CYTOKINE SIGNALING IN RENAL FIBROSIS

描述（改编自研究者摘要）：这是一个新的应用程序 由一位新的研究者长期研究细胞内 介导肾纤维化的细胞因子信号网络， 终末期肾病的发病机制。总体假设 是细胞内蛋白质的Smad家族成员整合了相反的 将促纤维化和抗纤维化信号传导途径转化为信号传导阈值 调节纤维化这一假设将通过研究 正介体Smad 2的功能、下游靶点和调节，以及 假定的负调节Smad 7，在相反的信号通路中， 促纤维化TGF β和抗纤维化TNF α。初步研究 表明Smad 2和Smad 7是有希望候选信号分子， 肾纤维化，具有相反的活动。第一个具体目标将测试 Smad 2通过激活纤维化基因促进纤维化的假说 对TGF β的反应。Smad 2缺陷对Smad 2基因敲除小鼠的影响 胚胎成纤维细胞对基质编码基因表达的影响 蛋白质，和对成纤维细胞增殖的影响将被确定。第二 Specific Aim将检验Smad 2作为阳性对照发挥作用的假设。 调节器和Smad 7作为重叠池的负调节器起作用。 纤维化基因为此，PI将识别并比较基因库， 在野生型小鼠胚胎成纤维细胞（MEFwt）中受到差异调节， Smad 2无效MEF（MEF 2-/-）和具有Smad 7组成性过表达的MEF (MEF7+），使用高通量微阵列方法进行比较分析， 基因表达。在MEF 2-/-和MEF 7+细胞中表达降低的基因 将鉴定和表征相对于MEFwt细胞。第三 Specific Aim将测试Smad 7是否通过抑制Smad 2发挥功能 Smad 2激活靶基因的激活或转录抑制 促进者，或两者。最后一个具体目标将检查是否TGF β和 TNF α信号通路会聚以调节Smad 7的转录。做 因此，PI克隆了功能性人Smad 7启动子区。启动子 缺失分析和电泳迁移率变动分析（EMSA）将在 用于鉴定Smad 7基因中的功能性顺式作用元件， 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.