PDGF GENE TRANSCRIPTION IN RENAL EPITHELIAL CELLS

血小板源性生长因子（PDGF）在肿瘤的发生发展中起着关键作用。 胚胎发育过程中细胞增殖的调节，细胞 分化和组织修复。 PDGF也是合成和分泌的 在异常细胞生长的部位，可能会刺激自主 通过自分泌或旁分泌机制的细胞增殖。 在 特别是PDGF可能在增生性病变中发挥病理作用， 在肾脏，系膜，内皮和上皮细胞表达 显著量的PDGF A链和B链。 我们已经确定 非转化的肾上皮细胞系（BSC-1;非洲绿色 猴），其表达可测量量的编码 同源PDGF链，以及显著水平的A链启动子 活动 我们在上游监管范围内有本地化的区域， 该基因的区域，其介导正转录和负转录 基因的活性。 提出了三个具体目标，以确定 介导受限但可测量速率的分子机制 PDGF A链基因转录的变化。 这些目的是 专注于定位和表征一个负面的 起始位点上游（-1029至-879）鉴定的调控元件 PDGF A-链转录。 第一，负面监管因素 (NRE)将被检查，以确定它是否发挥主导作用， 对两个相邻的转录增强子（-879至-634）的抑制作用 和-84至-62）。 NRE影响 这些增强子将在一组表达 在低水平与高水平下的PDGF A链，以确定是否转录 阻遏/去阻遏在介导相对 A链基因转录的基础速率。 第二，DNA反应 元件介导的转录沉默活性将被精细地 通过-1029至-879区域的定点诱变定位。 第三，以高亲和力和特异性结合到 最小NRE将通过电泳鉴定和表征， 迁移率变动分析（EMSA）和DNA足迹法。 DNA序列 NRE功能和因子绑定的要求将与 确定任何已识别消音器元件的功能重要性 NRE活性的结合蛋白。 拟议的实验提供了 可能确定控制的分子机制， PDGF在肾脏中的生理和病理生理表达， 可能是其他细胞类型。

Platelet-derived growth factor (PDGF) plays a critical role in the regulation of cell proliferation during embryonic development, cellular differentiation and tissue repair. PDGF is also synthesized and secreted at sites of abnormal cellular growth, and may act to stimulate autonomous cellular proliferation by an autocrine or paracrine mechanism. In particular, PDGF may play a pathological role in proliferative lesions of the kidney, where mesangial, endothelial and epithelial cells express significant quantities of the PDGF A- and B-chains. We have identified a non-transformed renal epithelial cell line (BSC-1; African green monkey) which expresses measurable quantities of the mRNA encoding the cognate PDGF chains, as well as significant levels of A-chain promoter activity. We have localized regions within the upstream regulatory region of this gene which mediate positive and negative transcriptional activity of the gene. Three specific aims are proposed to determine the molecular mechanisms which mediate the constrained but measurable rate of PDGF A-chain gene transcription in these cells. These aims are focused upon the localization and characterization of a negative regulatory element identified upstream (-1029 to -879) of the start site of PDGF A-chain transcription. First, the negative regulatory element (NRE) will be examined to determine whether it exerts a dominant repressing effect on two adjacent transcriptional enhancers (-879 to-634 and -84 to-62) within the PDGF A-chain promoter. NRE influence upon these enhancers will be evaluated in a panel of cell lines which express PDGF A-chain at low versus high levels to determine if transcriptional repression/derepression plays an important role in mediating relative basal rates of A-chain gene transcription. Second, the DNA response element which mediates transcriptional silencer activity will be finely localized by site-directed mutagenesis of the -1029 to-879 region. Third, nuclear factors which bind with high affinity and specificity to the minimal NRE will be identified and characterized by electrophoretic mobility shift assay (EMSA) and DNA footprinting. DNA sequence requirements for NRE function and factor binding will be compared to determine the functional importance of any identified silencer elements binding proteins for NRE activity. The proposed experiments offer the potential for identifying molecular mechanism which control the physiological and pathophysiological expression of PDGF in the kidney and possibly other cell types.