RETINOIDS AND COLLAGEN TRANSCRIPTION IN LUNG FIBROBLASTS

间质性肺纤维化(IPF)的特点是积聚 肺中的I型胶原蛋白。维甲酸，维生素A的衍生物，是 广泛用于抑制皮肤中胶原的形成，并已被证明 降低α2(I)胶原基因在体外的表达。我的初选 研究表明，维甲酸抑制I型胶原的形成 下调人肺成纤维细胞α1(I)胶原基因 抄写。与已描述的RA诱导基因的机制相反 调节，我的数据表明RA通过抑制α1(I)的表达 一种新合成的反式作用中间蛋白，与 在胶原蛋白启动子的近端。这项拨款建议 探讨维甲酸抑制I型胶原基因转录的机制 离子。 这项建议的具体目的是定义监管要素(S) 参与维甲酸对α1(I)胶原的抑制作用 基因，并鉴定与该元素结合的核蛋白(S)。 为了定位RA抑制元件(S)(RAIE)，活性900BP 将对α1(I)启动子/报告基因的构建进行缺失分析。 启动子活性将通过转基因研究进行评估。为了证明 即隔离RAIE赋予RA抑制作用，RAIE将被 亚克隆入异源SV40增强子驱动/荧光素酶报告基因 建造。体内和体外竞争分析将测试该功能 以及推测的RAIE的结合特异性。在体外，RAIE结合 特异性将使用DNA迁移率变化分析来确定 未标记的RAIE、无关的寡核苷酸和潜在突变的RAIE 甲基化干扰法检测蛋白质结合部位。在活体内，变化 互补和突变的RAIE浓度将被共转染 瞬时转染900bpAlpha1(I)/荧光素酶的细胞 建造。DNase I足迹研究将确定核蛋白 RAIE内的结合区。 为了表征与RAIE结合的蛋白质，序列如下 分析已知转录因子的共识结合基序。这个 定点突变对蛋白质与其中任何一种结合的影响 假定的转录因子元件将被确定。DNA迁移率 将使用抗体和寡核苷酸进行移位研究 与常见的转录因子互补。如果没有已知的共识 在RAIE中记录了结合序列，我将提取并表征 用生物素标记的RAIE亲和层析获得独特的蛋白质。 综上所述，这些数据将为胶原蛋白的调节提供深入的见解 肺，可能还有治疗特发性肺纤维化的新策略。

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.