Clock mutant promotes osteoarthritis by inhibiting the acetylation of NFκB（Clock突变体和骨关节炎研究）

G Yuan  ¹ , L Xu  ² , T Cai  ² , B Hua  ³ , N Sun  ⁴ , Z Yan  ³ , C Lu  ⁵ , R Qian  ⁶

• ¹ 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.
• ² Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.
• ³ Department of Orthopedics, Zhongshan Hospital, Fudan University, Shanghai 200032, China.
• ⁴ 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; State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai 200032, China.
• ⁵ 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; Shanghai Key Laboratory of Clinical Geriatric Medicine, Shanghai, China. Electronic address: luchao@shmu.edu.cn.
• ⁶ 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. Electronic address: rzqian@shmu.edu.cn.

Abstract

Objectives: To examine the effect of the circadian gene Clock on posttranscriptional function and pro-inflammatory mechanisms in osteoarthritis (OA).

Methods: 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.

Results: 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.

Conclusions: This study demonstrates the mutation of Clock promotes inflammatory activity by mediating the posttranscriptional regulation of NFκB in OA pathogenesis.

Keywords: Acetylation; CLOCK; NFκB; Osteoarthritis.