Clock mutant promotes osteoarthritis by inhibiting the acetylation of NFκB
Clock mutant promotes osteoarthritis by inhibiting the acetylation of NFκB
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时钟突变体通过抑制 NFκB 乙酰化促进骨关节炎
DOI：
10.1016/j.joca.2019.01.012
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发表时间：
2019
影响因子：
7
通讯作者：
Ruizhe Qian
中科院分区：
文献类型：
--
作者：
Gongsheng Yuan;Lun Xu;Tingting Cai;Bingxuan Hua;Ning Sun;Zuoqin Yan;Chao Lu;Ruizhe Qian
为研究Clock基因对骨关节炎（OA）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Clock基因突变小鼠软骨中关键促炎基因，并对Clock基因突变小鼠软骨进行组织学和促炎机制研究。采用免疫印迹法和IP法检测活化B细胞核因子κ轻链增强子（NFκB）的转位、翻译后状态和表达水平。通过使用染色质免疫沉淀（ChIP）和荧光素酶测定来评估软骨组织中Clock对转录的调节。使用免疫印迹和实时PCR定量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外源性Clock纳米颗粒治疗的效果，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 B核转位。昼夜节律蛋白CLOCK通过结合E-box结构域在转录水平上正调控NFκB。Clock突变显著抑制软骨中总赖氨酸乙酰化水平，抑制NFκB B在Lys 310残基的乙酰化，但促进Ser 276残基的磷酸化。Clock基因在体内的强制表达可通过增加乙酰化和降低磷酸化水平抑制NFκB的活化，从而减轻软骨损伤和炎症反应，本研究表明Clock基因的突变通过介导NFκB的转录后调节而促进炎症反应。
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.