Epigenetic Regulation in Cartilage Tissue

 描述(申请人提供)：这项研究的目标是确定表观遗传的从头DNA甲基转移酶Dnmt3b在调节出生后关节软骨内稳态中的功能作用。通过这项工作，我们希望为设计治疗骨关节炎(OA)的新策略确定新的目标，骨关节炎是美国医疗保险人群中最昂贵的疾病。目前对骨性关节炎的发病机制仍知之甚少，临床上尚需寻找新的治疗方法来减缓或阻止骨性关节炎的软骨退化。表观遗传学改变(如DNA甲基化、组蛋白修饰、microRNA介导的调节)与许多疾病有关，包括骨性关节炎。事实上，最近的全基因组甲基化图谱已经揭示了健康软骨和骨性关节炎软骨细胞中不同的甲基化位点。在两种脱氧核糖核酸甲基转移酶中，我们发现DNMT3b蛋白存在于正常小鼠和人关节软骨细胞中，而DNMT3A蛋白不存在。重要的是，我们还发现Dnmt3b在衰老/骨关节炎软骨中的表达减少，而软骨细胞中Dnmt3b功能丧失的转基因小鼠发生了自发性骨关节炎。对正常和Dnmt3b敲除软骨细胞产生的RNA-Seq和甲基-Seq数据的彻底分析表明，Dnmt3b的一个潜在下游靶点是代谢酶4-氨基丁酸氨基转移酶(ABAT)。ABAT的功能是将γ-氨基丁酸(GABA)代谢成琥珀酸，是细胞内线粒体代谢的关键调节因子。有趣的是，我们发现FDA批准的ABAT抑制剂药物Vigabatrin能够减弱体外诱导的Dnmt3b功能丧失软骨细胞的分解代谢基因表达。总之，我们的初步数据表明软骨细胞中存在Dnmt3b/Abat轴，调节这一轴可能是治疗骨性关节炎的一种有前途的治疗策略。在体外和体内的方法将被用来调节Dnmt3b或Abat的表达和/或功能，以确定它们调控关节软骨细胞的机制及其在骨关节炎发生中的作用。提出了两个主要的具体目标。具体目标1将涉及在生后体内消融软骨细胞中的DNMT3b，以确定小鼠在衰老过程中或在半月板韧带损伤导致的关节失稳后是否发生自发性骨性关节炎。体外实验将证明Abat是Dnmt3b在细胞代谢调节和关节软骨细胞向肥大/分解代谢表型分化中的关键下游靶点。特异性目标2将利用Dnmt3b在体外和体内的功能获得模型来确定Dnmt3b的过度表达是否对OA具有保护作用。Vigabatrin将用于确定体内抑制Abat是否可以延缓小鼠关节损伤后OA的发生。综上所述，该计划将DNMT3b介导的表观遗传学改变、ABAT功能和细胞代谢定义为OA发生发展中的一个新的途径轴。这项工作将加深我们对骨关节炎调节机制的理解，并为治疗骨关节炎的创新治疗方法提供新的靶点。

 DESCRIPTION (provided by applicant): The goals of this study are to determine the functional roles of the epigenetic de novo DNA methyltransferase enzyme, Dnmt3b, in regulating post-natal articular cartilage homeostasis. From this work, we expect to identify new targets for the design of novel strategies to treat osteoarthritis (OA), the most costly condition in the US Medicare population. The pathogenesis of OA is still poorly understood and there is an unmet clinical need to discover new therapies to slow down or stop the cartilage degradation that occurs in this disease. Epigenetic changes (e.g. DNA methylation, histone modifications, microRNA-mediated regulation) are implicated in many diseases, including OA. In fact, recent genome wide methylation profiling has revealed differentially methylated loci in cells of healthy and OA cartilage. Of the two de novo DNA methyltransferase enzymes, we found that Dnmt3b protein, but not Dnmt3a, was localized in chondrocytes of healthy murine and human articular cartilage. Importantly, we also showed that Dnmt3b expression decreased in aging/OA cartilage and that transgenic mice with Dnmt3b loss-of-function in chondrocytes developed spontaneous OA. Thorough analysis of RNA-Seq and Methyl-Seq data generated from normal versus Dnmt3b knock-down chondrocytes revealed that a potential downstream target of Dnmt3b is the metabolic enzyme, 4 aminobutyrate aminotransferase (Abat). Abat functions to metabolize gamma-aminobutyric acid (GABA) into succinate and is a key regulator of mitochondrial metabolism in the cell. Interestingly, we found that an FDA-approved Abat inhibitor drug, vigabatrin, was able to attenuate catabolic gene expression that was induced in Dnmt3b loss-of- function chondrocytes in vitro. Altogether, our preliminary data suggest the existence of a Dnmt3b/Abat axis in chondrocytes and that modulation of this axis may be a promising therapeutic strategy to treat OA. In vitro and in vivo approaches will be utilized to modulate Dnmt3b or Abat expression and/or function to define mechanisms involved in their regulation of articular chondrocytes and their role in the development OA. Two main Specific Aims are proposed. Specific Aim 1 will involve in vivo post-natal ablation of Dnmt3b in chondrocytes to determine if mice develop spontaneous OA during aging or following joint destabilization induced by meniscal ligament injury. In vitro experiments will be performed to show that Abat is a critical downstream target of Dnmt3b in the regulation of cell metabolism and in the differentiation of articular chondrocytes into a hypertrophic/catabolic phenotype. Specific Aim 2 will utilize Dnmt3b gain-of-function models in vitro and in vivo to determine if Dnmt3b over-expression confers protection against OA. Vigabatrin will be administered to determine if in vivo inhibition of Abat can delay the onset of OA in mice following joint injury. In summary, this program will define Dnmt3b-mediated epigenetic changes, Abat function and cell metabolism as a novel pathway axis in the development of OA. This work will enhance our understanding of mechanisms regulating OA and provide novel targets for innovative therapeutic approaches to treat OA.