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Am J Physiol Cell Physiol 284: C599-C606, 2003. First published October 3, 2002; doi:10.1152/ajpcell.00188.2002
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Vol. 284, Issue 3, C599-C606, March 2003

Rho kinase mediates serum-induced contraction in fibroblast fibers independent of myosin LC20 phosphorylation

Hiromi Nobe1, Koji Nobe1, Fabeha Fazal2, Primal de Lanerolle2, and Richard J. Paul1

1 Department of Molecular and Cellular Physiology, University of Cincinnati, College of Medicine, Cincinnati, Ohio, 45267-0576; and 2 Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612

Fibroblasts form fibers when grown in culture medium containing native type 1 collagen. The contractile forces generated can be precisely quantified and used to analyze the signal transduction pathways regulating fibroblast contraction. Calf serum (30%) induces a sustained contraction that is accompanied by a transient increase in intracellular calcium ([Ca2+]i). W-7, a calmodulin inhibitor, KN-62, an inhibitor of calcium/calmodulin-dependent protein kinase, and ML-7, a myosin light-chain kinase inhibitor, had no effects on either the contraction or the [Ca2+]i responses. Neither genistein, a tyrosine kinase inhibitor, nor calphostin C, a protein kinase C inhibitor, had major effects on force or [Ca2+]i. In contrast, the Rho kinase inhibitors (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) and HA1077 depressed the contraction in a dose-dependent manner without affecting the [Ca2+]i response. Stress fiber formation was also suppressed by Y-27632. Surprisingly, calf serum, Y-27632, and calf serum plus Y-27632 did not alter mono- or diphosphorylation of the myosin regulatory light chain (MRLC) compared with control untreated fibers. These results suggest that the sustained contraction of NIH 3T3 fibroblast fibers induced by calf serum is mediated by Rho kinase but is independent of a sustained increase in [Ca2+]i, calcium/calmodulin- or protein kinase C-dependent pathways, or increases in MRLC phosphorylation.

nonmuscle contractility; wound repair; calcium; stress fibers


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