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Tumor necrosis factor-? regulates distinct molecular pathways and gene networks in cultured skeletal muscle cells.

ABSTRACT: Skeletal muscle wasting is a debilitating consequence of large number of disease states and conditions. Tumor necrosis factor-? (TNF-?) is one of the most important muscle-wasting cytokine, elevated levels of which cause significant muscular abnormalities. However, the underpinning molecular mechanisms by which TNF-? causes skeletal muscle wasting are less well-understood.We have used microarray, quantitative real-time PCR (QRT-PCR), Western blot, and bioinformatics tools to study the effects of TNF-? on various molecular pathways and gene networks in C2C12 cells (a mouse myoblastic cell line). Microarray analyses of C2C12 myotubes treated with TNF-? (10 ng/ml) for 18h showed differential expression of a number of genes involved in distinct molecular pathways. The genes involved in nuclear factor-kappa B (NF-kappaB) signaling, 26s proteasome pathway, Notch1 signaling, and chemokine networks are the most important ones affected by TNF-?. The expression of some of the genes in microarray dataset showed good correlation in independent QRT-PCR and Western blot assays. Analysis of TNF-treated myotubes showed that TNF-? augments the activity of both canonical and alternative NF-?B signaling pathways in myotubes. Bioinformatics analyses of microarray dataset revealed that TNF-? affects the activity of several important pathways including those involved in oxidative stress, hepatic fibrosis, mitochondrial dysfunction, cholesterol biosynthesis, and TGF-? signaling. Furthermore, TNF-? was found to affect the gene networks related to drug metabolism, cell cycle, cancer, neurological disease, organismal injury, and abnormalities in myotubes.TNF-? regulates the expression of multiple genes involved in various toxic pathways which may be responsible for TNF-induced muscle loss in catabolic conditions. Our study suggests that TNF-? activates both canonical and alternative NF-?B signaling pathways in a time-dependent manner in skeletal muscle cells. The study provides novel insight into the mechanisms of action of TNF-? in skeletal muscle cells.

SUBMITTER: Bhatnagar S

PROVIDER: S-EPMC2953497 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Tumor necrosis factor-α regulates distinct molecular pathways and gene networks in cultured skeletal muscle cells.

Bhatnagar Shephali S Panguluri Siva K SK Gupta Sanjay K SK Dahiya Saurabh S Lundy Robert F RF Kumar Ashok A

PloS one 20101012 10

<h4>Background</h4>Skeletal muscle wasting is a debilitating consequence of large number of disease states and conditions. Tumor necrosis factor-α (TNF-α) is one of the most important muscle-wasting cytokine, elevated levels of which cause significant muscular abnormalities. However, the underpinning molecular mechanisms by which TNF-α causes skeletal muscle wasting are less well-understood.<h4>Methodology/principal findings</h4>We have used microarray, quantitative real-time PCR (QRT-PCR), West ...[more]

PMID: 20967264

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Project description:FBXL2 is an important ubiquitin E3 ligase component that modulates inflammatory signaling and cell cycle progression, but its molecular regulation is largely unknown. Here, we show that tumor necrosis factor alpha (TNF-α), a critical cytokine linked to the inflammatory response during skeletal muscle regeneration, suppressed Fbxl2 mRNA expression in C2C12 myoblasts and triggered significant alterations in cell cycle, metabolic, and protein translation processes. Gene silencing of Fbxl2 in skeletal myoblasts resulted in increased proliferative responses characterized by activation of mitogen-activated protein (MAP) kinases and nuclear factor kappa B and decreased myogenic differentiation, as reflected by reduced expression of myogenin and impaired myotube formation. TNF-α did not destabilize the Fbxl2 transcript (half-life [t1/2], ∼10 h) but inhibited SP1 transactivation of its core promoter, localized to bp -160 to +42 within the proximal 5' flanking region of the Fbxl2 gene. Chromatin immunoprecipitation and gel shift studies indicated that SP1 interacted with the Fbxl2 promoter during cellular differentiation, an effect that was less pronounced during proliferation or after TNF-α exposure. TNF-α, via activation of JNK, mediated phosphorylation of SP1 that impaired its binding to the Fbxl2 promoter, resulting in reduced transcriptional activity. The results suggest that SP1 transcriptional activation of Fbxl2 is required for skeletal muscle differentiation, a process that is interrupted by a key proinflammatory myopathic cytokine.IMPORTANCE Skeletal muscle regeneration and repair involve the recruitment and proliferation of resident satellite cells that exit the cell cycle during the process of myogenic differentiation to form myofibers. We demonstrate that the ubiquitin E3 ligase subunit FBXL2 is essential for skeletal myogenesis through its important effects on cell cycle progression and cell proliferative signaling. Further, we characterize a new mechanism whereby sustained stimulation by a major proinflammatory cytokine, TNF-α, regulates skeletal myogenesis by inhibiting the interaction of SP1 with the Fbxl2 core promoter in proliferating myoblasts. Our findings contribute to the understanding of skeletal muscle regeneration through the identification of Fbxl2 as both a critical regulator of myogenic proliferative processes and a susceptible gene target during inflammatory stimulation by TNF-α in skeletal muscle. Modulation of Fbxl2 activity may have relevance to disorders of muscle wasting associated with sustained proinflammatory signaling.

Project description:TNF-? levels are increased during muscle wasting and chronic muscle degeneration and regeneration processes, which are characteristic for primary muscle disorders. Pathologically increased TNF-? levels have a negative effect on muscle cell differentiation efficiency, while IGF1 can have a positive effect; therefore, we intended to elucidate the impact of TNF-? and IGF1 on gene expression during the early stages of skeletal muscle cell differentiation.This study presents gene expression data of the murine skeletal muscle cells PMI28 during myogenic differentiation or differentiation with TNF-? or IGF1 exposure at 0 h, 4 h, 12 h, 24 h, and 72 h after induction. Our study detected significant coregulation of gene sets involved in myoblast differentiation or in the response to TNF-?. Gene expression data revealed a time- and treatment-dependent regulation of signaling pathways, which are prominent in myogenic differentiation. We identified enrichment of pathways, which have not been specifically linked to myoblast differentiation such as doublecortin-like kinase pathway associations as well as enrichment of specific semaphorin isoforms. Moreover to the best of our knowledge, this is the first description of a specific inverse regulation of the following genes in myoblast differentiation and response to TNF-?: Aknad1, Cmbl, Sepp1, Ndst4, Tecrl, Unc13c, Spats2l, Lix1, Csdc2, Cpa1, Parm1, Serpinb2, Aspn, Fibin, Slc40a1, Nrk, and Mybpc1. We identified a gene subset (Nfkbia, Nfkb2, Mmp9, Mef2c, Gpx, and Pgam2), which is robustly regulated by TNF-? across independent myogenic differentiation studies.This is the largest dataset revealing the impact of TNF-? or IGF1 treatment on gene expression kinetics of early in vitro skeletal myoblast differentiation. We identified novel mRNAs, which have not yet been associated with skeletal muscle differentiation or response to TNF-?. Results of this study may facilitate the understanding of transcriptomic networks underlying inhibited muscle differentiation in inflammatory diseases.

Project description:BackgroundMuscle atrophy associated with various pathophysiological conditions represents a major health problem, because of its contribution to the deterioration of patient status and its effect on mortality. Although the involvement of pro-inflammatory cytokines in this process is well recognized, the role of sphingolipid metabolism alterations induced by the cytokines has received little attention.ResultsWe addressed this question both in vitro using differentiated myotubes treated with TNF-α, and in vivo in a murine model of tumor-induced cachexia. Myotube atrophy induced by TNF-α was accompanied by a substantial increase in cell ceramide levels, and could be mimicked by the addition of exogenous ceramides. It could be prevented by the addition of ceramide-synthesis inhibitors that targeted either the de novo pathway (myriocin), or the sphingomyelinases (GW4869 and 3-O-methylsphingomyelin). In the presence of TNF-α, ceramide-synthesis inhibitors significantly increased protein synthesis and decreased proteolysis. In parallel, they lowered the expression of both the Atrogin-1 and LC3b genes, involved in muscle protein degradation by proteasome and in autophagic proteolysis, respectively, and increased the proportion of inactive, phosphorylated Foxo3 transcription factor. Furthermore, these inhibitors increased the expression and/or phosphorylation levels of key factors regulating protein metabolism, including phospholipase D, an activator of mammalian target of rapamycin (mTOR), and the mTOR substrates S6K1 and Akt. In vivo, C26 carcinoma implantation induced a substantial increase in muscle ceramide, together with drastic muscle atrophy. Treatment of the animals with myriocin reduced the expression of the atrogenes Foxo3 and Atrogin-1, and partially protected muscle tissue from atrophy.ConclusionsCeramide accumulation induced by TNF-α or tumor development participates in the mechanism of muscle-cell atrophy, and sphingolipid metabolism is a logical target for pharmacological or nutritional interventions aiming at preserving muscle mass in pathological situations.

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Tumor necrosis factor-? regulates distinct molecular pathways and gene networks in cultured skeletal muscle cells.

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Tumor necrosis factor-α regulates distinct molecular pathways and gene networks in cultured skeletal muscle cells.

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