Tumor Necrosis Factor Alpha Regulates Skeletal Myogenesis by Inhibiting SP1 Interaction with -Acting Regulatory Elements within the Fbxl2 Gene Promoter.

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 mRNA expression in C2C12 myoblasts and triggered significant alterations in cell cycle, metabolic, and protein translation processes. Gene silencing of 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 transcript (half-life [], ∼10 h) but inhibited SP1 transactivation of its core promoter, localized to bp -160 to +42 within the proximal 5' flanking region of the gene. Chromatin immunoprecipitation and gel shift studies indicated that SP1 interacted with the 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 promoter, resulting in reduced transcriptional activity. The results suggest that SP1 transcriptional activation of is required for skeletal muscle differentiation, a process that is interrupted by a key proinflammatory myopathic cytokine. 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 core promoter in proliferating myoblasts. Our findings contribute to the understanding of skeletal muscle regeneration through the identification of 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 activity may have relevance to disorders of muscle wasting associated with sustained proinflammatory signaling.