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(The FASEB Journal. 2001;15:1139-1148.)
© 2001 FASEB

Extracellular ADP is a powerful osteolytic agent: evidence for signaling through the P2Y1 receptor on bone cells

ASTRID HOEBERTZ*,{ddagger}, SAJEDA MEGHJI{dagger}, GEOFFREY BURNSTOCK*,{ddagger} and TIMOTHY R. ARNETT*1

* Department of Anatomy and Developmental Biology,
{dagger} Cellular Microbiology Research Group, Eastman Dental Institute, and
{ddagger} Autonomic Neuroscience Institute, University College London, London WC1E 6BT, U.K.

1Correspondence: Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, U.K. E-mail: t.arnett{at}ucl.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There is increasing evidence that extracellular nucleotides act on bone cells via P2 receptors. This study investigated the action of ADP and 2-methylthioADP, a potent ADP analog with selectivity for the P2Y1 receptor, on osteoclasts, the bone-resorbing multinuclear cells. Using three different assays, we show that ADP and 2-methylthioADP at nanomolar to submicromolar levels caused up to fourfold to sixfold increases in osteoclastic bone resorption. On mature rat osteoclasts, cultured for 1 day on polished dentine disks, peak effects on resorption pit formation were observed between 20 nM and 2 µM of ADP. The same concentrations of ADP also stimulated osteoclast and resorption pit formation in 10-day mouse marrow cultures on dentine disks. In 3-day explant cultures of mouse calvarial bones, the stimulatory effect of ADP on osteoclast-mediated Ca2+ release was greatest at 5–50 µM and equivalent to the maximal effects of prostaglandin E2. The ADP effects were blocked in a nontoxic manner by MRS 2179, a P2Y1 receptor antagonist. Using in situ hybridization and immunocytochemistry, we found evidence for P2Y1 receptor expression on both osteoclasts and osteoblasts; thus, ADP could exert its actions both directly on osteoclasts and indirectly via P2Y1 receptors on osteoblasts. As a major ATP degradation product, ADP is a novel stimulator of bone resorption that could help mediate inflammatory bone loss in vivo.—Hoebertz, A., Meghji, S., Burnstock, G., Arnett, T. Extracellular ADP is a powerful osteolytic agent: evidence for signaling through the P2Y1 receptor on bone cells.


Key Words: P2 receptors • ADP • ATP • osteoclast • bone resorption • P2Y1


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BONE IS A dynamic tissue, being continuously remodeled by the coordinated actions of osteoclasts and of cells in the osteoblast lineage. Osteoclasts, cells responsible for bone resorption, are polarized multinuclear cells, derived from hematopoietic precursors of the monocyte-macrophage series. Osteoblasts, the bone-forming cells, originate from mesenchymal stem cells; osteocytes, cells thought to mediate mechanical responsiveness of bone, differentiate from osteoblasts to form a network of cells within bone matrix. In bone loss disorders, the normal remodeling process becomes unbalanced, which can result in excessive osteoclastic bone resorption and fragile bones. The complex mechanisms by which systemic and local factors influence osteoclastic formation and activation, key steps in the bone remodeling sequence, are still not well understood.

ATP and other extracellular nucleotides are now recognized as important messenger molecules for cell-cell communication (1) . It has recently become evident that extracellular nucleotides, signaling through P2 receptors, could play an important role in bone remodeling (2) . Receptors for nucleotides and nucleosides were originally divided into two groups: P1 receptors for which adenosine and AMP are major agonists, and P2 receptors for adenosine 5'-diphosphate (ADP), ATP, and uridine 5'-triphosphate (UTP). The P2 receptors are further classified into two main families: the ionotropic P2X receptors are a family of ligand-gated nonselective cation channels; in contrast, the metabotropic P2Y receptors are coupled to G-proteins, which activates signal transduction pathways involving inositol 1,4,5-trisphosphate-dependent mobilization of intracellular Ca2+. Nucleotides can be released into the extracellular fluid in a number of ways. ATP, the nucleotide with the widest spectrum of biological activity and present intracellularly at ~2–5 mM, could be released as a result of cell damage, via synaptic vesicles from nerve cells, by active secretion via "ATP binding cassette" (ABC) transport proteins and sulfonylurea receptors, or by release from activated platelets and leukocytes at a site of tissue injury and inflammation.

We have previously shown that ATP is capable of stimulating osteoclasts to perform their pathophysiological function, namely the formation of resorption pits (3) . In addition, we recently reported, using in situ hybridization and immunocytochemistry, that bone cells express several P2 receptors, of both the P2X and the P2Y families (4) . This result was consistent with earlier findings that both osteoclasts and osteoblasts respond to extracellular nucleotides with an increase in intracellular Ca2+ (5 6 7 8 9) and, in the case of osteoclasts, also with a decrease in intracellular pH (10) .

Few studies to date have related the presence of P2 receptors to specific functions of bone cells. The aim of this study was to investigate the effects of a wider range of P2 receptor agonists and antagonists on bone resorption and to determine which receptors were involved in mediating any effects. We show for the first time that extracellular ADP, the first degradation product of ATP, is a powerful stimulator of bone resorption and acts at nanomolar to submicromolar concentrations, as assessed by three independent methods in two different species. We also provide evidence that this stimulation of osteoclastic function is probably mediated via the P2Y1 receptor. This is the first study that links a specific P2 receptor to a key functional action of an extracellular nucleotide on bone and could point to a fundamental new mechanism in the local modulation of bone resorption.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Culture media and buffers were purchased from Life Technologies (Paisley, U.K.). Reagents for in situ hybridization experiments were purchased from Boehringer (Mannheim, Germany). All other reagents were purchased from Sigma (Poole, U.K.) unless stated otherwise. Stock solutions of nucleotides were prepared in PBS and stored at -80°C. Untreated elephant ivory was kindly provided by HM Customs and Excise (London Heathrow Airport).

Resorption pit formation assay
The effects of extracellular nucleotides on resorption pit formation by mature rat osteoclasts were studied by using modifications of an assay described previously (11) . All experiments were performed with minimum essential medium supplemented with Earle’s salts, 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin B (complete mixture abbreviated MEM). In most experiments, MEM was acidified by the direct addition of small amounts of concentrated hydrochloric acid (10 mEq/l H+, equivalent to 85 µl of 11.5 M HCl per 100 ml of medium). This has the effect of reducing HCO3- concentration and producing an operating pH close to 6.95 in a 5% CO2 environment, which is optimal for resorption pit formation (12) . Elephant ivory (dentine) was prepared by cutting 250-µm-thick transverse wafers using a low-speed diamond saw (Buehler, Coventry, U.K.); 5-mm-diameter disks were cut from wet dentine wafers by using a standard paper punch, washed extensively by sonication in distilled water, and stored dry at room temperature. Before use, dentine disks were sterilized by brief immersion in ethanol, after which they were allowed to dry and were then rinsed in sterile PBS.

Mixed cell populations containing osteoclasts were obtained by mincing rapidly the pooled long bones of 2-day-old Sprague-Dawley rat pups, killed by cervical dislocation (n = 5), in 5 ml MEM, followed by vortexing for 30 s. The resulting cell suspension was allowed to sediment for 45 min onto 5-mm dentine disks, prewetted with 50 µl of MEM, in 96-well plates (100 µl of cell suspension/disk). Disks were rinsed twice in PBS before transfer to the pre-equilibrated test culture media in a 6-well plate. Each test or control well contained 5 ml of acidified MEM and five replicate dentine disks; cultures were incubated for 26 h in a humidified atmosphere of 5% CO2/95% air. At the end of the experiment, medium pH and PCO2 were measured using a blood gas analyzer (Radiometer, Copenhagen, Denmark), with careful precautions to prevent CO2 loss. Dentine disks were removed and fixed in 2% glutaraldehyde and then were stained for tartrate-resistant acid phosphatase (TRAP), a cytochemical marker for mature osteoclasts (Sigma Kit 387-A). The numbers of TRAP-positive multinucleated osteoclasts (three or more nuclei) and the number of stromal cells were assessed "blind," by using transmitted light microscopy. Discrete resorption pits were counted blind by scanning the entire surface of each disk with a reflected light microscope after restaining in 1% toluidine blue in 1% sodium borate for 2 min.

Mouse calvarial bone resorption assay
The method, which measures bone resorption as Ca2+ release from neonatal mouse calvariae, was similar to that described in detail by Meghji et al. (13) . Briefly, 5-day-old MF1 mice were killed by cervical dislocation. The frontoparietal bones were removed and trimmed of any adhering connective tissue and interparietal bone, with care taken not to damage the periosteum. Dissected calvariae were pooled, washed free of blood and adherent brain tissue in HBSS, and then divided along the sagittal suture. Half-calvariae were cultured individually on 1-cm2 stainless steel grids (Minimesh, FDP quality, Expanded Metal, West Hartlepool, U.K.) in 6-well plates with 1.5 ml of BGJb medium, 5% heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, at the air-liquid interface in a humidified CO2 incubator. After an initial 24-h preincubation period, the medium was removed and replaced with control or test media. Prostaglandin E2 (PGE2) and indomethacin were dissolved in ethanol vehicle for use; the final concentration of ethanol in cultures did not exceed 1 part in 500. Each experimental group consisted of five individual cultures. The cultures were then incubated for 72 h without further medium changes, and without opening the incubator door, so as to ensure constant CO2 levels and minimize pH fluctuations. Culture medium acidification was achieved by adding small amounts of concentrated HCl to culture medium, as described before (11 , 12) , resulting in decreased HCO3- concentration (metabolic acidosis).

After 72 h, experiments were terminated by withdrawing culture medium and washing the bones once with PBS, followed by fixation in 95% ethanol/5% glacial acetic acid for 10 min. Incubator PCO2 was determined by immediately measuring a culture medium sample with a blood gas analyzer. The mean final pH of each treatment group was determined by removing and pooling a 100-µl sample from each replicate; the pooled samples were then re-equilibrated with CO2 in the incubator before measurement using the blood gas analyzer; slight differences in CO2 tension between groups were normalized to the initially measured value using pH-PCO2 calibration curves constructed for BGJb medium, as described previously (12) .

Ca2+ concentrations in culture medium at the end of experiments were measured colorimetrically via an autoanalyzer (Chem Lab Instruments, Essex, U.K.) using the procedure described earlier (13) . The basal Ca2+ concentration of the BGJb medium after addition of 5% heat inactivated FCS was 2.00 mM. All measurements were performed blind on coded samples.

After fixation and decalcification with 95% ethanol/5% glacial acetic acid, calvariae were stained for TRAP and mounted whole in melted glycerol jelly for transmitted light microscopy.

Osteoclast formation assays
Long bones of 8-wk-old MF1 mice (n = 2), killed by cervical dislocation, were fragmented in 5 ml of unmodified MEM, followed by vortexing for 1 min. The resulting cell suspension was allowed to sediment for 2 h onto sterile 5-mm-diameter dentine disks, prewetted with 50 µl of MEM, in 96-well plates (100 µl of cell suspension/disk). Dentine disks were then removed and placed in test or control medium in a 6-well plate. Each test or control well contained 5 ml of nonacidified MEM with 10 nM 1,25-dihydroxyvitamin D3, 10 nM dexamethasone, 20 ng/ml human recombinant macrophage colony-stimulating factor (M-CSF), 1 ng/ml RANKL (receptor activator of NF-{kappa}B ligand, also called OPGL, a kind gift of Dr. Colin Dunstan, Amgen, Thousand Oaks, CA), 100 nM PGE2, and six replicate dentine disks. Cultures were incubated for 10 days in a humidified atmosphere of 5% CO2/95% air, with medium changes every 2–3 days. For the first 7 days, NaOH was added to a running pH of 7.4, which has been shown to be required for optimal osteoclast formation (14) ; for the last 3 days, MEM was acidified by addition of HCl to ensure resorptive activity (14) . Medium pH and PCO2 were monitored during and at the end of experiments via a blood gas analyzer. After 10 days of incubation, the disks were fixed in 2% glutaraldehyde and were stained for TRAP (Sigma Kit 387-A). A control group of dentine disks was also removed, fixed, and stained after 3 days of incubation to check for the presence of any mature osteoclasts that might have been released during the initial cell preparation. The total number of TRAP-positive multinucleated osteoclasts and of discrete resorption pits was assessed blind by transmitted and reflected light microscopy.

As an alternative procedure, bone marrow cells were isolated from 8-wk-old MF1 mice, using a modification of a method described previously (15) . The marrow cavity of the long bones was flushed into a dish by slowly injecting MEM at one end of the bone using a sterile 25-gauge needle. The resulting suspension was washed twice and resuspended and incubated overnight in a 75-cm2 flask at a density of 3 x 106 cells/ml MEM containing M-CSF (5 ng/ml). After 24 h, nonadherent cells were harvested, washed, and resuspended (106/ml) in MEM containing M-CSF (30 ng/ml) and RANKL (10 ng/ml). This suspension was added to the wells of either 96-well plates containing dentine disks (100 µl) or 48-well plates (800 µl). After a 24-h preincubation period, dentine disks were transferred to 6-well plates (six replicates/well) and test media were added. Cultures were fed every 3 days by replacing half the medium with fresh medium and reagents. The functional absence of contaminating stromal cells was confirmed in cultures in which M-CSF was omitted; such cultures showed no cell growth. After 10 days of treatment, 48-well plates and dentine disks were fixed and assessed for TRAP or bone resorption as described above.

In situ hybridization and immunocytochemistry
Neonatal (2-day-old) Sprague-Dawley rats were killed by cervical dislocation. Long bones were removed immediately, frozen rapidly by immersion in isopentane at -70°C, and stored in liquid nitrogen. Cryostat sections of undecalcified, unfixed bone (10 µm) were prepared and collected on polysine-coated slides (BDH/Merck, Poole, Dorset, U.K.) for in situ hybridization. Tissues were kept frozen until used and were air-dried at room temperature prior to use.

Mixed cell suspensions containing osteoclasts, obtained from the long bones of 2-day-old neonatal Sprague-Dawley rats as described above, were allowed to sediment for 60 min onto LabTek 8-chamber slides. Chambers were rinsed twice with PBS before incubation with MEM for 4 h in a humidified atmosphere of 5% CO2/95% air. Cultures were fixed in 4% paraformaldehyde in 0.1 M PBS and processed for in situ hybridization or immunocytochemistry. Primary rat osteoblastic cells were obtained by sequential enzyme digestion of excised calvarial bones from 2-day-old rats using a three-step process as described previously (4). Cells were cultured in LabTek 8-chamber slides until confluence (up to 4 days) and fixed and processed as described above.

For immunofluorescent staining of the osteoclasts, fixed cells were treated with methanol at -20°C for 7 min. The cells were preincubated in 10% normal horse serum (NHS) and 0.1% Triton X-100 in PBS for 30 min at room temperature, followed by overnight incubation at 4°C with the affinity-purified anti-P2Y1 antibody (a kind donation of Carlos Matute, Leioa, Spain) (16) at 2 µg/ml in the same solution. Biotinylated donkey anti-rabbit immunoglobulin G (Jackson ImmunoResearch Lab, West Grove, PA), diluted 1:500 in 1% NHS in PBS and 0.1% Triton X-100, was applied for 1 h, followed by fluoresceinated streptavidin (Amersham, Bucks, U.K.) diluted 1:200 in PBS for 1 h at room temperature. Control experiments were carried out with antiserum that had been preadsorbed overnight at 4°C with the immunogenic peptide (15 µM = 31 µg/ml) (16) .

For in situ hybridization experiments, an antisense oligonucleotide (45 mer) directed against P2Y1 receptor subtype-specific sequence was designed. This sequence corresponds to the third extracellular domain of the rat P2Y1 subtype. The oligonucleotide sequence is as follows:

5'-AGGTGGCATAAACCCTGTCGTTGAAATCACACATTTCTGGGGTCT-3'

The above primer was labeled at the 3'-end with digoxigenin dUTP by using an oligonucleotide tailing kit. Digoxigenin, a naturally occurring plant steroid, is not found in animal tissues, so cytoplasmic localization of the immunoproduct is considered to be specific.

After fixation in 4% formaldehyde in PBS for 10 min, slides with cryostat sections or cultured and fixed cells were dehydrated in graded ethanol and air-dried. The hybridization buffer contained 2x SSC buffer, 0.1 mg/ml sheared and denatured salmon sperm DNA, 0.1 mg/ml tRNA, 50% deionized formamide, 1x Denhardt’s solution, and 1 ng/µl digoxigenin-labeled probe. Before hybridization, prehybridization was done at 37°C for 2 h in a humidified chamber without the digoxigenin-labeled probe. The slides were then incubated at the same temperature for 16 h with the digoxigenin-labeled probe.

After washing with decreasing salt solutions (twice with 2x SSC for 5 min at room temperature, twice with 2x SSC for 15 min at 37°C, twice with 1x SSC for 15 min at 37°C, and twice with 0.5x SSC for 30 min at 37°C), slides were blocked in 2% normal sheep serum in wash buffer (0.1 M Tris-HCl, 0.15 M NaCl; pH 7.4) for 2 h at room temperature. They were then incubated with anti-digoxigenin antibody (diluted 1:1000 in 2% normal sheep serum in wash buffer) conjugated with alkaline phosphatase for 2 h. The color reaction was made with 45 µl of 4-nitroblue tetrazolium salt, 35 µl of 5-bromo-4-chloro-3-indolyl-phosphate solution in 10 ml of detection buffer (0.1 M Tris-HCl, 0.1 M NaCl, 0.05 M MgCl2; pH 9.5) in the dark for up to 16 h. Negative controls were performed by hybridizing in the presence of 100-fold excess of unlabeled probe and by hybridizing without adding the labeled probe.

Statistics
Statistical comparisons were made by one-way analysis of variance or the Mann-Whitney test; representative data are presented as means ± SE for five or six replicates. Results are presented for representative experiments that were each repeated at least three times.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of ADP on mature rat osteoclasts
Extracellular ADP exerted a reproducible, biphasic effect on resorption pit formation by rat osteoclasts in acid-activated 26-h cultures. Figure 1 shows typical biphasic concentration results for the effects of ADP: in this experiment, high stimulatory effects were evident in the low nanomolar range (20–200 nM), with up to twofold increases in pit formation (Fig. 1) . In other experiments, ADP increased resorption pit formation up to threefold (Fig. 2 ). At higher ADP concentrations of 20–200 µM, there was no stimulatory effect on resorption. Numbers of osteoclasts and of mononuclear cells (i.e., cells of osteoblastic/fibroblastic morphology) were unaltered by ADP treatment.



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  		Figure 1. Effect of ADP on resorption pit formation by rat osteoclasts. Osteoclasts were cultured on 5-mm dentine disks in acidified medium (pH ~6.9) for 26 h. Large stimulatory effects were evident in the low nanomolar range (20–200 nM), at which ADP caused twofold increases in pit formation. Numbers of mononuclear cells and osteoclasts were unaltered by ADP treatment: treatment with 0, 0.002, 0.02, 0.2, 2, 20, or 200 µM ADP resulted in 2785 ± 198, 2726 ± 159, 2564 ± 241, 2491 ± 181, 2257 ± 281, 2642 ± 289, and 2782 ± 439 mononuclear cells and 60.4 ± 4.1, 35.4 ± 6.1, 39.6 ± 3.6, 37.8 ± 3.8, 43.6 ± 2.2, 46.4 ± 4.8, and 41.6 ± 6.7 osteoclasts/disk, respectively (all nonsignificant). Values are means ± SE (n = 5). Significantly different from control: *P < 0.05; **P < 0.01.



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  		Figure 2. Comparison of the effects of ADP on resorption pit formation by rat osteoclasts cultured in unmodified medium (pH ~7.1) or in acidified medium (pH ~6.8) for 26 h. The figure shows potentiation of ADP-stimulated resorption at low pH. The number of osteoclasts/disk was 42 ± 7.6, 57 ± 7.3, 77 ± 10, and 69.3 ± 8.4 and the number of mononuclear cells/disk was 3291 ± 210, 3473 ± 200, 3491 ± 393, and 3346 ± 303 for each treatment group, respectively (all nonsignificant). Values are means ± SE (n = 5). Significantly different from acidified control: **P < 0.01.

The stimulatory effect of ADP on rat osteoclast resorption pit formation was observable clearly only when culture medium (MEM) was acidified to a running pH of <=7.0 by addition of H+ as HCl (Fig. 2) . In the absence of ADP, acidification (pH reduction from 7.08 to 6.82) elicited a fourfold increase in resorption; in nonacidified MEM (pH 7.11), ADP caused a modest twofold stimulation. However, culturing osteoclasts in acidified MEM with addition of 1 µM ADP resulted in a 3-fold increase in the number of pits formed per osteoclast compared with an acidified control and a 13-fold increase compared with a nonacidified control, suggesting a synergy between the stimulatory effects of acidification and ADP. Similar acidification dependency was observed with all active P2 receptor agonists tested, and thus all further experiments were conducted at low pH.

2-MethylthioADP (2-MeSADP), a highly selective P2Y1 receptor agonist, was able to mimic the ADP effect (Fig. 3 ). At 200 nM, the effect of 2-MeSADP was somewhat greater than that of ADP, but the difference was not statistically significant. However, no stimulatory effect was observed with 1 µM 2-MeSADP, suggesting that this analog stimulates resorption effectively within a very narrow concentration range. Further degradation products of ADP, namely AMP and adenosine, had no significant effect on bone resorption, thus indicating that ADP itself is the signaling agent (Fig. 4 ).



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  		Figure 3. Effect of the selective P2Y1 agonist 2-MeSADP on resorption pit formation by rat osteoclasts. Osteoclasts were cultured on 5-mm dentine disks in acidified medium (pH ~6.9) for 26 h. 2-MeSADP mimicked the ADP effect with a peak effect at 0.2 µM, increasing resorption pit formation up to fourfold. The number of osteoclasts/disk was 12.2 ± 1.2, 13.8 ± 1.6, 15.2 ± 3.4, 13.8 ± 1.7, 17.2 ± 2.1, 13.2 ± 1.8, and 17.5 ± 4.5 and the number of mononuclear cells/disk was 3264 ± 89, 3724 ± 227, 3531 ± 221, 3759 ± 44, 2836 ± 271, 3339 ± 58, and 2494 ± 504 for each treatment group, respectively (all nonsignificant). Values are means ± SE (n = 5). Significantly different from control: *P < 0.05; **P < 0.01.



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  		Figure 4. Effects of further degradation products of ADP, namely, AMP and adenosine, on resorption pit formation by rat osteoclasts. Osteoclasts were cultured on 5-mm dentine disks in acidified medium (pH ~6.9) for 26 h. Adenosine and AMP had no effect on bone resorption compared with ADP at 0.2 µM, indicating that ADP itself is the signaling agent. The number of osteoclasts/disk was 43.6 ± 8.9, 53.6 ± 4.7, 48.8 ± 6.7, 37.2 ± 5.3, 38.6 ± 3.7, and 28.4 ± 3.2 and the number of mononuclear cells/disk was 3599 ± 290, 3994 ± 323, 2869 ± 311, 3455 ± 59, 3759 ± 168, 3372 ± 333, and 4192 ± 400 for each treatment group, respectively (all nonsignificant). Values are means ± SE (n = 5). Significantly different from control: **P < 0.01.

ADP is the major agonist at the P2Y1 receptor, but it is also a less potent agonist at the P2X1 receptor. We therefore investigated the action of P2X1 agonists, {alpha},ß-methylene-ATP ({alpha},ß-MeATP, EC50 1.5 µM) and ß,{gamma}-methylene-ATP (ß,{gamma}-MeATP, EC50 2 µM) (17) on bone resorption (Fig. 5 ). Neither {alpha},ß-MeATP nor ß,{gamma}-MeATP at concentrations of 0.5 and 5 µM had a significant stimulatory effect on bone resorption, thus suggesting a lack of involvement of the P2X1 receptor.



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  		Figure 5. Agonists for the P2X1 receptor subtype ({alpha},ß-MeATP and ß,{gamma}-MeATP) were unable to mimic the stimulatory effect of ADP on resorption pit formation, thus excluding the involvement of the P2X1 receptor. Osteoclasts were cultured on 5-mm dentine disks in acidified medium (pH ~6.9) for 26 h. The number of osteoclasts/disk was 24.4 ± 4.2, 19.5 ± 1.3, 19.8 ± 3.6, 26.8 ± 1.2, 23.8 ± 2.2, 27.2 ± 2.7, and 19.6 ± 2.5 and the number of mononuclear cells/disk was 3042 ± 212, 2846 ± 304, 3135 ± 145, 2568 ± 313, 2361 ± 397, 2212 ± 298, and 2514 ± 158 for each treatment group, respectively (all nonsignificant). Values are means ± SE (n = 5). Significantly different from control: *P < 0.05; **P < 0.01.

To further study the involvement of the P2Y1 receptor, we tested the compound MRS 2179 (N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate; a kind gift from Dr. K. A. Jacobson, National Institutes of Health, Bethesda, MD), the most potent P2Y1 receptor antagonist reported to date (18 , 19) . The twofold stimulatory effects of ADP at 0.2 µM could be blocked in a nontoxic manner by MRS 2179 at 0.02–20 µM (Fig. 6 ), whereas the control values were unchanged by this antagonist. Numbers of osteoclasts and mononuclear cells were unaltered by any treatment. Note that although baseline levels of resorption pit formation and cell numbers vary somewhat among individual assays, as would be expected for primary cell cultures of this nature, relative treatment/control effects were highly reproducible.



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  		Figure 6. Inhibition of ADP-stimulated resorption pit formation by the P2Y1 antagonist MRS 2179. ADP-induced stimulation of resorption pit formation at 0.2 µM was inhibited by MRS 2179 in a nontoxic manner. Control values were unchanged on addition of the antagonist. The number of mononuclear cells/disk was 4552 ± 572, 5134 ± 352, 4001 ± 439, 4311 ± 453, 3329 ± 57, 4572 ± 264, 4429 ± 345, and 4001 ± 413 and the number of osteoclasts/disk was 43.4 ± 6.8, 38 ± 3.5, 47.8 ± 6.7, 53.2 ± 2.8, 44 ± 4.7, 48.4 ± 4, 49 ± 4, and 47.2 ± 7 for each treatment group, respectively (all nonsignificant). Values are means ± SE (n = 5). Significantly different from control: **P < 0.01. Significantly different from ADP at 0.2 µM: #P < 0.05.

Effect of ADP on osteoclast-mediated Ca2+ release from mouse calvariae
In 72-h cultures of mouse calvariae, extracellular ADP also caused a dramatic increase in bone resorption. Peak effects were observed in the range 5–50 µM, but large stimulatory effects were observed at concentrations as low as 50 nM (0.05 µM). The peak effects of ADP in the mouse calvarial culture were observed at approximately 10-fold higher concentrations than in the disaggregated rat osteoclast system, reflecting the generally lower sensitivities exhibited by intact organ cultures. In the presence of 5 µM extracellular ADP, osteoclast-mediated Ca2+ release was increased up to sixfold compared with controls (Figs. 7 and 8 ). The peak stimulatory effects of ADP were equivalent to the maximal effects of PGE2 at 1 µM (Fig. 8) . Extracellular ADP appeared to be somewhat more effective than extracellular ATP, which stimulated Ca2+ release up to fourfold at 5 µM compared with the control (Fig. 7) . Similarly to the stimulatory effects on mature rat osteoclasts, the P2Y1 selective agonist 2-MeSADP mimicked the ADP effect and increased Ca2+ release fourfold compared with the control, with a peak effect at 5 µM (Fig. 7) . At 50 µM, 2-MeSADP increased Ca2+ release only twofold, again suggesting that 2-MeSADP effectively stimulates resorption in a relatively narrow concentration range. The selective P2Y1 antagonist MRS 2179 inhibited ADP-induced bone resorption (Fig. 8) . The stimulatory effect of ADP at 5 µM could be reduced 3.6-fold by MRS 2179 at 5 µM.



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  		Figure 7. Stimulatory effect of ADP, 2-MeSADP, and ATP on Ca2+ release from mouse half-calvariae cultured for 3 days in acidified medium. ADP increased osteoclast-mediated Ca2+ release up to sixfold, with peak effects close to 5 µM. The P2Y1 selective agonist 2-MeSADP was able to mimic the ADP effect. ADP appeared to be more potent than ATP. Values are means ± SE (n = 5). Significantly different from control: *P < 0.05; **P < 0.01; ***P < 0.001.



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  		Figure 8. Inhibition of the stimulatory action of ADP on Ca2+ release from mouse half-calvariae by MRS 2179 (MRS) and indomethacin (Ind) and equivalence of the stimulatory action of ADP to the maximal effects of PGE2 at 1 µM. Values are means ± SE (n = 5). Significantly different from control: **P < 0.01; ***P < 0.001. Significantly different from ADP at 5 µM: # #P < 0.01; # # #P < 0.001.

In addition, ADP-stimulated Ca2+ release was completely blocked by the cyclooxygenase inhibitor indomethacin at 0.1 and 1 µM (Fig. 8) , suggesting that the effect may be mediated by prostaglandins, as for other established resorption stimulators in this system.

Effect of ADP in mouse marrow cultures
In 10-day mouse marrow cultures on dentine disks, extracellular ADP at low concentrations reproducibly stimulated the formation of TRAP-positive osteoclasts and resorption pits (Fig. 9 ). The concentration of RANKL (1 ng/ml) in these experiments was chosen to permit relatively low level osteoclast formation without masking potential stimulatory effects of other agents. Effects of ADP were observed in the range 0.2–2 µM ADP. Lower concentrations were without effect. In the presence of 2 µM ADP, osteoclast formation was increased twofold, but resorption was increased up to fivefold compared with the control, presumably reflecting stimulation of newly formed mature osteoclasts and consistent with our findings for rat osteoclasts in short-term cultures. ATP at 2 µM was slightly more effective than ADP in stimulating osteoclast formation (2.4-fold increase) and showed a similar 5-fold stimulation of resorption. In the control groups that were fixed and stained for TRAP after 3 days of incubation, osteoclasts and resorption pits were never observed, indicating that the osteoclasts and resorption pits observed after 10 days of culture resulted entirely from formation of new osteoclasts.



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  		Figure 9. Effect of ADP and ATP on osteoclast formation and excavation of resorption pits in mouse marrow cultures maintained for 10 days on 5-mm dentine disks. Values are means ± SE (n = 6). Significantly different from control: *P < 0.05; **P < 0.01; ***P < 0.001.

In experiments with stromal cell-free marrow cultures derived from marrow cells that were initially nonadherent, ADP at concentrations of 0.2 µM (on dentine disks) and 2 µM (on plastic) stimulated the formation of TRAP-positive multinuclear cells up 2.7-fold. ADP at 20 µM was without effect. In cultures on dentine disks, resorption was also stimulated 4.2-fold by ADP at 0.2 µM, in line with results from the mixed cell cultures described above. Numbers of mononuclear hemopoietic cells were unchanged by ADP or ATP treatment (control: 1879 ± 266, ADP 0.2 µM: 2075 ± 216, ADP 2 µM: 2269 ± 335, ADP 20 µM: 2269 ± 300, ATP 0.2 µM: 2653 ± 309, ATP 2 µM: 2218 ± 312).

P2Y1 expression in bone cells
We investigated the expression of P2Y1 receptor mRNA in sections of rat long bone and in cultured rat osteoclasts and osteoblasts, and the expression of the P2Y1 receptor protein in cultured rat osteoclasts. In situ hybridization on long bone sections using P2Y1 receptor probe revealed intense, specific localization over osteoblasts on bone surfaces and over chondrocytes in the growth plate in long bones (Fig. 10E ). Negative controls of serial sections performed by hybridizing in the presence of 100-fold excess of unlabeled probe significantly reduced the signal (Fig. 10F ). On cultured rat osteoclasts, in situ hybridization and immunocytochemistry revealed intense specific localization of P2Y1 receptor mRNA and protein (Fig. 10A ,C ), whereas negative controls showed significantly less staining (Fig. 10B ). In addition, cultured primary calvarial rat osteoblasts showed strong specific staining for the P2Y1 receptor mRNA by in situ hybridization (Fig. 10D ), consistent with the osteoblast staining observed in long bone sections.



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  		Figure 10. Specific in situ localization of P2Y1 receptor probe on cultured rat osteoclasts (A) and cultured rat osteoblasts (D) and on osteoblasts (arrows) and chondrocytes (arrowheads) in frozen rat long bone section (E). Localization was reduced greatly in control preparations hybridized in the presence of excess unlabeled probe (B, F). Immunocytochemistry with P2Y1 antibody revealed strong specific staining in cultured rat osteoclasts (C). Also shown are representative micrographs of whole-mount mouse half-calvariae stained to demonstrate TRAP after 72 h of culture stimulated with ADP at 5 µM (G) and 50 µM (H); arrows point to examples of TRAP-positive osteoclasts. J) A representative photomicrograph of a 26-h culture of rat long bone cells on dentine disks shows ADP-stimulated, TRAP-stained multinuclear osteoclasts (arrows) with corresponding resorption pits (arrowheads), visualized by reflective light microscopy. Scale bars = 20 µm (A, B, C), 40 µm (J), 50 µm (D, E, F), and 200 µm (G, H).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The role of extracellular nucleotides signaling through P2 receptors in the bone remodeling process is still not well understood. This is the first study that links a specific P2 receptor to a key functional action of an extracellular nucleotide on bone.

We report that extracellular ADP, a potent agonist at the G-protein-coupled P2Y1 receptor, and 2-MeSADP, a selective P2Y1 receptor agonist, are potent stimulators of bone resorption at nanomolar concentrations, as assessed by three independent methods in two different species. Further degradation products of ADP, namely AMP and adenosine, had no significant effect on bone resorption. This result indicates that ADP itself is the signaling agent and excludes the involvement of P1 receptors for adenosine and AMP. In addition, the stimulatory ADP effect could be blocked in a nontoxic manner by the compound MRS 2179, the most potent P2Y1 receptor antagonist reported to date (18 , 19) . However, there is evidence that ADP can also act as an agonist at the P2X1 receptor, which is a nonselective cation channel (20 , 21) . To discriminate between the two receptor subtypes, we used subtype-selective agonists and histochemistry and provided evidence that the stimulatory ADP effect is mediated via the P2Y1 receptor rather than via the P2X1 receptor. P2Y1 receptor mRNA and protein were found to be expressed on both osteoclasts and osteoblasts. Using immunocytochemistry, we have recently studied the expression of P2X1–7 receptors on bone cells and found no evidence for expression of the P2X1 receptor on osteoclasts or osteoblasts (4) . Extracellular ADP could therefore stimulate resorption directly, while signaling through the receptor expressed on mature osteoclasts, or indirectly via receptors expressed on osteoblasts, which in turn release activators of osteoclastic bone resorption, or both.

We observed the osteolytic effects of ADP and 2-MeSADP at concentrations as low as 20 nM; effects of nucleotides on osteoclasts at such low concentrations have not been reported before. Two earlier studies investigated the actions of ADP at much higher concentrations on osteoclasts: ADP at 50 µM has been shown to increase intracellular Ca2+ levels (8) and ADP at 100 µM induced an intracellular pH decrease in rabbit osteoclasts, probably by enhancing the Cl-/HCO3- exchange across the osteoclast cell membrane (10) . However, the present results indicate that ADP exerts its major functional action on osteoclasts at concentrations ~1000-fold lower than in these previous studies and that ADP at concentrations between 20 and 200 µM is without effect.

Our evidence for expression of the P2Y1 receptor on osteoclasts appears to be consistent with recent electrophysiological data showing that 10–50 µM ATP, which is a partial agonist at the P2Y1 receptor, can activate a K+-selective outward current in rat osteoclasts that is dependent on P2Y receptor-mediated Ca2+ release from intracellular stores (9) . A more recent study from the same group reported that the ADP analog ADPßS at 100 µM also elicits a Ca2+-dependent K+ current in rabbit osteoclasts, consistent with the presence of the P2Y1 receptor (22) . Several studies have described effects of extracellular ADP on osteoblasts, in accordance with the histochemical evidence of P2Y1 receptor expression on osteoblasts reported in this study. ADP and 2-MeSADP in the submicromolar range caused a transient increase in intracellular Ca2+ in rat osteoblast-like UMR-106 cells (23 , 24) . This result is consistent with our findings that ADP and 2-MeSADP act at low concentrations. However, as with osteoclasts, the majority of studies on osteoblasts investigated the actions of ADP at concentrations of 10 µM and higher (5 , 25 , 26) .

Bone resorption requires both the formation of mature osteoclasts from hematopoietic progenitors and their subsequent activation to form resorption pits. The present data suggest that ADP stimulates both formation and activation of osteoclasts, resulting in striking increases in resorption in 10-day mouse marrow cultures. However, the observed positive effect of ADP on osteoclast formation in mouse marrow cultures could also be due to a prolongation of osteoclast life span, in addition to enhanced recruitment of osteoclasts from progenitors. Our results obtained using cultures of nonadherent, stromal cell-free marrow cells suggest that ADP can act directly on osteoclasts and their precursors, in addition to any effects that may be mediated via stromal cells or osteoblasts.

Perhaps the most striking effects of ADP, 2-MeSADP, and also ATP were observed in mouse calvarial bone organ cultures. We found that ADP was roughly as potent as PGE2, a reference osteolytic agent for this system, in activating osteoclastic resorption. However, resorption stimulated by ADP was blocked by the cyclooxygenase inhibitor indomethacin, suggesting a requirement for endogenous prostaglandin synthesis in this system, as is the case for other osteolytic agents such as protons (27 , 28) . It has long been known that adenine nucleotides can induce prostaglandin biosynthesis (29) .

A previous study showed that ATP at low concentrations (0.2–2 µM) is a potent stimulator of the activation and formation of rodent osteoclasts (3) , but this effect was not related to a specific P2 receptor subtype. The stimulatory effect on mature osteoclasts was evident only at low pH (~6.9), suggesting the possible involvement of the P2X2 receptor, the only P2 receptor subtype that needs extracellular acidification to show its full sensitivity to ATP (30 , 31) . However, we show here that a similarly low pH is also required for ADP to show its full stimulatory effects on osteoclastic resorption. This result is also consistent with studies showing that proresorptive effects of 1,25-dihydroxyvitamin D3, parathyroid hormone, PGE2, and RANKL are acid-dependent (12 , 32 , 33) . These findings point to a general dependency of osteolytic agents on slight local acidification, confirming that osteoclasts need to be "switched on" by low pH to resorb bone (11 , 34) .

To date the majority of studies on the effects of extracellular nucleotides on bone cells have focused on the actions of ATP, which is the nucleotide with the widest spectrum of biological activity and an agonist at all P2 receptor subtypes. On the basis of our findings that ADP appears to be somewhat more potent than ATP in stimulating mature osteoclasts, we suggest that, for several reasons, the P2Y1 receptor may also be responsible for at least part of the stimulatory effect observed with ATP. First, ATP has been considered a potent agonist of the P2Y1 receptor, based on studies using the cloned chick receptor (35) . However, recent studies suggest that pure ATP is in contrast a weak competitive antagonist at the mammalian P2Y1 receptor and that ATP actions were apparent only because of ADP contamination present or newly formed by ecto-ATPases (36 , 37) . This issue remains highly controversial; for example, potent ATP agonism at the P2Y1 receptor has now been demonstrated on mammalian neurons (38) and, in the case of 2-MeSATP, an ATP analog, also on rat hepatocytes (39) . Second, ATP could rapidly be hydrolyzed to ADP via ectonucleotidases present in the bone environment. Several studies have shown that ATP is rapidly hydrolyzed once present in the extracellular environment. For example, Sistare et al. showed that within 2 min approximately 25% of a 10 µM ATP solution is metabolized into ADP by rat osteoblast-like cells (24) . Similarly, white blood cells can quickly metabolize 10 µM ATP into ADP and into further degradation products, including AMP (40) . The presence of ectonucleotidases has also recently been demonstrated in bone marrow (41) . Third, commercial ATP samples are often contaminated with traces of about 1–5% ADP; thus, an ATP concentration of 2 µM would result in up to 0.02–0.1 µM ADP. As we report here, these concentrations are sufficient to exert a peak stimulatory effect on bone resorption.

There are a number of potential sources for extracellular nucleotides in the bone environment: ATP is a ubiquitous intracellular constituent (2–5 mM inside the cell), and thus any cell could potentially serve as a source of extracellular ATP. ATP can be released into the extracellular space from intact cells by vesicular exocytosis (e.g., from nerve endings), or a channel-like pathway (e.g., ABC-proteins), but also from damaged cells and during tissue injury. Platelets can also release ADP itself. The granules in platelets contain up to 40 nM ATP and ADP/mg protein, and plasma concentrations of ATP/ADP of 20 µM have been measured after platelet activation (42) . In addition, osteoblasts have been shown to release ATP under shear stress conditions (43) .

Our finding that ADP, signaling through the P2Y1 receptor, is a powerful activator of osteoclasts and may also induce recruitment of osteoclasts could be of relevance to several pathophysiological conditions that lead to increased bone resorption. First, inflammatory conditions such as rheumatoid arthritis lead to sustained systemic and localized bone loss, probably because of increased osteoclast activity. We show that ADP is as powerfully proresorptive as is PGE2, an osteolytic agent that is also implicated in inflammatory bone loss (44) . Release of nucleotides is increased under inflammatory conditions, suggesting an early role of extracellular nucleotides in the inflammatory process (45) ; thus, ADP could mediate a component of inflammatory bone loss. In addition, platelets play a key role in inflammation by being induced to release their granule contents, including adenine nucleotides (46) .

A second pathological condition in which ADP-mediated bone resorption could play a major role is the bone loss associated with cancer metastases. Tumor cells are important sources of extracellular ATP (47) ; thus, localized ATP/ADP release could recruit and stimulate osteoclasts. It is important to note that inflamed and cancerous tissues are also characterized by low extracellular pH, which would facilitate the osteolytic action of ADP and ATP.

Nucleotides may also be implicated in a wider spectrum of connective tissue destruction. For example, net loss of the cartilage extracellular matrix occurs in all forms of arthritis, and extracellular ATP has been shown to stimulate cartilage resorption by acting on P2 receptors (48 , 49) .

In conclusion, our study points to a fundamental new mechanism for the local modulation of bone resorption by extracellular nucleotides at nanomolar concentrations.


   ACKNOWLEDGMENTS
 
The authors would like to thank Drs. Brian King and Andrea Townsend-Nicholson for helpful advice, and Ms. Michelle Bardini for technical assistance. The support of Roche Bioscience, Palo Alto, CA, and the Arthritis Research Campaign (U.K.) is gratefully acknowledged. Astrid Hoebertz is the recipient of an Arthritis Research Campaign PhD studentship.

Received for publication August 31, 2000. Revision received November 30, 2000.
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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