Raptor and Rheb Negatively Regulate Skeletal Myogenesis through Suppression of Insulin Receptor Substrate 1 (IRS1)

Abstract

The mammalian target of rapamycin (mTOR) is essential for skeletal myogenesis through controlling distinct cellular pathways. 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 this study we have investigated the functions of raptor, S6K1, and Rheb in the differentiation of C2C12 mouse myoblasts. We 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 protein levels, and decreased Ser-307 (human Ser-312) 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.During embryonic skeletal myogenesis, cells in somites commit to myogenic lineage and become myoblasts, which differentiate and fuse to form multinucleated myofibers (1). This is a highly coordinated process where various environmental cues and signaling pathways integrate to regulate the formation of skeletal muscle (2, 3). This process is largely recapitulated by the in vitro differentiation of myoblasts, such as the C2C12 mouse satellite cell line. Upon growth factor withdrawal, these cells produce insulin-like growth factor II (IGF-II), 2 which in an autocrine fashion stimulates myogenic differentiation (4). One of the critical pathways downstream of myogenic IGF signaling is the PI3K-Akt pathway (5, 6), and insulin receptor substrate 1 (IRS1) is a well-established mediator of IGF receptor activation of downstream signaling (7). mTOR, the mammalian target of rapamycin, has long been recognized as a nutrient-and energy-sensing signaling hub regulating a wide spectrum of cellular processes including proliferation, growth, survival, differentiation, and metabolism (8). It nucleates two distinct biochemical complexes: the raptor-associated mTORC1 is acutely sensitive to rapamycin, and it targets S6K1 and 4E-BP1 to regulate translation initiation, among other functions; the rictor-associated mTORC2 is a kinase for the multifunctional kinase Akt and it also regulates cytoskeleton reorganization (9). Although mTORC2 was initially characte...

Role of the mTORC1 Complex in Satellite Cell Activation by RNA-Induced Mitochondrial Restoration: Dual Control of Cyclin D1 through MicroRNAs

“…Moreover, in contrast to the opposite effects of mTORC and Raptor on in vitro differentiation (19,20), we observed that KD of mTORC and Raptor have identical effects on early satellite cell differentiation in vivo, indicating that the mTORC1 kinase, rather than the individual components, mediates the timing of satellite cell activation. One explanation of these discrepancies is that the microenvironment of the myoblasts at the injury site being different from that in culture, energy-sensing mechanisms differ in their contribution to the differentiation program.…”

“…Rapamycin inhibits the proliferation of primary myoblasts in an S6K-independent manner (14), as well as the differentiation of cultured myoblasts to myotubes upon serum deprivation in vitro (15)(16)(17), but the inhibition can be rescued by a rapamycin-resistant mTORC through a process that involves insulin-like growth factor II (IGFII) but does not require its kinase activity (17,18). Knockdown (KD) of mTORC in myoblasts inhibits their differentiation to myotubes in vitro, but unexpectedly, knockdown of Raptor has the opposite effect (19,20); the role of Rictor, a subunit of the mTORC2 complex, is uncertain, with reports of either inhibition of differentiation (20) or no effect (19) of Rictor KD. From these studies, it is apparent that mTORC can regulate cell growth and division in vitro, that the effect on growth requires S6K whereas that on myoblast division does not, indicating the presence of additional (unknown) mTORC targets, and finally, that mTORC and Raptor regulate the differentiation of cultured myoblasts to myotubes in different ways that are still unexplained.…”

Mammalian Target of Rapamycin (mTOR) Signaling Network in Skeletal Myogenesis

“…Rheb most likely exerts this negative regulation through raptor/ mTOR and suppression of IRS1 (64). This is in contrast to a positive role of Rheb in stimulating muscle hypertrophy (73).…”

“…Knockdown of raptor, the defining component of mTORC1, was found to enhance, rather than inhibit, myoblast differentiation (52,64), and overexpression of raptor suppressed myogenic differentiation (64). Evidence suggests that the negative function of raptor in differentiation of C2C12 cells is through serine phosphorylation and destabilization of IRS1 by mTORC1 and subsequent inhibition of PI3K/Akt signaling (Fig.…”

“…The activator of mTORC1, Rheb, is also an inhibitor of myogenesis, as knockdown of Rheb enhances and overexpression of Rheb suppresses differentiation of C2C12 myoblasts (64). Rheb most likely exerts this negative regulation through raptor/ mTOR and suppression of IRS1 (64).…”