Characterization of inorganic phosphate transport in osteoclast-like cells

Mikiko Ito,1 Naoko Matsuka,1 Michiyo Izuka,1 Sakiko Haito,1 Yuko Sakai,1 Rie Nakamura,1 Hiroko Segawa,1 Masashi Kuwahata,1 Hironori Yamamoto,1 Wesley J. Pike,2 and Ken-ichi Miyamoto1

1Department of Molecular Nutrition, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima City, Japan; and 2Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin

Submitted 23 August 2004 ; accepted in final form 14 December 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Osteoclasts possess inorganic phosphate (Pi) transport systems to take up external Pi during bone resorption. In the present study, we characterized Pi transport in mouse osteoclast-like cells that were obtained by differentiation of macrophage RAW264.7 cells with receptor activator of NF-{kappa}B ligand (RANKL). In undifferentiated RAW264.7 cells, Pi transport into the cells was Na+ dependent, but after treatment with RANKL, Na+-independent Pi transport was significantly increased. In addition, compared with neutral pH, the activity of the Na+-independent Pi transport system in the osteoclast-like cells was markedly enhanced at pH 5.5. The Na+-independent system consisted of two components with Km of 0.35 mM and 7.5 mM. The inhibitors of Pi transport, phosphonoformic acid, and arsenate substantially decreased Pi transport. The proton ionophores nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone as well as a K+ ionophore, valinomycin, significantly suppressed Pi transport activity. Analysis of BCECF fluorescence indicated that Pi transport in osteoclast-like cells is coupled to a proton transport system. In addition, elevation of extracellular K+ ion stimulated Pi transport, suggesting that membrane voltage is involved in the regulation of Pi transport activity. Finally, bone particles significantly increased Na+-independent Pi transport activity in osteoclast-like cells. Thus, osteoclast-like cells have a Pi transport system with characteristics that are different from those of other Na+-dependent Pi transporters. We conclude that stimulation of Pi transport at acidic pH is necessary for bone resorption or for production of the large amounts of energy necessary for acidification of the extracellular environment.

Na+-dependent phosphate cotransporter; RAW264.7; phosphate uptake


OSTEOCLASTS ARE THE PRIMARY CELLS responsible for bone resorption. They arise by the differentiation of osteoclast precursors of the monocyte/macrophage lineage. These cells are required not only for the development of the skeleton but also for mineral homeostasis and normal remodeling of bone in adult animals (28, 36).

Bone resorption depends on the ability of the osteoclast to generate an acid extracellular compartment between itself and the bone surface (1). An acidic pH is essential for solubilization of the alkaline salts of bone minerals as well as for digestion of the organic bone matrix by acid lysosomal enzymes that are secreted by osteoclasts (6, 22). The primary cellular mechanism responsible for this acidification is active secretion of protons by the vacuolar-type H+-adenosine triphosphatase (V-type ATPase), which is localized in the ruffled border of the osteoclasts (1).

Inorganic phosphate (Pi) is the major anionic component of bone, and Pi released from bone may be transported into the osteoclast through a Pi transport system (8, 9). Pi influx has been reported to require extensive V-type ATPase activity and thus a large amount of energy. Pi may also help maintain the ATP content during the cyclical processes of migration, attachment, and resorption (8).

In mammals, the membrane transport of Pi is Na+ dependent because it is mediated by three classes of Na+-dependent phosphate (NaPi) cotransporters (types I, II, and III) (23). In studies using brush-border membrane vesicles, renal Pi transport is mediated by the type IIa NaPi cotransporter, which has a high affinity for Pi (0.1 mM) and is inhibited by acidic pH (18, 20). Interestingly, Gupta and colleagues (8, 9) showed that the differentiation of osteoclast-like cells from chicken and rabbit bone marrow and their Na+-dependent Pi transport through the type IIa NaPi cotransporter was induced in the presence of bone particles. The type IIa NaPi cotransporter is also expressed in primary osteoclasts as well as by mouse osteoclast-like cells generated from RAW264.7 cells by treatment with receptor activator of NF-{kappa}B (RANKL) (8, 10). Khadeer et al. (17) further demonstrated that the type III NaPi cotransporter is a functional Pi transporter in osteoclast-like cells. However, because it is difficult to isolate sufficient quantities of fully viable, fresh osteoclasts (12), the Pi transport system in osteoclast-like cells has not been well characterized. In addition, because osteoclastogenesis is influenced by various factors, the behavior of the isolated cells can differ between preparations.

To overcome these problems, we used an in vitro culture system in which mononuclear osteoclast precursors fuse with each other into multinuclear osteoclasts in the presence of RANKL. Hsu et al. (12) showed that the mouse monocyte/macrophage cell line RAW264.7 can be differentiated into osteoclasts with high yield by stimulation with RANKL. Treatment of RAW264.7 cells with RANKL readily stimulated differentiation into osteoclast-like, tartrate-resistant acid phosphatase (TRAP)-positive cells. The osteoclasts readily formed resorption lacunae on bone slices, verifying their osteoclast phenotype (4, 12). Therefore, in the present study, we used osteoclast-like cells derived from RAW264.7 cells treated with RANKL as a model of osteoclast Pi transport and compared the resulting data with data from untreated RAW264.7 cells (i.e., undifferentiated).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. The mouse monocyte/macrophage cell line RAW264.7 was obtained from the Riken Cell Bank (Tokyo, Japan). Dulbecco's modified Eagle's medium and {alpha}-minimum essential medium ({alpha}-MEM) without phenol red were obtained from Invitrogen (Carlsbad, CA). Recombinant human RANKL extracellular region (amino acids 137–316) fused to glutathione-S-transferase was expressed in Escherichia coli using the vector pGEX-3T (Amersham Biosciences, Piscataway, NJ) and purified by performing affinity chromatography using a glutathione-Sepharose column (Amersham Biosciences) (31). Recombinant murine macrophage colony-stimulating factor (M-CSF) was obtained from Genzyme/Techne (Cambridge, MA). The anion transporter inhibitor 4,4'-dinitrostilbene-2,2'-disulfonic acid disodium salt (DNDS) was obtained from Molecular Probes (Eugene, OR). All other chemicals were obtained from Sigma Chemical (St. Louis, MO). Bone particles derived from calf femur were a gift from Dr. Y. Ohba (University of Tokushima Graduate School, Tokushima, Japan) (25).

RAW264.7 cell culture. RAW264.7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-activated fetal bovine serum (FBS). Osteoclasts were generated from RAW264.7 cells with recombinant RANKL (160 ng/ml) without M-CSF. The cells were plated at 5 x 103/cm2 in {alpha}-MEM without phenol red supplemented with 10% charcoal-stripped FBS to remove endogenous steroids as described previously (31). The medium was changed on day 3 and replaced with fresh medium and mediators. After 7 days, multinucleated osteoclasts were identified using TRAP histochemical staining, and calcitonin receptor (CTR) mRNA was detected using reverse transcription polymerase chain reaction (RT-PCR) (39).

Primary osteoclastogenesis from murine bone marrow cells. Six-week-old C57BL/6 male mice were obtained from SLC (Shizuoka, Japan). The preparation of mouse bone marrow cells and the formation of osteoclasts were performed as described previously (31). Briefly, bone marrow cells were isolated from both the tibiae and femurs of mice and cultured in {alpha}-MEM with 10% FBS for 24 h. Nonadherent cells were isolated and enriched using a Ficoll density gradient and cultured at 9 x 104/cm2 in phenol red-free {alpha}-MEM supplemented with 10% charcoal-stripped FBS (31). For stimulation of osteoclast formation, cells were incubated with 10 ng/ml M-CSF and 300 ng/ml recombinant RANKL. The medium was refreshed on day 3, and osteoclast formation was assayed by counting the number of TRAP-positive cells per well between days 7 and 10.

Immunostaining. RAW264.7 cells were plated on glass coverslips at a density of 5 x 103/cm2 and analyzed by immunostaining after 24 h, 48 h, 96 h, and 7 days. Cells were fixed with 3% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. The cells were incubated with 1:50 anti-rat CTR affinity-purified antibody (gift from Asahikasei Parma, Tokyo, Japan) (14, 15), anti-Glvr-1 antibody (1:50) (3) (gift from Y. Taketani, University of Tokushima Graduate School, Tokushima, Japan), or an antibody to the COOH terminus of NaPi type IIa antibody by affinity purification (1:50 dilution) (30). The coverslips were incubated, followed by 1:200 Alexa Fluor 568-conjugated rabbit immunoglobulin G (IgG) combined with 1:200 Alexa Fluor 488 phalloidin (Molecular Probes, Eugene, OR), to detect actin filaments. The coverslips were mounted with Vectashield H-1000 (Vector Laboratories, Burlingame, CA). Confocal images were obtained using a Leica TCS-SL (Wetzlar, Germany) laser scanning microscope equipped with a x40 oil-immersion lens objective.

RT-PCR analysis. Total RNA was prepared using Isogen (Wako Pure Chemical, Tokyo, Japan) as described in the manufacturer's manual. A first-strand synthesis kit (Invitrogen) was first used to generate full-length cDNA from 1 µg of total RNA. The primers for amplification are shown in Fig. 1A.



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Fig. 1. Detection of the Na+-dependent Pi (NaPi) cotransporter. A: sequences of various primers used for detection of reverse transcriptase-polymerase chain reaction (RT-PCR). B: primer positions and combinations of type IIa NaPi cotransporter used for PCR amplification.

 
PCR Southern blot hybridization analysis. DNA was extracted from cultured cells and analyzed by PCR with oligonucleotide primers specific for a portion of the NaPi type IIa cotransporter. The PCR primers used were S3 and AS1, S4, and AS3 as described in Fig. 1A. PCR products were separated on a 2% agarose gel and transferred to a nylon membrane Hybond-N+ (Amersham Biosciences). Hybridization was performed with digoxigenin (DIG)-labeled PCR probe using S2 and AS3 primers and incubated for overnight in DIG Easy Hyb solution (Roche Diagnostics, Indianapolis, IN) at 42°C. Anti-DIG antibody conjugated with alkaline phosphatase (Roche Diagnostics) and a DIG detection kit (Roche Diagnostics) were used for probe detection. Further details of the PCR Southern blot hybridization method are described in the manufacturer's technical manual.

Pi and D-glucose transport measurements. Pi transport was studied in monolayers of RANKL-differentiated RAW264.7 cells in 12-well dishes. Cell densities were ~1.6 x 104 cells per well. The measurement of Pi transport was performed using a modification of a previously described procedure (27). Briefly, cell monolayers were gently washed three times with 0.5 ml of prewarmed (37°C) uptake solution (in mM: 137 NaCl, 5.4 KCl, 2.8 CaCl2, 1.2 MgCl2, and 10 HEPES-Tris, pH 7.4) in the presence or absence of various inhibitors. For pH 5.5 uptake solution, the HEPES-Tris buffer was replaced with MES-Tris buffer. Plates were incubated for 15 min at 37°C before the uptake assay was performed. The cells were preincubated with various inhibitors for 10 min. Pi transport was initiated by the addition of 0.7 ml of prewarmed (37°C) uptake solution containing 0.1 mM KH2PO4 and 1 µCi/ml 32Pi (PerkinElmer, Bridgeport, CT) in the presence or absence of various inhibitors. For kinetic analysis, cells were incubated with 1 µCi/ml 32Pi and increasing concentrations (0.05–12.8 mM) of K2HPO4/KH2PO4, such that the concentration of K+ was kept constant by the addition of KCl. Cells were incubated for 10 min at 37°C, and transport was terminated by the addition of 1 ml of ice-cold stop solution (in mM: 137 NaCl, 10 Tris·HCl, pH 7.2).

Glucose uptake measurements were performed to determine whether the changes in osteoclast-like cells were specific to Pi transport. For these experiments, osteoclast-like cells were mixed with 0.5 mM D-glucose and tracer amounts of 1 µCi/ml [14C]-D-glucose. After 10 min (which is within the linear rate phase), the reaction was terminated by addition of 1 ml of ice-cold stop solution.

In each experiment, after three additional washes with 1 ml of ice-cold stop solution, the cells were solubilized by the addition of 0.25 ml of 0.1 N NaOH at room temperature. The cell lysates were added to 2.5 ml of Aquasol-2 (Packard Instruments, Meriden, CT), and 32Pi or 14C radioactivity was determined using liquid scintillation counting. The protein concentration in lysates was determined using the BCA protein assay kit (Pierce Chemical, Rockford, IL). Pi and glucose transport were calculated as nanomoles of 32Pi or 14C per milligram of protein taken up in 10 min. To test the ion dependence of Pi transport, the NaCl in the uptake buffer was replaced with ChCl. Finally, for experiments to examine the effect of bone, cells were incubated for 1 h with 600 µg/ml bone particles (8). After cells were washed four times with prewarmed uptake solution, 32Pi uptake was assessed. All experiments were performed in triplicate and repeated two to four times.

Optical measurement of intracellular pH. Osteoclasts derived from RAW264.7 cells were washed three times with HEPES buffer (in mM: 153 NaCl, 5 KCl, 5 glucose, and 20 HEPES, pH 7.4) and then incubated with 5 µM BCECF-AM (Dojindo, Kumamoto, Japan) for 30 min at 37°C. The cells were then washed three times with the same buffer to remove unincorporated BCECF-AM. Single-cell measurements of intracellular pH (pHi) were performed using a fluorescence spectrophotometer (model 650-10MS; Hitachi, Tokyo, Japan) with excitation and emission wavelengths of 500 and 530 nm as described previously (21). pHi was measured at 5-min intervals after addition of 2 mM K2HPO4/KH2PO4, pH 7.4, in HEPES buffer. Time 0 measurements were performed just before the addition of K2HPO4/KH2PO4. The medium was replaced with Pi-free medium after 15 min. The resulting fluorescence-to-excitation ratios were converted to pHi values using the high-K+/nigericin technique (37). For Na+-free conditions, the NaCl in HEPES-loading buffer was replaced with an equivalent concentration of ChCl.

Statistical analysis. Results are reported as means ± SE for at least three samples. The statistical significance of differences between measured values was determined using ANOVA and InStat software (GraphPad, San Diego, CA). Kinetic parameters of uptake were determined using linear regression analysis of Lineweaver-Burk plots.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Characterization of osteoclast-like cells derived from RAW264.7 cells. To characterize the osteoclast-like cells, we first performed histochemical analysis of RANKL-treated RAW264.7 cells. After 7 days of RANKL treatment, the number of TRAP-positive and multinucleated cells was markedly increased compared with cells treated for 24 h (Fig. 2A). TRAP-positive cells were not detected in untreated RAW264.7 cells after 7 days (data not shown). RT-PCR for the CTR, which mediates calcitonin-stimulated bone resorption by mature osteoclasts, demonstrated ~90% differentiation to osteoclasts (Fig. 2B). Immunostaining of the osteoclast-like cells using a CTR-specific antibody showed staining at the cell surface (Fig. 2C).



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Fig. 2. Receptor activator of NF-{kappa}B ligand (RANKL)-induced osteoclastogenesis of macrophage-derived RAW264.7 cells. RAW264.7 cells were cultured with or without RANKL. A: osteoclast-like cells were detected using tartrate-resistant acid phosphatase (TRAP) staining after 24 h, 48 h, 96 h, and 7 days. B: on day 7, the formation of osteoclasts was also detected using RT-PCR for the calcitonin receptor (CTR). GAPDH was used as control for success of the RT-PCR reaction. C: CTR (red) and actin (green) staining in osteoclast-like cells. Right: merged image. Scale bar, 40 µm.

 
Expression of Na+-dependent Pi transporters in osteoclast-like cells. We investigated the expression of Na+-dependent Pi transporter transcripts in osteoclast-like cells. As shown in Fig. 3, RT-PCR analysis did not detect transcripts for the NaPi type I, type IIa, type IIb, or type IIc cotransporter in either the untreated or the RANKL-treated RAW264.7 cells. However, mRNA for the NaPi type III cotransporter (the amphotropic murine retrovirus, Ram-1, and the gibbon ape leukemia virus, Glvr-1) were expressed in both cells, but the levels of the transcript were the same in treated and untreated cells. Similar results were also found in primary osteoclast cells derived from mouse bone marrow cells (Fig. 3A). To detect of the type IIa NaPi cotransporter transcript in osteoclasts, we performed PCR Southern blot analysis using different combination of primers (Fig. 3B). The expression of type IIa transporter was detected in both primary and RAW264-derived osteoclast cells, but the levels of the transcript were the same in treated and untreated cells. Immunohistochemical analysis revealed the mouse type III transporter in the plasma membrane of the osteoclast-like cells, but the type IIa NaPi cotransporter was not detected (Fig. 3C). We further investigated whether the immunoreactive signals for the type IIa NaPi cotransporter could be observed in the plasma membrane using Western blot analysis. The mouse type IIa NaPi cotransporter did not react with the plasma membrane fraction when the antibodies to the NH2 or COOH terminus of type IIa NaPi cotransporter were used (data not shown).



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Fig. 3. Expression of various NaPi transporters in osteoclast-like cells. A: RT-PCR analysis of the NaPi cotransporter. RAW264.7 cell lines (RAW) and mouse bone marrow-derived cells (BMC) were cultured with or without RANKL for 7 days. Total RNA was extracted, and cDNA was synthesized by performing RT-PCR using specific primers for type I, type II (IIa, IIb, and IIc), and type III (the amphotropic murine retrovirus, Ram-1, and the gibbon ape leukemia virus, Glvr-1) NaPi cotransporters. The level of CTR mRNA was assessed as an indicator of differentiation to osteoclasts, and GAPDH was used as a control for the success of the RT-PCR reaction. K, mouse kidney; N, without template. B: type IIa NaPi detection using PCR Southern blot analysis. The PCR products using S3 and AS1 as well as S4 and AS3 primers were loaded onto a 2% agarose gel and then stained using ethidium bromide (top). The PCR product-derived mouse kidney (K10–3) was diluted 1:1,000. The type IIa NaPi cotransporter was detected by digoxigenin (DIG)-labeled PCR probe and DIG detection kit. C: osteoclast-like cells were cultured on glass coverslips and double-stained with NaPi cotransporter and actin. The coverslips were visualized using confocal sequential scanning. Top: type IIa NaPi (red) and actin (green) staining; bottom: Glvr-1 (red) and actin (green) staining; right: merged image. Scale bar, 40 µm.

 
Effect of RANKL on Pi transport in RAW264.7 cells. We next characterized how the differentiation of RAW264.7 cells to osteoclast-like cells by RANKL affected the activity and properties of Pi transport. Pi transport activity was assayed by measuring the uptake of 32Pi at pH 7.4. As shown in Fig. 4A, Pi transport activity was slightly increased (1.2-fold) in the presence of the Na+ ion in the RANKL-induced osteoclast-like cells compared with untreated RAW264.7 cells (control). In contrast, in the absence of Na+ ion (i.e., ChCl in place of NaCl), Pi transport activity was increased ~4.9-fold in the RANKL-treated cells compared with the untreated RAW264.7 cells. On the basis of the observation that Na+-independent Pi transport activity was increased in the RANKL-induced osteoclast-like cells, we suggest that types I, II, and III NaPi cotransporters are not involved in the upregulation of Pi transport in the osteoclast-like cells. In addition, the results suggest that Na+-dependent transport activity in untreated RAW264.7 cells may be contributed by the type III NaPi cotransporter.



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Fig. 4. Characterization of Pi transport in osteoclast-like cells. A: Na+ independence of Pi transport. Uptake of Pi was measured in cells treated with (solid bars) or without RANKL (open bars). The Pi influx into each cell was measured at 37°C in 137 mM NaCl or ChCl medium, pH 7.5, in the presence of 0.1 mM PO4. B and C: pH dependence of Pi uptake was measured in RANKL-treated (solid bars) and untreated (open bars) cells. B: pH dependence of Pi uptake in the presence of Na+. Pi uptake was measured in 137 mM NaCl uptake solution at pH 5.5, 6.5, 7.5, and 8.5. C: pH dependence of Pi uptake in Na+-free solution. Pi uptake was measured in 137 mM ChCl uptake solution at pH 5.5, 6.5, 7.5, and 8.5. D: typical time course of Pi uptake in osteoclast-like cells (circles) or untreated RAW264.7 cells (triangles). The Pi influx into each cell was measured at 37°C in 137 mM NaCl medium, pH 7.5, in the presence of 0.1 mM PO4. E: effects on osteoclast differentiation on D-glucose uptake. [14C]-D-glucose was measured in osteoclast-like cells in uptake solution containing 137 mM ChCl, pH 5.5, or in untreated RAW264.7 cells in uptake solution containing 137 mM NaCl, pH 6.5. F: stimulation of Pi uptake by RANKL in conjunction with differentiation. RAW264.7 cells were cultured with RANKL, and Pi transport was measured after 24 h, 48 h, 96 h, and 7 days. *P < 0.05 compared with 24-h measurement. In all experiments, values represent means ± SE; n = 3. Pi transport was calculated as nm of 32Pi per mg of protein taken up during a 10-min period. *P < 0.05.

 
In the absence and presence Na+ ion, Pi transport activity in osteoclast-like cells was markedly increased at pH 5.5 and gradually decreased as the pH became more alkaline (Fig. 4, B and C). In contrast, in untreated RAW264.7 cells, Pi transport activity was increased at pH 6.5 and 7.5 in the presence of Na+ ion. Thus the properties of Pi transport in the osteoclast-like cells were distinct from those of untreated RAW264.7 cells. Uptake of Pi in the presence or absence of Na+ ion was essentially linear for 30 min and reached a steady state after 40 min (Fig. 4D). As shown in Fig. 4E, uptake of 0.5 mM glucose in the RANKL-treated RAW264.7 cells was increased compared with the untreated cells, although not as substantially as Pi transport. The enhancement of Pi uptake by treatment with RANKL was remarkably time dependent (Fig. 4F), showing increases in parallel with the degree of osteoclastic cell differentiation (Fig. 2A).

Kinetics of Pi transport in the osteoclast-like cells. To further understand the mechanism of Pi transport in the osteoclast-like cells, we investigated the kinetic properties of Pi transport in these cells. As shown in Fig. 5A, we measured Pi transport at pH 5.5 in Na+-free solution using various Pi concentrations. Kinetic analysis revealed two components of Pi transport with high and low affinities for Pi. The Km for the high-affinity component was 0.35 mM (0–1.6 mM Pi) (Fig. 5B), and the Km for the low-affinity component was 7.5 mM (1.6–12.8 mM Pi) (Fig. 5C). These transport systems were not found in untreated RAW264.7 cells (data not shown).



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Fig. 5. Kinetics of Pi transport in osteoclast-like cells. The influx medium contained 137 mM ChCl, and the Pi concentration was varied from 0 to 12.8 mM by addition of K2HPO4/KH2PO4, pH 5.5. A: Michaelis-Menten curve of Pi uptake from 0 to 12.8 mM; inset: Michaelis-Menten curve of Pi uptake from 0 to 1.6 mM. B: Lineweaver-Burk plot for Pi concentrations between 0 and 2 mM. C: Lineweaver-Burk plot for Pi concentrations between 2 and 12.8 mM.

 
Monovalent cation selectivity for activation of Pi transport. Figure 6A shows the Pi influx into the osteoclast-like cells in the presence of various monovalent cations at equal Cl concentrations. When the cation was 137 mM Ch, K+, Na+, Li+, Rb+, or Cs+, K+ was able to activate the influx relative to the other cations. Therefore, we investigated the dependence of Pi transport on K+. As shown in Fig. 6B, the extracellular K+ ion stimulated Pi transport in a concentration-dependent manner. Furthermore, as shown in Fig. 6C, Pi transport activity was not affected by changes in the Cl concentration. These results suggest that Pi transport activity in the osteoclast-like cells is stimulated in the presence of increasing concentrations of K+ ions.



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Fig. 6. Activation of Pi influx by monovalent cations in osteoclast-like cells. A: monovalent cation activation of Pi influx was measured in an uptake solution, pH 5.5, wherein NaCl was replaced by ChCl, LiCl, RbCl, CsCl, or KCl. *P < 0.05, statistically significant difference vs. uptake in ChCl uptake solution. B: dependence of Pi uptake in osteoclast-like cells on the extracellular K+ concentration. The K+ concentration varied from 0 to 150 mM. The ionic strength was kept constant by maintaining the sum of [KCl] and [ChCl] at 150 mM. C: dependence of Pi uptake in osteoclast-like cells on Cl. The concentration of Cl was varied from 0 to 150 mM. The K+ concentration was kept constant by maintaining the sum of KCl and K+-gluconate concentrations at 150 mM. Values represent means ± SE; n = 3.

 
Effect of Pi transport inhibitors on Pi transport activity in the osteoclast-like cells. To further investigate the characteristics of Pi transport in osteoclast-like cells, we examined the effect of Pi transport-specific inhibitors. As shown in Fig. 7A, phosphonoformic acid (PFA; 1 mM), a competitive inhibitor of Pi transport, significantly inhibited Pi uptake in the osteoclast-like cells in the presence and absence of Na+ (56.3 and 32.6% inhibition, respectively). In the kidney and intestine, inhibition of Pi transport by PFA requires the presence of Na+ ion (19, 35). However, in the osteoclast-like cells, PFA affected Pi transport activity in the absence of Na+. We next examined the effect of arsenate (2 mM), a competitive inhibitor of Pi (5, 35), which also inhibited Pi uptake (89.3% inhibition) in the osteoclast-like cells (Fig. 7B). Furthermore, phosphonoacetic acid (PAA; 1 mM) inhibited (90% inhibition) Pi transport in the osteoclast-like cells (Fig. 7C). In contrast, PAA is only a weak inhibitor of the type IIa NaPi cotransporter (38). Similar effects were shown in the cells preincubated with these inhibitors for 30 min to 2 h before uptake (data not shown). Thus the effects of PAA and PFA on Pi inhibition in osteoclast cells are distinct from their effects on the type IIa NaPi cotransporter in kidney and intestine.



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Fig. 7. Effect of specific inhibitors of Pi transporters. A: effects phosphonoformic acid (PFA) on osteoclast-like cells. Pi uptake was measured in the presence of 0–5 mM PFA in uptake solution. Experiments were conducted at pH 5.5 in the presence of 0.1 mM PO4 and 137 mM ChCl or NaCl. B: inhibition of Pi uptake by arsenate. Various concentrations of arsenate were added to uptake solution containing ChCl, and Pi uptake was measured at pH 5.5. C: inhibition of Pi uptake by phosphonoacetic acid (PAA). Various concentrations of PAA were added to uptake solution containing ChCl, and Pi uptake was measured at pH 5.5. D: pharmacology of inhibition of Pi influx in osteoclast-like cells. The ability of various channel and transporter inhibitors to inhibit the Na+-free Pi uptake was assessed in osteoclast-like cells. Drug treatment included 0.2 mM N-ethyl maleimide (NEM), 1 µM bafilomycin A1, 0.5 mM acetazolamide, 0.2 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 1 mM amiloride, 1 mM 4,4'-dinitrostilbene-2,2'-disulfonic acid disodium salt (DNDS), 5 µM nigericin, 1 µM valinomycin, 50 µM FCCP, and 0.5 mM omeprazole for 10 min before uptake and upon uptake. The values of Pi uptake are indicated as the percentage of uptake in the absence of inhibitors. Values represent the means ± SE; n = 3. *P < 0.05. **P < 0.01.

 
Effect of proton ionophore on Pi transport in the osteoclast-like cells. To further investigate the connection between the Pi transport system and osteoclast function, we used several inhibitors of ion channels or transporters that are associated with osteoclast function (Fig. 7D). Pi transport activity in the osteoclast-like cells was not inhibited by acetazolamide, a specific inhibitor of carbonic anhydrase II. In addition, Pi uptake was inhibited by N-ethyl maleimide (NEM) but not by bafilomycin A1, which are specific inhibitors of the V-type ATPase. This suggests that the effects of NEM on Pi transport were mediated by components other than the V-type ATPase. Amiloride and omeprazole, which block epithelial proton-dependent transport, such as the Na+/H+ exchanger, slightly affected Pi transport activity. Furthermore, the anion transport inhibitors, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and DNDS, did not affect Pi transport activity. In contrast, the proton ionophores, nigericin and FCCP, and the K+ ionophore, valinomycin, significantly decreased Pi transport activity. These observations suggest that a proton gradient is necessary to transport Pi into the osteoclast-like cells.

Proton-dependent Pi transport in osteoclast-like cells. To further investigate whether the Pi transport system in the osteoclast-like cells is mediated by a proton-coupled transport system, we measured the pHi variations induced by Pi. For this purpose, we used the BCECF fluorescence technique (21, 37) to determine pHi changes in monolayers of RANKL-induced RAW264.7 cells. As shown in Fig. 8A, after an initial period of stabilization in NaCl buffer at pH 6.5, the addition 2 mM K2HPO4 induced a slight decrease in pHi within 5 min. When the initial external pH was 5.5, the changes in pHi were larger. In both cases, upon removal of K2HPO4, the pHi returned to the initial value. Although the decline of pHi in the presence of 2 mM K2HPO4 was Na+ independent, the recovery to the initial pH 6.5 was not as fast in the absence of Na+ (Fig. 8B). These results suggest that H+ flowed into osteoclast-like cells along with Pi in a manner that was unaffected by Na+.



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Fig. 8. Proton-dependent Pi transport. To measure H+-dependent Pi uptake, intracellular pH (pHi) changes were measured in osteoclast-like cells at pH 5.5 (squares) or 6.5 (circles) in Na+-containing (153 mM NaCl; A) and Na+-free (153 mM ChCl; B) HEPES-buffered solution. pHi was measured at 5-min intervals. The cells were first bathed in a Pi-free HEPES-buffered solution and then incubated in 2 mM Pi-containing HEPES-buffered solution. Time 0 represents the measurement before addition of the Pi-containing solution. After 15 min, the medium was replaced with fresh Pi-free HEPES-buffered solution.

 
Effect of bone particles on Pi transport in the osteoclast-like cells. Because it was previously shown that Na+-dependent Pi uptake was increased by adding bone particles (8), we next investigated whether H+-dependent Pi transport activity also would be increased by incubation with bone particles. As shown in Fig. 9, bone particles stimulated Pi transport activity in the osteoclast-like cells. Further analysis showed that the stimulation by bone particles was dose dependent, and a time course study demonstrated that Pi transport increased twofold within 10 min after the addition of the bone particles (data not shown).



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Fig. 9. Stimulation of Pi uptake activities by bone. Osteoclast-like cells were exposed to bone particles for 1 h before Pi uptake was measured. Cells that were not exposed to bone served as the untreated control. Pi uptake was measured for 10 min at pH 5.5 in ChCl uptake solution. Values represent the means ± SE; n = 3. *P < 0.05.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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In the present study, we have characterized the Pi transport properties of osteoclast-like cells that were derived from RAW264.7 cells by treatment with RANKL. The Pi transport system in osteoclast cells has not been well characterized, owing to the difficulty in obtaining sufficient numbers of mature, freshly isolated osteoclasts. In addition, osteoclastogenesis is influenced by a variety of factors that could differ between preparations, resulting in variability in cell cultures. We found that RANKL causes the time-dependent increase in the activity of the H+-dependent and voltage-sensitive Pi transport system. PFA, arsenate, and PAA, specific inhibitors of Pi transport, also inhibited the transport system in the osteoclast-like cells. In addition, these cells expressed two components (high and low affinity for Pi) of H+-dependent Pi transport. The values for the dissociation of Pi into H2PO4 and HPO42– are 2.1 and 7.2 pK, respectively. Therefore, below pH 6.0, most of the Pi is present as the monovalent H2PO4 species. In the present study of the pH dependency of Pi uptake into the osteoclast-like cells, we have demonstrated that transport rates are the highest between pH 5.0 and 5.5, where the H2PO4 species is predominant. This suggests that Pi is taken up as H2PO4 across the plasma membrane in the osteoclast-like cells.

Some initial findings were reported by Gupta and colleagues (8, 9), who showed that the type IIa NaPi cotransporter is expressed in various primary osteoclasts and osteoclast-like cells. In the present study, we examined the expression of the type IIa NaPi cotransporter in osteoclast-like cells that were generated by treatment of mouse RAW264.7 cells with RANKL. Expression of the type IIa NaPi cotransporter was detected in these cells or in murine bone marrow-derived cells. However, the subcellular distribution of type IIa NaPi cotransporter detected using immunohistochemical analysis and protein expression examined using Western blotting were negative. We do not know the reason for this apparent discrepancy with the previous work of Gupta and colleagues (8, 9). One possibility may be due to the culture conditions used in the present study (i.e., charcoal-stripped FBS). On the other hand, Npt2a–/– mouse studies have suggested that the type IIa NaPi cotransporter gene plays a role in osteoclasts (10, 16). Npt2a–/– mice showed several skeletal abnormalities, including reduced osteoclast number. These results suggest that the type IIa NaPi cotransporter is a major regulator of Pi homeostasis and is necessary for normal skeletal development. However, Npt2a–/– mice have decreased circulating levels of parathyroid hormone (PTH) relative to their wild-type littermates. Because PTH increases osteoclast number and activity, reduced serum PTH levels in Npt2a–/– mice may contribute to compromised osteoclast function and a consequent bone-remodeling defect. Further studies are needed to clarify the role of Npt2 in the mouse osteoclast.

In contrast to the type IIa NaPi cotransporter, the type III NaPi cotransporter may play a role in osteoclast-like cells (17). We also found that Na+-dependent Pi transport in the untreated RAW264.7 cells may be mediated by the type III NaPi cotransporter (Ram-1 and Glvr-1). However, the type III Na+-dependent Pi transporter was not increased by treatment of the RAW264.7 cells with RANKL. The present studies have indicated that the H+-dependent Pi transporter system, but not the type III NaPi cotransporter, is upregulated in the osteoclast-like cells.

On the other hand, the H+-dependent Pi transport system has been reported in sheep and goat intestine (13, 32). In ruminants, the salivary glands are the major organs responsible for endogenous Pi secretion entering the gut. The daily secretion rate is 10–16 L/day for sheep and 30–50 L/day in cows, containing 16–40 mmol/l orthophosphate, that is, ~200–300 mmol/day (13). This amount of Pi greatly exceeds that supplied by the diet (50–60 mmol/day), and therefore an efficient scavenging system for the absorption of Pi in the gut and secretion via saliva in ruminants are essential and of major significance (13). In these ruminants, the physiological relevance of the H+-dependent Pi transport system could be reflected by the lower pH values in the digesta of the upper small intestine (13). The H+-dependent Pi transport system is essential for efficiency in Pi intake. In the duodenum of goat, the transporter affinity for Pi (Km 0.32 ± 0.33 mM) was very similar to that of mouse osteoclasts (13). The existence of the H+ rather than Na+ coupling for Pi transport in the osteoclast may represent the use of an alternative cation substrate. Hirayama et al. (11) showed that H+ can substitute for Na+ in driving sugar transport through the intestinal Na+-glucose cotransporter (SGLT1). Therefore, it could be that the difference in the cation specificity of the Pi transporter in kidney (Na+ dependent) and osteoclast (H+ dependent) is a mechanistic adaptation and a structural modification in the cation binding sites of Pi transporters in response to extracellular pH (13).

What is a role of the H+-dependent Pi transport system in the osteoclast-like cells? One of the most important roles of the transport system is ATP production for the secretion of protons (28, 36). The osteoclast consumes large amounts of energy to drive HCl secretion, which produces Ca2+, water, and Pi from the strongly basic Ca2+ salt, hydroxyapatite, [Ca3(PO4)2]3Ca(OH)2, which comprises bone minerals. The source of the energy for this secretion is the oxidative phosphorylation of glucose in the mitochondria. Protons are derived from carbonic acid, a process facilitated by high expression of carbonic anhydrase II in osteoclasts. In addition, the extensive proton secretion in osteoclasts is mediated by high expression levels of the V-type ATPase (Fig. 10). Because this process is energy intensive, osteoclasts contain large numbers of mitochondria. Therefore, some of the Pi released by bone resorption may be taken up through osteoclast Pi transport processes and used for ATP production.



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Fig. 10. Hypothesis regarding Pi transporters in osteoclast-like cells. The osteoclast consumes large amounts of energy to drive HCl secretion, which produces Ca2+, water, and phosphate from the strongly basic CaPO4 salt hydroxyapatite, [Ca3(PO4)2]3Ca(OH)2, that comprises bone minerals. The primary cellular mechanism responsible for this acidification is active secretion of protons by the vacuolar-type H+-adenosine triphosphatase (V-type ATPase), which is localized in the ruffled border of the osteoclasts. Pi influx has been reported to require extensive V-type ATPase activity and thus a large amount of energy. Pi may also help maintain the ATP content during the cyclical processes of migration, attachment, and resorption. At least three NaPi transporters (type IIa, type III, and H+ dependent) are expressed in the plasma membrane of RANKL-induced osteoclast-like cells.

 
The low-affinity Pi transport system that we have identified in the present study may be involved in the removal of degraded products. The basic building blocks of bone are proteins, such as collagen, and hydroxyapatite, which is dissociated to Ca2+ and HPO42– under acidic conditions. Recent reports have indicated that the products of acidic bone degradation are trafficked by the osteoclast (24, 29, 34). Vesicular transcytosis is important in bone-resorbing osteoclasts. In contrast, during bone resorption, a large amount of Ca2+ (up to 40 mM) and Pi ion is generated within the osteoclast hemivacuole (26, 33). The precise mechanisms involved in the disposal of Ca2+ are not clear. Recently, Berger et al. (2) demonstrated that the Ca2+ produced in the resorption hemivacuole is continually transported out of the resorptive site. Moreover, Stenbeck and Horton (34) reported a critical role of the microtubule network in transport for trafficking events. These in situ studies suggest that in a bone-resorbing osteoclast, a relatively large amount of Ca2+ enters from the resorption hemivacuole into the cell and is continuously released at the basolateral plasma membrane. There are likely to be three routes of Ca2+ and Pi disposal: leakage, bulk transcytosis, and selective disposal involving channels and transporter (2, 7, 26, 29). We suspect that the low-affinity Pi transport system may be involved in the transcellular Pi transport system, providing Pi and degraded products for release as well as movement of Pi through the cytoplasm.

Finally, in the present study, we have demonstrated the characteristics of the Pi transport system in osteoclast-like cells. This transport system is activated at acidic pH and has Km for Pi of ~0.35 mM and 7.5 mM. The activity of the two transport components is enhanced by RANKL and bone particles. Further studies must be performed to determine the molecular identity of these transporters and their roles in Pi transport in the osteoclast.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by Grants 16790464 (to M. Ito) and 16390244 (to K. Miyamoto) from the Ministry of Education, Science, Sports and Culture of Japan and the Human Nutritional Science on Stress Control 21st Century Center of Excellence Program.


    ACKNOWLEDGMENTS
 
We thank Dr. Y. Kanai (Kyorin University School of Medicine, Tokyo, Japan) for helpful discussions and comments. We also thank Dr. Y. Ohba for providing bone particles, Dr. Y. Taketani for providing anti-Glvr-1 antibody, and Dr. M. Shono (University of Tokushima Graduate School, Tokushima, Japan) for pHi experiments.


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. Miyamoto, Dept. of Molecular Nutrition, Institute of Health Biosciences, The Univ. of Tokushima Graduate School, Kuramoto-cho 3-18-15, Tokushima City 770-8503, Japan (E-mail: miyamoto{at}nutr.med.tokushima-u.ac.jp)

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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