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.

Inflammation has important roles in tissue regeneration, autoimmunity, and cancer. Different inflammatory stimuli can lead to bone loss by mechanisms that are not well understood. We show that skin inflammation induces bone loss in mice and humans. In psoriasis, one of the prototypic IL-17A-mediated inflammatory human skin diseases, low bone formation and bone loss correlated with increased serum IL-17A levels. Similarly, in two mouse models with chronic IL-17A-mediated skin inflammation,K14-IL17A(ind)andJunB(Δep), strong inhibition of bone formation was observed, different from classical inflammatory bone loss where osteoclast activation leads to bone degradation. We show that under inflammatory conditions, skin-resident cells such as keratinocytes, γδ T cells, and innate lymphoid cells were able to express IL-17A, which acted systemically to inhibit osteoblast and osteocyte function by a mechanism involving Wnt signaling. IL-17A led to decreased Wnt signaling in vitro, and importantly, pharmacological blockade of IL-17A rescued Wnt target gene expression and bone formation in vivo. These data provide a mechanism where IL-17A affects bone formation by regulating Wnt signaling in osteoblasts and osteocytes. This study suggests that using IL-17A blocking agents in psoriasis could be beneficial against bone loss in these patients.

Inhibition of osteoclast (OC) activity has been associated with decreased tumor growth in bone in animal models. Increased recognition of factors that promote osteoclastic bone resorption in cancer patients led us to investigate whether increased OC activation could enhance tumor growth in bone. Granulocyte colony-stimulating factor (G-CSF) is used to treat chemotherapy-induced neutropenia, but is also associated with increased markers of OC activity and decreased bone mineral density (BMD). We used G-CSF as a tool to investigate the impact of increased OC activity on tumor growth in 2 murine osteolytic tumor models. An 8-day course of G-CSF alone (without chemotherapy) significantly decreased BMD and increased OC perimeter along bone in mice. Mice administered G-CSF alone demonstrated significantly increased tumor growth in bone as quantitated by in vivo bioluminescence imaging and histologic bone marrow tumor analysis. Short-term administration of AMD3100, a CXCR4 inhibitor that mobilizes neutrophils with little effect on bone resorption, did not lead to increased tumor burden. However, OC-defective osteoprotegerin transgenic (OPG Tg ) mice and bisphosphonatetreated mice were resistant to the effects of G-CSF administration upon bone tumor growth. These data demonstrate a G-CSF-induced stimulation of tumor growth in bone that is OC dependent.

osteoclasts, also rescued the osteoclast defect in CD47 À/À cells.We then examined the consequences of this osteoclast defect in bone metastasis. In a model of tumor metastasis to bone, bone tumor burden was decreased in the CD47 À/À mice compared with wild-type (WT) controls, with no decrease in s.c. tumor growth in CD47 À/À mice. There was decreased tumor-associated bone destruction in the CD47 À/À mice compared with WT controls, consistent with a defect in osteoclast function that was not rescued by the presence of tumor. Our data show that CD47 regulates osteoclastogenesis, in part, via regulation of NO production, and its disruption leads to a decrease in tumor bone metastasis. CD47 is a novel therapeutic target to strengthen bone mass and diminish metastatic tumor growth in bone.

The purpose of this work was to determine platelet and myeloid cell-specific requirements for beta3-containing integrins in hemostasis, bone resorption, and tumor growth. LoxP-flanked mice were generated to study the conditional deletion of beta3-integrin in platelets [knockout in platelets (KOP)] and myeloid cells [knockout in myeloid (KOM)]. Using the beta3KOP and beta3KOM strains of mice, we studied the role of beta3-integrin in hemostasis, bone resorption, and subcutaneous tumor growth. Tissue-specific deletion of platelet beta3-integrins in beta3KOP mice did not affect bone mass but resulted in a severe bleeding phenotype. No growth difference of tumor xenografts or in neoangiogenesis were found in beta3KOP mice, in contrast to the defects observed in germline beta3(-/-) mice. Conditional deletion of myeloid beta3-integrins in beta3KOM mice resulted in osteopetrosis but had no effect on hemostasis or mortality. Tumor growth in beta3KOM mice was increased and accompanied by decreased macrophage infiltration, without increase in blood vessel number. Platelet beta3-integrin deficiency was sufficient to disrupt hemostasis but had no effect on bone mass or tumor growth. Myeloid-specific beta3-integrin deletion was sufficient to perturb bone mass and enhance tumor growth due to reduced macrophage infiltration in the tumors. These results suggest that beta3-integrins have cell-specific roles in complex biological processes.-Morgan, E. A., Schneider, J. G., Baroni, T. E., Uluçkan, O., Heller, E., Hurchla, M. A., Deng, H., Floyd, D., Berdy, A., Prior, J. L., Piwnica-Worms, D., Teitelbaum, S. L., Ross, F. P., Weilbaecher, K. N. Dissection of platelet and myeloid cell defects by conditional targeting of the beta3-integrin subunit.