Based on the hypothesis that an important component of the fibrotic responses in lung fibrosis is an increased transcription of the type I and III collagen genes, this proposal seeks to determine the role of the cis-acting elements within these genes, and the function of the cellular transacting factors, which control the expression of these genes in fibroblasts. The proposed studies will first focus on the enhancer segment of the mouse alpha2(I) collagen gene, delineate its active sites, identify and purify the factors which bond to these sites, determine their function by several approaches including in vitro transcription, also generate cDNAs and specific antibodies for these factors. The properties of these factors will then be compared with those which interact with similar elements in the alpha1(I) and alpha(III) collagen genes. The second part of this proposal is based on the above studies and also on ongoing work in our laboratory that focuses on the promoter elements of the these genes and their regulation by growth factors (such as TGF-beta) and products of oncogenes. A mouse model will be used in which lung fibrosis as well as type I and III collagen synthesis in lung fibroblasts are induced by bleomycin. Since lung fibroblasts from bleomycin-treated animals lose their phenotype of increased type I collagen synthesis in culture, transgenic mice will be generated using a type I collagen promoter-enhancer-CAT chimeric gene. If, as expected, the transgene responds to bleomycin as the endogenous type I collagen genes, transgenic mice with mutations in specific cis-acting elements of the promoter-enhancer to type I collagen genes will be tested to see whether a specific mutation abolishes the response of the transgene to bleomycin. If such mutation is also known to inhibit the binding of a specific DNA binding protein, this will suggest that this DNA binding protein plays a role in the bleomycin-induced fibrotic response. If in addition this mutation fails to respond to a particular growth factor or other effector in DNA transfection experiments, a role for this growth factor in lung fibrosis will be suggested. In studies on biopsy samples and fibroblasts obtained from patients with lung fibrosis those specific cellular DNA binding factors most likely to be responsible for the fibrotic response will be examined using reagents developed in the first part of this proposal.
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