ADAMTS12 表达在骨关节炎中的表观遗传调控机制。
Epigenetic regulatory mechanism of ADAMTS12 expression in osteoarthritis.
机构信息
Department of Orthopedics, Hunan Provincial People's Hospital (The First-affiliated Hospital of Hunan Normal University), No. 61, Jiefang West Road, Furong District, Changsha, Hunan, 410005, People's Republic of China.
Department of Orthopedics, The First-affiliated Hospital of Hunan Normal University (Hunan Provincial People's Hospital), Changsha, Hunan, 410005, People's Republic of China.
出版信息
Mol Med. 2023 Jul 3;29(1):86. doi: 10.1186/s10020-023-00661-2.
BACKGROUND
Osteoarthritis (OA) is a degenerative joint disease with lacking effective prevention targets. A disintegrin and metalloproteinase with thrombospondin motifs 12 (ADAMTS12) is a member of the ADAMTS family and is upregulated in OA pathologic tissues with no fully understood molecular mechanisms.
METHODS
The anterior cruciate ligament transection (ACL-T) method was used to establish rat OA models, and interleukin-1 beta (IL-1β) was administered to induce rat chondrocyte inflammation. Cartilage damage was analyzed via hematoxylin-eosin, Periodic Acid-Schiff, safranin O-fast green, Osteoarthritis Research Society International score, and micro-computed tomography assays. Chondrocyte apoptosis was detected by flow cytometry and TdT dUTP nick-end labeling. Signal transducer and activator of transcription 1 (STAT1), ADAMTS12, and methyltransferase-like 3 (METTL3) levels were detected by immunohistochemistry, quantitative polymerase chain reaction (qPCR), western blot, or immunofluorescence assay. The binding ability was confirmed by chromatin immunoprecipitation-qPCR, electromobility shift assay, dual-luciferase reporter, or RNA immunoprecipitation (RIP) assay. The methylation level of STAT1 was analyzed by MeRIP-qPCR assay. STAT1 stability was investigated by actinomycin D assay.
RESULTS
The STAT1 and ADAMTS12 expressions were significantly increased in the human and rat samples of cartilage injury, as well as in IL-1β-treated rat chondrocytes. STAT1 is bound to the promoter region of ADAMTS12 to activate its transcription. METTL3/ Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) mediated N6-methyladenosine modification of STAT1 promoted STAT1 mRNA stability, resulting in increased expression. ADAMTS12 expression was reduced and the IL-1β-induced inflammatory chondrocyte injury was attenuated by silencing METTL3. Additionally, knocking down METTL3 in ACL-T-produced OA rats reduced ADAMTS12 expression in their cartilage tissues, thereby alleviating cartilage damage.
CONCLUSION
METTL3/IGF2BP2 axis increases STAT1 stability and expression to promote OA progression by up-regulating ADAMTS12 expression.
背景
骨关节炎（OA）是一种退行性关节疾病，缺乏有效的预防靶点。解整合素金属蛋白酶 12（ADAMTS12）是 ADAMTS 家族的成员，在 OA 病理组织中上调，但分子机制尚不清楚。
方法
采用前交叉韧带切断（ACL-T）法建立大鼠 OA 模型，给予白细胞介素 1β（IL-1β）诱导大鼠软骨细胞炎症。通过苏木精-伊红、过碘酸希夫、番红 O-快绿、骨关节炎研究协会国际评分和微计算机断层扫描检测软骨损伤。通过流式细胞术和 TdT dUTP 缺口末端标记检测软骨细胞凋亡。采用免疫组织化学、定量聚合酶链反应（qPCR）、Western blot 或免疫荧光法检测信号转导和转录激活因子 1（STAT1）、ADAMTS12 和甲基转移酶样 3（METTL3）水平。通过染色质免疫沉淀-qPCR、电泳迁移率变动分析、双荧光素酶报告基因或 RNA 免疫沉淀（RIP）实验验证结合能力。通过 MeRIP-qPCR 实验分析 STAT1 的甲基化水平。通过放线菌素 D 实验研究 STAT1 的稳定性。
结果
在软骨损伤的人及大鼠样本和 IL-1β 处理的大鼠软骨细胞中，STAT1 和 ADAMTS12 的表达均显著增加。STAT1 与 ADAMTS12 启动子区域结合，激活其转录。METTL3/胰岛素样生长因子 2 mRNA 结合蛋白 2（IGF2BP2）介导 STAT1 的 N6-甲基腺苷修饰促进 STAT1 mRNA 稳定性，导致表达增加。沉默 METTL3 可降低 ADAMTS12 的表达，并减轻 IL-1β 诱导的炎症性软骨细胞损伤。此外，在 ACL-T 产生的 OA 大鼠中敲低 METTL3 可降低其软骨组织中 ADAMTS12 的表达，从而减轻软骨损伤。
结论
METTL3/IGF2BP2 轴通过增加 STAT1 稳定性和表达来促进 OA 进展，从而上调 ADAMTS12 的表达。
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