N-cadherin, a member of the Ca2+-dependent cell–cell adhesion molecule family, plays an essential role in skeletal muscle cell differentiation. We show that inhibition of N-cadherin–dependent adhesion impairs the upregulation of the two cyclin-dependent kinase inhibitors p21 and p27, the expression of the muscle-specific genes myogenin and troponin T, and C2C12 myoblast fusion. To determine the nature of N-cadherin–mediated signals involved in myogenesis, we investigated whether N-cadherin–dependent adhesion regulates the activity of Rac1, Cdc42Hs, and RhoA. N-cadherin–dependent adhesion decreases Rac1 and Cdc42Hs activity, and as a consequence, c-jun NH2-terminal kinase (JNK) MAPK activity but not that of the p38 MAPK pathway. On the other hand, N-cadherin–mediated adhesion increases RhoA activity and activates three skeletal muscle-specific promoters. Furthermore, RhoA activity is required for β-catenin accumulation at cell–cell contact sites. We propose that cell–cell contacts formed via N-cadherin trigger signaling events that promote the commitment to myogenesis through the positive regulation of RhoA and negative regulation of Rac1, Cdc42Hs, and JNK activities.

The Rho family of GTP-binding proteins plays a critical role in a variety of cellular processes, including cytoskeletal reorganization and activation of kinases such as p38 and C-jun N-terminal kinase (JNK) MAPKs. We report here that dominant negative forms of Rac1 and Cdc42Hs inhibit the expression of the muscle-specific genes myogenin, troponin T, and myosin heavy chain in L6 and C2 myoblasts. Such inhibition correlates with decreased p38 activity. Active RhoA, RhoG, Rac1, and Cdc42Hs also prevent myoblast-to-myotube transition but affect distinct stages: RhoG, Rac1, and Cdc42Hs inhibit the expression of all muscle-specific genes analyzed, whereas active RhoA potentiates their expression but prevents the myoblast fusion process. We further show by two different approaches that the inhibitory effects of active Rac1 and Cdc42Hs are independent of their morphogenic activities. Rather, myogenesis inhibition is mediated by the JNK pathway, which also leads to a cytoplasmic redistribution of Myf5. We propose that although Rho proteins are required for the commitment of myogenesis, they differentially influence this process, positively for RhoA and Rac1/Cdc42Hs through the activation of the SRF and p38 pathways, respectively, and negatively for Rac1/Cdc42Hs through the activation of the JNK pathway. INTRODUCTIONThe development of skeletal muscle is a multistep process in which pluripotent mesodermal cells give rise to myoblasts that subsequently withdraw from the cell cycle and differentiate into plurinucleated myotubes. The earliest known markers for the skeletal muscle lineage are the four myogenic basic helix-loop-helix (bHLH) factors MyoD, Myf5, myogenin, and MRF4 (Molkentin and Olson, 1996;Arnold and Winter, 1998). During differentiation, myogenic bHLH factors activate muscle-specific genes (such as myosin light and heavy chains, troponin T, etc.) and coordinate withdrawal from the cell cycle (Lassar et al., 1994). In culture, differentiation of myocytes is induced when cells are exposed to medium containing a low concentration of mitogens (differentiation medium). These myogenic bHLH factors are targets of growth factor-signaling pathways that either negatively or positively regulate myogenic differentiation. In vivo experiments in mice have shown that sonic hedgehog and Wnt proteins coming from the dorsal neural tube control Myf5 expression, whereas MyoD is controlled by factors from the dorsal ectoderm (Munsterberg et al., 1995;Cossu et al., 1996;Stern et al., 1997). In addition, negative regulation by proteins such as BMP4, released by the lateral mesoderm, or proteins of the TGF␤ and FGF families plays an important role in this process (Pourquie et al., 1996).The Rho family of Ras-like GTPases are clustered in two distinct subgroups: the Rac/Cdc42 subgroup, which includes Rac1, Rac2, and Rac3, RhoG, Cdc42Hs, TC10, chp, and RhoH, and the Rho subgroup, in which RhoA, -B and -C, RhoD, RhoL, and Rnd1, Rnd2, and Rnd3 are found. Specific substitutions based on Ras studies result in the expression of proteins tha...

RhoG is a member of the Rho family of GTPases that shares 72% and 62% sequence identity with Rac1 and Cdc42Hs, respectively. We have expressed mutant RhoG proteins fused to the green fluorescent protein and analyzed subsequent changes in cell surface morphology and modifications of cytoskeletal structures. In rat and mouse fibroblasts, green fluorescent protein chimera and endogenous RhoG proteins colocalize according to a tubular cytoplasmic pattern, with perinuclear accumulation and local concentration at the plasma membrane. Constitutively active RhoG proteins produce morphological and cytoskeletal changes similar to those elicited by a simultaneous activation of Rac1 and Cdc42Hs, i.e., the formation of ruffles, lamellipodia, filopodia, and partial loss of stress fibers. In addition, RhoG and Cdc42Hs promote the formation of microvilli at the cell apical membrane. RhoG-dependent events are not mediated through a direct interaction with Rac1 and Cdc42Hs targets such as PAK-1, POR1, or WASP proteins but require endogenous Rac1 and Cdc42Hs activities: coexpression of a dominant negative Rac1 impairs membrane ruffling and lamellipodia but not filopodia or microvilli formation. Conversely, coexpression of a dominant negative Cdc42Hs only blocks microvilli and filopodia, but not membrane ruffling and lamellipodia. Microtubule depolymerization upon nocodazole treatment leads to a loss of RhoG protein from the cell periphery associated with a reversal of the RhoG phenotype, whereas PDGF or bradykinin stimulation of nocodazole-treated cells could still promote Rac1- and Cdc42Hs-dependent cytoskeletal reorganization. Therefore, our data demonstrate that RhoG controls a pathway that requires the microtubule network and activates Rac1 and Cdc42Hs independently of their growth factor signaling pathways.