TGF-BETA INDUCED COLLAGEN TRANSCRIPTION AND LUNG FIBROSIS

某些肺损伤诱导结缔组织含量的大量增加， 尤其是胶原蛋白，导致纤维化。在损伤过程中，细胞 暴露于效应物质如转化生长因子-β （TGF-β），其调节基质组分的产生。这个项目 重点是建立刺激过程中涉及的机制， 通过TGF-β的胶原蛋白转录。 我们的初步工作表明，TGF-β刺激肺 成纤维细胞积累I型胶原和α（I）mRNA。TGF-beta 通过一种独特的蛋白质复合物结合到一种 顺式调节区称为TAE。此元素位于上游1.6 kb处 从转录起始位点开始。双链共转染 dsTAE消除TGF-β应答。当TAE在体内发生突变时， 启动子，基础活性增加到TGF-β刺激的水平 这表明TAE是一个阻遏元件。TAE作为TGF-β 克隆在胶原基底（224 bp）启动子上游的应答元件， 但当克隆到胸苷激酶（TK）启动子上游时则不存在。此外，本发明还提供了一种方法， 用含有胶原蛋白3.6kb的小鼠进行的转基因动物研究 氯霉素乙酰转移酶基因上游启动子 证明成纤维细胞亚群具有增加的CAT表达 或者注射TGF-β的时候。因此，其他 转录调控区是胶原基因的重要组成部分 在其他细胞中表达。事实上，数据显示， 第一个内含子中的元素。 我们假设，在TGF-β刺激成纤维细胞的过程中， 修饰后，进入细胞核，与细胞核中的TAE序列结合， α（I）胶原启动子通过以下途径释放转录抑制 蛋白结合近端顺式作用位点。体内某些肌成纤维细胞 人群对TGF-β的反应类似，而其他人群似乎需要， 另外的调节区域以打开染色质结构和/或用于 在纤维化之前的急性阶段的刺激。 该提案的具体目标是1.执行现场导向 大鼠α（I）近端Nor和内含子的诱变以建立关键的 TGF-β反应的基础2：表征与TAE结合的蛋白质 并分析TGF-β反应机制。我们最近克隆了 潜在的TAE结合蛋白。mrnA在细胞和肺组织中的表达 组织将被确定。将表达全长克隆， 决定功能。我们将鉴定与TAE相关的蛋白质。我们 将检测TAE结合中磷酸化变化的存在 蛋白质，并确定转录因子是否转移到 在TGF-β刺激期间的细胞核。3：确定是否转基因 具有突变的胶原启动子的动物，有和没有其他调节性基因， 区域在博来霉素诱导的纤维化过程中增加CAT转录4： 进一步表征dsTAE对TGF-β反应的抑制 寡核苷酸继续评价TAE的抑制程度， 评估dsTAE作为抗纤维化药物的用途。

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.