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Tumor Necrosis Factor Alpha Regulates Skeletal Myogenesis by Inhibiting SP1 Interaction with cis-Acting Regulatory Elements within the Fbxl2 Gene Promoter.
Molecular and Cellular Biology ( IF 5.3 ) Pub Date : 2020-05-28 , DOI: 10.1128/mcb.00040-20
Michael E O'Brien 1 , James Londino 2 , Marcus McGinnis 1 , Nathaniel Weathington 1 , Jessica Adair 2 , Tomeka Suber 1 , Valerian Kagan 1 , Kong Chen 1 , Chunbin Zou 1 , Bill Chen 1 , Jessica Bon 1 , Rama K Mallampalli 3
Affiliation  

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 [t 1/2], ∼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.

中文翻译:

肿瘤坏死因子α通过抑制SP1与Fbxl2基因启动子内顺式作用调节元件的相互作用来调节骨骼肌新生。

FBXL2是重要的泛素E3连接酶成分,可调节炎症信号传导和细胞周期进程,但其分子调控很大程度上未知。在这里,我们显示肿瘤坏死因子α(TNF-α)是与骨骼肌再生过程中的炎症反应相关的关键细胞因子,抑制了C2C12成肌细胞中Fbxl2 mRNA的表达并触发了细胞周期,代谢和蛋白质翻译过程的显着改变。骨骼肌成肌细胞中Fbxl2的基因沉默导致增殖反应增加,其特征在于有丝分裂原激活的蛋白(MAP)激酶和核因子kappa B的激活以及成肌分化的降低,这反映在成肌蛋白表达减少和肌管形成受损中。TNF-α不会破坏Fbxl2转录本的稳定性(半衰期[t 1/2],约10小时),但抑制了SP1核心启动子的反式激活,该启动子位于Fbxl2基因近端5'侧翼区域内的bp -160至+42。染色质的免疫沉淀和凝胶迁移研究表明,SP1在细胞分化过程中与Fbxl2启动子相互作用,这种作用在增殖过程中或在TNF-α暴露后并不明显。TNF-α通过JNK的激活介导SP1的磷酸化,从而削弱SP1与Fbxl2启动子的结合,从而导致转录活性降低。结果表明,骨骼肌分化需要Fbxl2的SP1转录激活,这一过程被关键的促炎性肌病细胞因子打断。重要信息骨骼肌的再生和修复涉及在生肌分化形成肌纤维的过程中退出细胞周期的常驻卫星细胞的募集和增殖。我们证明,泛素E3连接酶亚基FBXL2通过其对细胞周期进程和细胞增殖信号的重要作用,对于骨骼肌新生是必不可少的。此外,我们表征了一种新的机制,通过该机制,主要的促炎细胞因子TNF-α的持续刺激通过抑制SP1与成肌细胞中Fbxl2核心启动子的相互作用来调节骨骼肌新生。我们的发现通过鉴定Fbxl2作为肌源性增生过程的关键调节剂以及骨骼肌中TNF-α炎症刺激过程中的易感基因靶标,有助于对骨骼肌再生的理解。Fbxl2活性的调节可能与与持续的促炎性信号传导相关的肌肉萎缩症有关。
更新日期:2020-03-23
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