Human skin fibroblasts in tissue culture have provided a useful tool by which to study connective tissue alterations in various disease processes. In this application, we are proposing studies designed to delineate molecular mechanisms which regulate collagen accumulation and synthesis by skin fibroblasts. For this purpose, fibroblast cultures established from patients with various forms of dermal fibrotic conditions (keloids, scleroderma, familial cutaneous collagenoma, connective tissue nevus of the collagen type), and which are collagen over-producers will be compared to control cells. Also, fibroblast cultures subjected to all-trans-retinoic acid treatment will be studied, since retinoic acid suppresses collagen production. Utilizing these two models of collagen production, we will first establish the correlation between Type I procollagen biosynthesis rates and the abundance of type I collagen specific mRNA levels, using Northern blot and dot blot hybridization techniques. If altered collagen mRNA levels are found, the rate of nuclear transcription of the type I collagen gene will be measured and the turnover of collagen mRNA transcripts will be determined by pulse-chase experiments. Should altered transcriptional rates be found, studies of DNASE I hypersensitivity and level of gene methylation will be initiated, using specific restriction endonucleases. Finally, the type I collagen gene copy number will be determined in fibroblast cultures established from patients with conditions of dermal fibrosis. Using these experimental approaches, it is hoped that specific levels of regulation of collagen gene expression can be determined. This knowledge will aid us in understanding fibrotic disease processes and may enable us to develop pharmacologic means by which to minimize the debilitating effects of these diseases.
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