Genetic Programming in Progressive Renal Disease

进行性肾病的基因编程

• 批准号: 8010742
• 负责人: Erwin P. Bottinger
• 金额: $ 10.68万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-15 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8010742
• 关键词: Adaptor Signaling Protein; Apoptosis; Binding; CD2-associated protein; Cell Survival; Complex; Diabetic Nephropathy; Diabetic mouse; Disease susceptibility; Down-Regulation; Doxycycline; Epithelial; Epithelial Cells; Genetic Programming; Genotype; Grant; Human; In Vitro; Injury; Kidney; Kidney Diseases; Knockout Mice; Lesion; Life; Ligands; Mediating; Modeling; Molecular; Mus; Mutation; Parietal; Pathway interactions; Phosphatidylinositols; Phosphotransferases; Receptor Signaling; Regulation; Renal glomerular disease; Role; Signal Transduction; Structural Models; TGF-beta type I receptor; Technology; Testing; Transgenes; Transgenic Organisms; Tubular formation; Up-Regulation; Work; base; diabetic; glomerulosclerosis; in vivo; men; mouse model; novel; podocyte; prevent; protein complex; protein expression; protein function; receptor; slit diaphragm

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)).

足细胞耗竭是进行性肾小球硬化症和糖尿病肾病的标志。 CD2- 相关蛋白（CD2AP）是一种广泛表达的细胞质接头蛋白，之前已被 参与足细胞狭缝隔膜蛋白复合物的形成。 CD2AP 缺失小鼠发育进行性 生命早期的肾小球硬化和 CD2AP 单倍体不足与肾小球疾病相关 人类的易感性。然而，目前尚不清楚 CD2AP 功能的丧失如何导致 肾小球硬化。在这个更新应用程序中，我们将扩展我们的工作来测试 CD2AP 的假设 通过作为抑制 TGF-β 的分子开关来促进肾上皮细胞存活 I 型受体 (Tgfbr1)/Smad3 依赖性凋亡途径并激活 Tgfbrl/磷酸肌醇-3- 激酶 (PI3K) 依赖性生存途径。如果得到证实，我们的研究将提供一种分子机制 解释 CD2AP 的缺失如何增强足细胞凋亡和耗竭，从而促进肾小球疾病 基于 CD2AP 在 TbRI- 中作为多途径调节剂的新的、重要的功能作用 诱导信号转导。最近的观察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，CD2AP 表达的功能性下调是一个 小鼠模型和人类肾小球疾病中的常见发现。 具体目标： 1. 确定分子决定因素并表征 TGF-β 诱导因子的调节 CD2AP 和细胞质 Tgfbrl 的相互作用。我们将 a) 确定序列基序和结构模型 Tgfbrl 与 CD2AP 相互作用的基础，b) 将其与 Smad 结合模型进行比较 由 Tgfbrl 的 GS 区和 L45 环介导。 2. 研究CD2AP如何介导负调控 Tgfbr1/Smad3 信号传导。我们将检查 a) CD2AP 是否与 SmadS 竞争结合 激活的Tgfbrl； b) CD2AP 靶向内化的 Tgfbrl 复合物，以降解和终止 发信号。 3. 定义足细胞特异性TGF-b受体信号在足细胞凋亡中的作用和 体内消耗，并确定这种足细胞特异性机制是否是足细胞进展的基础 CD2AP-/- 小鼠肾小球损伤导致肾小球硬化？我们将在体内确定是否 a) 有条件 使用 cre/lox 技术删除足细胞中的 TGF-b 受体 II (Tgfbr2) 可预防足细胞 CD2AP-/- 小鼠的细胞凋亡、耗竭和/或肾小球硬化； b) 是否是足细胞特异性的， TGF-(3 受体信号传导的受控激活足以诱导足细胞凋亡，足细胞 使用多西环素诱导的组成型活性表达进行体内耗竭和肾小球硬化 Tgfbrl（Tgfbrl（AAD））。

项目成果

Smad2 and myocardin-related transcription factor B cooperatively regulate vascular smooth muscle differentiation from neural crest cells.

• DOI: 10.1161/circresaha.113.301921
• 发表时间: 2013-09-27
• 期刊: Circulation research
• 影响因子: 20.1
• 作者: Xie WB;Li Z;Shi N;Guo X;Tang J;Ju W;Han J;Liu T;Bottinger EP;Chai Y;Jose PA;Chen SY
• 通讯作者: Chen SY

The molecular and functional phenotype of glomerular podocytes reveals key features of contractile smooth muscle cells.

• DOI: 10.1152/ajprenal.00559.2007
• 发表时间: 2008-10
• 期刊: American journal of physiology. Renal physiology
• 作者: Saleem MA;Zavadil J;Bailly M;McGee K;Witherden IR;Pavenstadt H;Hsu H;Sanday J;Satchell SC;Lennon R;Ni L;Bottinger EP;Mundel P;Mathieson PW
• 通讯作者: Mathieson PW