Mechanisms of mTOR Signal Transduction Regulating Cell Growth and Differentiation

131 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2009.In chapter V, among the list of microRNAs from the microarray analysis, we focus on the muscle-specific miR-1, which is up-regulated during differentiation. mTOR is essential for miR-1 expression in C2C12 cells and in vivo in regenerating myofibers. It regulates the transcription of miR-1 through its upstream enhancers, and this regulation is at least partially mediated by the myogenic transcription factor MyoD. To delineate the pathway downstream of mTOR-miR-1 in myogenesis, we have tested the reported miR-1 target in skeletal muscle-HDAC4, and separately, a myogenic factor that is reportedly inhibited by HDACs-follistatin. Anti-miR-1 LNA dampens follistatin mRNA level, which is rescued by HDAC inhibitor trichostatin A (TSA). Concurrently, anti-miR-1 LNA also inhibits myotube fusion, which could be rescued by either introducing recombinant follistatin, or inducing the expression of endogenous follistatin through TSA. These data strongly support an HDAC4-follistatin pathway as the functional target of miR-1 in myogenesis. Importantly, rapamycin enhances the expression of HDAC4 and decreases follistatin mRNA level during myoblast differentiation. Furthermore, myotube growth and maturation, which requires a late-stage myogenic fusion after nascent myotube formation, is rescued by either follistatin or TSA from rapamycin inhibition both in C2C12 cells and in skeletal muscle regeneration in vivo, suggesting that follistatin is most likely the long-sought fusion factor regulated by mTOR in muscle growth. Taken together, this study has revealed for the first time a link between mTOR signaling and microRNA, and identified a novel mTOR-miR-1-HDAC4-follistatin pathway that regulates myocyte fusion critical for myotube maturation and skeletal muscle growth.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD