Am J Physiol Cell Physiol 292: C526 –C534, 2007.
First published September 13, 2006; doi:10.1152/ajpcell.00357.2006.
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
1
Department 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
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
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: [email protected]).
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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 VATPase 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-␬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
HPO2⫺
4 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
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0363-6143/07 $8.00 Copyright © 2007 the American Physiological Society
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Ito M, Haito S, Furumoto M, Uehata Y, Sakurai A, Segawa H,
Tatsumi S, Kuwahata M, Miyamoto K. Unique uptake and efflux
systems of inorganic phosphate in osteoclast-like cells. Am J Physiol
Cell Physiol 292: C526 –C534, 2007. First published September 13,
2006; doi:10.1152/ajpcell.00357.2006.—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-␬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)ethaneN,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 EFFLUX IN OSTEOCLASTS
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
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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 ␮Ci/ml
14
C-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 ␮Ci/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 ␮Ci/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
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 RANKLtreated 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
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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 ␣-minimum essential medium (␣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).
14
C-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 ⫻ 103/cm2 in phenol red-free
␣-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 ␣-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 ␮Ci/ml 32Pi (PerkinElmer, 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 䡠 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
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PHOSPHATE EFFLUX IN OSTEOCLASTS
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-␬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.
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).
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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.
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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
PHOSPHATE EFFLUX IN OSTEOCLASTS
absence of Na⫹ there was little uptake of PFA in osteoclastlike 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 RANKLtreated 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 mitochonAJP-Cell Physiol • VOL
drial 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)ethaneN,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.
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.
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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
32
Pi/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.
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PHOSPHATE EFFLUX IN OSTEOCLASTS
DISCUSSION
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
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.
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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.
PHOSPHATE EFFLUX IN OSTEOCLASTS
C531
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, BaracNieto et al. (1) demonstrated the presence of a Pi-anion ex-
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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 ␮Ci/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 ␮Ci/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.
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
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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 ␮M), amiloride (1 mM), N-ethylmaleimide (NEM; 0.2 mM),
nigericin (5 ␮M), carbonyl cyanide p-trifluoromethoxyphenylhydrazone
(FCCP; 50 ␮M), and valinomycin (1 ␮M). Values represent means ⫾ SE (n ⫽
3), and results are representative of 3 independent experiments. *P ⬍ 0.05 vs.
vehicle control.
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PHOSPHATE EFFLUX IN OSTEOCLASTS
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⫹
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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.
change 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 H2PO⫺
4 for each 1 mole of extracellular
HPO2⫺
4 . 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
AJP-Cell Physiol • VOL
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 ␮M A-23187, 150 ␮M SKF-96365, 50 ␮M
1,2-bis(2-aminophenoxy)ethane-N,N,N⬘,N⬘-tetraacetic acid (BAPTA)-AM,
100 ␮M 2-aminoethoxydiphenyl borate (2-APB), 50 ␮M bongkrekic acid, 1
␮M U-73122, 1 ␮M 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.
292 • JANUARY 2007 •
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PHOSPHATE EFFLUX IN OSTEOCLASTS
GRANTS
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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