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‐β (TGF‐β), representative members of the TGF‐β family, during myotube differentiation in C2C12 cells. Smad phosphorylation at the C‐terminal serines was monitored, since TGF‐β 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 I 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‐β‐responsive Smad, was higher 8–16 days after differentiation stimulation. A‐83‐01, an inhibitor of TGF‐β type I receptor, increased the expression of Myf5 and MyoD, and enhanced myotube formation. The present results reveal that endogenous activities of the TGF‐β 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. © 2010 Wiley‐Liss, Inc.