The Regulation of Fibronectin Matrix Assembly in Wound Healing

纤连蛋白基质组装在伤口愈合中的调控

• 批准号: 6983836
• 负责人: SIOBHAN A CORBETT
• 金额: $ 33.05万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6983836
• 关键词: CHO cells; JUN kinase; biological signal transduction; cell cycle; cell growth regulation; cell proliferation; complementary DNA; cytoskeletal proteins; enzyme activity; extracellular matrix; fibrin; fibronectins; guanosinetriphosphatases; immunoprecipitation; integrins; intermolecular interaction; mitogen activated protein kinase; myosin light chain kinase; phosphorylation; platelet derived growth factor; protein biosynthesis; protein structure function; protein transport; scars; wound healing

DESCRIPTION (provided by applicant): Fibronectin (FN) is an indispensable extracellular matrix (ECM) protein that is required for tissue repair and remodeling. In the wound, cells assemble newly synthesized FN into a three-dimensional (3D) fibrillar matrix that regulates a variety of cell behaviors including contractility. The receptor responsible for FN matrix assembly is the integrin a5b1. Although the structural features of FN required for matrix assembly have been examined, the specific receptor requirements have not been well defined. During the previous funding period, we demonstrated the novel finding that the a5 integrin cytoplasmic domain regulates FN matrix assembly and as a consequence, confers strong intercellular cohesion to 3D cellular aggregates. In this new proposal, we show for the first time that FN matrix assembly is regulated by endocytosis, leading us to hypothesize that regulated internalization of a5b1 and its recycling to the plasma membrane are required for the initiation and stabilization of the FN matrix. To test this hypothesis, we will accomplish the following Specific Aims: 1) The mechanisms of a5b1 receptor trafficking, in the presence or absence of endogenous FN matrices, will be determined using pharmacologic blockade, immunofluorescent imaging and molecular biological techniques. Stimulation or inhibition of receptor trafficking will be used to correlate FN matrix formation with receptor endocytosis; 2) Recombinant FN proteins will be used to analyze how FN structure regulates a5b1 receptor trafficking. The role of the actin and tubulin cytoskeletons in a5b1 receptor trafficking will be determined using pharmacologic agents that alter actin organization and microtubule stabilization, respectively; 3) Sites on the a5 integrin cytoplasmic domain that regulate receptor trafficking will be mapped by generating point mutations in wild type or chimeric a5 cDNAs that block interactions with chaperone proteins involved in endocytosis; 4) The effect of de novo a5b1 expression on the trafficking of the av|33 integrin will be analyzed and correlated with changes in avb3 receptor function. Excessive FN matrix deposition is associated in vivo with pathologic fibrosis and scarring. Therefore, an understanding of the molecular mechanisms that regulate FN matrix assembly will be important to the design of new therapies to prevent abnormal wound repair and to improve functional recovery following injury.

描述（由申请人提供）：纤连蛋白（FN）是一种必不可少的细胞外基质（ECM）蛋白，是组织修复和重塑所需的。在伤口中，细胞将新合成的FN组装成三维（3D）原纤维基质，该基质调节了包括收缩力在内的各种细胞行为。负责FN基质组件的受体是整联蛋白A5B1。尽管已经检查了矩阵组装所需的FN的结构特征，但特定的受体要求尚未得到很好的定义。在上一个资金期间，我们证明了新的发现，即A5整联蛋白细胞质结构域调节FN基质组件，因此，对3D细胞聚集体赋予了强大的细胞间内聚力。在这项新提案中，我们首次表明FN基质组件受内吞作用调节，这使我们假设A5B1的内在化及其回收到质膜需要进行FN Matrix的启动和稳定。为了检验这一假设，我们将完成以下具体目的：1）在存在或不存在内源性FN矩阵的情况下，A5B1受体运输的机制将使用药理阻断，免疫荧光成像和分子生物学技术来确定。刺激或抑制受体运输将用于将FN基质形成与受体内吞作用相关。 2）重组FN蛋白将用于分析FN结构如何调节A5B1受体运输。肌动蛋白和微管蛋白细胞骨架在A5B1受体运输中的作用将使用分别改变肌动蛋白组织和微管稳定的药理剂确定。 3）在A5整合素细胞质结构域上调节受体运输的位点，将通过在野生型或嵌合A5 CDNA中产生点突变来映射，该突变阻断与参与内吞作用的伴侣蛋白相互作用的相互作用； 4）DE NOVO A5B1表达对AV | 33整合素的运输的影响将被分析并与AVB3受体功能的变化相关。过度的FN基质沉积与病理纤维化和疤痕有关。因此，对调节FN基质组件的分子机制的理解对于设计新疗法以防止伤口修复并改善损伤后功能恢复至关重要。