G-proteïne-gekoppelde-receptor 22 (GPR22): een nieuw doelwit in osteoartrose?

Osteoarthritis (OA) is the most prevalent joint disease and affects millions of people worldwide. OA targets single or multiple joints, occurs mostly in large joints such as knees and hips but also affects hands, feet, shoulders and the spine. The risk factors for OA are multi-factorial and include age, female gender, injury, malalignment, joint overuse and overweight in connection with a strong genetic component. OA is a socio-economic burden because of the high healthcare costs associated with the disease and its impact is predicted to further increase in the future due to ageing of the population and a global rise of obesity. In the normal joints, cartilage covers the ends of the bone providing a smooth and gliding surface for optimal joint movement. In OA, the homeostasis of the joint is challenged, leading to changes in the gene expression profile of articular chondrocytes, thereby triggering both cell death as well as reactivation of an endochondral ossification process characterized by chondrocyte hypertrophy and changes in extracellular matrix composition. This leads to progressive breakdown of cartilage, the underlying bone deforms, bone spurs are formed, an inflammatory process is induced in the surrounding synovium, the ligaments and menisci are degenerating and the joint capsule may thicken. These characteristic OA changes cause joint pain, stiffness and limited range of motion. There is no cure or effective treatment against OA. Treatments are limited to symptomatic management against joint pain, stiffness and swelling and life style changes such as maintaining a healthy weight, improving joint mobility and strengthening muscles by physical exercise. In severe cases, total replacement of the joint or corrective operations are needed to improve the quality of life. At present, the molecular mechanisms involved in OA initiation and progression are poorly understood. There is a need to elucidate these mechanisms and identify specific druggable targets. This will enable the development of disease modifying drugs, which are able to stop the disease or repair the damaged joint. A genome wide association study showed the association of OA with the chromosome 7q22 locus. In this locus six different genes are found. One of these is GPR22, a G protein coupled receptor (GPCR). GPCRs are interesting therapeutic targets as one third of the marketed drugs are targeting these types of receptors. Previously, our group showed the expression of GPR22 in mouse models of OA: GPR22 is present in damaged cartilage but absent in healthy cartilage. During this PhD project, we aimed to reveal the functional role of GPR22 in the joint and to elucidate the consequences of gain and loss of function of GPR22 with a translational perspective on OA. In addition, we tried to identify pharmacological compounds that regulate GPR22 signaling. In the first part, we analyzed the expression of GPR22 in human cartilage samples. Our data confirmed the presence of GPR22 protein in OA cartilage while it was absent in the healthy human cartilage. This observation suggests a role for GPR22 in osteoarthritis. We therefore studied the effect of GPR22 overexpression in an in vitro 3D micromass model of chondrogenesis. The in vitro data provided evidence that GPR22 overexpression shifts the chondrocyte phenotype towards hypertrophy. We investigated the contributing role of GPR22 towards cartilage damage in in vivo instability induced OA models. While gain of function Gpr22V385A/V385A mice showed increased cartilage damage in the two instability OA models, Gpr22 -/- mice had less articular cartilage degeneration compared to wild-type control mice. These data confirm a role for GPR22 in the osteoarthritic process and suggest GPR22 could be a therapeutic target. Next, we looked for possible antagonists to control GPR22 activity, taking advantage of GPCR homology structure analysis. We validated the potential modulating effect of cholecystokinin and purinoceptor antagonists on GPR22 signaling and outcome during cartilage differentiation. The cholecystokinin antagonist AG-041R showed the highest partial antagonizing effect on GPR22 signaling. The GPR22 hypertrophic shift observed in chondrogenic marker expression and matrix composition was partially antagonized by addition of AG-041R. These experiments suggest that AG-041R could be used to target GPR22. The second part of this search for antagonists assessed the effect of suramin, a broad purinergic receptor antagonist, in the ATDC5 micromass and human chondrocyte pellet in vitro models and in a translational model of cartilage damage. Suramin treatment increased the levels of anti-catabolic protein TIMP3 and thereby decreased the activity of matrix catabolic enzymes MMPs and ADAMTS, resulting in an increased proteoglycan content. The addition of anti-TIMP3 antibody could block this beneficial event of suramin. Fluorimetric analysis showed the interaction between suramin and TIMP3. Intra-articular suramin injections showed a decrease in articular proteoglycan loss compared to control mice with decreased MMP and ADAMTs activity and increased TIMP3 protein levels. These results were confirmed in human articular chondrocyte pellets from OA patients and non-OA controls. We found that suramin can possibly be repositioned to prevent post-traumatic articular cartilage damage and to block subsequent OA progression. In the third part, we investigated more specifically the link of the purinoceptor group P2Y12-14 to GPR22 as predicted by the GPCR database. Treatments with a P2-Y and -X group purinoceptor antagonist, PPADS, and the P2X purinoceptor antagonist, iso-PPADS, showed that blocking the P2Y group has the most promising antagonizing effect on GPR22. We further used specific P2Y receptor antagonists for P2Y6 and P2Y12 (MRS 2578 and PSB 0739 respectively). P2Y6 has the highest homology to the extracellular and transmembrane region of GPR22. MRS 2578 showed a higher potential to inhibit GPR22 signaling compared to the P2Y12 antagonist, PSB 0739. In summary, this work provides further insights into the relevance of GPR22 in the molecular regulation of joint biology and disease. Our obtained data suggest GPR22 could be a novel therapeutic target for the treatment of OA. Future work should focus on exploring the effect of antagonists in in vivo models and on the identification of factors triggering active GRP22 signaling.

Publication year:2018

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