TGF BETA--TRANSCRIPTIONAL CONTROL

描述（直接摘自申请人摘要） 在患有进行性肾小球疾病的儿童中， 从基质合成和降解之间平衡的转变， 有利于细胞外基质（ECM）的积累。 数据越来越多地支持 TGF-β 1在这一过程中起着重要作用。 然而，细胞内 TGF-β 1刺激细胞积聚基质的机制并不 很好理解。 用TGF-β 1处理的系膜细胞呈现出一种 由细胞结构变化定义的基质积累表型， 活动以及ECM营业额。 ECM mRNA表达的变化 蛋白质、蛋白酶和蛋白酶抑制剂之前， c-Ets 2和c-jun mRNA的表达。 这些转录的结合位点 因子（分别为Ets和AP-1），以及另一种转录因子 已在ECM相关基因的启动子中鉴定出Sp1因子 周转 我们假设Ets表达或活性的变化， AP-1和Sp1转录因子介导TGF-β 1诱导的细胞凋亡的变化。 系膜细胞ECM周转与硬化有关。 为了解决这个 假设，我们提出了两个具体目标。 第一，确定是否 TGF-β 1调节AP-1、Ets或Sp1转录的表达或活性 在系膜细胞中的因子，它们将用TGF-β 1处理并检查 用于以下：特异性转录因子mRNA和蛋白质 表达;核提取物与AP-1、Ets或 Sp1转录因子; AP-1磷酸化和Ets转录 因子;和转录因子活性，在细胞中瞬时评估 表达特异性结合位点-胸苷激酶启动子-荧光素酶 报告构建体。 TGF-B1诱导的变化的时间与以下因素有关： ECM营业额变化的时间。 第二个具体目标涉及 特定转录因子的激活是否可介导TGF-β 1 诱导与ECM周转相关的基因表达的变化。 研究将 鉴定可能介导TGF-β 1调节 转录因子活性，如细胞膜蛋白酪氨酸 激酶、细胞MAP激酶或TGF-β特异性Smad转换器家族。 将检查这些途径的抑制剂抑制TGF-β 1的能力 在系膜细胞中诱导转录因子活性。 最后， TGF-B1诱导的转录活性变化的抑制剂将是 评价阻断TGF-β 1诱导的系膜细胞变化的能力 基质周转率 这些研究将阐明细胞机制， 进行性肾小球硬化，并可能建议治疗方法， 这些机制在进行性儿童肾病中的作用。

DESCRIPTION (Taken directly from applicant's abstract) In children with progressive glomerular disease, glomerulosclerosis results from a shift in the balance between matrix synthesis and degradation to favor extracellular matrix (ECM) accumulation. Data increasingly support a mediating role for TGF-B1 in this process. However, the intracellular mechanism(s) by which TGF-B1 stimulates cells to accumulate matrix are not well understood. Mesangial cells treated with TGF-B1 assume a matrix-accumulating phenotype defined by changes in cell structure and activity as well as in ECM turnover. Changes in expression of mRNAs for ECM proteins, proteases and protease inhibitors are preceded by changes in expression of c-Ets2 and c-jun mRNA. Binding sites for these transcription factors (Ets and AP-1, respectively), as well as for another transcription factor, Sp1, have been identified in the promoters of genes related to ECM turnover. We hypothesize that changes in expression or activity of Ets, AP-1 and Sp1 transcription factors mediate TGF-B1 induced changes in mesangial cell ECM turnover related to sclerosis. To address this hypothesis, we propose two specific aims. First, to determine whether TGF-B1 modulates expression or activity of AP-1, Ets or Sp1 transcription factors in mesangial cells, they will be treated with TGF-B1 and examined for the following: specific transcription factor mRNA and protein expression; nuclear extract binding to DNA binding sites for AP-1, Ets or Sp1 transcription factors; phosphorylation of AP-1 and Ets transcription factors; and transcription factor activity, evaluated in cells transiently expressing specific binding site-thymidine kinase promoter-luciferase reporter constructs. Timing of changes induced by TGF-B1 will be related to timing of changes in ECM turnover. The second Specific Aim addresses whether activation of specific transcription factors may mediate TGF-B1 induced changes in gene expression related to ECM turnover. Studies will identify signaling pathways that could mediate TGF-B1 modulation of transcription factor activity, such as cell membrane protein tyrosine kineses, cellular MAP kineses, or the TGF-B specific Smad transducer family. Inhibitors of these pathways will be examined for ability to inhibit TGF-B1 induced transcription factor activity in mesangial cells. Finally, inhibitors of TGF-B1 induced changes in transcriptional activity will be evaluated for ability to block TGF-B1 induced changes in mesangial cell matrix turnover. These studies will elucidate cellular mechanisms of progressive glomerulosclerosis, and may suggest therapeutic approaches to these mechanisms in progressive childhood renal disease.