The Clock^Δ19 mutation promotes osteoarthritis via impairing SIRT3-mediated mitochondrial homeostasis in mice.

Abstract

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage breakdown and disrupted joint homeostasis. Mitochondrial dysfunction in chondrocytes contributes to cartilage degeneration, but the mechanisms linking circadian dysregulation to mitochondrial homeostasis in OA remain incompletely understood. Clock^Δ19 mice are a well-established model of impaired CLOCK-dependent circadian transcriptional regulation and exhibit metabolic and mitochondrial abnormalities. Two Gene Expression Omnibus datasets of human OA cartilage were integrated for bioinformatics analysis. OA severity was assessed in Clock^Δ19 mice and wild-type littermates subjected to destabilization of the medial meniscus (DMM). Primary chondrocytes were treated with interleukin-1β to mimic the inflammatory microenvironment of OA. Mitochondrial membrane potential, reactive oxygen species production, and NAD⁺/NADH ratio were evaluated by JC-1 staining, DHE staining, and biochemical assay, respectively. Protein expression and relative SOD2 K68 acetylation were examined by western blotting, immunoprecipitation, and immunostaining. CLOCK binding to the Sirt3 promoter was analyzed by chromatin immunoprecipitation. The role of SIRT3 was further examined by lentiviral overexpression in Clock^Δ19 chondrocytes. Integrated analysis of public datasets revealed circadian rhythm- related transcriptional alterations in human OA cartilage. Clock^Δ19 mice developed more severe cartilage degeneration after DMM surgery than wild-type controls. In vitro and in vivo, Clock^Δ19 mutation was associated with reduced SIRT3 expression and increased relative SOD2 K68 acetylation or Ac-SOD2 K68 immunoreactivity. In Clock^Δ19 chondrocytes, these changes were accompanied by elevated oxidative stress, decreased mitochondrial membrane potential, and a reduced NAD⁺/NADH ratio under interleukin-1β stimulation. Mechanistically, CLOCK occupied the Sirt3 promoter, and Clock^Δ19 chondrocytes showed reduced Sirt3 expression. Moreover, SIRT3 overexpression alleviated mitochondrial dysfunction and oxidative stress in Clock^Δ19 chondrocytes. CLOCK contributes to mitochondrial homeostasis in chondrocytes by regulating Sirt3 transcription and modulating the SIRT3-SOD2 K68 acetylation pathway. Disruption of this regulatory pathway may represent a mechanism linking circadian dysfunction to OA progression.