A p160ROCK-specific inhibitor, Y-27632, attenuates rat hepatic stellate cell growth.
OBJECTIVE
p160ROCK, a serine/threonine protein kinase, is a direct RhoA target mediating RhoA-induced assembly of focal adhesions and stress fibers. Recently, Rho signaling pathways were reported to play an important role in the activation of rat hepatic stellate cells (HSC). The aim of this study was to investigate the mechanism of action of a p160ROCK-specific inhibitor, Y-27632, on cultured rat HSC.


METHODS
HSC were isolated from normal rat livers and cultured on fibronectin-coated dishes. The cell morphology and actin cytoskeleton were studied with phase contrast and fluorescence microscopy, respectively. Immunoblot analysis was used to examine phosphorylation of focal adhesion kinase and extracellular signal-regulated kinase, and the expression of cell cycle-associated proteins. HSC proliferation was measured by quantitating the percentage of cells that exhibited nuclear incorporation of 5-bromodeoxyuridine. Type I collagen gene expression and accumulation in HSC culture media were evaluated by Northern blot and enzyme-linked immunosorbent assay, respectively.


RESULTS
Y-27632 consistently blocked cell spreading and suppressed RhoA-induced formation of stress fibers in HSC. In addition, Y-27632 inhibited phosphorylation of focal adhesion kinase and extracellular signal-regulated kinase. Cells treated with Y-27632 failed to proliferate, in contrast to untreated spread cells. This shape-dependent block in cell proliferation correlated with a failure to increase cyclin D1 protein level and to down-regulate the cell cycle inhibitor p27. Y-27632 decreased type I collagen gene expression and accumulation in HSC culture media.


CONCLUSIONS
Our findings indicate that p160ROCK-mediated actin stress fiber assembly is involved in the pathophysiology of hepatic fibrogenesis and suggest that inhibitors of the RhoA-ROCK pathway might be useful therapeutically in liver fibrogenesis.
Iwamoto
H
H
Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan. gann@intmed3.med.kyushu-u.ac.jp
Nakamuta
M
M
Tada
S
S
Sugimoto
R
R
Enjoji
M
M
Nawata
H
H
D017667:Adipocytes / Q000166:cytology* / Q000187:drug effects*; D000577:Amides / Q000494:pharmacology*; D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D008099:Liver / Q000166:cytology* / Q000187:drug effects; D008297:Male; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D011725:Pyridines / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D054460:rho-Associated Kinases
D000577:Amides; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D011725:Pyridines; C108830:Y 27632; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases