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

在肝硬变中，丰富的细胞外基质和错乱的小叶结构使器官特异性功能恶化。肝星状细胞(HSCs)转化为肌成纤维细胞样细胞，通过产生过量的细胞外基质蛋白，收缩创面愈合，在肝纤维化形成过程中发挥重要作用。我们从CCI_4诱导的大鼠肝硬变HSC系的cDNA文库中克隆了一个丝氨酸/苏氨酸蛋白激酶。它与最近报道的Rho效应器之一Rho-Kinase相同，通过它调节几个基于肌动球蛋白的细胞过程，如黏附、胞质分裂和收缩。本研究旨在阐明Rho和Rho-Kinase在HSC中的作用。免疫印迹分析证实HSC能够表达Rho-Kinase，并被Rho刺激物溶血磷脂酸(LPA)调节。I型胶原晶格收缩实验显示，LPA以剂量依赖方式诱导HSCs收缩，Rho失活剂C3胞外酶的作用减弱。LPA或C3外酶不影响α-平滑肌肌动蛋白的蛋白表达，α-平滑肌肌动蛋白是活化的HSC的细胞骨架蛋白。将纤维粘连蛋白包被的小球与HSCs复合后，可在小球周围观察到焦点黏附复合体及相关蛋白如Rho和Rho-Kinase的亚质内组装，C3胞外酶取消了这种组装，由此推测Rho和Rho-Kinase可能参与了HSCs细胞外基质信号的细胞内转导。

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.