Molecular Mechanisms of Liver Fibrosis

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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.