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RETINOIDS AND COLLAGEN TRANSCRIPTION IN LUNG FIBROBLASTS

肺成纤维细胞中的类维生素A和胶原蛋白转录

基本信息

批准号：
2872871
负责人：
JOHN L BERK
金额：
$ 11.62万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-02-01 至 2000-06-30
项目状态：
已结题

项目摘要

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.

间质性肺纤维化（IPF）的特点是积聚肺中的 I 型胶原蛋白。类维生素A，维生素A衍生物，是广泛用于抑制皮肤中胶原蛋白的形成，并已被证明降低体外 α2(I) 胶原蛋白基因表达。我的初步研究表明，视黄酸可抑制 I 型胶原蛋白的形成通过下调 alpha1(I) 胶原基因来调节人肺成纤维细胞转录。与 RA 诱导基因的描述机制相反调节，我的数据表明 RA 通过抑制 alpha1(I) 表达一种新合成的反式作用中间蛋白，可与 RAIE 位于胶原蛋白启动子的近端部分。这笔赠款建议研究RA抑制I型胶原基因转录的机制离子。该提案的具体目标是定义监管要素参与视黄酸诱导的 α1(I) 胶原蛋白抑制基因，并表征与该元件结合的核蛋白。为了定位 RA 抑制元件 (RAIE)，活性 900 bp alpha1(I) 启动子/报告基因构建体将进行删除分析。将通过转染研究评估启动子活性。展示分离的 RAIE 赋予 RA 抑制作用，则 RAIE 将是亚克隆到异源 SV40 增强子驱动/荧光素酶报告基因中构造。体内和体外竞争测定将测试功能以及假定的 RAIE 的结合特异性。体外，RAIE 结合特异性将通过 DNA 迁移率变化测定来确定未标记的 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的蛋白质结合位点。在体内，不同互补和突变 RAIE 的浓度将被共转染进入用 900 bp alpha1(I)/荧光素酶瞬时转染的细胞中构造。 DNase I 足迹研究将明确核蛋白 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。这定点诱变对蛋白质与其中任何一个结合的影响将确定假定的转录因子元件。 DNA迁移率将使用抗体和寡核苷酸进行轮班研究与常见的转录因子互补。如果没有已知的共识结合序列在 RAIE 中注明，我将提取并表征使用生物素化 RAIE 通过亲和层析分析独特的蛋白质。总而言之，这些数据将为胶原蛋白调节提供见解。肺部，以及治疗 IPF 的可能新策略。