Mechanisms and Effects of Fibronectin Matrix Remodeling

描述(申请人提供)：细胞外基质(ECM)分子的沉积和降解之间的精确平衡，包括I型胶原和纤维连接蛋白，是正常组织功能所必需的，也是正常组织修复的关键组成部分。过多或不适当的ECM分子沉积会破坏正常的组织结构，导致组织力学改变和器官功能受损。控制ECM沉积和周转的机制还不完全清楚。我们的数据表明，纤维连接蛋白基质聚合调节包括I型胶原在内的几种细胞外基质分子的沉积和保留。此外，纤维连接蛋白基质聚合调节细胞-细胞外基质纤维粘连的组成和稳定性，增强细胞的收缩能力，并增加胶原基组织结构的机械强度。我们的研究还表明，细胞外基质、纤维连接蛋白和I型胶原的结构组织依赖于纤维连接蛋白基质的连续聚合。破坏纤维连接蛋白聚合的药物会促进纤维连接蛋白和I型胶原的周转；这些药物也会导致组织中纤维连接蛋白的周转。这一数据表明，ECM的周转在一定程度上受到纤维连接蛋白聚合本身的调节。我们的初步数据表明，纤维连接蛋白基质的更新涉及小窝蛋白-1介导的内吞作用和溶酶体的降解。在这个方案中，我们将研究纤维连接蛋白基质堆积的控制机制，并确定纤维连接蛋白基质重构的功能后果。我们将使用我们的体外模型系统，将纤维连接蛋白缺失的肌成纤维细胞与重组突变的纤维连接蛋白和纤维连接蛋白片段结合起来，以确定纤维连接蛋白基质聚合控制纤维连接蛋白周转和内吞的机制。我们还将测试纤维连接蛋白基质聚合如何调节培养细胞和组织中细胞-基质纤维黏附部位的形成和稳定性。我们还建立了一套拉伸测试系统，以定量确定组织和胶原基组织结构的机械性能。我们将使用这个系统来确定纤维连接蛋白基质周转对细胞收缩、细胞张力产生和组织机械强度的影响。确定纤维连接蛋白聚合调节ECM重塑和组织机械强度的机制，将为促进纤维化发展的因素以及可能导致纤维化患者正常基质结构恢复和器官功能改善的机制提供重要的见解。

DESCRIPTION (provided by applicant): A precise balance between the deposition and degradation of extracellular matrix (ECM) molecules, including collagen I and fibronectin, is required for normal tissue function, and is a key component of normal tissue repair. Excessive or inappropriate deposition of ECM molecules disrupts normal tissue architecture, leading to altered tissue mechanics and impaired organ function. The mechanisms that control ECM deposition and turnover are incompletely understood. Our data demonstrates that fibronectin matrix polymerization regulates the deposition and retention of several ECM molecules, including collagen I. Furthermore, fibronectin matrix polymerization regulates the composition and stability of cell-ECM fibrillar adhesions, enhances cell contractility, and increases the mechanical strength of a collagen-based tissue construct. Our studies also indicate that the structural organization of ECM fibronectin and collagen I depends upon the continuous polymerization of a fibronectin matrix. Agents that disrupt fibronectin polymerization trigger enhanced fibronectin and collagen I turnover; these agents also induce turnover of fibronectin in tissues. This data suggests that ECM turnover is regulated, in part, by fibronectin polymerization itself. Our preliminary data indicate that fibronectin matrix turnover involves caveolin-1 mediated endocytosis and lysosomal degradation. In this proposal, we will investigate the mechanisms by which fibronectin matrix accumulation is controlled, and determine the functional consequences of fibronectin matrix remodeling. We will use our in vitro model system employing fibronectin-null myofibroblasts in conjunction with recombinant mutant fibronectins and fibronectin fragments to determine the mechanisms by which fibronectin matrix polymerization controls the turnover and endocytosis of fibronectin. We will also test how fibronectin matrix polymerization regulates the formation and stability of cell-matrix fibrillar adhesion sites in cultured cells and in tissues. We have also established a tensile testing system to quantitatively determine the mechanical properties of tissues and collagen-based tissue constructs. We will use this system to determine the effects of fibronectin matrix turnover on cell contractility, cell tension generation, and the mechanical strength of tissues. Determining the mechanisms by which fibronectin polymerization regulates ECM remodeling and tissue mechanical strength will provide important insights into factors that contribute to the development of fibrosis, and into mechanisms that could lead to restoration of normal matrix architecture and improved organ function in individuals with fibrotic disorders.