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

Abstract

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 Ca²⁺ 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 µm²; both were significantly higher than those in the pH 7.4 medium group (all P<0.01). Inhibition of OGR1 using copper ion (Cu²⁺) 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 Ca²⁺ levels in osteoclasts; however, inhibition of OGR1 using Cu²⁺ effectively attenuated the acid-induced increase of Ca²⁺ in osteoclasts. In addition, the elevation in Ca²⁺ levels was inhibited when BAPTA, a cytoplasmic Ca²⁺ chelator with cellular permeability, was added to the cells; however, the extracellular Ca²⁺-chelating agent ethylene glycol tetraacetic acid did not inhibit the acid-stimulated increase in Ca²⁺. Treatment with the phospholipase C inhibitor U73122 also inhibited the acid-stimulated increase of Ca²⁺ 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 Cu²⁺ 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 Ca²⁺ elevation could have a crucial role in osteoclasts by regulating the Ca²⁺-calcineurin-NFATc1 signaling pathway and downstream signaling.

Keywords: acidosis; osteoclast; ovarian cancer G protein-coupled receptor 1; proton sensing receptor.