Background: The macrocyclic antibiotic rapamycin is a highly selective inhibitor of the mTORC1 complex (mTOR-raptor), and potently inhibits terminal myogenic differentiation of C2C12 myoblasts. However, the mechanism by which rapamycin attenuates myogenesis has proven elusive. Here we show that rapamycin inhibits terminal myogenesis by disrupting the mTORC2 (mTOR-rictor) complex. Methods: Mouse C2C12 myoblasts were routinely grown in antibiotic-free Dulbecco's modified Eagle's medium with 15% fetal calf serum (growth medium, GM). Cells were induced to differentiate over 3-7 d by growth in differentiation medium (DM, Dulbecco's modified Eagle's medium with 2% horse serum supplemented with 4 mM L-glutamine) at 37 °C and 5% CO2. Differentiation was determined by immunoblotting (MyHC, myogenin, MyoD), and confocal microscopy. Rictor was down regulated by lentivirus-delivered shRNA, and the phosphomimetic AKT(S473D) was expressed transiently in C2C12 myoblasts. Results: Rapamycin treatment did not inhibit induction of myogenic factors (myogenin, MyoD, MyHC), but prevented formation of multinucleated myotubes. Treatment for 1 d with rapamycin inhibited mTORC1 signaling, but also caused decreased p-AKT(S473). Consistent with these data immunoprecipitation of mTOR showed that rapamycin selectively disrupted the mTORC2 complex. We found that down regulation of rictor, a component of the mTORC2 complex, also prevented terminal differentiation (fusion) of C2C12 myoblasts. Lentivirus-delivered rictor shRNA, but not control virus significantly down regulated rictor. Importantly, down regulation of rictor inhibited TORC2 signaling without inhibiting mTORC1 signaling as determined by maintained levels of phospho-S6(S235/6) and pS6K1(T389) proteins. These results suggest that inhibition of mTORC1 by rapamycin may not be the cause for abrogated fusion. In support of this, expression of a phospho-mimetic mutant AKT(S473D) in rictor-deficient cells rescued myoblast fusion even in the presence of rapamycin. mTORC2 signaling to AKT appears necessary for down regulation of the Rho-associated kinase (ROCK-1) that occurs during myogenic differentiation. Rapamycin treatment prevented ROCK-1 inactivation that occurs under differentiation conditions. Expression of AKT(S473D) suppressed ROCK-1 activity during differentiation and restored myoblast fusion even in the presence of rapamycin. Further, the ROCK inhibitor Y-27632 restored terminal differentiation in rapamycin treated myoblasts. Conclusion: These results provide the first evidence that mTORC2 signaling regulates terminal myogenic cell fusion through a ROCK-1-dependent pathway. (Supported by USPHS grant CA77776)

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4762.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO