Modulation of acto-myosin contractility in skeletal muscle myoblasts uncouples growth arrest from differentiation.
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 G(0) 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.
Dhawan
Jyotsna
J
Center for Cellular and Molecular Biology, Uppal Road, Hyderabad, 500 007, India. jdhawan@ccmb.res.in
Helfman
David M
DM
D000205:Actomyosin / Q000737:chemistry*; D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D001381:Azepines / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002448:Cell Adhesion; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D002469:Cell Separation; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003599:Cytoskeleton / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D003931:Diacetyl / Q000031:analogs & derivatives* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D009119:Muscle Contraction; D018482:Muscle, Skeletal / Q000166:cytology* / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009218:Myosins / Q000037:antagonists & inhibitors; D009281:Naphthalenes / Q000494:pharmacology; D011108:Polymers / Q000737:chemistry / Q000378:metabolism; D016192:Resting Phase, Cell Cycle; D015398:Signal Transduction; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D013997:Time Factors; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D000963:Antimetabolites; D001381:Azepines; D019086:Bridged Bicyclo Compounds, Heterocyclic; C493957:Cdkn1a protein, mouse; C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004791:Enzyme Inhibitors; D009281:Naphthalenes; D011108:Polymers; D013844:Thiazoles; D053778:Thiazolidines; D025521:Tumor Suppressor Proteins; C070571:ML 7; D049452:Green Fluorescent Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; C004717:diacetylmonoxime; D004247:DNA; D000205:Actomyosin; D009219:Myosin-Light-Chain Kinase; D018995:Myosin Heavy Chains; D009218:Myosins; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D001973:Bromodeoxyuridine; D003931:Diacetyl; C037068:latrunculin B