Regulation of skeletal myogenesis by the mTOR signaling network

mTOR;myogenesis;myoblast differentiation;microRNA;miRNA;mammalian\rtarget of rapamycin (mTOR);Ribonucleic acid (RNA)

Ge, Yejing

关键词: mTOR;    myogenesis;    myoblast differentiation;    microRNA;    miRNA;    mammalian\rtarget of rapamycin (mTOR);    Ribonucleic acid (RNA);   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/32047/Ge_Yejing.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Skeletal muscle is composed of post-mitotic multinucleated myofibers. Duringembryonic skeletal myogenesis, cells in somites commit to myogenic lineage and differentiateinto myoblasts, which then fuse to form multinucleated myofibers. Post-natal growth, repair, andmaintenance of skeletal muscle are dependent on muscle satellite cells, which in response tostimuli differentiate and fuse to form new myofibers. Developmental or post-natal failure ofskeletal myogenesis results in diverse muscular dystrophies and atrophies, largely impairing lifequality and sometimes directly causing death. The myogenic process is guided by variousenvironmental cues and regulated by distinct signaling pathways, resulting in the activation ofspecific transcription factors and subsequent reprogramming of gene expression. The mammaliantarget of rapamycin (mTOR) is a Ser/Thr kinase that controls a wide spectrum of cellular anddevelopmental processes including regulation of skeletal myogenesis. In my dissertation work, Iinvestigate the mechanisms of myogenic regulation, with a focus on the signaling networkassembled by mTOR.Various processes of skeletal muscle differentiation and remodeling were known to beinhibited by the mTOR-specific inhibitor, rapamycin. In cultured myoblasts, the target ofrapamycin – mTOR – had been reported to regulate differentiation at different stages throughdistinct mechanisms, including one that is independent of mTOR kinase activity. However, therehad been no in vivo evidence to validate those mTOR myogenic mechanisms in vitro. In ChapterII, I show that rapamycin impairs injury-induced muscle regeneration. To validate the role ofmTOR with genetic evidence and to probe the mechanism of mTOR function, I have generatedand characterized transgenic mice expressing two mutants of mTOR under the control of humanskeletal 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 byRR-mTOR expression. In the RR/KI-mTOR mice, nascent myofiber formation during the earlyphase of regeneration proceeds in the presence of rapamycin, but growth of the regeneratingmyofibers is blocked by rapamycin. Igf2 mRNA levels increase drastically during earlyregeneration, which is sensitive to rapamycin in WT muscles but partially resistant to rapamycinin both RR- and RR/KI-mTOR muscles, consistent with mTOR regulation of Igf2 expression in akinase-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 kinaseiiidependent mechanisms at the stages of nascent myofiber formation and myofiber growth,respectively.MicroRNAs have emerged as key regulators of skeletal myogenesis, but our knowledgeof the identity of the myogenic miRNAs and their targets remains limited. In Chapter III, Idescribe the identification and characterization of a novel myogenic microRNA – miR-125b. Ifind that the levels of miR-125b decline during myogenesis, and that miR-125b negativelymodulates myoblast differentiation in culture and muscle regeneration in mice. My resultsidentify the insulin-like growth factor 2 (Igf2), a critical regulator of skeletal myogenesis, as adirect and major target of miR-125b in both myocytes and regenerating muscles, revealing for thefirst time a microRNA mechanism controlling IGF-II expression. In addition, I provide evidencesuggesting that miR-125b biogenesis is negatively controlled by kinase-independent mTORsignaling both in vitro and in vivo, as a part of a dual mechanism by which mTOR regulates theproduction 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 differentiatingmyoblasts and in mouse regenerating skeletal muscle. We have found that mTOR controlsMyoD-dependent transcription of miR-1 through its upstream enhancer, most likely by regulatingMyoD protein stability. Moreover, a functional pathway downstream of mTOR and miR-1 isdelineated, in which miR-1 suppression of HDAC4 results in production of follistatin andsubsequent myocyte fusion. Collective evidence strongly suggests that follistatin is the longsoughtmTOR-regulated fusion factor. In summary, these findings unravel yet another linkbetween mTOR and microRNA biogenesis, and identify an mTOR-miR-1-HDAC4-follistatinpathway that regulates myocyte fusion during myoblast differentiation in vitro and skeletalmuscle regeneration in vivo.The importance of the canonical mTOR complex 1 signaling components, includingraptor, 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. InChapter V, I report the investigation of the functions of raptor, S6K1, and Rheb in thedifferentiation of C2C12 mouse myoblasts. I find that although mTOR knockdown severelyimpairs myogenic differentiation as expected, the knockdown of raptor, as well as Rheb,enhances differentiation. Consistent with a negative role for these proteins in myogenesis, overexpressionof raptor or Rheb inhibits C2C12 differentiation. On the other hand, neitherknockdown nor overexpression of S6K1 has any effect. Moreover, the enhanced differentiationelicited by raptor or Rheb knockdown is accompanied by increased Akt activation, elevated IRS1ivprotein levels, and decreased Ser307 (human Ser312) phosphorylation on IRS1. Finally, IRS1knockdown 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. Inconclusion, the Rheb-mTOR/raptor pathway negatively regulates myogenic differentiation bysuppressing IRS1-PI3K-Akt signaling. These findings underscore the versatility of mTORsignaling in biological regulations and implicate the existence of novel mTOR complexes and/orsignaling mechanism in skeletal myogenesis.Lastly in Appendix B, I document the effort of an RNAi screening to search for novelmyogenic regulators among secreted factors. A few distinct groups of cytokines and chemokinesare found to either enhance or suppress myoblast differentiation and fusion when knocked down,suggesting that they may regulate myogenesis. Future characterization of these candidates willinclude assessing knockdown efficiency, identifying the exact processes that they regulate, anddissecting their regulatory pathways.