Molecular Mechanisms of Liver Fibrosis

描述(申请人提供)：肝硬变是肝脏损伤的晚期，预示着不良的预后。在肝星状细胞(HSC)质膜上整合蛋白家族的生物力学作用下，细胞外的可溶性纤维连接蛋白(FN)转化为不溶于基质的成分，这是肝硬变发生发展的早期和重要的一步。这种转化很重要，因为它加速了胶原基质沉积的后续步骤，这是典型的肝硬变。我们的初步数据表明，在酒精和非酒精诱导的肝纤维化的人和动物中，蛋白多糖神经毛细蛋白-1(NRP)的蛋白水平都增加，并促进FN基质的组装。从机制上讲，FN与NRP的结合促进了？依赖整合素的基质结合FN的生成。FN与NRP的结合也与FN与？的结合增加有关。并通过激活关键的细胞内运输蛋白c ABL.这些重要的初步观察促使我们提出了NRP促进？的中心假说。在肝纤维化中整合素依赖的FN基质组装通过双重机制涉及FN结合增加与？并增加c-abl介导的质膜浓缩。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？的相互作用。整合素，从而促进？基质结合的FN的活性和组装。在Subaim 1a中，我们将确定NRP的特定蛋白多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结合的机制，通过增加FN与？的结合，从而促进？介导的FN基质组装。2)c-ABL的NRP激活增加了？膜再分布。从而进一步促进FN矩阵组装。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是如何促进？整合素重新分布到质膜，增加FN基质组装。3)在体内，NRP促进纤维连接蛋白的组装和随后的肝纤维化。在这个目标中，我们将使用一组在HSC中缺乏NRP、β1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来确定体内抑制NRP如何防止酒精和非酒精诱导的FN基质的生成和肝纤维化。因此，AIMS 1和2将分别侧重于NRP如何作为细胞外“共受体”和作为整合素质膜靶向的调节器来调节整合素的功能。反过来，Aim 3将利用最先进的成像方法，重点研究该发现在体内的适用性。总之，这项建议，使用概念和技术上的创新方法，将解决一个新的假说，该假说涉及HSC对推动肝纤维化过程的基质动力学的早期、可逆和重要的调节步骤。

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.