1Department of Molecular Nutrition, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima City, Japan; and 2Department of Biochemistry, University of WisconsinMadison, Madison, Wisconsin
Submitted 23 August 2004 ; accepted in final form 14 December 2004
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ABSTRACT |
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Na+-dependent phosphate cotransporter; RAW264.7; phosphate uptake
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-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).
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MATERIALS AND METHODS |
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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 -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 -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
-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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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 (0.0512.8 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.
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RESULTS |
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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 (01.6 mM Pi) (Fig. 5B), and the Km for the low-affinity component was 7.5 mM (1.612.8 mM Pi) (Fig. 5C). These transport systems were not found in untreated RAW264.7 cells (data not shown).
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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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DISCUSSION |
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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 1016 L/day for sheep and 3050 L/day in cows, containing 1640 mmol/l orthophosphate, that is, 200300 mmol/day (13). This amount of Pi greatly exceeds that supplied by the diet (5060 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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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.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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