Rho kinase mediates serum-induced contraction in fibroblast fibers independent of myosin LC20 phosphorylation.
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 ([Ca(2+)](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 [Ca(2+)](i) responses. Neither genistein, a tyrosine kinase inhibitor, nor calphostin C, a protein kinase C inhibitor, had major effects on force or [Ca(2+)](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 [Ca(2+)](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 [Ca(2+)](i), calcium/calmodulin- or protein kinase C-dependent pathways, or increases in MRLC phosphorylation.
Nobe
Hiromi
H
Department of Molecular and Cellular Physiology, University of Cincinnati, College of Medicine, Ohio 45267-0576, USA.
Nobe
Koji
K
Fazal
Fabeha
F
De Lanerolle
Primal
P
Paul
Richard J
RJ
D016475:3T3 Cells; D000818:Animals; D001798:Blood Proteins / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002147:Calmodulin / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D018613:Microscopy, Confocal; D018994:Myosin Light Chains / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D022502:Stress Fibers / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D013500:Surface Tension / Q000187:drug effects; D020741:rho GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D054460:rho-Associated Kinases
D001798:Blood Proteins; D002147:Calmodulin; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C; D020741:rho GTP-Binding Proteins; D002118:Calcium