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.
