Vertebrate collagenase, a neutral metalloendoproteinase, cleaves native interstitial collagen into specific three-quarter and one- quarter length fragments which are then susceptible to further proteolysis. In the skin, excessive production of collagenase is thought to contribute to the pathogenesis of the inherited blistering disorder, dystrophic epidermolysis bullosa (RDEB). The dermal fibroblast has been considered to be the primary source of collagenase in human skin and the enzyme has been extensively studied. We have recently documented the production of collagenase by human keratinocytes in culture. The keratinocyte collagenase appears functionally and structurally similar to the fibroblast enzyme, although modulation of production may be dissimilar. The goal of this proposal is to purify, further characterize, and evaluate the modulation of collagenase production in normal and RDEB keratinocytes. Collagenase will be purified from conditioned medium from phorbol-stimulated keratinocytes and characterized in regards to the isoelectric point, calcium requirements, thermal stability and partial amino acid sequence data. Antibodies to human skin collagenase will be produced in rabbits and an ELISA developed. Substrate specificity of the purified enzyme will be evaluated with type IV and type VII collagen. Modulation of collagenase production will be studied with known stimulators and inhibitors of fibroblast collagenase, protein kinase C pathway analogues and growth factors. The effect of cell-cell and cell-matrix interactions, cellular migration and cellular injury on synthesis of collagenase by keratinocytes will be assessed. The production and modulation of collagenase by RDEB keratinocytes will be studied and immunocytochemical localization of collagenase in wounded human skin and skin equivalent models evaluated.
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