赖氨酸甲基转移酶Smyd2对纤毛形成的调控及其在ADPKD中的作用与机制

Autosomal dominant polycystic kidney disease (ADPKD) is a hereditary kidney disease, which is caused by mutations in one of two genes, PKD1 or PKD2 encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. So far, there is no any effective treatment for ADPKD . There is an urgent need for all PKD researchers to understand novel mechanisms and then discover the novel treatment for ADPKD. However, ADPKD cannot be fully understood in terms of the constrained genetic setting, especially, in families with the same genetic mutations but variable disease severity. Epigenetic regulation has emerged as a critical driver of cell fate and survival that targets many pathways. Especially, epigenetic drift can occur even in genetically identical humans, which may be an alternative means of explaining PKD-associated alterations. In our recent study, we found that lysine methyltransferase Smyd2, an epigenetic regulator, promotes cyst formation in ADPKD via the methylation of its novel non-histone substrates, STAT3 and NF-κB. Our preliminary study that tubulin, a major component of the eukaryotic cytoskeleton and primary cilia, may also be a novel substrate of Smyd2, suggesting a role of Smyd2 in ciliopathy. In this study, we will investigate the roles and mechanisms of Smyd2 in the ciliogenesis of ADPKD .We will also investigate the roles of Smyd2 mediated tubulin methylation in mitosis, cytokinesis and abnormal centrosome amplification in different ADPKD mouse models. In addition, we will determine the changes of the methylation of tubulin and the epigenetic signatures in ADPKD samples compared to those in normal human kidney samples. This is the first study to investigate the methylation of α-tubulin on ciliogenesis, which should have high impact on ciliopathy related human diseases, including ADPKD, and should also provide rationale for the development of the novel therapeutic strategy for ADPKD.

显性遗传性多囊肾病（ADPKD）是一种由PKD1或PKD2基因突变所致的肾脏疾病，目前临床上无针对ADPKD的有效治疗方案。但研究发现ADPKD病程严重程度在相同遗传突变背景的家庭成员之间有重大临床差异，提示表观遗传调控可能是导致PKD病人间表型多样性的新机制。我们前期研究发现表观遗传调控因子赖氨酸甲基转移酶Smyd2可促进ADPKD囊肿的形成，进一步研究发现微管蛋白是Smyd2新的作用底物，而微管蛋白是真核生物细胞骨架和初级纤毛的主要组成成分，提示Smyd2在纤毛形成以及细胞有丝分裂中发挥重要作用。本项目拟研究Smyd2在纤毛形成以及ADPKD病理中的作用，以期找到Smyd2参与ADPKD病程的更多机理。本项目是第一个探讨微管蛋白甲基化在纤毛病中作用的研究，将会对包括ADPKD在内的纤毛相关性疾病产生重要影响，也将为开发ADPKD新的治疗策略提供重要依据。

常染色体显性遗传性多囊肾病（Autosomal dominant polycystic kidney disease，ADPKD)是一种遗传性基因缺陷，主要由多囊蛋白1（PKD1）或多囊蛋白2（PKD2）的基因突变造成， 以肾脏的多发性液性囊泡形成以及间质纤维化为主要特征，最终导致终末期肾病的发生（end-stage renal disease，ESRD)，因此成为构成肾衰竭的第四大病因。PKD 基因突变造成ADPKD 的病人中以及PKD 的小鼠模型中赖氨酸甲基转移酶SMYD2 的表达上调。细胞周期依赖性激酶CDK4和CDK6在肿瘤和以及许多增殖相关的人类疾病中的异常表达非常常见，通过抑制CDK4/CDK6 的活性可以限制肿瘤的生长，但是对于隐藏在其中的机制尚不清楚。在这个研究中，我们发现SMYD2 作为激酶CDK4/CDK6 的新的催化底物，并且与CDK4/CDK6 之间形成正反馈调节网络，CDK4/CDK6 调节SMYD2 的磷酸化水平以及酶活性，而SMYD2同样正性调节CDK4/CDK6 的表达，由此将CDK4/CDK6 与表观遗传调控联系起来。 我们同样鉴定出alpha-tubulin 是SMYD2 的新型甲基化底物， 从而将CDK4/CDK6 -SMYD2与微管的动态调节联系起来。而纤毛作为微管动态变化的一种代表，本身与细胞周期密切相关，而其聚合及解聚过程被CDK4/CDK6-SMYD2 信号调节。我们发现基因敲除SMYD2， 或是抑制剂针对CDK4/CDK6 或是SMYD2 均促进纤毛的聚合，由此将CDK4/CDK6-SMYD2 与纤毛发生以及细胞周期联系起来。 在临床疾病中，比如乳腺癌和多囊肾病， 上调的CDK4/CDK6以及SMYD2 导致纤毛发生异常，而靶向性抑制CDK4/CDK6 或是SMYD2 能恢复肿瘤细胞或是囊性细胞中的纤毛的形成。另一方面，肾脏纤维化是ADPKD 的重要特征，那么SMYD2 是否以及怎样参与肾脏纤维化的形成也不明确，我们发现细胞因子TGF-beta 通过经典的TGF-beta-SMAD3的信号通路诱导SMYD2 的表达; 而反过来,下调SMYD2 的表达或是抑制其活性同样能调节纤维化相关蛋白,比如 alpha-SMA 蛋白和Fibronectin 蛋白的表达， 以及SMYD2 介导的组蛋白H3K4的甲基