TGF-BETA-INDUCED COLLAGEN TRANSCRIPTION & LUNG FIBROSIS

某些肺损伤诱导结缔组织大量增加 尤其是胶原蛋白，导致纤维化。 在受伤期间， 细胞暴露于效应物质， 基质成分如转化生长因子-β（TGF-β）。 这项建议的重点是建立有关机制， 通过TGF-β刺激胶原蛋白转录。 我们的前期工作 表明TGF-β刺激肺成纤维细胞积聚型 I胶原和α 1（I）mRNA。 TGF-β升高α 1（I） 转录涉及一个顺式调节区，称为TGF-β， β激活元件（TAE），距离转录起始点1.6 Kb 绝佳的价钱 尽管该位点类似于核因子I（NF-1）的一致性， TAE位点，而不是NF-1，消除了胶原蛋白的TGF-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位点含有激活蛋白-2（AP-2） NF-1样结合位点内的结合位点。 当细胞生长时， 增加剂量的TGF-β、核TAE、NF-1和AP-2 DNA结合 蛋白质含量增加，细胞质DNA含量相应减少 结合蛋白 我们假设用TGF-β治疗会导致 特异性核结合蛋白的磷酸化， 从细胞质转移到细胞核。 这种核蛋白 与α 1（I）胶原启动子中的TAE序列结合， 转录。 该提案将表征TAE结合蛋白 并研究TGF-β改变其活性的机制。 此外，使用全长的转基因小鼠的初步数据显示， 胶原启动子CAT构建体的CAT活性增加了8倍， 博来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极好的模型 指导纤维化中的胶原转录。 该提案将进一步 研究TGF-β应答顺式和反式激活元件 使用细胞培养物刺激胶原蛋白转录所必需的， 转基因动物 该提案的具体目标是表征蛋白质， 绑定到TAE。 细胞核和细胞质结合蛋白均来源于 将检查用和不用TGF-β刺激的细胞，以分析 TGF-β反应的机制。 我们将确定 磷酸化状态和/或转录因子的定位 随着TGF-β处理的变化，以进一步表征抑制 TGF-β的反应。 我们将确定双链 (ds)TAE阻断蛋白质的核结合活性增加，或 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TGF-β 作用，对大鼠α 1（I）启动子进行定点诱变 建立TGF-β反应的关键基础，并确定 其它区域参与TGF-β作用。 这将继续我们的 利用转染入人细胞定位顺式作用应答元件的努力 肺成纤维细胞 当确定定点删除时， 消除转染细胞中的启动子TGF-β反应，然后 将制备转基因动物以在体内测试构建体。

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.