Molecular Mechanisms of Liver Fibrosis

描述（由申请人提供）：肝硬化是肝损伤谱中的晚期，预示着预后不良。肝硬化发展的早期和重要步骤是细胞外可溶性纤连蛋白（FN）通过整合素家族蛋白对肝星状细胞（HSC）质膜的生物力学作用转化为不溶性基质结合成分。这种转化是重要的，因为它加速了胶原基质沉积的后续步骤，这是肝硬化的典型特征。我们的初步数据表明，蛋白多糖神经纤毛蛋白-1（NRP）的蛋白水平增加，在人类和动物与酒精和非酒精诱导的肝纤维化，促进FN矩阵组装。从机制上讲，我们表明，FN与NRP结合促进？基质结合FN的整合素依赖性产生。FN与NRP的结合也与FN与NRP的结合增加有关。并激活一种关键的细胞内运输蛋白， 消融导管这些重要的初步观察促使我们提出了核心假设，即NRP促进了？整合素依赖性FN基质组装在肝纤维化中的双重机制，包括增加FN结合与？增加c-abl介导的质膜富集。为了验证我们的假设，我们提出以下具体目的：1）NRP增强FN与？整合素，从而促进？基质结合FN的活性和组装。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结构域如何促进其与FN的结合。在Subaim 1b中，我们将研究NRP与FN结合增加FN与？结合的机制，从而促进了？介导的FN基质组装。2)NRP激活c-abl增加细胞膜的再分布？从而进一步促进FN基质组装。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c-abl如何促进？整合素重新分布到质膜和增加FN基质组装。3)NRP促进FN组装和随后的体内肝纤维化。在这个目标中，我们将使用一种缺乏NRP的转基因小鼠，？1整联蛋白或c-abl结合确定NRP结构-功能关系的分子干预，以确定体内NRP抑制如何防止酒精和非酒精诱导的FN基质产生和肝纤维化。因此，目标1和2将分别关注NRP如何调节整合素作为细胞外“共受体”和作为整合素质膜靶向的调节剂的功能。反过来，Aim 3将重点关注使用最先进的成像方法发现的体内适用性。总的来说，这项提案，使用概念和技术创新的方法将解决一个新的假设有关的一个早期，可逆的，重要的一步，HSC调节基质动力学，驱动肝纤维化的过程。

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 ???? integrin dependent generation of matrix-bound FN. FN binding with NRP is also associated with increased binding of FN with ???? 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 ???? integrin dependent FN matrix assembly in liver fibrosis by a dual mechanism that involves both increased FN binding with ???? and increased c-abl mediated plasma membrane enrichment of ????. To examine our hypothesis, we propose the following Specific Aims: 1) NRP enhances FN interaction with ???? integrin thereby promoting ???? 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 ????, thereby promoting ???? mediated assembly of FN matrix. 2) NRP activation of c-abl increases membrane redistribution of ???? 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 ???? 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, ?1 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.