Tumor necrosis factor-α regulates skeletal myogenesis by inhibiting SP1 interaction with cis -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 TNFα, 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. TNFα did not destabilize the Fbxl2 transcript (t ½ ∼10 h), but inhibited SP1 transactivation of its core promoter, localized to 160/+42 base pairs 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 less pronounced during proliferation or after TNFα exposure. TNFα, 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 pro-inflammatory myopathic cytokine. Importance Skeletal muscle regeneration and repair involves 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 pro-inflammatory cytokine, TNFα, 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 TNFα in skeletal muscle. Modulation of Fbxl2 activity may have relevance to disorders of muscle wasting associated with sustained pro-inflammatory signaling.