Activated Raf inhibits avian myogenesis through a MAPK-dependent mechanism

Chronic overexpression of the oncogenic form of Ras is a potent inhibitor o f skeletal myogenesis. However, the intracellular signaling pathways that m ediate the repressive actions of Ras on myogenic differentiation have yet t o be identified. We examined the role of Raf-mediated signaling as a modula tor of avian myogenesis. Raf overexpression elicited pronounced effects on both myoblasts and mature myocytes. Most notably, the embryonic chick myobl asts overexpressing a constitutively active form of Raf (RCAS-Raf CAAX or R CAS-Raf BXB) fail to form the large multinucleated myofibers characteristic of myogenic cultures. While residual myofibers were apparent in the RCAS-R af BXB and RCAS-Raf CAAX infected cultures, these fibers had an atrophic ph enotype, The altered morphology is not a result of reinitiation of the myon uclei cell cycle nor is it due to apoptosis, Furthermore, the mononucleated myoblasts misexpressing Raf BXB are differentiation-defective due to overt MAPK activity. Supplementation of the culture media with the MAPK kinase ( MEK) inhibitor, PD98059, caused a reversal of the phenotype and allowed the formation of multinucleated myofibers at levels comparable to controls. Ou r results indicate that the Raf/MEK/MAPK axis is intact in chick myoblasts and that persistent activation of this signaling cascade is inhibitory to m yogenesis.