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.

Figure 1

Number of osteoclasts cultured in different pH cell media for 3 days (sample size, n=6). (A) More TRAP-positive multinucleated osteoclasts were formed in an acidic environment, at pH 6.8. (B) In the neutral medium, there were fewer TRAP-positive multinucleated osteoclasts. (C) Inhibition of OGR1 with Cu²⁺ (CuCl₂, 100 µM) reversed the acid-induced increase in osteoclasts. (D) Inhibition of OGR1 with Cu²⁺ in neutral medium. (E) Histogram of the number of osteoclasts. ^**P<0.01 vs. pH 7.4; ^#P<0.05 vs. pH 6.8. Cu²⁺, copper ion; OGR1, ovarian cancer G protein-coupled receptor 1; TRAP, tartrate-resistant acid phosphatase.

Figure 2

Resorption pit assay (n=6). (A) Osteoclasts form more bone pits in acidic medium at pH 6.8. (B) In the neutral medium, there were fewer resorption pits. (C) Treatment with Cu²⁺ inhibited acid-stimulated osteoclast resorption. (D) Inhibition of OGR1 with Cu²⁺ in neutral medium. (E) Analysis of bone resorption area. ^**P<0.01 vs. pH 7.4; ^#P<0.05 vs. pH 6.8. Cu²⁺, copper ion; OGR1, ovarian cancer G protein-coupled receptor 1.

Figure 3

OGR1 is involved in the acid-induced increase of Ca²⁺ levels in osteoclasts (n=6). (A) Representative fluorescence change showing the elevation of Ca²⁺ levels in acidic medium at pH 6.8. (B) Chelation of extracellular calcium with EGTA (0.5 mM) did not inhibit acid-induced Ca²⁺ increase. (C) Restrain acid-induced elevation of Ca²⁺ levels with BAPTA-AM (50 mM). (D) Acid-induced elevation of Ca²⁺ levels in osteoclasts was reduced by the OGR1 inhibitor. (E) Inhibition of phospholipase C with U73122 (10 mM) suppressed the acid-induced rise of Ca²⁺.

Figure 4

Relative mRNA expression levels of genes in osteoclasts cultured in different pH cell culture media (n=5). mRNA expression levels of (A) NFATc1, (B) OSCAR, (C) integrin β3, (D) TRAP, (E) MMP-9 and (F) cathepsin K in osteoclasts after 3 days of treatment with different pH cell culture media. ^*P<0.05, ^**P<0.01 vs. pH 7.4; ^#P<0.05, ^##P<0.01 vs. pH 6.8. Cu²⁺, copper ion; MMP-9, matrix metalloproteinase-9; NFATc1, nuclear factor-activated T cell 1; OSCAR, osteoclast-related receptor; TRAP, tartrate-resistant acid phosphatase.

Figure 5

Effects of extracellular acidification on the calcineurin-NFATc1 signaling pathway of osteoclast differentiation (n=5). (A) mRNA expression levels of calcineurin following treatment with an OGR1 inhibitor. (B) Effects of an OGR1 inhibitor on the protein expression levels of calcineurin in osteoclasts. (C) mRNA expression levels of NFATc1 in osteoclasts treated with the inhibitor of calcineurin cyclosporine A. ^*P<0.05 vs. pH 7.4; ^#P<0.05 vs. pH 6.8. NFATc1, nuclear factor-activated T cell 1; OGR1, ovarian cancer G protein-coupled receptor 1.