Background Osteoarthritis (OA) is the most common type of arthritis and its prevalence increases in the ageing population. A genome-wide association study (GWAS) identified an OA susceptibility locus of approximately 500 kb on chromosome 7q22. This locus contains a linkage disequilibrium block of six genes that includes G protein coupled receptor protein 22 (GPR22). GPR22 signalling inhibits protein kinase A (PKA) activity but its ligand is unknown. GPR22 shares 37% sequence homology with the Cholecystokinin B receptor (CCK-BR) and is predicted to belong to the purinergic P2Y12 family. Here we aim to further study the role of GPR22 in cartilage.
Methods We used the ATDC5 chondrogenic cell line as model system. Stably overexpressing GPR22 cells (GPR22+) were cultured in triplicate as high density micromasses (200 000 cells/10 µl). Cells were treated daily with ITS in DMEM/F12 medium for 14 days to induce chondrogenesis. Mineralization was triggered with beta-glycerol-phosphate in α-MEM medium till day 21. Different chemical compounds were added: CCK-BR antagonist AG-041R (1 and 10 µM), broad-spectrum purinoreceptor antagonist suramin (10, 33 and 100 µM) and PKA activator 8-Br-cAMP (10 and 33 µM). Gene expression of CCK-BR and chondrogenic markers, including Aggrecan (Agg), Decorin (Dco), Versican (Vcan), Collagen type 2 (Col2a1) and type 10 (Col10a1) and Matrix metalloprotease 13 (MMP13), were analysed. Sulfated proteoglycan, mineralization and collagen content were quantified by Alcian Blue, Alizarin Red, Safranin O and Picosirius Red staining.
Result Overexpression of GPR22 stimulates chondrocyte hypertrophy and accelerates calcification. CCK-BR expression was not detected in ATDC5 cells. AG-041R 1 µM increased Agg, Col2a1, Col10a1 expression at day 1 compared to unchallenged cells. Additionally, on day 21 the amount of proteoglycan and calcium deposition was increased. The use of 10 µM AG-041R and of 100 µM suramin was toxic for cells. However, 10 µM suramin treatment lead to increased decorin and versican expression. This translated into increased proteoglycan (and collagen) levels in the extracellular matrix (ECM). 8-Br-cAMP increased mRNA expression of Agg, Col2a1 and Col10a1.
Conclusion A cholecystokinin antagonist and the broad spectrum purinoreceptor antagonist slightly compensate for the effect of GPR22 overexpression on chondrogenesis. The PKA activator, 8-Br-cAMP did not change the GPR22 phenotype at the ECM level, but induced subtle transcriptomic changes.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.