Genetic Programming in Progressive Renal Disease

进行性肾病的基因编程

• 批准号: 7213943
• 负责人: Erwin P. Bottinger
• 金额: $ 34.75万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7213943
• 关键词: Adaptor Signaling Protein; Apoptosis; Binding; CD2-associated protein; Cell Survival; Complex; Diabetic Nephropathy; Disease susceptibility; Down-Regulation; Doxycycline; Epithelial Cells; Genetic Programming; Human; Injury; Kidney; Kidney Diseases; Knockout Mice; Life; Mediating; Modeling; Molecular; Mus; Mutation; Nodular glomerulosclerosis; Pathway interactions; Phosphatidylinositols; Phosphotransferases; Proteins; Receptor Signaling; Regulation; Renal glomerular disease; Role; Signal Transduction; Structural Models; TGF-beta type I receptor; Technology; Testing; Work; base; glomerulosclerosis; in vivo; men; mouse model; novel; podocyte; prevent; protein expression; protein function; receptor; slit diaphragm

DESCRIPTION (provided by applicant): Podocyte depletion is a hallmark of progressive glomerulosclerosis and diabetic nephropathy. CD2- associated protein (CD2AP) is a widely-expressed cytoplasmic adaptor protein that has previously been implicated in podocyte slit diaphragm protein complex formation. CD2AP null mice develop progressive glomerulosclerosis early in life and CD2AP haploinsufficiency is associated with glomerular disease susceptibility in humans. However, it remains unclear how loss of CD2AP function may cause glomerulosclerosis. In this renewal application, we will extend our work to test the hypothesis that CD2AP promotes renal epithelial cell survival by functioning as a molecular switch that suppresses the TGF-beta receptor type I (Tgfbr1)/Smad3-dependent apoptosis pathway and activates a Tgfbrl/phosphoinositide-3- kinase (PI3K) -dependent survival pathway. If confirmed, our studies will provide a molecular mechanism to explain how loss of CD2AP may enhance podocyte apoptosis and depletion to promote glomerular disease in mice and men based on a novel, essential functional role for CD2AP as multi-pathway regulator in TbRI- induced signal transduction. The relevance of this work is further underscored by recent observations suggesting that in the absence of direct genetic defects, functional downregulation of CD2AP expression is a common finding in mouse models and human glomerular disease. Specific Aims: 1. Identify the molecular determinants and characterize the regulation of the TGF-p-inducible interaction of CD2AP and cytoplasmic Tgfbrl. We will a) determine the sequence motif and structural model of Tgfbrl that underlies the interaction with CD2AP, and b) compare it with the Smad-binding model mediated by the GS region and L45 loop of Tgfbrl. 2. Investigate how CD2AP mediates negative regulation of Tgfbr1/Smad3 signaling. We will examine whether a) CD2AP competes with SmadS for binding to activated Tgfbrl; and b) CD2AP targets internalized Tgfbrl complexes for degradation and termination of signaling. 3. Define the role of podocyte-specific TGF-b receptor signaling in podocyte apoptosis and depletion in vivo, and determine whether this podocyte-specific mechanism underlies the progression of glomerular injury to glomerulosclerosis in CD2AP-/- mice? We will determine in vivo whether a) conditional deletion of TGF-b receptor type II (Tgfbr2) in podocytes using cre/lox technology prevents podocyte apoptosis, depletion, and/or glomerulosclerosis in CD2AP-/- mice; and b) whether podocyte-specific, controlled activation of TGF-(3 receptor signaling is sufficient to induce podocyte apoptosis, podocyte depletion and glomerulosclerosis in vivo using doxycycline-inducible expression of constitutively active Tgfbrl (Tgfbrl (AAD)).

描述（由申请人提供）：足细胞耗竭是进行性肾小球硬化症和糖尿病肾病的标志。CD 2相关蛋白（CD 2AP）是一种广泛表达的细胞质衔接蛋白，以前曾参与足细胞狭缝隔膜蛋白复合物的形成。CD 2AP敲除小鼠在生命早期发展为进行性肾小球硬化，并且CD 2AP单倍不足与人类肾小球疾病易感性相关。然而，目前尚不清楚CD 2AP功能的丧失如何导致肾小球硬化。在此更新申请中，我们将扩展我们的工作，以测试CD 2AP通过作为抑制TGF-β受体I型（Tgfbr 1）/Smad 3依赖性凋亡途径并激活Tgfbr 1/磷酸肌醇-3-激酶（PI 3 K）依赖性存活途径的分子开关发挥作用来促进肾上皮细胞存活的假设。如果得到证实，我们的研究将提供一种分子机制，以解释CD 2AP的丢失如何增强足细胞凋亡和耗竭，从而促进小鼠和男性肾小球疾病，这是基于CD 2AP作为TbRI诱导的信号转导中的多途径调节剂的新的基本功能作用。最近的观察结果进一步强调了这项工作的相关性，表明在没有直接遗传缺陷的情况下，CD 2AP表达的功能性下调是小鼠模型和人类肾小球疾病中的常见发现。具体目标：1。鉴定分子决定簇并表征CD 2AP和细胞质Tgfbrl的TGF-β诱导的相互作用的调节。我们将a）确定Tgfbrl的序列基序和结构模型，其是与CD 2 AP相互作用的基础，和B）将其与由Tgfbrl的GS区和L45环介导的Smad结合模型进行比较。2.研究CD 2AP如何介导Tgfbr 1/Smad 3信号的负调控。我们将检查a）CD 2AP是否与SmadS竞争结合活化的Tgfbrl;和B）CD 2AP是否靶向内化的Tgfbrl复合物以降解和终止信号传导。3.明确足细胞特异性TGF-β受体信号在体内足细胞凋亡和耗竭中的作用，并确定这种足细胞特异性机制是否是CD 2AP-/-小鼠肾小球损伤进展为肾小球硬化的基础？我们将在体内确定a）使用cre/lox技术在足细胞中有条件地缺失TGF-β受体II型（Tgfbr 2）是否防止CD 2AP-/-小鼠中足细胞凋亡、耗竭和/或肾小球硬化;和B）足细胞特异性的、受控的TGF-β 1活化是否β受体信号传导足以诱导足细胞凋亡，使用强力霉素诱导的组成型活性Tgfbrl（Tgfbrl（AAD））的表达在体内进行足细胞耗竭和肾小球硬化。