The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling
Xinming Su, Desiree H. Floyd, Alun Hughes, Jingyu Xiang, Jochen G. Schneider, Ozge Uluckan, Emanuela Heller, Hongju Deng, Wei Zou, Clarissa S. Craft, Kaiming Wu, Angela C. Hirbe, Dorota Grabowska, Mark C. Eagleton, Sarah Townsley, Lynne Collins, David Piwnica-Worms, Thomas H. Steinberg, Deborah V. Novack, Pamela B. Conley, Michelle A. Hurchla, Michael Rogers, Katherine N. Weilbaecher
Xinming Su, Desiree H. Floyd, Alun Hughes, Jingyu Xiang, Jochen G. Schneider, Ozge Uluckan, Emanuela Heller, Hongju Deng, Wei Zou, Clarissa S. Craft, Kaiming Wu, Angela C. Hirbe, Dorota Grabowska, Mark C. Eagleton, Sarah Townsley, Lynne Collins, David Piwnica-Worms, Thomas H. Steinberg, Deborah V. Novack, Pamela B. Conley, Michelle A. Hurchla, Michael Rogers, Katherine N. Weilbaecher
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Research Article Bone biology

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 αIIbβ3 integrin activation. Likewise, we found that ADP stimulation induced RAP1 activation in WT and integrin β3 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.

Authors

Xinming Su, Desiree H. Floyd, Alun Hughes, Jingyu Xiang, Jochen G. Schneider, Ozge Uluckan, Emanuela Heller, Hongju Deng, Wei Zou, Clarissa S. Craft, Kaiming Wu, Angela C. Hirbe, Dorota Grabowska, Mark C. Eagleton, Sarah Townsley, Lynne Collins, David Piwnica-Worms, Thomas H. Steinberg, Deborah V. Novack, Pamela B. Conley, Michelle A. Hurchla, Michael Rogers, Katherine N. Weilbaecher

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Figure 2

ADP enhances ex vivo OC bone resorption primarily through the P2RY12 receptor.

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ADP enhances ex vivo OC bone resorption primarily through the P2RY12 rec...
(A) Quantitative real-time PCR of P2ry12 expression on day 0 through day 5 of OC differentiation. (B) WT and P2ry12–/– OCs stained for the OC marker protein TRAP on days 3–5 of OC differentiation. Scale bar: 300 μm. (C–E) Quantitative real-time RT-PCR of OC differentiation markers; nuclear factor of activated T cells, cytoplasmic 1α (Nfatc1α), TRAP, and cathepsin K (Ctsk), normalized to levels of gapdh, comparing WT (black bars) and P2ry12–/– (white bars) cells on days 0–5 of OC culture. (F) WT and P2ry12–/– macrophages were cultured with 50 ng/ml M-CSF and 50 ng/ml RANKL and treated with or without 1 μM ADP for 3 days. Day 3 OC culture was stained for the OC marker protein TRAP. Scale bar: 300 μm. (G) Quantitation of OC number (≥ 3 nuclei). (H) BMMs derived from 8-month-old WT or P2ry12–/– mice were cultured with RANKL and MCSF on bone slices for 5 days. ADP was added at a final concentration of 1 μM where indicated. Scale bar: 100 μm. (I and J) Quantification of bone pit area. Data represent mean ± SD. n = 3 per condition. *P < 0.05; **P < 0.01.