RhoE controls myoblast alignment prior fusion through RhoA and ROCK.

Differentiation of skeletal myoblasts into multinucleated myotubes is a multi-step process orchestrated by several signaling pathways. The Rho small G protein family plays critical roles both during myogenesis induction and myoblast fusion. We report here that in C2C12 myoblasts, expression of RhoE, an atypical member of this family, increases until the onset of myoblast fusion before resuming its basal level once fusion has occurred. We show that RhoE accumulates in elongated, aligned myoblasts prior to fusion and that its expression is also increased during injury-induced skeletal muscle regeneration. Moreover, although RhoE is not required for myogenesis induction, it is essential for myoblast elongation and alignment before fusion and for M-cadherin expression and accumulation at the cell–cell contact sites. Myoblasts lacking RhoE present with defective p190RhoGAP activation and RhoA inhibition at the onset of myoblast fusion. RhoE interacts also with the RhoA effector Rhoassociated kinase (ROCK)I whose activity must be downregulated to allow myoblast fusion. Consistently, we show that pharmacological inactivation of RhoA or ROCK restores myoblast fusion in RhoE-deficient myoblasts. RhoE physiological upregulation before myoblast fusion is responsible for the decrease in RhoA and ROCKI activities, which are required for the fusion process. Therefore, we conclude that RhoE is an essential regulator of myoblast fusion. Cell Death and Differentiation (2008) 15, 1221–1231; doi:10.1038/cdd.2008.34; published online 28 March 2008 The Rho family of Ras-like GTPases consists of signaling molecules involved in cytoskeleton remodeling, membrane recycling and gene transcription. Rho GTPases play essential roles in controlling cell adhesion, migration, cell proliferation and differentiation 1 and particularly in skeletal myogenesis. 2–10 The Rho subfamily contains 21 members clustered in four subgroups that comprise RhoA, RhoB and RhoC; Rnd1, Rnd2 and Rnd3/RhoE; RhoBTB1, RhoBTB2 and RhoBTB3; and Rac1, Rac2, Rac3, Cdc42, TC10, TCL, RhoG, Chp1 and Chp2/Wrch-1. The remaining proteins (RhoD, Rif and RhoH/TTF) delineate distinct stems in the phylogenetic tree. Rho GTPases act as molecular switches that convert extracellular signals into multiple cellular effects by cycling between an inactive GDP-bound and an active GTP-bound state. Their activation is controlled by guanine nucleotide-exchange factors (GEFs), whereas their inactivation is promoted by GTPase-activating proteins (GAPs). The members of the Rnd subfamily (Rnd1, Rnd2 and Rnd3 also called RhoE) and RhoH are an exceptions, as they are always bound to GTP, are not regulated by GEFs and GAPs and have very low, if any, intrinsic GTPase activity. 11–14 Although 21 members are known in mammals, only few of them have been studied extensively. In skeletal muscle cells, the role of RhoA, Rac1 and TC10 has been analyzed. RhoA and Rac1 act both in myogenesis induction and myoblast fusion. RhoA positively regulates MyoD expression and skeletal muscle cell differentiation, as it is required for serum response factormediated activation of several muscle-specific gene promoters. 2 Conversely, Rac1 inhibits myogenesis induction by preventing myoblast withdrawal from the cell cycle. 3,15 Later