The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling

Abstract

The adenosine diphosphate (ADP) receptor P2RY12 (purinergic receptor P2Y, G protein coupled, 12) plays a critical role in platelet aggregation, and P2RY12 inhibitors are used clinically to prevent cardiac and cerebral thrombotic events. Extracellular ADP has also been shown to increase osteoclast (OC) activity, but the role of P2RY12 in OC biology is unknown. Here, we examined the role of mouse P2RY12 in OC function. Mice lacking P2ry12 had decreased OC activity and were partially protected from age-associated bone loss. P2ry12 -/-OCs exhibited intact differentiation markers, but diminished resorptive function. Extracellular ADP enhanced OC adhesion and resorptive activity of WT, but not P2ry12 -/-, OCs. In platelets, ADP stimulation of P2RY12 resulted in GTPase Ras-related protein (RAP1) activation and subsequent α IIb β 3 integrin activation. Likewise, we found that ADP stimulation induced RAP1 activation in WT and integrin β 3 gene knockout (Itgb3 -/-) OCs, but its effects were substantially blunted in P2ry12 -/-OCs. In vivo, P2ry12 -/-mice were partially protected from pathologic bone loss associated with serum transfer arthritis, tumor growth in bone, and ovariectomy-induced osteoporosis: all conditions associated with increased extracellular ADP. Finally, mice treated with the clinical inhibitor of P2RY12, clopidogrel, were protected from pathologic osteolysis. These results demonstrate that P2RY12 is the primary ADP receptor in OCs and suggest that P2RY12 inhibition is a potential therapeutic target for pathologic bone loss. IntroductionOsteoclasts (OCs) are multinucleated myeloid lineage cells that are the principal source of bone resorptive activity (1). Enhanced OC activity, bone loss, and fractures are associated with rheumatoid arthritis, postmenopausal osteoporosis, and bone metastases (2). Modulation of osteoclastic bone resorption represents an attractive point of therapeutic intervention for the treatment of such conditions.Numerous purinergic G-protein-coupled nucleotide receptors are expressed in the bone microenvironment (3, 4). For example, uridine diphosphate-activated (UDP-activated) P2Y6 has been reported to increase NF-κB activation and OC survival (5), while P2Y2 (an ATP receptor) expression on osteoblasts (OBs) blocks bone mineralization (6, 7). Hoebertz et al. demonstrated that extracellular adenosine diphosphate (ADP) stimulates OC bone resorption in vitro, in part through the ADP receptor P2Y1 on OC (8); however, other ADP receptors, including purinergic receptor P2Y, G protein coupled, 12 (P2RY12), which is the target of the widely prescribed antiplatelet drug clopidogrel (Plavix), have not been evaluated for their roles in osteoclastic bone resorption.

Purinergic signalling in the musculoskeletal system

“…This decrease in bone mass was unexpected given the previously suggested role for the P2Y 1 receptor in osteoclast activity. However, a recent study found that ADP still stimulated osteoclast formation and resorption in cells derived from P2Y 1 receptor knockout mice [120], suggesting the involvement of another P2Y receptor. Based on pharmacology, the logical alternative candidate for mediating the effects of ADP on osteoclasts was the P2Y 12 receptor.…”

“…Analysis of P2Y 1 , P2Y 2 , P2Y 6 , P2Y 12 , P2Y 13 and P2X7 knockout mice has provided evidence for the important role of purinergic signalling in bone remodelling in vivo [115][116][117][118][119][120][121][122]. Previous reviews concerned with purinergic signalling on bone have focussed on different aspects, including bone marrow cells [123]; bisphosphonates [124][125][126] Widespread expression of P2X receptors on osteoblasts has also been reported.…”

“…Reduced trabecular bone was also observed in ovariectomised mice treated with clopidogrel for 4 weeks [168]. In contrast, a paper by Su et al [120] demonstrated increased bone in mice treated with clopidogrel. However, the two investigations used very different dosing regimes (oral dosing compared to clopidogreltreated water), and thus, the divergent results may reflect variations in the final dose received by the test subjects.…”

“…Furthermore, expression of the P2Y 12 receptor by osteoclasts has only recently been reported [119]. There is now evidence to suggest that the P2Y 12 receptor plays a key role in mediating the effects of ADP on osteoclast formation and activity since cells derived from P2Y 12 knockout mice display impaired responses to ADP [120]. Consistent with effects mediating osteoclast activity, P2Y 12 receptor knockout mice have increased trabecular bone and decreased arthritis associated bone loss [120].…”

“…There is now evidence to suggest that the P2Y 12 receptor plays a key role in mediating the effects of ADP on osteoclast formation and activity since cells derived from P2Y 12 knockout mice display impaired responses to ADP [120]. Consistent with effects mediating osteoclast activity, P2Y 12 receptor knockout mice have increased trabecular bone and decreased arthritis associated bone loss [120]. Furthermore, clopidogrel, a P2Y 12 receptor antagonist, inhibits osteoclast formation, viability and resorptive activity [168].…”