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Published on: July 10, 2019
A p160ROCK-specific inhibitor, Y-27632, attenuates rat hepatic stellate cell growth
H Iwamoto1, M Nakamuta, S Tada
1Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan. gann@intmed3.med.kyushu-u.ac.jp
Background/Aims:
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.
Insights
Y-27632, a p160ROCK inhibitor, blocks hepatic stellate cell (HSC) activation and collagen production. This suggests RhoA-ROCK pathway inhibitors may treat liver fibrogenesis.
Area of Science:
- Cell Biology
- Biochemistry
- Hepatology
Background:
- p160ROCK is a serine/threonine protein kinase and a direct RhoA target.
- Rho signaling pathways are crucial in activating rat hepatic stellate cells (HSC).
Purpose of the Study:
- To investigate the mechanism of action of the p160ROCK-specific inhibitor Y-27632 on cultured rat HSC.
- To explore the role of the RhoA-ROCK pathway in hepatic fibrogenesis.
Main Methods:
- HSC isolation and culture on fibronectin-coated dishes.
- Microscopy for cell morphology and actin cytoskeleton analysis.
- Immunoblotting for protein phosphorylation and cell cycle markers.
- Assays for cell proliferation, collagen gene expression, and protein accumulation.
Main Results:
- Y-27632 inhibited HSC cell spreading, stress fiber formation, and focal adhesion kinase/extracellular signal-regulated kinase phosphorylation.
- Y-27632 blocked HSC proliferation by affecting cyclin D1 and p27 levels.
- Y-27632 reduced type I collagen gene expression and accumulation.
Conclusions:
- p160ROCK-mediated actin stress fiber assembly is implicated in hepatic fibrogenesis.
- Inhibitors of the RhoA-ROCK pathway show potential therapeutic value for liver fibrogenesis.
