Our hypotheses are that the interactions of EB keratinocytes with connective tissue components (matrix) are altered compared with normal keratinocytes and that these altered cell-matrix interactions account for poor adherence between the epidermis and dermis, the poor wound healing potential, and high risk of cancer invasion in this family of diseases. In performing these comparisons, we shall learn about the biology of normal keratinocyte locomotion and collagenase expression, two areas of importance to wound healing in general. Our preliminary data show that keratinocyte locomotion and expression of collagenase and tissue inhibitor of metalloproteases (TIMP) are dramatically influenced by extracellular matrix. We shall examine human keratinocyte locomotion and compare normal adult, fetal and EB keratinocytes using recently developed keratinocyte migration assays that employ quantitative, computer-assisted morphometry and discriminate between cell movement and cell proliferation. We shall assess the influence of purified connective tissue molecules and biologically active domains on keratinocyte locomotion. We shall examine keratinocyte locomotion when the cells are apposed to whole laminin, fibronectin and collagen types I and IV, as well as purified proteolytic fragments of these molecules such as fibronectin's collagen-binding, cell- binding and heparan-binding domains and laminin's thrombin, pepsin, and elastase fragments. We shall perform similar experiments with synthesized peptides such as the RGDS sequence in fibronectin and YIGSR sequence in laminin. We shall examine the expression of types I and IV collagenases and TIMP by human keratinocytes (normal adult, fetal and EB) when they are migrating on these substrata. In addition to the standard functional assay for collagenase we shall perform quantitative collagenase ELISAs and immunoprecipitations of collagenase and TIMP and visualize and quantitate precipitated products with densitometry. We shall also examine keratinocyte-derived collagenase and TIMP at the level of mRNA expression after the cells have been plated on the various substrata and migration has been quantitated. This work has important implications for understanding the nature of cell- matrix interactions during fetal skin development, wound healing in normal circumstances and in the aberrant wound healing that characterizes the severe forms of EB. In addition, these studies will promote an understanding of normal mechanisms of re-epithelialization and cancer invas n.
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