Molecular Mechanisms of Liver Fibrosis

描述（由申请人提供）：肝硬化是肝损伤谱系中的晚期，预示着预后不良。肝星状细胞（HSC）质膜上整合素家族蛋白的生物力学作用将细胞外可溶性纤维连接蛋白（FN）转化为不溶性基质结合成分，是肝硬化发展的早期重要步骤。这种转化很重要，因为它加速了肝硬化典型的胶原基质沉积的后续步骤。我们的初步数据表明，在酒精和非酒精性肝纤维化的人和动物中，蛋白多糖neuropilin-1 （NRP）的蛋白水平升高，并促进FN基质的组装。从机制上讲，我们发现FN与NRP结合促进α5ß - 1整合素依赖性基质结合FN的产生。FN与NRP结合还与FN与α5ß1结合增加以及细胞内关键运输蛋白c-abl的激活有关。这些重要的初步观察结果促使我们提出了中心假设，即NRP通过双重机制促进α5ß1整合素依赖的FN基质在肝纤维化中的组装，这一机制既包括增加FN与α5ß1的结合，也包括增加c-abl介导的α5ß1质膜富集。为了验证我们的假设，我们提出了以下具体目标：1)NRP增强FN与α5ß1整合素的相互作用，从而促进α5ß1活性和基质结合FN的组装。在Subaim 1a中，我们将确定NRP的特异性蛋白聚糖修饰和配体结合域如何促进其与FN的结合。在Subaim 1b中，我们将研究NRP与FN结合增加FN与α5ß1结合的机制，从而促进α5ß1介导的FN基质组装。2) NRP活化c-abl增加了α5ß1的膜重分布，从而进一步促进FN基质的组装。在Subaim 2a中，我们将确定NRP如何激活c-abl。在Subaim 2b中，我们将确定NRP激活的c-abl如何促进α5ß1整合素重新分布到质膜并增加FN基质组装。3) NRP在体内促进FN的组装并导致肝纤维化。在本研究中，我们将使用HSC中缺乏NRP、ß1整合素或c-abl的转基因小鼠，结合确定NRP结构-功能关系的分子干预，以确定体内NRP抑制如何阻止酒精和非酒精诱导的FN基质和肝纤维化的产生。因此，Aims 1和Aims 2将分别关注NRP如何调节整合素作为细胞外“共受体”的功能，以及作为整合素质膜靶向的调节剂。反过来，Aim 3将关注使用最先进的成像方法的发现在体内的适用性。总而言之，该建议使用概念和技术上的创新方法，将解决一个关于HSC调节驱动肝纤维化过程的基质动力学的早期、可逆和重要步骤的新假设。

DESCRIPTION (provided by applicant): Cirrhosis is the advanced stage in the spectrum of liver injury and portends a poor prognosis. An early and important step in the development of cirrhosis is the conversion of extracellular, soluble fibronectin (FN) into an insoluble matrix-bound constituent by the biomechanical actions of the integrin family of proteins on the plasma membrane of hepatic stellate cells (HSC). This conversion is important because it accelerates the subsequent steps of collagen matrix deposition that typify cirrhosis. Our preliminary data demonstrate that protein levels of the proteoglycan neuropilin-1 (NRP) are increased in both humans and animals with alcohol and non-alcohol induced liver fibrosis and promotes FN matrix assembly. Mechanistically, we show that FN binding with NRP promotes α5ß1 integrin dependent generation of matrix-bound FN. FN binding with NRP is also associated with increased binding of FN with α5ß1 and with activation of a key intracellular trafficking protein, c-abl. These important initial observations have stimulated us to propose the central hypothesis that NRP promotes α5ß1 integrin dependent FN matrix assembly in liver fibrosis by a dual mechanism that involves both increased FN binding with α5ß1 and increased c-abl mediated plasma membrane enrichment of α5ß1. To examine our hypothesis, we propose the following Specific Aims: 1) NRP enhances FN interaction with α5ß1 integrin thereby promoting α5ß1 activity and assembly of matrix-bound FN. In Subaim 1a, we will determine how specific proteoglycan modifications and ligand binding domains of NRP promote its binding with FN. In Subaim 1b, we will examine the mechanism by which NRP binding with FN increases binding of FN with α5ß1, thereby promoting α5ß1 mediated assembly of FN matrix. 2) NRP activation of c-abl increases membrane redistribution of α5ß1 thereby further promoting FN matrix assembly. In Subaim 2a, we will ascertain how NRP activates c-abl. In Subaim 2b, we will identify how NRP activated c-abl promotes α5ß1 integrin redistribution to the plasma membrane and increased FN matrix assembly. 3) NRP promotes FN assembly and ensuing liver fibrosis in vivo. In this Aim, we will use a compliment of genetically modified mice that lack NRP, ß1 integrin, or c-abl in HSC, in combination with molecular interventions that ascertain NRP structure- function relationships to determine how NRP inhibition in vivo prevents alcohol and non-alcohol induced generation of FN matrix and liver fibrosis. Thus, Aims 1 and 2 will focus on how NRP regulates integrin function both as an extracellular "co-receptor", and as a regulator of integrin plasma membrane targeting, respectively. In turn, Aim 3 will focus on in vivo applicability of the finding using state-of-the-art imaging approaches. In total, this proposal, using conceptually and technically innovative approaches will address a novel hypothesis pertaining to an early, reversible, and significant step in the HSC regulation of matrix dynamics that drive the process of liver fibrosis.