Background/Purpose:

Circadian clocks are key regulators of cell behaviour. Disruption to cell clocks is implicated in a range of diseases. Expression of two core clock components, PER2 and BMAL1, is altered in chondrocytes in osteoarthritis (OA). Both in vitro and in vivo studies indicate that this altered expression contributes to OA-associated changes in chondrocyte activity and cartilage loss. Why the chondrocyte clock is altered in OA is unclear. N-methyl-D-aspartate receptors (NMDAR) are critical for regulating the more well studied light-sensitive clock present in the hypothalamus. Chondrocytes also express NMDAR and the type of NMDAR expressed changes in OA. The purpose of this study was to determine if NMDAR regulate the chondrocyte clock and if the change in NMDAR expression in OA chondrocytes is responsible for disease-associated changes in the circadian clock and cell phenotype.

Methods: Chondrocytes were isolated from macroscopically-normal (MN) and osteoarthritic cartilage from patients with OA. Cells were treated with NMDAR antagonists or transfected with siRNA targeting NMDAR subunits. The effect on clock gene expression, and chondrocyte phenotypic markers, was assessed by RT-qPCR and ELISA. Effects of GluN2B expression were determined by ectopic expression in the H5 chondrocyte cell line.

Results:

In OA chondrocytes, NMDAR inhibition restored PER2 and BMAL1 expression to levels similar to MN chondrocytes and resulted in reduced RNA and protein levels of MMP13 (the major enzyme implicated in the cartilage loss in OA) and COLX (type X collagen, a marker of the OA chondrocyte phenotype). Paradoxically, NMDAR inhibition in MN chondrocytes resulted in a similar reduction in BMAL1 and increase in PER2, BMAL1, MMP13 and COLX expression as seen in OA. OA chondrocytes expressed both GluN2A and GluN2B NMDAR subunits whereas MN chondrocytes only expressed GluN2A. Knockdown of GluN2A in MN chondrocytes caused OA-associated changes in PER2, BMAL1 and chondrocyte phenotypic marker expression. In OA chondrocytes, GluN2A knockdown further exacerbated disease-associated increases in PER2, MMP13 and COLX. Conversely, inhibition or knockdown of GluN2B in OA chondrocytes restored expression of PER2, BMAL1 and phenotypic markers to levels similar to MN chondrocytes. Overexpression of GluN2B in the H5 chondrocyte cell line resulted in a similar reduction in BMAL1 and increase in PER2 expression as seen in OA and led to OA-associated changes in phenotypic markers. Knockdown of PER2 mitigated the effects of GluN2B overexpression on chondrocyte phenotype. Notably, it completely ablated the GluN2B-induced increase in MMP13 expression. This indicates that GluN2B causes disease-associated changes in chondrocyte phenotype in part, by altering expression of circadian clock components.

Conclusion:

NMDAR regulate the chondrocyte clock. Different NMDAR subtypes have different effects on the clock. The change in NMDAR sub-type expression in chondrocytes in OA, (from GluN2A only to GluN2A and GluN2B), may contribute to disease pathogenesis by promoting OA-associated changes in chondrocyte phenotype by altering expression of core clock components.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/aberrant-expression-of-the-glun2b-n-methyl-d-aspartate-receptor-subunit-in-osteoarthritic-chondrocytes-causes-disease-associated-changes-in-chondrocyte-phenotype-through-altered-expression-of-core-com/