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

描述(由申请人提供)：Valeriu Cebotaru，医学博士，约翰霍普金斯大学肾病学系讲师。他寻求获得以基础科学为指导的研究职业发展奖，以获得基本技能和指导研究经验，以便在多囊肾病领域成为一名独立的内科科学家。该研究提案详细介绍了一项五年计划，其中包括分子医学、统计学和流行病学方面的课程，威廉·古吉诺博士的指导，以及分子生物学和生物化学方面的基础科学研究培训，主要重点是常染色体显性多囊肾病(ADPKD)。ADPKD是一种遗传性疾病，影响1：1000至1：500的人，其特征是肾小管形成充满液体的囊肿。ADPKD是由PKD1或PKD2基因突变引起的，这两个基因分别编码多囊蛋白1(PC1)和多囊蛋白2(PC2)。尽管已经对PC1和PC2进行了深入的研究，但关于它们如何发挥促进小管形成的功能，或者这两种蛋白的功能障碍如何导致包囊形成的信息仍在不断涌现。在PKD1突变的肾上皮细胞中，组蛋白脱乙酰酶6(HDAC6)的表达和活性增加，我们发现在PKD2基因敲除的MEF细胞中HDAC6的水平增加，这表明需要一个完整的PC1-PC2复合体来调节HDAC6。在PKD中，囊性上皮的增殖指数增加；有趣的是，HDAC6的过表达促进了癌细胞的贴壁非依赖性增殖，而HDAC6的敲除则抑制了癌细胞的贴壁非依赖性增殖。我们提出了一个新的概念，即由于PKD1或PKD2基因突变而导致PC1-PC2复合体的破坏导致HDAC6水平/活性的增加和随后的包囊形成；靶向HDAC6活性可以防止包囊的形成或可能减缓包囊的生长。研究议程的具体目的是：1)确定PC1、PC2和HDAC6之间的相互关系在包囊形成中的作用。这一目标的总体假设是，在没有PC1或PC2的情况下，HDAC6活性增加将促进包囊形成。2)评估HDAC6与PC2绑定的影响。这一目标的总体假设是，PC2的异常降解和运输受HDAC6和PC1调控的额外信号转导系统的调控。3)明确HDAC6在肾小管功能中的作用。这一目标的总体假设是，HDAC6活性的增加将导致ADPKD中钙信号和细胞极性的异常，这两个关键的上皮功能在ADPKD中处于失调状态。目前，ADPKD还没有治愈方法。一种有希望的治疗方法包括血管加压素V2受体拮抗剂，它可以抑制ADPKD动物模型中的囊肿发展，已经在人类身上进行了测试，但尚未被批准作为一种治疗方法。然而，考虑到ADPKD中许多通路调节不当，针对不同途径的联合治疗可能更有效。我们的假设是，由于PKD1或PKD2基因突变而导致的PC1-PC2复合体的破坏将导致HDAC6水平/活性的增加，并可能随后导致包囊形成；靶向HDAC6活性可以防止包囊形成或可能减缓包囊的生长。这项翻译前的提案将研究在3D培养和PKD动物模型中，PC1-PC2复合体如何通过HDAC6调节包囊的形成。HDAC6抑制剂在各种恶性肿瘤的治疗中正在进行密集的试验，未来可能被考虑作为ADPKD的治疗方法。

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.