J Physiol Volume 511, Number 2, 495-500, September 1, 1998
The Journal of Physiology (1998), 511.2, pp. 495-500
© Copyright 1998 The Physiological Society
ATP is a potent stimulator of the activation and formation of rodent osteoclasts
Matthew S. Morrison *, Luca Turin *, Brian F. King *¹, Geoffrey Burnstock *¹ and Timothy R. Arnett *
* Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT and ¹ Autonomic Neuroscience Institute, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, UK
MS 8167 Received 28 April 1998; accepted after revision 7 July 1998.
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ABSTRACT |
- There is increasing evidence that extracellular ATP acts directly on bone cells via P2 receptors. In normal rat osteoclasts, ATP activates both non-selective cation channels and Ca2+-dependent K+ channels. In this study we investigated the action of ATP on the formation of osteoclasts and on the ultimate function of these cells, namely resorption pit formation.
- We found that ATP stimulated resorption pit formation up to 5·6-fold when osteoclast-containing bone cell populations from neonatal rats were cultured for 26 h on ivory discs, with a maximum effect occurring at relatively low concentrations (0·2-2 µM). The stimulatory effect of ATP was amplified greatly when osteoclasts were activated by culture in acidified media (pH 6·9-7·0). Pit formation by acid-activated osteoclasts in the absence of ATP was inhibited by apyrase, an ecto-ATPase and by suramin, an antagonist of P2 receptors.
- Over the same concentration range at which rat osteoclast activation occurred (0·2-2 µM), ATP also enhanced osteoclast formation in 10 day mouse marrow cultures, by up to 3·3-fold, with corresponding increases in resorption pit formation. Higher concentrations of ATP (20-200 µM) reduced or blocked osteoclast formation. Adenosine, a P1 receptor agonist, was without effect on either osteoclast activation or formation.
- These results suggest that low levels of extracellular ATP may play a fundamental role in modulating both the resorptive function and formation of mammalian osteoclasts.
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INTRODUCTION |
ATP and other extracellular nucleotides are now recognized as important messenger molecules for cell-cell communication. Cell membrane receptors for ATP are classified into two main groups: the P2Y receptor family couples to G-proteins to stimulate phospholipases, activating a series of intracellular signalling pathways, including IP3-dependent mobilization of intracellular Ca2+; the P2X receptor family gates cation channels permeable to calcium, sodium, potassium and, most probably, hydrogen ions. Seven main subtypes of each family have been identified to date (Burnstock & King, 1996). ATP is released into the extracellular space via synaptic vesicles from nerve cells, as the result of cell damage and also by active secretion via 'ATP binding cassette' transport proteins such as P-glycoproteins and sulphonylurea receptors (reviewed by Burnstock, 1997).
There is increasing evidence that ATP may play an important role in bone as a signalling agent. In osteoclasts, the polarized multinucleate cells responsible for the resorption of bone and other mineralized tissues, exogenous ATP induces an intracellular Ca2+ pulse (Yu & Ferrier, 1993, 1994) and causes a transient intracellular pH decrease that is Ca2+ independent (Yu & Ferrier, 1995). Recent electrophysiological experiments have provided indirect evidence for the co-expression on osteoclasts of both P2Y and P2X receptors for ATP (Weidema et al. 1997). Histochemical evidence exists for the expression of the P2Y2 (P2U) receptor on osteoclasts derived from human giant cell tumours (Bowler et al. 1995, 1998a). Bowler and co-workers have also shown that ATP exerts a small stimulatory effect on resorption pit formation by giant cell tumour osteoclasts but that this effect is not mediated by P2Y2 receptors (Bowler et al. 1998a). Following our initial reports in abstract form (Morrison & Arnett, 1997, 1998a), the present study provides the first description of the potent stimulatory effects of ATP on the formation and resorptive function of normal mammalian osteoclasts.
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METHODS |
Materials
1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) was provided by Dr K. W. Colston (St George's Hospital Medical School, London). Culture media were purchased from Gibco. All other reagents were from Sigma. Fresh stock solutions of ATP, adenosine, apyrase and suramin were prepared in phosphate-buffered saline (PBS) for each experiment; stock solutions of dexamethasone and 1,25(OH)2D3 were prepared in ethanol and stored for short periods at -20°C. ATP and adenosine solutions, both of which are acidic, were titrated to pH 7·0 with NaOH immediately before use to avoid unwanted pH effects on osteoclast function (Arnett & Spowage, 1996). Untreated elephant ivory was kindly provided by HM Customs and Excise (London Heathrow Airport).
Resorption pit formation assay
The effects of extracellular ATP and adenosine on resorption pit formation by mature rat osteoclasts were studied using modifications of an assay described previously (Arnett & Spowage, 1996). All experiments were performed using standard minimum essential medium supplemented with Earle's salts, 10 % fetal calf serum, 2 mM L-glutamine, 100 U ml-1 penicillin, 100 µg ml-1 streptomycin and 0·25 µg ml-1 amphotericin B (complete mixture abbreviated to 'MEM'). In some experiments, MEM was acidified by the direct addition of small amounts of concentrated hydrochloric acid (10 mequiv l-1 H+, equivalent to 85 µl of 11·5 M HCl per 100 ml 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 (Murrills et al. 1998). Elephant ivory was prepared by cutting 250 µm thick transverse wafers using a low speed diamond saw (Buehler, Coventry, UK); 5 mm diameter discs were cut from wet ivory wafers using a standard paper punch, washed extensively by sonication in distilled water and stored dry at room temperature. Before use, ivory discs were sterilized by brief immersion in ethanol, allowed to dry and then rinsed in sterile PBS.
Mixed cell populations containing osteoclasts were obtained by mincing rapidly the pooled long bones of 2-day-old rat pups, killed by cervical dislocation (n = 5), in 5 ml MEM, followed by vortexing for 20 s. The resulting cell suspension was allowed to sediment for 45 min onto 5 mm ivory discs, pre-wetted with 50 µl MEM, in 96-well plates (100 µl cell suspension per disc). Discs 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 4 ml of acidified MEM and five replicate ivory discs; 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 analyser (Radiometer, Copenhagen, Denmark), taking careful precautions to prevent CO2 loss. Ivory discs were removed and fixed in 2 % glutaraldehyde, then stained for tartrate-resistant acid phosphatase (TRAP) using Sigma kit 387-A. The numbers of TRAP-positive multinucleated osteoclasts (two or more nuclei), and the number of stromal cells were assessed 'blind' using transmitted light microscopy. Discrete resorption pits were counted 'blind' by scanning the entire surface of each disc using reflected light microscopy after restaining in 1 % Toluidine Blue in 1 % sodium borate for 2 min.
Osteoclast formation assay
Long bones of 8-week-old mice (n = 2), killed by cervical dislocation, were fragmented in 5 ml unmodified MEM, followed by vortexing for 1 min. The resulting cell suspension was allowed to sediment for 24 h onto sterile 5 mm diameter ivory discs, pre-wetted with 50 µl MEM, in 96-well plates (100 µl cell suspension per disc). Ivory discs were then removed and placed in test or control medium in a 6-well plate. Each test or control well contained 4 ml of non-acidified MEM with 10 nM 1,25(OH)2D3 and 10 nM dexamethasone, and six replicate ivory discs; cultures were incubated in a humidified atmosphere of 5 % CO2- 95 % air, with medium changes every 2 days. MEM was not acidified for these experiments because low pH conditions are inhibitory for osteoclast formation (Morrison & Arnett, 1998b). Medium pH and PCO2 were monitored during and at the end of experiments using a blood gas analyser, as above. After 10 days incubation, the discs were fixed in 2 % glutaraldehyde, and stained for tartrate-resistant acid phosphatase (TRAP) using Sigma kit 387-A. A control group of ivory discs was also removed, fixed and stained after 3 days incubation to check for the presence of any mature osteoclasts that might have been released during the initial cell preparation. The total area occupied by TRAP-positive multinucleated osteoclasts and resorption pits was assessed 'blind' by transmitted and reflected light microscopy, via a colour video image output, using standard 'dot count' morphometry. Area measurements, rather than discrete cell and pit counts, were necessary because this assay system requires high cell densities to function, resulting in large, semi-contiguous groups of TRAP-positive osteoclasts associated with extensive, often conjoined areas of resorption.
Statistics
Statistical comparisons were made by one-way analysis of variance of log-transformed data, using Bonferroni's correction for multiple comparisons; representative data are presented as means ± S.E.M. for five or six replicates. Significance was assumed at P < 0·05. Each experiment was repeated 3 or 4 times.
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RESULTS |
Effect of ATP on mature rat osteoclasts
Extracellular ATP exerted a reproducible, biphasic effect on resorption pit formation by rat osteoclasts in low density, acid-activated 26 h cultures (pH 6·94 ± 0·016). At low concentrations (0·2 and 2 µM ATP) striking stimulations were observed, with up to a 3·5-fold increase in pit number (Fig. 1).
At higher concentrations, resorption was progressively reduced (Fig. 1); this was largely due to a selective cytotoxic effect of ATP (associated with cell vacuolation) on osteoclasts. However, numbers of mononuclear cells (i.e. cells of osteoblastic/fibroblastic morphology) were unaltered by ATP: treatment with 0, 0·2, 2, 20, 200 and 2000 µM ATP resulted in 1230 ± 95, 1323 ± 213, 1200 ± 114, 1228 ± 167, 1390 ± 151 and 1001 ± 143 mononuclear cells per ivory disc. Time lapse video microscopy, using methods similar to those described by Arnett et al. (1996), revealed that 60-90 min following addition of 2 mM ATP to freshly isolated mature rat osteoclasts, the cell abruptly retracts and dies in a manner reminiscent of apoptosis (data not shown). Adenosine, over a similar concentration range, was without significant effect on either resorption pit formation or osteoclast numbers except at the highest dose (2 mM), where some cytotoxicity was observed (data not shown).
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Figure 1. Biphasic effect of ATP on resorption pit formation by rat osteoclasts
Osteoclasts were cultured on 5 mm dentine discs in acidified medium (pH 6·937 ± 0·016) for 26 h. The stimulatory effect of ATP was evident at lower concentrations and a selective cytotoxic effect at the highest concentration. Values are means ± S.E.M. (n = 5); * P < 0·05; ** P < 0·01; *** P < 0·001 with respect to control.
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The interaction between the effects of pH and extracellular ATP was investigated using rat osteoclast cultures incubated for 26 h with 0 or 2 µM ATP in either control (non-acidified) or acidified MEM (Fig. 2). In the absence of ATP, acidification (pH reduction from 7·166 to 6·945) elicited a modest, 3-fold stimulation of resorption pit formation; and in non-acidified medium ATP caused a 2·5-fold stimulation of pit formation. However, when osteoclasts were cultured with 2 µM ATP in acidified MEM, resorption was stimulated 17-fold compared with control. Thus, the stimulatory effect of ATP was enhanced greatly at low pH and vice versa.
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Figure 2. Comparison of the effects of ATP on resorption pit formation by rat osteoclasts cultured in unmodified medium (Control) or in acidified medium (Acid) for 26 h
The figure shows potentiation of ATP-stimulated resorption at low pH. Values are means ± S.E.M. (n = 5); ** P < 0·01; *** P < 0·001 with respect to control.
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To study further the possible dependence of the acid activation of resorption on low levels of extracellular ATP, osteoclasts were cultured for 26 h with apyrase, which hydrolyses extracellular ATP to AMP. In this experiment, acidification resulted in a 3·5-fold stimulation of pit formation; apyrase inhibited acid-activated resorption, with a near-maximal effect at 0·1 units ml-1 (Fig. 3). There was no evidence that apyrase was cytotoxic at any dose. A similar, non-toxic, inhibitory effect was also observed reproducibly when acid-activated osteoclasts were cultured with suramin, an antagonist of P2 receptors (resorption levels at pH 7·216, pH 7·013 and pH 7·009 + 10 µM suramin were 4·40 ± 0·67, 15·40 ± 3·39 and 6·20 ± 1·39 * pits per dentine disc, respectively; *P < 0·05 vs. pH 7·013 group; osteoclast and mononuclear cell numbers were unaltered by any treatment).
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Figure 3. Stimulation of resorption pit formation by rat osteoclasts cultured for 26 h in acidified medium and inhibition of acid-stimulated pit formation by apyrase
Values are means ± S.E.M. (n = 5). a P < 0·01; b P < 0·001 with respect to pH 7·113 control value. * P < 0·05; ** P < 0·01; *** P < 0·001 with respect to pH 6·926 control value.
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Effect of ATP on osteoclast formation in mouse marrow cultures
In 10 day mouse marrow cultures, extracellular ATP at low concentrations stimulated the formation of TRAP-positive osteoclasts and resorption pits reproducibly. As was the case in the mature rat osteoclast assay, peak effects were observed in the range 0·2-2 µM ATP. In the presence of 2 µM ATP, osteoclast formation was stimulated 3·3-fold and resorption increased 4-fold compared with control. Higher concentrations either inhibited (20 µM) or completely blocked (200 µM) osteoclast formation and resorption (Fig. 4). Adenosine over the same concentration range was without significant effect.
In the control groups that were fixed and stained for TRAP after 3 days incubation, osteoclasts and resorption pits were never observed, indicating that the osteoclasts and resorption pits observed after 10 days in culture resulted entirely from formation of new osteoclasts.
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DISCUSSION |
The modulation of bone resorption by local factors is a complex process that is still not well understood (Roodman, 1996). Our results indicate that extracellular ATP, which causes striking stimulation of both resorption pit formation and osteoclast recruitment at submicromolar concentrations, must be considered as a potentially important paracrine or autocrine agent for bone.
It is unclear from the present data whether exogenous ATP stimulates resorption pit formation via direct effects on mature osteoclasts, indirect effects on cells such as osteoblasts, or both. Electrophysiological evidence (Yu & Ferrier, 1993, 1994, 1995; Weidema et al. 1997) and histochemical evidence (Bowler et al. 1995, 1998a) exists for the expression of P2X and P2Y receptors on normal or tumour-derived osteoclasts. P2Y receptors are also present on osteoblast-like cells (Bowler et al. 1995, 1998b). Pure populations of normal mammalian osteoclasts are not routinely available for study and multinucleate osteoclasts constitute only about 1-2 % of the cells in the low density rat bone cell cultures used in our experiments. Additionally, our results do not distinguish the mode of action of ATP in increasing osteoclast formation in 10 day marrow cultures. ATP could be acting directly on osteoclast precursor cells, as well as indirectly on the stromal component of marrow cultures; it is possible also that the stimulatory effects of ATP in these long term cultures were due, in some degree, to enhanced osteoclast survival.
The rapid, selective cytotoxic action of ATP at millimolar concentrations on osteoclasts is in agreement with the findings of Nijweide et al. (1995); this effect may be mediated by the P2Z (P2X7) receptor for ATP4-, activation of which results in the formation of cytolytic pores (reviewed by Burnstock, 1997). Interestingly, we found that resorption pit formation by osteoclasts derived from late chick embryos was not enhanced by ATP, although the high dose inhibitory effect was still evident (M. S. Morrison & T. R. Arnett, unpublished observations). Chick osteoclasts also differ from mammalian osteoclasts in that they resorb bone more avidly and fail to show an inhibitory response to calcitonin (Arnett & Dempster, 1987). The lack of effect of adenosine suggests that P1 receptors are not involved in the modulation of osteoclastic function, in line with the earlier findings of Yu & Ferrier (1993), who demonstrated that adenosine exerted no significant effect on intracellular Ca2+ in osteoclasts.
Previous work has demonstrated the remarkable sensitivity of rat osteoclasts to stimulation by extracellular protons (Arnett & Dempster, 1986; Arnett & Spowage, 1996). The pH response occurs within a relatively limited range, such that shifts in the range pH 7·25-7·00 act as a reversible 'off- on switch' for resorptive activity, with the major change associated with a pH difference of as little as 0·1 unit (Arnett & Spowage, 1996). Embryonic chick osteoclasts exhibit similar responses (Arnett & Dempster, 1987), as do mouse calvarial cultures (Meghji et al. 1996) and osteoclasts formed from mouse marrow cultures (Morrison & Arnett, 1998b). An important feature of H+-stimulated resorption is that it does not exhibit desensitization: indeed, the magnitude of the response of mature rat osteoclasts appears to increase with time in culture, such that after 7 days, pH drops of 
0·1 unit are associated with 15- to 20-fold enhancements of pit formation (M. S. Morrison & T. R. Arnett, in preparation). The present work demonstrates that there is a powerful synergy between the stimulatory effects of low-dose ATP and protons on the resorptive activity of mature rat osteoclasts, i.e. the full sensitivity of osteoclasts to activation by ATP is only evident at low pH (6·9-7·0), and vice versa. In the case of the osteoclast formation experiments, ATP appeared to enhance resorptive efficiency only slightly; this was probably a consequence of the non-acidified medium (pH 7·2) used to maximize osteoclast recruitment in this assay (Morrison & Arnett, 1998b).
The effect of apyrase (which hydrolyses ATP to AMP) on acid-stimulated pit formation was to inhibit resorption without affecting cell viability or morphology, suggesting that the 'pH effect' may be dependent on trace levels of either free or bound extracellular ATP. The concentrations of ATP in question may be quite low, given the powerful stimulatory effect seen in the presence of 200 nM ATP. There are two main possibilities for the origin of extracellular ATP in osteoclast-containing bone cell cultures. First, ATP (intracellular concentration 2 mM) could be released from cells that were damaged during the relatively vigorous procedures used to isolate osteoclasts from bones. Second, ATP may be exported from intact cells via transport proteins; for example, osteoclasts have been reported to express a novel member of the ATP binding cassette superfamily (Wagstaff et al. 1998). There is also recent evidence for constitutive release of ATP by osteoblast-like cells, a process that appears to be stimulated by fluid shear forces (Bowler et al. 1998c). Interestingly, McSheehy & Chambers (1986) reported that osteoblasts released an unknown soluble factor of relatively low molecular weight that stimulated osteoclasts.
Striking recent experiments have shown that extracellular acidification is required for the P2X2 receptor subtype to show its full sensitivity to extracellular ATP (King et al. 1996; Wildman et al. 1997). Indeed, the pH-activation profile of the recombinant P2X2 receptor expressed in Xenopus is remarkably similar to that for resorption pit formation by rat osteoclasts (Arnett & Spowage, 1996). No other ATP receptors of the P2X or P2Y families are known to exhibit such pH sensitivity. Although our data do not distinguish conclusively between P2 receptor type(s) which may be mediating the stimulatory effects of low concentrations of ATP on rodent osteoclasts, the apparent lack of desensitization to ATP, suramin antagonism and acid activation are consistent with the possible involvement of the P2X2 receptor subtype. When the recombinant P2X2 receptor is activated by ATP and extracellular H+, it opens a non-selective cation/proton channel. Opening such a channel in the osteoclast (dorsal/basolateral) cell membrane could increase critically the intracellular availability of H+ (which is then actively pumped out across the ruffled border), so as to facilitate resorption pit formation (Arnett & King, 1997). Such a mechanism would augment the intracellular proton supply thought to be derived via osteoclastic carbonic anhydrase. Alternatively, the putative 'pH receptor' on osteoclasts may be distinct from ATP receptors.
In addition to increasing resorption, ATP may additionally exert negative effects on bone formation. Recent experiments have shown that ATP, albeit at concentrations somewhat higher than those optimal for stimulation of resorption, inhibits appositional bone formation by cultured primary osteoblasts (Jones et al. 1997). ATP also induces cartilage resorption in vitro (Leong et al. 1994). In inflamed tissue, ATP may be released from damaged cells, mast cells or platelets (King et al. 1996); inflammation is also associated with local acidification (Burnstock, 1997), as is the case at sites of fracture and surgery (Brueton et al., 1993). It is conceivable that ATP could also play in role in tumour osteolysis, given the propensity of transformed cells to release ATP (Burnstock, 1997) and cause local acidification. The present findings suggest a new mechanism by which localized bone destruction could occur when ATP is released in acidified tissues.
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Acknowledgements
We are grateful to the Arthritis Research Campaign for support. M. S. Morrison is the recipient of a Medical Research Council PhD studentship.
Corresponding author
T. R. Arnett: Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK.
Email: t.arnett{at}ucl.ac.uk
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Top
Introduction
