Molecular Mechanisms of Liver Fibrosis

项目摘要/摘要： 酒精和其他原因造成的肝损伤可最终导致肝硬变，并伴有显著的相关发病率。 和死亡率。肝星状细胞(HSC)激活包括增强的表型 迁移、增殖和基质沉积。迁移是协调放置HSC for Matrix的关键 肝硬变的沉积和发展。我们的长期目标是了解分子 以确定治疗靶点为目标的导致肝硬变的HSC生物学基础。最近， 我们确定了联结蛋白在HSC迁移和纤维化过程中的关键作用。聚合素是一种胞浆蛋白 介导信号转导、囊泡运输和最终基因表达的蛋白质。西奈克汀是 选择这一方案作为值得详细研究的重要蛋白质是因为，正如我们所证明的那样， 它在人类肝硬变中上调，是小鼠肝纤维化所必需的。从机制上讲，我们认为 联结蛋白通过短期受体信号和长期表观遗传在HSC迁移中的作用 基因表达网络的调控。我们的初步数据显示，HSC联结蛋白的消耗减少了 受体酪氨酸激酶下游的迁移信号，血小板衍生生长因子受体α (PDGFRα)；减弱一组肝星状细胞激活基因的转录，包括编码 多功能信号蛋白IGFBP3(胰岛素生长因子结合蛋白-3)； 体内纤维化。这些重要的观察结果使我们提出了联结蛋白的中心假说 通过促进血小板衍生生长因子受体α信号转导和调节基因网络促进造血干细胞迁移和纤维化 这包括IGFBP3。这一假说的具体目的如下：1)聚合素促进肝星状细胞 通过调节PDGFRα靶向和信号激活进行迁移。Aim 1a将决定Sycertin如何招募员工 并结合特定的囊泡运输蛋白，维持PDGFRα蛋白水平，靶向该蛋白 内体和激活迁移信号。目标1b将揭示连接蛋白功能中断是如何导致 血小板衍生生长因子受体α的自噬降解和减弱的造血干细胞迁移。2)聚合素表观遗传调控 IGFBP3基因表达促进HSC迁移。目标2a将确定联结蛋白如何调节特定的 组蛋白甲基转移酶，EZH2以及它如何通过组蛋白甲基化来调控IGFBP3基因的表达。 Aim 2b将确定IGFBP3的产生如何刺激HSC迁移。3)聚合素对体内纤维化有调节作用。 Aim 3a将使用一种新的小鼠纤维化回归模型，该模型对HSC选择性地修饰连接素和 α以进一步确定这些蛋白质在体内的拟议作用。AIM 3b将使用连接 与磁共振弹性成像(MRE)协同作用的选择性靶向HSC的中和肽 成像，以及IGFBP3基因缺失的小鼠，以阐明联结素如何促进体内的纤维化。总的来说， 这项提案将利用概念和技术上的创新方法和概念来测试一部小说 关于联结蛋白是导致迁移和纤维化的HSC信号的“主调节器”的假说。

PROJECT SUMMARY/ABSTRACT: Liver injury from alcohol and other etiologies can culminate in cirrhosis with significant associated morbidity and mortality. Hepatic stellate cell (HSC) activation encompasses a phenotype that includes enhanced migration, proliferation, and matrix deposition. Migration is critical for coordinately situating HSC for matrix deposition and development of cirrhosis. Our Long-Term Objectives are to understand the molecular underpinnings of HSC biology that lead to cirrhosis with the goal of identifying therapeutic targets. Recently, we identified a critical role of synectin in the process of HSC migration and fibrosis. Synectin is a cytosolic protein that mediates signal transduction, vesicle trafficking, and ultimately gene expression. Synectin is chosen for this proposal as a significant protein warranting detailed investigation because, as we demonstrate, it is upregulated in human cirrhosis and is required for murine fibrogenesis. Mechanistically, we implicate synectin in HSC migration through both short term receptor signaling as well as longer term epigenetic regulation of gene expression networks. Our preliminary data show that depletion of HSC synectin reduces migration signaling downstream of the receptor tyrosine kinase, platelet derived growth factor receptor alpha (PDGFRα); attenuates transcription of a set of HSC activation genes including one that encodes the multifunctional signaling protein, IGFBP3 (insulin growth factor binding protein-3); and abrogates murine fibrosis in vivo. These important observations have led us to propose the central hypothesis that synectin increases HSC migration and fibrosis by promoting PDGFRα signaling and by regulating a network of genes that include IGFBP3. This hypothesis leads to the following Specific Aims: 1) Synectin promotes HSC migration by regulating PDGFRα targeting and signal activation. Aim 1a will determine how synectin recruits and binds specific vesicle trafficking proteins that maintain PDGFRα protein levels, target the protein to endosomes and activate migration signaling. Aim 1b will uncover how disruption of synectin function leads to autophagic degradation of PDGFRα and attenuated HSC migration. 2) Synectin epigenetically controls IGFBP3 gene expression to promote HSC migration. Aim 2a will identify how synectin regulates a specific histone methyl transferase, EZH2 and how this governs IGFBP3 gene expression through histone methylation. Aim 2b will determine how IGFBP3 production stimulates HSC migration. 3) Synectin regulates fibrosis in vivo. Aim 3a will use a novel fibrosis regression model in mice with HSC selective modifications to synectin and PDGFRα to further ascertain the proposed role of these proteins in vivo. Aim 3b will use a synectin neutralizing peptide that selectively targets HSC in coordination with magnetic resonance elastography (MRE) imaging, and mice with genetic deletion of IGFBP3 to elucidate how synectin promotes fibrosis in vivo. In total, this proposal will utilize conceptually and technically innovative approaches and concepts to test a novel hypothesis pertaining to synectin as a “master regulator” of HSC signals that lead to migration and fibrosis.