TGF-BETA INDUCED COLLAGEN TRANSCRIPTION AND LUNG FIBROSIS

某些肺损伤会导致结缔组织含量大幅增加， 尤其是胶原蛋白，导致纤维化。在损伤过程中，细胞 暴露于转化生长因子-β等效应性物质 (转化生长因子-β)，调节基质成分的生产。这个项目 重点是建立在刺激过程中涉及的机制 通过转化生长因子-β转录胶原。 我们的初步工作表明，转化生长因子-β刺激肺 成纤维细胞积聚I型胶原和α(I)mRNA。转化生长因子-β 通过与一种独特的蛋白复合体结合来增加α(I)转录 顺式调控区域称为TAE。该元素位于上游1.6kb 来自转录起始点。双链共转染法 DsTAE消除了转化生长因子-β反应。当Tae在 启动子，基础活性增加到转化生长因子-β刺激水平 提示Tae是一种抑制因子。TAE扮演转化生长因子-β的角色 反应元件克隆在胶原基(224个碱基)启动子上游， 但当克隆胸苷激酶(TK)上游启动子时则不是这样。此外, 3.6kb胶原蛋白转基因小鼠的动物实验 氯霉素乙酰转移酶基因上游启动子(CAB 证明成纤维细胞亚群增加了CAT的表达 在纤维化或注射转化生长因子-β期间。因此，其他 转录调节区是胶原基因的重要调控区域 在其他单元格中表达。事实上，数据表明，可能会有回应 第一个内含子中的元素。 我们假设，在转化生长因子-β刺激成纤维细胞的过程中，蛋白质 都被修饰了，进入细胞核并与TAE序列结合在一起 α(I)胶原蛋白启动子通过 Prot结合到近端的顺式作用位点。体内某些肌成纤维细胞 人们对转化生长因子-β的反应类似，而其他人似乎需要， 开放染色质结构和/或用于 纤维化前急性期的刺激。 这项建议的具体目的是：1.现场指导 诱变大鼠α(I)近端NOR和内含子以建立临界 转化生长因子-β反应的基础2：与TAE结合的蛋白质的特性 并分析了转化生长因子-β的反应机制。我们最近克隆了 潜在的Tae结合蛋白。基因在细胞和肺组织中的表达 组织将被确定。全长克隆将表达为 确定功能。我们将鉴定与TAE相关的蛋白质。我们 将检测Tae结合中是否存在磷酸化变化 蛋白质，并确定转录因子是否转移到 在转化生长因子-β刺激过程中的细胞核。3：确定是否转基因 具有和不带有其他调节因子的胶原启动子突变的动物 博莱霉素诱导的肝纤维化过程中CAT转录增强的区域4： 进一步表征dsTAE对转化生长因子-β反应的抑制作用 寡核苷酸。继续评估抑制的程度 评估dsTAE作为抗纤维化药物的使用。

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.