Polycystin function in zebrafish (PA-06-158)

斑马鱼中的多囊蛋白功能 (PA-06-158)

• 批准号: 8299124
• 负责人: IAIN A. DRUMMOND
• 金额: $ 36.36万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8299124
• 关键词: Accounting; Adult; Affect; Aneurysm; Animal Model; Automobile Driving; Autosomal Dominant Polycystic Kidney; Basement membrane; Biological Assay; Biological Models; Blood Vessels; C-terminal; CD29 Antigen; Calcium; Calcium Channel; Cell Differentiation process; Cell Maturation; Cells; Cerebral Aneurysm; Chondrogenesis; Cilia; Collagen; Collagen Gene; Cystic Kidney Diseases; Defect; Development; Disease; Dominant-Negative Mutation; Down-Regulation; Embryo; Environment; Etiology; Event; Extracellular Matrix; Failure; Feedback; Focal Adhesions; Gene Expression; Gene Expression Regulation; Genes; Genetic; Hernia; Heterogeneous Nuclear RNA; Kidney; Larva; Length; Life; Link; Measures; Messenger RNA; MicroRNAs; Modeling; Mutation; Organ; PKD2 protein; PTK2 gene; Pathology; Pathway interactions; Pericardial effusion; Phenocopy; Phenotype; Phospho-Specific Antibodies; Polycystic Kidney Diseases; Production; Proteins; Pump; Regulation; Reporting; Role; Signal Pathway; Signal Transduction; Signaling Pathway Gene; Signaling Protein; Site; System; Testing; Tissues; Transcript; Transgenes; Transgenic Organisms; Work; Zebrafish; abdominal wall; base; fetal programming; gain of function; genetic manipulation; human disease; improved; in vivo; inhibitor/antagonist; loss of function; mRNA Expression; mRNA Stability; mutant; notochord; polycystic kidney disease 1 protein; promoter; public health relevance; sensor

DESCRIPTION (provided by applicant): Mutations in Polycystin-1 (PKD1) and Polycystin-2 (PKD2) account for all cases of Autosomal Dominant Polycystic Kidney Disease (ADPKD), the most common heritable human disease. In addition to their localization and function in primary cilia, polycystins are localized at sites of cell-matrix interactions, indicating they may act there as mechanosensors of the cellular environment. We have modeled polycystic kidney disease in the zebrafish by disrupting the function of polycystin1 and polycystin2. Our results demonstrate that Polycystins function in signaling pathways that sense the compostion of extracellular matrix and generate feedback signals that terminate "fetal" programs of gene expression. These signals are likely to involve PI3K and intracellular calcium stores since PI3K or SERCA pump inhibitors can phenocopy polycystin loss of function. Specifically, our findings that developmental collagen expression persists abnormally in older larvae lacking polycystins is directly relevant to defects associated with extracellular matrix seen in a variety of ADPKD pathologies including cystic kidney disease, vascular aneurysm, abdominal wall hernia, pericardial effusion, and defects in chondrogenesis. In this proposal we plan to exploit our ability to generate compound loss of function as well as gain of function conditions in zebrafish embryos to dissect signaling pathways by which polycystins function as sensors of the cellular environment. We hypothesize that Polycystin1, functioning at sites of cell matrix contact, interacts with focal adhesion proteins to generate signals involving PI3K that downregulate collagen gene expression. We will test whether beta1 integrin, FAK, or ILK are required in this pathway. We will also test whether the C-terminal product of Polycystin1 GPS cleavage or other subdomains of polycystin1 are sufficient to rescue polycystin1 morphants. The role of ER calcium stores in signaling will be explored by measuring ER calcium in polycystin deficient embryos and assaying for genetic interactions of polycystin1 and polycystin2 with stim1 and orai1, the recently discovered store operated calcium channels. Although it has long been appreciated that defects in extracellular matrix were prominent in ADPKD pathology, a direct role for Polycystins as regulators of matrix production has yet to be demonstrated. If sucessful, our work will provide a direct link between Polycystins as signaling proteins and the control of extracellular matrix production that may be common to all ADPKD pathologies. PUBLIC HEALTH RELEVANCE: Polycystic kidney disease is a common life-threatening disorder for which there is no cure. We intend to show how mutations in Polycystic kidney disease genes result in structural defects in affected organs by causing misregulation of collagen gene expression. Understanding the cellular basis of this disease will create new opportunities to improve the lives of those that suffer from it.

描述（由申请人提供）：Polycystin-1（PKD1）和Polycystin-2（PKD2）中的突变考虑了所有常染色体显性多囊肾脏病（ADPKD）的病例，这是最常见的人类疾病。除了它们在原发性纤毛中的定位和功能外，多囊菌素还位于细胞 - 矩阵相互作用的部位，表明它们可以在那里充当细胞环境的机械传感器。我们通过破坏斑马鱼的功能和polycystin2的功能来对斑马鱼中的多囊肾脏疾病进行建模。我们的结果表明，多囊素在信号通路中发挥作用，这些信号通路感知细胞外基质的构成并生成终止基因表达“胎儿”程序的反馈信号。这些信号可能涉及PI3K和细胞内钙存储，因为PI3K或SERCA泵抑制剂可以表苯甲酸多囊霉素的功能丧失。具体而言，我们的发现是，缺乏多囊蛋白的较老的幼虫中发育性胶原蛋白表达持续存在，这与与各种ADPKD病理学中的细胞外基质有关的缺陷直接相关，包括囊性肾脏疾病，血管性动脉动脉瘤，腹部壁，腹膜疝气，腹膜上毛发，抑制作用和抑制作用。在此提案中，我们计划利用我们产生功能复合损失的能力以及斑马鱼胚胎中功能条件的增益，以剖析polycystins用作细胞环境传感器的信号通路。我们假设PolyCystin1在细胞基质接触的位点起作用，与局灶性粘附蛋白相互作用，以生成涉及PI3K的信号，涉及pi3K，从而下调胶原基因表达。我们将测试该途径中是否需要Beta1整合素，FAK或ILK。我们还将测试PolycyStin1 GPS裂解的C末端产物或PolyCystin1的其他子域是否足以挽救PolycyStin1形态。 ER钙存储在信号传导中的作用将通过测量多囊蛋白缺乏的胚胎中的ER钙以及对PolycyStin1和Polycystin2与STIM1和ORAI1的遗传相互作用的ER钙（最近发现的最近发现的商店操作的钙通道）的遗传相互作用。尽管长期以来一直认为，细胞外基质中的缺陷在ADPKD病理学中是显着的，但多囊菌作为基质产生的调节剂的直接作用尚未证明。如果成功的话，我们的工作将提供多囊素作为信号蛋白和控制所有ADPKD病理可能共同的细胞外基质产生的直接联系。 公共卫生相关性：多囊性肾脏疾病是一种常见的威胁生命的疾病，无法治愈。我们打算展示多囊性肾脏疾病基因中的突变如何通过导致胶原基因表达的不正体来导致受影响器官的结构缺陷。了解该疾病的细胞基础将为改善患者的生活创造新的机会。