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

Abstract 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.