Extracellular acidification stimulates OGR1 to modify osteoclast differentiation and activity through the Ca2+‑calcineurin‑NFATc1 pathway.

The aim of the present study was to explore the role of ovarian cancer G protein-coupled receptor 1 (OGR1) in osteoclast differentiation and activity induced by extracellular acid. The impact of extracellular acidification on osteoclasts was investigated. Briefly, osteoclasts were generated from RAW 264.7 cells using 100 ng/ml receptor activator of nuclear factor-κB ligand in cell culture media at pH 6.8 or 7.4. Tartrate-resistant acid phosphatase (TRAP) staining and the bone resorption pit assay were used to detect the effects of extracellular acid on the number and absorptive capacity of osteoclasts. Intracellular Ca levels were analyzed using laser scanning confocal microscopy. Reverse transcription-quantitative PCR was used to detect the expression levels of genes associated with osteoclast formation and bone erosion. The role of OGR1 in the acid-stimulated formation and bone resorption of osteoclasts was also investigated. The results showed that in the pH 6.8 medium group the number of osteoclasts was 511.2±54.72 and the area of bone absorption was 4,184.88±277.14 µm; both were significantly higher than those in the pH 7.4 medium group (all P<0.01). Inhibition of OGR1 using copper ion (Cu) reduced the number of osteoclasts and the area of bone resorption in the pH 6.8 medium group (all P<0.05). Furthermore, extracellular acid (pH 6.8) was able to induce a transient increase of Ca levels in osteoclasts; however, inhibition of OGR1 using Cu effectively attenuated the acid-induced increase of Ca in osteoclasts. In addition, the elevation in Ca levels was inhibited when BAPTA, a cytoplasmic Ca chelator with cellular permeability, was added to the cells; however, the extracellular Ca-chelating agent ethylene glycol tetraacetic acid did not inhibit the acid-stimulated increase in Ca. Treatment with the phospholipase C inhibitor U73122 also inhibited the acid-stimulated increase of Ca in osteoclasts. Furthermore, the mRNA expression levels of TRAP, matrix metalloproteinase-9, osteoclast-related receptor, nuclear factor-activated T cell 1 (NFATc1), cathepsin K and integrin β3 were elevated in the pH 6.8 medium group compared with those in the pH 7.4 medium group (all P<0.05). By contrast, the inhibition of OGR1 using Cu partially reduced the effects of the acidic environment on osteoclast differentiation and activity-related gene expression (all P<0.05). In addition, the mRNA and protein expression levels of calcineurin were increased in osteoclasts in the pH 6.8 group compared with those in the pH 7.4 group (P<0.05), whereas blocking OGR1 suppressed the expression of acid-induced calcineurin. The mRNA expression levels of NFATc1 in osteoclasts were also increased in the pH 6.8 medium group compared with those in the pH 7.4 medium group (P<0.05). By contrast, the specific calcineurin inhibitor cyclosporine A significantly inhibited the acid-induced expression of NFATc1 in osteoclasts. In conclusion, the present study revealed that extracellular acidification may increase osteoclast differentiation and bone resorption activity. Furthermore, OGR1-mediated Ca elevation could have a crucial role in osteoclasts by regulating the Ca-calcineurin-NFATc1 signaling pathway and downstream signaling.