TGF-BETA-INDUCED COLLAGEN TRANSCRIPTION & LUNG FIBROSIS

某些肺损伤会导致结缔组织的大量增加 含量，尤其是胶原蛋白，会导致纤维化。在受伤期间， 细胞暴露在调节细胞生成的效应物质中 基质成分，如转化生长因子-β(转化生长因子-β)。 这项建议的重点是建立在 转化生长因子-β对胶原转录的刺激作用。我们的前期工作 证明转化生长因子-β刺激肺成纤维细胞聚集型 I型胶原和α1(I)mRNA。转化生长因子-β升高的α1(I) 涉及顺式调控区域的转录，称为转化生长因子- β激活元件(TAE)，从转录开始，为1.6kb 地点。虽然这个部位类似于核因子I(NF-1)共识 TAE，而不是NF-1，可抑制胶原的转化生长因子-β反应 氯霉素乙酰转移酶(CAT)启动子共转染 转化成肺成纤维细胞。Tae位点有一个激活蛋白-2(AP-2) 核因子-1类结合部位内的结合部位。当细胞与 随着转化生长因子-β剂量的增加，核内TAE、NF-1和AP-2的DNA结合 蛋白质增加，细胞质DNA相应减少 结合蛋白。我们假设用转化生长因子-β治疗会导致 一种特定的核结合蛋白的磷酸化 从细胞质转位到细胞核。这种核蛋白 与α1(I)胶原启动子中的Tae序列结合以激活 抄写。这项提议将描述Tae结合蛋白的特征 并研究转化生长因子-β改变其活性的机制。 此外，使用全长转基因小鼠的初步数据 胶原蛋白启动子CAT构建后CAT活性增加8倍 博莱霉素处理的动物的肺表明这些小鼠将 为研究顺式激活元件提供了一个极好的模型 引导纤维化中的胶原转录。这项提议将进一步 研究转化生长因子-β反应顺式元件和反式激活元件 使用细胞培养刺激胶原蛋白转录所必需的 转基因动物。 这项提案的具体目标是表征 绑定到Tae。核质结合蛋白均来源于 将检测经和不经转化生长因子-β刺激的细胞，以分析 转化生长因子-β反应的机制。我们将确定是否 转录因子的磷酸化状态和/或定位 用转化生长因子-β治疗改变，以进一步表征抑制作用 TAE对转化生长因子-β反应的影响。我们将确定双链是否 (DS)TAE阻断蛋白质或蛋白质核结合活性的增加 内源性胶原转录。我们将确定dsTAE是否进入 细胞核或通过细胞质机制抑制转化生长因子-β 大鼠α1(I)启动子的定点突变作用 为转化生长因子-β反应建立关键基础并确定是否 其他区域也参与了转化生长因子-β的作用。这将继续我们的 利用基因转染法定位顺式反应元件的努力 肺成纤维细胞。当建立站点定向删除时， 在转基因细胞中消除启动子转化生长因子-β反应，然后 转基因动物将被用来在体内测试构建物。

Certain lung injuries induce large increases in connective tissue content, particularly collagen, resulting in fibrosis. During injury, cells are exposed to effector substances that regulate production of matrix components such as transforming growth factor-beta (TGF-beta). this proposal focuses on establishing the mechanisms involved during the stimulation of collagen transcription by TGF-beta. Our preliminary work demonstrated that TGF-beta stimulated lung fibroblasts to accumulate type I collagen and alpha1(I) mRNA. TGF-beta increased alpha1(I) transcription involving a cis-regulatory region, referred to as a TGF- beta activating element (TAE), 1.6 Kb from the transcriptional start site. Although this site resembled a nuclear factor I (NF-1) consensus site, TAE, but not NF-1, abrogated the TGF-beta response of a collagen promoter driving chloramphenicol acetyl transferase (CAT) co-transfected into lung fibroblasts. The TAE site had an activator protein-2 (AP-2) binding site within a NF-1-like binding site. When cells were grown with increasing doses of TGF-beta, the nuclear TAE, NF-1 and AP-2 DNA-binding protein increased with a corresponding decrease in cytoplasmic DNA binding protein. We hypothesize that treatment with TGF-beta results in the phosphorylation of a specific nuclear binding protein which translocates from the cytoplasm to the nucleus. This nuclear protein binds to the TAE sequence in the alpha1(I) collagen promoter to activate transcription. This proposal will characterize the TAE binding proteins and examine the mechanism whereby TGF-beta alters its activity. In addition, preliminary data using transgenic mice with a full length collagen promoter CAT construct had 8 fold increased CAT activity in lungs of bleomycin treated animals suggesting that these mice will provide an excellent model for examining cis-activating elements directing collagen transcription in fibrosis. This proposal will further investigate TGF-beta responsive cis and trans-activating elements necessary for stimulated collagen transcription using cell cultures and transgenic animals. The specific aims of this proposal are to characterize the proteins that bind to TAE. Both nuclear and cytoplasmic binding proteins derived from cells stimulated with and without TGF-beta will be examined, to analyze the mechanism of the TGF-beta response. We will determine if the phosphorylation state and/or the localization of transcription factors change with TGF-beta treatment, to characterize further the inhibition of TGF-beta response by TAE. We will determine if the double stranded (ds) TAE blocks the increased nuclear binding activity of proteins or endogenous collagen transcription. We will determine if dsTAE enters the nucleus or acts through a cytoplasmic mechanism to inhibit TGF-beta action, to perform site directed mutagenesis of rat alpha1(I) promoter to establish critical bases for TGF-beta response and to determine if other regions are involved in TGF-beta action. This will continue our efforts to map cis-acting response elements using transfection into human lung fibroblasts. When site directed deletions are established that obliterate the promoter TGF-beta response in transfected cells, then transgenic animals will be made to test constructs in vivo.