Bone Abstracts

Extracellular nucleotides, signalling through P2 receptors, play a significant role in bone biology. ATP and ADP act via the P2Y₁ or P2Y₁₂ receptors to promote osteoclast formation and activity. Bone cells express the P2Y₂ receptor and, in osteoblasts, it plays a role in regulating bone mineralisation. This investigation examined the role of the P2Y₂ receptor in osteoclasts. Primary osteoclasts were isolated from the bone marrow of 8-week WT or P2Y₂ receptor knockout mice (P2Y₂R^−/−) and cultured on dentine discs for 9 days in the presence of 150 ng/ml M-CSF and 2 ng/ml RANKL. UTP (≧0.1 μM), the principal P2Y₂ receptor agonist, increased bone resorption by 40% (P≤0.05). P2Y₂R^-/- osteoclasts displayed a 65% reduction in resorptive activity (P≤0.001); osteoclast number was unchanged. Osteoclasts constitutively release ATP into the extracellular environment where it acts locally to stimulate resorption. We found that ATP release was reduced 60% in P2Y₂R^−/− osteoclasts (P≤0.001). To investigate whether decreased levels of extracellular ATP were causing the reduction in osteoclast function, P2Y₂R^−/− cells were cultured with exogenous ATP (1–10 μM). Addition of ATP to the culture medium fully rescued the bone resorption defect in P2Y₂R^-/- osteoclasts. We found that UTP, (≧10 μM), stimulated ATP release from osteoclasts by up to fourfold (P≤0.001). The enhanced ATP release was evident from 10 min after UTP treatment and was sustained for up to 90 min. The stimulatory action of UTP on ATP release was mediated via the P2Y₂ receptor, since UTP failed to induce ATP release in P2Y₂R^−/− osteoclasts. MicroCT analysis of P2Y₂R^−/− mice demonstrated an increased trabecular bone volume (20%, P<0.001) and trabecular number (25%, P<0.01) in the long bones. Taken together, these data suggest the P2Y₂ receptor regulates osteoclast function indirectly by promoting ATP release. Once released, ATP and its breakdown product, ADP, can act via other P2Y receptors to increase bone resorption.