Osteopontin and the fibrogenic response to liver injury

摘要 门静脉纤维化发生于慢性肝病，其中最初的原发性损害是小叶中心，但 涉及的机制仍不清楚。骨桥蛋白（OPN）是一种在细胞中组成性表达的细胞因子 在门静脉周围区域内，并且在肝损伤中高度诱导。我们认为OPN使细胞能够感知 与肝损伤相关的分子模式，并触发卵圆细胞扩张所需的信号， 小管反应和纤维形成。在本次竞争性更新中，我们将重点测试中央 假设氧化应激介导的肝损伤将上调OPN，从而诱导卵圆细胞 扩张、小管反应和I型胶原表达，促进肝纤维化的发展。 具体而言，我们假设：1）氧化应激介导的肝损伤将诱导OPN表达; 2）OPN 将上调星状细胞中的胶原I，作为前馈机制促进瘢痕形成; 3）OPN将 驱动卵圆细胞扩增和小管反应，以及4）Opn-/-小鼠将通过减少卵圆细胞扩增和小管反应来避免肝纤维化。 细胞扩增、小管反应和限制体内胶原I沉积。提出了四个具体目标 来解决这些假设。在目标1中，我们将在体外评估活性氧的分子基础， 物种信号传导上调卵圆细胞、胆管上皮细胞和肝星状细胞中的OPN 硫代乙酰胺诱导的肝纤维化。目的2研究OPN对小鼠I型胶原上调的影响 星状细胞，我们将确定膜蛋白从事骨桥蛋白和近端信号 在结合时激活的分子/应激敏感激酶，其触发促纤维化级联。目标3： 为了研究OPN在卵圆细胞扩张和小管反应中的作用，将用 重组OPN和卵圆细胞增殖和分化为胆管上皮细胞，以及 还将评价可能影响星状细胞纤维化反应的相关因素。在目标4中， OPN诱导对卵圆细胞增殖、小管反应和纤维化的体内生理作用 将使用野生型和Opn-/-小鼠和两种良好建立的 肝纤维化模型。卵圆细胞扩张的生化和免疫组织学参数，小管 反应、炎症、坏死、肝细胞复制停滞和纤维化将作为读数进行评价 OPN对肝纤维化的作用。公共卫生相关性：肝纤维化影响数百万人 在美国，许多患者进展为肝硬化和肝细胞癌。这个目标 建议是调查OPN信号在这一过程中的作用，并评估是否靶向OPN调节 网络可能是预防或治疗肝纤维化的有用策略。

ABSTRACT Portal fibrosis develops in chronic liver disease in which the initial primary insult is centrilobular, but the mechanism involved still remains unclear. Osteopontin (OPN) is a cytokine constitutively expressed in cells within the periportal region and it is highly induced in liver injury. We believe that OPN enables cells to sense molecular patterns associated with liver injury, and triggers signals that are required for oval cell expansion, ductular reaction, and fibrogenesis to occur. In this Competitive Renewal we will focus on testing the Central Hypothesis ¿Oxidative stress-mediated liver injury will up-regulate OPN, which in turn, will induce oval cell expansion, ductular reaction, and collagen I expression, contributing to the development of liver fibrosis¿. Specifically, we hypothesize that: 1) Oxidative stress-mediated liver injury will induce OPN expression; 2) OPN will up-regulate collagen I in stellate cells acting as a feed-forward mechanism to promote scarring; 3) OPN will drive oval cell expansion and ductular reaction, and 4) Opn-/- mice will avert liver fibrosis by decreasing oval cell expansion, ductular reaction, and limiting collagen I deposition in vivo. Four Specific Aims are proposed to address these hypotheses. In Aim 1, we will evaluate in vitro the molecular basis whereby reactive oxygen species signaling up-regulates OPN in oval cells, biliary epithelial cells, and hepatic stellate cells in thioacetamide-induced liver fibrosis. In Aim 2, to study the effects of OPN on collagen I up-regulation in stellate cells, we will identify the membrane proteins engaged by OPN and the proximal signaling molecules/stress-sensitive kinases activated upon binding that trigger the pro-fibrogenic cascade. In Aim 3, to dissect the role of OPN in oval cell expansion and ductular reaction, primary oval cells will be treated with recombinant OPN and oval cell proliferation and differentiation to biliary epithelial cells, as well as the potential factors involved that may also impact on the stellate cell fibrogenic response, will be evaluated. In Aim 4, the in vivo physiological contribution of OPN induction to oval cell proliferation, ductular reaction, and the fibrogenic response will be tested in time-course experiments using wild-type and Opn-/- mice and two well-established models of liver fibrosis. Biochemical and immunohistological parameters of oval cell expansion, ductular reaction, inflammation, necrosis, hepatocyte replicative arrest, and fibrogenesis will be evaluated as read-outs for the contribution of OPN to liver fibrosis. Public Health Relevance: Liver fibrosis affects several million people in the U.S. and progresses to cirrhosis and hepatocellular carcinoma in many patients. The Goal of this Proposal is to investigate the role of OPN signaling in this process, and to evaluate whether targeting the OPNregulated network may be a useful strategy for preventing or treating liver fibrosis.