CYTOKINE SIGNALING IN RENAL FIBROSIS

描述(改编自《调查者摘要》)：这是一个新的应用程序 由一位具有长期目标的新研究员研究细胞内 介导肾纤维化的细胞因子信号网络，一个主要的 终末期肾病的发病过程。总体假设 是Smad细胞内蛋白家族的成员整合了相反的 促纤维化和抗纤维化的信号通路进入信号阈值 来调节纤维化的形成。这一假设将通过研究 正向调节因子Smad2的功能、下游靶点和调控 推测的负调控因子Smad7，在与之相反的信号通路中 促纤维化的转化生长因子β和抗纤维化的肿瘤坏死因子α。初步研究 提示Smad2和Smad7是很有希望的候选信号分子 肾脏纤维化，具有相反的活动。第一个明确的目标是测试 Smad2通过激活致纤维化基因促进纤维化的假说 作为对TGFbeta的回应。Smad2基因缺失对Smad2基因缺失小鼠的影响 胚胎成纤维细胞对基质编码基因表达的影响 蛋白质，以及对成纤维细胞增殖的影响将被确定。第二 明确的目标将检验Smad2发挥积极作用的假设 调节器和Smad7作为重叠池的负调节器 导致纤维化的基因。为此，PI将识别和比较基因库，这些基因库 在野生型小鼠胚胎成纤维细胞(MEFwt)中差异调节， Smad2缺失MEF(MEF2-/-)和结构性过表达Smad7的MEF (MEF7+)，使用高通量微阵列方法进行比较分析 基因表达。MEF2-/-和MEF7+细胞中表达降低的基因 相对于MEFwt，将对细胞进行鉴定和表征。第三 特定目的将测试Smad7是否通过抑制Smad2发挥作用 Smad2激活的靶基因的激活或转录抑制 推动者，或者两者兼而有之。最后一个具体目标将检查TGFbeta和 肿瘤坏死因子α信号通路汇聚，调节Smad7的转录。去做 这，PI克隆了人Smad7启动子的功能区。推动者 缺失分析和电泳迁移率改变分析(EMSA)将 用于鉴定Smad7基因中的功能顺式作用元件和 它们的同源转录因子。

DESCRIPTION (Adapted from Investigator's Abstract): This is a new application by a new investigator with the long-term objectives to study intracellular networks of cytokine signaling which mediate renal fibrogenesis, a major process in the pathogenesis of end stage renal disease. The overall hypothesis is that members of the Smad family of intracellular proteins integrate opposing pro-fibrogenic and anti-fibrogenic signaling pathways into signaling thresholds that regulate fibrogenesis. This hypothesis will be tested by studying the function, downstream targets and regulation of the positive mediator Smad2, and the putative negative regulator Smad7, in the opposing signaling pathways of pro-fibrogenic TGFbeta and anti-fibrogenic TNFalpha. Preliminary studies indicate that Smad2 and Smad7 are promising candidate signaling molecules in renal fibrogenesis, with opposite activities. The first Specific aim will test the hypothesis that Smad2 promotes fibrosis by activation of fibrogenic genes in response to TGFbeta. The effects of Smad2 deficiency in Smad2 null mouse embryonic fibroblasts on the expression of selected genes encoding matrix proteins, and on fibroblast proliferation will be determined. The second Specific Aim will test the hypothesis that Smad2 function as a positive regulator and Smad7 functions as a negative regulator of an overlapping pool of fibrogenic genes. To do this the PI will identify and compare gene pools which are differentially-regulated in wild type mouse embryonic fibroblasts (MEFwt), Smad2 null MEFs (MEF2-/-) and MEFs with constitutive over expression of Smad7 (MEF7+), using high-throughput microarray methods for comparative analysis of gene expression. Genes with reduced expression in both MEF2-/- and MEF7+ cells relative to MEFwt cells will be identified and characterized. The third Specific Aim will test whether Smad7 functions by inhibition of Smad2 activation, or transcriptional repression of Smad2-activated target gene promoters, or both. The last Specific Aim will examine whether TGFbeta and TNFalpha signaling pathways converge to regulate transcription of Smad7. To do this, the PI has cloned the functional human Smad7 promoter region. Promoter deletion analyses and electrophoretic mobility shift assays (EMSAs) will be utilized to identify the functional cis-acting elements in the Smad7 gene and their cognate transcription factors.