CONTROL OF MATRIX PROTEOLYSIS BY INTEGRIN A3B1 IN SKIN

描述(改编自申请人的摘要)：皮肤地下室 膜是一种特殊的细胞外基质(ECM)，它将 真皮上的表皮。在皮肤发育和伤口修复过程中， 基底膜在结构和成分上经历了广泛的变化 与细胞迁移、信号转导和基因的变化有关 表情。整合素α3beta1是一种表皮黏附受体 基底膜中层粘连蛋白-5结合的角质形成细胞。基因突变 小鼠的alpha3基因揭示了alpha3beta1在组织中的关键作用 发育中的皮肤中的基底膜。细胞侵袭过程，如 促进皮肤发育、伤口愈合和癌症侵袭的因素是 细胞外ECM的蛋白分解，整合素在其中起作用 基质金属蛋白酶(MMPs)等的功能调节 ECM降解酶。Alpha3beta1在创伤过程中可能起到这样的作用 修复，在角质形成细胞上的高表达与 ECM降解酶的激活。然而，Alpha3beta1的机制 在皮肤发育和伤口愈合中控制ECM重塑的机制尚不清楚。这个 拟议的研究利用了Alpha3beta1基因缺陷的小鼠和角质形成细胞 从它们派生的线条，作为定义这些线条的唯一模型系统 机械装置。这些研究的初步数据显示，alpha3beta1是 角质形成细胞需要不同的蛋白水解酶功能和表达 编码已知的ECM蛋白水解酶或蛋白水解酶抑制剂的基因。生化 和分子方法，从正常或培养的皮肤和角质形成细胞 Alpha3beta1缺陷小鼠，将被用来识别 由字母3beta1调节。候选蛋白水解酶将被直接检测吗？ 恢复依赖于alpha3beta1的函数的能力 α3β1缺陷型角质形成细胞的转染。要定义 整合素介导的信号通路调节ECM重塑、信号转导 以依赖于α3β1的方式被激活的蛋白质将是 用免疫生物化学方法鉴定。这些基因的显性-负性突变 然后将蛋白质导入表达Alpha3beta1的角质形成细胞，并 检测是否抑制内源性蛋白水解酶活性或表达。这 研究将为整合素在体内的作用提供重要的见解 从基因表达和蛋白水解酶水平调控ECM重塑 功能，并可能有助于开发治疗方法 控制伤口愈合或癌症侵袭。

DESCRIPTION (Adapted from applicant's abstract): The cutaneous basement membrane is a specialized extracellular matrix (ECM) that separates the epidermis from the dermis. During skin development and wound repair, the basement membrane undergoes extensive changes in structure and composition that are associated with changes in cell migration, signal transduction, and gene expression. Integrin alpha3beta1 is an adhesion receptor on epidermal keratinocytes that binds laminin-5 in the basement membrane. Mutation of the alpha3 gene in mouse revealed a critical role for alpha3beta1 in organization of the basement membrane in developing skin. Cell invasive processes such as skin development, wound healing, and carcinoma invasion are facilitated by proteolysis of the pericellular ECM, in which integrins contribute to functional regulation of matrix metalloproteinases (MMPs) or other ECM-degrading proteinases. Alpha3beta1 may have such a role during wound repair, where its high expression on keratinocytes coincides with the activation of ECM-degrading enzymes. However, mechanisms whereby alpha3beta1 controls ECM remodeling in skin development and wound healing are unknown. The proposed research exploits alpha3beta1-deficient mice, and keratinocyte cell lines derived from them, as a unique model system for defining these mechanisms. Preliminary data for these studies show that alpha3beta1 is required in keratinocytes for distinct proteinase function and for expression of genes encoding known ECM proteinases or proteinase inhibitors. Biochemical and molecular methods, with skin and cultured keratinocytes from normal or alpha3beta1-deficient mice, will be used to identify proteinases that are regulated by alpha3beta1. Candidate proteinases will them be tested directly for their abilities to restore alpha3beta1-dependend functions through transfection of alpha3beta1-deficient keratinocytes. To define integrin-mediated signaling pathways that regulate ECM remodeling, signaling proteins that are activated in an alpha3beta1-dependent manner will be identified using immunobiochemical methods. Dominant-negative mutants of these proteins will then be transfected into alpha3beta1-expressing keratinocytes and tested for inhibition of endogenous proteinase activity or expression. This research will provide important insights into the roles of integrins in regulating ECM remodeling at the levels of gene expression and proteinase function, and may contribute to the development of therapeutic approaches to control wound healing or carcinoma invasion.