The differentiation and maturation of skeletal muscle require interactions between signaling pathways activated by hormones and growth factors and an intrinsic regulatory network controlled by myogenic transcription factors. Insulin-like growth factors (IGFs) play key roles in muscle development in the embryo and in regeneration in the adult. To study mechanisms of IGF action in muscle, we developed a myogenic cell line that overexpresses IGF-binding protein-5. C2BP5 cells remain quiescent in low serum differentiation medium until the addition of IGF-I. Here we use this cell line to identify signaling pathways controlling IGF-mediated differentiation. Induction of myogenin by IGF-I and myotube formation were prevented by the phosphatidylinositol (PI) 3-kinase inhibitor, LY294002, even when included 2 days after growth factor addition, whereas expression of active PI 3-kinase could promote differentiation in the absence of IGF-I. Differentiation also was induced by myogenin but was blocked by LY294002. The differentiation-promoting effects of IGF-I were mimicked by a modified membrane-targeted inducible Akt-1 (iAkt), and iAkt was able to stimulate differentiation of C2 myoblasts and primary mouse myoblasts incubated with otherwise inhibitory concentrations of LY294002. These results show that an IGF-regulated PI 3-kinaseAkt pathway controls muscle differentiation by mechanisms acting both upstream and downstream of myogenin.Skeletal muscle development is a multi-step process that begins with the determination of myogenic precursors from pluripotent mesodermal stem cells and concludes with the terminal differentiation of committed myoblasts, which is characterized by cell cycle withdrawal, expression of muscle-specific proteins, and formation and maturation of myofibers (1-3). The muscle-restricted basic helix-loop-helix transcription factors MyoD, MRF4, myogenin, and myf5 were identified initially based on their ability to confer a myogenic fate on non-muscle cells (4, 5). These proteins activate genes that are required for muscle determination or differentiation through formation of heterodimers with other ubiquitous basic helix-loop-helix factors and binding to DNA control elements termed E boxes that are found in the promoter regions of muscle-restricted genes (3-5). Several genes regulated by myogenic basic helix-loophelix proteins also contain binding sites for members of the MEF2 family (6), and MEF2 proteins have been shown to function as accessory transcription factors to enhance muscle gene expression and to facilitate muscle differentiation (7).Environmental cues also modulate muscle differentiation (6, 8 -10). Many peptide growth factors are able to stimulate myoblast proliferation and also prevent differentiation through signal transduction pathways induced upon growth factor binding to specific high affinity cell surface receptors (9, 10). The Ras-Raf-Mek-Erk pathway has been implicated in growth factor-stimulated muscle cell proliferation and the coordinate inhibition of differentiation (11)(12)(13)...
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