Role of TET1 Mediatd 5hmC in Osteoarthritis [RRHP]

No disease-modifying drugs exist to treat osteoarthritis (OA), a degenerative disease of the joint.  The complexity of OA necessitates a combinational and broad therapeutic approach. Epigenetic regulators are able to control large programs of genes, and recent work from our group and others have showcased systemic epigenetic dysregulation in OA. Previously, we demonstrated that OA chondrocytes accumulate 5-fold more 5-hydroxymethylcytosine (5hmC), an oxidized derivative of methylcytosine (5mC) associated with gene activation, at disease relevant sites. To test if 5hmC has a role in the early onset of OA, we utilized a mouse model of surgically induced OA, destabilization of the medial meniscus (DMM), and found that DMM mice gained ~40,000 differentially hydroxymethylated sites. Genetic loss of TET1, the enzyme responsible for 5hmC deposition, prevented pathologic gain of 5hmC, activation of many OA pathways, and protected mice from OA development. To test the clinical potential of a TET1 based OA therapy, we injected 2-hydroxyglutarate (2-HG), a TET inhibitor, into the joint after DMM induction and  observed stalled disease progression. Collectively, these data show that TET1 mediated 5hmC deposition regulates multiple OA pathways and that its modulation can be a powerful clinical tool for OA. DMM was induced on male mice 3-4 months of age. DNA was extracted from the joint at 6 post surgery. Age-matched sham controls are also included. All groups contain 3 biological replicates. 5hmC was measured at CCGG sites using reduced representation hydroxymethylation sequencing (RRHP) from Zymo. A negative control (Neg_control) without treatment with the B-Galc enzyme is included to give the background signal for this technique.

目前尚无治疗骨关节炎（osteoarthritis, OA）——一种关节退行性疾病——的疾病修饰药物。骨关节炎的病理复杂性要求采用联合且广谱的治疗策略。表观遗传调控因子可调控大规模基因表达程序，本团队及其他课题组近期的研究均证实骨关节炎中存在系统性表观遗传失调。此前我们已证明，在疾病相关位点处，骨关节炎软骨细胞内的5-羟甲基胞嘧啶（5-hydroxymethylcytosine, 5hmC）——一种与基因激活相关的甲基胞嘧啶（methylcytosine, 5mC）氧化衍生物——的积累量较正常水平高出5倍。

为探究5hmC在骨关节炎早期发病中的作用，我们采用了手术诱导骨关节炎的小鼠模型：内侧半月板失稳术（destabilization of the medial meniscus, DMM），结果发现DMM模型小鼠体内出现约40000个差异羟甲基化位点。敲除负责催化5hmC生成的TET1基因，可阻断病理性5hmC积累、激活多条骨关节炎相关通路，并保护小鼠免于发生骨关节炎。

为验证基于TET1的骨关节炎治疗方案的临床潜力，我们在DMM术后向小鼠关节腔内注射了TET抑制剂2-羟基戊二酸（2-hydroxyglutarate, 2-HG），观察到疾病进展被阻滞。综上，本研究数据表明，TET1介导的5hmC沉积调控了多条骨关节炎相关通路，对其进行干预有望成为骨关节炎极具前景的临床治疗手段。

实验设计与方法：本研究选用3~4月龄的雄性小鼠构建DMM模型，于术后6周提取关节组织DNA。同时设置同月龄的假手术对照组。所有实验组均设置3个生物学重复。采用Zymo公司的简化代表性羟甲基化测序（reduced representation hydroxymethylation sequencing, RRHP）技术，在CCGG位点处检测5hmC水平。本研究设置了未添加β-半乳糖苷酶（β-Galc）的阴性对照（Neg_control），以获取该技术的背景信号。