Modulation of acto-myosin contractility in skeletal muscle myoblasts uncouples growth arrest from differentiation

Abstract

Cell-substratum interactions trigger key signaling pathways that modulate growth control and tissue-specific gene expression. We have previously shown that abolishing adhesive interactions by suspension culture results in Go arrest of myoblasts. We report that blocking intracellular transmission of adhesion-dependent signals in adherent cells mimics the absence of adhesive contacts. We investigated the effects of pharmacological inhibitors of acto-myosin contractility on growth and differentiation of C2C12 myogenic cells. ML7 (5-iodonaphthalene-1-sulfonyl homopiperazine) and BDM (2,3, butanedione monoxime) are specific inhibitors of myosin light chain kinase, and myosin heavy chain ATPase, respectively. ML7 and BDM affected cell shape by reducing focal adhesions and stress fibers. Both inhibitors rapidly blocked DNA synthesis in a dose-dependent, reversible fashion. Furthermore, both ML7 and BDM suppressed expression of MyoD and myogenin, induced p27(kip1) but not p21(cip1), and inhibited differentiation. Thus, as with suspension-arrest, inhibition of acto-myosin contractility in adherent cells led to arrest uncoupled from differentiation. Over-expression of inhibitors of the small GTPase RhoA (dominant negative RhoA and C3 transferase) mimicked the effects of myosin inhibitors. By contrast, wild-type RhoA induced arrest, maintained MyoD and activated myogenin and p21. expression. The Rho effector kinase ROCK did not appear to mediate Rho's effects on MyoD. Thus, ROCK and MLCK play different roles in the myogenic program. Signals regulated by MLCK are critical, since inhibition of MLCK suppressed MyoD expression but inhibition of ROCK did not. Inhibition of contractility suppressed MyoD but did not reduce actin polymer levels. However, actin depolymerization with latrunculin B inhibited MyoD expression. Taken together, our observations indicate that actin polymer status and contractility regulate MyoD expression. We suggest that in myoblasts, the Rho pathway and regulation of acto-myosin contractility may define a control point for conditional uncoupling of differentiation and the cell cycle.