Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110
Submitted 26 August 2003 ; accepted in final form 26 March 2004
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ABSTRACT |
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macrophage fusion; P2X receptor; purinergic receptor; receptor activator nuclear factor-B
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 <1015 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-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.
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MATERIALS AND METHODS |
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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 (1620 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 35 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. -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 (
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.
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RESULTS |
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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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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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DISCUSSION |
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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 (911). 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 551582), 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" (573590) (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.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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