TGF-BETA INDUCED COLLAGEN TRANSCRIPTION AND LUNG FIBROSIS

点击翻译按钮获取中文摘要

Certain lung injuries induce large increases in connective tissue content, particularly collagen, resulting in fibrosis. During injury, cells are exposed to effector substances such as transforming growth factor-beta (TGF-beta), that regulate production of matrix components. This project focuses on establishing the mechanisms involved during the stimulation of collagen transcription by TGF-beta. Our preliminary work demonstrates that TGF-beta stimulates lung fibroblasts to accumulate type I collagen and alpha(I) mRNA. TGF-beta increases alpha(I) transcription by a unique protein complex binding to a cis-regulatory region termed TAE. This element is located 1.6 kb upstream from the transcriptional start site. Co-transfection with double stranded dsTAE abrogates the TGF-beta response. When TAE is mutated within the promoter, the basal activity increases to TGF-beta stimulated levels suggesting that TAE is a repressor element. TAE acts as a TGF-beta response element cloned upstream of a collagen basal (224 bp) promoter, but not when cloned upstream thymidine kinase (TK) promoter. In addition, transgenic animal studies with mice containing the collagen 3.6 kb promoter upstream of chloramphenicol acetyl transferase gene (CAB demonstrate that subset of fibroblasts have increased CAT expression during fibrosis or injections with TGF-beta. Therefore, other transcriptional regulatory regions are important for collagen gene expression in other cells. Indeed, data suggests there may be a response element in the first intron. We hypothesize that, during TGF-beta stimulation of fibroblasts, proteins are modified, enter the nucleus an bind to the TAE sequence in the alpha(I) collagen promoter releasing repression of transcription through prot binding to proximal cis-acting sites. In vivo certain myofibroblast populations respond similarly to TGF-beta whereas others seem to require, additional regulatory regions to open chromatin structure and/or for stimulation during acute stages preceding fibrosis. The specific aims of this proposal are to 1. perform site directed mutagenesis of rat alpha(I) proximal Nor and intron to establish critical bases for TGF-beta response 2: characterize the proteins binding to TAE and analyze the TGF-beta response mechanism. We have recently cloned potential TAE binding proteins. The mrnA expression in cells and lung tissue will be determined. Full length clones will be expressed to determine function. We will identify the proteins associated with TAE. We will detect the presence of phosphorylation changes in TAE binding proteins and determine if transcription factors are translocated into nucleus during TGF-beta stimulation. 3: determine whether transgenic animals with mutated collagen promoters with and without other regulatory regions increase CAT transcription during bleomycin induced fibrosis 4: characterize further the inhibition of TGF-beta response by dsTAE oligonucleotides. Continue evaluating the extent of inhibition t TAE to assess the use of dsTAE as an antifibrotic agent.