Rho/Rho-associated kinase signal regulates myogenic differentiation via myocardin-related transcription factor-A/Smad-dependent transcription of the Id3 gene.

RhoA is known to be involved in myogenic differentiation, but whether it acts as a positive or negative regulator is controversial. To resolve this issue, we investigated the differentiation stage-specific roles of RhoA and its effector, Rho-associated kinase, using C2C12 myoblasts. We found that proliferating myoblasts show high levels of RhoA and serum-response factor activities and strong expression of the downstream target of RhoA, myocardin-related transcription factor-A (MRTF-A or MAL); these activities and expression are markedly lower in differentiating myocytes. We further demonstrated that, in proliferating myoblasts, an increase in MRTF-A, which forms a complex with Smad1/4, strikingly activates the expression level of the Id3 gene; the Id3 gene product is a potent inhibitor of myogenic differentiation. Finally, we found that during differentiation, one of the forkhead transcription factors translocates into the nucleus and suppresses Id3 expression by preventing the association of the MRTF-A-Smad complex with the Id3 promoter, which leads to the enhancement of myogenic differentiation. We conclude that RhoA/Rho-associated kinase signaling plays positive and negative roles in myogenic differentiation, mediated by MRTF-A/Smad-dependent transcription of the Id3 gene in a differentiation stage-specific manner.