1. ABSTRACT

Skeletal development and bone remodeling depend on the coordinated activity of osteoblasts and osteoclasts, which are responsible for bone formation and resorption, respectively. Mature osteoclasts result from the fusion of precursor cells, and they are large, multinucleated, highly specialized cells. Cellular release of ATP and UTP occurs in response to a variety of stimuli including mechanical stimulation, which occurs in the bone environment. ATP and UTP or their metabolites can then act on P2 receptors in the plasma membrane to induce various responses in bone cells. The influence of these receptors on osteoclast physiology and bone physiology in general is beginning to be understood, but much work is still required. This review focuses on P2 receptors in osteoclasts, their expression, signaling and function in the regulation of osteoclast formation, resorptive activity and survival.

2. INTRODUCTION

2.1. Osteoclast biology

Osteoclasts originate from the fusion of mononucleated precursor cells which derive from bone marrow cells of the monocyte/macrophage lineage (1). Even though many regulatory signals are involved in this complex process of osteoclast formation (osteoclastogenesis), macrophage colony stimulating factor (M-CSF) and receptor activator of NF-k B (RANK) ligand (RANKL) have been identified as key factors (2). Exposure of monocytes to a combination of M-CSF and RANKL is sufficient to induce osteoclastogenesis in vitro (3). Many other signaling molecules acting downstream of these key factors are necessary for osteoclastogenesis. For example, mice lacking functional NF-k B display osteopetrosis, as the result of complete inhibition of osteoclast formation (4, 5). After differentiation, mature osteoclasts continue to be regulated by multiple extracellular signals, which influence functions such as migration, bone resorption and, ultimately, programmed cell death.

Osteoclasts are highly motile cells whose motion depends on the extension and retraction of pseudopodia/lamellipodia (6-8). Reversible integrin-mediated adhesion of pseudopodia to the extracellular matrix allow the generation of contractile force (9). The main integrin expressed by osteoclasts is a _{vb 3} (10-12). Osteoclasts isolated from mice lacking the integrin b ₃ subunit display significant impairment of pseudopodia spreading and deficient resorptive activity, giving rise to an osteopetrotic phenotype (13, 14). Chemotactic factors influence osteoclast migration (7, 15, 16). A resorption trail is often observed behind a resorbing osteoclast as it migrates along the bone surface in vitro, indicating that migration and bone resorption are not mutually exclusive processes (17).

At sites of resorption, osteoclasts attach to the surface of bone, forming a circumferential zone - the sealing zone - where plasma membrane proteins (e.g., a _{vb 3} integrins) bind to the extracellular matrix (18). This region of the cell is characterized by the presence of a ring of actin filaments, which delimits a specialized membrane referred to as the ruffled membrane due to its multiple invaginations (18, 19). The osteoclast isolates the region of bone surface that lies below the ruffled membrane - the so-called resorption lacuna - where resorption takes place. This process involves dissolution of the bone mineral as well as enzymatic degradation of the organic matrix. Dissolution of the mineral is enabled by acidification of the resorption lacuna, which is mediated by vacuolar-type ATP-driven proton pumps that are highly localized at the ruffled membrane. Degradation of the organic components is carried out by secreted proteases, such as cathepsin K (19).

The overall rate of bone resorption depends on the number of osteoclasts present in the bone and the intensity of their resorptive activity. An increase in osteoclast survival (prolonged life span) results in more resorptive osteoclasts present in bone and higher overall rate of resorption (20). Apoptosis is tightly regulated by signals received by the cell, which eventually activate an internal death program. In this regard, it is known that osteoclasts undergo apoptosis when exposed to a variety of stimuli (21-23). Indeed, subtle changes in the rate of osteoclast apoptotic death are proposed to have significant impact on overall bone resorption and remodeling (20, 21, 23).

2.2. P2 receptors

P2 receptors are activated by purines and pyrimidines (ATP, ADP, UTP, UDP, UDP-glucose) and are classified into two families, the metabotropic P2Y receptors and the ionotropic P2X receptors. These receptors are present in a variety of cell types (24), including osteoclasts (25-27). P2Y are G protein-coupled receptors, with seven transmembrane domains. P2X receptor channels are formed by three subunits, assembled either as homo- or heterotrimers. A number of excellent reviews have summarized P2 receptor biology and physiological functions (24, 28-31).

3. Expression, signaling and function of P2X receptors in osteoclasts

Seven members of the P2X receptor family are currently known: P2X_1-7. Gating of a non-selective cation channel occurs upon binding of extracellular ATP. The crystal structure of the Danio rerio (zebrafish) P2X₄ homotrimeric receptor has recently been solved (Fig. 1) (32). This outstanding discovery will certainly boost our understanding of P2X receptors. The structure reveals a membrane pore formed by the three transmembrane helices (TM2; one from each subunit of the trimer). The three suggested intersubunit ATP binding sites are ~ 45 Å from the ion channel domain. Conserved residues located at the interface between the transmembrane domain and the extracellular domain are proposed to propagate conformational changes from the ATP binding sites to the gate.

Among the mechanisms involved in P2X receptor signaling are changes in membrane potential, protein-protein interactions and Ca²⁺ influx through the channel (28). Although there is evidence for the expression of P2X_2,4,7 in osteoclasts (Table 1), electrophysiological studies only support the functional expression of P2X₄ and P2X₇ (33, 34).

3.1. P2X₂ receptors

Expression of P2X₂ receptors in rat osteoclasts has been shown by immunocytochemistry and in situ hybridization (35). However, there is no electrophysiological evidence for functional expression of this receptor at the plasma membrane of osteoclasts. Thus, it is possible that P2X₂ receptors are located in intracellular compartments. In this regard, it has been reported recently that another subtype of P2X receptor (P2X₄) resides mainly within intracellular compartments in rodent microglia and macrophages, where it may play important roles in organelles such as phagosomes. Trafficking of P2X₄ receptors between intracellular compartments and the plasma membrane was observed and it was enhanced by microglial activation with lipopolysaccharide (36). Hence, it is possible that - as happens with P2X₄ receptors in microglia and macrophages - P2X₂ receptors may be present intracellularly in osteoclasts.

3.2. P2X₄ receptors

Rat osteoclasts show expression of P2X₄ receptors by immunocytochemistry and in situ hybridization (35). Transcripts coding for P2X₄ were also detected by RT-PCR in rabbit osteoclasts purified by micromanipulation (33).

Naemsch et al. monitored currents activated by ATP and other purinergic agonists in rabbit osteoclasts using the whole-cell patch-clamp technique (33). When the cells were held at a negative potential (-30 mV), a biphasic pattern of ATP-activated currents was observed. A transient inward current was activated first, followed by a transient outward current (Fig. 2A). The outward current was dissociated from the initial current (i.e., only outward current was activated) by stimulation with the P2Y agonist ADPb S (Fig. 2B). Additional experiments showed that this latter component is a Ca²⁺-dependent K⁺ current activated by P2Y receptor-mediated release of Ca²⁺ from intracellular stores (33, 37).

The initial transient inward current reversed near 0 mV when the pipette and bath solutions contained physiological concentrations of K⁺ and Na⁺ (Fig. 2A and C), indicating that the current was non-selective for Na⁺ and K⁺. Furthermore, when extracellular Na⁺ concentration was lowered from 135 mM to 5 mM, there was a dramatic shift (~ 30 mV) in the reversal potential towards more negative values (33). This is expected for channels with high Na⁺ conductance. Reversal potential of the current did not change when chloride in the pipette solution was replaced by aspartate, indicating that no significant anion conductance was activated by ATP. Moreover, replacement of K⁺ by Cs⁺ in the pipette solution did not affect the reversal potential of the transient inward current, indicating that no Cs⁺-sensitive K⁺ channels were involved. Hence, these experiments established the expression of ATP-activated, non-selective cation channels in osteoclasts.

P2X subtypes display diverse current kinetics in response to ATP. Activation of the current occurs rapidly after ATP exposure in most subtypes. However, when exposure is prolonged, a rapid decline to basal levels is observed in some subtypes (P2X_1,3 - fast desensitization), whereas it is persistent in other subtypes (P2X_5,7 - slow desensitization). The inward current described in rabbit osteoclasts had a time course of desensitization that was well described by a single exponential, with an intermediate time constant of ~ 4 seconds (33). This value falls close to those reported for the P2X₄ receptor expressed in HEK-293 cells (38, 39).

The osteoclast current was elicited by 100 m M ATP, ATPg S or ADP, but not by 100 m M ADPb S, UTP or a ,b -methylene-ATP (33). This agonist profile rules out the involvement of some P2X receptors - such as P2X₁ or P2X₃ - but does not discriminate between P2X₂ and P2X₄ (40). In addition, the current was potentiated by Zn²⁺. However, both P2X₂ and P2X₄ receptors are potentiated by Zn²⁺ (40, 41).

Experiments making use of the P2X receptor antagonists Cibacron blue (CB) and suramin gave further evidence for the involvement of P2X₄ receptors (33). Both CB and suramin (100 m M each) failed to inhibit the current. CB is a weak antagonist of P2X₄ receptors (IC₅₀ >300 m M), but a potent antagonist of P2X₂ receptors (IC₅₀ 0.6-0.8 m M) in HEK-293 cells (42). Thus, the lack of inhibition by CB points to P2X₄ receptors rather than P2X₂ receptors. In the same way, it is well known that suramin is a poor antagonist at the recombinant P2X₄ receptors (IC₅₀ >300 m M), whereas it is a more potent antagonist of recombinant P2X₂ receptors (IC₅₀ 8-10 m M) (40). Taken together, these results established that the transient inward ATP-activated current described in rabbit osteoclasts by Naemsch et al. is mediated by P2X₄ receptors.

3.2.1. Surprising lack of Ca²⁺ permeability of P2X₄ receptors in osteoclasts

Weidema et al. investigated whether P2X₄ receptors induce Ca²⁺ entry when activated in rat and rabbit osteoclasts (43). Their conclusion, supported by three convincing lines of evidence, was that Ca²⁺ entry through P2X₄ receptors in osteoclasts is null or negligible.

First, it was observed that elevation in the concentration of cytosolic free Ca²⁺ ((Ca²⁺)_i) elicited by 20 m M ATP was abolished by 100 m M CB. This concentration of ATP is sufficient to activate osteoclast P2X₄ receptors (33) as well as P2Y receptors (44). Since 100 m M CB is unable to block the P2X₄-mediated current (33), then Ca²⁺ entry through P2X₄ channels should still be expected in the presence of CB (provided that the P2X₄ channels are permeable to Ca²⁺). Since no Ca²⁺ response was observed in the presence of CB, the authors concluded that there was no Ca²⁺ entry through P2X₄ receptor channels.

Second, the Ca²⁺ responses to agonists which activate P2X₄ receptors as well as P2Y receptors (e.g., 2-methylthioadenosine 5'-triphosphate, 2-MeSATP) were virtually identical in the presence and absence of extracellular Ca²⁺ (37, 43). This result indicates negligible Ca²⁺ entry through P2X₄ receptors.

Third, when osteoclasts were stimulated with 20 m M ATP, both P2X₄ and P2Y receptors were activated. To eliminate the contribution of P2Y receptors to the Ca²⁺ response elicited by ATP, osteoclasts were incubated with the phospholipase C inhibitor U73122. By means of simultaneous measurement of membrane currents and (Ca²⁺)_i, it was observed that osteoclasts incubated with U73122 and stimulated with 20 m M ATP did not show Ca²⁺ responses. However, they still displayed the typical P2X₄-mediated inward current (Fig. 3). This was a direct demonstration that, even when P2X₄ receptor channels are functional, there is no detectable entry of Ca²⁺. The reason why P2X₄ receptors in rat and rabbit osteoclasts have negligible Ca²⁺ permeability is not clear. The P2X₄ receptor cloned from rat brain has high Ca²⁺ permeability when expressed in a heterologous system (45). An interesting hypothesis to account for this observation is the existence of cell-specific endogenous regulators of P2X₄ ion permeability.

3.3. P2X₇ receptors

In comparison with other P2X subtypes, P2X₇ receptors exhibit distinctive features, such as the requirement for relatively high concentrations of ATP for activation and 10-30 times more potent activation by 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) than by ATP itself. Formation of large pores that allow permeation of hydrophilic molecules - up to 900 Da - is another feature of P2X₇ receptor activation in many cell types, although this has also been reported for P2X₂ and P2X₄ receptors (28).

Expression of P2X₇ receptors in osteoclasts is well documented. In human osteoclasts, mRNA coding for P2X₇ receptor has been detected by RT-PCR (46-48) and expression of the receptor at the protein level has been shown by immunocytochemistry (47-50). As well, in rabbit (34) and rat (35) osteoclasts, P2X₇ expression has been shown by immunocytochemistry.

3.3.1. Electrophysiology

In rabbit osteoclasts, Naemsch et al. characterized P2X₇-mediated currents using the whole-cell, patch-clamp technique (34). The patch pipette solution contained CsCl to avoid contamination by Ca²⁺-dependent K⁺ current. Application of 100 m M ATP induced a current that activated and desensitized quite rapidly (Fig. 4A and B), which was probably mediated by P2X₄ receptors. In contrast, when 100 m M BzATP or a higher concentration of ATP (1 mM) was applied, another current with distinctive characteristics was observed. In these experiments, ATP or BzATP still evoked the initial transient P2X₄ current, but it was followed by current that progressively increased in amplitude with successive stimulations (Fig. 4C), resembling the activity-dependent behavior of P2X₇ receptors (51, 52).

This activity-dependent current was inwardly rectifying and had a reversal potential close to 0 mV (Fig. 4D), as expected for the non-selective cation pore of P2X₇. When ATP or BzATP stimulation was maintained for several seconds, the current did not desensitize but was sustained (34) - a typical feature of P2X₇-mediated current (28). In contrast to the behavior of the P2X₄-mediated current, the activity-dependent current was inhibited by Zn²⁺ (as well as Ca²⁺ and Mg²⁺) (34), resembling P2X₇ receptors (53). Oxidized ATP or pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (100 m M) inhibited the activity-dependent current (34), consistent with P2X₇ receptors but not P2X₄ receptors (40).

In another study, osteoclasts were isolated from control mice and mice in which the gene encoding P2X₇ (P2rx7) had been disrupted (54). Electrophysiological responses to BzATP or high concentrations of ATP were then compared. A current similar to that described in rabbit osteoclasts was found in the control (wild-type) murine osteoclasts, but it was completely absent in osteoclasts isolated from P2rx7-deficient (P2rx7^-/-, knockout) animals (Fig. 5).

3.3.2. Permeabilization of the plasma membrane

ATP-induced permeabilization of the plasma membrane to molecules up to 900 Da is a hallmark of P2X₇ expression. However, there are some exceptions where P2X₇ receptors are functionally expressed, but pore formation is not observed (55-57). The molecular identity of the permeation pathway responsible for this phenomenon remains controversial. Pore formation is usually evidenced by cellular uptake of normally impermeant fluorescent dyes (such as ethidium or YO-PRO^®) or by means of electrophysiology. In the latter case, an increase in permeation to large cations (such as N-methyl D-glucamine⁺, NMDG⁺) is manifested by time-dependent shifts in the reversal potential of the current.

ATP-induced fluorescent dye uptake has been reported in human (47, 48) and mouse (58, 59) osteoclasts. However, in rabbit osteoclasts, the reversal potential of the BzATP-activated current does not change over time in the presence of NMDG⁺ (34).

3.3.3. Signaling

3.3.3.1. Ca²⁺ influx

Ca²⁺ influx through P2X₇ receptors may contribute importantly to signaling in osteoclasts. Simultaneous patch-clamp and Ca²⁺ fluorescence recordings showed that successive stimulation of rabbit osteoclasts with BzATP resulted in progressively increasing elevations of (Ca²⁺)_i. These Ca²⁺ signals were strongly correlated with the activity-dependent current mediated by the P2X₇ receptor (34). Indeed, removal of extracellular Ca²⁺ abolished these increases in (Ca²⁺)_i. The P2X₇ receptor antagonists oxidized ATP and PPADS inhibited both the activity-dependent Ca²⁺ responses and currents elicited by BzATP (34). Since P2X₇ and P2Y-mediated Ca²⁺ signals are expected to have different time courses and amplitudes, divergent cellular responses may arise from activation of P2X₇ or P2Y receptors in osteoclasts.

3.3.3.2. Signaling through PKC

Protein kinase C (PKC) comprises a family of serine/threonine protein kinases which play important roles in intracellular signaling. The conventional isoforms, such as PKCa and PKCb 1, depend on increases in (Ca²⁺)_i for activation. Upon activation, PKC is recruited to lipid membranes, where it phosphorylates proteins involved in signaling. Armstrong et al. investigated whether P2X₇ receptors induce activation of PKC in osteoclasts (60). Mouse bone marrow cells and macrophage RAW 264.7 cells were differentiated into multinucleated osteoclasts and osteoclast-like cells, respectively. Confocal imaging was used to localize enhanced green fluorescent protein (EGFP)-tagged PKC. Stimulation of these cells with BzATP (150 m M) induced transient translocation of PKCa and PKCb I to the plasma membrane (Fig. 6), but did not induce translocation of the novel, Ca²⁺-independent isoform PKCd (60).

Low concentration of ATP or UTP (150 m M) failed to induce PKC translocation, ruling out the involvement of P2Y receptors or other members of the P2X family, apart from P2X₇. In addition, BzATP failed to induce PKC translocation in osteoclasts derived from the bone marrow of P2rx7^-/- mice, confirming that P2X₇ receptors mediate this response. Time courses of BzATP-induced elevations in (Ca²⁺)_i and translocation of PKC were tightly correlated. Moreover, chelation of external Ca²⁺ with EGTA abolished PKC translocation.

In summary, the evidence strongly supports that P2X₇-mediated Ca²⁺ influx is responsible for activation of conventional PKCa and PKCb I isoforms in osteoclasts (60). However, the signaling functions of PKC downstream of the P2X₇ receptor in osteoclasts are not yet well understood.

3.3.3.3. Signaling through NF-k B

The transcription factor NF-k B resides in the cytoplasm in its inactive form. After activation, it translocates to the cell nucleus, where it regulates transcription of several genes involved in immune responses, inflammation, cell survival and cancer (61). This transcription factor plays an important role in osteoclast development (4, 5). Korcok et al. studied whether P2X₇ acts upstream of NF-k B activation in osteoclasts isolated from mice and rabbits (62). Stimulation with BzATP induced nuclear translocation of NF-k B in both rabbit and mouse cells. NF-k B translocation effect was transient, reaching a maximum about 30 minutes after exposure to the agonist and returning to basal levels an hour later. Low concentration of ATP (10 m M) or adenosine (10 m M) failed to reproduce the effects of BzATP, consistent with the involvement of P2X₇ receptors. Furthermore, the BzATP-induced translocation was not observed in osteoclasts isolated from P2rx7^-/- mice.

3.3.4. Physiological roles of P2X₇ receptors in osteoclasts

3.3.4.1. Role of P2X₇ in the formation of multinucleated osteoclasts

Fusion of precursor cells to form multinucleated osteoclasts is a complex phenomenon not completely understood. The discovery that a high expression level of P2X₇ receptors in macrophages promotes multinucleated giant cell formation (63) encouraged the idea that these receptors are involved in the formation of mature osteoclasts. Evidence in support of this possibility includes: 1) osteoclast formation can be prevented by incubation with an anti-P2X₇ antibody (48); 2) prolonged exposure to ATP results in extensive internalization of P2X₇ receptors and blocks the ability of RAW 264.7 cells to fuse into multinucleated osteoclast-like cells (59); and 3) some P2X₇ receptor antagonists block the formation of osteoclast-like cells from human blood monocytes in vitro (64).

However, observations using P2rx7^-/- mice indicated that P2X₇ receptors do not play an essential role in the formation of these cells in vivo and in vitro. Two independent groups have reported that knockout animals have multinucleated osteoclasts (54, 65). In fact, the number of osteoclasts in trabecular bone is higher in knockout compared to wild-type animals (54). Since P2X₇ expression renders some cells more susceptible to apoptosis, this observation may be related to potentiation of osteoclast survival rather than enhanced osteoclastogenesis. In addition, no differences were observed between wild-type and knockout mice regarding the ability of osteoclast precursors to differentiate and fuse into multinucleated osteoclasts in vitro (54, 65).

3.3.4.2. Role of P2X₇ in osteoblast-osteoclast and osteoclast-osteoclast communication

Signaling between osteoblasts and osteoclasts is important to maintain the balance between bone formation and resorption (66). ATP released during mechanical stimulation could play an important role in intercellular communication. In this regard, it has been suggested that P2X₇ receptors in osteoclasts mediate mechanically induced intercellular signaling between osteoblasts and osteoclasts, and among osteoclasts (47). It was proposed that mechanical stimulation causes ATP release, which induces (Ca²⁺)_i elevation in adjoining cells. Lack of desensitization and sensitivity of the response to oxidized ATP led to the suggestion that this response involves P2X₇ receptors in osteoclasts. However, oxidized ATP is not a specific P2X₇ antagonist (28), making it difficult to rule out the possible involvement of other subtypes of Ca²⁺-mobilizing P2 receptors. Regardless of which P2 receptors underlie the responses, these findings are important and more research is clearly needed in this area.

3.3.4.3. Role of P2X₇ in regulation of the osteoclast cytoskeleton

Remodeling of the actin cytoskeleton has been recently proposed as a novel function for osteoclast P2X₇ receptors. Stimulation of osteoclasts with a high concentration of ATP or with BzATP rapidly (within a few minutes) induced disruption of filamentous actin belts, whereas low concentrations of ATP had no significant effect (50, 67). This disruption was transient and actin reorganization was observed afterwards. Hazama and coworkers also reported that actin reorganization was accompanied by microtubule deacetylation, resulting in enhanced osteoclastic bone resorption (50).

3.3.4.4. Skeletal phenotype of P2rx7 knockout mice

Ke et al. described the skeletal phenotype resulting from disruption of the gene encoding the P2X₇ receptor in mice (54). Femurs and tibias of these mice and matched littermate controls were analyzed in detail. In femurs, it was observed that knockout animals had significant reduction in periosteal circumference (bone diameter), but not in length (Fig. 7A and B). This result indicates that P2X₇ has a role in regulating radial bone growth (periosteal bone formation) and expansion of the bone marrow cavity, but not longitudinal bone growth. In addition, femurs displayed significant reduction in cortical bone content and total bone content, as evaluated by peripheral quantitative computed tomography. In tibias, trabecular bone displayed an increased number of osteoclasts and a significant reduction in mass. This result suggests that loss trabecular bone mass was caused by increased osteoclast-mediated resorption. In addition, periosteal bone formation was significantly decreased (Fig. 7C).

Although both male and female knockouts showed these features, they were more pronounced in males. This suggests a link between sex hormones and P2X₇ signaling. In this regard, an electrophysiological study indicates that the P2X₇ receptor is inhibited by 17b -estradiol (68). It has also been reported that estrogen attenuates P2X₇ receptor-mediated apoptosis of uterine cervical cells by blocking calcium influx (69).

Ke and coworkers also studied whether osteoclastogenesis was affected in P2X₇-deficient mice. As mentioned above, osteoclasts with normal appearance were isolated from their long bones. In addition, when bone marrow cells were treated with M-CSF and RANKL, no significant difference in osteoclast formation was observed in wild-type and P2rx7^-/- cultures (54).

Interestingly, the skeletal phenotype of a second P2rx7^−/− mouse model has been described by Gartland and coworkers (65). In contrast to the findings of Ke et al. (54), these mice showed no overt skeletal phenotype with the exception of thicker cortical bones than wild-type controls. This discrepancy may be due to the presence of a splice variant that escaped deletion in these P2rx7^−/− mice, resulting in inadvertent tissue-specific expression of functional P2X₇ receptors (70, 71). However, it has yet to be reported whether escape from deletion occurs in osteoclasts from these knockout animals.

Based on findings from the P2rx7 knockout mouse described by Ke and coworkers (which have no functional P2X₇ receptors), P2X₇ receptors appear to be required for normal skeletal growth and anabolic responses to mechanical stimulation (72). Although P2X₇ receptors do not appear to be essential for osteoclastogenesis or bone resorption, the balance between bone formation and resorption may be affected in the knockout animals. Since P2X₇ plays a role in osteogenesis (73) and may contribute to osteoblast-osteoclast communication (47), the absence of this receptor could result in misbalance between the activities of these cell types.

3.3.4.5. P2X₇ polymorphisms in humans

Several genetic polymorphisms have been reported for the P2X₇ receptor. In particular, the Glu496Ala polymorphism results in a receptor which is deficient in ATP-induced pore formation (ethidium uptake) and promotion of apoptotic cell death (74). However, its function as an ion channel remains unaffected (75). The Glu496Ala polymorphism affects function but does not affect trafficking of the receptor to the plasma membrane (74). In contrast, another polymorphism, Ile568Asn, prevents normal trafficking and function of the receptor (76). Ohlendorff et al. have reported that 10-year fracture incidence in postmenopausal women is significantly associated with the Glu496Ala and Ile568Asn polymorphisms of the P2X₇ receptor (77). The authors also show that the Glu496Ala polymorphism results in decreased susceptibility of osteoclasts to ATP-induced apoptotic death. Impaired osteoclast apoptosis may enhance overall bone resorption, consistent with the increase in facture incidence observed by Ohlendorff and coworkers (77) and with the phenotype of the P2rx7^-/- mouse described by Ke et al. (54). More details can be found in this special issue's review on P2 receptor polymorphisms and bone (78).

4. Expression, signaling and function of P2Y receptors in osteoclasts

Currently, the P2Y receptor family is known to be composed of eight members: P2Y_{1,2,4,6,11,12,13,14}. They are G protein-coupled receptors and, in many cases, activate phospholipase C as a downstream effector for the formation of inositol 1,4,5-trisphosphate, which in turn causes release of Ca²⁺ from intracellular stores. Different P2Y receptors can be distinguished by their pharmacological properties. For example, P2Y₁ receptors are activated preferentially by ADP. On the other hand, P2Y₂ receptors are activated by ATP and UTP with approximately the same potency, and P2Y₆ receptors by UDP (44). When ATP or UTP are used as agonists, the results should be interpreted cautiously because the responses could be mediated by the nucleotides themselves or by their metabolites (e.g., ADP, UDP) generated by ectonucleotidase activity.

The precise roles of P2Y receptors in osteoclasts remain to be fully established. Nevertheless, biochemical and functional evidence indicates their expression in osteoclasts (Table 2). Korcok et al. investigated P2Y receptor expression by RT-PCR (79). Transcripts for P2Y_{1, 2, 6,11} as well as alkaline phosphatase (an osteoblast marker) were observed in samples obtained from cultured rabbit bone marrow cells - containing osteoclasts and other cell types. In contrast, when an additional step was carried out to purify osteoclasts, only P2Y_1,2,6 transcripts were observed (Fig. 8A). It is worth noting that expression of P2Y₄ was not investigated in this work. However, a previous study using in situ hybridization indicated that rat osteoclasts do not express P2Y₄ (35). Therefore, these results indicate that P2Y_1,2,6 receptors are expressed by rabbit osteoclasts, whereas P2Y₁₁ and alkaline phosphatase transcripts are the result of contamination by other cell types.

4.1. Physiological evidence for the presence of P2Y receptors in osteoclasts

4.1.1. Ca²⁺ signals

In many cases, activation of P2Y receptors induces Ca²⁺ release from intracellular stores. Early studies in rabbit osteoclasts revealed that ATP-induced elevations in (Ca²⁺)_i occurred even in the absence of extracellular Ca²⁺ (80, 81). Osteoclasts loaded with GDPb S (a blocker of G protein activation) failed to generate these Ca²⁺ responses (81), indicating that G protein-coupled receptors were involved.

When studied using fluorescent Ca²⁺ dyes, ADP, but not AMP or adenosine, elicited elevations in (Ca²⁺)_i in rabbit and rat osteoclasts (43). This result rules out the participation of adenosine receptors in Ca²⁺ signaling. The response to ADP was probably mediated by P2Y₁ receptors, because it was very similar to responses elicited by the potent P2Y₁ agonist ADPb S. Interestingly, application of the P2Y₁ selective agonists ADPb S or 2-MeSATP resulted in oscillations in (Ca²⁺)_i in some cells (Fig. 9C). The oscillations persisted even in the absence of extracellular Ca²⁺ (37). Therefore, these oscillations depend on dynamic Ca²⁺ release from intracellular stores.

(Ca²⁺)_i elevations were also observed in response to UTP (10 m M) (43), an agonist of P2Y₂ receptors (Fig. 8B and Fig. 9). Furthermore, prolonged application of UTP elicited a transient response that declined to basal levels after a couple of minutes. The time course of the response is determined by P2Y₂ receptor desensitization due to prolonged exposure to the agonist (82). When the initial UTP response had declined to basal levels, application of ATP in the continued presence of UTP elicited a large transient elevation in (Ca²⁺)_i (Fig. 9D). This result indicates that individual osteoclasts can express more than one subtype of P2Y receptor.

4.1.2. Activation of Ca²⁺-dependent K⁺ channels

P2Y receptor-induced Ca²⁺ release from intracellular stores activates Ca²⁺-dependent K⁺ channels in osteoclasts. Electrophysiological studies in rat and human osteoclasts indicate that this ATP-induced current: 1) is blocked by intracellular Cs⁺; 2) has a reversal potential very close to the predicted K⁺ equilibrium potential; 3) has a single-channel conductance about 50 pS; and 4) closely follows changes in (Ca²⁺)_i (37, 83). The time course of this current depends on the purinergic agonist employed. When ATP was used, it was usually preceded by a P2X₄-mediated transient inward current (Fig. 2A). When ADPb S was used, only the transient outward calcium-dependent K⁺ current was observed (Fig. 2B). This is because ADPb S does not activate P2X₄ receptors (33). In contrast, when 2-MeSATP was used, oscillations in the amplitude of this K⁺ current could be observed in some cells. These oscillations closely followed the oscillations in (Ca²⁺)_i induced by 2-MeSATP (37). Activation of this current hyperpolarizes osteoclasts, whereas activation of P2X receptors has the opposite effect on membrane potential. Thus, it is possible that these channels contribute to the fine tuning of membrane potential in osteoclasts.

4.2. P2Y₁ receptors

P2Y₁ receptor mRNA has been detected by RT-PCR in human (47) and rabbit (79) osteoclasts. Expression of the P2Y₁ receptor has also been demonstrated by immunocytochemistry and in situ hybridization in rat osteoclasts (84). Agonists of P2Y₁ - such as ADP, ADPb S and 2-MeSADP - induce Ca²⁺ responses in osteoclasts (Figs. 8B and 9C).

ADP enhances osteoclastic resorption (84). Concentrations as low as 20 nM had a stimulatory effect, consistent with the high sensitivity of P2Y₁ receptors for ADP (44). Furthermore, MRS-2179 - a P2Y₁ receptor antagonist - blocked this stimulatory effect on resorption. Degradation products of ADP (AMP and adenosine) had no effect. ADP also stimulated the differentiation of precursor cells into osteoclasts (84); thus, ADP could stimulate resorption by enhancing osteoclastogenesis. Since osteoblasts also express P2Y₁, it is not clear whether the observed effects were due to a direct action of ADP on osteoclast P2Y₁ receptors or an indirect effect mediated by P2Y₁ receptors on osteoblasts (84). Activation of osteoblast P2Y₁ receptors could induce the expression of RANKL or other factors that stimulate osteoclasts.

4.3. P2Y₂ receptors

The P2Y₂ receptor was cloned from a human osteoclastoma cDNA library. Moreover, mRNA coding for P2Y₂ was detected by RT-PCR in osteoclast-like cells obtained from an osteoclastoma (85). Expression of P2Y₂ receptors in rat osteoclasts was also shown by in situ hybridization (35, 86).

Very little is known about the role of P2Y₂ receptors in osteoclasts. In fact, it has been reported by some authors that UTP - a potent P2Y₂ agonist - fails to elicit Ca²⁺ responses in osteoclasts (47, 81, 86), whereas others have reported Ca²⁺ responses (37, 79, 87) (see also Figs. 8 and 9). The reason for this discrepancy is not clear; it may be due to differences among species or different experimental conditions.

4.4. P2Y₆ receptors

Korcok et al. studied the role of P2Y₆ receptors signaling through NF-k B in the regulation of osteoclast survival (79). A stable analog of diuridine 5'-triphosphate - INS48823 - was used in this study, because it is a selective agonist at P2Y₆ receptors (88). Stimulation of osteoclasts with the P2Y₆ receptor agonists UDP or INS48823 elicited a Ca²⁺ response. Even though agonists at other subtypes of P2Y receptors (ADP and UTP) also elicited Ca²⁺ responses, only UDP and INS48823 were able to induce translocation of NF-k B from the cytosol to the nuclei (Fig. 10A). This result suggests that an increase in (Ca²⁺)_i alone is not sufficient for NF-k B activation; other conditions must be met. Furthermore, stimulation with UDP or INS48823 (but not ADP or UTP) significantly increased osteoclast survival (79). This increase in survival was mediated by NF-k B, since SN50 - a cell-permeable peptide inhibitor of NF-k B - abolished the effect. This peptide also abolished translocation of NF-k B to the nuclei induced by the P2Y₆ agonists. Therefore, the P2Y₆ receptor stimulates osteoclast survival by a mechanism dependent on NF-k B.

As mentioned above, an extension in osteoclast life span may have important physiological consequences (20, 21, 23). P2X₇ receptors seem to promote apoptosis in osteoclasts (77), whereas P2Y₆ receptors have the opposite action (79). The net effect of P2 receptor activation on osteoclast survival could be determined by integration of these signals.

5. SUMMARY AND perspective

A number of different P2 receptors are functionally expressed in osteoclasts. Currently, most evidence points to P2X₄, P2X₇, P2Y₁, P2Y₂ and P2Y₆; however, it is likely that a wider picture involving more P2 receptor subtypes will emerge in the future. Despite the important advances made in the field, a better understanding of the mechanisms involved and the roles that P2 receptors play in osteoclasts is still necessary.

P2 receptors are likely to influence osteoclast physiology at many, if not all, stages of their life cycle, from formation to death. Regarding formation, an early report indicates that low ATP concentrations (0.2-2 m M) stimulate formation, whereas higher concentrations (20-200 m M) reduce or block it (89). This result suggests that P2X₇ receptors, which are activated by high ATP concentrations, suppress osteoclast formation or survival. Other receptors, such as P2Y₁ might account for the stimulatory effect observed. Other reports suggest that P2X₇ receptors regulate osteoclast formation (48, 59, 64); however, it is clear that P2X₇ receptors are not essential for osteoclastogenesis since P2rx7^-/- mice possess multinucleated osteoclasts (54, 65).

Resorption by osteoclasts is modulated by P2 receptors, but the detailed mechanisms are not completely understood. ATP stimulates resorption up to about six-fold with a maximum effect occurring at low concentrations (0.2-2 m M) (89). It is possible that at least part of this stimulatory effect is mediated by P2Y₁ receptors, since ADP has a potent stimulatory effect on resorption, which is blocked by the P2Y₁ antagonist MRS-2179 (84). The P2X₇ receptor appears to have the opposite effect on resorption. It is possible that induction of apoptosis accounts for the reduction in overall resorptive activity. Furthermore, signs of increased resorptive activity are observed when P2X₇ receptor activity is reduced by genetic modification in mice (54), or loss-of-function polymorphisms in humans (77).

Osteoclast death is a tightly regulated phenomenon. In this regard, evidence to date indicates that P2 receptors play a role in regulating osteoclast survival. P2X₇ receptors promote osteoclast death by apoptosis (77) and P2Y₆ receptors suppress osteoclast apoptosis by a mechanism dependent on NF-k B (79).

Taking into account the influence that P2 receptors have on osteoclasts, there is considerable excitement about the potential of P2 receptors as targets for drugs that inhibit resorption (with relevance to diseases such as osteoporosis, periodontitis, rheumatoid arthritis and tumor-induced osteolysis). Previous reviews have considered these possibilities (25, 90-92). However, there are still no clear solutions and more research is needed.

6. ACKNOWLEDGMENT

Studies from the authors' laboratories described in this review are supported by the Canadian Institutes of Health Research (CIHR).

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Abbreviations: ADPb S: adenosine 5'-O-(2-thiodiphosphate); ATPg S: adenosine 5'-O-(3-thiotriphosphate); BzATP: 2',3'-O-(4-benzoylbenzoyl)-ATP; (Ca²⁺)_i: concentration of cytosolic free Ca²⁺; CB: Cibacron blue; EGFP: enhanced green fluorescent protein; EGTA: ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid; GDPb S: guanosine-5'-O-(2-thiodiphosphate); IC₅₀: half maximal inhibitory concentration; M-CSF: macrophage colony stimulating factor; 2-MeSADP: 2-methylthioadenosine 5'-diphosphate; 2-MeSATP: 2-methylthioadenosine 5'-triphosphate; NF-k B: nuclear factor κ-light-chain-enhancer of activated B cells; NMDG⁺: N-methyl D-glucamine⁺; PKC: protein kinase C; PPADS: pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; RANKL: receptor activator of NF-κB (RANK) ligand; RT-PCR: reverse transcription polymerase chain reaction.

Key Words: Apoptosis, ATP, Bone, Calcium, Calcium-activated potassium channels, Electrophysiology, Ion channel, NF-k B, Nucleotide, Osteoclastogenesis, Osteoclasts, P2 receptors, P2RX7, P2X receptors, P2X₇, P2Y receptors, Patch clamp, Protein kinase C, Purinergic, Resorption, Signaling, Survival, Review

Send correspondence to: S. Jeffrey Dixon, Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario, Canada, N6A 5C1, Tel: 519-661-3769, Fax: 519-850-2459, E-mail:jeff.dixon@schulich.uwo.ca