Purpose A genome-wide association study (GWAS) identified an association between osteoarthritis and a gene cluster on chromosome 7 in which we proposed a role for G protein coupled receptor (GPR)22. This orphan receptor was found in OA cartilage while not in healthy cartilage. Activated GPR22 signals through the G inhibitory pathway resulting in inhibition of adenylcyclase and consequently decreases protein kinase A (PKA) activity. The cholecystokinin (CCK) receptors A and B are paralogues of this orphan receptor.
Methods The ATDC5 chondrogenic cell line was used as model system. ATDC5 cells stably overexpressing Gpr22 were cultured as high density micromasses (200,000 cells/10 µl) in triplicate. The chemical compound AG-041R (at 1, 3 or 10 µM) was used as a potential antagonist. Gene expression of Ccka and Cckb, of chondrogenic markers was analysed in both undifferentiated and differentiated ATDC5 cells. Sulfated proteoglycan, mineralization and collagen content were quantified. PKA activity was determined with a fluorescence-based assay. Real-time PKA concentrations and kinetics were measured by fluorescence resonance energy transfer (FRET) imaging by expressing FRET-based PKA sensors in ATDC5 cells.
Results ATDC5 cells stably overexpressing Gpr22 showed a 30% increased in mineralization and a 50% decrease in proteoglycan content after 21 days of differentiation in the micromass model. Gene expression studies demonstrated a decrease in Agg and Cola1 mRNA at day 1, 7, 14 and 21. Col10a1 and Mmp13 expression were increased suggesting an earlier and enhanced shift of the cells towards hypertrophy. PKA activity was 40% reduced in the Gpr22 overexpressing cells. The effect of Gpr22 overexpression in the differentiating cells within the micromass was antagonised by AG-041R treatment. FRET imaging confirmed the neutralising effect of AG-041R on intracellular PKA concentration in Gpr22 overexpressing cells using a forskolin titration model.
Conclusions GPR22, an orphan G-coupled protein receptor has been linked to osteoarthritis. Overexpression of this receptor reduces PKA activity in differentiating chondrocytes and shifts the differentiation program towards hypertrophy, a deleterious outcome in the context of osteoarthritis. The effects of GPR22 can be antagonised by cholecystokinin antagonist AG-041R.