: This renewal application proposes focused studies on the regulation of collagen gene expression towards delineation of the molecular mechanisms leading to accumulation of collagen in dermal fibrotic diseases. Our significant progress has allowed us to formulate the hypothesis that altered growth factor modulation of collagen gene expression, with particular emphasis on the antagonistic effects of TGF-B and TNF-a on type I collagen, is the critical event leading to fibrotic skin diseases. To test this hypothesis, we propose to elucidate regulatory mechanisms of extracellular matrix gene expression in fibroblasts on the transcriptional and post-transcriptional levels. The first specific aim will concentrate on growth factor modulation of gene expression, with particular emphasis on type I and VI collagen, and decorin genes in transient transfections of promoter/reporter gene constructs. We will also dissect the cytokine signaling pathways with particular emphasis on AP-1 and TGF-B/Smad towards identification of novel target genes by combined cDNA expression microarray/promoter transactivation assays. We will also take advantage of AP-1 (c-jun-/- c-fos-/-, and JunB-/-) knock-out mouse embryonic fibroblasts (ko-MEFs) so as to elucidate the role of AP-1 in the regulation of type I collagen gene expression by TGF-B. Finally, we will elucidate the role of Smad-independent pathways in TGF-B regulation utilizing Smad3 and Smad4 ko-MEFs and cDNA array. The feasibility of this aim is attested to by our exceptional progress in this project over the past five years. The second specific aim will explore the regulation of collagen gene expression through post-transcriptional modulation of mRNA stability by proposing highly innovative and novel technologies for identification and cloning of mRNA binding proteins by gel shift assays and the yeast two- and three-hybrid systems, using the 3'-untranslated regions of al(I), a2(I), and al(III) collagen mRNAs as targets. In fact, our preliminary data provide evidence for the role of novel mRNA binding proteins that will be characterized in further detail. Collectively, we expect that these experimental approaches will allow us to define the pathways operative in the physiological regulation of collagen gene expression, and to identify the mechanisms of collagen accumulation in fibrotic skin diseases, with pharmacologic implications.
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