Cry2 prompts clock oscillation and temporomandibular joint homeostasis under mechanical loading.

Aims: Cartilage metabolism exhibits circadian rhythms (CR), and disruption of these often correlates with cartilage degeneration. Mechanical stimulation is a vital zeitgeber of CR in cartilage. However, the effects of mechanical loading on CR and cartilage homeostasis have not been explored. In this study, we aimed to explore the involvement of CR in mediating temporomandibular joint (TMJ) cartilage homeostasis under mechanical loading. Methods: We introduced the mechanical loading models of both rat TMJ chondrocytes and condyle explants. The mechanical loading was applied through rhythmic compression (12 hrs on and 12 hrs off), while the mechanical unloading model was set as control through static culture of cells and explants. Results: One-week static culture led to imbalanced cartilage metabolism and accelerated cartilage degeneration. This was accompanied by diminished levels of core clock genes (bmal1, clock, per1, and cry2), compared with oscillations of these genes in one-day static cultured chondrocytes and condyle explants. Rhythmic physiological mechanical loading for one week not only sustained the oscillations of CR genes, but also partially maintained homeostasis of cartilage. The mechanistic delineation confirmed that one week of mechanical loading promoted the nuclear-cytoplasmic shuttling of Per1 and Cry2, which were obvious in one-day static cultured chondrocytes but weakened in one-week static cultured cells. Furthermore, nuclear translocation of Cry2, but not Per1, was dependent upon mechanical loading-induced Rho-associated protein kinase (ROCK) activation and actin polymerization. Inhibition of ROCK caused actin depolymerization and partially blocked Cry2 nuclear-cytoplasmic trafficking. Finally, down-regulation of Cry2 in both chondrocytes and condyle explants attenuated mechanical loading-sustained circadian oscillations and cartilage homeostasis. Conclusion: This study elucidated the involvement of CR in mediating mechanical loading-related cartilage homeostasis, as well as the critical role of ROCK-actin-Cry2 in regulating normal CR and homeostasis of TMJ cartilage under rhythmic mechanical loading.