Crosstalk of DNMT1 and Sirt1 in Autosomal Dominant Polycystic Kidney Disease

项目摘要 常染色体显性遗传性多囊肾病(ADPKD)的囊性形成机制仍然存在 对ADPKD的认识尚不完全清楚，缺乏有效的临床治疗方法。我的长期生活 职业目标是建立一个独立的翻译研究实验室，在那里我将阐明 研究囊腔形成和生长的机制，并发现ADPKD的新治疗策略。这 NIDDK导师研究科学家开发(K01)应用程序提出了一个多学科的5- 年培训计划为应聘者夏舟博士提供经验和资源 开始一个成功的职业生涯所必需的。培训计划是与主要导师Dr。 李小刚和共同导师詹姆斯·卡尔维博士将加强我以前在表观遗传学方面的专业知识 通过提高我在DNA研究方面的知识和技术，进行ADPKD研究 甲基化和组蛋白乙酰化。堪萨斯大学非常激动人心的科学环境 医疗中心不仅为我提供成功所需的专业知识和设施 完成这个项目，也将为我顺利过渡到独立学院做好准备 位置。这项拟议研究的目的是了解一个关键的表观遗传学的功能作用。 ADPKD中的调节因子，DNA甲基转移酶1(DNMT1)。我们的初步研究表明 DNMT1在PKD1突变的肾上皮细胞和组织中表达上调， DNMT1抑制剂5-氮胞苷和肼并肼延缓PKD1条件性基因敲除中的囊性生长 老鼠。我们先前的研究表明，SIRT1的上调通过 在ADPKD动物模型中调节囊性肾上皮细胞的增殖和死亡 使用烟酰胺(维生素B3)治疗ADPKD有希望的治疗策略。SIRT1有 已发现可使DNMT1脱乙酰化，并提高其甲基转移酶活性。因此，我们假设 DNMT1上调通过激活STAT3调节囊性肾上皮细胞增殖 并通过P53信号转导调控细胞凋亡，靶向DNMT1延缓小鼠体内肾囊肿生长 ADPKD同种小鼠模型及DNMT1与SIRT1协同调控囊壁生长的研究 在ADPKD中，同时靶向SIRT1和DNMT1将进一步延缓ADPKD中的囊性生长。我们会 用三个具体的目标来检验这一假设。在具体目标1中，我们将1)调查DNMT1 SHP-1介导的磷酸化和磷酸化调控囊性肾上皮细胞增殖 2)研究DNMT1是否通过P53调控细胞凋亡 依赖途径。在特定的目标2中，我们将1)测试DNMT1基因敲除是否会延迟肾囊肿 2)检测5-氮胞苷或5-氮胞苷对DNMT1的抑制作用 联苯肼延缓PKD1基因敲除小鼠模型中的肾囊肿生长。在具体目标3中，我们将1) 研究SIRT1是否调节囊性肾上皮细胞DNMT1的活性和稳定性； 2)研究SIRT1是否调节SHP-1的甲基化状态，以及DNMT1是否 调节SHP-1的组蛋白乙酰化；以及3)检测是否同时抑制DNMT1和SIRT1 协同延缓PKD1基因敲除小鼠的囊性生长。这是第一次定义功能性的研究 DNMT1和DNMT1介导的信号通路在ADPKD囊变过程中的作用 不仅加深了我们对包囊发育的了解，而且为使用DNMT1提供了理论基础 抑制剂作为ADPKD的治疗方法。此外，本研究还将揭示DNMT1- 介导的甲基化和SIRT1介导的乙酰化在调节包囊发育中的作用，并将有助于 促进针对DNMT1和SIRT1的新治疗方法的开发。

Project Summary The mechanisms of cyst formation in autosomal dominant polycystic kidney disease (ADPKD) remain incompletely understood and the effective clinical therapies for ADPKD are not available. My long-term career goal is to establish an independent translational research laboratory, where I will elucidate the mechanisms of cyst formation and growth, and discover novel therapeutic strategies for ADPKD. This NIDDK Mentored Research Scientist Development (K01) application proposes a multidisciplinary 5- year training program to provide the candidate Dr. Xia Zhou with the experience and resources necessary to launch a successful career. The training plan, developed closely with primary mentor Dr. Xiaogang Li and co-Mentor Dr. James Calvet will strengthen my previous expertise in the epigenetics and ADPKD research by advancing my knowledge and technologies related to the study of DNA methylation and histone acetylation. The very stimulating scientific environment at University of Kansas Medical Center will not only provide me with the expertise and facilities necessary for successful completion of this project, but will also prepare me to transition smoothly into an independent faculty position. The objective of the proposed study is to understand the functional roles of a key epigenetic regulator, DNA methyltransferase 1 (DNMT1), in ADPKD. Our preliminary studies indicated that DNMT1 was upregulated in Pkd1 mutant renal epithelial cells and tissues, and that treatment with DNMT1 inhibitors, 5-azacytidine and hydralazine, delayed cyst growth in Pkd1 conditional knockout mice. Our previous study showing that upregulation of SIRT1 contributes to cyst development through regulating cystic renal epithelial cell proliferation and death in ADPKD animal models suggests a promising therapeutic strategy for ADPKD treatment by using nicotinamide (vitamin B3). SIRT1 has been found to deacetylate DNMT1 and increase its methyltransferase activity. Thus, we hypothesize that upregulation of DNMT1 regulates cystic renal epithelial cell proliferation through STAT3 activation and regulates apoptosis through p53 signaling, and targeting DNMT1 delays renal cyst growth in vivo in orthologous murine models of ADPKD, and that DNMT1 synergizes with SIRT1 to regulate cyst growth in ADPKD, and targeting both SIRT1 and DNMT1 would delay cyst growth further in ADPKD. We will test this hypothesis with three specific aims. In specific aim 1, we will 1) investigate whether DNMT1 regulates cystic renal epithelial cell proliferation through SHP-1 mediated phosphorylation and activation of STAT3; and 2) investigate whether DNMT1 regulates apoptosis through the p53 dependent pathway. In specific aim 2, we will 1) test whether knockout of DNMT1 delays renal cyst growth in Pkd1 knockout mice; and 2) test whether inhibition of DNMT1 with 5-azacytidine or hydralazine delays renal cyst growth in Pkd1 knockout mouse models. In specific aim 3 we will 1) investigate whether SIRT1 regulates the activity and stability of DNMT1 in cystic renal epithelial cells; and 2) investigate whether SIRT1 regulates the methylation status of SHP-1, and whether DNMT1 regulates histone acetylation of SHP-1; and 3) test whether inhibition of both DNMT1 and SIRT1 synergistically delays cyst growth in Pkd1 knockout mice. This is the first study to define the functional roles of DNMT1 and DNMT1-mediated signaling pathways in cyst development in ADPKD, which will not only further our understanding of cyst development but also provide a rationale for using DNMT1 inhibitors as a therapy for ADPKD. In addition, this study will reveal the relationship between DNMT1- mediated methylation and SIRT1-mediated acetylation in regulating cyst development, and will help promote the development of novel therapeutic approaches targeting both DNMT1 and SIRT1.