This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, J.-P. Articles by Okabe, K. Search for Related Content PubMed PubMed Citation Articles by Li, J.-P. Articles by Okabe, K.

Three Na⁺/Ca²⁺ Exchanger (NCX) Variants Are Expressed in Mouse Osteoclasts and Mediate Calcium Transport during Bone Resorption

Department of Physiological Science and Molecular Biology (J.-P.L., H.K., F.O., A.N., K.O.), Fukuoka Dental College, Fukuoka 8140193, Japan; Department of Pharmacology (T.I.), School of Medicine, Fukuoka University, Fukuoka 8140180 Japan; and Department of Periodontics and Oral Medicine (J.-P.L.), Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou 510060, People’s Republic of China

Address all correspondence and requests for reprints to: H. Kajiya, Ph.D., Department of Physiological Science and Molecular Biology Fukuoka Dental College Tamura 2-15-1, Sawara-ku, Fukuoka 8140193, Japan. E-mail: kajiya{at}college.fdcnet.ac.jp.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The plasma membrane Na⁺/Ca²⁺ exchanger (NCX) is a bidirectional transporter that mediates the exchange of Na⁺ for Ca²⁺ depending on the electrochemical gradients. Mammalian NCXs form a multigene family comprising NCX1, NCX2, and NCX3 isoforms. Although it has been known that NCX1 in rat osteoclasts is coupled with the Na⁺/ H⁺ exchanger for regulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i), it is unclear what kind of NCX1 variants are expressed and whether the other two NCX isoforms are also present in mouse osteoclasts. To clarify the role of NCXs during bone resorption, we investigated the expression of NCXs, the ion transport via NCXs, and the effects of NCX inhibitors on bone-resorbing activity in mouse osteoclasts. Using RT-PCR, immunocytochemical, and Western blot methods, we detected three splice variants of NCX1 and NCX3, namely NCX1.3, NCX1.41, and NCX3.2. Of these, NCX1.41 is a newly identified splice variant. Low extracellular sodium ([Na⁺]_o) solution increases the intracellular Ca²⁺ concentration via NCX transporter in fura-2-loaded osteoclasts. The [Na⁺]_o-free solution-induced [Ca²⁺]_i increase was suppressed by benzyloxyphenyl NCX inhibitors. Bidirectional NCX currents in mouse osteoclasts were recorded using the patch clamp method and could be suppressed with NCX inhibitors. NCX inhibitors also decreased the resorption pit area surrounding osteoclasts in a dose-dependent manner. Furthermore, small interference RNAs targeted against NCX1.3, NCX1.41, and NCX3.2 expressed in mouse osteoclasts suppressed osteoclastic pit formation. These results show that three NCX variants are expressed in mouse osteoclasts and play an important role for Ca²⁺ transport and regulation during osteoclastic bone resorption.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
OSTEOCLASTS ARE MULTINUCLEATED cells that adhere to the bone surface via integrin receptors and form ruffled borders with a highly polarized cytoplasmic organization. After cytoskeleton formation, osteoclasts extrude protons and chloride ions onto the bone surface to dissolve inorganic components of the bone matrix. Diverse agents, such as calcitonin, IL-4, and ipriflavone have been shown to increase intracellular Ca²⁺ concentration ([Ca²⁺]_i), modulating bone resorption of mammalian osteoclasts (1, 2, 3). We previously reported that cell membrane depolarization and protein tyrosine kinase inhibitors increase [Ca²⁺]_i via Ca²⁺ influx, disrupt the cytoplasmic organization, and inhibit bone resorption (4, 5). However it remained unclear what kind of Ca²⁺ transport mechanism was responsible for this modulation of bone resorption.

The Na⁺/Ca²⁺ exchanger (NCX) is a bidirectional transporter that plays an important role for Ca²⁺ homeostasis in a variety of tissues and cell populations (6). NCX is a multigene family of homologous proteins consisting of three isoforms: NCX1, NCX2, and NCX3 (7, 8). NCX1 and NCX3 isoforms have several tissue-specific splice variants differing in a small region of the large intracellular loop (9, 10, 11). NCX1 in rat osteoclasts is coupled with the Na⁺/H⁺ exchanger for regulation of cytosolic Ca²⁺ (12). However, it is unclear what kind of NCX1 variants are expressed in mouse osteoclasts and whether the other two NCX isoforms are also present in these cells. Therefore, we focused on NCXs as possible candidates for Ca²⁺ transport pathways during osteoclastic bone resorption.

In the present study, we demonstrate that three types of NCX variants are expressed in mouse osteoclasts and that these play an important role for bone resorption. The NCXs are likely involved in the Ca²⁺ signaling pathway across the osteoclast cell membrane and in calcium disposal from the resorption lacunae.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
All procedures concerning animal experiments were approved by the Council on Animal Care of the Fukuoka Dental College.

Osteoclast culture
Mouse bone marrow cells (5 x 10⁵ cells/ml) were obtained from tibiae of 4- to 6-wk-old ddy mice. Primary osteoblastic cells (5 x 10⁴ cells/ml) were prepared from calvaria of newborn ddy mice (13). These cells were cocultured in -MEM (Life Technologies, Inc., Grand Island, NY) containing 10% fetal bovine serum (Life Technologies), 10 nM 1,25-dihydroxyvitamin D₃, and 1 µM prostaglandin E₂ in culture dishes precoated with a collagen gel matrix (Nitta Gelatin, Osaka, Japan). After 7 d of culture, mature osteoclasts were released from the dishes by treatment with 0.2% collagenase and collected by centrifugation at 250 x g for 5 min. In some experiments, osteoclasts were purified according to Tezuka’s method (14). Briefly, crude osteoclasts were cultured on 100-mm-diameter dishes for 2 h and then subjected to PBS containing 0.001% pronase E and 0.02% EDTA at 37 C for 10 min. After washing twice with PBS, multinuclear osteoclasts (purified osteoclast preparation) could be isolated from all other cells. These multinuclear cells (cells having at least three nuclei) had been proved to be capable of forming resorption pits on calcium phosphate-coated coverslips or dentine slices. In some experiments, bone marrow cells (1 x 10⁶ cells/ml) were cultured for 7 d in -MEM containing 10% fetal bovine serum, 10 ng/ml macrophage colony-stimulating factor, and 50 ng/ml soluble receptor activator of nuclear factor-B ligand, in the absence of primary osteoblastic cells, to obtain pure osteoclasts. For proper identification of osteoclasts after each experiment, cells were stained for tartrate-resistant acid phosphatase (TRAP) using an acid phosphatase kit (Sigma Chemical Co., St. Louis, MO).

RNA isolation and RT-PCR
Total RNA was extracted from purified osteoclast preparation using TRIzol reagent (Life Technology, Rockville, MD). First-strand cDNA was synthesized with Superscript First Chain Synthesis Kit according to manufacturer’s instructions (Invitrogen, Carlsbad, CA). To examine mRNA expression of mouse NCX, we designed isoform- and variant-specific primers based on the reported cDNA sequences information. The PCR conditions were 94 C for 30 sec, 48–53 C for 30 sec, and 72 C for 45 sec (36 cycles). The PCR primers, amplicon size, and corresponding annealing temperatures were as follows: NCX1, 5'-CCT TGT GCA TCT TAG CAA TG and 5'-TCT CAC TCA TCT CCA CCA GA, 437 bp, 52 C; NCX2, 5'-CTC AGG GAA TGG AGA CAA GA and 5'-GAA CGC AGG CGA ATA GAA, 341 bp, 52 C; NCX3, 5'-GGT GTG TGG TCA CGG GTT C and 5'-TCG GTG TTT ATC TGT GCG GTA T, 352 bp, 53 C; NCX1A, 5'-GGG TCT GAT TAT GAA TTC ACG GAA and 5'-AAT GAA GAA GGT CTT GTT TTT CT, 513 bp, 49 C; NCX1B, 5'-GGG TCT GAT TAT GAA TTC ACG GAA and 5'-TCA TAT TCC TCA CGG TCA AAT ATT, 491 bp, 49 C; NCX3A, 5'-GGG GGA GAT ATA TCC AAG ACC ATG and 5'-CAT CTC AAT GAC GTA ATT CTT GTT, 591 bp, 48 C; NCX3B, 5'-GGG GGA GAT ATA TCC AAG ACC ATG and 5'-AAG GGC AAT GAA GAA ATT CTC TTG, 591 bp, 50 C; and GAPDH, 5'-CAG GAG CGA GAC CCC ACT AAC and 5'-CGG ACA CAT TGG GGG TAG GA, 493 bp, 55 C. Total RNA from brain and skeleton muscle was used as positive control because brain had been reported to express all the NCX isoforms except NCX3A, which is present only in skeletal muscle cells.

Extraction of total cellular protein and membrane fraction
For total cellular protein extraction, the purified osteoclast preparation was harvested by scraping the culture dish with a Teflon policeman. The sample lysed in TNT buffer (20 mM Tris-HCl, 200 mM NaCl, 0.1% Triton X-100, 200 µM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, and 1 mg/ml aprotinin, pH 7.4). After 15,000 x g centrifugation, the supernatant was collected, and its protein concentration was determined with a BCA kit (Pierce, Hercules, CA). For membrane fraction isolation, the osteoclast pellet was resuspended in 1 ml lysis buffer (20 mM Tris-HCl, 200 mM NaCl, 10 g/ml pepstatin, 2 g/ml aprotinin, and 10 g/ml leuptin, pH 7.4) and subjected to sonification. The lysate was centrifuged at 700 x g for 10 min to remove nuclei and cell debris. Supernatant was further centrifuged at 40,000 x g for 30 min at 4 C. The resulting membrane-enriched pellets were dissolved in a solubilizing buffer (50 mM Tris-HCl, 0.15 M NaCl, 2 mM CaCl₂, 5 mM KCl, 5 mM MgCl₂, 4 mM EDTA, 20% glycerol, and the proteinase inhibitors, pH 7.5) before being stored at –80 C. Under these conditions, the membrane fraction preparations remained stable for at least 1 month.

Immunoprecipitation
Total cellular protein (100 µg) was incubated with rabbit anti-NCX polyclonal antibodies for 1 h at 4 C. Then 20 µl Protein A/G Plus-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA) was added and incubated at 4 C overnight. After centrifugation, the pellet was washed three times with TNT buffer, each time followed by 1000 x g centrifugation. The final pellet was resuspended in 20 µl electrophoresis sample buffer for SDS-PAGE and Western blot analysis.

Western blot analysis
Protein samples were separated in 7.5% SDS-PAGE gel and electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane. After blocking with 5% milk, the membrane was incubated overnight with monoclonal anti-NCX1, anti-NCX2, and anti-NCX3 antibodies. The blots were then incubated for 1 h with horseradish-peroxidase-conjugated antimouse IgG and visualized with an ECL system (Amersham, Arlington Heights, IL).

Immunochemistry
Osteoclasts plated on glass or dentine slices were fixed with 4% formaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min. After endogenous peroxidase activity was suppressed by 3% H₂O₂ and nonspecific binding sites were blocked with 10% normal goat or horse serum for 30 min at room temperature, fixed cells were incubated successively first with primary antibodies (monoclonal anti-NCX1, -NCX2, or -NCX3, respectively) at 4 C overnight and then with secondary antimouse IgG antibody at room temperature for 2 h and visualized using fluorescein isothiocyanate (FITC) or Alexa fluor488.

PCR cloning and DNA sequence
Nucleotide full coding sequences of mouse NCX1 and -3 cDNA (nearly 3 kb) were amplified by PCR and inserted into pcDNA 3.1 plasmid. The sequences of the inserts were then determined by DNA sequencing. The following primers were used for PCR cloning: NCX 1, 5'-gTA ccG cgg AcC ATG gTT CGA TTA AGT CTC and 5'-gTT GTg gaT ccA GAA GCC CTT TAT GTG GC, and NCX3, 5'-gAG AAC CTg cGg Ccg cac cAT GGC GTG GTT ACG G and 5'-gCC GGg GaT cCA GAA CCC CTT GAT GTA G. The primers had been modified to include Kozak consensus sequence and restriction endonuclease recognition sites, as indicated by the lowercase letters within the primer sequences.

[Ca²⁺]_i measurements
Osteoclasts adhering to glass coverslips were incubated for 40 min at 25 C with 2 µM fura-2/AM in a physiological salt solution (PSS) containing (in mM) 134 NaCl, 6 KCl, 10 HEPES, 2.5 CaCl₂, 0.5 MgCl₂, and 10 glucose, pH adjusted to 7.3 with Tris. The coverslips were then mounted on a temperature-controlled chamber (1 ml volume) and superfused continuously with PSS at 1 ml/min. Excitation wavelengths of 340 and 380 nm were applied via an inverted microscope equipped with a x40 fluor lens (Nikon, Tokyo, Japan). Emitted fluorescence was monitored by a photomultiplier (SPEX Industries, Edison, NJ). All experiments were performed at 33–34 C. Emission fluorescence intensities due to excitation at fluorescence intensity at 340 (F340) and 380 (F380) nm were measured. The ratio (F340/F380) was calibrated to [Ca²⁺]_i at the end of each experiment by exposing the cells to 10 µM ionomycin to assess the Ca²⁺-saturated ratio (R_max), followed by 5 mM EGTA to determine the nominal Ca²⁺-free ratio (R_min). Finally, MnCl₂ (5 mM) was added to estimate autofluorescence, which was subtracted from the experimental values. [Ca²⁺]_i was calculated using the method of Grynkiewicz et al. (15). At the end of the experiments, all cells were stained for TRAP to conclusively identify them as osteoclasts using an acid phosphatase kit (Sigma). In some experiments, we calculated the rate of [Ca²⁺]_i increase by dividing the increase of fluorescence ratio (df) by the elapsed time (dt) to estimate the efficacy of Ca²⁺ influx via NCX.

Patch-clamp recording
Coverslips with adherent osteoclasts were placed in a recording chamber (volume 1 ml) attached to an inverted microscope and superfused (3 ml/min) with extracellular solution containing (in mM) 140 NaCl, 2 CaCl₂, 1 MgCl₂, 10 glucose, 0.02 ouabain, and 10 HEPES, adjusted to pH 7.3 with Tris. Patch pipette solution contained (in mM) 120 CsCl, 20 NaCl, 5 MgATP, 20 1,2-bis-(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), 13 CaCl₂, 10 HEPES, adjusted to pH 7.3 with Tris. Free Ca²⁺ concentration in the patch pipette solution ([Ca²⁺]_p) was 475 nM at 34 C (estimated by MAXC software). In the present study, K⁺ conductances were eliminated by Cs⁺ in the patch pipette solution and omission of K⁺ in the extracellular solution. Osmolarity of all extracellular solutions was adjusted to that of the patch pipette solution by adding mannitol. The equilibrium potential of the Na⁺/Ca²⁺ exchanger (E_NCX) was estimated using the following equation: E_NCX = 3E_Na– 2E_Ca (at 3:1 stoichiometry) where E_Na and E_Ca are the equilibrium potentials of Na⁺ and Ca²⁺, respectively. Predicted E_NCX at above condition (140 mM [Na⁺]_o and 2 mM [Ca²⁺]_o in the extracellular solution and 20 mM [Na⁺]_p and 475 nM [Ca²⁺]_p in the patch pipette solution) was –67 mV. To set E_NCX to different values, an extracellular solution containing 0.1 or 0.5 mM instead of 2 mM CaCl₂ was used. Osmolarity of all solutions was adjusted and measured with a freezing-point depression osmometer (Osmometer Automatic; Knauer, Berlin, Germany). All solutions for electrophysiological experiments were kept in the range of 33–35 C. Membrane currents were recorded using the patch-clamp whole-cell configuration with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Currents were filtered at 1 kHz and digitized at a sampling frequency of 2–5 kHz. Data acquisition and analysis were performed with pCLAMP8 software (Axon Instruments). The pipette resistance was 3–5 M when filled with patch pipette solution. Series resistance (70–90%) was compensated to reduce the voltage error. Whole-cell capacitance was determined by digital integration of capacitive transients. An Ag-AgCl reference electrode was connected to the extracellular solution through a 3 M KCl-agar salt bridge. The zero current potential before formation of the gigaseal was taken as 0 mV. Membrane potential was held at predicted E_NCX, and voltage ramps were applied at 10-sec intervals to determine the current-voltage (I-V) relationships. These voltage ramps started at a hyperpolarized potential of –120 mV and then depolarized the cell membrane to +90 mV at a rate of 420 mV/sec.

Small interference (si)RNA synthesis and transfection
Three siRNA molecules targeting, respectively, the mouse NCX1A exon, NCX1B exon, and NCX3B exon were designed according to Reynolds’ rationale (16) and synthesized with the Silencer siRNA construction kit as described in its instruction book. The siRNA sequences are as follows: N1A sense siRNA, 5'-UAU CAG UCA AGG UAA UCG AUU; N1A antisense siRNA, 5'-AGC UAA UGG AAC UGA CUA UAA; N1B sense siRNA, 5'-UAU UUG ACC GUG AGG AAU AUU; N1B antisense siRNA, 5'-AUA AGG AGU GCC AGU UUA UAA; N3B sense siRNA, 5'-UAG UAG AUG AGG AGG AGU AUU; N3B antisense siRNA, 5'-AUG AGG AGG AGU AGA UGA UAA; nonsense sense siRNA, 5'-UAC GUA CUA UCG CGC GGA UTT; nonsense antisense siRNA, 5'-A UCC GCG CGA UAG UAC GUA TT. We used nonsense siRNA as negative control. All siRNA molecules were used at 20 or 100 nM concentrations and were transfected into osteoclasts by means of Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). We used semiquantitative RT-PCR to validate the silencing effect of these siRNAs.

Bone resorption assay
Osteoclast preparations were placed on dentine slices (4 mm diameter) and incubated for 15 h with or without NCX inhibitors. About 450–500 osteoclasts were placed on each dentine slice. At the end of the culture period, osteoclasts on the dentine slices were fixed with 10% formaldehyde and stained for TRAP, and the number of osteoclasts was counted. The osteoclasts were then removed from the dentine slices by ultrasonication in 0.25 M ammonium hydroxide. The exposed resorption pits were stained with Mayer’s hematoxylin solution. Finally, the total pit areas in dentine slices were measured using an image analysis system (NIH Image, version 1.66). The ratio of resorption pit area (mm²) per total number of osteoclasts was used as an indicator of osteoclast resorption activity. In parallel experiments, osteoclasts on dentine slices were transfected with NCX variant-specific siRNAs for 48 h by means of Lipofectamine 2000.

Chemicals
Fura-2/AM was obtained from Dojindo Laboratory (Kumamoto, Japan). Ouabain was purchased from Wako Pure Chemical (Osaka, Japan). SEA0400, SN-6, and KB-R7943 were kindly provided by Taisho Pharmaceutical Co. Ltd. (Saitama, Japan), Senju Pharmaceutical Co. Ltd. (Kobe, Japan), and Nippon Organon (Osaka, Japan), respectively. Rabbit anti-NCX1, -NCX2, and -NCX3 antibodies used in the experiments were the antibodies for NCXs previously reported by Iwamoto et al. (17). FITC-conjugated goat antimouse IgG was obtained from Molecular Probes Inc. (Eugene, OR). All other chemicals were obtained from Sigma. Hydrophobic drugs were dissolved in dimethylsulfoxide (DMSO; Sigma) and later diluted to their final concentration in PSS, in which DMSO was not more than 0.1%. This concentration of DMSO had no effect on [Ca²⁺]_i measurements, pit formation, or osmolarity of the solution.

Statistics
Data are expressed as mean values with SE (± SEM) from number of cells (n). Statistical differences were analyzed by one-way ANOVA. P values < 0.05 were considered to be significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of NCX isoforms in mouse osteoclasts
Mammalian cells express three types of NCX isoforms: NCX1, NCX2, and NCX3. First, we examined the existence of these NCX isoforms in mouse osteoclasts compared with that in brain and heart. In mouse brain, the expression of all three NCX isoforms was confirmed by RT-PCR. On the other hand, mouse osteoclasts expressed only NCX1 and NCX3, whereas NCX2 was absent in these cells (Fig. 1A). Similar results were obtained using immunoprecipitation. NCX1 and NCX3, but not NCX2, were detected in osteoclastic total proteins as bands of about 100–120 kDa (Fig. 1B). Additional bands of smaller size in heart and brain samples probably represent proteolytic products of NCX protein. Such degradation products of NCX1 and NCX3 have previously been described in both cardiomyocytes and neurons (7, 18). Next, we confirmed that NCX localizes in the isolated membrane fraction of osteoclasts. NCX1 and NCX3 were expressed in the osteoclast as well as brain and heart membrane fraction (Fig. 1C). Using immunochemical staining, we found that NCX1 and NCX3 are mostly localized in the plasma membrane of osteoclasts, both in vertical bone sections and cells plated on glass coverslips (Fig. 1D). These results demonstrate that NCX1 and NCX3 isoforms are expressed in mouse osteoclasts.

View larger version (36K): [in this window] [in a new window]   FIG. 1. Expression of NCX isoforms in mouse osteoclasts. A, Total RNAs from cultured mouse osteoclasts (OC) were reverse transcribed and amplified by PCR using specific primers for NCX1, NCX2, NCX3, and GAPDH mRNA. Total RNA from mouse brain (BR) was used as a positive control. B, Total proteins (100 µg) from osteoclasts (OC) were precipitated by polyclonal anti-NCX antibodies, transferred to PVDF membrane, and detected by corresponding monoclonal antibodies. Samples from heart (HT) and brain (BR) of mouse were used as positive controls. C, Crude membrane fraction was subjected to urea-SDS-PAGE, transferred to PVDF membrane, and detected by monoclonal NCX1 or NCX3 antibodies. Samples from heart (HT) and brain (BR) were used as positive controls. D, Osteoclasts cultured on bone slices (upper graph, vertical section) and glass coverslips (lower graph) were stained with monoclonal anti-NCX1 or -NCX3 antibodies and visualized with Alexa 488- or FITC-conjugated antimouse antibodies. Arrowheads indicate osteoclasts. Bar, 50 µm.

View larger version (52K): [in this window] [in a new window]   FIG. 2. Expression of splice variants for NCX1 and NCX3 in osteoclasts. A, Total RNAs of mouse osteoclasts were amplified by RT-PCR using primers specifically designed to bind different sites of NCX mRNA. H, Head of NCX coding sequences; A, sequence from exon A; B, sequence from exon B. B, The coding sequences of NCX1 and NCX3 cDNA were cloned into pcDNA3.1 plasmid, and sequenced. Names of variants are indicated on the left. C, The sequence of the new splice variant NCX1.41 includes a stop codon within the open reading frame. D, Total cellular proteins (40 µg) from osteoclasts (OC) were subjected to monoclonal NCX1 antibodies using Western blot analysis.

Effects of NCX inhibitors on intracellular Ca²⁺ concentration
Because NCX is a bidirectional transporter, Ca²⁺ transport via NCX can mediate Ca²⁺ influx under conditions of low extracellular sodium and/or high intracellular sodium in the presence of ouabain, a sodium-potassium pump inhibitor. To evaluate the transport function of NCX, especially Ca²⁺ influx mode, we examined effects of benzyloxyphenyl NCX inhibitors on Ca²⁺ influx by measuring intracellular Ca²⁺ concentration ([Ca²⁺]_i) in mouse osteoclasts. Removal of extracellular sodium [Na⁺]_o in the presence of ouabain (200 µM) increased [Ca²⁺]_i (Fig. 3A). This [Ca²⁺]_i elevation was abolished in a Ca²⁺-free external solution, indicating that the increase in [Ca²⁺]_i was due to Ca²⁺ influx (Fig. 3A). The [Na⁺]_o-free-induced [Ca²⁺]_i increase was partially and reversibly reduced by application of KB-R7943 (30 µM), an NCX inhibitor (Fig. 3B) (20). [Ca²⁺]_i decreased back to control levels when the normal [Na⁺]_o-containing extracellular solution was reapplied (Fig. 3B). Recently it was reported that KB-R7943 also inhibits nonselective and calcium channels other than NCX, such as N-methyl-D-aspartate acid (NMDA) channels (21, 22). Therefore, we also examined the effects of other NCX selective inhibitors: SN-6 and SEA0400 (20, 23). At 30 µM, both showed inhibitory action similar to KB-R7943 on the [Na⁺]_o-free-induced [Ca²⁺]_i increase (Fig. 3C). Combined application of KB-R7943 and SEA0400 showed further inhibition of [Ca²⁺]_i compared with SEA0400 alone but no significant difference from KB-R7943 or SN-6 alone. We also assessed the effects of NCX inhibitors on the rate of [Ca²⁺]_i increase induced by [Na⁺]_o-free solution to estimate the rate of Ca²⁺ influx via NCX. All three inhibitors significantly decreased the rate of [Na⁺]_o-free-induced [Ca²⁺]_i increase (Fig. 3D). These results strongly suggest that NCX inhibitors suppress Ca²⁺ influx, resulting in decreased [Ca²⁺]_i in mouse osteoclasts.

View larger version (27K): [in this window] [in a new window]   FIG. 3. Effects of NCXs inhibitors on [Na+]o-free-induced [Ca2+]i increase via NCX in osteoclasts. A, Extracellular Na+-free solution with ouabain (200 µM) increased [Ca2+]i. This increase was completely reversible with Ca2+-free solution. B, In contrast, NCX inhibitor KB-R7943 only partially reversed the [Na+]o-free-induced [Ca2+]i increase. C, Summary of NCX inhibitors (30 µM) KB-R7943, SN-6, and SEA0400 on the Na+-free-induced [Ca2+]i increase in presence of ouabain (200 µM). KB-R+SEA indicates application of KB-R7943 plus SEA0400. The [Ca2+]i increase before application of inhibitors was normalized to 100% for each cell (dotted line in C) and used as control level. [Ca2+]i after application of each NCX inhibitor was expressed in percent relative to this control level. D, Effects of three NCX inhibitors (30 µM) on the rates of [Ca2+]i increase induced by [Na+]o-free solution in the presence of ouabain. The inset diagram illustrates the calculation of the rate of [Ca2+]i increase df, Increase of ratio; dt, elapsed time. Data are expressed as means ± SEM, and numbers of experiments for each condition are shown in parentheses.

View larger version (27K): [in this window] [in a new window]   FIG. 4. Effects of NCX inhibitors on whole-cell currents in mouse osteoclasts. A, I-V relationships recorded before (control) and 3 min after application of 100 µM KB-R7943 (KB-R) at 2 mM (a) or 0.1 mM (b) of [Ca2+]o in the presence of 20 µM ouabain. The [Na+]o, [Na+]p, and [Ca2+]p were, respectively, 140, 20, and 475 nM. Predicted ENCX is shown in each graph. Membrane potential was held at predicted ENCX, and I-V curves were recorded in response to voltage ramps from –120 to +90 mV over 500 msec in the absence (control) and presence of KB-R7943 (KB-R). The KB-R-sensitive difference current (diff) is shown in each graph. Arrows indicate the reversal potential of the KB-R-sensitive current. B, I-V curves were recorded before (control) and 4 min after application of 50 µM SEA0400 (SEA) at the same ionic condition as in Aa. Arrow indicates the reversal potential of the SEA-sensitive current (diff). C, Relationship between the reversal potential of KB-R-sensitive () and SEA-sensitive () currents and predicted ENCX. The reversal potentials of KB-R- and SEA-sensitive currents were measured using 2, 0.5, and 0.1 mM [Ca2+]o and compared with the predicted ENCX of –67, –30, and 13 mV, respectively. Each data point is the mean of n = 3 experiments. The straight line indicates a least-squares fit of the data (R2 = 0.97).

View larger version (30K): [in this window] [in a new window]   FIG. 5. Effects of NCX inhibitors on osteoclastic resorption activity. A, Osteoclasts were placed on dentine slices and cultured for 12–15 h in the presence or absence (control) of NCX inhibitors KB-R7943 (KB-R), SEA0400 (SEA), and SN-6. After removing cells, resorption pits formed on the dentine slice (black dots) were stained with hematoxylin solution. B, Dose-dependent effect of NCX inhibitors on the number of TRAP-positive multinuclear cells (upper graph) and pit formation (lower graph). The osteoclastic bone-resorbing activity was calculated as the ratio of total pit area size/number of osteoclasts and expressed in percent relative to control. Data are expressed as means ± SEM (n = 5–8). C, Combined application of KB-R and SEA at concentrations of 3 or 5 µM significantly inhibited pit formation. Each column indicates mean and SEM, and the numbers of experiments are shown in parentheses. **, P < 0.05.

View larger version (46K): [in this window] [in a new window]   FIG. 6. Effects of siRNAs targeting for three NCX variants on osteoclast resorption activity. A, Osteoclasts were pretreated for 48 h with or without 100 nM siRNAs (NCX1A, NCX1B, and NCX3B) and then incubated on dentine for 15 h. After cells had been removed, pits formed on dentine slices were stained for visualization. B, The ratios of total pit area size/osteoclast number in the presence of each siRNA are expressed in percent of the value in the absence of siRNA (control). Each column indicates mean and SEM, and the numbers of experiments for each condition are shown in parentheses. C, Osteoclasts were incubated with each NCX siRNA (100 nM) and nonsense siRNA (NS), and osteoclastic total RNAs were analyzed by RT-PCR. Numbers below the gels (upper trace) indicate the intensity of NCX variants’ mRNA relative to GAPDH mRNA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we demonstrate the expression and function of three NCX variants in mouse osteoclasts. Our data suggest that these NCX variants in mouse osteoclasts play an important role as Ca²⁺ regulators and transporters during bone resorption.

Expression of NCX isoforms and variants in osteoclasts
Three NCX isoforms, NCX1, NCX2, and NCX3, have been described in mammalian tissues. NCX1 has been found ubiquitously expressed in most tissues including brain, heart, skeletal muscle, smooth muscle, kidneys, eye secretor, and blood cells (24, 25). In contrast, NCX2 and NCX3 are expressed predominantly in neuronal and skeletal muscle tissues (11, 26). At least 12 NCX1 and three NCX3 variants, generated through alternative splicing of the primary nuclear transcript, have been reported in rabbit (10). These variants are selectively expressed in different tissues and cell populations of the rat (11). In rat osteoclasts, NCX1 appears to be coupled with the Na⁺/H⁺ exchanger for regulation of cytosolic Ca²⁺ (1). However, it was unclear which NCX1 isoforms and variants are expressed in mouse osteoclasts. We found that mouse osteoclasts express not only NCX1 but also NCX3 variants, in particular NCX1.3, NCX1.41, and NCX3.2. Of these, NCX1.41 represents a novel splice variant, which includes both A and B exons. Previously it had been thought that A and B exons are mutually exclusive (19). However, the insertion of exon B between A and D shifts the reading frame, resulting in a stop codon in the B exon. As a result, NCX1.41 is a truncated protein. We found the NCX1.41 mRNAs were expressed in brain, skeletal muscle, and heart muscle as well as osteoclasts (data not shown). This truncated form (theoretically 74 kDa) includes only the first five transmembrane segments of NCX plus a large portion of the intracellular loop and 29 extra amino acids resulting from frame shift. This protein thus lacks the COOH-terminal hydrophobic domain and therefore the conservative 2 repeat, which is thought to be important for ion transport (17). Therefore, one might question whether this truncated NCX1.41 is functional or not. It was reported that a similar truncation within the splicing region of NCX1 produces a functional protein with weakened Na⁺/Ca²⁺ exchange ability (27). It seems possible that the two NH₂-terminal halves of the truncated NCX1 protein might come together forming a dimer within the membrane and thereby creating an ion-binding and -transport pocket composed of two 1 repeats instead of the normal 1/2 structure (27). Our data clearly show that NCX proteins are essential for Ca²⁺ transport during osteoclastic bone resorption. NCX1.41 is expressed in these cells, but in lower amounts than the other NCX proteins, and its role remains unclear.

Role of NCX isoforms and variants in osteoclastic function
We showed that the [Na⁺]_o-free-induced [Ca²⁺]_i increase was abolished by removal of extracellular Ca²⁺ and partially suppressed by three benzyloxyphenyl NCX inhibitors, KB-R7943, SN-6, and SEA0400. These NCX inhibitors also reduced bone resorption in a dose-dependent manner. Furthermore, we recorded KBR- and SEA-sensitive NCX currents in osteoclasts. These were dependent on [Ca²⁺]_o in the presence of ouabain, and their reversal potentials were very close to the expected equilibrium potential of NCX. These data indicate that NCXs express and transport Ca²⁺ in mouse osteoclasts. The three NCX inhibitors used in this study differ in their potency for the NCX isoforms. We found that the inhibition by SEA0400 of [Na⁺]_o-free-induced [Ca²⁺]_i increase was less than that by KB-R7943 and SN-6, probably because KB-R7943 and SN-6 inhibit both NCX1 and NCX3, whereas SEA0400 is a selective NCX1 inhibitor with almost no effect on NCX3 (28, 29). Similarly, KB-R7943 also exhibited a stronger inhibition than SEA0400 on pit formation on dentine slices. All three siRNAs targeted for NCX1A, NCX1B, and NCX3B effectively suppressed pit formation, whereas nonsense siRNA showed no effect. These results indicate that both NCX1 and NCX3 variants are functional in mouse osteoclasts.

To our knowledge, there is no published information regarding the effect of NCX gene knockout on bone formation and structure in mice. Homozygous NCX1 knockout mice died from deficiency of cardiac development between embryonic d 9 and 10 (29). Heterozygous NCX1 knockout mice survived: the expression of NCX1 as well as the Na⁺/Ca²⁺ exchange activity was decreased by approximately 50% in heart, kidney, aorta, and smooth muscle cells (30). NCX3 knockout mice were reported to exhibit reduced motor activity, weakness of forelimb muscles, and fatigability (31). In preliminary experiments, we studied osteoclasts from heterozygous NCX1 knockout mice. These cells exhibited NCX1 mRNA with an expression level reduced to about 50% of the wild-type cells and also showed a decrease of the [Na⁺]_o-free-induced [Ca²⁺]_i increase compared with wild-type cells (unpublished observations). In the present study, we demonstrate that NCX1 and NCX3 variants are expressed in mouse osteoclasts and play an important role during bone resorption. The significance of the coexistence of these NCX variants and any functional differences remain unclear. Future studies using NCX knockout mice and/or overexpressing cells are needed to address these questions.

Transportation mode of NCX in resorbing osteoclasts
NCX currents depend on membrane potential and transmembrane gradients of Na⁺ and Ca²⁺ (24, 25, 30). If membrane potential is more negative than the reversal potential of NCX, it operates in Ca²⁺ efflux mode. If membrane potential rises above the reversal potential of NCX, the current reverses and mediates Ca²⁺ influx. In the experiments presented here, we succeeded for the first time in recording bidirectional NCX currents from mouse osteoclasts. These currents directly demonstrate that NCX can mediate both Ca²⁺ efflux and Ca²⁺ influx. Osteoclasts change their morphological and functional properties between the resorbing and nonresorbing/motile state of their resorption cycle (32, 33). Resorbing osteoclasts exhibiting a so-called actin ring have a resting membrane potential of –10 to approximately –20 mV (5, 34). On the other hand, nonresorbing/motile osteoclasts exhibiting pseudopodia but no actin ring have a resting membrane potential of –50 to approximately –70 mV. Mouse osteoclasts plated on glass coverslips form actin rings and no pseudopodia, and their membrane potential is around –20 mV (35). We calculated the reversal potential of NCX according to published equations (21, 36). If NCX protein is located on the ruffled border of the osteoclast, then because of the extremely high Ca²⁺ concentration (40 mM) in the resorption lacunae, the calculated reversal potential of NCX would be as low as –70 to about –150 mV, which is far less than the cell membrane potential. Therefore, it can be predicted that NCX on the ruffled border operates in the Ca²⁺ influx mode in resorbing osteoclasts. If NCX indeed operates in Ca²⁺ influx mode during the resorption state, then NCX likely plays a very important role for the regulation of [Ca²⁺]_i and bone resorption.

Transcellular Ca²⁺ transport from resorption lacunae via osteoclasts
During bone resorption, osteoclasts dissolve mineral components of the bone matrix resulting in Ca²⁺ accumulation in the resorption lacunae. It often is assumed that Ca²⁺ in resorption lacunae is removed by transcytosis; however, this assumption cannot explain the observation that Ca²⁺ flux at the basolateral surface starts quickly within minutes after the seeding of osteoclasts on the bone surface, whereas the cellular collagen trafficking occurs only after several hours (37). More recently, the transient receptor potential channel V5 (TRPV5) has been located in the ruffled border membrane of mouse osteoclasts and was suggested to transport Ca²⁺ ions from the lacunae into the cytosol of the osteoclasts (38). If the membrane potential in mouse osteoclasts depolarizes from around –60 mV (during the motile phase) to –15 mV (resorbing phase), then the driving force of Ca²⁺ influx via TRPV5 decreases because the reversal membrane potential of TRPV5 is nearly 0 mV. Therefore, TRPV5 in the ruffled border is not a good candidate for Ca²⁺ uptake during bone resorption. Significantly, TRPV5 knockout mice did not develop osteopetrosis (38). On the other hand, because the relationship between reversal potential of NCX and membrane potential in resorbing osteoclasts is opposite that of TRPV5, NCX expressed at the ruffled border membrane are predicted to operate in reverse mode, mediating Ca²⁺ influx. The inhibition of osteoclastic bone resorption by NCX inhibitors and siRNAs is therefore due to reduced Ca²⁺ influx. Thus, NCX is a very interesting candidate for disposal of Ca²⁺ from the osteoclastic resorption lacunae.

We conclude that NCX variants 1.3, 1.41, and 3.2 are expressed in osteoclasts and play an important role as Ca²⁺ regulators and/or transporters during bone resorption.

	Acknowledgments

 
We thank Dr. Andreas Carl for English editing.

	Footnotes

 
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports Science, and Technology of Japan (No. 17591957 and No. 18592054), Grant-in-Aid 18059031 for Scientific Research on Priority Areas, and Frontier Research Grant.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 22, 2007

Abbreviations: DMSO, Dimethylsulfoxide; FITC, fluorescein isothiocyanate; I-V, current-voltage; NCX, Na⁺/Ca²⁺ exchanger; PSS, physiological salt solution; PVDF, polyvinylidene difluoride; si, small interference; TRAP, tartrate-resistant acid phosphatase; TRPV5, transient receptor potential channel V5.

Received September 26, 2006.

Accepted for publication February 12, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Moonga BS, Alam AS, Bevis PJ, Avaldi F, Soncini R, Huang CL, Zaidi M 1992 Regulation of cytosolic free calcium in isolated rat osteoclasts by calcitonin. J Endocrinol 132:241–249[Abstract/Free Full Text]
2. Albanese CV, Cudd A, Argentino L, Zambonin-Zallone A, MacIntyre I 1994 Ipriflavone directly inhibits osteoclastic activity. Biochem Biophys Res Commun 199:930–936[CrossRef][Medline]
3. Bizzarri C, Shioi A, Teitelbaum SL, Ohara J, Harwalkar VA, Erdmann JM, Lacey DL, Civitelli R 1994 Interleukin-4 inhibits bone resorption and acutely increases cytosolic Ca²⁺ in murine osteoclasts. J Biol Chem 269:13817–13824[Abstract/Free Full Text]
4. Kajiya H, Okabe K, Okamoto F, Tsuzuki T, Soeda H 2000 Protein tyrosine kinase inhibitors increase cytosolic calcium and inhibit actin organization as resorbing activity in rat osteoclasts. J Cell Physiol 183:83–90[CrossRef][Medline]
5. Kajiya H, Okamoto F, Fukushima H, Takada K, Okabe K 2003 Mechanism and role of high-potassium-induced reduction of intracellular Ca²⁺ concentration in rat osteoclasts. Am J Physiol Cell Physiol 285:C457–C466
6. Philipson KD, Nicoll DA, Ottolia M, Quednau BD, Reuter H, John S 2002 The Na⁺/Ca²⁺ exchange molecule: an overview. Ann NY Acad Sci 976:1–10[CrossRef][Medline]
7. Philipson KD, Longoni S, Ward R 1988 Purification of the cardiac Na⁺-Ca²⁺ exchange protein. Biochim Biophys Acta 945:298–306[Medline]
8. Nicoll DA, Hryshko LV, Matsuoka S, Frank JS, Philipson KD 1996 Mutation of amino acid residues in the putative transmembrane segments of the cardiac sarcolemmal Na⁺-Ca²⁺ exchanger. J Biol Chem 271:13385–13391[Abstract/Free Full Text]
9. Nakasaki Y, Iwamoto T, Hanada H, Imagawa T, Shigekawa M 1993 Cloning of the rat aortic smooth muscle Na⁺/Ca²⁺ exchanger and tissue-specific expression of isoforms. J Biochem (Tokyo) 114:528–534[Abstract/Free Full Text]
10. Kofuji P, Lederer WJ, Schulze DH 1994 Mutually exclusive and cassette exons underlie alternatively spliced isoforms of the Na/Ca exchanger. J Biol Chem 269:5145–5149[Abstract/Free Full Text]
11. Quednau BD, Nicoll DA, Philipson KD 1997 Tissue specificity and alternative splicing of the Na⁺/Ca²⁺ exchanger isoforms NCX1, NCX2, and NCX3 in rat. Am J Physiol 272:C1250–C1261
12. Moonga BS, Davidson R, Sun L, Adebanjo, OA, Moser J, Abedin M, Zaidi N, Huang, CL, Zaidi M 2001 Identification and characterization of a sodium/calcium exchanger, NCX-1, in osteoclasts and its role in bone resorption. Biochem Biophys Res Commun 283:770–775[CrossRef][Medline]
13. Takahashi N, Akatsu T, Sasaki T, Nicholson GC, Moseley JM, Martin TJ, Suda T 1988 Osteoblastic cells are involved in osteoclast formation. Endocrinology 123:2600–2602[Abstract/Free Full Text]
14. Tezuka K, Sato T, Kamioka H, Nijweide PJ, Tanaka K, Matsuo T, Ohta M, Kurihara N, Hakeda Y, Kumegawa M 1992 Identification of osteopontin in isolated rabbit osteoclasts. Biochem Biophys Res Commun 186:911–917[CrossRef][Medline]
15. Grynkiewicz G, Poenie M, Tsien RY 1985 A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450[Abstract/Free Full Text]
16. Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, Khvorova A 2004 Rational siRNA design for RNA interference. Nat Biotechnol 22:326–330[CrossRef][Medline]
17. Iwamoto T, Pan Y, Nakamura TY, Wakabayashi S, Shigekawa M 1998 Protein kinase C-dependent regulation of Na⁺/Ca²⁺ exchanger isoforms NCX1 and NCX3 does not require their direct phosphorylation. Biochemistry 37:17230–17238[CrossRef][Medline]
18. Bano D, Young KW, Guerin CJ, LeFeuvre R, Rothwell NJ, Naldini L, Rizzuto R, Carafoli E, Nicotera P 2005 Cleavage of the plasma membrane Na⁺/Ca²⁺ exchanger in excitotoxicity. Cell 120:275–285[CrossRef][Medline]
19. Nicoll DA, Quednau BD, Qui Z, Xia YR, Lusis AJ, Philipson, KD 1996 Cloning of a third mammalian Na⁺-Ca²⁺ exchanger, NCX3. J Biol Chem 271:24914–24921[Abstract/Free Full Text]
20. Iwamoto T 2004 Forefront of Na⁺/Ca²⁺ exchanger studies: molecular pharmacology of Na⁺/Ca²⁺ exchange inhibitors. J Pharmacol Sci 96:27–32[CrossRef][Medline]
21. Sokolow S, Manto M, Gailly P, Molgo J, Vandebrouck C, Vanderwinden JM, Herchuelz A, Schurmans S 2004 Impaired neuromuscular transmission and skeletal muscle fiber necrosis in mice lacking Na/Ca exchanger 3. J Clin Invest 113:265–273[CrossRef][Medline]
22. Reuter H, Henderson SA, Han T, Matsuda T, Baba A, Ross RS, Goldhaber JI, Philipson KD 2002 Knockout mice for pharmacological screening: testing the specificity of Na⁺-Ca²⁺ exchange inhibitors. Circ Res 91:90–92[Abstract/Free Full Text]
23. Matsuda T, Arakawa N, Takuma K, Kishida Y, Kawasaki Y, Sakaue M, Takahashi K, Takahashi T, Suzuki T, Ota T, Hamano-Takahashi A, Onishi M, Tanaka Y, Kameo K, Baba A 2001 SEA0400, a novel and selective inhibitor of the Na⁺-Ca²⁺ exchanger, attenuates reperfusion injury in the in vitro and in vivo cerebral ischemic models. J Pharmacol Exp Ther 298:249–256[Abstract/Free Full Text]
24. Blaustein MP, Lederer WJ 1999 Sodium/calcium exchange: its physiological implications. Physiol Rev 79:763–854[Abstract/Free Full Text]
25. Annunziato L, Pignataro G., Di Renzo GF 2004 Pharmacology of brain Na⁺/Ca²⁺ exchanger: from molecular biology to therapeutic perspectives. Pharmacol Rev 56:633–654[Abstract/Free Full Text]
26. Lee SL, Yu AS, Lytton J 1994 Tissue-specific expression of Na⁺-Ca²⁺ exchanger isoforms. J Biol Chem 269:14849–14852[Abstract/Free Full Text]
27. Li XF, Lytton J 1999 A circularized sodium-calcium exchanger exon 2 transcript. J Biol Chem 274:8153–8160[Abstract/Free Full Text]
28. Iwamoto T, Kita S, Uehara A, Imanaga I, Matsuda T, Baba A, Katsuragi T 2004 Molecular determinants of Na⁺/Ca²⁺ exchange (NCX1) inhibition by SEA0400. J Biol Chem 279:7544–7553[Abstract/Free Full Text]
29. Wakimoto K, Kobayashi K, Kuro O, Yao A, Iwamoto T, Yanaka N, Kita S, Nishida A, Azuma S, Toyoda Y, Omori K, Imahie H, Oka T, Kudoh S, Kohmoto O, Yazaki Y, Shigekawa M, Imai Y, Nabeshima Y, Komuro I 2000 Targeted disruption of Na⁺/Ca²⁺ exchanger gene leads to cardiomyocyte apoptosis and defects in heartbeat. J Biol Chem 275:36991–36998[Abstract/Free Full Text]
30. Philipson KD, Nicoll DA 2000 Sodium-calcium exchange: a molecular perspective. Annu Rev Physiol 62:111–133[CrossRef][Medline]
31. Shigekawa M, Iwamoto T 2001 Cardiac Na⁺-Ca²⁺ exchange: molecular and pharmacological aspects. Circ Res 88:864–876[Abstract/Free Full Text]
32. Arkett SA, Dixon SJ, Sims SM 1992 Substrate influences rat osteoclast morphology and expression of potassium conductances. J Physiol 458:633–653[Abstract/Free Full Text]
33. Kanehisa J, Yamanaka T, Doi S, Turksen K, Heersche JN, Aubin JE, Takeuchi H 1990 A band of F-actin containing podosomes is involved in bone resorption by osteoclasts. Bone 11:287–293[Medline]
34. Sims SM, Dixon SJ 1989 Inwardly rectifying K⁺ current in osteoclasts. Am J Physiol 256:C1277–C1282
35. Jimi E, Nakamura I, Duong LT, Ikebe T, Takahashi N, Rodan GA Suda T 1999 Interleukin 1 induces multinucleation and bone-resorbing activity of osteoclasts in the absence of osteoblasts/stromal cells. Exp Cell Res 247:84–93[CrossRef][Medline]
36. Dong H, Dunn J, Lytton J 2002 Electrophysiological studies of the cloned rat cardiac NCX1.1 in transfected HEK cells: a focus on the stoichiometry. Ann NY Acad Sci 976:159–165[Medline]
37. Datta HK, Horrocks BR 2003 Mechanisms of calcium disposal from osteoclastic resorption hemivacuole. J Endocrinol 176:1–5[Abstract]
38. van der Eerden BC, Hoenderop JG., de Vries TJ, Schoenmaker T, Buurman CJ, Uitterlinden AG., Pols HA, Bindels RJ, Leeuwen JP 2005 The epithelial Ca²⁺ channel TRPV5 is essential for proper osteoclastic bone resorption. Proc Natl Acad Sci USA 102:17507–17512[Abstract/Free Full Text]

This article has been cited by other articles:

				H. Fukushima, A. Nakao, F. Okamoto, M. Shin, H. Kajiya, S. Sakano, A. Bigas, E. Jimi, and K. Okabe The Association of Notch2 and NF-{kappa}B Accelerates RANKL-Induced Osteoclastogenesis Mol. Cell. Biol., October 15, 2008; 28(20): 6402 - 6412. [Abstract] [Full Text] [PDF]

				H K Datta, W F Ng, J A Walker, S P Tuck, and S S Varanasi The cell biology of bone metabolism J. Clin. Pathol., May 1, 2008; 61(5): 577 - 587. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, J.-P. Articles by Okabe, K. Search for Related Content PubMed PubMed Citation Articles by Li, J.-P. Articles by Okabe, K.