Department of Kinesiology, Human Performance Laboratory, College of Health and Human Performance, East Carolina University, Greenville, North Carolina; Department of Biochemistry and Molecular Biology, Department of Physiology, Brody School of Medicine, East Carolina University, Greenville, North Carolina; and East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, North Carolina.
Department of Kinesiology, Human Performance Laboratory, College of Health and Human Performance, East Carolina University, Greenville, North Carolina; East Carolina Diabetes and Obesity Institute, East Carolina University, Greenville, North Carolina.
Am J Physiol Cell Physiol. 2014 Aug 1;307(3):C278-87. doi: 10.1152/ajpcell.00391.2013. Epub 2014 Jun 11.
Muscle-specific RING finger-1 (MuRF-1), a ubiquitin ligase and key regulator of proteasome-dependent protein degradation, is highly expressed during skeletal muscle atrophy. The transcription factor forkhead box O3 (FoxO3) induces MuRF-1 expression, but the direct role of other major atrophy-related transcription factors, such as SMAD3, is largely unknown. The goal of this study was to determine whether SMAD3 individually regulates, or with FoxO3 coordinately regulates, MuRF-1 expression. In cultured myotubes or human embryonic kidney cells, MuRF-1 mRNA content and promoter activity were increased by FoxO3 but not by SMAD3 overexpression. However, FoxO3 and SMAD3 coexpression synergistically increased MuRF-1 mRNA and promoter activity. Mutation of the SMAD-binding element (SBE) in the proximal MuRF-1 promoter or overexpression of a SMAD3 DNA-binding mutant attenuated FoxO3-dependent MuRF-1 promoter activation, showing that SMAD binding to DNA is required for optimal activation of FoxO3-induced transcription of MuRF-1. Using chromatin immunoprecipitation, SMAD3 DNA binding increased FoxO3 abundance and SBE mutation reduced FoxO3 abundance on the MuRF-1 promoter. Furthermore, SMAD3 overexpression dose-dependently increased FoxO3 protein content, and coexpression of FoxO3 and SMAD3 synergistically increased FoxO-dependent gene transcription [assessed with a FoxO response element (FRE)-driven reporter]. Collectively, these results show that SMAD3 regulates transcription of MuRF-1 by increasing FoxO3 binding at a conserved FRE-SBE motif within the proximal promoter region, and by increasing FoxO3 protein content and transcriptional activity. These data are the first to indicate that two major transcription factors regulating protein degradation, FoxO3 and SMAD3, converge to coordinately and directly regulate transcription of MuRF-1.
肌肉特异性环指蛋白 1（MuRF-1）是一种泛素连接酶，也是蛋白酶体依赖性蛋白降解的关键调节因子，在骨骼肌萎缩过程中高度表达。转录因子叉头框 O3（FoxO3）诱导 MuRF-1 的表达，但其他主要的萎缩相关转录因子（如 SMAD3）的直接作用在很大程度上尚不清楚。本研究的目的是确定 SMAD3 是否单独调节，或与 FoxO3 协调调节 MuRF-1 的表达。在培养的肌管或人胚肾细胞中，FoxO3 而非 SMAD3 过表达增加 MuRF-1 mRNA 含量和启动子活性。然而，FoxO3 和 SMAD3 共表达协同增加 MuRF-1 mRNA 和启动子活性。MuRF-1 启动子近端的 SMAD 结合元件（SBE）突变或 SMAD3 DNA 结合突变体的过表达减弱了 FoxO3 依赖性 MuRF-1 启动子激活，表明 SMAD 与 DNA 的结合对于 FoxO3 诱导的 MuRF-1 转录的最佳激活是必需的。通过染色质免疫沉淀，SMAD3 DNA 结合增加了 FoxO3 的丰度，SBE 突变降低了 MuRF-1 启动子上 FoxO3 的丰度。此外，SMAD3 过表达呈剂量依赖性增加 FoxO3 蛋白含量，FoxO3 和 SMAD3 共表达协同增加 FoxO 依赖性基因转录[用 FoxO 反应元件（FRE）驱动的报告基因评估]。总之，这些结果表明，SMAD3 通过增加 FoxO3 在近端启动子区域内保守的 FRE-SBE 基序上的结合，以及增加 FoxO3 蛋白含量和转录活性，调节 MuRF-1 的转录。这些数据首次表明，两种主要的调节蛋白降解的转录因子 FoxO3 和 SMAD3 协同作用，直接调节 MuRF-1 的转录。
