RhoA 在血小板衍生生长因子-BB 诱导的大鼠肝星状细胞迁移中的作用。
Role of RhoA in platelet-derived growth factor-BB-induced migration of rat hepatic stellate cells.
机构信息
Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai, China.
出版信息
Chin Med J (Engl). 2010 Sep;123(18):2502-9.
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, Rac1 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, Rac1 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 migration.
背景
尽管肝星状细胞（HSCs）的迁移对于肝纤维化反应至关重要，但涉及的详细机制仍知之甚少。本研究旨在探讨Rho GTPases（尤其是 RhoA）在血小板衍生生长因子（PDGF-BB）诱导的 HSCs 迁移中的作用。
方法
使用 Transwell Boyden 室评估原代大鼠 HSCs 的迁移，通过细胞内肌动蛋白和纽蛋白的免疫荧光染色观察细胞骨架变化。使用定量实时 PCR 和 Western blot 分析检测 HSCs 中 Rho GTPases（RhoA、Rac1 和 Cdc42）的表达，并通过谷胱甘肽 S-转移酶下拉测定法测定其激活。最后，使用稳定转染组成型激活（CA，Q63L）或显性负（DN，T19N）RhoA 突变体的 HSC-T6 细胞分析 RhoA 对 PDGF-BB 诱导的细胞迁移和细胞骨架重塑的影响。使用 SPSS 16.0 软件分析数据。使用 Student's t 检验分析两组之间的差异，使用单向方差分析（ANOVA）分析多组之间的差异。
结果
PDGF-BB 对 HSCs 进行趋化（间接）和趋化（直接）刺激后，快速的细胞骨架重塑导致原代大鼠 HSCs 的运动性显著增加。PDGF-BB 导致总蛋白和活性 RhoA 蛋白水平急剧升高。然而，Rho GTPases 的 mRNA 水平，包括 RhoA、Rac1 和 Cdc42，不受影响。此外，在转染 CA-RhoA 的 HSC-T6 细胞中，PDGF-BB 诱导应激纤维和焦点黏附的形成增加，但在转染 DN-RhoA 的 HSC-T6 细胞中则没有。令人惊讶的是，与对照相比，在不存在或存在 PDGF-BB 的情况下，转染 CA 和 DN-RhoA 的 HSC-T6 细胞的迁移潜力均降低。
结论
PDGF-BB 诱导大鼠 HSCs 细胞骨架重塑，并通过调节细胞内 RhoA 促进其迁移。RhoA 可能是 PDGF-BB 诱导 HSC 迁移的决定因素之一。
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