Matrilysin in Lung Epithelial Cell Migration

描述（由申请人提供）： 上皮脱落是急性肺损伤、哮喘和其他常见肺部疾病的显著病理特征，阐明调节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。 目前的建议解决了一个潜在的机制，重点是基质溶解素（MMP-7），分泌的基质金属蛋白酶（MMP），在肺上皮细胞迁移的功能。 虽然许多蛋白酶，包括基质金属蛋白酶，表达损伤的上皮细胞，具体的功能和在体内底物尚未确定。 先前的工作已经证明，基质溶素表达诱导肺损伤在气道和肺泡上皮细胞在人类和小鼠中，和基质溶素活性促进肺上皮细胞迁移修复过程中，并促进脱落的细胞-细胞连接蛋白E-钙粘蛋白的细胞外胞外域从肺上皮细胞在体外和体内。 由于上皮细胞迁移所必需的细胞-细胞连接的重塑，拟议的研究将测试的假设，即基质溶解素被交付到细胞-细胞连接在受伤的上皮细胞，它促进细胞迁移的切割E-钙粘蛋白。 互补的形态学和生物化学策略将被用来确定肺上皮细胞迁移中基质溶解素作用的分子机制，并在人类疾病的动物模型中验证这些机制。 具体目标1中的研究将使用上皮损伤的体外和体内模型，定位与含E-钙粘蛋白的细胞-细胞连接相关的基质溶解素的空间和时间分泌，并将表征基质溶解素活性诱导的细胞-细胞连接的形态和结构变化。 具体目标2中的研究将通过在无细胞和基于细胞的测定中绘制切割位点，直接评估E-钙粘蛋白是否为基质溶解素蛋白水解活性的底物。 特异性目标3将确立基质溶解素在促进细胞-细胞连接解体和细胞迁移中切割E-钙粘蛋白的特异性。 这些研究的结果应提供新的见解MMP功能上皮修复和形成的基础上，进一步调查到肺上皮细胞损伤反应的调节。 与威廉·帕克斯博士合作，作为MMP生物学和上皮修复领域的资深研究者，McGuire博士将有机会在分子生物学、细胞生物学和蛋白质生物化学的基本技术和方法应用于肺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.