Scleroderma (PSS) is a serious disease of unknown cause characterized by excessive accumulation of collagen and other connective tissue components in skin and internal organs. The mechanisms responsible for such accumulation are not known. In previous work form our laboratories we have demonstrated that the TSK (tight skin) mutant mouse strain displays connective tissue abnormalities that closely resemble those present in patients with PSS including increased collagen tissue deposition, and increased biosynthesis in skin organ cultures and cultured fibroblasts. Furthermore, we demonstrated a coordinated increase in the levels of Types I and III collagen mRNAS these cells. These results suggests that the TSK mice are an ideal experimental model for study of the connective tissue alterations in PSS. The purpose of the work proposed in this application will be to continue our in depth and exhaustive studies of the mechanisms responsible for the connective tissue alterations displayed by TSK mice. Particular emphasis will be placed on the study of the regulation of collagen synthesis in TSK cultured fibroblasts applying STATE of the ART biosynthetic and biochemical methods as well as recently developed recombinant DNA techniques currently used in our laboratories. We will examine the mechanisms responsible for increased collagen gene expression by measuring transcription rates and mRNA stability. In addition, we will attempt to identify regulatory defects by mapping pertinent regions of one collagen gene by nuclease sensitivity and by transient transfection experiments utilizing a shuttle vector to introduce normal and deleted putative regulatory sequences into normal and TSK fibroblasts. Biosynthesis of collagen in skin organ cultures and tissue cultures from normal and TSK dermal fibroblasts will be continued and the biosynthesized products will be characterized. Differences in the content or structure of the various components present in the tissues or synthesized in the TSK cultures will be identified. Special emphasis will be placed on the identification of the minor collagen types (V and VI) present and in the quantitation of their relative proportions. Several other differences in the biological characteristics of normal and TSK cells and tissues will be explored in further detail. It is expected that the knowledge gained from these studies will be of direct relevance to understand the pathogenesis of the excessive collagen deposition characteristic of PSS and will permit a more rational approach to develop possible modes of therapy for this incurable and devastating disease.
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