IL-1ß induces changes in expression of core circadian clock components PER2 and BMAL1 in primary human chondrocytes through the NMDA receptor/CREB and NF-κB signalling pathways.

The circadian clock is a specialised cell signalling circuit present in almost all cells. It controls the timing of key cell activities such as proliferation and differentiation. In osteoarthritis, expression of two components of the circadian clock, BMAL1 and PER2 is altered in chondrocytes and this change has been causally linked with the increase in proliferation and altered chondrocyte differentiation in disease. IL-1ß, an inflammatory cytokine abundant in OA joints, has previously been shown to induce changes in BMAL1 and PER2 expression in chondrocytes. The purpose of this study is to identify the mechanism involved. We found IL-1ß treatment of primary human chondrocytes led to activation of NMDA receptors as evidenced by an increase in phosphorylation of GluN1 and an increase in intracellular calcium which was blocked by the NMDAR antagonist MK801. Levels of phosphorylated CREB were also elevated in IL-1ß treated cells and this effect was blocked by co-treatment of cells with IL-1ß and the NMDAR antagonist MK-801. Knockdown of CREB or inhibition of CREB activity prevented the IL-1ß induced increase in PER2 expression in chondrocytes but had no effect on BMAL1. Phosphorylated p65 levels were elevated in IL-1ß treated chondrocytes indicating increased NF-κB activation. Inhibition of NF-κB activity prevented the IL-1ß induced reduction in BMAL1 expression and partially mitigated the IL-1ß induced increase in PER2 expression in chondrocytes. These data indicate that the NMDAR/CREB and NF-κB signalling pathways regulate the core circadian clock components PER2 and BMAL1 in chondrocytes. Given that changes in expression of these clock components have been observed in a wide range of diseases, these findings may be broadly relevant for understanding the mechanism leading to circadian clock changes in pathology.

Altered N-methyl D-aspartate receptor subunit expression causes changes to the circadian clock and cell phenotype in osteoarthritic chondrocytes.

The chondrocyte circadian clock is altered in osteoarthritis. This change is implicated in the disease-associated changes in chondrocyte phenotype and cartilage loss. Why the clock is changed is unknown. N-methyl-D-aspartate receptors (NMDAR) are critical for regulating the hypothalamic clock. Chondrocytes also express NMDAR and the type of NMDAR subunits expressed changes in osteoarthritis. OBJECTIVE: To determine if NMDAR regulate the chondrocyte clock and phenotype. DESIGN: Chondrocytes isolated from macroscopically-normal (MN) and osteoarthritic human cartilage were treated with NMDAR antagonists or transfected with GRIN2A or GRIN2B-targetting siRNA. H5 chondrocytes were transfected with GluN2B-expression plasmids. Clock genes and chondrocyte phenotypic markers were measured by RT-qPCR. RESULTS: PER2 amplitude was higher and BMAL1 amplitude lower in osteoarthritic compared to MN chondrocytes. In osteoarthritic chondrocytes, NMDAR inhibition restored PER2 and BMAL1 expression to levels similar to MN chondrocytes, and resulted in reduced MMP13 and COL10A1. Paradoxically, NMDAR inhibition in MN chondrocytes resulted in increased PER2, decreased BMAL1 and increased MMP13 and COL10A1. Osteoarthritic, but not MN chondrocytes expressed GluN2B NMDAR subunits. GluN2B knockdown in osteoarthritic chondrocytes restored expression of circadian clock components and phenotypic markers to levels similar to MN chondrocytes. Ectopic expression of GluN2B resulted in reduced BMAL1, increased PER2 and altered SOX9, RUNX2 and MMP13 expression. Knockdown of PER2 mitigated the effects of GluN2B on SOX9 and MMP13. CONCLUSIONS: NMDAR regulate the chondrocyte clock and phenotype suggesting NMDAR may also regulate clocks in other peripheral tissues. GluN2B expression in osteoarthritis may contribute to pathology by altering the chondrocyte clock.