RETINOIDS AND COLLAGEN TRANSCRIPTION IN LUNG FIBROBLASTS

间质性肺纤维化（IPF）的特征是 I型胶原蛋白。 维生素A衍生物， 广泛用于抑制皮肤中胶原蛋白的形成， 降低体外α 2（I）胶原基因表达。 我的初步 研究表明，视黄酸抑制I型胶原蛋白的形成， 下调α 1（I）胶原基因诱导人肺成纤维细胞凋亡 转录。 与RA诱导的基因表达的机制相反， 调节，我的数据表明，RA抑制α 1（I）的表达，通过 一种新合成的反式作用中间蛋白， RAIE在胶原促进剂的近端部分。 该基金建议 研究RA抑制I型胶原基因转录的机制 离子。 本提案的具体目的是确定监管要素 参与视黄酸诱导的α 1（I）胶原抑制 基因，并表征与该元件结合的核蛋白。 为了定位RA抑制元件（RAIE），将活性900 bp的 α 1（I）启动子/报告基因构建体将进行缺失分析。 将通过转染研究评估启动子活性。 证明 如果分离的RAIE赋予RA抑制作用，则RAIE将是 亚克隆到异源SV40增强子驱动的/荧光素酶报告基因中 构建体 体内和体外竞争测定将测试功能 和结合特异性。 体外，RAIE结合 特异性将使用DNA迁移率变动测定法测定， 未标记的RAIE、不相关的寡核苷酸和潜在突变的RAIE 通过甲基化干扰测定蛋白结合位点。 体内，不同 将不同浓度的互补RAIE和突变RAIE共转染 转化到用900 bp α 1（I）/荧光素酶瞬时转染的细胞中 构建体 DNA酶I足迹研究将指定核蛋白 RAIE内的结合区域。 为了表征结合RAIE的蛋白质，序列将是 分析已知转录因子的共有结合基序。 的 定点诱变对蛋白质与这些蛋白质中任何一种结合的影响 将确定推定的转录因子元件。 DNA迁移率 将使用抗体和寡核苷酸进行偏移研究 与常见的转录因子互补。 如果没有共识 RAIE中记录了结合序列，我将提取并表征 通过使用生物素化RAIE的亲和层析分离独特的蛋白质。 总之，这些数据将提供对胶原蛋白调节的见解， 肺，以及可能的治疗IPF的新策略。

Interstitial pulmonary fibrosis (IPF) is characterized by the accumulation of type I collagen in the lung. Retinoids, vitamin A derivatives, are widely used to suppress collagen formation in the skin and have been shown to decrease alpha2(I) collagen gene expression in vitro. My preliminary work indicates that retinoic acid inhibits type I collagen formation in human lung fibroblasts by down regulating alpha1(I) collagen gene transcription. In contrast to described mechanisms of RA-induced gene regulation, my data suggest that RA inhibits alpha1(I) expression through a newly synthesized trans-acting intermediary protein that binds to the RAIE in the proximal portion of the collagen promoter. This grant proposes to examine the mechanism by which RA inhibits type I collagen gene transcri ion. The Specific Aims of this proposal are to define the regulatory element(s) involved in retinoic acid-induced inhibition of the alpha1(I) collagen gene, and to characterize the nuclear protein(s) binding to that element. To localize the RA inhibitory elements(s) (RAIE), the active 900 bp alpha1(I) promoter/reporter gene construct will undergo deletion analysis. Promoter activity will be assessed by transfection studies. To demonstrate that the isolated RAIE confers the RA inhibitory effect, the RAIE will be subcloned into a heterologous SV40-enhancer driven/luciferase reporter construct. In vivo and in vitro competition assays will test the function and binding specificity of the putative RAIE. In vitro, RAIE binding specificity will be determined using DNA mobility shift assays with unlabelled RAIE, unrelated oligonucleotides, and RAIE mutated at potential protein binding sites by methylation interference assay. In vivo, varying concentrations of complementary and mutated RAIE will be co-transfected into cells transiently transfected with the 900 bp alpha1(I)/luciferase construct. DNase I footprinting studies will specify nuclear protein binding regions within the RAIE. To characterize the proteins that bind the RAIE, the sequence will be analyzed for consensus binding motifs for known transcription factors. The effect of site directed mutagenesis on protein binding to any of these putative transcription factor elements will be determined. DNA mobility shift studies will be performed with antibodies and oligonucleotides complementary to the common transcription factors. If no known consensus binding sequence is noted within the RAIE, I will extract and characterize the unique proteins by affinity chromatography using biotinylated RAIE. Taken together, these data will offer insights into collagen regulation in the lung, and possibly new strategies for treatment IPF.