Epigenetic Regulation in Cartilage Tissue

 描述（由申请人提供）：本研究的目标是确定表观遗传从头 DNA 甲基转移酶 Dnmt3b 在调节产后关节软骨稳态中的功能作用。通过这项工作，我们期望确定新的目标来设计治疗骨关节炎（OA）的新策略，骨关节炎是美国医疗保险人群中最昂贵的疾病。对 OA 的发病机制仍知之甚少，临床需求尚未得到满足，需要发现新的疗法来减缓或阻止这种疾病中发生的软骨退化。表观遗传变化（例如 DNA 甲基化、组蛋白修饰、microRNA 介导的调节）与许多疾病有关，包括 OA。事实上，最近的全基因组甲基化分析揭示了健康和 OA 软骨细胞中的差异甲基化位点。在这两种从头 DNA 甲基转移酶中，我们发现 Dnmt3b 蛋白（而非 Dnmt3a）定位于健康小鼠和人关节软骨的软骨细胞中。重要的是，我们还发现衰老/骨关节炎软骨中的 Dnmt3b 表达降低，并且软骨细胞中 Dnmt3b 功能丧失的转基因小鼠出现自发性骨关节炎。对正常软骨细胞与 Dnmt3b 敲低软骨细胞生成的 RNA-Seq 和甲基-Seq 数据进行彻底分析表明，Dnmt3b 的潜在下游靶标是代谢酶 4 氨基丁酸转氨酶 (Abat)。 Abat 的功能是将 γ-氨基丁酸 (GABA) 代谢为琥珀酸，是细胞中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 抑制剂药物氨己烯酸 (vigabatrin) 能够减弱体外 Dnmt3b 功能丧失软骨细胞中诱导的分解代谢基因表达。总而言之，我们的初步数据表明软骨细胞中存在 Dnmt3b/Abat 轴，并且对该轴的调节可能是治疗 OA 的一种有前途的治疗策略。将利用体外和体内方法来调节 Dnmt3b 或 Abat 表达和/或功能，以定义涉及它们调节关节软骨细胞的机制及其在 OA 发展中的作用。提出了两个主要具体目标。具体目标 1 将涉及出生后软骨细胞中 Dnmt3b 的体内消融，以确定小鼠在衰老过程中或半月板韧带损伤引起的关节不稳定后是否会出现自发性 OA。将进行体外实验以证明 Abat 是 Dnmt3b 在细胞代谢调节和关节软骨细胞分化为肥大/分解代谢表型方面的关键下游靶标。具体目标 2 将利用 Dnmt3b 体外和体内功能获得模型来确定 Dnmt3b 过度表达是否具有针对 OA 的保护作用。将给予氨己烯酸以确定 Abat 的体内抑制是否可以延迟小鼠关节损伤后 OA 的发作。总之，该计划将定义 Dnmt3b 介导的表观遗传变化、Abat 功能和细胞代谢作为 OA 发展的新途径轴。这项工作将增强我们对 OA 调节机制的理解，并为治疗 OA 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.