Matrilysin in Lung Epithelial Cell Migration

描述(由申请人提供)： 上皮脱屑是急性肺损伤、哮喘和其他常见肺部疾病的显著病理特征，阐明调控上皮修复的复杂分子机制对于开发合理的治疗方法至关重要。目前的建议通过关注基质溶素(MMP7)在肺上皮细胞迁移中的作用提出了一种潜在的机制。尽管包括MMPs在内的许多蛋白酶在损伤的上皮细胞中都有表达，但其特定的功能和体内底物尚不清楚。以往的工作表明，在人和小鼠的呼吸道和肺泡上皮中，基质溶素的表达都是由肺损伤诱导的，而且基质溶素的活性促进了修复过程中肺上皮细胞的迁移，并促进了细胞-细胞连接蛋白E-钙粘附素的胞外结构域在体外和体内从肺上皮细胞中脱落。由于细胞-细胞连接的重塑是上皮细胞迁移所必需的，因此拟议的研究将检验一种假设，即基质溶素被运送到受损上皮的细胞-细胞连接，在那里它通过裂解E-钙粘附素来促进细胞迁移。将使用互补的形态和生化策略来确定基质溶素在肺上皮细胞迁移中作用的分子机制，并在人类疾病的动物模型中验证这些机制。具体目标1的研究将利用体外和体内上皮损伤模型，定位基质溶素与含有E-钙粘素的细胞-细胞连接的空间和时间分泌，并将表征由基质溶素活性诱导的细胞-细胞连接的形态和结构变化。具体目标2中的研究将通过绘制无细胞和基于细胞的分析中的裂解位点来直接评估E-钙粘附素是否是基质溶酶蛋白分解活性的底物。具体目标3将确定基质溶素切割E-钙粘附素在促进细胞-细胞连接分解和细胞迁移方面的特异性。这些研究结果将对基质金属蛋白酶在上皮修复中的作用提供新的见解，并为进一步研究肺上皮损伤反应的调控奠定基础。与威廉·帕克斯博士合作，麦奎尔博士将有机会在将分子生物学、细胞生物学和蛋白质生物化学中的基本技术和方法应用于肺部生物学的基本问题方面积累经验和专业知识。

DESCRIPTION (provided by applicant): Epithelial desquamation is a prominent pathological feature of acute lung injury, asthma, and other common pulmonary diseases, and elucidating the complex molecular mechanisms that regulate epithelial repair is essential to developing rational therapies. The current proposal addresses one potential mechanism by focusing on the function of matrilysin (MMP-7), a secreted matrix metalloproteinase (MMP), in lung epithelial cell migration. Although many proteinases, including MMPs, are expressed by injured epithelium, the specific functions and in vivo substrates have not been identified. Previous work has demonstrated that matrilysin expression is induced by lung injury in both airway and alveolar epithelium in humans and in mice, and that matrilysin activity facilitates lung epithelial cell migration during repair and promotes shedding of the extracellular ectodomain of the cell-cell junction protein E-cadherin from lung epithelium both in vitro and in vivo. Because remodeling of cell-cell junctions is necessary for epithelial cell migration, the proposed studies will test the hypothesis that matrilysin is delivered to cell-cell junctions in injured epithelium where it promotes cell migration by cleaving E-cadherin. Complementary morphological and biochemical strategies will be used to determine the molecular mechanisms of matrilysin action in lung epithelial cell migration and to verify these mechanisms in animal models of human disease. The studies in Specific Aim 1 will localize the spatial and temporal secretion of matrilysin in relationship to E-cadherin containing cell-cell junctions using both in vitro and in vivo models of epithelial injury and will characterize the morphological and structural changes in cell-cell junctions induced by matrilysin activity. The studies in Specific Aim 2 will assess directly whether E-cadherin is a substrate of matrilysin proteolytic activity by mapping cleavage sites in cell-free and cell-based assays. Specific Aim 3 will establish the specificity for E-cadherin cleavage by matrilysin in promoting cell-cell junction disassembly and cell migration. The results of these studies should provide new insights into MMP function in epithelial repair and form the basis for further investigations into the regulation of lung epithelial responses to injury. Working with William Parks, Ph.D., an established investigator in MMP biology and epithelial repair, Dr. McGuire will have the opportunity to develop experience and expertise in the application of basic techniques and approaches in molecular biology, cell biology, and protein biochemistry to fundamental questions in pulmonary biology.