Ge Yejing, Wu Ai-Luen, Warnes Christine, Liu Jianming, Zhang Chongben, Kawasome Hideki, Terada Naohiro, Boppart Marni D, Schoenherr Christopher J, Chen Jie
Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, USA.
Am J Physiol Cell Physiol. 2009 Dec;297(6):C1434-44. doi: 10.1152/ajpcell.00248.2009. Epub 2009 Sep 30.
Rapamycin-sensitive signaling is required for skeletal muscle differentiation and remodeling. In cultured myoblasts, the mammalian target of rapamycin (mTOR) has been reported to regulate differentiation at different stages through distinct mechanisms, including one that is independent of mTOR kinase activity. However, the kinase-independent function of mTOR remains controversial, and no in vivo studies have examined those mTOR myogenic mechanisms previously identified in vitro. In this study, we find that rapamycin impairs injury-induced muscle regeneration. To validate the role of mTOR with genetic evidence and to probe the mechanism of mTOR function, we have generated and characterized transgenic mice expressing two mutants of mTOR under the control of human skeletal actin (HSA) promoter: rapamycin-resistant (RR) and RR/kinase-inactive (RR/KI). Our results show that muscle regeneration in rapamycin-administered mice is restored by RR-mTOR expression. In the RR/KI-mTOR mice, nascent myofiber formation during the early phase of regeneration proceeds in the presence of rapamycin, but growth of the regenerating myofibers is blocked by rapamycin. Igf2 mRNA levels increase drastically during early regeneration, which is sensitive to rapamycin in wild-type muscles but partially resistant to rapamycin in both RR- and RR/KI-mTOR muscles, consistent with mTOR regulation of Igf2 expression in a kinase-independent manner. Furthermore, systemic ablation of S6K1, a target of mTOR kinase, results in impaired muscle growth but normal nascent myofiber formation during regeneration. Therefore, mTOR regulates muscle regeneration through kinase-independent and kinase-dependent mechanisms at the stages of nascent myofiber formation and myofiber growth, respectively.
雷帕霉素敏感信号通路是骨骼肌分化和重塑所必需的。在培养的成肌细胞中，据报道哺乳动物雷帕霉素靶蛋白（mTOR）通过不同机制在不同阶段调节分化，包括一种独立于mTOR激酶活性的机制。然而，mTOR的激酶非依赖性功能仍存在争议，且尚无体内研究检验先前在体外鉴定出的那些mTOR的生肌机制。在本研究中，我们发现雷帕霉素会损害损伤诱导的肌肉再生。为了用遗传学证据验证mTOR的作用并探究mTOR功能的机制，我们构建并鉴定了在人骨骼肌肌动蛋白（HSA）启动子控制下表达两种mTOR突变体的转基因小鼠：雷帕霉素抗性（RR）和RR/激酶失活（RR/KI）。我们的结果表明，RR-mTOR的表达可恢复给予雷帕霉素的小鼠的肌肉再生。在RR/KI-mTOR小鼠中，再生早期新生肌纤维的形成在有雷帕霉素存在的情况下仍能进行，但雷帕霉素会阻断再生肌纤维的生长。Igf2 mRNA水平在再生早期急剧增加，这在野生型肌肉中对雷帕霉素敏感，但在RR-和RR/KI-mTOR肌肉中对雷帕霉素部分抗性，这与mTOR以激酶非依赖性方式调节Igf2表达一致。此外，mTOR激酶的靶标S6K1的全身敲除会导致肌肉生长受损，但再生过程中新生肌纤维的形成正常。因此，mTOR分别在新生肌纤维形成和肌纤维生长阶段通过激酶非依赖性和激酶依赖性机制调节肌肉再生。
