Regulation of differentiation of the BC₃H1 muscle cell line through cAMP-dependent and -independent pathways

Serum mitogens, fibroblast growth factor (FGF), and type β transforming growth factor (TGF-β) suppress differentiation of the mouse muscle cell line BC₃H1; however, the signal transduction pathways whereby these growth factors exert their effects on this system are unknown. The goal of this study was to determine whether the program for differentiation of BC₃H1 cells was susceptible to negative regulation by signaling pathways involving cAMP or protein kinase C and whether these intracellular effectors participate in the mechanism by which growth factors prevent establishment of the myogenic phenotype. Exposure of BC₃H1 cells to dibutyryl cAMP, 8-bromo-cAMP, or compounds that stimulate adenylate cyclase, i.e. forskolin, prostaglandin E₁, and cholera toxin, prevented upregulation of muscle-specific gene products following growth arrest in mitogen-deficient medium. Conversely, addition of cAMP to differentiated BC₃H1 myocytes caused down-regulation of muscle-specific mRNAs. In contrast to the ability of cAMP to block differentiation, chronic exposure to O-tetradecanoylphorbol-13-acetate, the potent activator of protein kinase C, exhibited no apparent effects on expression of muscle-specific gene products. The proto-oncogenes c-myc and c-fos were up-regulated rapidly by cAMP in a manner similar to that observed previously by serum, FGF, and TGF-β. However, these growth factors failed to increase intracellular cAMP levels, and they did not induce ornithine decarboxylase, which was subject to positive regulation by cAMP and O-tetradecanoyl-13-acetate. Together, these data indicate that differentiation of BC₃H1 cells is subject to negative regulation through a cAMP-dependent pathway and that serum mitogens, FGF, and TGF-β inhibit differentiation through a mechanism independent of cAMP or protein kinase C.