Regulation of skeletal myogenesis by the mTOR signaling network
Skeletal muscle is composed of post-mitotic multinucleated myofibers. During embryonic skeletal myogenesis, cells in somites commit to myogenic lineage and differentiate into myoblasts, which then fuse to form multinucleated myofibers. Post-natal growth, repair, and maintenance of skeletal muscle are dependent on muscle satellite cells, which in response to stimuli differentiate and fuse to form new myofibers. Developmental or post-natal failure of skeletal myogenesis results in diverse muscular dystrophies and atrophies, largely impairing life quality and sometimes directly causing death. The myogenic process is guided by various environmental cues and regulated by distinct signaling pathways, resulting in the activation of specific transcription factors and subsequent reprogramming of gene expression. The mammalian target of rapamycin (mTOR) is a Ser/Thr kinase that controls a wide spectrum of cellular and developmental processes including regulation of skeletal myogenesis. In my dissertation work, I investigate the mechanisms of myogenic regulation, with a focus on the signaling network assembled by mTOR. Various processes of skeletal muscle differentiation and remodeling were known to be inhibited by the mTOR-specific inhibitor, rapamycin. In cultured myoblasts, the target of rapamycin – mTOR – had been reported to regulate differentiation at different stages through distinct mechanisms, including one that is independent of mTOR kinase activity. However, there had been no in vivo evidence to validate those mTOR myogenic mechanisms in vitro. In Chapter II, I show that rapamycin impairs injury-induced muscle regeneration. To validate the role of mTOR with genetic evidence and to probe the mechanism of mTOR function, I 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) mTOR. My 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 WT 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 kinaseiii dependent mechanisms at the stages of nascent myofiber formation and myofiber growth, respectively. MicroRNAs have emerged as key regulators of skeletal myogenesis, but our knowledge of the identity of the myogenic miRNAs and their targets remains limited. In Chapter III, I describe the identification and characterization of a novel myogenic microRNA – miR-125b. I find that the levels of miR-125b decline during myogenesis, and that miR-125b negatively modulates myoblast differentiation in culture and muscle regeneration in mice. My results identify the insulin-like growth factor 2 (Igf2), a critical regulator of skeletal myogenesis, as a direct and major target of miR-125b in both myocytes and regenerating muscles, revealing for the first time a microRNA mechanism controlling IGF-II expression. In addition, I provide evidence suggesting that miR-125b biogenesis is negatively controlled by kinase-independent mTOR signaling both in vitro and in vivo, as a part of a dual mechanism by which mTOR regulates the production of IGF-II – a master switch governing the initiation of skeletal myogenesis. In Chapter IV, in collaboration with a former graduate student in the lab, Dr. Yuting Sun, I find that expression of another microRNA, miR-1, is regulated by mTOR both in differentiating myoblasts and in mouse regenerating skeletal muscle. We have found that mTOR controls MyoD-dependent transcription of miR-1 through its upstream enhancer, most likely by regulating MyoD protein stability. Moreover, a functional pathway downstream of mTOR and miR-1 is delineated, in which miR-1 suppression of HDAC4 results in production of follistatin and subsequent myocyte fusion. Collective evidence strongly suggests that follistatin is the longsought mTOR-regulated fusion factor. In summary, these findings unravel yet another link between mTOR and microRNA biogenesis, and identify an mTOR-miR-1-HDAC4-follistatin pathway that regulates myocyte fusion during myoblast differentiation in vitro and skeletal muscle regeneration in vivo. The importance of the canonical mTOR complex 1 signaling components, including raptor, S6K1, and Rheb, had been suggested in muscle maintenance, growth, and metabolism. However, the role of those components in myogenic differentiation is not entirely clear. In Chapter V, I report the investigation of the functions of raptor, S6K1, and Rheb in the differentiation of C2C12 mouse myoblasts. I find that although mTOR knockdown severely impairs myogenic differentiation as expected, the knockdown of raptor, as well as Rheb, enhances differentiation. Consistent with a negative role for these proteins in myogenesis, overexpression of raptor or Rheb inhibits C2C12 differentiation. On the other hand, neither knockdown nor overexpression of S6K1 has any effect. Moreover, the enhanced differentiation elicited by raptor or Rheb knockdown is accompanied by increased Akt activation, elevated IRS1 iv protein levels, and decreased Ser307 (human Ser312) phosphorylation on IRS1. Finally, IRS1 knockdown eliminated the enhancement in differentiation elicited by raptor or Rheb knockdown, suggesting that IRS1 is a critical mediator of the myogenic functions of raptor and Rheb. In conclusion, the Rheb-mTOR/raptor pathway negatively regulates myogenic differentiation by suppressing IRS1-PI3K-Akt signaling. These findings underscore the versatility of mTOR signaling in biological regulations and implicate the existence of novel mTOR complexes and/or signaling mechanism in skeletal myogenesis. Lastly in Appendix B, I document the effort of an RNAi screening to search for novel myogenic regulators among secreted factors. A few distinct groups of cytokines and chemokines are found to either enhance or suppress myoblast differentiation and fusion when knocked down, suggesting that they may regulate myogenesis. Future characterization of these candidates will include assessing knockdown efficiency, identifying the exact processes that they regulate, and dissecting their regulatory pathways.
mTOR, myogenesis, myoblast differentiation, microRNA, miRNA, mammalian target of rapamycin (mTOR), Ribonucleic acid (RNA)
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign