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Am J Physiol Cell Physiol 292: C526-C534, 2007. First published September 13, 2006; doi:10.1152/ajpcell.00357.2006
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CELLULAR METABOLISM

Unique uptake and efflux systems of inorganic phosphate in osteoclast-like cells

Mikiko Ito,1,2 Sakiko Haito,1,2 Mari Furumoto,1 Yoko Uehata,1 Aya Sakurai,1 Hiroko Segawa,1 Sawako Tatsumi,1,2 Masashi Kuwahata,1 and Ken-ichi Miyamoto1,2

1Department of Molecular Nutrition and 2Project Team of the Human Nutritional Science on Stress Control 21st Century Center of Excellence Program, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan

Submitted 27 June 2006 ; accepted in final form 16 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
During bone resorption, a large amount of inorganic phosphate (Pi) is generated within the osteoclast hemivacuole. The mechanisms involved in the disposal of this Pi are not clear. In the present study, we investigated the efflux of Pi from osteoclast-like cells. Pi efflux was activated by acidic conditions in osteoclast-like cells derived by the treatment of RAW264.7 cells with receptor activator of nuclear factor-{kappa}B ligand. Acid-induced Pi influx was not observed in renal proximal tubule-like opossum kidney cells, osteoblast-like MC3T3-E1 cells, or untreated RAW264.7 cells. Furthermore, Pi efflux was stimulated by extracellular Pi and several Pi analogs [phosphonoformic acid (PFA), phosphonoacetic acid, arsenate, and pyrophosphate]. Pi efflux was time dependent, with 50% released into the medium after 10 min. The efflux of Pi was increased by various inhibitors that block Pi uptake, and extracellular Pi did not affect the transport of [14C]PFA into the osteoclast-like cells. Preloading of cells with Pi did not stimulate Pi efflux by PFA, indicating that the effect of Pi was not due to transstimulation of Pi transport. Pi uptake was also enhanced under acidic conditions. Agents that prevent increases in cytosolic free Ca2+ concentration, including acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 2-aminoethoxydiphenyl borate, and bongkrekic acid, significantly inhibited Pi uptake in the osteoclast-like cells, suggesting that Pi uptake is regulated by Ca2+ signaling in the endoplasmic reticulum and mitochondria of osteoclast-like cells. These results suggest that osteoclast-like cells have a unique Pi uptake/efflux system and can prevent Pi accumulation within osteoclast hemivacuoles.

phosphate transporter; RAW264.7; proton dependent; acidification

OSTEOCLASTS ARE the primary cells responsible for bone resorption. They arise through 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 (33, 38). Bone resorption depends on the ability of the osteoclast to generate an acid extracellular compartment between it and the bone surface (2). An acidic pH is essential for solubilization of the alkaline salts of bone mineral hydroxyapatite ([Ca3(PO4)2]3Ca(OH)2) as well as for digestion of the organic bone matrix by acid lysosomal enzymes secreted by osteoclasts (9, 26). The primary cellular mechanism responsible for this acidification is active secretion of protons by vacuolar-type H+-ATPase (V-ATPase), which is localized in the ruffled border of osteoclasts (2). The energy requirement to drive this process is high and is manifested by the presence of abundant mitochondria.

Inorganic phosphate (Pi) is the major anionic component of bone. Pi uptake has been reported to require extensive V-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 (13). In osteoclasts, the type IIa sodium-dependent phosphate (Na-Pi) transporter is expressed in primary osteoclasts as well as in mouse osteoclast-like cells generated from RAW264.7 cells by treatment with receptor activator of nuclear factor-{kappa}B ligand (RANKL) (13, 14). RAW264.7-derived osteoclasts express Pit-1, a type III Na-Pi transporter that also acts as a Pi transporter (21), and the function of RANKL-treated RAW264.7 cells is affected by inhibitors of Na+-dependent Pi transport (13, 14). The feeding of low- and high-Pi diets also affects the function of osteoclasts in vivo (18, 23). Furthermore, in a previous report (16), we suggested that osteoclasts have an H+-dependent Pi transporter. Pi transport under acidic conditions may be necessary for bone resorption or for production of the large amounts of energy necessary for acidification of the extracellular environment.

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 osteoclasts (31, 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 (10). The precise mechanisms involved in the disposal of Ca2+ and Pi are not clear. The Ca2+ produced in the resorption hemivacuole is continually transported out of the resorptive site (4), and a relatively large amount of Ca2+ enters from the resorption hemivacuole into the cell and is continuously released at the basolateral plasma membrane (4). There are likely to be three routes of Ca2+ and Pi disposal: leakage, bulk transcytosis, and selective disposal involving channels and transporters.

In the present study, we demonstrate that osteoclast-like cells have a unique Pi efflux system. The characteristics and molecular identity of the Pi efflux system in various cells have not been well established. A few reports have examined the Pi efflux system in opossum kidney (OK) cells (1), pancreatic islets (11, 12), and vagus nerves (19). In the proximal tubule, the way in which Pi exits from the basolateral side is not well defined. In the present study, we investigated the Pi uptake and efflux system in osteoclast-like cells derived by RANKL treatment of RAW264.7 cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. The mouse monocyte/macrophage cell line RAW264.7 and the osteoblastic cell line MC3T3-E1 were obtained from the RIKEN Bioresource Center (Tokyo, Japan). Dulbecco's modified Eagle's medium (DMEM) 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 with the vector pGEX-3T (GE Healthcare Bio-Sciences, Piscataway, NJ) and purified by affinity chromatography using a glutathione-Sepharose column (GE Healthcare Bio-Sciences) (35). 14C-labeled phosphonoformic acid (PFA) was obtained from Moravek Biochemicals (Brea, CA). All other chemicals were obtained from Sigma Chemical (St. Louis, MO).

Cell Culture. RAW264.7 cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS). Osteoclasts were generated as previously described (16). Briefly, RAW264.7 cells were plated at 5 x 103/cm2 in phenol red-free {alpha}-MEM supplemented with recombinant RANKL (300 ng/ml) and 10% FBS that had been stripped with charcoal to remove endogenous steroids (35). The medium was changed on day 3 and replaced with fresh medium and mediators. After 7 days, multinucleated osteoclasts were identified with tartrate-resistant acidic phosphatase (TRAP), histochemical staining, and detection of calcitonin receptor mRNA by reverse transcription-polymerase chain reaction (RT-PCR) (16, 42).

MC3T3-E1 cells were cultured in {alpha}-MEM supplemented with 10% FBS, penicillin-streptomycin, and L-glutamine (17). OK cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in DMEM-Ham's F-12 (Sigma) supplemented with 10% FBS and antibiotics (15). For experiments, the cells were grown in 12-well dishes for 3 days to ~90% confluence.

Assay of Pi uptake and efflux with 32Pi. Pi transport was studied in monolayers of RANKL-differentiated or undifferentiated RAW264.7 cells in 12-well dishes. Cell densities were ~1.6 x 104 cells per well. Pi efflux was measured with a modification of a previously described procedure (1, 16, 32). The cell monolayers were gently washed three times with 0.5 ml of prewarmed (37°C) uptake solution (mM: 137 NaCl, 5.4 KCl, 2.8 CaCl2, 1.2 MgCl2, and 10 HEPES-Tris, pH 7.4) for Na+-dependent Pi uptake assays, and NaCl was replaced with choline chloride for Na+-independent Pi uptake assays. For the pH 5.5 uptake solution, the HEPES-Tris buffer was replaced with MES-Tris buffer. Pi transport was initiated by the addition of prewarmed (37°C) uptake solution containing 0.1 mM KH2PO4 and 1 µCi/ml 32Pi (Perkin-Elmer, Bridgeport, CT). For MC3T3-E1 and OK cells, the uptake assay was carried out with uptake solution containing NaCl and at pH 7.5 for 10 min. For efflux experiments, the cells were incubated for 30 min at 37°C and then washed three times with Pi-free uptake solution. The uptake solution was then replaced with efflux solution containing 0, 0.1, or 2 mM Pi in uptake solution. After 10 min, the supernatant was collected and mixed with 2.5 ml of Aquasol-2 (Packard Instruments, Meriden, CT). The remaining cell monolayer was washed three times in 1 ml of ice-cold stop solution (137 mM NaCl and 10 mM Tris·HCl, pH 7.2). After three additional washes with 1 ml of 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. 32Pi in the collected supernatants and cell lysates was measured by liquid scintillation counting. The protein concentration in lysates was determined with a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL). Pi transport was calculated as nanomoles of 32Pi per milligram of protein taken up in 10 min. The percentage of Pi efflux was calculated as the ratio of released 32Pi to total cellular 32Pi.

For efflux inhibition experiments, the inhibitors were added to the wash solution and the efflux assay solution. Glucose uptake measurements were performed to determine whether the changes in efflux in osteoclast-like cells were specific to Pi. For these experiments, osteoclast-like cells were mixed with 0.5 mM D-glucose and 1 µCi/ml 14C-labeled D-glucose. All experiments were performed in triplicate and repeated two to four times.

Assay of PFA uptake. PFA uptake was assayed with 0.1 mM PFA and 1 µCi/ml [14C]PFA. The uptake was linear with time for at least 60 min (data not shown). PFA transport was calculated as nanomoles of PFA per milligram of protein taken up in 10 min. For Pi exchange experiments, the cells were first loaded with 0.1 mM Pi for 10 min and then incubated with 0.1 mM PFA and 1 µCi/ml [14C]PFA. The cells were collected, and absorbed 14C radioactivity was measured. All experiments were performed in triplicate and repeated two to four times.

Statistics. Significant differences (P < 0.05) between means were determined with paired or unpaired t-tests.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
A unique efflux system for Pi in RANKL-induced RAW264.7 cells. In the present studies, we generated osteoclast-like cells by treating RAW264.7 cells with RANKL. Histochemical analysis showed that the numbers of TRAP-positive and multinucleated cells were markedly increased after 7 days of RANKL treatment compared with cells treated for 24 h. We observed ~90% differentiation of the RAW264.7 cells into osteoclast-like cells after 7 days. TRAP-positive cells were not detected in untreated RAW264.7 cells after 7 days. In addition, RT-PCR for the calcitonin receptor was markedly increased. Furthermore, immunostaining of the osteoclast-like cells with a calcitonin receptor-specific antibody showed staining at the cell surface (16).

In a previous study (16), we demonstrated that RANKL-treated RAW264.7 cells possess a H+-dependent Pi transport system. Here we confirmed that Pi uptake is markedly enhanced at pH 5.5 in the RANKL-treated cells (Fig. 1A). We also examined Pi efflux from untreated and RANKL-treated RAW264.7 cells that had been preloaded with 32Pi for 30 min at 37°C. As shown in Fig. 1B, Pi efflux was markedly higher at pH 5.5 than at pH 7.5 in the RANKL-treated RAW264.7 cells but not in the untreated RAW264.7 cells. Furthermore, we investigated the time course of Pi uptake and efflux in osteoclast-like cells. As shown in Fig. 2, Pi accumulation occurred at a relatively steady rate for ~30 min and reached a maximum at 40–60 min, whereas Pi release occurred at a high rate over the first 5 min, followed by a much slower rate.


Figure 1
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  		Fig. 1. Characterization of inorganic phosphate (Pi) transport in osteoclast-like cells. A: pH dependence of Pi uptake in the absence of Na+. Pi uptake was measured in 137 mM choline chloride (ChCl) uptake solution at pH 5.5, 6.5, 7.5, and 8.5. Uptake of Pi was measured in RAW264.7 cells treated with (gray bars) or without (open bars) receptor activator of nuclear factor-{kappa}B ligand (RANKL). B: efflux of Pi in RAW264.7 cells treated with [RAW(R+)] or without [RAW(R–)] RANKL. Pi efflux was measured with 32Pi, and the efflux is shown as % of the total Pi released. Open bars, pH 5.5; gray bars, pH 7.5. Values represent means ± SE (n = 3), and the results are representative of 3 independent experiments.

 

Figure 2
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  		Fig. 2. Time courses of Pi uptake and efflux in osteoclast-like cells. A: time course of Pi uptake in osteoclast-like cells. Pi uptake into RANKL-treated RAW264.7 cells was measured after 10 min at 37°C in 137 mM ChCl medium (pH 5.5) containing 0.1 mM PO4. B: Pi efflux was measured after 10 min at 37°C in 137 mM ChCl medium (pH 5.5) containing 0.1 mM PO4. Values represent means ± SE (n = 3), and results are representative of 3 independent experiments.

 
We next investigated whether the Pi efflux system is present in other cultured cell lines, including kidney proximal tubule (OK) cells and osteoblast-like (MC3T3-E1) cells. As shown in Fig. 3, Pi uptake at pH 7.5 in OK cells occurred at a higher rate than in MC3T3-E1, RAW264.7, or RANKL-treated RAW264.7 cells. In contrast, the rate of Pi uptake at pH 5.5 was higher in RANKL-treated RAW264.7 cells than in untreated RAW264.7 cells, OK cells, or MC3T3-E1 cells. Analysis of cells preloaded with 32Pi showed that RANKL-treated RAW264.7 cells released 45.6% of the Pi at pH 5.5, whereas untreated RAW264.7 cells released only 2.4% of the Pi (Fig. 3B). Efflux of Pi from OK and MC3T3E-1 cells was only 0.57% and 0.94%, respectively. A high Pi efflux at pH 7.5 was observed in OK cells, but increase at pH 5.5 was absent. These results show that, of the four cell types tested, only the RANKL-treated RAW264 cells have the acid-dependent high Pi efflux system.


Figure 3
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  		Fig. 3. Pi uptake and efflux by various cell types. A: uptake of Pi in RANKL-treated or untreated RAW264.7 cells, opossum kidney (OK) cells, and osteoblast-like MC3T3-E1 cells. Pi transport was calculated as nmol 32Pi/mg protein taken up in 10 min. Open bars, pH 5.5; gray bars, pH 7.5. B: Pi efflux by various cell types. Efflux of preloaded 32Pi was calculated as % of the total preloaded Pi. Open bars, pH 5.5; gray bars, pH 7.5. Values represent means ± SE (n = 3), and results are representative of 2 independent experiments. *P < 0.05.

 
Na+ and pH dependence of Pi efflux in osteoclast-like cells. To further characterize the Pi efflux system in RANKL-treated RAW264.7 cells, we analyzed the effect of pH and Na+. As shown in Fig. 4, the rate of Pi efflux was substantially affected by the external pH, with the highest rate at pH 5.5. Pi efflux, however, was not dependent on Na+. In Na+-free conditions, the activity was approximately fivefold higher at pH 5.5 than at pH 7.5. Interestingly, we previously reported (16) a similar sixfold higher rate of Pi uptake at pH 5.5 than at pH 7.5.


Figure 4
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  		Fig. 4. Dependence of Pi efflux on pH and Na+. RANKL-treated RAW264.7 cells were loaded with 32Pi at pH 5.5 for 30 min, and then Pi efflux was measured at 37°C in 137 mM NaCl or ChCl medium at pH 5.5, 6.5, 7.5, and 8.5 in the presence of 0.2 mM extracellular Pi. Values represent means ± SE (n = 3), and results are representative of 4 independent experiments.

 
Pi concentration dependence of Pi efflux in osteoclast-like cells. We next investigated the effect of external Pi levels on Pi efflux in RANKL-treated RAW264.7 cells at pH 5.5 (Fig. 5). Pi had a dose-dependent effect on the rate of efflux, with a Km of 0.078 mM. In contrast, Pi uptake at pH 5.5 (Fig. 5B) had a Km for Pi of 0.37 mM. In addition, Pi did not enhance the rate of glucose transport (Fig. 5C). Finally, Pi efflux was unaffected by preloading the cells with Pi (Fig. 5D), indicating that transstimulation did not occur.


Figure 5
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  		Fig. 5. Characterization of Pi efflux and uptake in RANKL-treated RAW264.7 cells. A: kinetics of Pi efflux. The efflux medium contained 137 mM ChCl, and the Pi concentration was varied from 0.1 to 2 mM by addition of K2HPO4/KH2PO4, pH 5.5. Inset: Lineweaver-Burk plot for Pi concentrations between 0 and 2 mM. S, 1/Pi (mM); V, 1/Pi efflux. B: kinetics of Pi uptake. The uptake medium contained 137 mM ChCl, and the Pi concentration was varied from 0 to 10 mM by addition of K2HPO4/KH2PO4, pH 5.5. C: effect of Pi on the efflux of D-glucose. Cells were loaded for 30 min with 14C-labeled D-glucose, and efflux after 10 min was measured in the presence of 0 mM (open bars) or 2 mM (gray bars) Pi. D: Pi transstimulation. Cells were preloaded with 0.1–10 mM Pi for 30 min, and Pi efflux was measured in the presence of 0.2 mM extracellular Pi. Values represent means ± SE (n = 3), and results are representative of 2 independent experiments.

 
Substrate specificity of Pi efflux in osteoclast-like cells. We examined whether various anions could affect Pi efflux in osteoclast-like cells (Fig. 6). The presence of 2 mM Pi enhanced Pi efflux at pH 5.5 by 3.2-fold compared with cells in Pi-free buffer (19.7% to 63.3%). Pi efflux was not affected by citrate, glutamate, succinate, chloride, gluconate, or bicarbonate compared with 2 mM Pi (Fig. 6A), but Pi analogs that are known to act as competitive inhibitors of Pi uptake increased Pi efflux (Fig. 6B). Specifically, Pi efflux was increased by 2 mM PFA (82.7% vs. control), phosphonoacetic acid (PAA; 97.7% vs. control), and arsenate (82.7% vs. control). Pyrophosphate, pyridoxal 5'-phosphate, and D-glucose-6-phosphate also enhanced Pi efflux, but to a lesser extent.


Figure 6
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  		Fig. 6. Substrate specificity for Pi efflux in RANKL-treated RAW264.7 cells. A: effect of Pi analogs on Pi efflux in osteoclast-like cells. Pi was replaced with various anions (1 mM). GluNa, sodium glutamate. B: effects of Pi analogs on Pi efflux, including known competitive inhibitors of Pi uptake in bone. G6P, glucose-6-phosphate; PLP, pyridoxal 5'-phosphate;PPi, pyrophosphate; PAA, phosphonoacetic acid; PFA, phosphonoformic acid. The value was calculated as a ratio compared with the value of Pi efflux of 2 mM extracellular Pi. Values represent means ± SE (n = 3), and results are representative of 3 independent experiments. *P < 0.05 vs. SO4.

 
Effect of PFA on phosphate efflux in osteoclast-like cells. The antiviral agent PFA is a known specific, competitive, and reversible inhibitor of Na-Pi cotransporter (24, 30). We next investigated whether the effect of PFA on Pi efflux is due to an exchange with P ion. As shown in Fig. 7A, in the presence or absence of Na+ there was little uptake of PFA in osteoclast-like cells at pH 5.5 or 7.5. The time course also showed a low-level Pi uptake for 30 min, after which it reached a plateau (data not shown). Furthermore, preloading the cells with Pi did not stimulate PFA uptake (Fig. 7B). Thus there was no transstimulation effect by Pi and PFA in osteoclast-like cells. Moreover, increasing the external Pi concentration did not inhibit PFA uptake. These results show that the stimulation of Pi efflux is not due to exchange with external PFA; rather, the binding of PFA to the membrane may stimulate the efflux of Pi.


Figure 7
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  		Fig. 7. Effect of PFA on Pi efflux in RANKL-treated RAW264.7 cells. A: uptake of PFA. PFA uptake was measured at pH 5.5 (open bars) or 7.5 (gray bars) in 137 mM NaCl or ChCl solution including 0.1 mM PFA and 1 µCi/ml [14C]PFA for 10 min. B: effect of Pi preloading on PFA uptake. Cells were preloaded with 0–10 mM Pi for 10 min and then treated with 0.1 mM PFA and 1 µCi/ml [14C]PFA. The cells were collected and measured for absorbed 14C. Values represent means ± SE (n = 3), and results are representative of 3 independent experiments.

 
Effects of various inhibitors on Pi uptake and efflux in osteoclast-like cells. We further investigated the effects of inhibitors of ion channels, Pi transporters, the pyrophosphate channel, and the anion exchanger on Pi efflux in RANKL-treated RAW264.7 cells. As shown in Fig. 8, Pi uptake was decreased to 32.6% of control value by PFA, 10.7% by arsenate (inhibitor of Pi transport), 48.2% by probenecid (inhibitor of the anion exchanger and the pyrophosphate channel ANK inhibitor), 16.8% by N-ethylmaleimide (inhibitor of mitochondrial Pi exchanger), 8.3% by nigericin (inhibitor of mitochondrial K+/H+ exchanger), 8.8% by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (H+ ionophore in mitochondria), and 36.8% by valinomycin (inhibitor of Na+-K+-ATPase). Interestingly, all compounds that inhibited Pi uptake appeared to stimulate Pi efflux (Fig. 8). For example, probenecid inhibited Pi uptake by 51.8% and stimulated Pi efflux by 179.5%. In general, however, the inhibition of Pi efflux did not parallel that of uptake.


Figure 8
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  		Fig. 8. Effect of specific inhibitors of Pi uptake and various transporters on Pi uptake (open bars) and efflux (gray bars) in RANKL-treated RAW264.7 cells. Cells were pretreated for 10 min, and efflux was measured in the presence of 0.1 mM extracellular Pi and 137 mM ChCl at pH 5.5. Pi uptake was measured in the presence of 0.1 mM extracellular Pi. A: effects of inhibitors of Pi transport (5 mM PFA or 2 mM arsenate) and anion exchange (2.5 mM probenecid). B: effects of inhibitors of various transporters or channels, including 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 0.2 mM), bafilomycin (1 µM), amiloride (1 mM), N-ethylmaleimide (NEM; 0.2 mM), nigericin (5 µM), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 50 µM), and valinomycin (1 µM). Values represent means ± SE (n = 3), and results are representative of 3 independent experiments. *P < 0.05 vs. vehicle control.

 
Effect of extracellular Ca2+ on Pi efflux in osteoclast-like cells. Figure 9A shows that Pi efflux in RANKL-treated RAW264.7 cells was dose-dependently enhanced by extracellular Ca2+. In Ca2+-free solution, the Km of Pi efflux was elevated from 0.078 to 0.24 mM (Figs. 5A and 9B). In Ca2+-free buffer, however, a high concentration of Pi (2 mM) stimulated Pi efflux even in the presence of Ca2+ (data not shown). These results suggest that external Ca2+ also regulates Pi efflux in osteoclast-like cells.


Figure 9
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  		Fig. 9. Effect of extracellular Ca2+ on Pi efflux in RANKL-treated RAW264.7 cells. A: Pi efflux in medium containing 0–5 mM Ca2+. Efflux was examined in the presence of 0.1 mM extracellular Pi. *P < 0.05 vs. no added Ca2+. B: kinetics of Pi efflux in Ca2+-free medium. The efflux medium contained 0.1–2 mM Pi from the addition of K2HPO4/KH2PO4, pH 5.5. Inset: Lineweaver-Burk plot for Pi concentrations between 0 and 2 mM. Values represent means ± SE (n = 3), and results are representative of 3 independent experiments.

 
Role of intracellular Ca2+ signaling pathways in Pi uptake and efflux. To investigate the connection between the Pi uptake/efflux system and intracellular Ca2+ signaling, we used several inhibitors or activators of intracellular Ca2+ signaling pathways (3, 22, 25) that participate in osteoclast function (Fig. 10A). Acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM; membrane-permeant calcium chelator), 2-aminoethoxydiphenyl borate (2-APB; antagonist of intracellular inositol triphosphate-induced Ca2+ release), and bongkrekic acid (inhibitor of the mitochondrial permeability transition pore) greatly reduced Pi uptake in RANKL-treated RAW264.7 cells. SKF-96365 (Ca2+ channel blocker) had only a weak effect on Pi uptake, whereas A-23187 (calcium ionophore), thapsigargin (inhibitor of inositol triphosphate-independent intracellular Ca2+ release), and U-73122 (specific inhibitor of receptor-mediated phospholipase C) did not affect Pi uptake (data not shown). Furthermore, BAPTA-AM, 2-APB, SKF-96365, thapsigargin, and U-73122 did not affect Pi efflux (Fig. 10B). These results show that inhibitors of Ca2+ release from the endoplasmic reticulum and mitochondria reduce Pi uptake in osteoclast-like cells.


Figure 10
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  		Fig. 10. Effect of inhibitors of Ca2+ signaling and Pi transport on Pi uptake and efflux in RANKL-treated RAW264.7 cells. A: effect of inhibitors of Ca2+ signaling and Pi transport on Pi uptake. Before uptake measurement, cells were pretreated for 10 min with 1 µM A-23187, 150 µM SKF-96365, 50 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM, 100 µM 2-aminoethoxydiphenyl borate (2-APB), 50 µM bongkrekic acid, 1 µM U-73122, 1 µM thapsigargin, or vehicle. B: effect of inhibitors of Ca2+ signaling and Pi transport on Pi efflux. Efflux was measured in the presence of inhibitor and 0.2 mM extracellular Pi. Values represent means ± SE (n = 3), and results are representative of 3 independent experiments. *P < 0.05 vs. vehicle control.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
During the resorption process, osteoclasts are tightly sealed to the bone surface, and hence the site of resorption is isolated from the extracellular fluid (7, 37). How osteoclasts handle the large amounts of inorganic material released during resorption has been unclear. Interestingly, fluorescence microscopy experiments have indicated that organic and inorganic materials are transported through the osteoclast during resorption, a process termed transcytosis (31); however, a close analysis of the data on the kinetics of Ca2+ disposal at the surface of the bone-resorbing osteoclast and its comparison with fluorescence microscopy experiments reveals some critical differences between the mechanisms of Ca2+ disposal and transcytosis (34). The most important and obvious difference is that, unlike collagen trafficking, which occurs after several hours, Ca2+ flux at the basolateral surface occurs within minutes after the seeding of osteoclasts on bone and occurs at an approximately constant rate (39, 41). These data suggest that the Ca2+ flux occurs by selective transport rather than transcytosis by the osteoclasts (10, 29). The selective transport route in osteoclasts may be able to prevent inorganic ion accumulation within the resorptive hemivacuole (5, 6), but the mechanisms and structures that are involved in the transport of inorganic materials from the basolateral surface remain unknown.

The Pi efflux of osteoclast-like cells can be divided into two phases: the rapid efflux phase during the first 5 min and the subsequent steady efflux phase. We examined efflux with various inhibitor assays at both phases (data not shown). All data shown are from the steady efflux phase. Our time course analysis showed that, at pH 5.5, nearly half of the intracellular Pi is released within 10 min. The stimulation of Pi efflux by external Pi was not due to increased exchange activity between external and internal Pi, in other words, by transstimulation of Pi efflux by Pi. Furthermore, analysis of [14C]PFA uptake indicates that PFA does not exchange with intracellular Pi, suggesting that PFA stimulates Pi efflux by a mechanism different from the Pi/PFA exchange system. In contrast, Barac-Nieto et al. (1) demonstrated the presence of a Pi-anion exchange mechanism across the basolateral membrane of proximal tubule-like cells. The basolateral membrane of renal proximal tubule has a transporter that is capable of exchanging 2 moles of intracellular H2PO4– for each 1 mole of extracellular HPO42–. Like other anion exchangers, this basolateral Pi exchanger is modulated by extracellular Na+ (40). The fact that Pi efflux is enhanced by extracellular Pi but not by chloride, citrate, lactate, succinate, or glutamate suggests that this anion transport system is specific to osteoclast-like cells. Moreover, the Km value for Pi in the efflux system is lower in the osteoclast-like cells than in renal proximal tubule cells, suggesting that a high-affinity Pi sensing system may be important for Pi efflux. Thus the Pi efflux system in the osteoclast-like cells is very different from the basolateral Pi exchanger in the kidney.

In a previous study (16), we demonstrated that the osteoclast Pi uptake system is coupled to acidification of the intracellular space at the ruffled border membrane. This H+-coupled Pi transport system contributes to reuptake of H+ in activated osteoclasts. In addition, Pi released by bone resorption may be taken up through osteoclast H+-dependent Pi transport and used for ATP production (13, 44). Interestingly, we demonstrated that pyrophosphate significantly inhibited H+-dependent Pi transporter activity but not Na+-dependent Pi transport activity (renal type II Na-Pi cotransporters; data not shown). In the present study, the Pi uptake and efflux system were affected by external pyrophosphate, suggesting that several bisphosphonates, which are analogs of pyrophosphate that potently inhibit osteoclast-mediated bone resorption, may affect Pi transport in osteoclasts (36, 45). However, whether these compounds affect Pi efflux directly or indirectly by modulating Pi uptake by the cells or through completely different mechanisms remain unknown. Further studies are needed to clarify the mechanisms.

Acidification has been shown to increase the bone resorption activity of osteoclasts cultured on bone (8, 20). When Ca2+ signaling is triggered by various hormones or cytokines in osteoclasts, Ca2+ enters the cytoplasm from both intracellular Ca2+ stores and the extracellular compartment (6, 28, 43). Several studies have demonstrated that the blockade of intracellular Ca2+ reuptake by inhibitors of the sarco(endo)plasmic reticulum Ca2+-ATPase and capacitive Ca2+ entry significantly decreases osteoclast survival and bone resorption (27). In osteoclasts, movement of Ca2+ between intracellular stores is closely connected to the regulation of osteoclast survival and bone resorption. Prevention of increases in intracellular Ca2+ with inhibitors of intracellular Ca2+ release (e.g., BAPTA-AM, 2-APB, or bongkrekic acid) significantly inhibits Pi uptake in osteoclast-like cells. Thus the regulation of H+-Pi transport may be linked to Ca2+ signaling pathways in osteoclasts. Interestingly, several inhibitors of H+-Pi transport stimulated Pi efflux in the osteoclast-like cells. This may be due to an increase in the extracellular Pi concentration by blocking of Pi uptake, which could activate Pi efflux. On the basis of these findings, we suggest that there is a system in the plasma membrane of osteoclast-like cells that senses the extracellular Pi concentration as well as Pi analogs (e.g., PFA, PAA, and arsenate) and that is modulated by the extracellular Ca2+ concentration.

Finally, we showed that the unique efflux and uptake systems of Pi are present in osteoclast-like cells. In acid-secreting osteoclasts, the Pi efflux system may participate in transcellular transport of Pi. The activation of Pi uptake is dependent on Ca2+ and associated with osteoclast function. Acid-secreting osteoclasts may have a unique Pi efflux system that is involved in the continuous release of Pi at the basolateral plasma membrane. These findings suggest that a selective Pi transport route rather than transcytosis mediates Pi disposal by osteoclasts.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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This work was supported by Grants 18590891 (to M. Ito) and 18390250 (to K. Miyamoto) and a Grant-in Aid for Scientific Research on Priority Area 17081013 (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.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Miyamoto, Dept. of Molecular Nutrition, Inst. of Health Biosciences, 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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
1. Barac-Nieto M, Alfred M, Spitzer A. Basolateral phosphate transport in renal proximal-tubule-like OK cells. Exp Biol Med (Maywood) 227: 626–631, 2002.[Abstract/Free Full Text]

2. Bekker PJ, Gay CV. Biochemical characterization of an electrogenic vacuolar proton pump in purified chicken osteoclast plasma membrane vesicles. J Bone Miner Res 5: 569–579, 1990.[Web of Science][Medline]

3. Bennett BD, Alvarez U, Hruska KA. Receptor-operated osteoclast calcium sensing. Endocrinology 142: 1968–1974, 2001.[Abstract/Free Full Text]

4. Berger CE, Rathod H, Gillespie JI, Horrocks BR, Datta HK. Scanning electrochemical microscopy at the surface of bone-resorbing osteoclasts: evidence for steady-state disposal and intracellular functional compartmentalization of calcium. J Bone Miner Res 16: 2092–2102, 2001.[CrossRef][Web of Science][Medline]

5. Blair HC. How the osteoclast degrades bone. Bioessays 20: 837–846, 1998.[CrossRef][Web of Science][Medline]

6. Blair HC, Robinson LJ, Zaidi M. Osteoclast signalling pathways. Biochem Biophys Res Commun 328: 728–738, 2005.[CrossRef][Web of Science][Medline]

7. Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature 423: 337–342, 2003.[CrossRef][Medline]

8. Bushinsky DA. Effects of medium acidification by alteration of carbon dioxide or bicarbonate concentrations on the resorptive activity of rat osteoclasts. J Bone Miner Res 9: 1839–1844, 1994.[Web of Science][Medline]

9. Carano A, Schlesinger PH, Athanasou NA, Teitelbaum SL, Blair HC. Acid and base effects on avian osteoclast activity. Am J Physiol Cell Physiol 264: C694–C701, 1993.[Abstract/Free Full Text]

10. Datta HK, Horrocks BR. Mechanisms of calcium disposal from osteoclastic resorption hemivacuole. J Endocrinol 176: 1–5, 2003.[Abstract]

11. Freinkel N, Younsi CE, Bonnar J, Dawson RM. Rapid transient efflux of phosphate ions from pancreatic islets as an early action of insulin secretagogues. J Clin Invest 54: 1179–1189, 1974.[Web of Science][Medline]

12. Freinkel N, Younsi CE, Dawson RM. Insulin release and phosphate ion efflux from rat pancreatic islets induced by L-leucine and its nonmetabolizable analogue, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid. Proc Natl Acad Sci USA 73: 3403–3407, 1976.[Abstract/Free Full Text]

13. Gupta A, Guo XL, Alvarez UM, Hruska KA. Regulation of sodium-dependent phosphate transport in osteoclasts. J Clin Invest 100: 538–549, 1997.[Web of Science][Medline]

14. Gupta A, Tenenhouse HS, Hoag HM, Wang D, Khadeer MA, Namba N, Feng X, Hruska KA. Identification of the type II Na+-Pi cotransporter (Npt2) in the osteoclast and the skeletal phenotype of Npt2–/– mice. Bone 29: 467–476, 2001.[Medline]

15. Ito M, Iidawa S, Izuka M, Haito S, Segawa H, Kuwahata M, Ohkido I, Ohno H, Miyamoto K. Interaction of a farnesylated protein with renal type IIa Na/Pi co-transporter in response to parathyroid hormone and dietary phosphate. Biochem J 377: 607–616, 2004.[CrossRef][Web of Science][Medline]

16. Ito M, Matsuka N, Izuka M, Haito S, Sakai Y, Nakamura R, Segawa H, Kuwahata M, Yamamoto H, Pike WJ, Miyamoto K. Characterization of inorganic phosphate transport in osteoclast-like cells. Am J Physiol Cell Physiol 288: C921–C931, 2005.[Abstract/Free Full Text]

17. Ito M, Sakai Y, Furumoto M, Segawa H, Haito S, Yamanaka S, Nakamura R, Kuwahata M, Miyamoto K. Vitamin D and phosphate regulate fibroblast growth factor-23 in K-562 cells. Am J Physiol Endocrinol Metab 288: E1101–E1109, 2005.[Abstract/Free Full Text]

18. Jara A, Lee E, Stauber D, Moatamed F, Felsenfeld AJ, Kleeman CR. Phosphate depletion in the rat: effect of bisphosphonates and the calcemic response to PTH. Kidney Int 55: 1434–1443, 1999.[CrossRef][Web of Science][Medline]

19. Jirounek P, Vitus J, Jones GJ, Pralong WF, Straub RW. Involvement of intracellular calcium in the phosphate efflux from mammalian nonmyelinated nerve fibers. J Membr Biol 79: 87–95, 1984.[CrossRef][Web of Science][Medline]

20. Karsdal MA, Henriksen K, Sorensen MG, Gram J, Schaller S, Dziegiel MH, Heegaard AM, Christophersen P, Martin TJ, Christiansen C, Bollerslev J. Acidification of the osteoclastic resorption compartment provides insight into the coupling of bone formation to bone resorption. Am J Pathol 166: 467–476, 2005.[Abstract/Free Full Text]

21. Khadeer MA, Tang Z, Tenenhouse HS, Eiden MV, Murer H, Hernando N, Weinman EJ, Chellaiah MA, Gupta A. Na+-dependent phosphate transporters in the murine osteoclast: cellular distribution and protein interactions. Am J Physiol Cell Physiol 284: C1633–C1644, 2003.[Abstract/Free Full Text]

22. Komarova SV, Pereverzev A, Shum JW, Sims SM, Dixon SJ. Convergent signaling by acidosis and receptor activator of NF-kappaB ligand (RANKL) on the calcium/calcineurin/NFAT pathway in osteoclasts. Proc Natl Acad Sci USA 102: 2643–2648, 2005.[Abstract/Free Full Text]

23. Koyama Y, Rittling SR, Tsuji K, Hino K, Salincarnboriboon R, Yano T, Taketani Y, Nifuji A, Denhardt DT, Noda M. Osteopontin deficiency suppresses high phosphate load-induced bone loss via specific modulation of osteoclasts. Endocrinology 147: 3040–3049, 2006.[Abstract/Free Full Text]

24. Loghman-Adham M, Levi M, Scherer SA, Motock GT, Totzke MT. Phosphonoformic acid blunts adaptive response of renal and intestinal Pi transport. Am J Physiol Renal Fluid Electrolyte Physiol 265: F756–F763, 1993.[Abstract/Free Full Text]

25. Matsuo K, Galson DL, Zhao C, Peng L, Laplace C, Wang KZ, Bachler MA, Amano H, Aburatani H, Ishikawa H, Wagner EF. Nuclear factor of activated T-cells (NFAT) rescues osteoclastogenesis in precursors lacking c-Fos. J Biol Chem 279: 26475–26480, 2004.[Abstract/Free Full Text]

26. Meghji S, Morrison MS, Henderson B, Arnett TR. pH dependence of bone resorption: mouse calvarial osteoclasts are activated by acidosis. Am J Physiol Endocrinol Metab 280: E112–E119, 2001.[Abstract/Free Full Text]

27. Mentaverri R, Kamel S, Brazier M. Involvement of capacitive calcium entry and calcium store refilling in osteoclastic survival and bone resorption process. Cell Calcium 34: 169–175, 2003.[CrossRef][Web of Science][Medline]

28. Moonga BS, Li S, Iqbal J, Davidson R, Shankar VS, Bevis PJ, Inzerillo A, Abe E, Huang CL, Zaidi M. Ca2+ influx through the osteoclastic plasma membrane ryanodine receptor. Am J Physiol Renal Physiol 282: F921–F932, 2002.[Abstract/Free Full Text]

29. Mulari M, Vaaraniemi J, Vaananen HK. Intracellular membrane trafficking in bone resorbing osteoclasts. Microsc Res Tech 61: 496–503, 2003.[CrossRef][Web of Science][Medline]

30. Murer H, Hernando N, Forster I, Biber J. Proximal tubular phosphate reabsorption: molecular mechanisms. Physiol Rev 80: 1373–1409, 2000.[Abstract/Free Full Text]

31. Nesbitt SA, Horton MA. Trafficking of matrix collagens through bone-resorbing osteoclasts. Science 276: 266–269, 1997.[Abstract/Free Full Text]

32. Reshkin SJ, Forgo J, Murer H. Functional asymmetry of phosphate transport and its regulation in opossum kidney cells: phosphate transport. Pflügers Arch 416: 554–560, 1990.[CrossRef][Web of Science][Medline]

33. Roodman GD. Mechanisms of bone metastasis. N Engl J Med 350: 1655–1664, 2004.[Free Full Text]

34. Salo J, Lehenkari P, Mulari M, Metsikko K, Vaananen HK. Removal of osteoclast bone resorption products by transcytosis. Science 276: 270–273, 1997.[Abstract/Free Full Text]

35. Shevde NK, Bendixen AC, Dienger KM, Pike JW. Estrogens suppress RANK ligand-induced osteoclast differentiation via a stromal cell independent mechanism involving c-Jun repression. Proc Natl Acad Sci USA 97: 7829–7834, 2000.[Abstract/Free Full Text]

36. Suzuki K, Takeyama S, Sakai Y, Yamada S, Shinoda H. Current topics in pharmacological research on bone metabolism: inhibitory effects of bisphosphonates on the differentiation and activity of osteoclasts. J Pharmacol Sci 100: 189–194, 2006.[CrossRef][Web of Science][Medline]

37. Teitelbaum SL. Bone resorption by osteoclasts. Science 289: 1504–1508, 2000.[Abstract/Free Full Text]

38. Teitelbaum SL, Ross FP. Genetic regulation of osteoclast development and function. Nat Rev Genet 4: 638–649, 2003.[CrossRef][Web of Science][Medline]

39. Vaaraniemi J, Halleen JM, Kaarlonen K, Ylipahkala H, Alatalo SL, Andersson G, Kaija H, Vihko P, Vaananen HK. Intracellular machinery for matrix degradation in bone-resorbing osteoclasts. J Bone Miner Res 19: 1432–1440, 2004.[CrossRef][Web of Science][Medline]

40. Van Aubel RA, Masereeuw R, Russel FG. Molecular pharmacology of renal organic anion transporters. Am J Physiol Renal Physiol 279: F216–F232, 2000.[Abstract/Free Full Text]

41. Yamaki M, Nakamura H, Takahashi N, Udagawa N, Ozawa H. Transcytosis of calcium from bone by osteoclast-like cells evidenced by direct visualization of calcium in cells. Arch Biochem Biophys 440: 10–17, 2005.[CrossRef][Web of Science][Medline]

42. Yan T, Riggs BL, Boyle WJ, Khosla S. Regulation of osteoclastogenesis and RANK expression by TGF-beta1. J Cell Biochem 83: 320–325, 2001.[CrossRef][Web of Science][Medline]

43. Zaidi M, Adebanjo OA, Moonga BS, Sun L, Huang CL. Emerging insights into the role of calcium ions in osteoclast regulation. J Bone Miner Res 14: 669–674, 1999.[CrossRef][Web of Science][Medline]

44. Zaidi M, Alam AS, Shankar VS, Bax BE, Bax CM, Moonga BS, Bevis PJ, Stevens C, Blake DR, Pazianas M. Cellular biology of bone resorption. Biol Rev Camb Philos Soc 68: 197–264, 1993.[Medline]

45. Zaidi M, Blair HC, Moonga BS, Abe E, Huang CL. Osteoclastogenesis, bone resorption, and osteoclast-based therapeutics. J Bone Miner Res 18: 599–609, 2003.[CrossRef][Web of Science][Medline]





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