ATP downregulates P2X7 and inhibits osteoclast formation in RAW cells

Jeffrey F. Hiken and Thomas H. Steinberg

Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

Submitted 26 August 2003 ; accepted in final form 26 March 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Multinucleated giant cells derive from fusion of precursor cells of the macrophage lineage. It has been proposed that the purinoreceptor P2X7 is involved in this fusion process. Prolonged exposure of macrophages to ATP, the ligand for P2X7, induces the formation of plasma membrane pores and eventual cell death. We took advantage of this cytolytic property to select RAW 264.7 (RAW) cells that lacked P2X7 function by maintaining them in ATP (RAW ATP-R cells). RAW ATP-R cells failed to fuse to form multinucleated osteoclasts in response to receptor activator nuclear factor-{kappa}B ligand, although they did become positive for the osteoclast marker enzyme tartrate-resistant acid phosphatase, and upregulated expression of other osteoclast marker genes. RAW ATP-R cells and wild-type RAW cells expressed similar amounts of P2X7 protein, but little P2X7 was present on the surface of RAW ATP-R cells. After ATP was removed from the medium of RAW ATP-R cells, the cells reexpressed P2X7 on the cell surface, regained sensitivity to ATP, and formed multinucleated osteoclasts. These results suggest that P2X7 or another protein that is downregulated in concert with P2X7 is involved either in the mechanics of cell fusion to form osteoclasts or in a signaling pathway proximal to this event. These results also suggest that P2X7 may be regulated by ligand-mediated internalization and that extracellular ATP may regulate the formation of osteoclasts and other multinucleated giant cells.

macrophage fusion; P2X receptor; purinergic receptor; receptor activator nuclear factor-{kappa}B


IN SEVERAL SITUATIONS, MONONUCLEAR phagocytes fuse to form multinucleated giant cells (2, 28). These include the formation of immune giant cells, foreign body giant cells, and osteoclasts. Although the mechanism of giant cell formation is not understood, several plasma membrane (PM) molecules have been implicated in this process, including SIRP-{alpha}, CD47, CD44, intercellular adhesion molecule-1, leukocyte function-associated antigen-1, and E-cadherin (15, 19, 22, 25). P2X7, a PM receptor for ATP, has also been implicated in giant cell formation. For example, J774 mouse macrophage clones selected for high expression of P2X7 spontaneously fuse to form multinucleated giant cells, whereas clones selected for low P2X7 expression do not (4). In addition, concanavalin A-induced formation of multinucleated giant cells from human monocytes is blocked by an inactivating monoclonal antibody directed against P2X7 (7). Although these studies support a role for P2X7 in giant cell multinucleation, a recent report showed that a P2X7 null mouse is able to form multinucleated osteoclasts in vitro and in vivo (16). Thus the role of P2X7 in osteoclast formation remains unclear.

The only known physiologically relevant ligand for P2X7 is ATP. In the presence of divalent cations, millimolar levels of ATP are required for receptor activation (24). Transient activation of P2X7 with ligand opens a cation channel. However, P2X7 displays an unusual property in that repeated or prolonged activation of the receptor with ligand is associated with the formation of a membrane pore that allows for the bidirectional passage of molecules up to ~900 Da in size (24, 26). The P2X7 channel/pore closes on removal of ATP, and macrophages exposed to millimolar levels of ATP for <10–15 min generally recover to functionally normal cells (24). Opening of the P2X7 pore in macrophages by prolonged incubation with ATP leads to cell lysis (31).

One well-established model for macrophage fusion and multinucleation is the formation of osteoclast-like cells by induction of RAW macrophage-like cells with receptor activator nuclear factor-{kappa}B ligand (RANKL), a key regulator of osteoclast differentiation and function (14). We took advantage of the cytolytic effect of prolonged ATP exposure to select RAW mouse macrophages that lack P2X7 pore-forming activity to further examine the role of extracellular ATP and P2X7 in osteoclast formation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Differentiation of osteoclast-like cells from RAW cells. RAW cell media contained 90% DMEM with 2 g/l sodium bicarbonate, 1 mM sodium pyruvate, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 10% fetal bovine serum (not heat inactivated). On day 0, RAW cells were scraped from 100-mm tissue culture dishes, seeded into multiwell plates (24-well: 10,000 cells in 1 ml of media; 6-well: 50,000 cells in 3 ml of media), and grown overnight. On day 1, soluble mouse RANKL (a generous gift from Jose Moreno and Mehrdad Tondravi) was added to a final concentration of 100 ng/ml. Cells were fed on day 3 of RANKL induction.

Dye uptake assay. Media (0.2 ml) containing 0.5 mg/ml lucifer yellow or 2 µM YO-PRO-1 (Molecular Probes, Eugene, OR), either with 2 mM ATP or without ATP, were added to RAW cells growing in 24-well plates. Cells were incubated 15 min at 37°C before being washed three times with DMEM and viewed by phase and fluorescence microscopy.

Tartrate-resistant acid phosphatase staining. Cells were fixed for 10 min in 4% paraformaldehyde before being stained for tartrate-resistant acid phosphatase (TRAP) by using a leukocyte acid phosphatase kit, according to the manufacturer's instructions (Sigma Diagnostics, St. Louis, MO; kit no. 387-A).

Generation of ATP-resistant RAW cells. Frozen 1-ml aliquots of RAW cells of the same passage were thawed for each experiment where ATP-resistant RAW cells were generated. RAW cells growing at ~70% confluence in 100-mm tissue culture dishes were fed with 10 ml of media containing 2 mM ATP (Roche Applied Science, Indianapolis, IN). ATP was freshly added to media from frozen stocks of 100 mM ATP prepared in DMEM and adjusted to pH 7.5. After overnight incubation (16–20 h), ~95% of cells were killed. Cells were fed with media and freshly added 2 mM ATP after the initial overnight incubation and every other day thereafter. After an additional 3–5 days, cells were scraped and seeded into multiwell plates in media, either with 2 mM ATP (RAW ATP-R) or without ATP (RAW ATP-S). After overnight incubation, cells were induced with RANKL, as needed.

P2 receptor agonists/antagonist. Stock solutions of adenosine, AMP, ADP, UTP, and 2'-3'-O-(4-benzoylbenzoyl)-ATP (bzATP) (from Calbiochem, San Diego, CA) were prepared in DMEM and adjusted to approximately pH 7. {alpha}{beta}-Methylene-ATP, 2-methylthio-ATP, and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS) were from Sigma-Aldrich (St. Louis, MO). Osteoclast number in cultures incubated with P2 agonists/antagonists was assessed by counting detectable multinucleated cells in a low-power field (x4).

Real-time PCR. RAW, RAW ATP-R, and RAW ATP-S cells were either untreated or treated for 3 days with RANKL. Total RNA was isolated by using TRIzol reagent (Invitrogen, Carlsbad, CA). DNase I-treated RNA (2 µg) was reverse transcribed by using random hexamer primers and SuperScript II reverse transcriptase (Invitrogen). cDNA corresponding to 10 ng of total RNA was used for real-time PCR analysis by using SYBRgreen I dye chemistry and the GeneAmp 5700 sequence detection system (Perkin Elmer Life Sciences). Primers corresponded to carbonic anhydrase II, cathepsin K, matrix metalloproteinase-9, nuclear factor of activated T cells 1, TRAP, and GADPH.

Biotinylation of cell surface proteins. Two 100-mm dishes each of RAW, RAW ATP-R, and RAW ATP-S cells (~80% confluent) were washed three times with cold PBS (pH 8.0). Five milliliters of PBS (pH 8.0) containing 0.5 mg/ml succinimidyl-6-(biotinamido) hexanoate (NHS-LC-biotin) (Pierce, Rockford, IL) were added, and cells were incubated for 2 h at 4°C with gentle shaking. The biotinylation reaction was quenched by washing cells once with cold PBS containing 50 mM Tris, pH 8.0. Cells were then washed twice with cold PBS (pH 8.0) before lysing with 0.5 ml RIPA buffer [40 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 1 mM Pefabloc SC, and 1/100th volume protease inhibitor cocktail for mammalian cell extracts (Sigma-Aldrich)]. Lysates were allowed to sit on ice for 30 min before microfuging at 15,000 rpm for 10 min at 4°C. Supernatants were collected and protein concentrations determined by using a bicinchoninic acid assay kit (Pierce). Immobilized Neutravidin (Pierce) was prepared by washing three times with RIPA buffer and then adding RIPA buffer to bring to a 50% slurry. Biotinylated proteins were affinity purified by adding 100 µl of prepared Neutravidin slurry to lysates (1 mg total protein in 250 µl RIPA) and incubating for 2 h at 4°C with mixing. Samples were then washed five times with RIPA buffer (4°C) and eluted by boiling for 3 min in 70 µl of 2x concentrated Laemmli SDS sample buffer. Twenty microliters of eluate were loaded per lane for SDS-PAGE. In addition, total lysates were run on SDS-PAGE gels (60 µg protein per lane). A lysate of human embryonic kidney (HEK)-293 cells stably expressing rat P2X7 (80 ng protein) was loaded on a lane of each gel to serve as a P2X7 molecular weight marker. BenchMark Prestained Protein Ladder was from Invitrogen. After SDS-PAGE, proteins were transferred to nitrocellulose by using a semidry blotter (Bio-Rad) for 2 h at 15 V, with transfer buffer containing 25 mM Tris, 192 mM glycine, and 20% methanol. For Western blotting, rabbit P2X7 antibody (Alomone, Jerusalem, Israel) was used at a dilution of 1:1,000, and Na-K-ATPase monoclonal antibody in tissue culture supernatant ({alpha}A6, developed by D. M. Fambrough and obtained from Developmental Studies Hybridoma Bank, Iowa City, IA) was used at a dilution of 1:100.

Subcellular fractionation. Subcellular fractionations were carried out essentially according to the method of Piper et al. (21). All operations were carried out at 4°C. Two near-confluent 100-mm dishes of cells were washed once with HES buffer [20 mM HEPES, pH 7.4, 1 mM EDTA, 255 mM sucrose, 1 mM Pefabloc SC, and 1/100th volume protease inhibitor cocktail for mammalian cell extracts (Sigma-Aldrich)]. Cells were then scraped with 5 ml of HES and homogenized six strokes in a stainless steel Dounce. Homogenates were centrifuged for 20 min at 13,000 g. The supernatant was centrifuged for 75 min at 175,000 g (50,000 rpm in a Beckmann MLA-80 rotor) to yield a high-speed pellet (HSP). The pellet from the 13,000 g spin was resuspended in 0.5 ml of HES and layered on top of a 3-ml sucrose cushion (1.12 M sucrose in HES). The samples were then centrifuged for 60 min at 77,000 g (27,000 rpm in a Beckmann MLS-50 swinging bucket rotor). The PM fraction, which floats on top of the sucrose cushion, was pipetted off and washed by adding to 7 ml of HES and centrifuging 20 min at 30,000 g. The mitochondria/nuclei (M/N) fraction pellets through the sucrose cushion. The membrane pellets were resuspended in HES as follows: 100 µl PM, 300 µl HSP, and 600 µl M/N. Protein concentrations were determined by the bicinchoninic acid assay, and 25 µg of protein of each fraction were loaded per well for SDS-PAGE. The 25 µg represented ~10% of the total PM fraction, ~2% of the total HSP fraction, and ~1% of the total M/N fraction. Molecular weight markers were from Cell Signaling Technology (Beverly, MA; Biotinylated Protein Marker Detection Pack).

Immunofluorescence. Cells growing on 12-mm glass coverslips were washed three times with PBS and fixed for 30 min in PLP (2% paraformaldehyde, 75 mM lysine, and 10 mM periodate in 35 mM phosphate buffer, pH 7.4). Cells were then washed twice for 10 min in PBS and permeabilized by washing twice for 10 min in PBS containing 0.1% saponin. Cells were then incubated for 1 h in blocking solution (PBS, 0.1% saponin, 10% heat-inactivated goat serum). Coverslips were incubated overnight at 4°C in blocking solution containing P2X7 polyclonal antibody (1:100 dilution, Alomone). Cells were washed twice for 10 min in blocking solution before a 2-h incubation at room temperature with secondary antibody (1:2,000 dilution, Alexa Fluor 568-conjugated goat anti-rabbit IgG; Molecular Probes, Portland, OR). Coverslips were then washed twice for 10 min with PBS containing 0.1% saponin and twice for 10 min with PBS before mounting.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
RAW 264.7 cells form multinucleated osteoclast-like cells and display P2X7 pore-forming activity. In the presence of RANKL, RAW cells fuse to form multinucleated giant cells that express TRAP activity, characteristic of osteoclasts (14). In our hands, very little cell fusion occurred on days 0–3 of RANKL induction (Fig. 1B). By day 4 of RANKL induction, however, cell fusion and an osteoclast-like morphology were clearly evident (Fig. 1D). The resultant giant cells had upwards of ~100 nuclei, usually distributed around the cell periphery. Thus a large amount of cell fusion occurred between days 3 and 4 after addition of RANKL. These multinucleated cells also expressed TRAP activity (Fig. 1).



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Fig. 1. Receptor activator nuclear factor-{kappa}B ligand (RANKL)-induced RAW cell osteoclast-like differentiation. RAW cells were grown on glass coverslips in the absence or presence of 100 ng/ml RANKL, fixed with paraformaldehyde, and stained for tartrate-resistant acid phosphatase (TRAP) activity (without counterstain). Little fusion was seen in the absence of RANKL (A and C), or after 3 days of induction with RANKL (B). D: by 4 days of RANKL induction, large multinucleated cells had formed. TRAP staining (dark pigment) was present in RAW cells induced 3 days with RANKL (B); however, staining was not as intense or uniform as in cells induced for 4 days (D). Scale bars = 75 µm.

 
P2X7 is a receptor for extracellular nucleotides that is expressed predominantly on leukocytes. This receptor is associated with an unusual pore-forming activity in response to high concentrations of its ligand, ATP. Opening of these pores, which allows the nonspecific passage of molecules up to ~900 Da, can be monitored by the uptake of fluorescent dyes (24, 26). We asked whether ATP-induced pore formation occurred in RAW cells and in RAW cell-derived osteoclast-like cells by monitoring uptake of the membrane-impermeant fluorescent dyes lucifer yellow (457 Da) or YO-PRO-1 (375 Da). Cells were incubated in medium containing 0.5 mM lucifer yellow in the presence or absence of 2 mM ATP at 37°C for 15 min and viewed by epifluorescence microscopy (Fig. 2). In the absence of extracellular ATP, RAW cells did not take up lucifer yellow, but cells incubated in medium containing ATP demonstrated diffuse cytosolic staining with the fluorescent dye (Fig. 2, A and B). RANKL-treated RAW cells that had undergone osteoclastic differentiation also became permeable to lucifer yellow in the presence, but not in the absence, of extracellular ATP (Fig. 2, C and D).



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Fig. 2. RAW cells and RAW osteoclast-like cells express P2X7 activity. RAW cells and RANKL-generated RAW osteoclast-like cells were incubated in medium containing the fluorescent dye lucifer yellow in the presence (+) or absence (–) of 2 mM ATP for 15 min, washed, and examined by phase and fluorescence microscopy. B and D: diffuse cytoplasmic fluorescence in RAW cells and RAW osteoclast-like cells was seen only in the presence of ATP. A: little fluorescence was detected in uninduced RAW cells in the absence of ATP. Lucifer yellow was detected in large endocytic vesicles along the periphery of some RAW osteoclast-like cells in the absence of ATP (for example, arrows in C). D: in addition, dye uptake by unfused RAW cells was evident in RANKL-induced samples in the presence of ATP. Scale bars = 100 µm.

 
RAW cells maintained in 2 mM ATP lose P2X7 activity. Prolonged opening of P2X7-associated pores ultimately leads to cell lysis. We took advantage of this property to select RAW cells that lacked P2X7 activity (Figs. 3 and 4). When RAW cells were incubated overnight in 2 mM ATP, ~95% of the cells died. The surviving cells, when maintained in 2 mM ATP, proliferated nearly normally but were resistant to permeabilization by ATP (RAW ATP-R cells, Fig. 4, A and B). This resistance to ATP permeabilization was reversible, because the cells regained their sensitivity to ATP permeabilization after overnight incubation without ATP (RAW ATP-S cells, Fig. 4, C and D).



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Fig. 3. Protocol for generating ATP-resistant RAW cells. RAW 264.7 cells were incubated overnight (16–20 h) in the presence of 2 mM ATP, during which time ~95% of the cells died. Cells were maintained in the presence of 2 mM ATP and allowed to expand over the following 3–5 days. Cells were then split and grown overnight either in the presence of 2 mM ATP (RAW ATP-R cells) or in the absence of ATP (RAW ATP-S cells), before addition of RANKL.

 


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Fig. 4. RAW cells maintained in 2 mM ATP lose P2X7 pore-forming activity, but regain activity after withdrawal of ATP. RAW ATP-R and RAW ATP-S cells were generated as described in Fig. 3 and MATERIALS AND METHODS. P2X7 pore-forming activity was assayed by incubating cells with 2 µM YO-PRO-1 in the absence or presence of 2 mM ATP for 15 min, washing, and examining by phase and fluorescence microscopy. Fluorescence was barely detectable in RAW ATP-R cells incubated either in the absence (A) or presence (B) of ATP, or in RAW ATP-S cells when incubated in the absence of ATP (C). D: by contrast, after withdrawal of 2 mM ATP and overnight incubation of RAW ATP-R cells, the resultant RAW ATP-S cells regained their ability to take up dye in response to ATP.

 
RAW ATP-R cells fail to fuse efficiently when induced by RANKL but upregulate expression of osteoclast markers. We next asked whether RAW ATP-R cells formed multinucleated osteoclasts when incubated in medium containing RANKL. RAW ATP-R cells incubated in ATP maintained their resistance to ATP permeabilization on each of days 0–4 of RANKL induction (data not shown). In contrast to RAW cells, RAW ATP-R cells failed to fuse efficiently to form multinucleated cells after being incubated with RANKL for 4 days (Fig. 5D). While some multinucleation was observed, the number of osteoclasts formed was reduced ~90% (Table 1), and those that did form contained far fewer nuclei. RAW ATP-S cells, which had regained ATP responsiveness after overnight withdrawal of ATP, efficiently formed multinucleated cells after 4 days in RANKL (Fig. 5F).



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Fig. 5. RAW ATP-R cells fail to fuse in response to RANKL. RAW, RAW ATP-R, and RAW ATP-S cells were incubated in the presence (B, D, and F) or absence (A, C, and E) of 100 ng/ml soluble RANKL for 4 days. Cells were then fixed and stained for TRAP activity. Untreated cells (Con) did not display multinucleation, whereas RANKL-treated RAW cells (B) formed large multinucleated cells that stained positive for TRAP activity (dark pigment). D: RANKL-treated RAW ATP-R cells failed to form large multinucleated cells but did develop staining for TRAP activity. F: RAW ATP-S cells, which had regained ATP sensitivity, formed multinucleated giant cells after RANKL induction, similar to those seen for RAW cells. Scale bars = 75 µm.

 

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Table 1. Effect of ATP degradation products and P2 agonists/antagonist on RAW cell fusion

 
Despite the lack of multinucleation, RANKL-treated RAW ATP-R cells still demonstrated TRAP activity (Fig. 5D). The presence of TRAP activity in these cells led us to ask whether the expression of other osteoclast markers was similarly upregulated during RANKL induction of RAW ATP-R cells. We used real-time PCR to quantitate the expression of five different osteoclast marker genes and found that they were similarly upregulated in RANKL-treated RAW, RAW ATP-S, and RAW ATP-R cells (Fig. 6). The increase in expression of these markers ranged from ~5-fold for carbonic anhydrase to ~300-fold for matrix metalloproteinase-9 and TRAP (Fig. 6). These results suggest that RAW ATP-R cells may be blocked in their ability to fuse, as opposed to their ability to undergo the osteoclast differentiation program per se. Further evidence supporting this notion was seen when ATP was withdrawn from RAW ATP-R cells on day 3 of RANKL induction. Withdrawal of ATP on day 3 of induction, a time at which little cell fusion is apparent in RAW cells, allowed formation of multinucleated osteoclast-like cells by day 4 (Fig. 7).



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Fig. 6. RANKL-induced RAW ATP-R cells upregulate expression of osteoclast markers. Total RNA was isolated from RAW, RAW ATP-R, and RAW ATP-S cells that were either untreated or treated for 3 days with RANKL. Real-time PCR was performed by using primers specific to matrix metalloproteinase-9 (MMP-9), nuclear factor of activated T cells 1 (NFATc1), carbonic anhydrase, cathepsin K, TRAP, and GADPH. Results were normalized to GADPH levels, and fold enhancement was calculated by dividing the RANKL-induced value by the uninduced value. Data are from a representative experiment and are presented as averages ± SD.

 


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Fig. 7. RAW ATP-R cells fuse in response to RANKL if ATP is removed at day 3 of induction. RAW ATP-R cells were induced with 100 ng/ml soluble RANKL in the presence of 2 mM ATP. After 3 days, the cells were washed and incubated for an additional 24 h in media with RANKL, either in the absence or presence of 2 mM ATP. The cells were then fixed and stained for TRAP activity. When ATP was maintained throughout the 4 days of RANKL induction, multinucleation failed to occur efficiently (left). Withdrawal of ATP after 3 days of RANKL induction allowed the formation of large multinucleated cells by day 4 (right). Scale bars = 75 µm.

 
Effect of ATP degradation products and other nucleotides on RANKL-induced fusion. Many cells, including macrophages, express ecto-nucleotidases that degrade ATP to ADP, AMP, and adenosine. It is possible that the block of RANKL-induced fusion seen in RAW ATP-R cells was due to the effects of one of these degradation products on other P2 or P1 receptors, rather than a loss of P2X7 function. We therefore induced RAW cells with RANKL in the presence of 1 mM ADP, AMP, or adenosine (Table 1). Neither AMP nor adenosine inhibited multinucleated osteoclast-like formation. ADP at a concentration of 1 mM killed the RAW cells, but, at 100 µM, had no effect on RANKL-induced fusion. Similarly, the P2Y agonist UTP (1 mM) had no effect on fusion (Table 1). Nor did the P2X1 and P2X3 agonists {alpha}{beta}-methylene ATP (10 µM) and 2-methylthio-ATP (10 µM) show an appreciable effect on RANKL-induced fusion (Table 1).

The ATP analog bzATP has become a widely used agonist for P2X7, because it generally shows greater potency at P2X7 than ATP. However, the kinetic properties of P2X7 can be affected significantly by its buffer environment (13). For example, NaCl and serum decrease the potency of bzATP at P2X7 receptors. We tested the dose response of RAW permeabilization to lucifer yellow in response to ATP and bzATP in whole media, because we were limited in our ability to alter conditions of the RAW cell osteoclast differentiation model. The RAW cells began to permeabilize in response to ATP at a concentration of 2 mM and to bzATP at a concentration of 1 mM (data not shown). When 2 mM ATP was replaced with 100 µM bzATP on day 0 of RANKL induction, RAW ATP-R cells formed multinucleated osteoclast-like cells by day 4 (Table 1). In contrast, when the 2 mM ATP was replaced with 1 mM bzATP, osteoclast formation was blocked (Table 1). Thus the ability of bzATP to block osteoclast formation correlated with its ability to induce permeabilizaton. This result is consistent with P2X7 loss of function mediating the failure of RAW ATP-R cells to fuse in response to RANKL.

Effect of PPADS on RANKL-induced fusion. PPADS is a nonselective P2 receptor antagonist, with the exception that it is only weakly effective at P2X4 receptors (IC50 > 300 µM) (20). The IC50 of PPADS on cloned mouse P2X7 receptors is ~10 µM when measured in NaCl-free buffer (3). As with bzATP, the presence of NaCl and BSA has been shown to decrease PPADS antagonist potency at P2X7 (13). Consistent with this observation, RAW cell osteoclast differentiation proceeded in the presence of 10 µM PPADS in NaCl and serum-containing medium (Table 1). By contrast, 100 µM PPADS was sufficient to inhibit RAW cell osteoclast formation (Table 1).

P2X7 receptor is not expressed at the surface of RAW ATP-R cells. We next sought to determine the mechanism by which RAW ATP-R cells had become resistant to ATP-mediated pore formation. First we analyzed lysates from RAW ATP-R cells by Western blotting to determine whether downregulation of P2X7 protein expression accounted for the loss of P2X7 function in these cells. RAW, RAW ATP-R, and RAW ATP-S cells all expressed similar levels of P2X7 protein (Fig. 8A, top).



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Fig. 8. P2X7 is not expressed on the cell surface in RAW ATP-R cells. A: RAW, RAW ATP-S, and RAW ATP-R cell surface proteins were covalently labeled with biotin, as described in MATERIALS AND METHODS. A portion of total cell lysate was subjected to Western blotting with anti-P2X7 antibody (top). Biotinylated (surface) proteins were affinity purified from another portion of total cell lysate and then subjected to Western blotting with anti-P2X7 antibody (middle) or antibody directed against the {alpha}-1 subunit of Na-K-ATPase (bottom). Lysate from human embryonic kidney cells stably transfected with rat P2X7 was also loaded on gels as a P2X7 molecular weight marker (P2X7 marker). Similar amounts of P2X7 were seen in total cell lysates of all samples (top), whereas the amount of biotinylated P2X7 in RAW ATP-R cells was dramatically reduced. Blotting with antibody targeting the known plasma membrane protein Na-K-ATPase indicates that biotin labeling of cell surface proteins was similarly efficient for all samples (bottom). B: subcellular fractionation of RAW, RAW ATP-S, and RAW ATP-R cells. Membranes were separated into plasma membrane (PM), high-speed pellet (HSP), and mitochondria/nuclei (M/N) fractions, as described in MATERIALS AND METHODS. Equal amounts of protein were loaded per lane (25 µg), corresponding to ~10, 2, and 1% of the total yield of PM, HSP, and M/N fractions, respectively. STDS, molecular weight standards; ATP-S, RAW ATP-S; ATP-R, RAW ATP-R.

 
Because P2X7 protein was present in RAW ATP-R cells, we asked whether internalization of P2X7 could account for the lack of activity. We assayed PM localization by surface biotinylation. Cells were incubated in medium containing 0.5 mg/ml NHS-LC-biotin for 4 h at 4°C. Unreacted biotin was quenched with 50 mM Tris, and cells were lysed in buffer containing 1% Nonidet P-40. Cell surface proteins were affinity purified from total cell lysates by using streptavidin-conjugated beads and analyzed by Western blotting. While P2X7 protein was present at the surface of RAW cells, expression was dramatically reduced at the surface of RAW ATP-R cells (Fig. 8A, middle). Furthermore, RAW ATP-S cells, which had regained their P2X7 function, expressed a similar level of P2X7 protein at their surface as RAW cells (Fig. 8A, middle). The surface expression of the known PM protein, Na-K-ATPase ({alpha}-subunit), was similar for RAW, RAW ATP-R, and ATP-S cells, indicating that the biotinylation reaction was similarly efficient for all samples (Fig. 8A, bottom). These results indicate that RAW ATP-R cells lack P2X7 function because they do not express the receptor at their cell surface. Furthermore, these results suggest that localization of the P2X7 receptor may be regulated by its ligand.

We subjected RAW, RAW ATP-R, and RAW ATP-S cells to subcellular fractionation as an alternate method for assessing P2X7 PM expression. Membrane pellets from a low-speed spin of cell homogenates (13,000 g) were resuspended in buffer, layered on top of a 1.12 M sucrose cushion, and centrifuged at 77,000 g. The PM fraction (Fig. 8B) floats on top of the sucrose buffer, while the M/N fraction pellets (Fig. 8B). In addition, the supernatant from the initial low-speed spin was centrifuged at 175,000 g to yield a HSP (Fig. 8B). In agreement with the surface biotinylation experiment, the subcellular fractionation indicates that the level of P2X7 expression at the PM of RAW ATP-R cells is dramatically reduced compared with RAW and RAW ATP-S cells (Fig. 8B). Note that 25 µg of protein were loaded per lane on gels for the subcellular fractionations, which represented ~10 and 2% of the total PM and HSP yields, respectively. Therefore, Fig. 7B underrepresents the difference between PM and HSP expression by about fivefold. Densitometry of the representative blots shown in Fig. 8B indicated that the band representing P2X7 in the RAW cell PM fraction was approximately equivalent to the band representing P2X7 in the HSP fraction (data not shown). This suggests that approximately five times more P2X7 were localized intracellulary than on the surface of RAW cells.

Assessment of P2X7 localization by immunofluorescence. We analyzed localization of P2X7 in RAW cells and in RAW ATP-R cells using immunofluorescence with a polyclonal antibody raised against the last 20 amino acids of the intracellular COOH-terminal tail. While we knew from our biotinylation and subcellular fractionation experiments that P2X7 was expressed at the surface of RAW cells (Fig. 8), the receptor was difficult to discern at the PM by immunofluorescence. Instead, what appeared to be vesicular punctae appeared throughout the cytoplasm (Fig. 9). This staining was eliminated by preadsorbing the antibody with peptide antigen (data not shown). The P2X7 labeling in RAW ATP-R cells appeared similar to that seen in RAW cells (Fig. 9). By immunofluorescence, the receptor showed no obvious colocalization with markers for early endosomes (early endosome antigen-1), endoplasmic reticulum (bip), Golgi (GM-130), or lysosomes (lamp-2) (data not shown).



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Fig. 9. P2X7 immunofluorescence in RAW and RAW ATP-R cells. RAW and RAW ATP-R cells were fixed and permeabilized with 0.1% saponin before labeling with antibody that targeted the final 20 amino acids of the P2X7 intracellular COOH-terminal tail. In both cell types, the majority of labeling appeared as vesicular punctae throughout the cytoplasm.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In this study, we examined the effect of extracellular ATP and of P2X7 loss of function on the formation of multinucleated osteoclast-like cells. Incubation of RAW mouse macrophages in ATP caused reversible downregulation of P2X7 receptors and loss of P2X7 pore formation. These RAW ATP-R cells failed to form mutinucleated osteoclast-like cells in response to RANKL (Fig. 5). Removal of ATP restored cell surface expression of P2X7, ATP-induced pore formation, and the ability to form multinucleated cells. The functional and pharmacological data presented here suggest that the lack of P2X7 activity was involved in the failure to multinucleate. Other cell surface proteins, such as leukocyte function-associated antigen-1, intercellular adhesion molecule-1, SIRP-{alpha}, CD47, CD44, CD9, and CD81, have been implicated in the process of macrophage multinucleation (12, 15, 25, 27). We cannot exclude the possibility that other PM molecules whose expression or function was altered in RAW ATP-R cells are involved in multinucleation. Regardless of the potential involvement of multiple molecules in the inhibition of multinucleation in our RAW cell model, our results point to a possible role for extracellular ATP in modulating osteoclast multinucleation and, hence, bone turnover. Indeed, extracellular ATP has recently been shown to promote NF-{kappa}B nuclear translocation in mouse and rabbit osteoclasts in a RANKL-independent manner (18).

Our results support the observations of Chiozzi et al. (4), who found a correlation between P2X7 expression and giant cell formation in macrophages, and the more recent study of Gartland et al. (8), in which an inactivating P2X7 monoclonal antibody prevented osteoclast formation from human peripheral blood monocytes. In contrast to these findings, Ke et al. (16) reported that a P2X7 null mouse formed multinucleated osteoclasts. Thus the role of P2X7 in multinucleation of mononuclear phagocytes remains unclear.

One possible explanation for the above findings is that another receptor, possibly also a member of the P2X family, compensates for loss of P2X7 function in the knockout mouse. ATP-induced pore formation, although primarily ascribed to P2X7 among P2X receptors, has more recently been suggested to occur with at least P2X2 and P2X4 (17, 29). We have identified P2X2, P2X4, and P2X6 protein in RAW cells, although biotinylation studies so far have not revealed regulation of cell surface expression of these receptors by ATP (preliminary data). Additionally, the RAW cell line may differ from primary bone marrow osteoclast precursors in an inability to utilize a possible compensatory mechanism for the loss of P2X7 function.

Another possibility is that P2X7 associates with or regulates the function of a molecule required for cell fusion, but that this molecule can function in the absence of P2X7. This molecule could physically associate with P2X7 in the PM, in which case P2X7 internalization induced by extracellular ATP (as in our RAW ATP-R cells) might cause internalization of this molecule and inhibit multinucleation by this means. This molecule could also be downstream of P2X7 in a signaling cascade, in which case P2X7 downregulation by extracellular ATP or binding of P2X7 by an inhibitory monoclonal antibody might inhibit activation of this molecule.

While RAW ATP-R cells did not fuse efficiently in response to RANKL, the cells did become positive for the osteoclast marker enzyme TRAP (Fig. 5) and upregulated expression of four other osteoclast marker mRNAs to an extent similar to RAW and RAW ATP-S cells (Fig. 6). Furthermore, when ATP was withdrawn from RAW ATP-R cells after 3 days of induction, the cells still fused by day 4 (Fig. 7). These results indicate that RANKL was able to promote osteoclast differentiation in the presence of ATP and in the absence of P2X7 pore-forming activity, and that extracellular ATP suppressed only later events (occurring between days 3 and 4), leading to cell fusion and multinucleation. Our results, therefore, suggest that P2X7 is involved either in the mechanics of cell fusion or in a signaling pathway proximal to this event.

Our model system selected RAW cells that lacked P2X7 activity and that were, consequently, resistant to the permeabilizing effect of ATP. Maintaining RAW ATP-R cells in 2 mM ATP did not appear to commit them to a nonosteoclastic pathway of terminal differentiation, or result in a global toxicity to the cells, because fusion of RAW ATP-R cells proceeded when ATP was withdrawn as late as 3 days of RANKL induction.

Resistance to ATP-induced pore formation in RAW ATP-R cells was reversible, because the cells regained their sensitivity to ATP-induced pore formation after overnight withdrawal of ATP (RAW ATP-S cells, Fig. 4). RAW, RAW ATP-R, and RAW ATP-S cells all expressed similar quantities of P2X7. However, ATP-resistant RAW cells lacked P2X7 pore-forming activity because the receptor was not expressed at the cell surface (Fig. 8). After removal of ATP from the cells, both P2X7 cell surface expression and P2X7 pore formation reappeared. These findings suggest that ligation of P2X7 regulates P2X7 activity via receptor internalization and that a latent pool of P2X7 can appear at the cell surface after withdrawal of the nucleotide. Thus P2X7 expression and activity appear to be regulated by receptor ligation.

The majority of P2X7 protein was localized intracellularly in RAW cells. The subcellular fractionation experiment shown in Fig. 8B provided a rough estimate of fivefold more P2X7 expressed intracellularly than on the cell surface (see RESULTS). Consistent with this finding, immunofluorescence of P2X7 in RAW cells suggested that the receptor is localized mostly in intracellular vesicles (Fig. 9). Several factors may account for why P2X7 was difficult to detect at the PM by immunofluorescence. The receptor may have been too diffuse in the PM for detection. It is also possible that cell surface P2X7 was lost during permeabilization with saponin, or that the COOH-terminal epitope was masked at the PM due to homomerization or interaction with other proteins.

The apparent low level of PM P2X7 expression suggests that surface localization of the receptor may be an important mechanism for regulation of its activity. There were no clearly discernable differences in immunofluorescence localization of P2X7 between RAW cells and RAW ATP-R cells (Fig. 9). However, because the proportion of P2X7 expressed at the surface was apparently small, it would be difficult to follow the fate of internalized P2X7. The similar level of P2X7 expression seen in RAW and RAW ATP-R cells by immunofluorescence and Western blotting suggests that the receptor is not, overall, degraded more rapidly in the RAW ATP-R cells.

A number of recent studies have examined the localization of P2X7. P2X7 expression in human peripheral blood monocytes is predominantly intracellular, but surface expression increases as the cells mature in vitro into macrophages (9–11). Mutations and polymorphisms that affect cell surface expression have been identified in the COOH-terminal tail of P2X7 (1, 6, 23, 30). Adriouch et al. (1) identified a Pro-451 to Leu natural polymorphism present in C57BL/6 and DBA/2 mouse strains that results in reduced P2X7 surface expression on T cells and concomitant reduction of ATP-induced phosphatidylserine exposure and ethidium bromide uptake. Smart et al. (23) defined a "pore-enabling domain" in the distal COOH-terminal tail of P2X7 (amino acids 551–582), where various truncations and point mutations result in loss of surface immunoreactivity and consequent loss of P2X7 channel and pore-forming activity in transfected HEK cells. The same group subsequently discovered a human polymorphism within this domain (Ile-568 to Asn), where heterozygotes displayed half-normal P2X7 surface expression and reduced P2X7 function (30). Transfection of the mutant P2X7 into HEK cells indicated a trafficking defect that resulted in the absence of surface expression. The pore-enabling domain overlaps with a previously identified putative functional domain of P2X7, the "LPS binding domain" (573–590) (5). Mutation of a dibasic motif in the LPS binding domain results in a trafficking defect in P2X7 that prevents surface expression (6). These studies point to surface localization of P2X7 as a potential mode of regulation of receptor function. It will be of interest to determine whether binding of ATP to P2X7 leads to posttranslation modifications or changes in the complement of proteins bound to the domains defined by the above studies and subsequent regulation of receptor localization.

In summary, these studies show that RAW cells maintained in extracellular ATP lose P2X7 surface expression and function and fail to form multinucleated osteoclasts in response to RANKL. They suggest that P2X7 is involved in the multinucleation process and that ligand-sensitive localization of P2X7 may underlie a mechanism by which ATP regulates bone remodeling by a local and transient effect on osteoclast formation.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Institutes of Health Grants DK-46686 and HD-37799. J. F. Hiken was supported by a fellowship from the W. M. Keck Foundation and by Infectious Disease/Basic Microbial Pathogenic Mechanisms Training Grant 5T32AI0717223 from Washington University.


    ACKNOWLEDGMENTS
 
We thank Drs. Mehrdad Tondravi and Jose Moreno for the generous gift of RANKL and Drs. F. Patrick Ross and Brian Bennett for helpful advice on the RAW cell model of osteoclast differentiation.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. Hiken, Dept. of Internal Medicine, Washington Univ. School of Medicine, 660 South Euclid Ave, St. Louis, Missouri 63110 (E-mail: hiken{at}id.wustl.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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