CONTROL OF MATRIX PROTEOLYSIS BY INTEGRIN A3B1 IN SKIN

描述（改编自申请人摘要）：皮肤基底 膜是一种专门的细胞外基质（ECM）， 表皮和真皮。在皮肤发育和伤口修复过程中， 基底膜的结构和组成发生了广泛的变化， 与细胞迁移、信号转导和基因表达的变化有关。 表情整合素α 3 β 1是表皮细胞上的粘附受体 在基底膜中结合层粘连蛋白-5的角质形成细胞。突变 小鼠的α 3基因揭示了α 3 β 1在组织中的关键作用 在发育中的皮肤的基底膜。细胞侵入过程，如 促进皮肤发育、伤口愈合和癌侵袭， 细胞周围ECM的蛋白水解，其中整合素有助于 基质金属蛋白酶（MMPs）或其他 ECM降解蛋白酶。α 3 β 1可能在创伤过程中起这样的作用 修复，其中其在角质形成细胞上的高表达与 ECM降解酶的活化。然而，α 3 β 1 控制皮肤发育和伤口愈合中的ECM重塑尚不清楚。的 一项研究计划利用α 3 β 1缺陷小鼠和角质形成细胞， 从它们衍生出来的线条，作为一个独特的模型系统，用于定义这些 机制等这些研究的初步数据显示， 在角质形成细胞中需要不同的蛋白酶功能和表达 编码已知ECM蛋白酶或蛋白酶抑制剂的基因。生化 和分子方法，皮肤和培养的角质形成细胞从正常或 α 3 β 1缺陷小鼠，将用于鉴定蛋白酶， 由α 3 β 1调节。候选蛋白酶将被直接检测 因为它们能够通过以下方式恢复α 3 β 1依赖性功能： α 3 β 1缺陷型角质形成细胞的转染。以限定 整合素介导的调节ECM重塑的信号传导途径， 以α 3 β 1依赖性方式激活的蛋白质将被 使用免疫生物化学方法鉴定。这些基因的显性阴性突变体 然后将蛋白质转染到表达α 3 β 1的角质形成细胞中， 测试内源性蛋白酶活性或表达的抑制。这 研究将提供重要的见解整合素的作用， 在基因表达和蛋白酶水平调节ECM重塑 功能，并可能有助于治疗方法的发展， 控制伤口愈合或癌细胞侵袭。

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.