Epigenetic Regulation in Cartilage Tissue

软骨组织的表观遗传调控

• 批准号: 9234475
• 负责人: Audrey McAlinden
• 金额: $ 33.55万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-01 至 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9234475
• 关键词: 4-Aminobutyrate aminotransferase; Ablation; Address; Affect; Age; Aging; Attenuated; Cartilage; Cartilage Diseases; Catabolic Process; Cell division; Cells; Cellular Metabolic Process; Characteristics; Chondrocytes; Clinical; DNA; DNA Methylation; DNA Modification Methylases; DNMT3B gene; DNMT3a; Data; Degenerative polyarthritis; Development; Disease; Doxycycline; Elderly; Energy Metabolism; Enzymes; Epigenetic Process; FDA approved; Family member; Gene Expression; Goals; Grant; Growth and Development function; Homeostasis; Human; Hypertrophy; In Vitro; Injury; Joints; Lead; Mediating; Mediator of activation protein; Medical; Medicare; Metabolic; Metabolism; Methylation; Methyltransferase; MicroRNAs; Mus; Operative Surgical Procedures; Pathogenesis; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Play; Population; Prevention; Process; Proteins; Regulation; Reporting; Resistance; Role; Succinates; Testing; Therapeutic; Tissues; Transgenic Mice; United States; Vigabatrin; Work; articular cartilage; cartilage degradation; clinically relevant; cost; cytokine; design; epigenetic regulation; experimental study; gain of function; gamma-Aminobutyric Acid; genome wide methylation; histone modification; in vitro Model; in vivo; inhibitor/antagonist; innovation; joint injury; knock-down; ligament injury; loss of function; methylation pattern; mitochondrial metabolism; novel; novel strategies; novel therapeutics; overexpression; premature; programs; protective effect; public health relevance; small molecule inhibitor; transcriptome sequencing

 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.

 描述（由申请方提供）：本研究的目的是确定表观遗传从头DNA甲基转移酶Dnmt 3b在调节出生后关节软骨稳态中的功能作用。从这项工作中，我们期望确定新的目标，设计新的策略来治疗骨关节炎（OA），这是美国医疗保险人群中最昂贵的疾病。OA的发病机制仍然知之甚少，并且存在未满足的临床需求，以发现新的疗法来减缓或停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（包括OA）有关。事实上，最近的全基因组甲基化分析已经揭示了健康和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Dnmt 3b蛋白定位于健康小鼠和人关节软骨的软骨细胞中，而不是Dnmt 3a。重要的是，我们还表明，Dnmt 3b的表达下降，在老化/OA软骨和转基因小鼠与Dnmt 3b功能丧失的软骨细胞自发性OA。从正常与Dnmt 3b敲低软骨细胞产生的RNA-Seq和Methyl-Seq数据的彻底分析揭示，Dnmt 3b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，并且是细胞中线粒体代谢的关键调节剂。有趣的是，我们发现FDA批准的Abat抑制剂药物氨己烯酸能够减弱体外Dnmt 3b功能丧失软骨细胞中诱导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Dnmt 3b/Abat轴，并且调节该轴可能是治疗OA的有希望的治疗策略。将利用体外和体内方法来调节Dnmt 3b或Abat表达和/或功能，以确定参与其对关节软骨细胞的调节的机制及其在OA发展中的作用。提出了两个主要的具体目标。具体目标1将涉及软骨细胞中Dnmt 3b的体内出生后消融，以确定小鼠是否在衰老期间或在由踝关节韧带损伤诱导的关节不稳定之后发展自发性OA。将进行体外实验以显示Abat是Dnmt 3b在细胞代谢调节和关节软骨细胞分化成肥大/分解代谢表型中的关键下游靶标。具体目标2将利用体外和体内的Dnmt 3b功能获得模型来确定Dnmt 3b过表达是否赋予针对OA的保护。将施用氨己烯酸以确定体内抑制Abat是否可以延迟关节损伤后小鼠中OA的发作。总之，该计划将Dnmt 3b介导的表观遗传变化，Abat功能和细胞代谢定义为OA发展中的新途径轴。这项工作将提高我们对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。