摘要
Background Although the migration of hepatic stellate cells （HSCs） is essential for hepatic fibrotic response, the detailed mechanisms involved are poorly understood. The aim of this study was to examine the role of Rho GTPases （especially RhoA） in platelet-derived growth factor （PDGF）-BB-induced migration of HSCs. Methods The migration of primary rat HSCs was evaluated using transwell Boyden chamber, while cytoskeletal changes were visualized by immunofluorescence staining of intracellular actins and vinculin. Quantitative real-time PCR and Western blotting analysis were used to detect the expression of Rho GTPases （RhoA, Racl and Cdc42） within HSCs and their activation was determined by glutathione S-transferase pull-down assay. Finally, the effects of RhoA on PDGF-BB-induced cell migration and cytoskeletal remodeling were analyzed using HSC-T6 cells stably transfected with constitutively active （CA, Q63L） or dominant negative （DN, T19N） RhoA mutants. Data were analyzed using SPSS 16.0 software. Student＇s t test was used to analyze differences between two groups and one-way analysis of variance （ANOVA） was used among multiple groups. Results Rapid cytoskeletal remodeling led to a significant increase in the motility of primary rat HSCs after haptotactic （direct） and chemotactic （indirect） stimulation by PDGF-BB： PDGF-BB caused a dramatic elevation in the levels of both total and active RhoA protein. However, the levels of mRNA for Rho GTPases, including RhoA, Racl and Cdc42, were unaffected. Furthermore, PDGF-BB induced increased formation of stress fibers and focal adhesions in HSC-T6 cells transfected with CA-RhoA, but not in HSC-T6 transfected with DN-RhoA. Surprisingly, both CA- and DN-RhoA-transfected HSC-T6 cells showed decreased migratory potential in the absence or presence of PDGF-BB compared with controls. Conclusions PDGF-BB induced cytoskeletal remodeling in rat HSCs and promoted their migration via regulation of intracellular RhoA. RhoA may be one of the determinants in PDGF-BB-induced HSC miaration.
Background Although the migration of hepatic stellate cells （HSCs） is essential for hepatic fibrotic response, the detailed mechanisms involved are poorly understood. The aim of this study was to examine the role of Rho GTPases （especially RhoA） in platelet-derived growth factor （PDGF）-BB-induced migration of HSCs. Methods The migration of primary rat HSCs was evaluated using transwell Boyden chamber, while cytoskeletal changes were visualized by immunofluorescence staining of intracellular actins and vinculin. Quantitative real-time PCR and Western blotting analysis were used to detect the expression of Rho GTPases （RhoA, Racl and Cdc42） within HSCs and their activation was determined by glutathione S-transferase pull-down assay. Finally, the effects of RhoA on PDGF-BB-induced cell migration and cytoskeletal remodeling were analyzed using HSC-T6 cells stably transfected with constitutively active （CA, Q63L） or dominant negative （DN, T19N） RhoA mutants. Data were analyzed using SPSS 16.0 software. Student＇s t test was used to analyze differences between two groups and one-way analysis of variance （ANOVA） was used among multiple groups. Results Rapid cytoskeletal remodeling led to a significant increase in the motility of primary rat HSCs after haptotactic （direct） and chemotactic （indirect） stimulation by PDGF-BB： PDGF-BB caused a dramatic elevation in the levels of both total and active RhoA protein. However, the levels of mRNA for Rho GTPases, including RhoA, Racl and Cdc42, were unaffected. Furthermore, PDGF-BB induced increased formation of stress fibers and focal adhesions in HSC-T6 cells transfected with CA-RhoA, but not in HSC-T6 transfected with DN-RhoA. Surprisingly, both CA- and DN-RhoA-transfected HSC-T6 cells showed decreased migratory potential in the absence or presence of PDGF-BB compared with controls. Conclusions PDGF-BB induced cytoskeletal remodeling in rat HSCs and promoted their migration via regulation of intracellular RhoA. RhoA may be one of the determinants in PDGF-BB-induced HSC miaration.
基金
This study was supported by a grant from the National Natural Science Foundation of China （No. 30300151 ）.
