GSK3B 过表达通过促进 DNMT1 介导的 NR4A3 启动子超甲基化缓解小鼠创伤性骨关节炎。
GSK3B Overexpression Alleviates Posttraumatic Osteoarthritis in Mice by Promoting DNMT1-Mediated Hypermethylation of NR4A3 Promoter.
机构信息
Orthopedic Resident Standardized Training, Qingdao Municipal Hospital (Group), Qingdao 266000, China.
Department of Orthopedic Trauma, Yantai Affiliated Hospital of Binzhou Medical University, Yantai 264000, China.
出版信息
Dis Markers. 2022 Jun 14;2022:4185489. doi: 10.1155/2022/4185489. eCollection 2022.
BACKGROUND
Glycogen synthase kinase 3 (GSK3B) is reported to be a protective factor for the degradation of chondrocytes by extracellular mechanisms. Nuclear receptor subfamily 4 group A member 3 (NR4A3) is a proinflammatory factor in osteoarthritis. Their regulation mechanism in posttraumatic osteoarthritis (PTOA) is not fully understood.
METHODS
GSK3B expression in the cartilage tissue of PTOA patients was analyzed by western blotting. IL-1-induced chondrocytes were transfected with pcDNA-GSK3B, and then, the cell viability, apoptosis, expression of the chondrocyte extracellular matrix degradation-related genes MMP13, aggrecan, and type II collagen, and secretion of inflammatory factors TNF- and IL-6 were detected. Co-IP was used to analyze the interaction between GSK3B and DNMT1. Ch-IP and methylation-specific PCR assays were used for methylation. Also, cells were transfected with pcDNA-GSK3B or together with pcDNA-NR4A3, as well as transfected with si-NR4A3, and then, cell functions were tested. Then, the mice subjected to destabilization of medial meniscus (DMM) surgery were intra-articular injected with 100 L of the following adeno-related virus vectors (empty vector, Ad-GSK3B, scrambled shRNA, and sh-NR4A3), respectively, and the virus titer was 2 × 10 TU/mL. Cartilage integrity was evaluated by safranin O/fast green staining, HE staining, and Osteoarthritis Research Society International (OARSI) score.
RESULTS
The expression of GSK3B protein was downregulated in PTOA patients. GSK3B overexpression alleviated IL-1-induced chondrocyte apoptosis and extracellular matrix degradation, as well as cartilage mineralization in PTOA model mice. NR4A3 overexpression reversed the effect of GSK3B on IL-1-induced chondrocyte functions. GSK3B could recruit DNMT1 to the NR4A3 promoter region to promote the methylation of NR4A3 and inhibit the expression of NR4A3 protein. Similarly, NR4A3 interference alleviated cartilage degradation under stimulating conditions by inhibiting the activation of the JAK2/STAT3 signaling pathway.
CONCLUSION
GSK3B recruits DNMT1 to the NR4A3 promoter region and inhibits the activation of the NR4A3-mediated JAK2/STAT3 signaling pathway, thereby alleviating PTOA.
背景
糖原合酶激酶 3（GSK3B）据报道是通过细胞外机制降解软骨细胞的保护因子。核受体亚家族 4 组 A 成员 3（NR4A3）是骨关节炎中的促炎因子。它们在创伤后骨关节炎（PTOA）中的调节机制尚未完全阐明。
方法
通过 Western blot 分析 PTOA 患者软骨组织中的 GSK3B 表达。用 pcDNA-GSK3B 转染 IL-1 诱导的软骨细胞，然后检测细胞活力、凋亡、软骨细胞细胞外基质降解相关基因 MMP13、聚集蛋白聚糖和 II 型胶原的表达以及炎症因子 TNF-α和 IL-6 的分泌。用 co-IP 分析 GSK3B 和 DNMT1 之间的相互作用。用 Ch-IP 和甲基化特异性 PCR 检测甲基化。此外，用 pcDNA-GSK3B 或与 pcDNA-NR4A3 共转染细胞，并转染 si-NR4A3，然后检测细胞功能。然后，用以下腺相关病毒载体（空载体、Ad-GSK3B、 scrambled shRNA 和 sh-NR4A3）分别对接受内侧半月板不稳定（DMM）手术的小鼠进行关节内注射，每种病毒滴度为 2×10TU/mL。通过番红 O/快绿染色、HE 染色和骨关节炎研究协会国际（OARSI）评分评估软骨完整性。
结果
PTOA 患者的 GSK3B 蛋白表达下调。GSK3B 过表达可减轻 IL-1 诱导的软骨细胞凋亡和细胞外基质降解，并减轻 PTOA 模型小鼠的软骨矿化。NR4A3 过表达逆转了 GSK3B 对 IL-1 诱导的软骨细胞功能的影响。GSK3B 可以将 DNMT1 募集到 NR4A3 启动子区域，促进 NR4A3 的甲基化，抑制 NR4A3 蛋白的表达。同样，NR4A3 干扰通过抑制 JAK2/STAT3 信号通路的激活来减轻刺激条件下的软骨降解。
结论
GSK3B 将 DNMT1 募集到 NR4A3 启动子区域并抑制 NR4A3 介导的 JAK2/STAT3 信号通路的激活，从而缓解 PTOA。
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