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，是约翰霍普金斯大学肾脏科的讲师。他寻求指导基础科学为导向的研究职业发展奖，以获得基本技能和指导研究经验，作为一个独立的职业生涯在多囊肾疾病领域的医生科学家。该研究提案详细介绍了一个五年计划，包括分子医学，统计学和流行病学课程，William Guggino博士的指导以及分子生物学和生物化学的基础科学研究培训，主要关注常染色体显性遗传性多囊肾病（ADPKD）。ADPKD是一种遗传性疾病，影响1：1000至1：500人，其特征是由肾小管产生的充满液体的囊肿。ADPKD由PKD 1或PKD 2基因突变引起，PKD 1或PKD 2基因分别编码基因产物多囊蛋白1（PC 1）和多囊蛋白2（PC 2）。虽然PC 1和PC 2已被深入研究，但关于它们如何促进小管形成或蛋白质故障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（HDAC 6）的表达和活性在Pkd 1突变的肾上皮细胞中增加，我们发现HDAC 6水平在Pkd 2敲除的MEF细胞中增加，这表明需要完整的PC 1-PC 2复合物来调节HDAC 6。在PKD中，囊性上皮具有增加的增殖指数;有趣的是，HDAC 6的过表达促进锚定非依赖性增殖，而HDAC 6的敲低抑制癌细胞系中的锚定非依赖性增殖。我们提出了一个新的概念，其中由于PKD 1或PKD 2基因突变导致PC 1-PC 2复合物的破坏导致HDAC 6水平/活性增加和随后的囊肿形成;靶向HDAC 6活性可以防止囊肿形成或可能减缓囊肿生长。研究议程的具体目标是：1）确定PC 1，PC 2和HDAC 6相互关系在囊肿形成中的作用。该目的的总体假设是，在不存在PC 1或PC 2的情况下，HDAC 6活性增加将促进囊肿形成。2)评估HDAC 6与PC 2结合的影响。该目的的总体假设是，PC 2的异常降解和运输受HDAC 6和PC 1调节的额外信号转导系统调节。3)明确HDAC 6在肾小管功能中的作用。该目的的总体假设是HDAC 6活性增加将导致ADPKD中钙信号传导和细胞极性异常，这是ADPKD中失调的两种关键上皮功能。目前，ADPKD没有治愈方法。一种有希望的治疗方法涉及加压素V2受体拮抗剂，它抑制ADPKD动物模型中的囊肿发育，并已在人体中进行了测试，但尚未被批准作为治疗方法。然而，考虑到ADPKD中多种途径被错误调节，靶向不同途径的联合治疗可能更有效。我们的假设是，由于PKD 1或PKD 2基因突变导致的PC 1-PC 2复合物的破坏将导致HDAC 6水平/活性增加，随后可能导致囊肿形成;靶向HDAC 6活性可以防止囊肿形成或可能减缓囊肿生长。这个翻译前的建议将研究PC 1-PC 2复合物如何通过HDAC 6在PKD的3D培养和动物模型中调节囊肿形成。HDAC 6抑制剂正在各种恶性肿瘤的治疗中进行密集测试，未来可能被视为ADPKD治疗。

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.