Signaling Pathways in Pathogenesis of Renal Fibrosis

描述(申请人摘要)：糖尿病动脉病变，糖尿病 肾病和许多形式的进行性肾脏疾病都是典型的 由于IV型胶原过度沉积。缺乏有效的治疗方法 这些形式的慢性组织损伤在很大程度上是由于缺乏 了解纤维化形成的基本机制。虽然转化生长因子-13 1已经 作为IV型胶原表达的主要决定因素，被认为 一种重要的促纤维化细胞因子--细胞内信号通路 通过转化生长因子-β1及转化生长因子-J3-1刺激转录的机制 IV型胶原基因在病理生物学状态下的作用尚不清楚。中心假说 在这一应用中要测试的是，转化生长因子-β1增加了 IV型胶原基因通过触发几个不同功能的细胞内 信号通路，涉及Smad蛋白和一个或多个有丝分裂原 活化的蛋白激酶级联反应。在具体目标1中，职能 IV型胶原启动子和侧翼区域的特征将是 完成。IV型胶原嵌合启动子-CAT载体的缺失构建 将用于定义特定于方向的序列 指导A1(IV)和A2(IV)胶原转录的激活区 基因。结合这些关键调控区域的核蛋白将是 由凝胶迁移率变化分析确定。在特定目标2中，转化生长因子-β1的作用 诱导IV型胶原转录的信号通路，稳态mRNA 表达，以及蛋白质的产生将被确定。大鼠肾小球系膜细胞和 血管平滑肌细胞将被转染有成分活性的或 显性否定Smad构式和与构式构成 激活ERK、INK和p38信号通路；这些信号通路的作用 对基础和TOP-B1刺激IV型胶原表达的干预将是 已经确定了。Smad和MAPK信号相互作用的潜在位点 将定义路径。在这些研究的基础上，转基因分析和凝胶 将使用迁移率变化分析来定义序列元素 提供转录反应的胶原N启动子或侧翼区域 转化生长因子-β1(转化生长因子-β1，特异靶向3)。最后，系膜细胞和血管 从转化生长因子-β1基因敲除动物中分离的平滑肌细胞将被用于 确定Smad和/或MAP激酶信号的激活是否级联 能激活非依赖转化生长因子-β1的IV型胶原基因转录 举止。IV型胶原信号通路的描绘将揭示基本的 进展性组织下纤维形成增强的细胞机制 可为新型建筑的设计提供合理的依据 针对特定识别信号的药物治疗干预 台阶。如果成功，这些干预措施可能会延缓组织的进展 对终末期疾病的伤害。

DESCRIPTION (Applicant's abstract): Diabetic arteriopathy, diabetic nephropathy, and many forms of progressive renal disease are all characterized by excessive deposition of collagen IV. The lack of effective therapies for these forms of chronic tissue injury are in large part due to a lack of understanding of basic mechanisms of fibrogenesis. Although TGF-13 1 has emerged as the dominant determinant of collagen IV expression and is considered an essential fibrogenic cytokine, the intracellular signaling pathways elicited by TGF-B 1 and the mechanism by which TGF-J3 1 stimulates transcription of the collagen IV genes in pathobiologic states is not known. The central hypothesis to be tested in this application is that TGF-B 1 increases transcription of the collagen IV genes by triggering several functionally distinct intracellular signaling pathways, involving the Smad proteins and one or more of the mitogen activated protein kinase cascades. In Specific Aim 1, functional characterization of the collagen IV promoter and flanking regions will be completed. Deletion constructs of chimeric collagen IV promoter-CAT vectors will be used to define sequences which function as orientation-specific activator regions that direct transcription of the a1(IV) and a2(IV) collagen genes. Nuclear proteins that bind these critical regulatory regions will be defined by gel mobility shift assays. In Specific Aim 2, the role of TGF-B 1 elicited signaling pathways on collagen IV transcription, steady state mRNA expression, and protein production will be ascertained. Rat mesangial cells and aortic smooth muscle cells will be transfected with constitutively active or dominant negative Smad constructs and with constructs to constitutively activate the ERK, iNK, and p38 signaling pathways; the role of these interventions on basal and TOP-B 1 stimulated collagen IV expression will be ascertained. Potential sites of interaction between the Smad and MAPK signaling pathways will be defined. Based on these studies, transfection analysis and gel mobility shift assays will be employed to define sequence elements within the collagen N promoter or flanking region that confer a transcriptional response to TGF-beta 1(TGF-B1, Specific Aim 3). Finally, mesangial cells and vascular smooth muscle cells isolated from TGF-B 1 knockout animals will be used to determine whether activation of the Smad and/or MAP kinase signaling cascades can activate transcription of the collagen IV genes in a TGF-B 1-independent manner. Delineation of the collagen IV signaling pathway will reveal basic cellular mechanisms of enhanced fibrogenesis underlying progressive tissue injury, and may provide the rational basis for the design of new pharmaco-therapeutic interventions targeted to specific identified signaling steps. If successful, these interventions may retard progression of tissue injury to end-stage disease.