|Atypical behavior of NFATc1 in cultured intercostal myofibers.|
Robison, P; Hernández-Ochoa, EO; Schneider, MF
The NFATc transcription factor family is responsible for coupling cytoplasmic calcium signals to transcription programs in a wide variety of cell types. In skeletal muscle, these transcription factors control the fiber type in response to muscle activity. This excitation-transcription (E-T) coupling permits functional adaptation of muscle according to use. The activity dependence of these transcription programs is sensitive to the firing patterns of the muscle, not merely the period of activity, enabling a nuanced adaptation to various functional tasks.Isolated skeletal muscle fibers expressing exogenous fluorescent NFATc1 were studied by confocal microscopy under stimulation both with and without pharmacological inhibitors. Western blots of whole muscle lysates were also used.This study investigates the activity dependent response of NFATc1 skeletal muscle fibers cultured from mice, comparing fibers of respiratory origin to muscles responsible for limb locomotion. Using patterns of stimulation known to strongly activate NFATc1 in the commonly cultured flexor digitorum brevis and soleus muscles, we have observed significant deactivation of NFATc1 in cultured intercostal muscle fibers. This effect is at least partially dependent on the action of JNK and CaMKII in intercostal fibers.Our findings highlight the role of lineage in the NFAT pathway, showing that the respiratory intercostal muscle fibers decode similar E-T coupling signals into NFAT transcriptional programs in a different manner from the more commonly studied locomotor muscles of the limbs.
|Kaposi's sarcoma herpesvirus K15 protein contributes to virus-induced angiogenesis by recruiting PLCγ1 and activating NFAT1-dependent RCAN1 expression.|
Bala, K; Bosco, R; Gramolelli, S; Haas, DA; Kati, S; Pietrek, M; Hävemeier, A; Yakushko, Y; Singh, VV; Dittrich-Breiholz, O; Kracht, M; Schulz, TF
Kaposi's Sarcoma (KS), caused by Kaposi's Sarcoma Herpesvirus (KSHV), is a highly vascularised angiogenic tumor of endothelial cells, characterized by latently KSHV-infected spindle cells and a pronounced inflammatory infiltrate. Several KSHV proteins, including LANA-1 (ORF73), vCyclin (ORF72), vGPCR (ORF74), vIL6 (ORF-K2), vCCL-1 (ORF-K6), vCCL-2 (ORF-K4) and K1 have been shown to exert effects that can lead to the proliferation and atypical differentiation of endothelial cells and/or the secretion of cytokines with angiogenic and inflammatory properties (VEGF, bFGF, IL6, IL8, GROα, and TNFβ). To investigate a role of the KSHV K15 protein in KSHV-mediated angiogenesis, we carried out a genome wide gene expression analysis on primary endothelial cells infected with KSHV wildtype (KSHVwt) and a KSHV K15 deletion mutant (KSHVΔK15). We found RCAN1/DSCR1 (Regulator of Calcineurin 1/Down Syndrome critical region 1), a cellular gene involved in angiogenesis, to be differentially expressed in KSHVwt- vs KSHVΔK15-infected cells. During physiological angiogenesis, expression of RCAN1 in endothelial cells is regulated by VEGF (vascular endothelial growth factor) through a pathway involving the activation of PLCγ1, Calcineurin and NFAT1. We found that K15 directly recruits PLCγ1, and thereby activates Calcineurin/NFAT1-dependent RCAN1 expression which results in the formation of angiogenic tubes. Primary endothelial cells infected with KSHVwt form angiogenic tubes upon activation of the lytic replication cycle. This effect is abrogated when K15 is deleted (KSHVΔK15) or silenced by an siRNA targeting the K15 expression. Our study establishes K15 as one of the KSHV proteins that contribute to KSHV-induced angiogenesis.
|Sustained postexercise increases in AS160 Thr642 and Ser588 phosphorylation in skeletal muscle without sustained increases in kinase phosphorylation.|
Schweitzer, GG; Arias, EB; Cartee, GD
Journal of applied physiology (Bethesda, Md. : 1985)
Prior exercise by rats can induce a sustained increase in muscle Akt substrate of 160 kDa (AS160) phosphorylation on Thr(642) (pAS160(Thr642)). Because phosphorylation of AS160 on both AS160(Thr642) and AS160(Ser588) is important for insulin-stimulated glucose transport (GT), we determined if exercise would also induce a sustained increase in pAS160(Ser588) concomitant with persistently elevated pAS160(Thr642) and GT. Given that the mechanisms for sustained postexercise (PEX) effects on pAS160 were uncertain, we also studied the four kinases known to phosphorylate AS160 (Akt, AMPK, RSK, and SGK1). In addition, because the serine/threonine phosphatase(s) that dephosphorylate muscle AS160 were previously unidentified, we assessed the ability of four serine/threonine phosphatases (PP1, PP2A, PP2B, and PP2C) to dephosphorylate AS160. We also evaluated exercise effects on posttranslational modifications (Tyr(307) and Leu(309)) that regulate PP2A. In isolated epitrochlearis muscles from rats, GT at 3hPEX with insulin significantly (P less than 0.05) exceeded SED controls. Muscles from 0hPEX vs. 0hSED and 3hPEX vs. 3hSED rats had greater pAS160(Thr642) and pAS160(Ser588). AMPK was the only kinase with greater phosphorylation at 0hPEX vs. 0hSED, and none had greater phosphorylation at 3hPEX vs. 3hSED. Each phosphatase was able to dephosphorylate pAS160(Thr642) and pAS160(Ser588) in cell-free assays. Exercise did not alter posttranslational modifications of PP2A. Our results revealed: 1) pAMPK as a potential trigger for increased pAS160(Thr642) and pAS160(Ser588) at 0hPEX; 2) PP1, PP2A, PP2B, and PP2C were each able to dephosphorylate AS160; and 3) sustained PEX-induced elevations of pAS160(Thr642) and pAS160(Ser588) were attributable to mechanisms other than persistent phosphorylation of known AS160 kinases or altered posttranslational modifications of PP2A.
|Divergent effects of GLP-1 analogs exendin-4 and exendin-9 on the expression of myosin heavy chain isoforms in C2C12 myotubes.|
Lina Wang,Feng Guo,Shi Wei,Ruqian Zhao
Exendin 1-39 amide (Ex-4) and its truncated form exendin 9-39 amide (Ex-9) are peptides of non-mammalian nature, which act as an agonist and antagonist, respectively, of the glucagon-like peptide-1 (GLP-1) receptor in mammals. GLP-1 is an intestinal peptide that plays an important role in the regulation of glucose metabolism and glucose uptake in skeletal muscle; however, the effects of its two analogs (Ex-4 and Ex-9) on myofiber properties are still unclear. Here, we report the effects of Ex-4 and Ex-9 alone or in combination on the myosin heavy chain (MyHC) type composition and the glucose uptake capacity in differentiated C2C12 myotubes. Neither Ex-4 nor Ex-9 altered basal glucose uptake, whereas Ex-9 significantly increased insulin-stimulated glucose uptake, suggesting enhanced insulin sensitivity. The mRNA expression of MyHC I and 2A as well as the percentage of MyHC I protein was remarkably increased in Ex-9-treated myotubes. In contrast, Ex-4, alone or in combination with Ex-9, caused a significant reduction in MyHC 2A mRNA expression and the percentage of MyHC I protein. Consistent with the MyHC type switching peroxisome proliferator-activated receptor-? coactivator (PGC)-1? expression in myotubes was remarkably increased by Ex-9 yet was significantly inhibited by Ex-4. In addition, intracellular concentrations of free Ca(2+) were increased in all treatment groups, but only Ex-9-treated myotubes showed higher calcineurin A protein content. Taken together, our data suggest that Ex-9 promotes oxidative differentiation in myotubes to improve cell insulin sensitivity, probably through calcineurin and PGC-1? mediated pathways.