The expression patterns of clock-controlled genes (ccgs) are regulated by circadian rhythm, which is a major regulatory and physiological mechanism tied to the solar day. Disruptions in circadian rhythm contribute to the development of cardiovascular diseases, cancer, metabolic syndromes, and aging. It has been reported that bone remodeling is also regulated by circadian rhythm. However, the molecular mechanism by which the circadian gene Clock regulates bone remodeling has yet to be elucidated. Here, we show that Clock mutant mice exhibit a significant reduction in bone density as well as increased apoptosis. Protein disulfide isomerase family A member 3 (PDIA3) is a 1,25-dihydroxy-vitamin D3 [1α,25(OH)2D3] receptor that can regulate bone formation and apoptosis. Using luciferase and ChIP assays, we confirmed that Pdia3 is a ccg. Clock activates Pdia3 transcription by binding the E-box promoter, and transcription is decreased in Clock mutant mice. Forced expression of Pdia3 or of Clock completely rescues the osteogenic disorders found in the mutant background and inhibits apoptosis in vivo and in vitro. Furthermore, ablation of PDIA3 via RNA interference completely blocks the compensatory effect of forced expression of Clock in osteoblasts. Our results demonstrate that the core circadian gene Clock regulates bone formation via transcriptional control of 1,2,5(OH)2D3 receptor PDIA3. © 2016 American Society for Bone and Mineral Research.

Objectives: To examine the effect of the circadian gene Clock on posttranscriptional function and proinflammatory 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 kappalight-chain-enhancer of activated B cells (NFkB) 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-1b, IL-6 and MCP-1. The Clock mutation significantly promoted NFkB nuclear translocation. The circadian protein CLOCK positively regulates NFkB at the transcriptional level by binding the E-box domain. The Clock mutation significantly inhibited the total lysine acetylation level in cartilage and inhibited NFkB acetylation at the Lys310 residue but promoted phosphorylation at the Ser276 residue. The forced expression of Clock in vivo inhibited NFkB 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 NFkB in OA pathogenesis.