Role of PC1, Pc2 and HDAC6 in cyst formation in ADPKD

简介（由申请人提供）：Valeriu Cebotaru，医学博士，约翰霍普金斯大学肾脏病学系讲师。他寻求指导基础科学导向的研究职业发展奖，以获得多囊肾病领域独立职业生涯的基本技能和指导研究经验。该研究计划详细说明了一个五年计划，包括分子医学、统计学和流行病学课程，由William Guggino博士指导，博士学位和分子生物学和生物化学基础科学研究培训，主要关注常染色体显性多囊肾病（ADPKD）。ADPKD是一种遗传性疾病，患病比例为1:100至1:500，其特征是肾小管产生充满液体的囊肿。ADPKD是由编码多囊蛋白1 （PC1）和多囊蛋白2 （PC2）的基因PKD1或PKD2突变引起的。尽管PC1和PC2已被深入研究，但关于它们如何促进小管形成或其中一种蛋白的功能障碍如何导致囊肿形成的信息仍在不断涌现。组蛋白去乙酰化酶6 （HDAC6）在Pkd1突变的肾上皮细胞中的表达和活性增加，我们发现HDAC6水平在Pkd2敲除的MEF细胞中增加，这表明需要一个完整的PC1-PC2复合物来调节HDAC6。在PKD中，囊性上皮的增殖指数增高；有趣的是，在癌细胞系中，过表达HDAC6促进锚定非依赖性增殖，而敲低HDAC6则抑制锚定非依赖性增殖。我们提出了一个新的概念，即PKD1或PKD2基因突变导致PC1-PC2复合物的破坏，导致HDAC6水平/活性增加，随后形成囊肿；靶向HDAC6活性可以预防囊肿形成或可能减缓囊肿生长。研究议程的具体目的是：1)确定PC1、PC2和HDAC6相互关系在囊肿形成中的作用。本Aim的总体假设是，在缺乏PC1或PC2的情况下，HDAC6活性的增加会促进囊肿的形成。2)评估HDAC6与PC2结合的影响。本研究的总体假设是，PC2的异常降解和转运是由HDAC6和由PC1调节的其他信号转导系统调节的。3)明确HDAC6在小管功能中的作用。本研究的总体假设是，HDAC6活性的增加将导致ADPKD中钙信号和细胞极性的异常，这两种关键的上皮功能在ADPKD中失调。目前，还没有治愈ADPKD的方法。一种有希望的治疗方法是抗利尿激素V2受体拮抗剂，它可以抑制ADPKD动物模型中的囊肿发育，并已在人类中进行了测试，但尚未被批准作为一种治疗方法。然而，考虑到ADPKD中多种通路被错误调控，针对不同通路的联合治疗可能更有效。我们的假设是，由于PKD1或PKD2基因突变导致PC1-PC2复合物的破坏将导致HDAC6水平/活性增加，并可能随后导致囊肿形成；靶向HDAC6活性可以预防囊肿形成或可能减缓囊肿生长。本研究将研究PC1-PC2复合物如何在3D培养和PKD动物模型中通过HDAC6调控囊肿形成。HDAC6抑制剂在治疗各种恶性肿瘤方面正在进行深入的测试，未来可能被视为一种ADPKD治疗方法。

DESCRIPTION (provided by applicant): Valeriu Cebotaru, MD, is an Instructor in the Division of Nephrology at Johns Hopkins University. He seeks a Mentored Basic Science-Oriented Research Career Development Award in order to obtain essential skills and mentored research experience for an independent career as a physician scientist in the field of polycystic kidney disease. The research proposal details a five-year plan consisting of coursework in Molecular Medicine, Statistics and Epidemiology, mentorship by Dr. William Guggino, PhD and basic science research training in molecular biology and biochemistry with primary focus on autosomal dominant polycystic kidney disease (ADPKD). ADPKD is a hereditary disorder that affects 1:1000 to 1:500 people and is characterized by fluid-filled cysts that arise from renal tubules. ADPKD results from mutations in either the PKD1 or PKD2 gene, which encode the gene products polycystin 1 (PC1) and polycystin 2 (PC2), respectively. Although PC1 and PC2 have been studied intensively, information on how they function to promote tubulogenesis or how a malfunction of either protein leads to cyst formation is still emerging. Histone deacetylase 6 (HDAC6) expression and activity are increased in Pkd1 mutant renal epithelial cells, and we have found that HDAC6 levels are increased in Pkd2 knockout MEF cells, suggesting that an intact PC1-PC2 complex is required to regulate HDAC6. In PKD, the cystic epithelium has an increased proliferative index; interestingly, overexpression of HDAC6 promotes anchorage-independent proliferation, whereas knockdown of HDAC6 inhibits anchorage-independent proliferation in cancer cell lines. We propose a novel concept in which disruption of the PC1-PC2 complex as a result of mutations in the PKD1 or PKD2 gene leads to increased HDAC6 levels/activity and subsequent cyst formation; targeting HDAC6 activity could prevent cyst formation or perhaps slow down cyst growth. The specific aims of the research agenda are to: 1) To determine the role of the PC1, PC2, and HDAC6 interrelationship in cyst formation. The overall hypothesis of this Aim is that in the absence of PC1 or PC2, increased HDAC6 activity will promote cyst formation. 2) To assess the impact of HDAC6 binding to PC2. The overall hypothesis of this Aim is that aberrant degradation and trafficking of PC2 is regulated by HDAC6 and by additional signal transduction systems regulated by PC1. 3) To define the role of HDAC6 in tubule function. The overall hypothesis of this Aim is that increased activity of HDAC6 will lead to aberrant calcium signaling and cell polarity in ADPKD, two critical epithelial functions that are dysregulated in ADPKD. Currently, there is no cure for ADPKD. One promising therapy involves the vasopressin V2 receptor antagonist, which inhibits cyst development in animal models of ADPKD and has been tested in humans but has not yet been approved as a therapy. However, given the multitude of pathways mis-regulated in ADPKD, a combination therapy that targets different pathways may be more efficacious. Our hypothesis is that disruption of the PC1-PC2 complex as a result of mutations in the PKD1 or PKD2 gene will lead to increased HDAC6 level/activity and could subsequently lead to cyst formation; targeting HDAC6 activity could prevent cyst formation or perhaps slow down cyst growth. This pre-translational proposal will study how the PC1-PC2 complex regulates cyst formation through HDAC6 in both 3D culture and animal models of PKD. HDAC6 inhibitors are being tested intensively in the treatment of various malignancies and could be considered as an ADPKD treatment in the future.