Role of Endogenous TGF-β Family in Myogenic Differentiation of C2C12 Cells

The present study evaluated endogenous activities and the role of BMP and transforming growth factor-beta (TGF-beta), representative members of the TGF-beta family, during myotube differentiation in C2C12 cells. Smad phosphorylation at the C-terminal serines was monitored, since TGF-beta family members signal via the phosphorylation of Smads in a ligand-dependent manner. Expression of phosphorylated Smad1/5/8, which is an indicator of BMP activity, was higher before differentiation, and rapidly decreased after differentiation stimulation. Differentiation-related changes were consistent with those in the expression of Ids, well-known BMP-responsive genes. Treatment with inhibitors of BMP type 1 receptors or noggin in C2C12 myoblasts down-regulated the expression of myogenic regulatory factors, such as Myf5 and MyoD, leading to impaired myotube formation. Addition of BMP-2 during the myoblast phase also inhibited myotube differentiation through the down-regulation of Myf5 and MyoD. In contrast to endogenous BMP activity, the phosphorylation of Smad2, a TGF-beta-responsive Smad, was higher 8-16 days after differentiation stimulation. A-83-01, an inhibitor of TGF-beta type I receptor, increased the expression of Myf5 and MyoD, and enhanced myotube formation. The present results reveal that endogenous activities of the TGF-beta family are changed during myogenesis in a pathway-specific manner, and that the activities are required for myogenesis. J. Cell. Biochem. 112: 614-624, 2011. (C) 2010 Wiley-Liss, Inc.