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-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.


RESULTS
We have used microarray, quantitative real-time PCR (QRT-PCR), Western blot, and bioinformatics tools to study the effects of TNF-α on various molecular pathways and gene networks in C2C12 cells (a mouse myoblastic cell line). Microarray analyses of C2C12 myotubes treated with TNF-α (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-α. 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-α augments the activity of both canonical and alternative NF-κB signaling pathways in myotubes. Bioinformatics analyses of microarray dataset revealed that TNF-α affects the activity of several important pathways including those involved in oxidative stress, hepatic fibrosis, mitochondrial dysfunction, cholesterol biosynthesis, and TGF-β signaling. Furthermore, TNF-α was found to affect the gene networks related to drug metabolism, cell cycle, cancer, neurological disease, organismal injury, and abnormalities in myotubes.


CONCLUSIONS
TNF-α 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-α activates both canonical and alternative NF-κB signaling pathways in a time-dependent manner in skeletal muscle cells. The study provides novel insight into the mechanisms of action of TNF-α in skeletal muscle cells.
Department of Anatomical Sciences and Neurobiology, University of Louisville School of Medicine, Louisville, Kentucky, United States of America.
Shephali
S
Bhatnagar
Siva K
SK
Panguluri
Sanjay K
SK
Gupta
Saurabh
S
Dahiya
Robert F
RF
Lundy
Ashok
A
Kumar
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D015536:Down-Regulation; D020869:Gene Expression Profiling; D053263:Gene Regulatory Networks; D051379:Mice; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*; D015854:Up-Regulation
D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha