Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China; Research Center on Aging and Medicine, Fudan University, Shanghai 200032, China.
Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.
Osteoarthritis Cartilage. 2019 Jun;27(6):922-931. doi: 10.1016/j.joca.2019.01.012. Epub 2019 Feb 2.
To examine the effect of the circadian gene Clock on posttranscriptional function and pro-inflammatory mechanisms in osteoarthritis (OA).
The cartilage from Clock mutant mice was assessed using histology, (OA) score, and real-time polymerase chain reaction (PCR) quantification of key pro-inflammatory genes. Nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) translocation, posttranslational state and expression levels during day and night conditions were assessed using immunoblot and IP. The regulation of transcription by Clock in cartilage tissue was assessed by using chromatin immunoprecipitation (ChIP) and luciferase assays. Total acetylation level and pattern over 24 h were quantified using immunoblot and real-time PCR. Finally, the effects of exogenous Clock nanoparticle treatment were quantified by histology and immunoblot.
The Clock mutation significantly promoted the degradation of cartilage and the expression of the key pro-inflammatory mediators, IL-1β, IL-6 and MCP-1. The Clock mutation significantly promoted NFκB nuclear translocation. The circadian protein CLOCK positively regulates NFκB at the transcriptional level by binding the E-box domain. The Clock mutation significantly inhibited the total lysine acetylation level in cartilage and inhibited NFκB acetylation at the Lys310 residue but promoted phosphorylation at the Ser276 residue. The forced expression of Clock in vivo inhibited NFκB activation by increasing acetylation and decreasing phosphorylation levels and by decreasing cartilage damage and inflammation.
This study demonstrates the mutation of Clock promotes inflammatory activity by mediating the posttranscriptional regulation of NFκB in OA pathogenesis.
研究生物钟基因 Clock 对骨关节炎（OA）中转录后功能和促炎机制的影响。
采用组织学、OA 评分和实时聚合酶链反应（PCR）定量检测关键促炎基因，评估 Clock 基因突变小鼠的软骨情况。采用免疫印迹和免疫沉淀（IP）检测核因子κ轻链增强子的 B 细胞激活因子（NFκB）转位、昼夜条件下的翻译后状态和表达水平。采用染色质免疫沉淀（ChIP）和荧光素酶检测评估 Clock 在软骨组织中的转录调控。采用免疫印迹和实时 PCR 定量检测 24 小时内的总乙酰化水平和模式。最后，通过组织学和免疫印迹定量分析外源性 Clock 纳米颗粒处理的效果。
Clock 突变显著促进软骨降解和关键促炎介质 IL-1β、IL-6 和 MCP-1 的表达。Clock 突变显著促进 NFκB 核易位。生物钟蛋白 CLOCK 通过结合 E 盒结构域，正向调节 NFκB 的转录水平。Clock 突变显著抑制软骨中的总赖氨酸乙酰化水平，抑制 NFκB 在 Lys310 残基的乙酰化，但促进 Ser276 残基的磷酸化。Clock 的体内强制表达通过增加乙酰化和降低磷酸化水平，减少软骨损伤和炎症，抑制 NFκB 激活。
本研究表明，Clock 突变通过调节 OA 发病机制中 NFκB 的转录后调控，促进炎症活性。
