Aberrant regulation of type I collagen gene expression is a hallmark of the fibrotic disorders which are often associated with rheumatoid arthritis. The long term objective of this proposal is to formulate a comprehensive conceptual framework to precisely delineate the DNA-protein interactions which underlie transcriptional regulation of the human Proalpha(I) collagen gene. The more immediate goal will be the molecular dissection of the cis- regulatory sequence motifs that comprise the Proalpha1(I) promoter/enhancer and the trans-acting factors which interact with these sequences. Chimeric collagen promoter/enhancer-reporter gene constructs will be introduced into cultured cells of mesenchymal and non-mesenchymal origin by transient or stable transfection protocols, and also incorporated into the genome of transgenic mice; the expression of the reporter gene will be assessed and compared between in vitro and in vivo models. Putative trans-acting factors involved in the constitutive and modulatory transcriptional regulation of the Proalpha1(I) gene will be extensively characterized and cDNA encoding these factors will be molecularly cloned. Mechanisms by which interactions between the cis-elements and the trans-acting factors ensure appropriate transcriptional regulation of the human Proalpha1(I) gene will be studied by cotransfection of the target promoter/enhancer- reporter constructs and expression vectors containing the putative trans- acting factor(s). Additionally, cell-free systems will be developed to investigate the detailed protein-DNA interactions which determine Proalpha1(I) collagen transcription. These studies will unravel the molecular interactions that orchestrate appropriate tissue and cell specificity of transcriptional regulation of human Proalpha1(I) collagen gene expression. A detailed understanding of the mechanisms of transcriptional control of type I collagen genes will enable us to devise therapeutic interventions to reduce the articular and extraarticular fibrotic manifestations associated with rheumatoid arthritis.
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