The proton-activated ovarian cancer G protein-coupled receptor 1 (OGR1) is responsible for renal calcium loss during acidosis.

Hypercalciuria is a common feature during metabolic acidosis and associates to nephrolithiasis and nephrocalcinosis. The mechanisms sensing acidosis and inducing increased urinary calcium excretion are still unknown. Here we tested whether mice deficient for proton-activated Ovarian cancer G-protein coupled receptor 1 (OGR1 or Gpr68) have reduced urinary excretion of calcium during chronic metabolic acidosis. In the kidney, OGR1 mRNA was found in cells of the glomerulus, proximal tubule, and interstitium including endothelial cells. Wild type (OGR1+/+) and OGR1 knockout (OGR1-/-) mice were given standard chow without (control) or loaded with ammonium chloride for one or seven days to induce acute or chronic metabolic acidosis, respectively. No differences in responding to the acid load were observed in the knockout mice, except for higher plasma bicarbonate after one day. Bone mineral density, resorption activity of osteoclasts, and urinary deoxypyridinoline were similar between genotypes. During metabolic acidosis the expression levels of key proteins involved in calcium reabsorption, i.e. the sodium/proton exchanger (NHE3), the epithelial calcium-selective channel TRPV5, and the vitamin D-dependent calcium binding protein calbindin-D28k were all higher in the knockout mice compared to wild type mice. This is consistent with the previous demonstration that OGR1 reduces NHE3 activity in proximal tubules of mice. Wild-type mice displayed a non-linear positive association between urinary proton and calcium excretion which was lost in the knockout mice. Thus, OGR1 is a pH sensor involved in the hypercalciuria of metabolic acidosis by controlling NHE3 activity in the proximal tubule. Hence, novel drugs modulating OGR1 activity may improve renal calcium handling.

Hypoxia stimulates osteoclast formation from human peripheral blood.

Active pathological bone destruction in humans often occurs in locations where oxygen tension (pO(2)) is likely to be low, for example, at the sites of tumours, inflammation, infections and fractures, or the poorly vascularized yellow fatty marrow of the elderly. We examined the effect of pO(2) on formation of osteoclasts, the cells responsible for bone resorption, in 14-day cultures of normal human peripheral blood mononuclear cells (hPBMCs) on ivory discs. Hypoxia (1-2% O(2)) caused threefold increases in the number of osteoclasts formed, compared with 20% O(2). Hypoxia also caused a twofold increase in the number of nuclei per osteoclast, leading to stimulations of resorption pit formation of up to 10-fold. Exposure to hypoxia led to stabilization of the hypoxia-inducible factors, HIF1alpha and HIF2alpha, and upregulation of vascular endothelial growth factor and interleukin-6 expression by hPBMCs. These findings help explain why extravasation of mononuclear precursors into relatively O(2)-deficient bone microenvironments could result in osteoclast formation and suggest a new mechanism for the bone loss associated with the pathophysiological conditions where hypoxia commonly occurs.

Extracellular nucleotides block bone mineralization in vitro: evidence for dual inhibitory mechanisms involving both P2Y2 receptors and pyrophosphate.

Extracellular nucleotides, signaling through P2 receptors, may act as local regulators of bone cell function. We investigated the effects of nucleotide agonists [ATP, ADP, uridine triphosphate (UTP), and uridine diphosphate] and pyrophosphate (PPi, a key physiological inhibitor of mineralization) on the deposition and mineralization of collagenous matrix by primary osteoblasts derived from rat calvariae. Our results show that extracellular ATP, UTP, and PPi strongly and selectively blocked the mineralization of matrix nodules; ADP and uridine diphosphate were without effect. Significant inhibition of mineralization occurred in the presence of relatively low concentrations of ATP, UTP, or PPi (1-10 microm), without affecting production of fibrillar or soluble collagen. In cultures treated with 10 microm ATP or UTP, the expression and activity of alkaline phosphatase, which promotes mineralization by hydrolyzing PPi, was inhibited. The potent inhibitory actions of ATP and UTP on bone mineralization are consistent pharmacologically with mediation by the P2Y(2) receptor, which is strongly expressed by mature osteoblasts. In support of this notion, we found 9-17% increases in bone mineral content of hindlimbs of P2Y(2)-deficient mice. We also found that osteoblasts express ectonucleotide phosphodiesterase/pyrophosphatase-1, an ectonucleotidase that hydrolyzes nucleotide triphosphates to yield PPi, and that addition of 10 microm ATP or UTP to osteoblast cultures generated 2 microm PPi within 10 min. Thus, a component of the profound inhibitory action of ATP and UTP on bone mineralization could be mediated directly by PPi, independently of P2 receptors.

The regulation of osteoblast function and bone mineralisation by extracellular nucleotides: The role of p2x receptors.

Extracellular nucleotides, signalling through P2 receptors, regulate the function of both osteoblasts and osteoclasts. Osteoblasts are known to express multiple P2 receptor subtypes (P2X2,5,7 and P2Y(1),(2,4,6)), levels of which change during differentiation. ATP and UTP potently inhibit bone mineralisation in vitro, an effect mediated, at least in part, via the P2Y(2) receptor. We report here that primary rat osteoblasts express additional, functional P2 receptors (P2X1, P2X3, P2X4, P2X6, P2Y(12), P2Y(13) and P2Y(14)). Receptor expression changed with cellular differentiation: e.g., P2X4 receptor mRNA levels were 5-fold higher in mature, bone-forming osteoblasts, relative to immature, proliferating cells. The rank order of expression of P2 receptor mRNAs in mature osteoblasts was P2X4>>P2Y(1)>P2X2>P2Y(6)>P2X1>P2Y(2)>P2Y(4)>P2X6>P2X5>P2X7>P2X3>P2Y(14)>P2Y(13)>P2Y(12). Increased intracellular Ca(2+) levels following stimulation with P2X-selective agonists indicated the presence of functional receptors. To investigate whether P2X receptors might also regulate bone formation, osteoblasts were cultured for 14days with P2X receptor agonists. The P2X1 and P2X3 receptor agonists, α,ß-meATP and ß,γ-meATP inhibited bone mineralisation by 70% and 90%, respectively at 1µM, with complete abolition at ≥25µM; collagen production was unaffected. Bz-ATP, a P2X7 receptor agonist, reduced bone mineralisation by 70% and 99% at 10µM and 100µM, respectively. Osteoblast alkaline phosphatase activity was similarly inhibited by these agonists, whilst ecto-nucleotide pyrophosphatase/phosphodiesterase activity was increased. The effects of α,ß-meATP and Bz-ATP were attenuated by antagonists selective for the P2X1 and P2X7 receptors, respectively. Our results show that normal osteoblasts express functional P2X receptors and that the P2X1 and P2X7 receptors negatively regulate bone mineralisation.

The P2X7 Receptor is an Important Regulator of Extracellular ATP Levels.

Controlled ATP release has been demonstrated from many neuronal and non-neuronal cell types. Once released, extracellular ATP acts on cells in a paracrine manner via purinergic receptors. Considerable evidence now suggests that extracellular nucleotides, signaling via P2 receptors, play important roles in bone homeostasis modulating both osteoblast and osteoclast function. In this study, we demonstrate that mouse osteoclasts and their precursors constitutively release ATP into their extracellular environment. Levels were highest at day 2 (precursor cells), possibly reflecting the high number of red blood cells and accessory cells present. Mature osteoclasts constitutively released ATP in the range 0.05-0.5 pmol/ml/cell. Both osteoclasts and osteoblasts express mRNA and protein for the P2X7 receptor. We found that in osteoclasts, expression levels are fourfold higher in mature cells relative to precursors, whilst in osteoblasts expression remains relatively constant during differentiation. Selective antagonists (0.1-100 µM AZ10606120, A438079, and KN-62) were used to determine whether this release was mediated via P2X7 receptors. AZ10606120, A438079, and KN-62, at 0.1-10 µM, decreased ATP release by mature osteoclasts by up to 70, 60, and 80%, respectively. No differences in cell viability were observed. ATP release also occurs via vesicular exocytosis; inhibitors of this process (1-100 µM NEM or brefeldin A) had no effect on ATP release from osteoclasts. P2X7 receptor antagonists (0.1-10 µM) also decreased ATP release from primary rat osteoblasts by up to 80%. These data show that ATP release via the P2X7 receptor contributes to extracellular ATP levels in osteoclast and osteoblast cultures, suggesting an important additional role for this receptor in autocrine/paracrine purinergic signaling in bone.

Clopidogrel (Plavix), a P2Y12 receptor antagonist, inhibits bone cell function in vitro and decreases trabecular bone in vivo.

Clopidogrel (Plavix), a selective P2Y(12) receptor antagonist, is widely prescribed to reduce the risk of heart attack and stroke and acts via the inhibition of platelet aggregation. Accumulating evidence now suggests that extracellular nucleotides, signaling through P2 receptors, play a significant role in bone, modulating both osteoblast and osteoclast function. In this study, we investigated the effects of clopidogrel treatment on (1) bone cell formation, differentiation, and activity in vitro; and (2) trabecular and cortical bone parameters in vivo. P2Y(12) receptor expression by osteoblasts and osteoclasts was confirmed using qPCR and Western blotting. Clopidogrel at 10 µM and 25 µM inhibited mineralized bone nodule formation by 50% and >85%, respectively. Clopidogrel slowed osteoblast proliferation with dose-dependent decreases in cell number (25% to 40%) evident in differentiating osteoblasts (day 7). A single dose of 10 to 25 µM clopidogrel to mature osteoblasts also reduced cell viability. At 14 days, ≥10 µM clopidogrel decreased alkaline phosphatase (ALP) activity by ≤70% and collagen formation by 40%, while increasing adipocyte formation. In osteoclasts, ≥1 µM clopidogrel inhibited formation, viability and resorptive activity. Twenty-week-old mice (n = 10-12) were ovariectomized or sham treated and dosed orally with clopidogrel (1 mg/kg) or vehicle (NaCl) daily for 4 weeks. Dual-energy X-ray absorptiometry (DXA) analysis showed clopidogrel-treated animals had decreases of 2% and 4% in whole-body and femoral bone mineral density (BMD), respectively. Detailed analysis of trabecular and cortical bone using micro-computed tomography (microCT) showed decreased trabecular bone volume in the tibia (24%) and femur (18%) of clopidogrel-treated mice. Trabecular number was reduced 20%, while trabecular separation was increased up to 15%. Trabecular thickness and cortical bone parameters were unaffected. Combined, these findings indicate that long-term exposure of bone cells to clopidogrel in vivo could negatively impact bone health.

Normal human osteoclasts formed from peripheral blood monocytes express PTH type 1 receptors and are stimulated by PTH in the absence of osteoblasts.

The prevailing view for many years has been that osteoclasts do not express parathyroid hormone (PTH) receptors and that PTH's effects on osteoclasts are mediated indirectly via osteoblasts. However, several recent reports suggest that osteoclasts express PTH receptors. In this study, we tested the hypothesis that human osteoclasts formed in vitro express functional PTH type 1 receptors (PTH1R). Peripheral blood monocytes (PBMC) were cultured on bone slices or plastic culture dishes with human recombinant RANK ligand (RANKL) and recombinant human macrophage colony-stimulating factor (M-CSF) for 16-21 days. This resulted in a mixed population of mono- and multi-nucleated cells, all of which stained positively for the human calcitonin receptor. The cells actively resorbed bone, as assessed by release of C-terminal telopeptide of type I collagen and the formation of abundant resorption pits. We obtained evidence for the presence of PTH1R in these cells by four independent techniques. First, using immunocytochemistry, positive staining for PTH1R was observed in both mono- and multi-nucleated cells intimately associated with resorption cavities. Second, PTH1R protein expression was demonstrated by Western blot analysis. Third, the cells expressed PTH1R mRNA at 21 days and treatment with 10(-7) M hPTH (1-34) reduced PTH1R mRNA expression by 35%. Finally, bone resorption was reproducibly increased by two to threefold when PTH (1-34) was added to the cultures. These findings provide strong support for a direct stimulatory action of PTH on human osteoclasts mediated by PTH1R. This suggests a dual regulatory mechanism, whereby PTH acts both directly on osteoclasts and also, indirectly, via osteoblasts.