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. There are three outstanding features involved in the pathogenesis of PSS: a) excessive collagen deposition; b) vascular abnormalities and c) alterations in humoral and cellular immunity. We will continue to investigate the role of each of these components employing state-of-the-art methods currently being used in our laboratories. We previously showed that PSS fibroblasts display increased biosynthesis of collagen and elevated levels of Types I and III procollagen mRNAs. We will extend these studies to examine the mechanisms responsible for these alterations employing in vitro transcription assays and determination of mRNA stability. In addition, we will examine the transcriptional regulation of collagen gene expression by transfection of chimeric alphal(I) procollagen-CAT gene and minigene constructs containing various deletions in the promoter and first intron regions of the gene. Transfection of these constructs into normal and PSS fibroblasts will allow to determine the elements within the gene responsible for normal regulation of expression and their interaction with transcriptional factors. These studies will yield information that may permit the identification of the levels of regulation of collagen gene expression that are abnormal in PSS. To investigate the role of immunologic alterations, we will continue our studies on the modulation of fibroblast collagen gene expression by gamma- interferon (gamma-IFN) and transforming growth factor-beta(TGFbeta) and will identify alterations in their production by PSS T cell subpopulations employing Northern hybridizations nad polymerase chain reaction amplification of reverse transcriptase-generated cDNAs. The mechanisms responsible for modulation of collagen synthesis by gamma-IFN and TGFbeta will be studied utilizing in vitro transcription and transient transfection experiments and by the identification of gamma-IFN and TGBbeta-induced transcriptional factors. To study the mechanisms involved in PSS vascular alterations we will examine the effect of gamma-IFN, TGFbeta and T cell products on microvascular endothelial cells. It is expected that this comprehensive approach will give further insight into the pathogenesis and possible treatment of this incurable disease.
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