Tumor Necrosis Factor-α Regulates Distinct Molecular Pathways and Gene Networks in Cultured Skeletal Muscle Cells

Background: Skeletal muscle wasting is a debilitating consequence of large number of disease states and conditions. Tumor necrosis factor-alpha (TNF-alpha) 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-alpha causes skeletal muscle wasting are less well-understood. Methodology/Principal Findings: We have used microarray, quantitative real-time PCR (QRT-PCR), Western blot, and bioinformatics tools to study the effects of TNF-alpha on various molecular pathways and gene networks in C2C12 cells (a mouse myoblastic cell line). Microarray analyses of C2C12 myotubes treated with TNF-alpha (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-alpha. 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-alpha augments the activity of both canonical and alternative NF-kappa B signaling pathways in myotubes. Bioinformatics analyses of microarray dataset revealed that TNF-alpha affects the activity of several important pathways including those involved in oxidative stress, hepatic fibrosis, mitochondrial dysfunction, cholesterol biosynthesis, and TGF-beta signaling. Furthermore, TNF-alpha was found to affect the gene networks related to drug metabolism, cell cycle, cancer, neurological disease, organismal injury, and abnormalities in myotubes. Conclusions: TNF-alpha 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-alpha activates both canonical and alternative NF-kappa B signaling pathways in a time-dependent manner in skeletal muscle cells. The study provides novel insight into the mechanisms of action of TNF-alpha in skeletal muscle cells.