The mTORC2 Complex Regulates Terminal Differentiation of C2C12 Myoblasts

Abstract

Rapamycin, a selective inhibitor of mTORC1 signaling, blocks terminal myoblast differentiation. We found that downregulation of rictor, a component of the mTORC2 complex, but not downregulation of raptor, a component of the mTORC1 complex, prevented terminal differentiation (fusion) of C2C12 myoblasts. Both rapamycin and rictor downregulation suppressed the phosphorylation of AKT(S 473 ), and rapamycin treatment of C2C12 myoblasts disrupted the mTORC2 complex. Importantly, downregulation of rictor inhibited TORC2 signaling without inhibiting mTORC1 signaling, suggesting that inhibition of mTORC1 by rapamycin may not be the cause of arrested differentiation. In support of this, expression of a phosphomimetic mutant AKT(S473D) in rictor-deficient cells rescued myoblast fusion even in the presence of rapamycin. mTORC2 signaling to AKT appears necessary for downregulation of the Rho-associated kinase (ROCK1) that occurs during myogenic differentiation. Rapamycin treatment prevented ROCK1 inactivation during differentiation, while suppression of ROCK1 activity during differentiation and myoblast fusion was restored through expression of AKT(S473D), even in the presence of rapamycin. Further, the ROCK inhibitor Y-27632 restored terminal differentiation in rapamycin-treated myoblasts. These results provide the first evidence of a specific role for mTORC2 signaling in terminal myogenic differentiation.Differentiation of skeletal muscle cells involves highly coordinated processes involving myogenic determination of pluripotent mesodermal precursors, withdrawal from the cell cycle, subsequent expression of myotube-specific genes, and cell fusion to form multinucleated myotubes (5,19,23,33). AKT represents a nodal point, signaling to several pathways to positively or negatively regulate growth, proliferation, survival, and myogenic differentiation (34,35). AKT phosphorylates the FoxO1a transcription factor required for myotube fusion of primary myoblasts (3), causing cytoplasmic localization. However, in primary myoblasts and C2C12 myoblasts, phosphorylation of FoxO1a appears partially independent of the PI3K/ AKT pathway, possibly regulated through the Rho-associated kinase ROCK1. Inactivation of ROCK1 has been shown to be necessary for FoxO1a nuclear translocation and C2C12 cell fusion (20). AKT also activates mTOR (mammalian target of rapamycin) through phosphorylation and inactivation of the tuberous sclerosis complex (36) and phosphorylation of PRAS40, an endogenous inhibitor of mTOR (31, 32). Recent studies have shown that the TOR kinase(s) exists in two complexes that are conserved (25,36). In mammalian cells, TORC1 comprises mTOR, raptor, and mLST8 and is part of a pathway that senses nutrient (amino acids, O 2 , AMP) and growth factor status. mTORC1 activates S6K1 and phosphorylates and inactivates 4E-BPs, promoting association of eIF4E, the RNA cap-binding protein, with the eIF4G scaffolding protein and assembly of the eIF4F preinitiation translation complex (36). Importantly, rapamycin in complex with the immuno...

mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for cell cycle exit and myogenic differentiation

“…In both cases, results are shown from a single experiment but similar data were obtained in at least 3 independent experiments. results were seen with extracts from cells allowed to differentiate in the presence of RAD001 (lanes [8][9][10][11][12][13]. Figure 2A shows that lambda phosphatase was able to dephosphorylate 4E-BP1 in extracts prepared at 48 hours ( Fig.…”

“…We and others have previously shown that rapamycin 10,12 or RAD001 26 potently inhibits myogenic differentiation in the C2C12 myoblast model system. Furthermore, we showed that although inhibition of mTORC1 reduced p70S6K activity, prevented the phosphorylation of rpS6, reduced protein synthesis rates and delayed myotube formation, paradoxically 4E-BP1 remained in a hyperphosphorylated form.…”

“…[5][6][7] Activation of mTORC1 and phosphorylation of its downstream targets are central to the control of protein synthesis in many cell types, but the role of mTOR activity and its downstream signaling pathways in regulating differentiation in C2C12 myoblasts remains unclear. [8][9][10][11][12][13] mTOR is contained within 2 distinct complexes: mTORC1 and mTORC2. [13][14][15] The former complex includes mTOR, raptor, mLST8, proline-rich Akt substrate 40 kDa (PRAS40), DEPdomain-containing partner of mTOR (DEPTOR), and scaffolds tti1/tel2 7,13,16 mTORC1 regulates the phosphorylation and activity of eIF4E-binding proteins (4E-BP1 5,7,14 ) and the ribosomal protein S6 kinase, p70S6K.…”

“…21 Down-regulation of rictor has been reported to inhibit the ability of mTORC2 to phosphorylate Akt on Ser473 and stimulates protein synthesis in C2C12 myocytes. 12 Protein synthesis is carried out in 3 stages (initiation, elongation and termination), with the initiation stage of translation generally accepted as a major site of regulation of gene expression in mammalian cells. 5,7,[22][23][24][25][26][27][28][29][30] This step in protein synthesis is regulated by a family of proteins, the initiation factors 5,23,24 which interact with each other and the mRNA.…”

Hepatic CD206-positive macrophages express amphiregulin to promote the immunosuppressive activity of regulatory T cells in HBV infection

“…Therefore, we concluded that macrophage-derived AREG promote T reg function through activation of mTORC1 signaling in T regs . However, although rapamycin is thought to inhibit mTORC1 signaling preferentially, evidence indicates that rapamcyin also blocks mTORC2 signaling [58,59]. mTORC2 regulates cellular metabolism and the cytoskeleton, and recent studies suggest its role in modulation of T reg generation and function as well.…”