RhoA/Rho-Associated Kinase Mediates Fibroblast Contractile Force Generation Hal F. Yee, Jr., Andrew C. Melton, and Binh N. Tran Department of Medicine and Department of Physiology, CURE Digestive Diseases Research Center, University of California Los Angeles School of Medicine, Los Angeles, California 90095 Received December 18, 2000 The intracellular signals governing contractile force generation by non-muscle cells remain uncertain. Our aim was to test the hypothesis that the rhoA/rho- associated kinase signaling pathway is a principal me- diator of contractile force generation in non-muscle cells. We measured myosin II regulatory light chain (MLC) phosphorylation and directly quantitated force generation by chicken embryo fibroblasts in the ab- sence and presence of selective inhibitors of rhoA, and its downstream effector, rho-associated kinase. Inac- tivation of rhoA, with C3 transferase, inhibited serum- stimulated MLC phosphorylation and contractile force generation. Y-27632, an inhibitor of rho- associated kinase, reduced basal contractile tension, and inhibited both serum and endothelin-1 stimulated MLC phosphorylation and contractile force genera- tion. The results of this study provide novel evidence indicating that the rhoA/rho-associated kinase signal- ing pathway is a principal mediator of MLC phosphor- ylation and consequent contractile force generation by non-muscle cells. © 2001 Academic Press Key Words: myosin; myosin light chain kinase; myo- sin regulatory light chain phosphorylation; rho- associated kinase; rhoA; contractile force generation; contraction; fibroblast. Although generation of contractile force by non- muscle cells, such as fibroblasts, is essential for im- portant biological processes such as chemotaxis, cy- tokinesis, and tissue remodeling (1–7), the signals regulating contractile force generation in these cell types are incompletely understood. For many years it was accepted that contractile force generation by non-muscle cells was principally mediated by myosin light chain kinase (MLCK) in a manner similar to that demonstrated in smooth muscle (8, 9). In the smooth muscle model, contraction is powered by the myosin II motor protein complex (myosin II), which is activated when the myosin II regulatory light chain (MLC) is phosphorylated by Ca 2+ -calmodulin stimulated MLCK. However, our laboratory has re- cently demonstrated Ca 2+ -independent MLC phos- phorylation and contractile force generation by fibro- blasts (10), which suggests that in non-muscle cells the Ca 2+ /MLCK signaling pathway is not a primary mediator of contractile force generation. Thus, the identity of the signal transduction pathways pre- dominantly responsible for the regulation of contrac- tile force generation by non-muscle cells remain to be clarified. Emerging evidence indicates that MLC phosphory- lation can be modulated by mechanisms that are inde- pendent of increases in cytosolic Ca 2+ concentration ([Ca 2+ ] i ) and consequent MLCK activation (10 –13). In particular, the monomeric GTPase, rhoA, acting through its downstream effector rho-associated kinase, has been shown to phosphorylate and thereby inhibit the myosin II regulatory light chain phosphatase (MLCP) resulting in an augmentation of net MLC phosphorylation, in both smooth muscle and non- muscle cells (13–15). In smooth muscle it is now recog- nized that this rhoA/rho-associated kinase-dependent inhibition of MLCP is largely responsible for modulat- ing Ca 2+ sensitization of contraction via its effects on MLC phosphorylation (14, 15). In contrast, the role that rhoA plays in the regulation of contractile force generation by non-muscle cells is uncertain. The aim of this study was to test the hypothesis that in non-muscle cells the rhoA/rho-associated kinase sig- naling pathway is a principal mediator of contractile force generation, by directly measuring isometric force generation by chicken embryo fibroblasts. Our results provide novel evidence demonstrating that generation of contractile force by non-muscle cells is mediated by rhoA via the effects of rho-associated kinase on MLC phosphorylation. Abbreviations used: MLC, myosin II regulatory light chain; MLCK, myosin II light chain kinase; [Ca 2+ ] i , cytosolic ionic calcium concentration; MLCP, myosin II light chain phosphatase; DMEM, Dulbecco’s modified Eagle medium. Biochemical and Biophysical Research Communications 280, 1340 –1345 (2001) doi:10.1006/bbrc.2001.4291, available online at http://www.idealibrary.com on 1340 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.