The Effect of GluN2B-containing NMDA Receptor Inhibition on Osteoarthritis Phenotypic Markers and Clock Gene Regulation

Abstract

Osteoarthritic (OA) chondrocytes express elevated levels of matrix-degrading enzymes. A disrupted chondrocyte clock (specifically increased expression of clock component Per2 and reduced expression of Bmal1) has been shown to have a role in this enzyme dysregulation. A clock-regulating receptor, N-methyl-D-aspartate receptors (NMDAR), is expressed in chondrocytes and the subunit composition is altered in osteoarthritis. The GluN2B NMDAR subunit is detected only in osteoarthritic chondrocytes. This study aimed to investigate the effect of GluN2B-selective inhibition on osteoarthritis phenotypic markers and clock gene regulation. The H5 mouse chondrocyte cell line, as well as primary human chondrocytes from macroscopically normal and osteoarthritic cartilage, were used in this study. NMDA receptor activity was assessed based on Ca2+-influx measured using Fura-2 assay. Two isomers of the GluN2B-selective inhibitor, ifenprodil, and their benzyl derivatives were used as the inhibitors. Western blot was performed to elucidate signalling pathway activity upon NMDA receptor activation. Expression of clock genes and OA phenotypic markers were assessed by real time RT-qPCR. Benzyl ifenprodil significantly inhibited Ca2+ influx in H5 cells transfected to express GluN2B and OA chondrocytes. However, this effect was also seen in empty vectortransfected cells (albeit with a lower magnitude) and normal chondrocytes, suggesting offtarget mechanisms. Level of the downstream signalling molecule, phosphorylated CREB (PCREB), were lower upon treatment of OA chondrocytes with benzyl erythro ifenprodil but not benzyl threo ifenprodil. P-CREB is known to activate transcription of Per2 and Mmp13, two genes upregulated in osteoarthritis. However, expression of Per2 was not affected whereas a possible trend towards increased Bmal1 expression and reduced Mmp13 expression was observed with benzyl erythro ifenprodil treatment in OA chondrocytes compared to DMSO controls. Although used at a low concentration (0.05%), addition of the drug solvent DMSO resulted in increased P-CREB, decreased Per2 and Bmal1 expression and a possible trend towards a reduction in Mmp13 expression, potentially confounding the assessment of the effects of the drugs. Together, this study shows that selective inhibition of GluN2B-containing NMDAR may affect transcription of clock genes and OA phenotypic marker however, further study is required to confirm this.