NMDA receptor function in osteoarthritic chondrocytes is dependent on β1 integrins

Ramage, L.; Hardingham, G.E.; Salter, D.M.

Abstract

Purpose: Osteoarthritis (OA) is associated with abnormal loading of articular joints. In normal joints physiological loading maintains structure and function of cartilage and chondrocytes within. This is mediated by a process known as mechanotransduction whereby the mechanical forces are recognized by the resident chondrocytes and transduced into biochemical and molecular responses. In chondrocytes a5b1 integrin is a major mechanoreceptor and activation triggers signaling events and autocrine/paracrine signaling via IL4 which lead to up-regulation of anabolic genes and down-regulation of catabolic activity. In OA however this mechanism is disrupted and mechanically stimulate chondrocytes express pro-inflammatory and catabolic IL1b. The reasons for this are unclear but recent research from our group has raised the possibility that differential involvement of ionotropic glutamate receptors, NMDARs in the mechanotransduction pathway may be important. NMDARs are associated with neurons, although non-neuronal tissues have been shown to express these receptors. In neurons, NMDAR interact with intracellular signaling proteins through the scaffold protein PSD-95. As potential roles for integrins in modifying NMDAR activity have also been shown we have now investigated the relationship between NMDAR signaling in chondrocytes and molecules that are involved in chondrocyte mechanotransduction.

Methods: Articular cartilage was removed from OA joints and chondrocytes released using sequential enzymatic digestion. Chondrocytes were maintained as non-confluent primary monolayer cultures at a concentration of 5×105 cells/ml. To measure the function of NMDAR cell membrane potentials of OA cells were measured by electrophysiology in resting cells, after treatment (integrin modifying or CD47 antibody (1 mg/ml)) and following stimulation with NMDA (50 mM). Chondrocytes seeded onto cell culture dishes coated with substrates (poly-L-lysine or fibronectin; 10 mg/ml) were used to assess cell membrane potential before and after NMDA treatment. Co-immunoprecipitation of PSD-95 with b1 integrin and CD47 using specific antibodies was analysed by Western blot.

Results: Incubation of OA chondroctytes with anti-b1 integrin and antiCD47 (integrin associated protein) function blocking antibodies inhibited the electrophysiologcal response to NMDA; antibodies to b3, b5 and anti-a integrin antibodies had no effect. Integrin dependency of NMDAR signaling with NMDA treatment was analysed; Chondrocytes adherent to poly-L-lysine (integrin independent attachment) showed no electrophysiological response to NMDA while cells adherent to fibronectin and type II collagen (b1 integrin dependent attachment) showed a membrane depolarization. Co-immunopreciptiation experiments of PSD-95 with b1 integrin and CD47 identified that these molecules are physically linked and therefore may be involved in regulating NMDAR function.

Conclusions: We identify that NMDAR function in chondrocytes is linked to b1 integrin and CD47, and that the electrophysiological response to NMDA requires integrin dependent cell-matrix interactions. Linkage of b1 integrins and CD47 to PSD-95, possibly as part of a complex that may contain NMDAR, appears to be important in NMDAR activity in chondrocytes. In neurons studies have shown integrins can be involved in regulating the expression and function of NMDAR, although the mechanisms by which this occurs have yet to be defined. The role of NMDAR in OA chondrocyte/cartilage function is yet to be fully understood, but parallel studies indicate that these receptors may participate in the signal cascade that results in anabolic/catabolic responses to applied mechanical forces

• Presentation Conference Type: Conference Paper (published)
• Acceptance Date: Mar 1, 2008
• Publication Date: 2008-09
• Deposit Date: Mar 25, 2017
• Publicly Available Date: Mar 28, 2017
• Journal: Osteoarthritis and Cartilage
• Print ISSN: 1063-4584
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 16
• Issue: S4
• Pages: S82
• DOI: https://doi.org/10.1016/s1063-4584%2808%2960207-1
• Keywords: Rheumatology; Orthopedics and Sports Medicine; Biomedical Engineering
• Public URL: http://researchrepository.napier.ac.uk/Output/819797
• Contract Date: Mar 25, 2017