Inactivation of Rho/ROCK signaling is crucial for the nuclear accumulation of FKHR and myoblast fusion.

Myoblast fusion is a critical process for the terminal differentiation of skeletal muscle. To elucidate the intracellular mechanisms regulating myoblast fusion, we studied the roles of signaling through the small GTPase Rho and its effector, the Rho-associated kinase ROCK, in myoblast fusion of mouse C2C12 cells. We found that Rho activity, which was high in proliferating myoblasts, decreased during myogenesis. Expression of a constitutively active form of Rho blocked myoblast fusion, but not the earlier steps of differentiation. Consistently, ROCK activity was also decreased in differentiating C2C12 cells, and an active ROCK mutant prevented their fusion. Furthermore, inactivation of ROCK by the specific inhibitor Y-27632 enhanced myoblast fusion, even in cells expressing the active Rho mutant. Thus, the down-regulation of Rho/ROCK signaling is required for myoblast fusion. We also found that Rho/ROCK signaling was required for retaining FKHR, a transcription factor implicated in myoblast fusion, in the cytoplasm and that inactivation of ROCK was essential for the nuclear accumulation of FKHR that took place just before the onset of myoblast fusion. Moreover, ROCK directly phosphorylated FKHR in vitro. We conclude that the inactivation of Rho/ROCK signaling is a prerequisite for FKHR nuclear translocation and myoblast fusion in C2C12 cells, providing evidence for a novel regulatory role of Rho/ROCK signaling in myogenic differentiation.