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.

Nonsurgical treatment options, such as hormonal therapy, chemotherapy, radiation, and bisphosphonate therapy, are undoubtedly improving outcomes for women with breast cancer; however, these therapies also carry significant skeletal side effects. For example, adjuvant hormonal treatments, such as aromatase inhibitors that disrupt the estrogen-skeleton axis, have the potential to cause decreased bone mineral density. Similarly, chemotherapy often induces primary ovarian failure in premenopausal women, resulting in decreased levels of circulating estrogen and subsequent osteopenia. In both cases, women receiving these therapies are at an increased risk for the development of osteoporosis and skeletal fracture. Furthermore, women undergoing radiation therapy to the upper body may have an increased incidence of rib fracture, and those receiving bisphosphonates may be vulnerable to the development of osteonecrosis of the jaw. Therefore, women with breast cancer who are undergoing any of these therapies should be closely monitored for bone mineral loss and advised of skeletal health maintenance strategies. Hormonal TherapyIn adults, the skeleton undergoes complete turnover every 10 years. Bone mass maintenance is a balance between the activity of osteoblasts, which form bone, and osteoclasts, which resorb it. Estrogen plays a key regulatory role in this cycle of bone remodeling by mediating effects through the estrogen receptor (ER) present on several cell types in the bone. Estrogen stimulates osteoblasts to produce osteoprotegerin, a decoy receptor for the receptor of activated nuclear factor-nB (1). Osteoprotegerin blocks the binding of receptor of activated nuclear factor-nB ligand to receptor of activated nuclear factornB on osteoclasts, leading to impaired osteoclast activity and decreased bone resorption. Additionally, estrogen is believed to directly induce apoptosis of bone-resorbing osteoclasts (2, 3). Thus, in premenopausal women, estrogen both inhibits bone remodeling and suppresses bone resorption, contributing to bone strength (Fig. 1). As estrogen levels decline in postmenopausal women, this regulation diminishes and bone resorption increases out of proportion to bone formation, leading to a net loss in bone and weakened bony microarchitecture. Despite the persistence of low levels of circulating estrogen in the postmenopausal state (produced by the conversion of peripheral tissue androgens to estrogen by the aromatase enzyme), bone mass can decrease by as much as 3% yearly in the first 5 years after menopause (4).The ER is expressed by 70% of breast tumors (5), and circulating estrogen can promote the growth of ER-positive tumors. Current breast cancer therapies exploit this relationship either by decreasing circulating estrogen levels or by blocking or down-regulating the receptor itself. Although some of the estrogen-mimicking agents seem to be bone sparing, others that disrupt the estrogen-skeleton axis cause adverse effects on bone remodeling, leading to decreased bone mineral density (BMD)...

CXCR4 regulates hematopoietic and tumor cell homing to bone, but its role during osteoclast (OC) development is unknown. We investigated the role of CXCR4 in osteoclastogenesis and in a model of bone metastasis. Compared with controls, mice reconstituted with CXCR4 null hematopoietic cells exhibited elevated markers of bone resorption, increased OC perimeter along bone, and increased bone loss. CXCR4؊/؊ OCs demonstrated accelerated differentiation and enhanced bone resorption in vitro. Furthermore, tumor growth specifically in bone was significantly increased in mice reconstituted with CXCR4؊/؊ hematopoietic cells. Finally, enhancement of bone tumor growth in the absence of CXCR4 was abrogated with the OC inhibitor, zoledronic acid. These data demonstrate that disruption of CXCR4 enhances osteoclastogenesis and suggest that inhibition of CXCR4 may enhance established skeletal tumor burden by increasing OC activity.bone metastasis ͉ osteoclast B one metastases are a significant cause of morbidity, causing pain, pathologic fractures, spinal cord compression, and hypercalcemia (1). Tumor cells that reach the bone marrow (BM) can stimulate one or both types of cells involved in bone remodeling, namely osteoblasts (stromal cell origin) or osteoclasts (OCs) (hematopoietic cell lineage), leading to new bone formation and osteoblastic lesions or bone loss and osteolytic lesions, respectively. OC resorption releases bone matrix-derived growth factors that can enhance local tumor growth (1-3). Disruption of OC resorption can decrease tumor growth in bone (3), and we have recently shown that pharmacologic enhancement of OC activity with granulocyte-colony stimulating factor increases tumor growth in bone in mice (4).CXCR4 is a seven-transmembrane G protein-coupled receptor expressed by many cell types, including hematopoietic, endothelial, stromal, and neuronal cells (5). Stromal cell-derived factor-1 (SDF-1) (or CXCL12) is the only known ligand for CXCR4 and is produced by multiple BM cell types, including stromal cells, osteoblasts, and OCs (6-8). CXCR4/SDF-1 are critical molecules in the process of hematopoietic stem cell (HSC) homing to and egress from the BM (9, 10). Gene-targeted disruptions of CXCR4 and SDF-1 are embryonic lethal, but examination of late-stage embryos reveal diminished HSC homing to the fetal BM (11-13). Additionally, inhibitors of either SDF-1 or CXCR4 have been shown to impair BM engraftment in transplant models (5). Small molecule inhibitors of CXCR4 mobilize HSCs and progenitor cells from the BM into the peripheral blood. Clinical trials are underway using CXCR4 inhibitors to mobilize and collect HSCs/hematopoietic progenitor cells from the peripheral blood in preparation for stem cell transplantation for malignant BM diseases (14).Signaling through CXCR4 has been shown to have both stimulatory and inhibitory roles on various hematopoietic cells, including promoting survival and proliferation of HSCs (5), inducing apoptosis in T cells (15), or causing quiescence in neural progenitors (16). The s...

Background Vaccination is an efficient strategy to control the COVID-19 pandemic. In north Cyprus, vaccine distribution started with CoronaVac followed by BNT162b2, and ChAdOx1 vaccines. An option to obtain a third booster dose with BNT162b2 or CoronaVac was later offered to people fully inoculated with CoronaVac. There are few simultaneous and comparative real-world antibody data for these three vaccines as well as boosters after CoronaVac vaccination. Our study was aimed at evaluating antibody responses after these vaccination schemes. Methods We did a prospective, longitudinal population-based study to measure SARS-CoV-2 anti-spike receptor binding domain (RBD) IgG concentrations, assessed by assaying blood samples collected, in participants in north Cyprus who had received the BNT162b2, ChAdOx1, or CoronaVac vaccine at 1 month and 3 months after the second dose. Participants were recruited when they voluntarily came to the laboratory for testing after vaccination, solicited from health-care access points, or from the general population. We also evaluated antibody responses 1 month after a booster dose of BNT162b2 or CoronaVac after primary CoronaVac regimen. Demographics, baseline characteristics, vaccination reactions, and percentage of antibody responders were collected by phone interviews or directly from the laboratory summarised by vaccine and age group. Antibody levels were compared between groups over time by parametric and non-parametric methods. Findings Recruitment, follow-up, and data collection was done between March 1 and Sept 30, 2021. BNT162b2 induced the highest seropositivity and anti-spike RBD IgG antibody titres, followed by ChAdOx1, and then by CoronaVac. In addition, the rate of decline of antibodies was fastest with CoronaVac, followed by ChAdOx1, and then by BNT162b2. For the older age group, the rate of seropositivity at 3 months after the second dose was 100% for BNT162b2, 90% for ChAdOx1, and 60% for CoronaVac. In the multivariate repeated measures model, lower antibody titres were also significantly associated with male sex, older age, and time since vaccination. Boosting a two-dose CoronaVac regimen at 6 months with a single BNT162b2 dose led to significantly increased titres of IgG compared with boosting with CoronaVac; for the 60 years and older age group, the geometric mean fold rise in antibody titre after the booster relative to 1 month post-baseline was 7·9 (95% CI 5·8–10·8) in the BNT162b2 boost group versus 2·8 (1·6–5·0) in the CoronaVac group. Interpretation These longitudinal data can help shape vaccination strategies. Given the low antibody titres and fast decline in the CoronaVac group in individuals 60 years or older, more potent vaccine options could be considered as the primary vaccination or booster dose in these high-risk populations to sustain antibody responses for longer. Funding Crowdfunded in north Cyprus.