Tabla espec. clave
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, R, M||EMSA, IP, WB||Rb||Purified||Polyclonal Antibody|
|Safety Information according to GHS|
|Material Size||100 µg|
Ficha datos de seguridad (MSDS)
|Cargo||Número de lote|
|Anti-NF kB p65, CT - JBC1395528||JBC1395528|
|Anti-NF#954;B p65, CT - 2123172||2123172|
|Anti-NF#954;B p65, CT - DAM1774705||DAM1774705|
|Anti-NFB p65, CT - 2040539||2040539|
|Anti-NFB p65, CT - 2189944||2189944|
|Anti-NFB p65, CT - 2203119||2203119|
|Anti-NFB p65, CT - 2356106||2356106|
|Anti-NFB p65, CT - DAM1687538||DAM1687538|
|Anti-NFB p65, CT - DAM1713190||DAM1713190|
|Anti-NFB p65, CT - DAM1745297||DAM1745297|
|Anti-NFB p65, CT - JBC1395528||JBC1395528|
|Anti-NFkappa;B p65, CT - 0701049995||0701049995|
|Anti-NFkappa;B p65, CT - 13485||13485|
|Anti-NFkappa;B p65, CT - 17802||17802|
|Anti-NFkappa;B p65, CT - 19404||19404|
|Anti-NFkappa;B p65, CT - 21977||21977|
|Anti-NFkappa;B p65, CT - 26471||26471|
|Anti-NFκB p65, CT - 2424728||2424728|
|Anti-NFκB p65, CT - 2452511||2452511|
|Anti-NFκB p65, CT - 2459636||2459636|
|Anti-NFκB p65, CT - 2508876||2508876|
|Anti-NFκB p65, CT - 2561980||2561980|
|Anti-NFκB p65, CT -2575269||2575269|
Referencias bibliográficas | 29 Disponible | Ver todas las referencias
|Visión general referencias||Aplicación||Pub Med ID|
|Calcitriol ameliorates renal damage in a pre-established proteinuria model. |
Maquigussa, E; Arnoni, CP; Pereira, LG; Boim, MA
Molecular medicine reports 12 1009-15 2015
Proteinuria is critical in the tubulointerstitial changes that ultimately lead to renal insufficiency. Increased protein filtration has direct toxic effects on tubular epithelial cells, leading to epithelial mesenchymal transition (EMT) to a myofibroblast phenotype. Angiotensin II and transforming growth factor (TGF)-β1 are the main mediators of EMT. Calcitriol may exert a potential renoprotective effect by reducing the activity of the renin angiotensin system by suppressing renin gene expression and also by inhibiting the proinflammatory nuclear factor-κB pathway. The present study investigated the benefits of calcitriol treatment in a puromycin-induced proteinuric nephropathy model. Uninephrectomized adult male Wistar rats received intraperitoneal administration of a single dose of puromycin (100 mg/kg) or vehicle. After eight weeks, the animals were divided into two groups and received vehicle or calcitriol (0.5 µg/kg) for four weeks. The vehicle-treated, proteinuric rats developed progressive proteinuria and tubulointerstitial fibrosis after 12 weeks. Increased collagen deposition and fibrosis were significantly ameliorated by calcitriol treatment. Calcitriol was effective in preventing an increase in the EMT markers, α-smooth muscle actin and fibroblast-specific protein 1, reducing macrophage infiltration as evidenced by levels of ED-1. In addition, calcitriol increased the anti-inflammatory cytokine interleukin-10 and reduced the pro-oxidant p47 phox enzyme. These effects were paralleled by a reduction in TGF-β/Smad3 expression. Calcitriol may have therapeutic potential in the proteinuric nephropathy model used in the present study by inhibiting the TGF-β1 axis.
|An activated unfolded protein response promotes retinal degeneration and triggers an inflammatory response in the mouse retina. |
Rana, T; Shinde, VM; Starr, CR; Kruglov, AA; Boitet, ER; Kotla, P; Zolotukhin, S; Gross, AK; Gorbatyuk, MS
Cell death & disease 5 e1578 2014
Recent studies on the endoplasmic reticulum stress have shown that the unfolded protein response (UPR) is involved in the pathogenesis of inherited retinal degeneration caused by mutant rhodopsin. However, the main question of whether UPR activation actually triggers retinal degeneration remains to be addressed. Thus, in this study, we created a mouse model for retinal degeneration caused by a persistently activated UPR to assess the physiological and morphological parameters associated with this disease state and to highlight a potential mechanism by which the UPR can promote retinal degeneration. We performed an intraocular injection in C57BL6 mice with a known unfolded protein response (UPR) inducer, tunicamycin (Tn) and examined animals by electroretinography (ERG), spectral domain optical coherence tomography (SD-OCT) and histological analyses. We detected a significant loss of photoreceptor function (over 60%) and retinal structure (35%) 30 days post treatment. Analysis of retinal protein extracts demonstrated a significant upregulation of inflammatory markers including interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and IBA1. Similarly, we detected a strong inflammatory response in mice expressing either Ter349Glu or T17M rhodopsin (RHO). These mutant rhodopsin species induce severe retinal degeneration and T17M rhodopsin elicits UPR activation when expressed in mice. RNA and protein analysis revealed a significant upregulation of pro- and anti-inflammatory markers such as IL-1β, IL-6, p65 nuclear factor kappa B (NF-kB) and MCP-1, as well as activation of F4/80 and IBA1 microglial markers in both the retinas expressing mutant rhodopsins. We then assessed if the Tn-induced inflammatory marker IL-1β was capable of inducing retinal degeneration by injecting C57BL6 mice with a recombinant IL-1β. We observed ~19% reduction in ERG a-wave amplitudes and a 29% loss of photoreceptor cells compared with control retinas, suggesting a potential link between pro-inflammatory cytokines and retinal pathophysiological effects. Our work demonstrates that in the context of an established animal model for ocular disease, the persistent activation of the UPR could be responsible for promoting retinal degeneration via the UPR-induced pro-inflammatory cytokine IL-1β.
|NF-κB plays a key role in inducing CD274 expression in human monocytes after lipopolysaccharide treatment. |
Huang, G; Wen, Q; Zhao, Y; Gao, Q; Bai, Y
PloS one 8 e61602 2013
CD274, one of two co-stimulatory ligands for programmed death 1 and widely expressed in the mononuclear phagocyte system (MPS), may co-stimulate T cells and regulates inflammatory responses. However, changes in CD274 gene expression and the underlying molecular mechanism are poorly understood during inflammatory responses. Therefore, delineation of the complex mechanisms regulating CD274 expression is critical to understand this immunoregulatory system during inflammatory responses. The purpose of this study was to assess the molecular mechanisms regulating CD274 expression in an in vitro monocyte model of inflammatory response. Firstly, CD274 expression levels in human primary monocytes after lipopolysaccharide (LPS) treatment were observed and correlated with NF-κB activation. Secondly, based on the distribution of putative NF-κB binding sites, 5' truncated human CD274 promoter reporters were constructed, transfected into U937 cells and critical promoter regions for basal (nt -570 to +94) and LPS-induced (nt -1735 to -570) transcription were identified by dual luciferase assays. Finally, a key NF-κB binding site (nt -610 to -601) for LPS-inducible CD274 transcriptional activity was characterized by point mutation analysis and chromatin immunoprecipitation analysis assays (ChIP). Thus, the present study establishes a molecular basis to understand the mechanisms governing CD274 expression in certain infections and inflammatory disorders.
|SIMPL enhancement of tumor necrosis factor-α dependent p65-MED1 complex formation is required for mammalian hematopoietic stem and progenitor cell function. |
Zhao, W; Breese, E; Bowers, A; Hoggatt, J; Pelus, LM; Broxmeyer, HE; Goebl, M; Harrington, MA
PloS one 8 e61123 2013
Significant insight into the signaling pathways leading to activation of the Rel transcription factor family, collectively termed NF-κB, has been gained. Less well understood is how subsets of NF-κB-dependent genes are regulated in a signal specific manner. The SIMPL protein (signaling molecule that interacts with mouse pelle-like kinase) is required for full Tumor Necrosis Factor-α (TNFα) induced NF-κB activity. We show that SIMPL is required for steady-state hematopoiesis and the expression of a subset of TNFα induced genes whose products regulate hematopoietic cell activity. To gain insight into the mechanism through which SIMPL modulates gene expression we focused on the Tnf gene, an immune response regulator required for steady-state hematopoiesis. In response to TNFα SIMPL localizes to the Tnf gene promoter where it modulates the initiation of Tnf gene transcription. SIMPL binding partners identified by mass spectrometry include proteins involved in transcription and the interaction between SIMPL and MED1 was characterized in more detail. In response to TNFα, SIMPL is found in p65-MED1 complexes where SIMPL enhances p65/MED1/SIMPL complex formation. Together our results indicate that SIMPL functions as a TNFα-dependent p65 co-activator by facilitating the recruitment of MED1 to p65 containing transcriptional complexes to control the expression of a subset of TNFα-induced genes.
|The response of human macrophages to β-glucans depends on the inflammatory milieu. |
Municio, C; Alvarez, Y; Montero, O; Hugo, E; Rodríguez, M; Domingo, E; Alonso, S; Fernández, N; Crespo, MS
PloS one 8 e62016 2013
β-glucans are fungal cell wall components that bind to the C-type lectin-like receptor dectin-1. Polymorphisms of dectin-1 gene are associated with susceptibility to invasive fungal infection and medically refractory ulcerative colitis. The purpose of this study has been addressing the response of human macrophages to β-glucans under different conditions mimicking the composition of the inflammatory milieu in view of the wide plasticity and large range of phenotypical changes showed by these cells, and the relevant role of dectin-1 in several pathophysiological conditions.Serum-differentiated macrophages stimulated with β-glucans showed a low production of TNFα and IL-1β, a high production of IL-6 and IL-23, and a delayed induction of cyclooxygenase-2 and PGE2 biosynthesis that resembled the responses elicited by crystals and those produced when phagosomal degradation of the phagocytic cargo increases ligand access to intracellular pattern recognition receptors. Priming with a low concentration of LPS produced a rapid induction of cyclooxygenase-2 and a synergistic release of PGE2. When the differentiation of the macrophages was carried out in the presence of M-CSF, an increased expression of dectin-1 B isoform was observed. In addition, this treatment made the cells capable to release arachidonic acid in response to β-glucan.These results indicate that the macrophage response to fungal β-glucans is strongly influenced by cytokines and microbial-derived factors that are usual components of the inflammatory milieu. These responses can be sorted into three main patterns i) an elementary response dependent on phagosomal processing of pathogen-associated molecular patterns and/or receptor-independent, direct membrane binding linked to the immunoreceptor tyrosine-based activation motif-bearing transmembrane adaptor DNAX-activating protein 12, ii) a response primed by TLR4-dependent signals, and iii) a response dependent on M-CSF and dectin-1 B isoform expression that mainly signals through the dectin-1 B/spleen tyrosine kinase/cytosolic phospholipase A2 route.
|miR-146a-5p circuitry uncouples cell proliferation and migration, but not differentiation, in human mesenchymal stem cells. |
Hsieh, JY; Huang, TS; Cheng, SM; Lin, WS; Tsai, TN; Lee, OK; Wang, HW
Nucleic acids research 41 9753-63 2013
Administration of mesenchymal stem cells (MSCs) has the potential to ameliorate degenerative disorders and to repair damaged tissues. The homing of transplanted MSCs to injured sites is a critical property of engraftment. Our aim was to identify microRNAs involved in controlling MSC proliferation and migration. MSCs can be isolated from bone marrow and umbilical cord Wharton's jelly (BM-MSCs and WJ-MSCs, respectively), and WJ-MSCs show poorer motility yet have a better amplification rate compared with BM-MSCs. Small RNA sequencing revealed that miR-146a-5p is significantly overexpressed and has high abundance in WJ-MSCs. Knockdown of miR-146a-5p in WJ-MSCs inhibited their proliferation yet enhanced their migration, whereas overexpression of miR-146a-5p in BM-MSCs did not influence their osteogenic and adipogenic potentials. Chemokine (C-X-C motif) ligand 12 (CXCL12), together with SIKE1, which is an I-kappa-B kinase epsilon (IKKε) suppressor, is a direct target of miR-146a-5p in MSCs. Knockdown of miR-146a-5p resulted in the down-regulation of nuclear factor kappa-B (NF-κB) activity, which is highly activated in WJ-MSCs and is known to activate miR-146a-5p promoter. miR-146a-5p is also downstream of CXCL12, and a negative feedback loop is therefore formed in MSCs. These findings suggest that miR-146a-5p is critical to the uncoupling of motility and proliferation of MSCs. Our miRNome data also provide a roadmap for further understanding MSC biology.
|BCL-3 attenuation of TNFA expression involves an incoherent feed-forward loop regulated by chromatin structure. |
Walker, T; Adamson, A; Jackson, DA
PloS one 8 e77015 2013
Induction of genes is rarely an isolated event; more typically occurring as part of a web of parallel interactions, or motifs, which act to refine and control gene expression. Here, we define an Incoherent Feed-forward Loop motif in which TNFα-induced NF-κB signalling activates expression of the TNFA gene itself and also controls synthesis of the negative regulator BCL-3. While sharing a common inductive signal, the two genes have distinct temporal expression profiles. Notably, while the TNFA gene promoter is primed to respond immediately to activated NF-κB in the nucleus, induction of BCL3 expression only occurs after a time delay of about 1h. We show that this time delay is defined by remodelling of the BCL3 gene promoter, which is required to activate gene expression, and characterise the chromatin delayed induction of BCL3 expression using mathematical models. The models show how a delay in inhibitor production effectively uncouples the rate of response to inflammatory cues from the final magnitude of inhibition. Hence, within this regulatory motif, a delayed (incoherent) feed-forward loop together with differential rates of TNFA (fast) and BCL3 (slow) mRNA turnover provide robust, pulsatile expression of TNFα . We propose that the structure of the BCL-3-dependent regulatory motif has a beneficial role in modulating expression dynamics and the inflammatory response while minimising the risk of pathological hyper-inflammation.
|Inhibition of endoplasmic reticulum stress alleviates lipopolysaccharide-induced lung inflammation through modulation of NF-κB/HIF-1α signaling pathway. |
Kim, HJ; Jeong, JS; Kim, SR; Park, SY; Chae, HJ; Lee, YC
Scientific reports 3 1142 2013
Lipopolysaccharide (LPS) is involved in a variety of inflammatory disorders. Under stress conditions, endoplasmic reticulum (ER) loses the homeostasis in its functions, which is defined as ER stress. Little is known how ER stress is implicated in LPS-induced lung inflammation. In this study, effects of inhibition of ER stress on LPS-induced lung inflammation and transcriptional regulation were examined. An ER stress regulator, 4-phenylbutyrate (PBA) reduced LPS-induced increases of various ER stress markers in the lung. Furthermore, inhibition of ER stress reduced the LPS-induced lung inflammation. Moreover, LPS-induced increases of NF-κB and HIF-1α activity were lowered by inhibition of ER stress. These results suggest that inhibition of ER stress ameliorates LPS-induced lung inflammation through modulation of NF-κB/IκB and HIF-1α signaling pathway.
|Sirtuin 1 is a key regulator of the interleukin-12 p70/interleukin-23 balance in human dendritic cells. |
Alvarez, Y; Rodríguez, M; Municio, C; Hugo, E; Alonso, S; Ibarrola, N; Fernández, N; Crespo, MS
The Journal of biological chemistry 287 35689-701 2012
Stimulation of human dendritic cells with the fungal surrogate zymosan produces IL-23 and a low amount of IL-12 p70. Trans-repression of il12a transcription, which encodes IL-12 p35 chain, by proteins of the Notch family and lysine deacetylation reactions have been reported as the underlying mechanisms, but a number of questions remain to be addressed. Zymosan produced the location of sirtuin 1 (SIRT1) to the nucleus, enhanced its association with the il12a promoter, increased the nuclear concentration of the SIRT1 co-substrate NAD(+), and decreased chromatin accessibility in the nucleosome-1 of il12a, which contains a κB-site. The involvement of deacetylation reactions in the inhibition of il12a transcription was supported by the absence of Ac-Lys-14-histone H3 in dendritic cells treated with zymosan upon coimmunoprecipitation of transducin-like enhancer of split. In contrast, we did not obtain evidence of a possible effect of SIRT1 through the deacetylation of c-Rel, the central element of the NF-κB family involved in il12a regulation. These data indicate that an enhancement of SIRT1 activity in response to phagocytic stimuli may reduce the accessibility of c-Rel to the il12a promoter and its transcriptional activation, thus regulating the IL-12 p70/IL-23 balance and modulating the ongoing immune response.
|Myh7b/miR-499 gene expression is transcriptionally regulated by MRFs and Eos. |
Yeung, F; Chung, E; Guess, MG; Bell, ML; Leinwand, LA
Nucleic acids research 40 7303-18 2012
The sarcomeric myosin gene, Myh7b, encodes an intronic microRNA, miR-499, which regulates cardiac and skeletal muscle biology, yet little is known about its transcriptional regulation. To identify the transcription factors involved in regulating Myh7b/miR-499 gene expression, we have mapped the transcriptional start sites and identified an upstream 6.2 kb region of the mouse Myh7b gene whose activity mimics the expression pattern of the endogenous Myh7b gene both in vitro and in vivo. Through promoter deletion analysis, we have mapped a distal E-box element and a proximal Ikaros site that are essential for Myh7b promoter activity in muscle cells. We show that the myogenic regulatory factors, MyoD, Myf5 and Myogenin, bind to the E-box, while a lymphoid transcription factor, Ikaros 4 (Eos), binds to the Ikaros motif. Further, we show that through physical interaction, MyoD and Eos form an active transcriptional complex on the chromatin to regulate the expression of the endogenous Myh7b/miR-499 gene in muscle cells. We also provide the first evidence that Eos can regulate expression of additional myosin genes (Myosin 1 and β-Myosin) via the miR-499/Sox6 pathway. Therefore, our results indicate a novel role for Eos in the regulation of the myofiber gene program.
|Glutamine modulates lipopolysaccharide-induced activation of NF-κB via the Akt/mTOR pathway in lung epithelial cells. |
Hou, YC; Chiu, WC; Yeh, CL; Yeh, SL
American journal of physiology. Lung cellular and molecular physiology 302 L174-83 2012
Lung epithelial cells are important barriers in the respiratory system that provoke inflammatory responses through nuclear factor (NF)-κB activation to prevent pathogens from invading the body. Lipopolysaccharide (LPS) is a common pathogen-associated stimulus that activates IκB kinase (IKK) to regulate NF-κB-mediated inflammation through modulating nuclear translocation and phosphorylation of NF-κB. Previously, it was shown that Akt and the mammalian target of rapamycin (mTOR) are involved in the phosphorylation of IKK to activate NF-κB. Herein, we demonstrate that glutamine (GLN) modulated LPS-induced activation of NF-κB through the Akt/mTOR/IKK pathway in BEAS-2B cells. BEAS-2B cells in submerged culture were placed in medium containing different concentrations of GLN (0, 0.5, 1, and 2.5 mM) with 1 μg/ml LPS. Results showed that GLN deprivation induced phosphorylation of Akt/mTOR/IKK signaling, increased levels of NF-κB nuclear translocation and phosphorylated NF-κB, and upregulated NF-κB-dependent transcriptional activity, which was suppressed by GLN administration. Expressions of NF-κB-targeted genes were also reduced by supplemental GLN. GLN administration improved cell viability, whereas 0.5 mM GLN had a greater extent of inhibition on the Akt/mTOR/IKK/NF-κB signaling cascade. The inhibitory effects of GLN on NF-κB activation were also observed in cells cultured under air-liquid interface condition. These results indicate that GLN deprivation increased LPS-induced NF-κB activation and transcriptional activity, which was reversed by GLN administration. The findings provide potential mechanisms of GLN's modulation of LPS-induced NF-κB activation in lung epithelial cells and imply that maintaining a physiological concentration of GLN is essential in preventing LPS-induced lung inflammation.
|IKKα and alternative NF-κB regulate PGC-1β to promote oxidative muscle metabolism. |
Bakkar, N; Ladner, K; Canan, BD; Liyanarachchi, S; Bal, NC; Pant, M; Periasamy, M; Li, Q; Janssen, PM; Guttridge, DC
The Journal of cell biology 196 497-511 2012
Although the physiological basis of canonical or classical IκB kinase β (IKKβ)-nuclear factor κB (NF-κB) signaling pathway is well established, how alternative NF-κB signaling functions beyond its role in lymphoid development remains unclear. In particular, alternative NF-κB signaling has been linked with cellular metabolism, but this relationship is poorly understood. In this study, we show that mice deleted for the alternative NF-κB components IKKα or RelB have reduced mitochondrial content and function. Conversely, expressing alternative, but not classical, NF-κB pathway components in skeletal muscle stimulates mitochondrial biogenesis and specifies slow twitch fibers, suggesting that oxidative metabolism in muscle is selectively controlled by the alternative pathway. The alternative NF-κB pathway mediates this specificity by direct transcriptional activation of the mitochondrial regulator PPAR-γ coactivator 1β (PGC-1β) but not PGC-1α. Regulation of PGC-1β by IKKα/RelB also is mammalian target of rapamycin (mTOR) dependent, highlighting a cross talk between mTOR and NF-κB in muscle metabolism. Together, these data provide insight on PGC-1β regulation during skeletal myogenesis and reveal a unique function of alternative NF-κB signaling in promoting an oxidative metabolic phenotype.
|Notch- and transducin-like enhancer of split (TLE)-dependent histone deacetylation explain interleukin 12 (IL-12) p70 inhibition by zymosan. |
Alvarez, Y; Municio, C; Hugo, E; Zhu, J; Alonso, S; Hu, X; Fernández, N; Sánchez Crespo, M
The Journal of biological chemistry 286 16583-95 2011
The fungal analog zymosan induces IL-23 and low amounts of IL-12 p70. This study addresses the molecular mechanisms underlying this cytokine pattern in human monocyte-derived dendritic cells. The transcriptional regulation of il23a, one of the chains of IL-23, depended on the activation of c-Rel and histone H3 phosphorylation, as judged from the association of c-Rel with the il23a promoter and the correlation between IL-23 production and Ser-10-histone H3 phosphorylation. Consistent with its reduced ability to produce IL-12 p70, zymosan induced a transient occupancy of the il12a promoter by c-Rel, blocked the production of IL-12 p70 and the transcription of il12a induced by other stimuli, and triggered the expression and nuclear translocation of the transcriptional repressors of the Notch family hairy and enhancer of split (Hes)-1, Hes5, hairy/enhancer-of-split related with YRPW motif protein (Hey)-1, and transducin-like enhancer of split (TLE). Zymosan also induced the interaction of Hes1 and TLE with histone H3 phosphorylated on Ser-10 and deacetylated on Lys-14. Inhibition of class III histone deacetylases increased the production of IL-12 p70 and partially blunted the inhibitory effect of zymosan on the production of IL-12 p70 elicited by LPS and IFN-γ. These results indicate that the selective induction of IL-23 by β-glucans is explained by the activation of c-Rel associated with Ser-10-histone H3 phosphorylation in the il23a promoter mediated by mitogen- and stress-activated kinase and/or protein kinase A and inhibition of il12a transcription by a mechanism involving activation of several corepressors with the ability to bind TLE and to promote histone deacetylation.
|p50 (NF-κB1) is an effector protein in the cytotoxic response to DNA methylation damage. |
Schmitt, AM; Crawley, CD; Kang, S; Raleigh, DR; Yu, X; Wahlstrom, JS; Voce, DJ; Darga, TE; Weichselbaum, RR; Yamini, B
Molecular cell 44 785-96 2011
The functional significance of the signaling pathway induced by O(6)-methylguanine (O(6)-MeG) lesions is poorly understood. Here, we identify the p50 subunit of NF-κB as a central target in the response to O(6)-MeG and demonstrate that p50 is required for S(N)1-methylator-induced cytotoxicity. In response to S(N)1-methylation, p50 facilitates the inhibition of NF-κB-regulated antiapoptotic gene expression. Inhibition of NF-κB activity is noted to be an S phase-specific phenomenon that requires the formation of O(6)-MeG:T mismatches. Chk1 associates with p50 following S(N)1-methylation, and phosphorylation of p50 by Chk1 results in the inhibition of NF-κB DNA binding. Expression of an unphosphorylatable p50 mutant blocks inhibition of NF-κB-regulated antiapoptotic gene expression and attenuates S(N)1-methylator-induced cytotoxicity. While O(6)-MeG:T-induced, p50-dependent signaling is not sufficient to induce cell death, this pathway sensitizes cells to the cytotoxic effects of DNA breaks.
|Proteinase-activated receptors induce interleukin-8 expression by intestinal epithelial cells through ERK/RSK90 activation and histone acetylation. |
Wang, H; Moreau, F; Hirota, CL; MacNaughton, WK
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 24 1971-80 2010
Proteinase-activated receptors (PARs) are involved in both inflammation and tumorigenesis in epithelial cells. Interleukin (IL)-8 is a potent chemoattractant and is also involved in angiogenesis. The molecular mechanism whereby PARs induce epithelial IL-8 expression is not known. In HT-29 colonic epithelial cells, PAR(1) or PAR(2) agonists stimulated the expression of IL-8 through a NF-kappaB-dependent pathway without inducing IkappaB degradation and disassociation of IkappaB from NF-kappaB. Further studies revealed that PAR activation induced the phosphorylation of p65 at Ser-276 in the nucleus, which increased the recruitment of histone acetyltransferase (HAT) p300 to p50. Inhibition of ERK activation completely blocked PAR-induced IL-8 expression, phosphorylation of p65 and HAT activity. We also demonstrated that RSK p90 was the downstream kinase that mediated ERK-induced nuclear p65 phosphorylation. In conclusion, activation of either PAR(1) or PAR(2) stimulated the transcriptional up-regulation of IL-8 in HT-29 colonic epithelial cells through a pathway that involved ERK/RSK p90, NF-kappaB phosphorylation, and HAT activity. These studies provide evidence of a new role for serine proteinases and PARs in the regulation of gene expression in colonic inflammation and tumorigenesis.
|Retinoic acid protects human breast cancer cells against etoposide-induced apoptosis by NF-kappaB-dependent but cIAP2-independent mechanisms. |
Jiménez-Lara, AM; Aranda, A; Gronemeyer, H
Molecular cancer 9 15 2010
Retinoids, through their cognate nuclear receptors, exert potent effects on cell growth, differentiation and apoptosis, and have significant promise for cancer therapy and chemoprevention. These ligands can determine the ultimate fate of target cells by stimulating or repressing gene expression directly, or indirectly through crosstalking with other signal transducers.Using different breast cancer cell models, we show here that depending on the cellular context retinoids can signal either towards cell death or cell survival. Indeed, retinoids can induce the expression of pro-apoptotic (i.e. TRAIL, TNF-Related Apoptosis-Inducing Ligand, Apo2L/TNFSF10) and anti-apoptotic (i.e. cIAP2, inhibitor of apoptosis protein-2) genes. Promoter mapping, gel retardation and chromatin immunoprecipitation assays revealed that retinoids induce the expression of this gene mainly through crosstalk with NF-kappaB. Supporting this crosstalk, the activation of NF-kappaB by retinoids in T47D cells antagonizes the apoptosis triggered by the chemotherapeutic drugs etoposide, camptothecin or doxorubicin. Notably apoptosis induced by death ligands (i.e. TRAIL or antiFAS) is not antagonized by retinoids. That knockdown of cIAP2 expression by small interfering RNA does not alter the inhibition of etoposide-induced apoptosis by retinoids in T47D cells reveals that stimulation of cIAP2 expression is not the cause of their anti-apoptotic action. However, ectopic overexpression of a NF-kappaB repressor increases apoptosis by retinoids moderately and abrogates almost completely the retinoid-dependent inhibition of etoposide-induced apoptosis. Our data exclude cIAP2 and suggest that retinoids target other regulator(s) of the NF-kappaB signaling pathway to induce resistance to etoposide on certain breast cancer cells.This study shows an important role for the NF-kappaB pathway in retinoic acid signaling and retinoic acid-mediated resistance to cancer therapy-mediated apoptosis in breast cancer cells, independently of cIAP2. Our data support the use of NF-kappaB pathway activation as a marker for screening that will help to develop novel retinoids, or retinoid-based combination therapies with improved efficacy.
|BCL2 and CASP8 regulation by NF-kappaB differentially affect mitochondrial function and cell fate in antiestrogen-sensitive and -resistant breast cancer cells. |
Nehra, R; Riggins, RB; Shajahan, AN; Zwart, A; Crawford, AC; Clarke, R
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 24 2040-55 2010
Resistance to endocrine therapies remains a major problem in the management of estrogen receptor-alpha (ER)-positive breast cancer. We show that inhibition of NF-kappaB (p65/RELA), either by overexpression of a mutant IkappaB (IkappaBSR) or a small-molecule inhibitor of NF-kappaB (parthenolide; IC(50)=500 nM in tamoxifen-resistant cells), synergistically restores sensitivity to 4-hydroxytamoxifen (4HT) in resistant MCF7/RR and MCF7/LCC9 cells and further sensitizes MCF-7 and MCF7/LCC1 control cells to 4HT. These effects are independent of changes in either cell cycle distribution or in the level of autophagy measured by inhibition of p62/SQSTM1 expression and cleavage of LC3. NF-kappaB inhibition restores the ability of 4HT to decrease BCL2 expression, increase mitochondrial membrane permeability, and induce a caspase-dependent apoptotic cell death in resistant cells. Each of these effects is reversed by a caspase 8 (CASP8)-specific inhibitor that blocks enzyme-substrate binding. Thus, increased activation of NF-kappaB can alter sensitivity to tamoxifen by modulating CASP8 activity, with consequent effects on BCL2 expression, mitochondrial function, and apoptosis. These data provide significant new insights into how molecular signaling affects antiestrogen responsiveness and strongly suggest that a combination of parthenolide and tamoxifen may offer a novel therapeutic approach to the management of some ER-positive breast cancers.
|Decoy receptor 3, upregulated by Epstein-Barr virus latent membrane protein 1, enhances nasopharyngeal carcinoma cell migration and invasion. |
Ho, CH; Chen, CL; Li, WY; Chen, CJ
Carcinogenesis 30 1443-51 2009
Decoy receptor 3 (DcR3), a member of tumor necrosis factor receptor superfamily, has been implicated in tumorigenesis through its abilities to modulate immune responses and induce angiogenesis. Epstein-Barr virus (EBV), a ubiquitous gamma-herpesvirus, is associated with malignancies including nasopharyngeal carcinoma (NPC). Previous studies show that DcR3 is overexpressed in EBV-positive lymphomas and Rta, an EBV transcription activator, can upregulate DcR3 in Burkitt lymphoma cell lines. However, DcR3 expression has not been demonstrated in EBV-associated NPC nor have there been any EBV latent genes linked to DcR3 upregulation. Here, we showed DcR3 was overexpressed in NPC. Higher DcR3 expression score and DcR3-positive rate were found in metastatic NPC than in primary NPC tissues, suggesting DcR3 may enhance cell metastatic potential. This hypothesis is supported by our observation that NPC HONE-1 cells overexpressing DcR3 exhibited significant higher migration and invasion abilities in vitro. We found besides Rta, EBV latent membrane protein (LMP) 1 can upregulate DcR3 via nuclear factor-kappaB and phosphatidylinositol 3-kinase-signaling events. Approximate 75% of LMP1-positive NPC tissues overexpressed DcR3, suggesting LMP1 may enhance DcR3 expression in vivo. Data herein suggested that increasing DcR3 expression by LMP1 not only helps EBV-associated cancer cells gain survival advantage by preventing host immune detection but also increases the chance of cancer metastasis by enhancing cell migration and invasion. All these DcR3-mediated events facilitate normal cells to gain cancer hallmarks.
|Control of stochastic gene expression by host factors at the HIV promoter. |
Burnett, JC; Miller-Jensen, K; Shah, PS; Arkin, AP; Schaffer, DV
PLoS pathogens 5 e1000260 2009
The HIV promoter within the viral long terminal repeat (LTR) orchestrates many aspects of the viral life cycle, from the dynamics of viral gene expression and replication to the establishment of a latent state. In particular, after viral integration into the host genome, stochastic fluctuations in viral gene expression amplified by the Tat positive feedback loop can contribute to the formation of either a productive, transactivated state or an inactive state. In a significant fraction of cells harboring an integrated copy of the HIV-1 model provirus (LTR-GFP-IRES-Tat), this bimodal gene expression profile is dynamic, as cells spontaneously and continuously flip between active (Bright) and inactive (Off) expression modes. Furthermore, these switching dynamics may contribute to the establishment and maintenance of proviral latency, because after viral integration long delays in gene expression can occur before viral transactivation. The HIV-1 promoter contains cis-acting Sp1 and NF-kappaB elements that regulate gene expression via the recruitment of both activating and repressing complexes. We hypothesized that interplay in the recruitment of such positive and negative factors could modulate the stability of the Bright and Off modes and thereby alter the sensitivity of viral gene expression to stochastic fluctuations in the Tat feedback loop. Using model lentivirus variants with mutations introduced in the Sp1 and NF-kappaB elements, we employed flow cytometry, mRNA quantification, pharmacological perturbations, and chromatin immunoprecipitation to reveal significant functional differences in contributions of each site to viral gene regulation. Specifically, the Sp1 sites apparently stabilize both the Bright and the Off states, such that their mutation promotes noisy gene expression and reduction in the regulation of histone acetylation and deacetylation. Furthermore, the NF-kappaB sites exhibit distinct properties, with kappaB site I serving a stronger activating role than kappaB site II. Moreover, Sp1 site III plays a particularly important role in the recruitment of both p300 and RelA to the promoter. Finally, analysis of 362 clonal cell populations infected with the viral variants revealed that mutations in any of the Sp1 sites yield a 6-fold higher frequency of clonal bifurcation compared to that of the wild-type promoter. Thus, each Sp1 and NF-kappaB site differentially contributes to the regulation of viral gene expression, and Sp1 sites functionally "dampen" transcriptional noise and thereby modulate the frequency and maintenance of this model of viral latency. These results may have biomedical implications for the treatment of HIV latency.
|Mechanism of UV-induced IkappaBalpha-independent activation of NF-kappaB. |
Csaba F László, Shiyong Wu, Csaba F László, Shiyong Wu, Csaba F László, Shiyong Wu, Csaba F László, Shiyong Wu, Csaba F László, Shiyong Wu
Photochemistry and photobiology 84 1564-8 2008
Nuclear factor-kappa B (NF-kappaB) plays an important role in UV-induced skin tumorigenesis. Activation of NF-kappaB by UV-irradiation is composed of two phases. The early phase culminates with maximal levels of DNA binding ability at 4 h postirradiation and is dependent on translational inhibition. The late-phase activation of NF-kappaB occurs between 16 and 48 h post-irradiation and the mechanism is not clear due to the fact that NF-kappaB was activated in the presence of high level of IkappaBalpha. In this report, we provide evidence that without translational inhibition, the transcription of IkappaBalpha was induced by UV-irradiation. In the late-phase of UV-induced NF-kappaB activation, the IkappaBalpha depletion is the combined result of regulation at both transcriptional and translational levels. Neither ubiquitination nor proteasomal degradation have detectable attributions to IkappaBalpha breakdown. We also demonstrate that UV only induced phosphorylation of p65(S276), while tumor necrosis factor-alpha induced phosphorylation at both Ser276 and 536 sites of p65. Based upon our results, we propose a novel mechanism for translation-regulated IkappaBalpha depletion and MSK-mediated NF-kappaB activation at 24 h post-UV-irradiation.Artículo Texto completo
|NF-kappaB represses E-cadherin expression and enhances epithelial to mesenchymal transition of mammary epithelial cells: potential involvement of ZEB-1 and ZEB-2. |
Chua, HL; Bhat-Nakshatri, P; Clare, SE; Morimiya, A; Badve, S; Nakshatri, H
Oncogene 26 711-24 2007
The transcription factor nuclear factor kappa B (NF-kappaB) is constitutively active in both cancer cells and stromal cells of breast cancer; however, the precise role of activated NF-kappaB in cancer progression is not known. Using parental MCF10A cells and a variant that expresses the myoepithelial marker p63 stably overexpressing the constitutively active p65 subunit of NF-kappaB (MCF10A/p65), we show that NF-kappaB suppresses the expression of epithelial specific genes E-cadherin and desmoplakin and induces the expression of the mesenchymal specific gene vimentin. P65 also suppressed the expression of p63 and the putative breast epithelial progenitor marker cytokeratin 5/6. MCF10A/p65 cells were phenotypically similar to cells undergoing epithelial to mesenchymal transition (EMT). MCF10A/p65 cells failed to form characteristic acini in three-dimensional Matrigel. Analysis of parental and MCF10A/p65 cells for genes previously shown to be involved in EMT revealed elevated expression of ZEB-1 and ZEB-2 in MCF10A/p65 cells compared to parental cells. In transient transfection assays, p65 increased ZEB-1 promoter activity. Furthermore, MCF10A cells overexpressing ZEB-1 showed reduced E-cadherin and p63 expression and displayed an EMT phenotype. The siRNA against ZEB-1 or ZEB-2 reduced the number of viable MCF10A/p65 but not parental cells, suggesting the dependence of MCF10A/p65 cells to ZEB-1 and ZEB-2 for cell cycle progression or survival. MCF10A cells chronically exposed to tumor necrosis factor alpha (TNFalpha), a potent NF-kappaB inducer, also exhibited the EMT-like phenotype and ZEB-1/ZEB-2 induction, both of which were reversed following TNFalpha withdrawal.
|IkappaB kinase alpha-mediated derepression of SMRT potentiates acetylation of RelA/p65 by p300. |
Hoberg, JE; Popko, AE; Ramsey, CS; Mayo, MW
Molecular and cellular biology 26 457-71 2006
Over the last several years, significant progress has been made in identifying chromatin-regulated events that govern NF-kappaB transcription. Using either laminin attachment or tumor necrosis factor alpha as a physiological stimulus of NF-kappaB activation, we demonstrate that IkappaB kinase alpha (IKKalpha) is recruited to chromatin in distinct phases. In the initial phase, IKKalpha is responsible for derepressing the silencing mediator for retinoic acid and thyroid hormone receptor (SMRT)-histone deacetylase 3 (HDAC3) corepressor complex from the p50 homodimer. However, in the latter phase, chromatin-bound IKKalpha coordinates the simultaneous phosphorylation of RelA/p65(S536) and SMRT(S2410) as detected by chromatin immunoprecipitation (ChIP) assays. Although phosphorylated SMRT remains bound to the active p50-RelA/p65 heterodimer of NF-kappaB, derepression of SMRT is evidenced by the loss of chromatin-associated HDAC3 activity. ChIP and re-ChIP analysis demonstrates that phosphorylation of RelA/p65(S536) and SMRT(S2410) occurs prior to acetylation of RelA/p65 at K310. Moreover, IKKalpha-induced phosphorylation of RelA/p65(S536) displaces corepressor activity, allowing p300-mediated acetylation of RelA/p65. Introduction of nonphosphorylatable mutants of RelA/p65 and SMRT proteins or the inhibition of IKK activity results in active repression of NF-kappaB promoters by tethering the SMRT-HDAC3 complex. Similar to phosphorylation within the Rel homology domain of RelA/p65, which governs an exchange of HDAC1 for CBP/p300 acetyltransferases, we demonstrate that phosphorylation within the transactivation domain of RelA/p65(S536) displaces SMRT-HDAC3 repressor activity, allowing p300 to acetylate RelA/p65.
|Nuclear factor-kappaB induced by doxorubicin is deficient in phosphorylation and acetylation and represses nuclear factor-kappaB-dependent transcription in cancer cells. |
Ho, WC; Dickson, KM; Barker, PA
Cancer research 65 4273-81 2005
The primary goal of chemotherapy is to cause cancer cell death. However, a side effect of many commonly used chemotherapeutic drugs is the activation of nuclear factor-kappaB (NF-kappaB), a potent inducer of antiapoptotic genes, which may blunt the therapeutic efficacy of these compounds. We have assessed the effect of doxorubicin, an anthracycline in widespread clinical use, on NF-kappaB activation and expression of antiapoptotic genes in breast cancer cells. We show that doxorubicin treatment activates NF-kappaB signaling and produces NF-kappaB complexes that are competent for NF-kappaB binding in vitro. Surprisingly, these NF-kappaB complexes suppress, rather than activate, constitutive- and cytokine-induced NF-kappaB-dependent transcription. We show that doxorubicin treatment produces RelA, which is deficient in phosphorylation and acetylation and which blocks NF-kappaB signaling in a histone deacetylase-independent manner, and we show that NF-kappaB activated by doxorubicin does not remain stably bound to kappaB elements in vivo. Together these data show that NF-kappaB signaling induced by doxorubicin reduces expression of NF-kappaB-dependent genes in cancer cells.
|COMMD proteins, a novel family of structural and functional homologs of MURR1. |
Burstein, E; Hoberg, JE; Wilkinson, AS; Rumble, JM; Csomos, RA; Komarck, CM; Maine, GN; Wilkinson, JC; Mayo, MW; Duckett, CS
The Journal of biological chemistry 280 22222-32 2005
MURR1 is a multifunctional protein that inhibits nuclear factor kappaB (NF-kappaB), a transcription factor with pleiotropic functions affecting innate and adaptive immunity, apoptosis, cell cycle regulation, and oncogenesis. Here we report the discovery of a new family of proteins with homology to MURR1. These proteins form multimeric complexes and were identified in a biochemical screen for MURR1-associated factors. The family is defined by the presence of a conserved and unique motif termed the COMM (copper metabolism gene MURR1) domain, which functions as an interface for protein-protein interactions. Like MURR1, several of these factors also associate with and inhibit NF-kappaB. The proteins designated as COMMD or COMM domain containing 1-10 are extensively conserved in multicellular eukaryotic organisms and define a novel family of structural and functional homologs of MURR1. The prototype of this family, MURR1/COMMD1, suppresses NF-kappaB not by affecting nuclear translocation or binding of NF-kappaB to cognate motifs; rather, it functions in the nucleus by affecting the association of NF-kappaB with chromatin.
|Expression of the GTP-binding protein (Galphas) is repressed by the nuclear factor kappaB RelA subunit in human myometrium. |
Chapman, NR; Smyrnias, I; Anumba, DO; Europe-Finner, GN; Robson, SC
Endocrinology 146 4994-5002 2005
In humans, the factors that govern the switch from myometrial quiescence to coordinated contractions at the initiation of labor are not well defined. The onset of parturition is itself associated with increases in a number of proinflammatory mediators, many of which are regulated by the nuclear factor kappaB (NF-kappaB) family of transcription factors. Recently, we have provided evidence that the RelA NF-kappaB subunit associates with protein kinase A in pregnant myometrial tissue, suggesting links with the Galphas/cAMP/protein kinase A pathway. TNFalpha is a potent activator of NF-kappaB, and levels of this cytokine are increased within the myometrium at term. In the current study, using primary cultures of myometrial cells, TNFalpha was observed to repress expression of Galphas while, at the same time, stimulating NF-kappaB activity. Furthermore, this effect could be replicated by exposure to bacterial lipopolysaccharide and exogenous expression of RelA. Moreover, TNFalpha was seen to repress endogenous Galphas mRNA expression as judged by RT-PCR analyses. Using the chromatin immunoprecipitation assay, we show that RelA did not bind directly to the Galphas promoter. Significantly, expression of a coactivator protein, cAMP response element binding protein binding protein, relieved RelA-induced down-regulation of Galphas expression. Together, these data suggest that, in human myometrium, repression of the Galphas gene by NF-kappaB occurs through a non-DNA binding mechanism involving competition for limiting amounts of cellular coactivator proteins including cAMP response element binding protein binding protein.
|Ineffectiveness of histone deacetylase inhibitors to induce apoptosis involves the transcriptional activation of NF-kappa B through the Akt pathway |
Mayo, M. W., et al
J Biol Chem, 278:18980-9 (2003) 2003
|Chromatin Immunoprecipitation (ChIP)||12649266|
|Apoptotic action of 17beta-estradiol in raloxifene-resistant MCF-7 cells in vitro and in vivo. |
Hong Liu, Eun-Sook Lee, Csaba Gajdos, Sandra Timm Pearce, Bin Chen, Clodia Osipo, Jessica Loweth, Kevin McKian, Alexander De Los Reyes, Laura Wing, V Craig Jordan
Journal of the National Cancer Institute 95 1586-97 2003
BACKGROUND: Resistance to tamoxifen, a selective estrogen receptor modulator (SERM), involves changes that prevent apoptosis and enhance cell proliferation and survival. Paradoxically, estrogen treatment inhibits the growth of long-term tamoxifen-treated breast tumors. Because of the increasing use of raloxifene, another SERM, to prevent osteoporosis and potentially reduce breast cancer risk, some women will develop raloxifene-resistant breast cancer. We developed a raloxifene-resistant MCF-7 cell model (MCF-7/Ral) and investigated the nature of raloxifene-resistant breast cancer and its response to estradiol. METHODS: Raloxifene resistance and hormone responsiveness were assessed by proliferation assays and cell cycle analysis in parental MCF-7 and MCF-7/Ral cells. Nuclear factor kappaB (NF-kappaB) activity was investigated with a transient transfection assay. Apoptosis was investigated by annexin V staining, mRNA was measured by real-time polymerase chain reaction, and protein was measured by western blotting. Tumorigenesis was studied by injecting MCF-7 or MCF-7/Ral cells into ovariectomized athymic mice (10 per group) and monitoring tumor size weekly. All statistical tests were two-sided. RESULTS: Basal NF-kappaB activity was higher in MCF-7/Ral cells (1.6 U, 95% confidence interval [CI] = 1.2 to 2.0 U) than in MCF-7 cells (0.8 U, 95% CI = 0.4 to 1.1 U; P =.004). When cultured with 1 microM raloxifene, MCF-7/Ral cells grew statistically significantly (P.001) faster than MCF-7 cells. Estradiol treatment of MCF-7/Ral cells arrested cells in G(2)/M phase of the cell cycle, decreased NF-kappaB activity (0.2 U, 95% CI = 0.2 to 0.3 U; P.001), increased expression of Fas protein and mRNA (4.5-fold, 95% CI = 2.8- to 6.3-fold versus 0.5-fold, 95% CI = 0.3- to 0.8-fold for control treatment; P.001), and induced apoptosis. Treatment with either raloxifene or tamoxifen stimulated MCF-7/Ral tumor growth, suggesting that such tumors were resistant to both drugs. When a 9-week raloxifene or tamoxifen treatment was followed by a 5-week estradiol treatment, estradiol statistically significantly reduced the size of tumors stimulated by raloxifene or tamoxifen (at week 14, P =.004 for raloxifene and P.001 for tamoxifen). CONCLUSIONS: Growth of raloxifene-resistant MCF-7/Ral cells in vitro and in vivo is repressed by estradiol treatment by a mechanism involving G2/M-phase arrest, decreased NF-kappaB activity, and increased Fas expression to induce apoptosis.
|Generation of destabilized green fluorescent protein as a transcription reporter |
Li, X., et al
J Biol Chem, 273:34970-5 (1998) 1998
|A family of serine proteases expressed exclusively in myelo-monocytic cells specifically processes the nuclear factor-kappa B subunit p65 in vitro and may impair human immunodeficiency virus replication in these cells. |
Franzoso, G, et al.
J. Exp. Med., 180: 1445-56 (1994) 1994
Two groups of U937 promonocytic cells were obtained by limiting dilution cloning which differed strikingly in their ability to support human immunodeficiency virus 1 (HIV-1) replication. "Plus" clones replicated the virus efficiently, whereas "minus" clones did not. We examined these clones for differences in nuclear factor (NF)-kappa B activity which might account for the observed phenomenon. Stimulation of plus clones liberated the classical p50-p65 complex from cytoplasmic pools, whereas minus clones produced an apparently novel, faster-migrating complex, as judged by electrophoretic mobility shift assays. It is surprising that the faster-migrating complex was composed also of p50 and p65. However, the p65 subunit was COOH-terminally truncated, as shown by immunoprecipitation. The truncation resulted from limited proteolysis of p65 during cellular extraction which released particular lysosomal serine proteases, such as elastase, cathepsin G, and proteinase 3. These specific proteases are coordinately expressed and were present exclusively in the minus U937 clones, but not in the plus clones, as demonstrated in the case of cathepsin G. In addition, these proteases were detected in certain subclones of THP-1 and HL-60 cells and in primary monocytes, in each case correlating with the truncated from of p65. We demonstrate in vitro cleavage of p65 by purified elastase and cathepsin G. It is possible that particular serine proteases may have inhibiting effects on the replication of HIV-1 in myelo-monocytic cells. The data also demonstrate that special precautions must be taken when making extracts from myelo-monocytic cells.