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-κB 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
- proton dependent
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-κB 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
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 α-minimum essential medium (α-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).
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 × 103/cm2 in phenol red-free α-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 α-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 × 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.
Significant differences (P < 0.05) between means were determined with paired or unpaired t-tests.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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