) vs. 11±4/high power field (hpf), n=7 sections, Pless than 0.05). Taken together, these results provide strong evidence that Notch1 promotes SMC differentiation of CDC through an RBPJ-dependent signaling pathway in vitro, which may have important implications for progenitor cell-mediated angiogenesis.
|Effect of acute administration of Pistacia lentiscus L. essential oil on rat cerebral cortex following transient bilateral common carotid artery occlusion. |
Quartu, Marina, et al.
Lipids Health Dis, 11: 8 (2012) 2012
Ischemia/reperfusion leads to inflammation and oxidative stress which damages membrane highly polyunsaturated fatty acids (HPUFAs) and eventually induces neuronal death. This study evaluates the effect of the administration of Pistacia lentiscus L. essential oil (E.O.), a mixture of terpenes and sesquiterpenes, on modifications of fatty acid profile and endocannabinoid (eCB) congener concentrations induced by transient bilateral common carotid artery occlusion (BCCAO) in the rat frontal cortex and plasma.
|Stability of reference proteins in human placenta: general protein stains are the benchmark. |
D Lanoix,J St-Pierre,A-A Lacasse,M Viau,J Lafond,C Vaillancourt,A A Lacasse
Placenta 33 2012
The stability of reference proteins in semi-quantitative Western blot experiments in normal and diseased placenta has never been studied. This study aims to determine the stability of five reference proteins and two general protein stains in placentas from preeclampsia, gestational diabetes mellitus and matched control pregnancies. The stability of the reference proteins was analysed using indicators of inter-group (P value) and intra-group (coefficient of variation) stability. The effect of different normalization strategies was determined by normalizing serotonin transporter (SERT) expression against the different reference protein markers. Results show significant expression variability of β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1), peptidylprolyl isomerase A (PPIA) and α-tubulin, and that amido black staining is the most stable reference protein marker. Furthermore, results show that SERT expression significantly differs according to the reference protein markers used for its normalization. The present study demonstrated the importance of using stable reference protein markers and normalization strategy in order to get correct results in semi-quantitative Western blot experiments in placental tissues.
|JMJD5, a Jumonji C (JmjC) domain-containing protein, negatively regulates osteoclastogenesis by facilitating NFATc1 protein degradation. |
Youn, MY; Yokoyama, A; Fujiyama-Nakamura, S; Ohtake, F; Minehata, K; Yasuda, H; Suzuki, T; Kato, S; Imai, Y
The Journal of biological chemistry 287 12994-3004 2012
Osteoclastogenesis is a highly regulated process governed by diverse classes of regulators. Among them, nuclear factor of activated T-cells calcineurin-dependent 1 (NFATc1) is the primary osteoclastogenic transcription factor, and its expression is transcriptionally induced during early osteoclastogenesis by receptor activation of nuclear factor κB ligand (RANKL), an osteoclastogenic cytokine. Here, we report the novel enzymatic function of JMJD5, which regulates NFATc1 protein stability. Among the tested Jumonji C (JmjC) domain-containing proteins, decreased mRNA expression levels during osteoclastogenesis were found for JMJD5 in RAW264 cells stimulated by RANKL. To examine the functional role of JMJD5 in osteoclast differentiation, we established stable JMJD5 knockdown cells, and osteoclast formation was assessed. Down-regulated expression of JMJD5 led to accelerated osteoclast formation together with induction of several osteoclast-specific genes such as Ctsk and DC-STAMP, suggesting that JMJD5 is a negative regulator in osteoclast differentiation. Although JMJD5 was recently reported as a histone demethylase for histone H3K36me2, no histone demethylase activity was detected in JMJD5 in vitro or in living cells, even for other methylated histone residues. Instead, JMJD5 co-repressed transcriptional activity by destabilizing NFATc1 protein. Protein hydroxylase activity mediated by the JmjC domain in JMJD5 was required for the observed functions of JMJD5. JMJD5 induced the association of hydroxylated NFATc1 with the E3 ubiquitin ligase Von Hippel-Lindau tumor suppressor (VHL), thereby presumably facilitating proteasomal degradation of NFATc1 via ubiquitination. Taken together, the present study demonstrated that JMJD5 is a post-translational co-repressor for NFATc1 that attenuates osteoclastogenesis.
|Consumption of oxygen: a mitochondrial-generated progression signal of advanced cancer. |
Cook, CC; Kim, A; Terao, S; Gotoh, A; Higuchi, M
Cell death & disease 3 e258 2012
Changes in mitochondrial genome such as mutation, deletion and depletion are common in cancer and can determine advanced phenotype of cancer; however, detailed mechanisms have not been elucidated. We observed that loss of mitochondrial genome reversibly induced overexpression and activation of proto-oncogenic Ras, especially K-Ras 4A, responsible for the activation of AKT and ERK leading to advanced phenotype of prostate and breast cancer. Ras activation was induced by the overexpression of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), the rate-limiting enzyme of the mevalonate pathway. Hypoxia is known to induce proteasomal degradation of HMGR. Well differentiated prostate and breast cancer cells with high mitochondrial DNA content consumed a large amount of oxygen and induced hypoxia. Loss of mitochondrial genome reduced oxygen consumption and increased in oxygen concentration in the cells. The hypoxic-to-normoxic shift led to the overexpression of HMGR through inhibiting proteasomal degradation. Therefore, reduction of mitochondrial genome content induced overexpression of HMGR through hypoxic to normoxic shift and subsequently the endogenous induction of the mevalonate pathway activated Ras that mediates advanced phenotype. Reduction of mitochondrial genome content was associated with the aggressive phenotype of prostate cancer in vitro cell line model and tissue specimens in vivo. Our results elucidate a coherent mechanism that directly links the mitochondrial genome with the advanced progression of the disease.
|Human immunodeficiency virus type 1 envelope proteins traffic toward virion assembly sites via a TBC1D20/Rab1-regulated pathway. |
Nachmias, D; Sklan, EH; Ehrlich, M; Bacharach, E
Retrovirology 9 7 2012
The cellular activity of many factors and pathways is required to execute the complex replication cycle of the human immunodeficiency virus type 1 (HIV-1). To reveal these cellular components, several extensive RNAi screens have been performed, listing numerous 'HIV-dependency factors'. However, only a small overlap between these lists exists, calling for further evaluation of the relevance of specific factors to HIV-1 replication and for the identification of additional cellular candidates. TBC1D20, the GTPase-activating protein (GAP) of Rab1, regulates endoplasmic reticulum (ER) to Golgi trafficking, was not identified in any of these screens, and its involvement in HIV-1 replication cycle is tested here.Excessive TBC1D20 activity perturbs the early trafficking of HIV-1 envelope protein through the secretory pathway. Overexpression of TBC1D20 hampered envelope processing and reduced its association with detergent-resistant membranes, entailing a reduction in infectivity of HIV-1 virion like particles (VLPs).These findings add TBC1D20 to the network of host factors regulating HIV replication cycle.
|The carboxyl-terminal region of erythroid-specific 5-aminolevulinate synthase acts as an intrinsic modifier for its catalytic activity and protein stability. |
Senkottuvelan Kadirvel,Kazumichi Furuyama,Hideo Harigae,Kiriko Kaneko,Yoshiko Tamai,Yoji Ishida,Shigeki Shibahara
Experimental hematology 40 2012
Erythroid-specific 5-aminolevulinate synthase (ALAS2) is essential for hemoglobin production, and a loss-of-function mutation of ALAS2 gene causes X-linked sideroblastic anemia. Human ALAS2 protein consists of 587 amino acids and its carboxyl(C)-terminal region of 33 amino acids is conserved in higher eukaryotes, but is not present in prokaryotic ALAS. We explored the role of this C-terminal region in the pathogenesis of X-linked sideroblastic anemia. In vitro enzymatic activity was measured using bacterially expressed recombinant proteins. In vivo catalytic activity was evaluated by comparing the accumulation of porphyrins in eukaryotic cells stably expressing each mutant ALAS2 tagged with FLAG, and the half-life of each FLAG-tagged ALAS2 protein was determined by Western blot analysis. Two novel mutations (Val562Ala and Met567Ile) were identified in patients with X-linked sideroblastic anemia. Val562Ala showed the higher catalytic activity in vitro, but a shorter half-life in vivo compared to those of wild-type ALAS2 (WT). In contrast, the in vitro activity of Met567Ile mutant was about 25% of WT, while its half-life was longer than that of WT. However, in vivo catalytic activity of each mutant was lower than that of WT. In addition, the deletion of 33 amino acids at C-terminal end resulted in higher catalytic activity both in vitro and in vivo with the longer half-life compared to WT. In conclusion, the C-terminal region of ALAS2 protein may function as an intrinsic modifier that suppresses catalytic activity and increases the degradation of its protein, each function of which is enhanced by the Met567Ile mutation and the Val562Ala mutation, respectively.
|Rap1 can bypass the FAK-Src-Paxillin cascade to induce cell spreading and focal adhesion formation. |
Ross, Sarah H, et al.
PLoS ONE, 7: e50072 (2012) 2012
We developed new image analysis tools to analyse quantitatively the extracellular-matrix-dependent cell spreading process imaged by live-cell epifluorescence microscopy. Using these tools, we investigated cell spreading induced by activation of the small GTPase, Rap1. After replating and initial adhesion, unstimulated cells exhibited extensive protrusion and retraction as their spread area increased, and displayed an angular shape that was remodelled over time. In contrast, activation of endogenous Rap1, via 007-mediated stimulation of Epac1, induced protrusion along the entire cell periphery, resulting in a rounder spread surface, an accelerated spreading rate and an increased spread area compared to control cells. Whereas basal, anisotropic, spreading was completely dependent on Src activity, Rap1-induced spreading was refractory to Src inhibition. Under Src inhibited conditions, the characteristic Src-induced tyrosine phosphorylations of FAK and paxillin did not occur, but Rap1 could induce the formation of actomyosin-connected adhesions, which contained vinculin at levels comparable to that found in unperturbed focal adhesions. From these results, we conclude that Rap1 can induce cell adhesion and stimulate an accelerated rate of cell spreading through mechanisms that bypass the canonical FAK-Src-Paxillin signalling cascade.
|Interleukin-1 participates in the classical and alternative activation of microglia/macrophages after spinal cord injury. |
Sato, A; Ohtaki, H; Tsumuraya, T; Song, D; Ohara, K; Asano, M; Iwakura, Y; Atsumi, T; Shioda, S
Journal of neuroinflammation 9 65 2012
Microglia and macrophages (MG/MΦ) have a diverse range of functions depending on unique cytokine stimuli, and contribute to neural cell death, repair, and remodeling during central nervous system diseases. While IL-1 has been shown to exacerbate inflammation, it has also been recognized to enhance neuroregeneration. We determined the activating phenotype of MG/MΦ and the impact of IL-1 in an in vivo spinal cord injury (SCI) model of IL-1 knock-out (KO) mice. Moreover, we demonstrated the contribution of IL-1 to both the classical and alternative activation of MG in vitro using an adult MG primary culture.SCI was induced by transection of the spinal cord between the T9 and T10 vertebra in wild-type and IL-1 KO mice. Locomotor activity was monitored and lesion size was determined for 14 days. TNFα and Ym1 levels were monitored to determine the MG/MΦ activating phenotype. Primary cultures of MG were produced from adult mice, and were exposed to IFNγ or IL-4 with and without IL-1β. Moreover, cultures were exposed to IL-4 and/or IL-13 in the presence and absence of IL-1β.The locomotor activity and lesion area of IL-1 KO mice improved significantly after SCI compared with wild-type mice. TNFα production was significantly suppressed in IL-1 KO mice. Also, Ym1, an alternative activating MG/MΦ marker, did not increase in IL-1 KO mice, suggesting that IL-1 contributes to both the classical and alternative activation of MG/MΦ. We treated primary MG cultures with IFNγ or IL-4 in the presence and absence of IL-1β. Increased nitric oxide and TNFα was present in the culture media and increased inducible NO synthase was detected in cell suspensions following co-treatment with IFNγ and IL-1β. Expression of the alternative activation markers Ym1 and arginase-1 was increased after exposure to IL-4 and further increased after co-treatment with IL-4 and IL-1β. The phenotype was not observed after exposure of cells to IL-13.We demonstrate here in in vivo experiments that IL-1 suppressed SCI in a process mediated by the reduction of inflammatory responses. Moreover, we suggest that IL-1 participates in both the classical and alternative activation of MG in in vivo and in vitro systems.
|N-methyl-D-aspartate receptor- and calpain-mediated proteolytic cleavage of K+-Cl- cotransporter-2 impairs spinal chloride homeostasis in neuropathic pain. |
Zhou, HY; Chen, SR; Byun, HS; Chen, H; Li, L; Han, HD; Lopez-Berestein, G; Sood, AK; Pan, HL
The Journal of biological chemistry 287 33853-64 2012
Loss of synaptic inhibition by γ-aminobutyric acid and glycine due to potassium chloride cotransporter-2 (KCC2) down-regulation in the spinal cord is a critical mechanism of synaptic plasticity in neuropathic pain. Here we present novel evidence that peripheral nerve injury diminishes glycine-mediated inhibition and induces a depolarizing shift in the reversal potential of glycine-mediated currents (E(glycine)) in spinal dorsal horn neurons. Blocking glutamate N-methyl-D-aspartate (NMDA) receptors normalizes synaptic inhibition, E(glycine), and KCC2 by nerve injury. Strikingly, nerve injury increases calcium-dependent calpain activity in the spinal cord that in turn causes KCC2 cleavage at the C terminus. Inhibiting calpain blocks KCC2 cleavage induced by nerve injury and NMDA, thereby normalizing E(glycine). Furthermore, calpain inhibition or silencing of μ-calpain at the spinal level reduces neuropathic pain. Thus, nerve injury promotes proteolytic cleavage of KCC2 through NMDA receptor-calpain activation, resulting in disruption of chloride homeostasis and diminished synaptic inhibition in the spinal cord. Targeting calpain may represent a new strategy for restoring KCC2 levels and tonic synaptic inhibition and for treating chronic neuropathic pain.
|The synaptic accumulation of hyperphosphorylated tau oligomers in Alzheimer disease is associated with dysfunction of the ubiquitin-proteasome system. |
Tai, HC; Serrano-Pozo, A; Hashimoto, T; Frosch, MP; Spires-Jones, TL; Hyman, BT
The American journal of pathology 181 1426-35 2012
In Alzheimer disease (AD), deposition of neurofibrillary tangles and loss of synapses in the neocortex and limbic system each correlate strongly with cognitive impairment. Tangles are composed of misfolded hyperphosphorylated tau proteins; however, the link between tau abnormalities and synaptic dysfunction remains unclear. We examined the location of tau in control and AD cortices using biochemical and morphologic methods. We found that, in addition to its well-described axonal localization, normal tau is present at both presynaptic and postsynaptic terminals in control human brains. In AD, tau becomes hyperphosphorylated and misfolded at both presynaptic and postsynaptic terminals, and this abnormally posttranslationally modified tau is enriched in synaptoneurosomal fractions. Synaptic tau seems to be hyperphosphorylated and ubiquitinated, and forms stable oligomers resistant to SDS denaturation. The accumulation of hyperphosphorylated tau oligomers at human AD synapses is associated with increased ubiquitinated substrates and increased proteasome components, consistent with dysfunction of the ubiquitin-proteasome system. Our findings suggest that synaptic hyperphosphorylated tau oligomers may be an important mediator of the proteotoxicity that disrupts synapses in AD.
|Critical role of B cell lymphoma 10 in BAFF-regulated NF-κB activation and survival of anergic B cells. |
Yu, M; Chen, Y; He, Y; Podd, A; Fu, G; Wright, JA; Kleiman, E; Khan, WN; Wen, R; Wang, D
Journal of immunology (Baltimore, Md. : 1950) 189 5185-93 2012
Anergy is a key physiological mechanism for restraining self-reactive B cells. A marked portion of peripheral B cells are anergic B cells that largely depend on BAFF for survival. BAFF activates the canonical and noncanonical NF-κB pathways, both of which are required for B cell survival. In this study we report that deficiency of the adaptor protein B cell lymphoma 10 (Bcl10) impaired the ability of BAFF to support B cell survival in vitro, and it specifically increased apoptosis in anergic B cells in vivo, dramatically reducing anergic B cells in mice. Bcl10-dependent survival of self-reactive anergic B cells was confirmed in the Ig hen egg lysozyme/soluble hen egg lysozyme double-transgenic mouse model of B cell anergy. Furthermore, we found that BAFF stimulation induced Bcl10 association with IκB kinase β, a key component of the canonical NF-κB pathway. Consistently, Bcl10-deficient B cells were impaired in BAFF-induced IκBα phosphorylation and formation of nuclear p50/c-Rel complexes. Bcl10-deficient B cells also displayed reduced expression of NF-κB2/p100, severely reducing BAFF-induced nuclear accumulation of noncanonical p52/RelB complexes. Consequently, Bcl10-deficient B cells failed to express Bcl-x(L), a BAFF-induced NF-κB target gene. Taken together, these data demonstrate that Bcl10 controls BAFF-induced canonical NF-κB activation directly and noncanonical NF-κB activation indirectly. The BAFF-R/Bcl10/NF-κB signaling axis plays a critical role in peripheral B cell tolerance by regulating the survival of self-reactive anergic B cells.
|Neurodegeneration and early lethality in superoxide dismutase 2-deficient mice: a comprehensive analysis of the central and peripheral nervous systems. |
Oh, SS; Sullivan, KA; Wilkinson, JE; Backus, C; Hayes, JM; Sakowski, SA; Feldman, EL
Neuroscience 212 201-13 2012
The contribution of oxidative stress to diabetic complications including neuropathy is widely known. Mitochondrial and cellular damage are associated with the overproduction of reactive oxygen species and decreased levels or function of the cellular antioxidant mitochondrial manganese superoxide dismutase (SOD2). We hypothesized that targeted SOD2 deletion in the peripheral nervous system using cre-lox technology under control of the nestin promoter would accelerate neuropathy in a type 2 model of diabetes, the BKS.db/db mouse. SOD2-deficient mice, however, demonstrated severe gait deformities and seizures and died by 20 days of age. Examination of SOD2 expression levels revealed that SOD2 was lost in brain and reduced in the spinal cord, but appeared normal in dorsal root ganglia and peripheral nerves in SOD2-deficient mice. These findings indicate incomplete targeted knockout of SOD2. Morphological examination revealed cortical lesions similar to spongiform encephalopathy in the brain of SOD2-deficient mice. No lesions were evident in the spinal cord, but changes in myelin within the sciatic and sural nerves including a lack of cohesion between layers of compact myelin were observed. Together, these results indicate that targeted neuronal SOD2 knockout using the nestin promoter results in severe central nervous system degeneration and perinatal lethality in mice. A specific peripheral nervous system-targeting construct is required to examine the consequences of SOD2 knockout in diabetic neuropathy.
|Tp53-induced glycolysis and apoptosis regulator (TIGAR) protects glioma cells from starvation-induced cell death by up-regulating respiration and improving cellular redox homeostasis. |
Wanka, C; Steinbach, JP; Rieger, J
The Journal of biological chemistry 287 33436-46 2012
Altered metabolism in tumor cells is increasingly recognized as a core component of the neoplastic phenotype. Because p53 has emerged as a master metabolic regulator, we hypothesized that the presence of wild-type p53 in glioblastoma cells could confer a selective advantage to these cells under the adverse conditions of the glioma microenvironment. Here, we report on the effects of the p53-dependent effector Tp53-induced glycolysis and apoptosis regulator (TIGAR) on hypoxia-induced cell death. We demonstrate that TIGAR is overexpressed in glioblastomas and that ectopic expression of TIGAR reduces cell death induced by glucose and oxygen restriction. Metabolic analyses revealed that TIGAR inhibits glycolysis and promotes respiration. Further, generation of reactive oxygen species (ROS) levels was reduced whereas levels of reduced glutathione were elevated in TIGAR-expressing cells. Finally, inhibiting the transketolase isoenzyme transketolase-like 1 (TKTL1) by siRNA reversed theses effects of TIGAR. These findings suggest that glioma cells benefit from TIGAR expression by (i) improving energy yield from glucose via increased respiration and (ii) enhancing defense mechanisms against ROS. Targeting metabolic regulators such as TIGAR may therefore be a valuable strategy to enhance glioma cell sensitivity toward spontaneously occurring or therapy-induced starvation conditions or ROS-inducing therapeutic approaches.
|The intermediate-activity (L597V)BRAF mutant acts as an epistatic modifier of oncogenic RAS by enhancing signaling through the RAF/MEK/ERK pathway. |
Andreadi, C; Cheung, LK; Giblett, S; Patel, B; Jin, H; Mercer, K; Kamata, T; Lee, P; Williams, A; McMahon, M; Marais, R; Pritchard, C
Genes & development 26 1945-58 2012
(L597V)BRAF mutations are acquired somatically in human cancer samples and are frequently coincident with RAS mutations. Germline (L597V)BRAF mutations are also found in several autosomal dominant developmental conditions known as RASopathies, raising the important question of how the same mutation can contribute to both pathologies. Using a conditional knock-in mouse model, we show that endogenous expression of (L597V)Braf leads to approximately twofold elevated Braf kinase activity and weak activation of the Mek/Erk pathway. This is associated with induction of RASopathy hallmarks including cardiac abnormalities and facial dysmorphia but is not sufficient for tumor formation. We combined (L597V)Braf with (G12D)Kras and found that (L597V)Braf modified (G12D)Kras oncogenesis such that fibroblast transformation and lung tumor development were more reminiscent of that driven by the high-activity (V600E)Braf mutant. Mek/Erk activation levels were comparable with those driven by (V600E)Braf in the double-mutant cells, and the gene expression signature was more similar to that induced by (V600E)Braf than (G12D)Kras. However, unlike (V600E)Braf, Mek/Erk pathway activation was mediated by both Craf and Braf, and ATP-competitive RAF inhibitors induced paradoxical Mek/Erk pathway activation. Our data show that weak activation of the Mek/Erk pathway underpins RASopathies, but in cancer, (L597V)Braf epistatically modifies the transforming effects of driver oncogenes.
|Pressure mediated hypertrophy and mechanical stretch up-regulate expression of the long form of leptin receptor (ob-Rb) in rat cardiac myocytes. |
Matsui, H; Yokoyama, T; Tanaka, C; Sunaga, H; Koitabashi, N; Takizawa, T; Arai, M; Kurabayashi, M
BMC cell biology 13 37 2012
Hyperleptinemia is known to participate in cardiac hypertrophy and hypertension, but the relationship between pressure overload and leptin is poorly understood. We therefore examined the expression of leptin (ob) and the leptin receptor (ob-R) in the pressure-overloaded rat heart. We also examined gene expressions in culture cardiac myocytes to clarify which hypertension-related stimulus induces these genes.Pressure overload was produced by ligation of the rat abdominal aorta, and ob and ob-R isoform mRNAs were measured using a real-time polymerase chain reaction (PCR). We also measured these gene expressions in neonatal rat cardiac myocytes treated with angiotensin II (ANGII), endothelin-1 (ET-1), or cyclic mechanical stretch. Leptin and the long form of the leptin receptor (ob-Rb) gene were significantly increased 4 weeks after banding, but expression of the short form of the leptin receptor (ob-Ra) was unchanged. ob-Rb protein expression was also detected by immunohistochemistry in hypertrophied cardiac myocytes after banding. Meanwhile, plasma leptin concentrations were not different between the control and banding groups. In cultured myocytes, ANGII and ET-1 increased only ob mRNA expression. However, mechanical stretch activated both ob and ob-Rb mRNA expression in a time-dependent manner, but ob-Ra mRNA was unchanged by any stress.We first demonstrated that both pressure mediated hypertrophy and mechanical stretch up-regulate ob-Rb gene expression in heart and cardiac myocytes, which are thought to be important for leptin action in cardiac myocytes. These results suggest a new local mechanism by which leptin affects cardiac remodeling in pressure-overloaded hearts.
|Tumor-promoting circuits that regulate a cancer-related chemokine cluster: dominance of inflammatory mediators over oncogenic alterations. |
Leibovich-Rivkin, T; Buganim, Y; Solomon, H; Meshel, T; Rotter, V; Ben-Baruch, A
Cancers 4 55-76 2012
Here, we investigated the relative contribution of genetic/signaling components versus microenvironmental factors to the malignancy phenotype. In this system, we took advantage of non-transformed fibroblasts that carried defined oncogenic modifications in Ras and/or p53. These cells were exposed to microenvironmental pressures, and the expression of a cancer-related chemokine cluster was used as readout for the malignancy potential (CCL2, CCL5, CXCL8, CXCL10). In cells kept in-culture, synergism between Ras hyper-activation and p53 dysfunction was required to up-regulate the expression of the chemokine cluster. The in vivo passage of RasHigh/p53Low-modified cells has led to tumor formation, accompanied by potentiation of chemokine release, implicating a powerful role for the tumor microenvironment in up-regulating the chemokine cluster. Indeed, we found that inflammatory mediators which are prevalent in tumor sites, such as TNFa and IL-1β, had a predominant impact on the release of the chemokines, which was substantially higher than that obtained by the oncogenic modifications alone, possibly acting through the transcription factors AP-1 and NF-kB. Together, our results propose that in the unbiased model system that we were using, inflammatory mediators of the tumor milieu have dominating roles over oncogenic modifications in dictating the expression of a pro-malignancy chemokine readout.
|Targeted overexpression of amelotin disrupts the microstructure of dental enamel. |
Lacruz, RS; Nakayama, Y; Holcroft, J; Nguyen, V; Somogyi-Ganss, E; Snead, ML; White, SN; Paine, ML; Ganss, B
PloS one 7 e35200 2012
We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel) gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL) and ameloblastin (AMBN) was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM) was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms.
|Co-expression of α9β1 integrin and VEGF-D confers lymphatic metastatic ability to a human breast cancer cell line MDA-MB-468LN. |
Majumder, M; Tutunea-Fatan, E; Xin, X; Rodriguez-Torres, M; Torres-Garcia, J; Wiebe, R; Timoshenko, AV; Bhattacharjee, RN; Chambers, AF; Lala, PK
PloS one 7 e35094 2012
Lymphatic metastasis is a common occurrence in human breast cancer, mechanisms remaining poorly understood. MDA-MB-468LN (468LN), a variant of the MDA-MB-468GFP (468GFP) human breast cancer cell line, produces extensive lymphatic metastasis in nude mice. 468LN cells differentially express α9β1 integrin, a receptor for lymphangiogenic factors VEGF-C/-D. We explored whether (1) differential production of VEGF-C/-D by 468LN cells provides an autocrine stimulus for cellular motility by interacting with α9β1 and a paracrine stimulus for lymphangiogenesis in vitro as measured with capillary-like tube formation by human lymphatic endothelial cells (HMVEC-dLy); (2) differential expression of α9 also promotes cellular motility/invasiveness by interacting with macrophage derived factors; (3) stable knock-down of VEGF-D or α9 in 468LN cells abrogates lymphangiogenesis and lymphatic metastasis in vivo in nude mice.A comparison of expression of cyclo-oxygenase (COX)-2 (a VEGF-C/-D inducer), VEGF-C/-D and their receptors revealed little COX-2 expression by either cells. However, 468LN cells showed differential VEGF-D and α9β1 expression, VEGF-D secretion, proliferative, migratory/invasive capacities, latter functions being stimulated further with VEGF-D. The requirement of α9β1 for native and VEGF-D-stimulated proliferation, migration and Erk activation was demonstrated by treating with α9β1 blocking antibody or knock-down of α9. An autocrine role of VEGF-D in migration was shown by its impairment by silencing VEGF-D and restoration with VEGF-D. 468LN cells and their soluble products stimulated tube formation, migration/invasiveness of HMVEC-dLy cell in a VEGF-D dependent manner as indicated by the loss of stimulation by silencing VEGF-D in 468LN cells. Furthermore, 468LN cells showed α9-dependent stimulation of migration/invasiveness by macrophage products. Finally, capacity for intra-tumoral lymphangiogenesis and lymphatic metastasis in nude mice was completely abrogated by stable knock-down of either VEGF-D or α9 in 468LN cells.Differential capacity for VEGF-D production and α9β1 integrin expression by 468LN cells jointly contributed to their lymphatic metastatic phenotype.
|Reduced susceptibility of DNA methyltransferase 1 hypomorphic (Dnmt1N/+) mice to hepatic steatosis upon feeding liquid alcohol diet. |
Kutay, H; Klepper, C; Wang, B; Hsu, SH; Datta, J; Yu, L; Zhang, X; Majumder, S; Motiwala, T; Khan, N; Belury, M; McClain, C; Jacob, S; Ghoshal, K
PloS one 7 e41949 2012
Methylation at C-5 (5-mdC) of CpG base pairs, the most abundant epigenetic modification of DNA, is catalyzed by 3 essential DNA methyltransferases (Dnmt1, Dnmt3a and Dnmt3b). Aberrations in DNA methylation and Dnmts are linked to different diseases including cancer. However, their role in alcoholic liver disease (ALD) has not been elucidated.Dnmt1 wild type (Dnmt1(+/+)) and hypomorphic (Dnmt1(N/+)) male mice that express reduced level of Dnmt1 were fed Lieber-DeCarli liquid diet containing ethanol for 6 weeks. Control mice were pair-fed calorie-matched alcohol-free liquid diet, and Dnmtase activity, 5-mdC content, gene expression profile and liver histopathology were evaluated. Ethanol feeding caused pronounced decrease in hepatic Dnmtase activity in Dnmt1(+/+) mice due to decrease in Dnmt1 and Dnmt3b protein levels and upregulation of miR-148 and miR-152 that target both Dnmt1 and Dnmt3b. Microarray and qPCR analysis showed that the genes involved in lipid, xenobiotic and glutathione metabolism, mitochondrial function and cell proliferation were dysregulated in the wild type mice fed alcohol. Surprisingly, Dnmt1(N/+) mice were less susceptible to alcoholic steatosis compared to Dnmt1(+/+) mice. Expression of several key genes involved in alcohol (Aldh3b1), lipid (Ppara, Lepr, Vldlr, Agpat9) and xenobiotic (Cyp39a1) metabolism, and oxidative stress (Mt-1, Fmo3) were significantly (Pless than 0.05) altered in Dnmt1(N/+) mice relative to the wild type mice fed alcohol diet. However, CpG islands encompassing the promoter regions of Agpat9, Lepr, Mt1 and Ppara were methylation-free in both genotypes irrespective of the diet, suggesting that promoter methylation does not regulate their expression. Similarly, 5-mdC content of the liver genome, as measured by LC-MS/MS analysis, was not affected by alcohol diet in the wild type or hypomorphic mice.Although feeding alcohol diet reduced Dnmtase activity, the loss of one copy of Dnmt1 protected mice from alcoholic hepatosteatosis by dysregulating genes involved in lipid metabolism and oxidative stress.
|Mechanism of chemical activation of Nrf2. |
Li, Y; Paonessa, JD; Zhang, Y
PloS one 7 e35122 2012
NF-E2 related factor-2 (Nrf2) promotes the transcription of many cytoprotective genes and is a major drug target for prevention of cancer and other diseases. Indeed, the cancer-preventive activities of several well-known chemical agents were shown to depend on Nrf2 activation. It is well known that chemopreventive Nrf2 activators stabilize Nrf2 by blocking its ubiquitination, but previous studies have indicated that this process occurs exclusively in the cytoplasm. Kelch-like ECH-associated protein 1 (Keap1) binds to Nrf2 and orchestrates Nrf2 ubiquitination, and it has been a widely-held view that inhibition of Nrf2 ubiquitination by chemopreventive agents results from the dissociation of Nrf2 from its repressor Keap1. Here, we show that while the activation of Nrf2 by prototypical chemical activators, including 5,6-dihydrocyclopenta-1,2-dithiole-3-thione (CPDT) and sulforaphane (SF), results solely from inhibition of its ubiquitination, such inhibition occurs predominantly in the nucleus. Moreover, the Nrf2 activators promote Nrf2 association with Keap1, rather than disassociation, which appears to result from inhibition of Nrf2 phosphorylation at Ser40. Available evidence suggests the Nrf2 activators may block Nrf2 ubiquitination by altering Keap1 conformation via reaction with the thiols of specific Keap1 cysteines. We further show that while the inhibitory effects of CPDT and SF on Nrf2 ubiquitination depend entirely on Keap1, Nrf2 is also degraded by a Keap1-independent mechanism. These findings provide significant new insight about Nrf2 activation and suggest that exogenous chemical activators of Nrf2 enter the nucleus to exert most of their inhibitory impact on Nrf2 ubiquitination and degradation.
|Nuclear distributions of NUP62 and NUP214 suggest architectural diversity and spatial patterning among nuclear pore complexes. |
Kinoshita, Y; Kalir, T; Dottino, P; Kohtz, DS
PloS one 7 e36137 2012
The shape of nuclei in many adherent cultured cells approximates an oblate ellipsoid, with contralateral flattened surfaces facing the culture plate or the medium. Observations of cultured cell nuclei from orthogonal perspectives revealed that nucleoporin p62 (NUP62) and nucleoporin 214 (NUP214) are differentially distributed between nuclear pore complexes on the flattened surfaces and peripheral rim of the nucleus. High resolution stimulated emission depletion (STED) immunofluorescence microscopy resolved individual NPCs, and suggested both heterogeneity and microheterogeneity in NUP62 and NUP214 immunolabeling among in NPC populations. Similar to nuclear domains and interphase chromosome territories, architectural diversity and spatial patterning of NPCs may be an intrinsic property of the nucleus that is linked to the functions and organization of underlying chromatin.
|The PARN deadenylase targets a discrete set of mRNAs for decay and regulates cell motility in mouse myoblasts. |
Lee, JE; Lee, JY; Trembly, J; Wilusz, J; Tian, B; Wilusz, CJ
PLoS genetics 8 e1002901 2012
PARN is one of several deadenylase enzymes present in mammalian cells, and as such the contribution it makes to the regulation of gene expression is unclear. To address this, we performed global mRNA expression and half-life analysis on mouse myoblasts depleted of PARN. PARN knockdown resulted in the stabilization of 40 mRNAs, including that encoding the mRNA decay factor ZFP36L2. Additional experiments demonstrated that PARN knockdown induced an increase in Zfp36l2 poly(A) tail length as well as increased translation. The elements responsible for PARN-dependent regulation lie within the 3' UTR of the mRNA. Surprisingly, changes in mRNA stability showed an inverse correlation with mRNA abundance; stabilized transcripts showed either no change or a decrease in mRNA abundance. Moreover, we found that stabilized mRNAs had reduced accumulation of pre-mRNA, consistent with lower transcription rates. This presents compelling evidence for the coupling of mRNA decay and transcription to buffer mRNA abundances. Although PARN knockdown altered decay of relatively few mRNAs, there was a much larger effect on global gene expression. Many of the mRNAs whose abundance was reduced by PARN knockdown encode factors required for cell migration and adhesion. The biological relevance of this observation was demonstrated by the fact that PARN KD cells migrate faster in wound-healing assays. Collectively, these data indicate that PARN modulates decay of a defined set of mRNAs in mammalian cells and implicate this deadenylase in coordinating control of genes required for cell movement.
|Pigment epithelium-derived factor (PEDF) protects cortical neurons in vitro from oxidant injury by activation of extracellular signal-regulated kinase (ERK) 1/2 and induction of Bcl-2. |
Sanchez, A; Tripathy, D; Yin, X; Luo, J; Martinez, J; Grammas, P
Neuroscience research 72 1-8 2012
Mitigating oxidative stress-induced damage is critical to preserve neuronal function in diseased or injured brains. This study explores the mechanisms contributing to the neuroprotective effects of pigment epithelium-derived factor (PEDF) in cortical neurons. Cultured primary neurons are exposed to PEDF and H₂O₂ as well as inhibitors of phosphoinositide-3 kinase (PI3K) or extracellular signal-regulated kinase 1/2 (ERK1/2). Neuronal survival, cell death and levels of caspase 3, PEDF, phosphorylated ERK1/2, and Bcl-2 are measured. The data show cortical cultures release PEDF and that H₂O₂ treatment causes cell death, increases activated caspase 3 levels and decreases release of PEDF. Exogenous PEDF induces a dose-dependent increase in Bcl-2 expression and neuronal survival. Blocking Bcl-2 expression by siRNA reduced PEDF-induced increases in neuronal survival. Treating cortical cultures with PEDF 24 h before H₂O₂ exposure mitigates oxidant-induced decreases in neuronal survival, Bcl-2 expression, and phosphorylation of ERK1/2 and also reduces elevated caspase 3 level and activity. PEDF pretreatment effect on survival is blocked by inhibiting ERK or PI3K. However, only inhibition of ERK reduced the ability of PEDF to protect neurons from H₂O₂-induced Bcl-2 decrease and neuronal death. These data demonstrate PEDF-mediated neuroprotection against oxidant injury is largely mediated via ERK1/2 and Bcl-2 and suggest the utility of PEDF in preserving the viability of oxidatively challenged neurons.
|Periostin modulates myofibroblast differentiation during full-thickness cutaneous wound repair. |
Elliott, CG; Wang, J; Guo, X; Xu, SW; Eastwood, M; Guan, J; Leask, A; Conway, SJ; Hamilton, DW
Journal of cell science 125 121-32 2012
The matricellular protein periostin is expressed in the skin. Although periostin has been hypothesized to contribute to dermal homeostasis and repair, this has not been directly tested. To assess the contribution of periostin to dermal healing, 6 mm full-thickness excisional wounds were created in the skin of periostin-knockout and wild-type, sex-matched control mice. In wild-type mice, periostin was potently induced 5-7 days after wounding. In the absence of periostin, day 7 wounds showed a significant reduction in myofibroblasts, as visualized by expression of α-smooth muscle actin (α-SMA) within the granulation tissue. Delivery of recombinant human periostin by electrospun collagen scaffolds restored α-SMA expression. Isolated wild-type and knockout dermal fibroblasts did not differ in in vitro assays of adhesion or migration; however, in 3D culture, periostin-knockout fibroblasts showed a significantly reduced ability to contract a collagen matrix, and adopted a dendritic phenotype. Recombinant periostin restored the defects in cell morphology and matrix contraction displayed by periostin-deficient fibroblasts in a manner that was sensitive to a neutralizing anti-β1-integrin and to the FAK and Src inhibitor PP2. We propose that periostin promotes wound contraction by facilitating myofibroblast differentiation and contraction.
|Metformin reduces hepatic expression of SIRT3, the mitochondrial deacetylase controlling energy metabolism. |
Buler, M; Aatsinki, SM; Izzi, V; Hakkola, J
PloS one 7 e49863 2012
Metformin inhibits ATP production in mitochondria and this may be involved in the anti-hyperglycemic effects of the drug. Sirtuin 3 (SIRT3) is a mitochondrial protein deacetylase that regulates the function of the electron transport chain and maintains basal ATP yield. We hypothesized that metformin treatment could diminish mitochondrial ATP production through downregulation of SIRT3 expression. Glucagon and cAMP induced SIRT3 mRNA in mouse primary hepatocytes. Metformin prevented SIRT3 induction by glucagon. Moreover, metformin downregulated constitutive expression of SIRT3 in primary hepatocytes and in the liver in vivo. Estrogen related receptor alpha (ERRα) mediates regulation of Sirt3 gene by peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). ERRα mRNA expression was regulated in a similar manner as SIRT3 mRNA by glucagon, cAMP and metformin. However, a higher metformin concentration was required for downregulation of ERRα than SIRT3. ERRα siRNA attenuated PGC-1α mediated induction of SIRT3, but did not affect constitutive expression. Overexpression of the constitutively active form of AMP-activated protein kinase (AMPK) induced SIRT3 mRNA, indicating that the SIRT3 downregulation by metformin is not mediated by AMPK. Metformin reduced the hepatocyte ATP level. This effect was partially counteracted by SIRT3 overexpression. Furthermore, metformin decreased mitochondrial SIRT3 protein levels and this was associated with enhanced acetylation of several mitochondrial proteins. However, metformin increased mitochondrial mass in hepatocytes. Altogether, our results indicate that metformin attenuates mitochondrial expression of SIRT3 and suggest that this mechanism is involved in regulation of energy metabolism by metformin in the liver and may contribute to the therapeutic action of metformin.
|Alpha-synuclein aggregation involves a bafilomycin A 1-sensitive autophagy pathway. |
Klucken, J; Poehler, AM; Ebrahimi-Fakhari, D; Schneider, J; Nuber, S; Rockenstein, E; Schlötzer-Schrehardt, U; Hyman, BT; McLean, PJ; Masliah, E; Winkler, J
Autophagy 8 754-66 2012
Synucleinopathies like Parkinson disease and dementia with Lewy bodies (DLB) are characterized by α-synuclein aggregates within neurons (Lewy bodies) and their processes (Lewy neurites). Whereas α-synuclein has been genetically linked to the disease process, the pathological relevance of α-synuclein aggregates is still debated. Impaired degradation is considered to result in aggregation of α-synuclein. In addition to the ubiquitin-proteasome degradation, the autophagy-lysosomal pathway (ALP) is involved in intracellular degradation processes for α-synuclein. Here, we asked if modulation of ALP affects α-synuclein aggregation and toxicity. We have identified an induction of the ALP markers LAMP-2A and LC3-II in human brain tissue from DLB patients, in a transgenic mouse model of synucleinopathy, and in a cell culture model for α-synuclein aggregation. ALP inhibition using bafilomycin A 1 (BafA1) significantly potentiates toxicity of aggregated α-synuclein species in transgenic mice and in cell culture. Surprisingly, increased toxicity is paralleled by reduced aggregation in both in vivo and in vitro models. The dichotomy of effects on aggregating and nonaggregating species of α-synuclein was specifically sensitive to BafA1 and could not be reproduced by other ALP inhibitors. The present study expands on the accumulating evidence regarding the function of ALP for α-synuclein degradation by isolating an aggregation specific, BafA1-sensitive, ALP-related pathway. Our data also suggest that protein aggregation may represent a detoxifying event rather than being causal for cellular toxicity.
|Epistatic rescue of Nkx2.5 adult cardiac conduction disease phenotypes by prospero-related homeobox protein 1 and HDAC3. |
Risebro, CA; Petchey, LK; Smart, N; Gomes, J; Clark, J; Vieira, JM; Yanni, J; Dobrzynski, H; Davidson, S; Zuberi, Z; Tinker, A; Shui, B; Tallini, YI; Kotlikoff, MI; Miquerol, L; Schwartz, RJ; Riley, PR
Circulation research 111 e19-31 2012
Nkx2.5 is one of the most widely studied cardiac-specific transcription factors, conserved from flies to man, with multiple essential roles in both the developing and adult heart. Specific dominant mutations in NKX2.5 have been identified in adult congenital heart disease patients presenting with conduction system anomalies and recent genome-wide association studies implicate the NKX2.5 locus, as causative for lethal arrhythmias ("sudden cardiac death") that occur at a frequency in the population of 1 in 1000 per annum worldwide. Haploinsufficiency for Nkx2.5 in the mouse phenocopies human conduction disease pathology yet the phenotypes, described in both mouse and man, are highly pleiotropic, implicit of unknown modifiers and/or factors acting in epistasis with Nkx2.5/NKX2.5.To identify bone fide upstream genetic modifier(s) of Nkx2.5/NKX2.5 function and to determine epistatic effects relevant to the manifestation of NKX2.5-dependent adult congenital heart disease.A study of cardiac function in prospero-related homeobox protein 1 (Prox1) heterozygous mice, using pressure-volume loop and micromannometry, revealed rescue of hemodynamic parameters in Nkx2.5(Cre/+); Prox1(loxP/+) animals versus Nkx2.5(Cre/+) controls. Anatomic studies, on a Cx40(EGFP) background, revealed Cre-mediated knock-down of Prox1 restored the anatomy of the atrioventricular node and His-Purkinje network both of which were severely hypoplastic in Nkx2.5(Cre/+) littermates. Steady state surface electrocardiography recordings and high-speed multiphoton imaging, to assess Ca(2+) handling, revealed atrioventricular conduction and excitation-contraction were also normalized by Prox1 haploinsufficiency, as was expression of conduction genes thought to act downstream of Nkx2.5. Chromatin immunoprecipitation on adult hearts, in combination with both gain and loss-of-function reporter assays in vitro, revealed that Prox1 recruits the corepressor HDAC3 to directly repress Nkx2.5 via a proximal upstream enhancer as a mechanism for regulating Nkx2.5 function in adult cardiac conduction.Here we identify Prox1 as a direct upstream modifier of Nkx2.5 in the maintenance of the adult conduction system and rescue of Nkx2.5 conduction disease phenotypes. This study is the first example of rescue of Nkx2.5 function and establishes a model for ensuring electrophysiological function within the adult heart alongside insight into a novel Prox1-HDAC3-Nkx2.5 signaling pathway for therapeutic targeting in conduction disease.
|Metabotropic actions of the volatile anaesthetic sevoflurane increase protein kinase M synthesis and induce immediate preconditioning protection of rat hippocampal slices. |
Wang, J; Meng, F; Cottrell, JE; Sacktor, TC; Kass, IS
The Journal of physiology 590 4093-107 2012
Anaesthetic preconditioning occurs when a volatile anaesthetic, such as sevoflurane, is administered before a hypoxic or ischaemic insult; this has been shown to improve neuronal recovery after the insult. We found that sevoflurane-induced preconditioning in the rat hippocampal slice enhances the hypoxic hyperpolarization of CA1 pyramidal neurons, delays and attenuates their hypoxic depolarization, and increases the number of neurons that recover their resting and action potentials after hypoxia. These altered electrophysiological effects and the improved recovery corresponded with an increase in the amount of a constitutively active, atypical protein kinase C isoform found in brain, protein kinase M zeta (PKMζ). A selective inhibitor of this kinase, zeta inhibitory peptide (ZIP), blocked the increase in the total amount of PKMζ protein and the amount of the activated form of this kinase, phospho-PKMζ (p-PKMζ); it also blocked the altered electrophysiological effects and the improved recovery. We found that both cycloheximide, a general protein synthesis inhibitor, and rapamycin, a selective inhibitor of the mTOR pathway for regulating protein synthesis, blocked the increase in p-PKMζ, the electrophysiological changes, and the improved recovery due to sevoflurane-induced preconditioning. Glibenclamide, a KATP channel blocker, when present only during the hypoxia, prevented the enhanced hyperpolarization, the delayed and attenuated hypoxic depolarization, and the improved recovery following sevoflurane-induced preconditioning. To examine the function of persistent PKMζ and KATP channel activity after the preconditioning was established, we administered 4% sevoflurane for 30 min and then discontinued it for 30 min before 10 min of hypoxia. When either tolbutamide, a KATP channel blocker, or ZIP were administered at least 15 min after the washout of sevoflurane, there was little recovery compared with sevoflurane alone. Thus, continuous KATP channel and PKMζ activity are required to maintain preconditioning protection. We conclude that sevoflurane induces activation of the mTOR pathway, increasing the new protein synthesis of PKMζ, which is constitutively phosphorylated to its active form, leading to an increased KATP channel-induced hyperpolarizaton. This hyperpolarization delays and attenuates the hypoxic depolarization, improving the recovery of neurons following hypoxia. Thus, sevoflurane acts via a metabotropic pathway to improve recovery following hypoxia.
|Increased smooth muscle contractility in mice deficient for neuropilin 2. |
Bielenberg, DR; Seth, A; Shimizu, A; Pelton, K; Cristofaro, V; Ramachandran, A; Zwaans, BM; Chen, C; Krishnan, R; Seth, M; Huang, L; Takashima, S; Klagsbrun, M; Sullivan, MP; Adam, RM
The American journal of pathology 181 548-59 2012
Neuropilins (NRPs) are transmembrane receptors that bind class 3 semaphorins and VEGF family members to regulate axon guidance and angiogenesis. Although expression of NRP1 by vascular smooth muscle cells (SMCs) has been reported, NRP function in smooth muscle (SM) in vivo is unexplored. Using Nrp2(+/LacZ) and Nrp2(+/gfp) transgenic mice, we observed robust and sustained expression of Nrp2 in the SM compartments of the bladder and gut, but no expression in vascular SM, skeletal muscle, or cardiac muscle. This expression pattern was recapitulated in vitro using primary human SM cell lines. Alterations in cell morphology after treatment of primary visceral SMCs with the NRP2 ligand semaphorin-3F (SEMA3F) were accompanied by inhibition of RhoA activity and myosin light chain phosphorylation, as well as decreased cytoskeletal stiffness. Ex vivo contractility testing of bladder muscle strips exposed to electrical stimulation or soluble agonists revealed enhanced tension generation of tissues from mice with constitutive or SM-specific knockout of Nrp2, compared with controls. Mice lacking Nrp2 also displayed increased bladder filling pressures, as assessed by cystometry in conscious mice. Together, these findings identify Nrp2 as a mediator of prorelaxant stimuli in SMCs and suggest a novel function for Nrp2 as a regulator of visceral SM contractility.
|GEP100/Arf6 is required for epidermal growth factor-induced ERK/Rac1 signaling and cell migration in human hepatoma HepG2 cells. |
Hu, Z; Du, J; Yang, L; Zhu, Y; Yang, Y; Zheng, D; Someya, A; Gu, L; Lu, X
PloS one 7 e38777 2012
Epidermal growth factor (EGF) signaling is implicated in the invasion and metastasis of hepatoma cells. However, the signaling pathways for EGF-induced motility of hepatoma cells remain undefined.We found that EGF dose-dependently stimulated the migration of human hepatoma cells HepG2, with the maximal effect at 10 ng/mL. Additionally, EGF increased Arf6 activity, and ectopic expression of Arf6 T27N, a dominant negative Arf6 mutant, largely abolish EGF-induced cell migration. Blocking GEP100 with GEP100 siRNA or GEP100-△PH, a pleckstrin homology (PH) domain deletion mutant of GEP100, blocked EGF-induced Arf6 activity and cell migration. EGF also increased ERK and Rac1 activity. Ectopic expression GEP100 siRNA, GEP100-△PH, or Arf6-T27N suppressed EGF-induced ERK and Rac1 activity. Furthermore, blocking ERK signaling with its inhibitor U0126 remarkably inhibited both EGF-induced Rac1 activation as well as cell migration, and ectopic expression of inactive mutant form of Rac1 (Rac1-T17N) also largely abolished EGF-induced cell migration.Taken together, this study highlights the function of the PH domain of GEP100 and its regulated Arf6/ERK/Rac1 signaling cascade in EGF-induced hepatoma cell migration. These findings could provide a rationale for designing new therapy based on inhibition of hepatoma metastasis.
|Hepatitis C virus infection suppresses GLUT2 gene expression via downregulation of hepatocyte nuclear factor 1α. |
Matsui, C; Shoji, I; Kaneda, S; Sianipar, IR; Deng, L; Hotta, H
Journal of virology 86 12903-11 2012
Hepatitis C virus (HCV) infection causes not only intrahepatic diseases but also extrahepatic manifestations, including type 2 diabetes. We previously reported that HCV replication suppresses cellular glucose uptake by downregulation of cell surface expression of glucose transporter 2 (GLUT2) (D. Kasai et al., J. Hepatol. 50:883-894, 2009). GLUT2 mRNA levels were decreased in both HCV RNA replicon cells and HCV J6/JFH1-infected cells. To elucidate molecular mechanisms of HCV-induced suppression of GLUT2 gene expression, we analyzed transcriptional regulation of the GLUT2 promoter using a series of GLUT2 promoter-luciferase reporter plasmids. HCV-induced suppression of GLUT2 promoter activity was abrogated when the hepatocyte nuclear factor 1α (HNF-1α)-binding motif was deleted from the GLUT2 promoter. HNF-1α mRNA levels were significantly reduced in HCV J6/JFH1-infected cells. Furthermore, HCV infection remarkably decreased HNF-1α protein levels. We assessed the effects of proteasome inhibitor or lysosomal protease inhibitors on the HCV-induced reduction of HNF-1α protein levels. Treatment of HCV-infected cells with a lysosomal protease inhibitor, but not with a proteasome inhibitor, restored HNF-1α protein levels, suggesting that HCV infection promotes lysosomal degradation of HNF-1α protein. Overexpression of NS5A protein enhanced lysosomal degradation of HNF-1α protein and suppressed GLUT2 promoter activity. Immunoprecipitation analyses revealed that the region from amino acids 1 to 126 of the NS5A domain I physically interacts with HNF-1α protein. Taken together, our results suggest that HCV infection suppresses GLUT2 gene expression via downregulation of HNF-1α expression at transcriptional and posttranslational levels. HCV-induced downregulation of HNF-1α expression may play a crucial role in glucose metabolic disorders caused by HCV.
|The mitochondrial heme exporter FLVCR1b mediates erythroid differentiation. |
Chiabrando, D; Marro, S; Mercurio, S; Giorgi, C; Petrillo, S; Vinchi, F; Fiorito, V; Fagoonee, S; Camporeale, A; Turco, E; Merlo, GR; Silengo, L; Altruda, F; Pinton, P; Tolosano, E
The Journal of clinical investigation 122 4569-79 2012
Feline leukemia virus subgroup C receptor 1 (FLVCR1) is a cell membrane heme exporter that maintains the balance between heme levels and globin synthesis in erythroid precursors. It was previously shown that Flvcr1-null mice died in utero due to a failure of erythropoiesis. Here, we identify Flvcr1b, a mitochondrial Flvcr1 isoform that promotes heme efflux into the cytoplasm. Flvcr1b overexpression promoted heme synthesis and in vitro erythroid differentiation, whereas silencing of Flvcr1b caused mitochondrial heme accumulation and termination of erythroid differentiation. Furthermore, mice lacking the plasma membrane isoform (Flvcr1a) but expressing Flvcr1b had normal erythropoiesis, but exhibited hemorrhages, edema, and skeletal abnormalities. Thus, FLVCR1b regulates erythropoiesis by controlling mitochondrial heme efflux, whereas FLVCR1a expression is required to prevent hemorrhages and edema. The aberrant expression of Flvcr1 isoforms may play a role in the pathogenesis of disorders characterized by an imbalance between heme and globin synthesis.
|Endothelial derived factors inhibit anoikis of head and neck cancer stem cells. |
Campos, MS; Neiva, KG; Meyers, KA; Krishnamurthy, S; Nör, JE
Oral oncology 48 26-32 2012
Recent evidence demonstrated that cancer stem cells reside in close proximity to blood vessels in human head and neck squamous cell carcinomas (HNSCC). These findings suggest the existence of a supporting perivascular niche for cancer stem cells. The purpose of this study was to evaluate the effect of endothelial cell-secreted factors on the behavior of head and neck cancer stem-like cells (HNCSC). HNCSC were identified by sorting UM-SCC-22A (cell line derived from a primary squamous cell carcinoma of the oropharynx) and UM-SCC-22B (derived from the metastatic lymph node of the same patient) for CD44 expression and ALDH (aldehyde dehydrogenase) activity. HNCSC (ALDH+CD44+) and control (ALDH-CD44-) cells were cultured in ultra-low attachment plates in presence of conditioned medium from primary human endothelial cells. ALDH+CD44+ generated more orospheres than control cells when cultured in suspension. The growth factor milieu secreted by endothelial cells protected HNCSC against anoikis. Mechanistic studies revealed that endothelial cell-secreted vascular endothelial growth factor (VEGF) induces proliferation of HNCSC derived from primary UM-SCC-22A, but not from the metastatic UM-SCC-22B. Likewise, blockade of VEGF abrogated endothelial cell-induced Akt phosphorylation in HNCSC derived from UM-SCC-22A while it had a modest effect in Akt phosphorylation in HNCSC from UM-SCC-22B. This study revealed that endothelial cells initiate a crosstalk that protect head and neck cancer stem cells against anoikis, and suggest that therapeutic interference with this crosstalk might be beneficial for patients with head and neck cancer.
|The sirtuin 2 inhibitor AK-7 is neuroprotective in Huntington's disease mouse models. |
Chopra, V; Quinti, L; Kim, J; Vollor, L; Narayanan, KL; Edgerly, C; Cipicchio, PM; Lauver, MA; Choi, SH; Silverman, RB; Ferrante, RJ; Hersch, S; Kazantsev, AG
Cell reports 2 1492-7 2012
Inhibition of sirtuin 2 (SIRT2) deacetylase mediates protective effects in cell and invertebrate models of Parkinson's disease and Huntington's disease (HD). Here we report the in vivo efficacy of a brain-permeable SIRT2 inhibitor in two genetic mouse models of HD. Compound treatment resulted in improved motor function, extended survival, and reduced brain atrophy and is associated with marked reduction of aggregated mutant huntingtin, a hallmark of HD pathology. Our results provide preclinical validation of SIRT2 inhibition as a potential therapeutic target for HD and support the further development of SIRT2 inhibitors for testing in humans.
|Sarcospan-dependent Akt activation is required for utrophin expression and muscle regeneration. |
Marshall, JL; Holmberg, J; Chou, E; Ocampo, AC; Oh, J; Lee, J; Peter, AK; Martin, PT; Crosbie-Watson, RH
The Journal of cell biology 197 1009-27 2012
Utrophin is normally confined to the neuromuscular junction (NMJ) in adult muscle and partially compensates for the loss of dystrophin in mdx mice. We show that Akt signaling and utrophin levels were diminished in sarcospan (SSPN)-deficient muscle. By creating several transgenic and knockout mice, we demonstrate that SSPN regulates Akt signaling to control utrophin expression. SSPN determined α-dystroglycan (α-DG) glycosylation by affecting levels of the NMJ-specific glycosyltransferase Galgt2. After cardiotoxin (CTX) injury, regenerating myofibers express utrophin and Galgt2-modified α-DG around the sarcolemma. SSPN-null mice displayed delayed differentiation after CTX injury caused by loss of utrophin and Akt signaling. Treatment of SSPN-null mice with viral Akt increased utrophin and restored muscle repair after injury, revealing an important role for the SSPN-Akt-utrophin signaling axis in regeneration. SSPN improved cell surface expression of utrophin by increasing transportation of utrophin and DG from endoplasmic reticulum/Golgi membranes. Our experiments reveal functions of utrophin in regeneration and new pathways that regulate utrophin expression at the cell surface.
|Clinical data and characterization of the liver conditional mouse model exclude neoplasia as a non-neurological manifestation associated with Friedreich's ataxia. |
Martelli, A; Friedman, LS; Reutenauer, L; Messaddeq, N; Perlman, SL; Lynch, DR; Fedosov, K; Schulz, JB; Pandolfo, M; Puccio, H
Disease models & mechanisms 5 860-9 2012
Friedreich's ataxia (FRDA) is the most common hereditary ataxia in the caucasian population and is characterized by a mixed spinocerebellar and sensory ataxia, hypertrophic cardiomyopathy and increased incidence of diabetes. FRDA is caused by impaired expression of the FXN gene coding for the mitochondrial protein frataxin. During the past ten years, the development of mouse models of FRDA has allowed better understanding of the pathophysiology of the disease. Among the mouse models of FRDA, the liver conditional mouse model pointed to a tumor suppressor activity of frataxin leading to the hypothesis that individuals with FRDA might be predisposed to cancer. In the present work, we investigated the presence and the incidence of neoplasia in the largest FRDA patient cohorts from the USA, Australia and Europe. As no predisposition to cancer could be observed in both cohorts, we revisited the phenotype of the liver conditional mouse model. Our results show that frataxin-deficient livers developed early mitochondriopathy, iron-sulfur cluster deficits and intramitochondrial dense deposits, classical hallmarks observed in frataxin-deficient tissues and cells. With age, a minority of mice developed structures similar to the ones previously associated with tumor formation. However, these peripheral structures contained dying, frataxin-deficient hepatocytes, whereas the inner liver structure was composed of a pool of frataxin-positive cells, due to inefficient Cre-mediated recombination of the Fxn gene, that contributed to regeneration of a functional liver. Together, our data demonstrate that frataxin deficiency and tumorigenesis are not associated.
|The CUL3-KLHL18 ligase regulates mitotic entry and ubiquitylates Aurora-A. |
Moghe, S; Jiang, F; Miura, Y; Cerny, RL; Tsai, MY; Furukawa, M
Biology open 1 82-91 2012
The cullin-RING family of ubiquitin ligases regulates diverse cellular functions, such as cell cycle control, via ubiquitylation of specific substrates. CUL3 targets its substrates through BTB proteins. Here we show that depletion of CUL3 and the BTB protein KLHL18 causes a delay in mitotic entry. Centrosomal activation of Aurora-A, a kinase whose activity is required for entry into mitosis, is also delayed in depleted cells. Moreover, we identify Aurora-A as a KLHL18-interacting partner. Overexpression of KLHL18 and CUL3 promotes Aurora-A ubiquitylation in vivo, and the CUL3-KLHL18-ROC1 ligase ubiquitylates Aurora-A in vitro. Our study reveals that the CUL3-KLHL18 ligase is required for timely entry into mitosis, as well as for the activation of Aurora-A at centrosomes. We propose that the CUL3-KLHL18 ligase regulates mitotic entry through an Aurora-A-dependent pathway.
|Isocitrate dehydrogenase (IDH) mutations promote a reversible ZEB1/microRNA (miR)-200-dependent epithelial-mesenchymal transition (EMT). |
Grassian, AR; Lin, F; Barrett, R; Liu, Y; Jiang, W; Korpal, M; Astley, H; Gitterman, D; Henley, T; Howes, R; Levell, J; Korn, JM; Pagliarini, R
The Journal of biological chemistry 287 42180-94 2012
Mutations in the genes encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in a variety of tumor types, resulting in production of the proposed oncometabolite, 2-hydroxyglutarate (2-HG). How mutant IDH and 2-HG alter signaling pathways to promote cancer, however, remains unclear. Additionally, there exist relatively few cell lines with IDH mutations. To examine the effect of endogenous IDH mutations and 2-HG, we created a panel of isogenic epithelial cell lines with either wild-type IDH1/2 or clinically relevant IDH1/2 mutations. Differences were noted in the ability of IDH mutations to cause robust 2-HG accumulation. IDH1/2 mutants that produce high levels of 2-HG cause an epithelial-mesenchymal transition (EMT)-like phenotype, characterized by changes in EMT-related gene expression and cellular morphology. 2-HG is sufficient to recapitulate aspects of this phenotype in the absence of an IDH mutation. In the cells types examined, mutant IDH-induced EMT is dependent on up-regulation of the transcription factor ZEB1 and down-regulation of the miR-200 family of microRNAs. Furthermore, sustained knockdown of IDH1 in IDH1 R132H mutant cells is sufficient to reverse many characteristics of EMT, demonstrating that continued expression of mutant IDH is required to maintain this phenotype. These results suggest mutant IDH proteins can reversibly deregulate discrete signaling pathways that contribute to tumorigenesis.
|Fibroblast growth factor receptor 3 effects on proliferation and telomerase activity in sheep growth plate chondrocytes. |
Smith, LB; Belanger, JM; Oberbauer, AM
Journal of animal science and biotechnology 3 39 2012
Fibroblast growth factor receptor 3 (FGFR3) inhibits growth-plate chondrocyte proliferation and limits bone elongation. Gain-of-function FGFR3 mutations cause dwarfism, reduced telomerase activity and shorter telomeres in growth plate chondroyctes suggesting that FGFR3 reduces proliferative capacity, inhibits telomerase, and enhances senescence. Thyroid hormone (T3) plays a role in cellular maturation of growth plate chondrocytes and a known target of T3 is FGFR3. The present study addressed whether reduced FGFR3 expression enhanced telomerase activity, mRNA expression of telomerase reverse transcriptase (TERT) and RNA component of telomerase (TR), and chondrocyte proliferation, and whether the stimulation of FGFR3 by T3 evoked the opposite response.Sheep growth-plate proliferative zone chondrocytes were cultured and transfected with siRNA to reduce FGFR3 expression; FGFR3 siRNA reduced chondrocyte FGFR3 mRNA and protein resulting in greater proliferation and increased TERT mRNA expression and telomerase activity (p less than 0.05). Chondrocytes treated with T3 significantly enhanced FGFR3 mRNA and protein expression and reduced telomerase activity (p less than 0.05); TERT and TR were not significantly reduced. The action of T3 at the growth plate may be partially mediated through the FGFR3 pathway.The results suggest that FGFR3 inhibits chondrocyte proliferation by down-regulating TERT expression and reducing telomerase activity indicating an important role for telomerase in sustaining chondrocyte proliferative capacity during bone elongation.
|Tumour suppressors miR-1 and miR-133a target the oncogenic function of purine nucleoside phosphorylase (PNP) in prostate cancer. |
Kojima, S; Chiyomaru, T; Kawakami, K; Yoshino, H; Enokida, H; Nohata, N; Fuse, M; Ichikawa, T; Naya, Y; Nakagawa, M; Seki, N
British journal of cancer 106 405-13 2012
Our recent analyses of miRNA expression signatures showed that miR-1 and miR-133a were significantly reduced in several types of cancer. Interestingly, miR-1 and miR-133a are located on the same chromosomal locus in the human genome. We examined the functional significance of miR-1 and miR-133a in prostate cancer (PCa) cells and identified the novel molecular targets regulated by both miR-1 and miR-133a.The expression levels of miR-1 and miR-133a were significantly downregulated in PCa compared with non-PCa tissues. Restoration of miR-1 or miR-133a in PC3 and DU145 cells revealed significant inhibition of proliferation, migration, and invasion. Molecular target identification by genome-wide gene expression analysis and luciferase reporter assay showed that purine nucleoside phosphorylase (PNP) was directly regulated by both miRNAs. Silencing of the PNP gene inhibited proliferation, migration, and invasion in both PC3 and DU145 cells. Immunohistochemistry detected positive staining of PNP in PCa specimens.Downregulation of miR-1 and miR-133a was a frequent event in PCa and both function as tumour suppressors. The PNP is a novel target gene of both miRNAs and potentially functions as an oncogene. Therefore, identification of novel molecular networks regulated by miRNAs may provide new insights into the underlying causes of PCa oncogenesis.
|Recruitment of OCRL and Inpp5B to phagosomes by Rab5 and APPL1 depletes phosphoinositides and attenuates Akt signaling. |
Bohdanowicz, M; Balkin, DM; De Camilli, P; Grinstein, S
Molecular biology of the cell 23 176-87 2012
Sealing of phagosomes is accompanied by the disappearance of phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2)) from their cytoplasmic leaflet. Elimination of PtdIns(4,5)P(2), which is required for actin remodeling during phagosome formation, has been attributed to hydrolysis by phospholipase C and phosphorylation by phosphatidylinositol 3-kinase. We found that two inositol 5-phosphatases, OCRL and Inpp5B, become associated with nascent phagosomes. Both phosphatases, which are Rab5 effectors, associate with the adaptor protein APPL1, which is recruited to the phagosomes by active Rab5. Knockdown of APPL1 or inhibition of Rab5 impairs association of OCRL and Inpp5B with phagosomes and prolongs the presence of PtdIns(4,5)P(2) and actin on their membranes. Even though APPL1 can serve as an anchor for A