Extracellular matrix in liver cirrhosis : its abnormality and influence on liver functions.

在肝硬化中，丰富的细胞外基质和紊乱的小叶结构恶化了器官特异性功能。肝星状细胞（hepatic stellate cells，HSCs）可转化为肌纤维母细胞样细胞，通过分泌过量的细胞外基质蛋白和收缩愈合的伤口，在肝纤维化的发生中发挥重要作用。我们从CCl_4诱导的大鼠肝硬化的HSC细胞系的cDNA文库中克隆了一个丝氨酸/苏氨酸蛋白激酶。它与新近报道的Rho-激酶相同，Rho-激酶是Rho效应器之一，通过其调节几种基于肌动蛋白的细胞过程，如粘附、胞质分裂和收缩。本研究旨在阐明Rho和Rho激酶在HSC中的功能，免疫印迹分析证实了Rho激酶在HSC中的蛋白表达和自身磷酸化，以及Rho刺激剂溶血磷脂酸（LPA）对它们的调节作用。使用I型胶原晶格的收缩实验表明，LPA以剂量依赖的方式诱导HSC收缩，并且其作用被加入Rho的灭活剂C3外切酶减弱。α-平滑肌肌动蛋白（活化HSC中的细胞骨架蛋白）的蛋白表达不受LPA或C3外切酶的影响。当HSC与纤连蛋白包被的微球共培养时，可观察到黏着斑复合物及相关蛋白Rho和Rho激酶在微球周围的质膜下组装，而C3外切酶可阻断这种组装，推测Rho和Rho激酶可能参与HSC细胞外基质信号的细胞内传递。

In cirrhotic liver, abundant extracellular matrices and deranged lobular architecture deteriorate the organ-specific function. Hepatic stellate cells (HSCs) transform into myofibroblast-like cells and play an important role in the hepatic fibrogenesis by producing an excess amount of extracellular matrix proteins and contracting the healing wound. We have cloned a serine/threonine protein kinase from a cDNA library prepared from HSC line derived from a CCI_4 induced cirrhotic rat liver. It is identical to the recently-reported Rho-kinase, one of Rho effectors, through which several actomyosin-based cellular processes such as adhesion, cytokinesis, and contraction are regulatcd. This study mainly aimed to clarify the functions of Rho and Rho-kinase in HSCs.Immunoblot analysis confirmed the protein expression and auto-phosphorylation of Rho-kinase by HSCs, as well as their regulation by Rho-stimulator lysophosphatidic acid (LPA). Contraction assay using type I collagen lattice showed that LPA induced contraction of HSCs in a dose-dependent manner, and its effect was reduced by the addition of C3 exoenzyme, an inactivator of Rho. Protein expression of alpha-smooth muscle actin, a cytoskeletal protein in activated HSCs, was not affected by addition of LPA or C3 exoenzyme. When HSCs were added with fibronectin-coated beads, the subplasmalemmal assembly of focal adhesion complex and related proteins including Rho and Rho-kinase were observed arround the beads, and C3 exoenzyme abrogated the assembly.From these data, Rho and Rho-kinase might be involved in the intracellular transduction of the signal from extracellular matrices in HSCs.