TGF BETA--TRANSCRIPTIONAL CONTROL

描述(直接取自申请人的摘要) 进行性肾小球疾病的儿童，肾小球硬化 从基质合成和降解之间的平衡转移到 有利于细胞外基质(ECM)的积累。数据日益支持 转化生长因子-β1在这一过程中的中介作用。然而，细胞内的 转化生长因子-β1刺激细胞积累基质的机制(S)未见报道 很好理解。转化生长因子-β1处理的肾小球系膜细胞 基质积累表型由细胞结构的变化和 活动和ECM成交量也是如此。细胞外基质mRNAs表达的变化 在蛋白质、蛋白水解酶和蛋白水解酶抑制剂之前， C-Ets2和c-Jun基因的表达。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 在细胞外基质相关基因的启动子中发现了Sp1因子 营业额。我们假设ETS的表达或活性的变化， AP-1和Sp1转录因子介导转化生长因子-β1诱导的血管内皮细胞的变化 系膜细胞ECM周转与硬化有关。要解决这个问题 假设，我们提出了两个具体的目标。首先，确定是否 转化生长因子-β1调节AP-1、Ets或Sp1转录的表达或活性 系膜细胞中的因子，它们将被转化生长因子-β1处理并检测 用于以下内容：特定转录因子mRNA和蛋白质 表达；核提取物与AP-1、Ets或 SP1转录因子；AP-1的磷酸化和Ets转录 因子；和转录因子活性，在细胞中瞬时评估 胸苷激酶启动子荧光素酶特异性结合位点的表达 记者构建。转化生长因子-β1引起的变化的时间将与 ECM成交量变化的时机。第二个具体目标是 特异性转录因子的激活是否可能介导转化生长因子-β1 诱导细胞外基质周转相关基因表达的变化。研究将会 识别可能介导转化生长因子-β1调控的信号通路 转录因子活性，如细胞膜蛋白酪氨酸 运动学、细胞映射运动学或转化生长因子-β特异性Smad转导家族。 这些途径的抑制剂将被检测抑制转化生长因子-β1的能力。 诱导系膜细胞转录因子活性。最后， 转化生长因子-β1诱导转录活性改变的抑制剂将是 评价阻断转化生长因子-β1诱导的系膜细胞改变的能力 矩阵周转。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脏疾病中起作用。

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.