Intercellular Calcium Signaling Occurs between Human Osteoblasts and Osteoclasts and Requires Activation of Osteoclast P2X7 Receptors*

Signaling between osteoblasts and osteoclasts is important in bone homeostasis. We previously showed that human osteoblasts propagate intercellular calcium signals via two mechanisms: autocrine activation of P2Y receptors, and gap junctional communication. In the current work we identified mechanically induced intercellular calcium signaling between osteoblasts and osteoclasts and among osteoclasts. Intercellular calcium responses in osteoclasts required P2 receptor activation but not gap junctional communication. Pharmacological studies and reverse transcriptase-PCR amplification demonstrated that human osteoclasts expressed functional P2Y1 receptors, but, unexpectedly, desensitization of P2Y1 did not block calcium signaling to osteoclasts. We also found that osteoclasts expressed functional P2X7 receptors and showed that pharmacological inhibition of these receptors blocked calcium signaling to osteoclasts. Thus these studies show that calcium signaling between osteoblasts and osteoclasts occurs via activation of P2 receptors, but that different families of P2 receptors are required for calcium signaling in these two cell types. were performed with a Zeiss confocal microscopy system using a 40 (cid:3) water immersion objective (40/1.2 Zeiss), 4 (cid:3) zoom, and Cy3 excitation with a 543-nm laser.

Signaling between osteoblasts and osteoclasts is important in bone homeostasis. We previously showed that human osteoblasts propagate intercellular calcium signals via two mechanisms: autocrine activation of P2Y receptors, and gap junctional communication. In the current work we identified mechanically induced intercellular calcium signaling between osteoblasts and osteoclasts and among osteoclasts. Intercellular calcium responses in osteoclasts required P2 receptor activation but not gap junctional communication. Pharmacological studies and reverse transcriptase-PCR amplification demonstrated that human osteoclasts expressed functional P2Y1 receptors, but, unexpectedly, desensitization of P2Y1 did not block calcium signaling to osteoclasts. We also found that osteoclasts expressed functional P2X7 receptors and showed that pharmacological inhibition of these receptors blocked calcium signaling to osteoclasts. Thus these studies show that calcium signaling between osteoblasts and osteoclasts occurs via activation of P2 receptors, but that different families of P2 receptors are required for calcium signaling in these two cell types. Intercellular calcium signaling among bone cells is therefore amenable to pharmacological manipulation that will specifically affect only bone-forming or bone-resorbing cells. P2 receptors may be important drug targets for the modulation of bone turnover.
Osteoclasts are the cells responsible for bone resorption, whereas osteoblasts deposit new bone throughout a lifetime. Bone resorption and formation are coordinated, and in adult life are maintained in a balance, so that no significant bone loss occurs. In later life, especially in women after menopause, osteoclast activity is increased relative to osteoblast activity, and this unbalanced cellular activity causes increased relative bone resorption, and in turn, bone loss and osteoporosis. Both endocrine and paracrine factors modulate osteoclast activity, including calciotropic, growth, sex, and adrenal hormones as well as cytokines, growth factors, electrolytes, and mechanical forces. Most of these paracrine and endocrine factors affect osteoclast activity indirectly, acting on osteoblasts. Thus, boneresorptive signals must be transmitted from osteoblasts to osteoclasts via mechanisms of cell-to-cell communication between the two lineages.
We have previously shown that mechanical stimulation of human osteoblasts in vitro generates a calcium signal that is communicated to other osteoblasts (1,2). The propagation of this signal involves two different mechanisms. One is the autocrine action of ATP on plasma membrane purinergic receptors of the P2Y subtype, and the other involves the passage of a soluble messenger through gap junctions, leading to influx of extracellular calcium.
Recent studies have revealed the presence of nucleotide receptors in osteoclasts. 1 ATP can act on two different classes of receptors. The P2Y receptors are G-protein-coupled and stimulate phospholipases, subsequently activating the inositol 1,4,5-triphosphate pathway releasing calcium from intracellular stores. Members of the P2X family are non-selective cation channels permeable to Na ϩ , K ϩ , Ca 2ϩ , and H ϩ . Receptors from both classes have been identified on bone-resorbing cells (3,4), as determined both by pharmacological profiles and effector functions. Thus, ATP induces both a non-selective cation current (P2X-mediated) and release of calcium from intracellular stores (P2Y-mediated) (3). P2Y2 mRNA has been demonstrated in giant cells from a human osteoclastoma by RT-PCR, 2 although the significance of this finding is unclear because P2Y2mediated responses were not present as UTP failed to increase intracellular calcium concentration ([Ca 2ϩ ] i ) (5,6). Other investigators have identified nucleotide-mediated responses in osteoclastic cells. ATP has been shown to cause a transient decrease in intracellular pH, possibly related to activation of P2X cation channels (7). This response might favor formation of osteoclastic resorption pits and increased bone resorption. Supporting this hypothesis, ATP induces osteoclast activation and resorption in rat osteoclasts (8) and in giant cells from human osteoclastoma (6). The effect on osteoclast formation is, however, biphasic, because low concentrations increase osteoclast formation while high concentrations decrease mouse osteoclast formation (8).
In this study we have investigated the propagation of calcium transients from primary osteoblasts to osteoclasts from human bone marrow and among osteoclasts. This intercellular signal propagation might represent a mechanism by which signals initiated by mechanical stimulation of bone cells, primarily osteocytes, are diffused through the bone tissue to surface osteoblasts and in turn to osteoclasts, thus regulating bone remodeling. We report herein that a calcium signal can be communicated from osteoblasts to osteoclasts and among osteoclasts. Whereas calcium signals among osteoblasts involve activation of P2Y receptors, we found unexpectedly that P2X nucleotide receptors, probably of the P2X7 subtype, are required for osteoclast calcium signals activation.

EXPERIMENTAL PROCEDURES
Cells-Human bone marrow (5-10 ml) was obtained from healthy volunteers (aged 20 -34) by puncture of the posterior iliac spine. The marrow material was collected in a 50-ml tube, containing 15 ml of heparinized (100 units/ml) minimum essential medium. The mononuclear fraction of the cells was then isolated by centrifugation on a Lymphoprep gradient. The cells were plated in T-75 culture flasks with 10 ϫ 10 6 cells/flask in RPMI 1640 medium supplemented with glutamine (Invitrogen, Grand Island, NY)) and 30% horse serum (Sigma Chemical Co., St. Louis, MO). The culture was incubated in a humidified atmosphere of 5% CO 2 and 37°C. The following day, to separate adherent and non-adherent cell populations, the medium was collected, flushing the bottom of the flask a couple of times.
The adherent cell population was transferred to minimum essential medium and 10% fetal calf serum (Invitrogen) and maintained by completely changing the medium once a week. After 3-4 weeks of culture, the osteoblastic cells reached 80% confluence and were used to co-culture with the tartrate-resistant acid phosphatase-positive giant cells (osteoclastic cells). The non-adherent cells were counted, and 5% osteoblastic cells were added to the cell suspension. The cells were then replated in 6-well plates, each well containing a 25-mm no. 1 glass coverslip, at 2 ϫ 10 6 osteoclastic cells/well. The co-culture was maintained in RPMI 1640 medium supplemented with glutamine (Invitrogen) and 30% horse serum (Sigma). Initially and at every medium change 10 Ϫ8 M 1,25(OH) 2 vitamin-D 3 (Roche Molecular Biochemicals) was added to the medium. After 2-3 weeks, multinucleated cells started to appear, and the co-cultures were ready for video imaging experiments. The day before experiments were performed, the co-culture was washed twice with PBS to remove the non-adherent cells.
The methods for obtaining osteoblastic and osteoclastic cells in human bone marrow co-cultures have been described previously (9,10). These methods allow the production of cells of both (a) osteoblastic cells, as demonstrated by high alkaline phosphatase activity, secretion of osteocalcin, production of procollagen type I, presence of parathyroidhormone receptors, and ability to produce mineralized matrix in the presence of ␤-glycerophosphate and ascorbic acid (9,11,12), and (b) multinucleated, giant cells staining positively for tartrate-resistant acid phosphatase activity (TRAP), which are able to resorb bone in vitro (10,11).
Osteoclasts for RNA extraction were isolated from human bone marrow as described above, but instead of co-culturing with osteoblastic cells, the non-adherent cells were plated in 6-cm plates and maintained in minimum essential medium and 10% fetal calf serum (Invitrogen) supplemented with 200 ng/ml human macrophage-colony stimulating factor (R&D Systems) and 20 ng/ml human RANKL (PeproTech), to promote osteoclast differentiation and maturation. A similar method has been shown to be able to produce osteoclasts from human peripheral blood (13,14). This method yields a high number of multinucleated, giant cells that stain positively for TRAP, as shown in Fig. 1. The study was approved by the local Ethics Committee, and study participants signed informed consent prior to participation.
Chemicals-The fluorescent calcium indicator fura-2 was purchased from Molecular Probes Inc. (Eugene, OR). Thapsigargin, an inhibitor of the calcium-ATPase of the endoplasmic reticulum, was from Calbiochem (San Diego, CA) and was used in a final concentration of 50 nM. The ATP-receptor antagonist suramin was purchased from Research Biochemicals Inc. (Natick, MA), and nucleotides were from Roche Mo-lecular Biochemicals (Mannheim, Germany). All other chemicals were from Sigma (St. Louis, MO). The gap junctional inhibitor heptanol was freshly made each day as a 1:4 heptanol:ethanol solution and used in a final concentration of 3.5 mM.
Calcium Imaging-Measurement of the intracellular calcium concentration was done using the calcium indicator dye fura-2. Co-cultures of osteoblastic and osteoclastic cells in monolayers adherent to noncoated glass coverslips were incubated at 37°C in medium containing 5 M fura-2/AM for 30 min and then incubated for an additional 20 min in medium without dye. Coverslips were affixed to a Teflon chamber and mounted in a PDMI-2 open perfusion microincubator (Medical Systems Corp., Greenvale, NY) maintained at 37°C with superfused CO 2 on a Zeiss Axiovert 35 inverted microscope (Carl Zeiss Inc., Thornwood, NY). Imaging was performed with the Metamorph/Metafluor system (Universal Imaging, West Chester, PA) with excitation wavelengths of 340 and 380 nm for acquiring ratio images of fura-2. Probenecid at a concentration of 1 mM was added throughout the experiment to prevent dye leakage from the cytoplasm of the cells (15). Ratio images were calibrated using buffers of known calcium concentrations (Molecular Probes). Calcium waves were induced by stimulating a single cell with a borosilicate glass micropipette affixed to an Eppendorf 5171 micromanipulator (Eppendorf Inc., Madison, WI).
Nucleotide-induced Dye Uptake-We assessed nucleotide-induced plasma membrane permeabilization by measuring uptake of the membrane-impermeant fluorescent dye Lucifer Yellow (Sigma). Cell monolayers were incubated at 37°C in medium containing 0.5 mg/ml Lucifer Yellow in the presence of different ATP analogs for 10 min. Thereafter, monolayers were washed with PBS. Cells demonstrating diffuse cytoplasmic staining with Lucifer Yellow were identified by fluorescence microscopy, and images were recorded. Images of the same cells were also recorded by phase contrast microscopy to identify the cell type.
RT-PCR-Almost pure osteoclast cultures were used as described above. After 2 weeks of culture, total RNA was extracted from the osteoclasts using the RNeasy Mini kit from Qiagen. The RNA was DNase-treated using an RNase-free DNase set also from Qiagen.
Reverse transcription was done using the Carboxydothermus hydrogenofomans. Polymerase kit supplied by Roche Molecular Biochemicals. Briefly, cDNA was synthesized from 1 g of total RNA, using oligo(dT) primers, and the reaction was incubated for 30 min at 60°C. PCR was done using the Expand High Fidelity PCR System kit supplied by Roche Molecular Biochemicals. The forward primer used for P2Y1 was 5Ј-G-TGTACATGTTCAATTTGGCTCT and the reverse primer 5Ј-GTTGAG-ACTTGCTAGACCTCTTG. The primers used for P2X7 were: forward primer 5Ј-AGATCGTGGAGAATGGAGTG and reverse primer 5Ј-TTC-TCGTGGTGTAGTTGTGG. All oligonucleotides were synthesized by Invitrogen. The PCR reactions were set up using 5 l of the cDNA template previously synthesized. The PCR was performed in a PTC-100 programmable thermal controller (MJ Research, Inc.). Initially, the template was denatured for 2 min at 94°C, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 55°C (P2Y1)/53°C (P2X7) for 30 s, elongation at 72°C for 1 min., and ended with a prolonged elongation at 72°C for 7 min. Finally, the PCR products were separated in a 2% agarose gel, the P2Y1 product at a length of 685 bp and the P2X7 product at a length of 399 bp.
Histochemical Staining of TRAP-Osteoclasts were cultured on glass slides and treated as described above in the RNA extraction section. After 2 weeks of culture, the flask was removed from the slide, and the slide was washed briefly in PBS and then fixed in 70% ethanol for 5 min. Then the cells were incubated for 15 min in TRAP solution (naphthol-as-bi-phosphate, N,N-methylformamid, pararosanilin, sodium nitrite, and tartrate in Michaelis buffer, pH 5.0), washed with tap water, and then counterstained with Meyers hematoxylin for 10 s. Finally, preparations were mounted in aqueous mounting medium.
Immunocytochemistry-Osteoclasts were cultured on glass slides and treated as described above. They were fixed in ethanol for 10 min and incubated for 15 h with P2X7 receptor antibody, developed in rabbit (Sigma), diluted 1:50 with TBS containing 2% bovine serum albumin. After incubation the slides were washed 3 ϫ 5 min in TBS. Then the cells were incubated for 1 h with a Cy3-conjugated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch), diluted 1:50 with TBS containing 2% bovine serum albumin. Finally the cells were washed 3 ϫ 5 min in TBS, and preparations were mounted with mounting medium. Observations and images were performed with a Zeiss confocal microscopy system using a 40 ϫ water immersion objective (40/1.2 Zeiss), 4 ϫ zoom, and Cy3 excitation with a 543-nm laser.

Intercellular Calcium Waves Can Be Communicated Bi-directionally between Osteoblasts and
Osteoclasts-We have previously shown that human osteoblastic cells can communicate intercellular calcium waves by two mechanisms: one involving autocrine action of ATP and one dependent on gap junctional communication (2). In this study, we asked whether the intercellular calcium signal, which can be elicited in osteoblast networks, is also communicated to osteoclasts, the other major cell type effecting bone remodeling. Co-cultures of human osteoblastic and osteoclastic cells were loaded with the calcium indicator fura-2, and a single osteoblastic cell was stimulated mechanically with a glass micropipette. The stimulated cell showed an initial increase in [Ca 2ϩ ] i , spreading to adjacent osteoblasts and, with a 10-to 15-s time lag, to adjacent osteoclasts (n ϭ 34) (Fig. 2). Interestingly, when one osteoclast was mechanically stimulated, [Ca 2ϩ ] i rapidly increased in this cell, and a calcium wave was initiated and propagated to adjacent osteoblasts as well as osteoclasts (n ϭ 48 of 52 experiments), in a similar fashion as it occurred with mechanical stimulation of one osteoblast (Fig. 3). Hence, intercellular calcium waves can be elicited by mechanical perturbation of either osteoblasts or osteoclasts and can be communicated bi-directionally between cells of the two lineages, as well as among cells of each lineage.
Osteoclast Intercellular Calcium Waves Require Activation of P2 Receptors but Do Not Require Gap Junctional Communication-We next asked whether propagation of calcium signals to osteoclasts required either gap junctional communication or activation of P2 receptors. We used the gap junction inhibitor heptanol to test whether gap junctional communication is required for the propagation of osteoclast-initiated calcium waves. First, a single cell in the fura-2 loaded monolayer was mechanically stimulated to confirm the ability of the cells to propagate a calcium signal upon mechanical stimulation. After 5 min incubation with heptanol (3.5 mM), another cell was mechanically stimulated. Despite the inhibition of gap junctional communication, the stimulated osteoclast was still able to initiate a calcium pulse to the neighboring osteoclasts (n ϭ 5) (Fig. 4). Thus, gap junctional communication is not required for the propagation of intercellular calcium signals generated by mechanical perturbation of osteoclasts (Table I).
In similar experiments we used the P2 receptor antagonist suramin to determine whether P2 receptors were required for calcium signal propagation to osteoclasts, because they are in osteoblast-to-osteoblast signaling (2). One single osteoblast in a fura-2-loaded monolayer of an osteoblast-osteoclast co-culture was stimulated mechanically, and a calcium signal was propagated to neighboring osteoblasts and osteoclasts as described above. We then added suramin in a final concentration of 100 M, and after a short incubation period, we stimulated an osteoblast mechanically. No calcium waves were seen, either to osteoblasts or osteoclasts, in five of six experiments (Table I). Thus, P2 receptors are involved in the propagation of intercellular calcium signals to osteoclasts.
Human Osteoclasts Express Functional P2Y1 Receptors-Having demonstrated the involvement of a P2Y receptor in osteoclast calcium wave propagation, we next functionally characterized the member of this receptor superfamily that may be present in our human osteoclast preparations, by measuring nucleotide-induced calcium transients in osteoblast-osteoclast monolayers, as summarized in Table II. We have previously shown that UTP and ATP induce increases in [Ca 2ϩ ] i in human osteoblasts, consistent with the expression of P2Y2 receptors in the osteoblasts.
Monolayers of osteoblast/osteoclast co-cultures were loaded with fura-2, mounted on the stage of the calcium imaging system, and the effect of different nucleotides on the osteoclas-  (Fig. 5). Based on nucleotide specificity of the different purinergic receptors, this pharmacological profile is most consistent with the presence of functional P2Y1 receptors on these cells. Osteoblasts sometimes exhibited a weak calcium response after addition of ADP, again consistent with P2Y2 expression in these cells.
We next confirmed the expression of P2Y1 receptors by RT-PCR amplification of mRNA from human osteoclasts. For these experiments, monocultures of human osteoclasts were produced from human bone marrow mononuclear cells by stimulation with RANKL and M-CSF. RT-PCR using primers specific for the P2Y1 coding sequence and total RNA extracted from isolated osteoclastic cells as a template revealed a clear 685-bp product corresponding to P2Y1 (Fig. 6).
Desensitization of P2Y Receptors Does Not Inhibit Osteoclast Intercellular Calcium Signaling-P2Y receptors, like many other G-protein-coupled receptors, undergo desensitization after ligand binding. Accordingly, we found that a second addition of ADP to osteoclasts was not accompanied by a cytosolic calcium transient (n ϭ 5) (Fig. 5). We used this homologous desensitization to ask whether activation of P2Y receptors on human osteoclasts was required for the propagation of intercellular calcium waves to these cells. After demonstrating the ability to propagate waves from osteoblasts to osteoclasts, we desensitized the osteoclast P2Y1 receptors with ADP and saw an increase in [Ca 2ϩ ] i . Surprisingly, when another mechanical stimulation was applied, intercellular calcium signaling to osteoclasts was unperturbed by the desensitization (Table I). Thus, although osteoclasts express functional P2Y1 receptors, these receptors were not required for the communication of calcium waves from osteoblasts to osteoclasts.
In separate experiments we also used ATP to desensitize P2Y receptors. In all cases both osteoblasts and osteoclasts responded to ATP stimulation with an increase in [Ca 2ϩ ] i . Subsequent mechanical stimulation of an osteoblast resulted in a calcium transient in the stimulated cell. Whereas intracellular calcium did not increase in neighboring osteoblasts, as expected, neighboring osteoclasts propagated a calcium wave as they did before ATP desensitization (Table I). Thus, P2Y receptors are required for intercellular calcium signaling among osteoblasts but not for calcium waves among osteoclasts.

FIG. 4. The gap junction inhibitor heptanol does not affect the propagation of a calcium signal to osteoclasts in response to mechanical stimulus.
The left-hand panel shows a monolayer of osteoclastic cells before stimulation. In the middle panel one single osteoclast is stimulated mechanically with a glass micropipette, and a calcium wave is generated to neighboring osteoclasts (as shown in the right-hand panel), even in the presence of heptanol. Osteoclasts are obtained by the RANKL and M-CSF method as described in the text.
Osteoclasts Express Functional P2X7 Receptors, Which Are Required for Osteoclast Calcium Signaling-The pharmacological studies revealed that, although P2Y1 receptors are present in osteoclasts, they are not involved in propagation of mechanically induced calcium waves among osteoclasts. Because several P2X receptors, namely P2X2, P2X4, and P2X7, have been detected in osteoclastic cells, we then asked whether members of this class of P2 receptors may be responsible for interosteoclast calcium waves. We first tested for the presence of functional P2X receptors in the osteoblast-osteoclast co-cultures using BzATP, a P2X agonist with relative specificity for P2X7. BzATP (100 M) increased [Ca 2ϩ ] i of osteoclasts in all experiments (n ϭ 9) (Fig. 5) but had no effect on cells of the osteoblastic lineage. When we preincubated cell monolayers with the P2X7 receptor antagonist, oxidized ATP (oATP), the BzATP-induced calcium increase was blocked (n ϭ 4) (Fig. 5). These experiments demonstrated that osteoclasts but not osteoblasts expressed functional P2X7 receptors and showed that osteoclast P2X7 activity could be inhibited by oATP.
Activation of P2X7 receptors with ATP results in formation of a plasma membrane pore that allows molecules Ͻ900 Da to traverse the plasma membrane. To confirm P2X7 activity in the osteoclastic cells, we assessed ATP-induced uptake of Lucifer Yellow in the co-cultures by incubation with Lucifer Yellow in the presence of 100 M BzATP for 10 min. In the presence of BzATP, Lucifer Yellow uptake occurred in osteoclastic, but not osteoblastic cells, demonstrating ATP-induced pore formation selectively in osteoclasts. In the absence of the agonist, cytoplasmic dye uptake of Lucifer Yellow was not seen (Fig. 7). Thus, human osteoclasts express functional P2X7 receptors in the plasma membrane, as do monocytes and macrophages. We then determined the expression of P2X7 mRNA and protein by osteoclasts. As described above, human osteoclast monocultures induced by RANKL were used to selectively detect the presence of P2X7 in these cells. P2X7 mRNA was clearly detected by RT-PCR using primers specific for the P2X7 coding sequence of total osteoclast RNA, (Fig. 8). Immunochemical staining using a Cy3-labeled anti-P2X7 antibody confirmed the presence of this receptor on the surface of osteoclastic cells (Fig. 9) but only faint staining in the osteoblastic cells.
Osteoblast-Osteoclast Intercellular Calcium Signaling Requires Functional P2X Receptors-We next asked whether P2X7 receptors mediate intercellular calcium signaling between osteoblasts and osteoclasts. After an initial mechanically induced wave, we incubated cells in medium containing 300 M oATP, which did not result in an increase in the [Ca 2ϩ ] i . A second mechanical stimulation was applied to an osteoblast, and a calcium signal was propagated to surrounding osteoblasts in 11 of 12 experiments. In contrast, in only 2 of 18 experiments the signal was propagated to a single osteoclast in the field of view, whereas in 16 experiments the signal was not propagated to osteoclasts at all. Thus, P2X7 receptors seem to be responsible for the propagation of intercellular calcium signaling between osteoblasts and osteoclasts (Table I).
To verify that the addition of oATP did not affect the cells P2Y1 response, we first added oATP to the cell culture. As expected, the intracellular calcium concentration was not affected. Subsequently we added ADP to stimulate P2Y1 receptors, and the osteoclasts showed an increase in intracellular calcium concentration (n ϭ 3; Fig. 5). Thus oATP did not inhibit the P2Y1-mediated response. We also confirmed that the cal-

FIG. 5. P2 receptor agonist and antagonist responses on intracellular calcium concentrations in cultures of human osteoclastic cells.
a, ADP is added to a monolayer of osteoclastic cells to desensitize P2Y1 receptors. A second ADP addition induces no increase in intracellular calcium concentration, supporting the desensitization of the P2Y1 receptor. In contrast, the response to BzATP is not affected by ADP treatment, indicating that ADP does not desensitize P2X7 receptors. b, osteoclastic cells treated with BzATP without any pretreatment respond with an increase in intracellular calcium concentration. c, the relatively specific P2X7 antagonist, oATP, was added to a culture of osteoclastic cells. No change in intracellular calcium was seen. A subsequent addition of BzATP was now unable to increase calcium, indicating the ability of oATP to block P2X7 receptor activation. In contrast, adding ADP after oATP, cells increased in intracellular calcium concentration, showing that the response of ADP binding to P2Y1 receptors is not affected by oATP action. The scale bar shows intracellular calcium concentrations in micromolar. Osteoclasts were obtained from human bone marrow, and osteoclast phenotype was induced by RANKL and M-CSF as described in the text. BzATP, benzoyl-benzyl-ATP; oATP, oxidized ATP. cium response elicited by P2X receptor activation was not subject to desensitization by ADP. Although, as mentioned above, incubation in medium containing ADP prevented a second ADP-induced calcium transient, ADP did not prevent a subsequent calcium transient in response to BzATP (Fig. 5).
Osteoclast-Osteoclast Calcium Signaling Also Requires P2X but Not P2Y Receptors-We finally wanted to determine the mechanism by which osteoclasts communicate calcium signals to each other. First, we examined the role of purinergic receptors by examining areas in the co-cultures with two or more osteoclastic cells in close proximity to each other. One single osteoclast was mechanically stimulated, and this generated the expected increase in [Ca 2ϩ ] i that rapidly propagated to the neighboring osteoclasts. To desensitize receptors of the P2Y type, ATP was added (100 M to 1 mM), and an increase in [Ca 2ϩ ] i was seen. The mechanical stimulation was repeated, and a calcium wave was again propagated to the neighboring osteoclasts (n ϭ 8). The experiment was also done with ADP (100 M), and no inhibition of the calcium signaling was seen either (n ϭ 4; Table I). These results demonstrate that inter-cellular calcium signaling between osteoclasts does not depend on receptors of the P2Y type. By contrast, in the presence of oATP at a final concentration of 300 M, mechanical stimulation of osteoclasts did not induce a calcium wave, because a wave was seen only in 16 of 18 experiments (Table I). Thus, P2X7 receptors are involved in the propagation of intercellular calcium signals to osteoclast upon mechanical stimulation of either osteoblasts or osteoclasts. DISCUSSION These studies demonstrate that intercellular calcium signaling can occur between osteoblasts and osteoclasts and among osteoclasts. In addition, they reveal that the mechanism by which osteoclasts respond to calcium signals is different from the mechanisms we previously identified for calcium signaling among osteoblasts. Whereas osteoblast intercellular calcium signaling is propagated either by gap junctional communication or by autocrine activation of P2Y2 receptors, intercellular calcium signaling to osteoclasts requires activation of P2X7 receptors. Thus, these studies not only demonstrate that intercellular calcium signaling is a mechanism by which bone-forming and -resorbing cells can directly communicate, but they also reveal that different molecular mechanisms are responsible for this form of communication between and among the two major bone cell types.
In these studies, summarized in Table II, we found that human osteoclastic cells respond to nucleotide stimulation with an increase in intracellular calcium concentration. The cells express both purinergic receptors of the P2Y and P2X subclasses, as determined by nucleotide affinity. The pharmacological profiles of the nucleotide receptors as well as the presence of P2Y1 and P2X7 mRNA and P2X7 protein is consistent with expression of P2Y1 and P2X7 on osteoclasts, but other P2 receptors, especially other P2X receptors, may also be present. We also found that human osteoblasts-osteoclasts networks can propagate calcium transients in response to mechanical stimulation and that propagation of these signals involves the action of ATP or other nucleotides on membrane-bound purinergic receptors. These calcium signals can also be transmitted between osteoclasts by P2X7 receptors. Finally, we show that prolonged ATP stimulation of the osteoclastic cells induces permeabilization of the plasma membrane, presumably via pore formation of the P2X7 receptor, a phenomenon previously described for macrophages (16), lymphocytes (17), and dendritic cells (18).
It is commonly believed that signaling from osteocytes through their processes in the canaliculi to surface osteoblasts or lining cells is the mechanism by which mechanical signals are diffused through the bone tissue and modulate bone cell activity. The propagation of calcium transients from osteoblasts to osteoclasts represents an additional mechanism of intercellular communication that allows signals to be transmitted from cells of the osteogenic lineage to bone-resorptive, osteoclastic cells. Our work demonstrated that osteoblasts utilize the autocrine action of ATP and specific purinergic receptors to propagate calcium signals to osteoclasts, and it is known that rat osteoclasts (3,4) and human osteoclastoma giant cells (6) respond to ATP with an increase in intracellular calcium concentrations. Thus, P2 receptor-mediated signaling between osteoblasts and osteoclasts may represent a mechanism by which bone remodeling is controlled.
The effect of ATP on osteoclasts seems to be biphasic. At relatively low concentrations (0.2-2.0 M), ATP increased osteoclast formation and resorption pit formation up to 5.6-fold in neonatal rat co-cultures of osteoblasts and osteoclasts, as well as in a model system based on mouse bone marrow (8). The slowly hydrolyzable ATP analog ATP␥S can also induce bone resorption in human osteoclastoma giant cells at 10 M (6). Conversely, at higher concentrations (20 -200 M), ATP reduces or even blocks osteoclast formation by a selective cytotoxic effect that does not alter osteoblast viability (8). This correlates well with other studies finding a cytotoxic effect of ATP in millimolar concentrations on other cells of hemopoietic origin, e.g. macrophages (19,20). Osteoclasts are part of the mononuclear phagocyte system with precursors present in both bone marrow and in the circulating blood and share precursors with macrophages (21). In macrophages, the P2X7 receptor is involved in the control of cell death (22). In the present study, we detected the presence of this receptor in osteoclastic cells. This may be a mechanism for high dose ATP-induced cell death in osteoclasts. The presence of multiple P2Y and P2X receptors in osteoclasts may allow generation of different responses to ATP and perhaps to other nucleotides, depending upon ligand concentration and/or receptor density in different stimulatory conditions, thus accounting for the seemingly inconsistent effect of ATP on bone resorption reported by others (8).
It is known that lowering the pH in the osteoclast cytoplasm increases the bone-resorbing activity. In vitro studies have shown that ATP stimulation can decrease the intracellular pH in rabbit osteoclasts (7). This might occur by opening of P2X receptors/channels, thereby letting protons enter the cytoplasm. This could occur if the osteoclastic cells had P2X receptors of other subtypes than P2X7, or if the ATP concentration was low enough not to induce large pore formation in the P2X7 molecule.
However, it is important to keep in mind that the model system used did not necessarily mirror the in vivo mechanisms. This model is based on an ex vivo system, because there is not yet a good method available to study these events in vivo.
Factors that might affect the degree of bias are the lack of cell-matrix interaction, the relatively non-physiological stimulus of cell poking, and the limitations of pharmacological methods. Though a lot of effort has been put into culturing cells as close to "in vivo" osteoclasts and osteoblasts as possible, in vitro cultured cells still lack some of the exact phenotypic characteristics and stimuli present in vivo.
In conclusion, we provide evidence for a novel mechanism by which calcium signals can be transmitted from osteoblasts to osteoclasts by means of activation of P2X7 receptors. Although the ultimate physiological role of P2X7 receptor activation for osteoclast function remains to be determined, the ability to exchange short-range calcium signals in the bone microenvironment via paracrine or autocrine secretion of nucleotides may be important for osteoblast modulation of osteoclast function. Furthermore, the cell type-specific roles of P2Y2 and P2X7 receptors in osteoblasts and osteoclasts, respectively, and their unique pharmacological profiles in theory allows selective modulation of signal diffusion to either osteoblasts or osteoclasts by pharmacological agents, thus offering novel therapeutic strategies for modulation of bone turnover.