1 Department of Oral and Craniofacial Biological Sciences, University of Maryland, Baltimore, Maryland 21201; 2 Departments of Pediatrics and Human Genetics, Montreal Children's Hospital Research Institute, McGill University, Montreal, Quebec, Canada H3Z 2Z3; 3 Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, Maryland 20892; 4 Physiologisches Institut, Universität Zürich-Irchel, Zürich, CH-8057, Switzerland; and 5 Department of Medicine, University of Maryland, Baltimore, Maryland 21201
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ABSTRACT |
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We previously demonstrated that inhibition of Na-dependent phosphate (Pi) transport in osteoclasts led to reduced ATP levels and diminished bone resorption. These findings suggested that Na/Pi cotransporters in the osteoclast plasma membrane provide Pi for ATP synthesis and that the osteoclast may utilize part of the Pi released from bone resorption for this purpose. The present study was undertaken to define the cellular localization of Na/Pi cotransporters in the mouse osteoclast and to identify the proteins with which they interact. Using glutathione S-transferase (GST) fusion constructs, we demonstrate that the type IIa Na/Pi cotransporter (Npt2a) in osteoclast lysates interacts with the Na/H exchanger regulatory factor, NHERF-1, a PDZ protein that is essential for the regulation of various membrane transporters. In addition, NHERF-1 in osteoclast lysates interacts with Npt2a in spite of deletion of a putative PDZ-binding domain within the carboxy terminus of Npt2a. In contrast, deletion of the carboxy-terminal TRL amino acid motif of Npt2a significantly reduced its interaction with NHERF-1 in kidney lysates. Studies in osteoclasts transfected with green fluorescent protein-Npt2a constructs indicated that Npt2a colocalizes with NHERF-1 and actin at or near the plasma membrane of the osteoclast and associates with ezrin, a linker protein associated with the actin cytoskeleton, likely via NHERF-1. Furthermore, we demonstrate by RT/PCR of osteoclast RNA and in situ hybridization that the type III Na/Pi cotransporter, PiT-1, is also expressed in mouse osteoclasts. To examine the cellular distribution of PiT-1, we infected mouse osteoclasts with a retroviral vector encoding PiT-1 fused to an epitope tag. PiT-1 colocalizes with actin and is present on the basolateral membrane of the polarized osteoclast, similar to that previously reported for Npt2a. Taken together, our data suggest that association of Npt2a with NHERF-1, ezrin, and actin, and of PiT-1 with actin, may be responsible for membrane sorting and regulation of these Na/Pi cotransporters in the osteoclast.
phosphate transport; osteoclasts; Npt2a; NHERF-1; PiT-1
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INTRODUCTION |
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PHOSPHATE (Pi) is the
major anionic component of the mineralized bone matrix
(2). Previously, we had hypothesized that during the
process of bone resorption, part of the Pi released from
bone may be utilized by the osteoclast to maintain cellular ATP content
during the cyclical processes of migration, attachment, and resorption
(10). In addition, we provided evidence for the expression
of the type II family of Na/Pi cotransporters in the osteoclast, identical or closely related to the type IIa cotransporter (Npt2a) in the renal proximal tubule (11). The Npt2a
protein mediates the rate-limiting step in renal Pi
reabsorption (27). The importance of Npt2a in the
regulation of extracellular Pi was suggested by recent
studies in which the Npt2a gene was inactivated in mice by
targeted mutagenesis (3). Previously, we examined the
impact of Npt2a gene ablation on the skeletal phenotype in mice. At weaning, Npt2a/
mice showed several skeletal
abnormalities, including retarded secondary ossification, increased
trabecular thickness, retention of growth plate proteoglycan in
trabecular bone, and a reduction in osteoclast number. However, with
increasing age, there was a correction of the skeletal phenotype
(11).
We demonstrated that the Npt2a-immunoreactive protein is localized exclusively on the basolateral membrane (BLM) and in areas contiguous to the sealing zone of the polarized osteoclast (10). Disruption of the actin cytoskeleton in polarized osteoclasts with cytochalasin D inhibited Pi uptake by ~80% (10). Because Na/Pi cotransport in the osteoclast is regulated by the actin cytoskeleton, we hypothesized that the Npt2a-immunoreactive protein in the osteoclast associates indirectly with the actin cytoskeleton and that this interaction may be a determinant of the plasma membrane localization and activity of Npt2a in the osteoclast.
The first aim of the present study was to identify a linker protein that would provide a bridge between the actin cytoskeleton and the Npt2a protein. In the kidney, Gisler et al. (9) identified a protein-binding V/ATXL domain in the COOH terminus of the Npt2a protein, representing a potential PDZ (PSD-95/Dlg/ZO-1)-interacting motif. PDZ domains are conserved protein modules that mediate protein-protein interactions associated with the plasma membrane (7). One such PDZ protein, Na/H exchanger regulatory factor (NHERF-1), has been found to participate in regulation of phosphorylation, targeting, endocytic retrieval, and trafficking of several membrane transporters, including Npt2a in the kidney (9, 26). In the present study, we undertook to determine whether the Npt2a protein also associates with NHERF-1 in the osteoclast, because this association may play a role in the cellular localization of Npt2a.
Previous studies have shown that Npt2a knockout (Npt2a/
)
mice exhibit an age-dependent increase in type III Na/Pi
cotransporter mRNA expression in the kidney as part of an effort to
compensate for the loss of the Npt2a gene (12).
We hypothesized that the expression and regulation of the type III
family of Na/Pi cotransporters in the osteoclast could
account for reversal of the skeletal phenotype in the
Npt2a
/
mice (11). Thus, in the present
study, we sought to examine the expression and cellular localization of
the type III family of Na/Pi cotransporters in murine
osteoclasts. The type III Na/Pi cotransporters, originally
identified as retroviral receptors for the gibbon ape leukemia virus
(galv), are ubiquitously expressed and thought to function as
housekeeping Na/Pi cotransporters (14, 15).
One such galv receptor, PiT-1 (also known as Glvr-1), is expressed at
high levels in bone marrow, osteoblasts, and chondrocytes (21,
23).
Our study suggests that association of Npt2a with a PDZ protein such as NHERF-1, and of PiT-1 with actin, may play a role in membrane sorting and regulation of these Na/Pi cotransporters in the osteoclast.
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MATERIALS AND METHODS |
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Reagents.
The enhanced green fluorescent protein (EGFP)-Npt2a constructs encoding
for the wild type (WT) and COOH-terminal TRL deletions (Npt2a-TRL)
were prepared as previously described (13). Briefly, the
mouse type IIa (mIIa) Na/Pi cotransporters were fused to
the COOH terminus of the EGFP by inserting the Npt2a (
WT and
TRL) cDNAs into the pEGFP-C1 vector. Dr. Vijaya Ramesh (Molecular
Neurogenetics Unit, Massachusetts General Hospital, Charlestown, MA)
provided the glutathione S-transferase (GST)-NHERF-1 fusion
protein. A rabbit osteoclast cDNA library was the gift of Dr. M. Kumegawa (Meikai University, Sakado, Japan) (28). The
murine macrophage (mMa) cDNA library was obtained from Stratagene (La
Jolla, CA). Recombinant osteoprotegerin ligand (OPGL) was prepared as
previously described (5). Most reagents were of analytical
grade and were purchased from Sigma (St. Louis, MO) unless otherwise indicated.
Mice.
Mice (C57BL/6NHsd, 6-7-wk-old) were purchased from Harlan
(Indianapolis, IN). Npt2a knockout mice were established by
homologous recombination (3). Wild-type
(Npt2a+/+) and homozygous mutant (Npt2a/
) mice, generated by crossing
heterozygous (Npt2a+/
) male and female mice, were
genotyped by PCR amplification of genomic DNA as previously described
(3).
Murine osteoclast culture.
The tibiae and femurs of 7-wk-old mice were used to isolate bone cells,
as previously described (4, 11). Bone marrow cells were
suspended in -minimal essential medium (
MEM, GIBCO-BRL) supplemented with 10% fetal bovine serum (
-10 MEM) and cultured at
37°C in a 5% CO2 incubator. After 24 h, nonadhered
cells were layered on Histopaque-1077 (Sigma) and centrifuged at 300 g for 15 min at room temperature. The cell layer between the
Histopaque and the medium was removed and washed with
-10 MEM and
then centrifuged at 2,000 rpm for 7 min. mCSF-1 (R & D Systems,
Minneapolis, MN) was added to the cultures at a concentration of 10 ng/ml, and OPGL was added at 50-100 ng/ml. The multinucleated
osteoclasts were seen to form and mature after day 4. Their
viability was routinely assessed by trypan blue dye exclusion, and the
percentage of tartrate-resistant acid phosphatase
(TRAP)-positive cells was assessed to be ~99%, as previously
described (11).
Preparation of the GST-Npt2a fusion proteins.
EGFP-Npt2a constructs, encoding the WT and COOH-terminal TRL deletion
(TRL) cDNAs, were prepared as previously described (13). These constructs served as templates for generation
of the GST-Npt2a-WT and GST-Npt2a-
TRL fusion proteins. Npt2a-WT and
Npt2a-
TRL fragments were PCR amplified by using the following primers: WT Npt2a: forward,
5'-CCGGAATTCATGTCCTGCAGAGCCGAA-3', and reverse,
5'-CCGCTCGAGTAGAGGCGGGTAGCATT-3'; Npt2a-
TRL:
forward, 5'-CCGGAATTCATGTCCTGCAGAGCCGAA-3', and reverse,
5'-CCGCTCGAGTAGCATTGTGGTGAGCAG-3'. Amplification was carried out using
a hot start method and Taq DNA polymerase (Life
Technologies) with denaturation at 94°C for 1 min, annealing at
50°C for 1 min, and extension at 72°C for 1 min. At the end of 35 cycles, an extension cycle for 10 min at 72°C was performed. Both the
Npt2a-WT (82 amino acids, 246 bp) and the Npt2a-
TRL (79 amino acids,
234 bp) fragments were directionally cloned into pGEX-5X-1 (Amersham
Biosciences, Piscataway, NJ) at the EcoR1 and
Xho1 sites. Positive clones were identified and confirmed by
sequencing with pGEX-5' end primer. Subsequently, these clones were
transformed in BL-21 cells. The GST-NHERF-1 (1-358 amino acids)
fusion protein was provided in a pGex/BL21 construct by Dr. Vijaya
Ramesh and was purified using a similar protocol.
Purification of GST-Npt2a fusion proteins.
Overnight cultures of both the recombinant GST-Npt2a in BL-21 cells
were transferred into larger volumes of LB/Amp, grown for 2 h
until the OD600 was determined to be 1.0, and subsequently induced with 1.0 mM isopropyl--D-thiogalactopyranoside
(IPTG) for an additional 2 h at 37°C. Thereafter, the cultures
were spun down at 2,500 rpm for 20 min. The pellets were resuspended in 3.6 ml of binding buffer that comprised 50 mM Tris-Cl, pH 8.0, 120 mM
NaCl, 0.5% NP-40, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Then, 0.4 ml of lysozyme (10 µg/ml) was added, and the mixture was allowed to stand at room temperature for 20 min.
The bacteria were lysed by repeated freeze-thaw cycles. The lysates
were centrifuged at 14,000 rpm for 20 min. The supernatants were
collected (~4 ml) and incubated with 200 µl of washed
GST-Sepharose; these were rocked at room temperature for 1 h. The
Sepharose beads were washed five times for 10 min each using 10 volumes
of binding buffer. An aliquot of ~10 µl of GST-bound proteins was
taken and separated on 8% SDS-PAGE. After the extraction was
confirmed, a 50% suspension of this extract was prepared with binding
buffer and stored at 4°C.
Preparation of kidney homogenate and osteoclast cell lysates. Kidneys were homogenized in lysis buffer (10 mM Tris-Cl, pH 7.6, 150 mM NaCl, 1% NP-40, 10% glycerol plus protease inhibitor cocktail) for 1 h at 4°C. Osteoclast cell lysates were prepared in modified RIPA buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 1% deoxycholic acid, 0.1% SDS, and 1% NP-40). The cells were spun down at 14,000 rpm at 4°C for 10 min. The supernatants were collected and bound to fusion proteins as detailed below.
GST-sepharose and nickel-agarose affinity-precipitation assays.
To perform affinity-precipitation of NHERF-1, GST-beads (Qiagen,
Valencia, CA) bound to fusion proteins (Npt2a-WT or Npt2a-TRL) or
GST (control) were incubated with either (~500 µg) murine
osteoclast or rat kidney (~100 µg) lysates at 4°C for 2-4 h.
The beads were pelleted at 500 g for 2 min, washed four
times with binding buffer, resuspended in 1-2 volumes of sodium
dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample
buffer, and boiled before fractionation on SDS-PAGE. The proteins were
then transferred to polyvinylidene difluoride membranes (PVDF; Bio-Rad
Laboratories, Hercules, CA) for Western analyses with a polyclonal
antibody to NHERF-1 at a dilution of 1:1,000. A similar protocol was
followed for incubation of murine osteoclast lysates with the
GST-NHERF-1 fusion protein for affinity-precipitation of Npt2a. For the
nickel (Ni)-agarose affinity-precipitation assays, the following
conditions were used: Ni-agarose (Qiagen, Valencia, CA) beads (15 µl)
were incubated with 500 µl of the 6XHis-tagged NHERF-1 fusion protein
(0.2 mg/ml) for 1 h and subsequently incubated with ~500 µg of
murine osteoclast lysates overnight at 4°C. The beads were washed
with washing buffer (50 mM NaH2PO4, 300 mM
NaCl, 20 mM imidazole, pH 8.0) three times for 10 min each. The beads
were collected after centrifugation at 500 g for 2 min.
Elution buffer (20 µl, 50 mM NaH2PO4,
300 mM NaCl, 250 mM imidazole, pH 8.0) was added and incubated at 4°C
for 1 h. The negative control for the Ni-agarose
affinity-precipitation was performed by incubating osteoclast lysates
with Ni-agarose beads alone in the absence of the 6XHis-tagged NHERF
fusion protein. SDS-sample buffer was added to the beads and boiled,
and the affinity precipitates were separated on SDS-PAGE. After
transfer to PVDF membranes, the blots were probed with a polyclonal
antibody to Npt2a at a 1:5,000 dilution, as previously described
(10, 11).
Western analysis of osteoclast and kidney lysates. Murine osteoclasts were used after day 5 in culture for preparation of lysates. After two quick rinses with ice-cold phosphate-buffered saline (PBS), the cells were lysed in a buffer containing 10 mM Tris · HCl, pH 7.05, 50 mM NaCl, 0.5% Triton X-100, 30 mM sodium pyrophosphate, 5 mM NaF, 0.1 mM Na3VnO4, 5 mM ZnCl2, and 2 mM PMSF. Lysates were pelleted by centrifugation at 15,000 rpm for 15 min at 4°C. The supernatant was transferred into a fresh microfuge tube and held on ice. Protein concentrations were measured using the Bio-Rad protein assay reagent kit (Bio-Rad) so that equal amounts of protein were loaded per lane (5-50 µg) and analyzed by SDS-PAGE on 8% gels. Western blot analyses were done as previously described (10, 11). The gel was then electroblotted onto a PVDF membrane by wet-dry transfer (Enprotech, Integrated Separation Systems). Nonspecific protein binding was blocked with 5% nonfat dry milk powder dissolved in PBS containing 0.1% Tween 20. The blots were incubated overnight with a 1:5,000 dilution of the primary antibody to the Npt2a cotransporter. This was followed by detection with a horseradish peroxidase (HRP)-conjugated secondary goat anti-rabbit antibody (Sigma; 1:1,000 dilution). The secondary antibody (HRP-conjugated goat anti-rabbit antibody) was detected by the enhanced chemiluminescence (ECL) detection kit (Amersham Biosciences, Piscataway, NJ) following the manufacturer's instructions, and as previously described (10, 11).
Transient transfection of murine osteoclasts with EGFP-Npt2a-WT
and Npt2a-TRL.
For transfection of osteoclasts, the osteoclast culture medium was
replaced with serum-free (SF) MEM medium (SF-MEM). Approximately 0.8 µg of each DNA (EGFP-Npt2a-WT/-Npt2a-
TRL) was used to transfect the cells with the transfection reagent Lipofectamine 2000 (Life Technologies) following the manufacturer's instructions. The DNA and
Lipofectamine reagent complex was then added to the osteoclasts, and
the plates were rocked gently before incubation for 4 h at 37°C,
5% CO2. After the incubation, the SF-MEM medium was
replaced with 10% serum-containing MEM (
-10 MEM) and incubated for
48 h at 37°C, 5% CO2. These osteoclasts were then
fixed and processed for immunostaining, as detailed below.
RT-PCR in the osteoclast and screening of cDNA library for PiT-1. Primers for amplification of murine PiT-1 were as follows: forward, 5'-CACCCATATGGCTTCTGCTT-3', and reverse, 5'-CAGGAATTCATAGCCCAGGA-3'. The primers were chosen from the murine (kidney) PiT-1 sequence (GenBank accession no. M73696). The PCR reactions were performed using cDNA templates from RT reactions performed with equal quantities of total RNA isolated from murine osteoclasts and rat kidneys. The conditions for PCR-amplification of PiT-1 were as follows: an initial denaturation at 94°C for 4 min, followed by 35 cycles of 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min. A final extension was done for 10 min at 72°C. Two cDNA libraries (100 ng of template) were screened for PiT-1, one from rabbit osteoclasts (28), and the other from murine macrophages (Stratagene), using the same primer pairs and PCR conditions mentioned above. The expected size of the PiT-1 amplicons was 600 bp, which was subsequently confirmed by sequencing.
In situ hybridization of PiT-1 in the murine osteoclast. In situ hybridization for PiT-1 mRNA in osteoclasts was performed, as previously described (27). Murine osteoclasts were seeded on coverslips. The cells were washed in diethyl pyrocarbonate (DEPC)-PBS and fixed in 4% paraformaldehyde at room temperature for 20 min. The osteoclasts were permeabilized with 0.3% Triton X-100 for 15 min at room temperature. After the cells were rinsed in PBS, they were treated with proteinase K (5 µg/ml) for 30 min at 37°C. To inactivate proteinase K, the osteoclasts were rinsed for 5 min at room temperature in 0.1 M glycine and PBS. The endogenous peroxidase was inhibited with 2% H2O2 in PBS for 30 min at room temperature. The osteoclasts were post fixed in 4% paraformaldehyde for 20 min at room temperature and acetylated for 5 min at room temperature with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0). Subsequently, the cells were rinsed with DEPC water and prehybridized at 48°C for 2 h in a humidified chamber. At the end of prehybridization, the osteoclasts were incubated successively with 50, 70, 95, and 100% ethanol; the dehydrated cells were denatured with 50% formamide solution in 2× SSC for 10 min at 60°C and air-dried briefly. The cells were incubated overnight at 48°C with hybridization solution (50% formamide, 5× Denhardt's, 0.5 M NaCl, 50 mM dibasic sodium phosphate, 5 mM EDTA, 10 mM DTT, 0.5% SDS, 1% milk powder, and 0.1 mg/ml salmon sperm DNA) solution, containing heat denatured digoxigenin sense and antisense PiT-1 probes (2.5 ng/µl) under humidified conditions. A separate set of osteoclasts was incubated with hybridization solution without any probes to serve as negative control. The cells were then washed in 2× SSC for 5 min and with STE (500 mM NaCl, 1 mM EDTA and 20 mM Tris-Cl, pH 7.5) for 1 min at room temperature. The osteoclasts were washed with 50% formamide in 2× SSC for 30 min at room temperature. The cells were then placed in buffer 1 (100 mM maleic acid, 100 mM NaCl, and 0.3% Triton X-100, pH 7.5) for 5 min and blocked with buffer 1 containing 10% normal horse serum for 1 h at room temperature. For antibody visualization of digoxigenin, the osteoclasts were further incubated with anti-digoxigenin alkaline phosphatase-conjugated antibody (diluted 1:500) in blocking solution at 4°C overnight in a humidified chamber. These cells were rinsed successively in buffer 1 and buffer 3 (100 mM Tris, 100 mM NaCl, and 50 mM MgCl2, pH 9.5). To each of the wells containing osteoclasts on the coverslips, 1 ml of NBT-BCIP (Roche Diagnostics) staining solution (that was prepared just before use) was added and incubated overnight at room temperature in the dark. After 24 h, cells were washed in DEPC-PBS, mounted, and visualized using a Nikon E800 microscope equipped with a SPOT camera (Diagnostic Instruments, Alexandria, VA).
Construction of the retroviral vector encoding for PiT-1. PiT-1 was tagged at the carboxy terminus by subcloning PiT-1 cDNA into the pSC2-HA plasmid containing a double HA epitope tag (YPYDVPDYA), derived from influenza hemagglutinin (6). Using PCR mutagenesis, a segment of PiT-1 cDNA was amplified using primers 5'-CCATGGCAATATGTGGCATGCC-3' (sense) and 5'-CAAACAGAGCTCCATTCTGAGGATGACC-3' (antisense), resulting in the removal of the stop codon and the introduction of a SacI site (underlined). The modified PiT-1 cDNA was subcloned into the pSC2-HA plasmid between HindIII and SacI in the same reading frame as the HA tag. The HA-tagged PiT-1 cDNA was then digested with HindIII and StuI and subcloned into the retroviral vector pLNSX between HindIII and ClaI, which was filled in using T4 DNA polymerase to create the pLNSPiT1-HA plasmid. Vector particles were produced, as previously described (8).
Immunostaining of osteoclast cultures. Murine osteoclasts were rinsed twice in ice-cold PBS, fixed with 3% paraformaldehyde, and permeabilized with PBS containing 0.2% Triton X-100. The cells were blocked with PBS containing 2% bovine serum albumin (BSA) overnight. The cells were incubated for 2 h in a 1:500 dilution of the primary antibody at room temperature in the blocking solution described above. In experiments to determine the cellular distribution of NHERF-1 and Npt2a, NHERF-1 was detected with a chicken (IgY) polyclonal antibody, whereas Npt2a was detected with a rabbit (IgG) polyclonal COOH-terminal antibody. After two 15-min rinses with PBS containing 0.1% Tween 20, the cells were further incubated with rhodamine phalloidin (Sigma) to stain the actin cytoskeleton, as previously described (10, 11). The transfected pool of Npt2a was detected by fluorescence of EGFP. Epifluorescence was detected with either a Nikon Eclipse 800 microscope equipped with a SPOT camera (Diagnostic Instruments, Alexandria, VA) or a Bio-Rad Radiance 2100 confocal microscope. Confocal microscopy (Bio-Rad Radiance 2100) for immunostaining of Npt2a and NHERF-1 was performed with an Alexa-568-conjugated goat anti-rabbit antibody and Alexa-488-conjugated goat anti-chicken antibody (Molecular Probes), respectively. All images shown were taken with a Plan Apo ×60/1.4 na oil-immersion objective lens. The lateral (xy) resolution at this magnification was 0.12-0.17 µm.
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RESULTS |
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Validation of Npt2a protein expression in normal mouse osteoclasts.
We have previously demonstrated that an Npt2a-like protein was present
in the osteoclast, which was either closely related or identical to
that in the kidney (10, 11). In the current study, we have
definitively established the identity of the Npt2a protein in the
osteoclast by Western analysis of osteoclast lysates isolated from
Npt2a+/+ and Npt2a/
mice. Using a
COOH-terminal antibody to Npt2a, we detected an 88-kDa protein in
osteoclast lysates from Npt2a+/+ but not in
Npt2a
/
mice (Fig. 1A, lanes 1 and
2; ~80 µg protein/lane).
As controls for the Western blots, murine kidney homogenates (~25
µg protein/lane) were isolated from Npt2a+/+ and
Npt2a
/
mice (Fig. 1B, lanes 1 and
2). As expected, there was no detectable signal in
Npt2a
/
mice. The individual Western blots for Npt2a in
osteoclast and kidney lysates were reprobed for actin to demonstrate
protein loading for each lane.
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Cellular distribution of Npt2a, actin, and NHERF-1 in the
osteoclast.
We previously showed that Npt2a is localized almost exclusively on the
basolateral membrane, near the sealing zone of the polarized osteoclast
on bone (10, 11). In the present study, we examined the
nature of the association of Npt2a with the actin cytoskeleton. Murine
osteoclasts, cultured on glass, were immunostained for Npt2a (FITC,
green), while actin was stained with rhodamine phalloidin (red). The
localization of Npt2a is contiguous to the actin cytoskeleton, as shown
in Fig. 2A. Significant
colocalization of Npt2a with the actin ring near the periphery of the
osteoclast was evident, as indicated in yellow. NHERF-1 is associated
with various membrane transporters (31) and is generally
classified as a membrane-cytoskeletal adaptor protein. We showed that
NHERF-1 can be readily detected in the murine osteoclast by Western
blotting (Fig. 2B, lane 2) using a previously
characterized polyclonal antibody to NHERF-1 (30). We used
rat kidney homogenates (Fig. 2B, lane 1) as a
positive control for the Western blot. The NHERF-1 antibody recognizes
an ~50-kDa protein in several species, including rat, human, and
mouse (25, 30).
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Interaction of NHERF-1 with Npt2a: affinity-precipitation assays.
Having established that Npt2a and NHERF-1 are present in the
osteoclast, we examined whether Npt2a associates with the adaptor protein. NHERF-1 was produced as a GST fusion protein and incubated with murine osteoclast lysates, and the affinity precipitates with
GST-NHERF-1 were Western blotted for Npt2a. The signal for Npt2a in
murine osteoclast lysates is shown (Fig. 3A, lane
1); there was no signal obtained for
Npt2a when the GST protein itself was incubated with murine osteoclast
lysates (Fig. 3A, lane 2). The Npt2a protein
could be detected when rat kidney lysates were incubated with
GST-NHERF-1 (Fig. 3A, lane 3). A signal for Npt2a was also detected when murine osteoclast lysates were incubated with
the GST-NHERF-1 fusion protein (Fig. 3A, lane 4).
These findings were further corroborated using 6XHis-tagged NHERF-1
bound to Ni-agarose beads and incubated with murine osteoclast lysates (Fig. 3B). In the absence of 6XHis-tagged NHERF-1, there was
very little binding of Npt2a to Ni-agarose (Fig. 3B,
lane 1); however, Npt2a was bound to the
6XHis-tagged NHERF-1 (Fig. 3B, lane 2). The
signal for Npt2a in the osteoclast lysates is also shown (Fig. 3B, lane 3). Taken together, these experiments
suggest an association of Npt2a with NHERF-1 in the osteoclast.
|
Cellular distribution of Npt2a-WT and Npt2a-TRL in murine
osteoclasts.
NHERF-1 has been implicated in the membrane sorting of several
transporters (26). We next asked whether there were any
differences in sorting of the Npt2a-WT and Npt2a-
TRL proteins in the
osteoclast. Although osteoclasts are notorious for being resistant to
conventional methods of transfection, transfection can be achieved in a
small percentage of cells in culture (18). First, we
plated murine osteoclasts on glass coverslips, which were then
transfected with either the Npt2a-WT or the Npt2a-
TRL constructs in
an EGFP vector, as shown in Fig.
4A, lanes
1-3. As a control for the EGFP-derived fluorescence, murine osteoclasts were also transfected with the empty
EGFP vector. The osteoclasts were subsequently processed for
EGFP-derived immunofluorescence 48 h later. As shown in the EGFP-control cells, there was a low level of diffuse fluorescence due
to the EGFP alone (Fig. 4A, lane 1). In
osteoclasts that were transfected with EGFP-Npt2a-WT, the cellular
distribution of EGFP-Npt2a was seen as both perinuclear and peripheral
near the plasma membrane, as indicated by the arrow (Fig.
4A, lane 2). In contrast, the cellular
distribution of EGFP-Npt2a-
TRL was apparent as mostly perinuclear
and weak staining at the plasma membrane, as indicated by the arrow
(Fig. 4A, lane 3).
|
Expression of PiT-1 in osteoclasts.
Previous studies demonstrated that hypophosphatemia in Npt2a knockout
mice (Npt2a/
) resulted in an age-dependent increase in
type III Na/Pi cotransporter mRNA expression in the kidney as part of an effort to compensate for the loss of the Npt2a
gene (12). Given the evidence that expression of other
Na/Pi cotransporters can increase in the
Npt2a
/
mice, we asked whether the type III family of
Na/Pi cotransporters is expressed in the osteoclast. As
previously mentioned, PiT-1 is one such galv receptor that is highly
expressed in bone marrow, osteoblasts, and chondrocytes (19, 20,
22, 29). In our preliminary studies, we demonstrated the
expression of PiT-1 in murine osteoclasts by RT-PCR, using total RNA
isolated from murine osteoclasts, as shown in Fig.
5A, lanes
1-4. A 600-bp amplicon was obtained for both the
murine osteoclast PiT-1 (Fig. 5A, lane 2) and the
rat kidney PiT-1 (Fig. 5A, lane 3). In the
absence of RT, no product was obtained from kidney RNA (Fig.
5A, lane 4). The sequence identity between the rat kidney and the murine osteoclast PiT-1 was 98%. Previously, we had
successfully established the presence and identity of the Npt2a gene by
PCR-based cloning of a rabbit osteoclast and a mouse macrophage cDNA
library (10, 11). In the current study, to further
establish the presence of PiT-1 in osteoclasts, we detected PiT-1 by
PCR-screening of the cDNA library generated from rabbit osteoclasts
(rOC cDNA), as shown (Fig. 5B, lane 2); PiT-1
could also be detected in a murine macrophage cDNA library (Fig.
5B, lane 3). There was no detectable signal for
PiT-1 in the absence of cDNA template (Fig. 5B, lane
4). Therefore, these data suggest that both the osteoclast and the
macrophage express the PiT-1 transporter. To prove that the RT-PCR
signal for PiT-1 was indeed originating from osteoclastic cells, we
decided to perform in situ hybridization for PiT-1 in murine
osteoclasts in culture. No signal was detected in osteoclasts
hybridized with sense digoxigenin-labeled riboprobes for PiT-1 (Fig.
5A, lanes 1-3). However,
a signal was detected in osteoclasts hybridized with antisense
digoxigenin-labeled riboprobes for PiT-1, as shown in the brown-yellow
color (Fig. 5B, lanes 1-3).
Osteoclasts that were not hybridized with either sense or antisense
riboprobes for PiT-1 served as negative controls (Fig. 5C).
Therefore, our in situ results, combined with those obtained from
RT-PCR and PCR-based amplification of PiT-1 from (osteoclast and
macrophage) cDNA libraries, suggest that PiT-1 is expressed in the
osteoclast and osteoclast-like cells.
|
Cellular distribution of PiT-1 in osteoclasts.
Currently, there are no antibodies available for PiT-1. To circumvent
this limitation, we infected murine osteoclasts with a retroviral
vector carrying a replication-deficient retrovirus that contains a
HA-tagged PiT-1 cDNA, as shown in Fig.
6A. This provides a high-
efficiency gene transfer of a HA-tagged PiT-1 into osteoclasts. Murine
osteoclasts plated on glass coverslips were assayed for
immunofluorescence 48 h postinfection with the retrovirus. Actin
was labeled with rhodamine phalloidin, whereas PiT-1 immunofluorescence
was detected using an HA antibody. As can be seen, the punctate
cellular distribution of PiT-1 is contiguous to that of actin, at or
near the plasma membrane, as indicated in yellow. Similar results were
obtained with two different dilutions of the retrovirus encoding for
PiT-1 (1:1,000 and 1:500). Next, murine osteoclasts were plated on
1-µm-thick CaPO4-coated Osteologic discs, as previously
described (10). Osteoclasts become polarized and avidly
resorb the matrix (10). Murine osteoclasts were infected with the retrovirus-encoded HA-tagged PiT-1 for 48 h. PiT-1 was seen to colocalize with the actin ring, as shown in the representative osteoclast (Fig. 6B). Next, we used confocal microscopy
to assess the polarity of the PiT-1 distribution, as shown in an
xz (cross section) view of the osteoclast (Fig.
6C, lane 1). The transporter was found to
localize almost exclusively to the basolateral membrane and the sealing
zone, similar to that previously reported for the Npt2a cotransporter.
The planar optical section that was used to generate the
cross-sectional view of the osteoclast is shown (Fig. 6C,
lane 2). The cellular distribution of PiT-1 is punctate on
the basolateral surface of the polarized osteoclast.
|
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DISCUSSION |
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We previously demonstrated that an Npt2a-like Na/Pi cotransporter is expressed in the osteoclast and is exclusively localized to the basolateral membrane of the polarized osteoclast (10, 11). In addition, we showed that phosphonoformic acid, a selective inhibitor of Na/Pi cotransport, greatly diminished the resorptive capacity of osteoclasts and that disruption of the actin cytoskeleton in osteoclasts greatly diminished Na-dependent Pi uptake (10).
In the present study, we demonstrate that Npt2a protein in the osteoclast is identical to that in the kidney as previously suggested (10, 11) and is absent from Npt2a-null osteoclasts. We also demonstrate, using GST-fusion protein affinity-precipitation assays, that the Npt2a protein in the osteoclast interacts with the PDZ-domain protein NHERF-1. Previous studies using the yeast two-hybrid system indicated that the TRL motif of Npt2a is critical for binding to NHERF-1 as well as other PDZ-domain proteins in the kidney (9). Some of these PDZ proteins are localized in the brush border or in the subapical compartment of proximal tubular cells. These proteins include NaPi-Cap1 and NaPi-Cap2, identical to diphor-1 and PDZK1 (16, 17). It has been suggested that because PDZ proteins also interact with the actin cytoskeleton, they contribute to the stabilization of proteins at or near the plasma membrane (26).
The yeast two-hybrid studies also suggested that removal of the
COOH-terminal amino acid residues, TRL, blunted the interaction of the
Npt2a cotransporter with NaPi-Cap1, NaPi-Cap2,
NHERF-1, and NHERF-2 (9). This conclusion was based on the
lack of -galactosidase activation by the TRL-truncated COOH-terminal
tail of Npt2a; however, no GST affinity-precipitation assays were
performed with either the full-length or the truncated Npt2a construct.
In our studies, we subcloned fragments of the Npt2a cDNA that spanned
82 amino acids from the COOH terminus (the GST-Npt2a-WT fusion
protein), or 79 amino acids that lacked the terminal TRL domain at the
COOH terminus (the GST-Npt2a-
TRL fusion protein). Our in vitro GST affinity-precipitation studies indicate that the TRL motif in Npt2a is
not the sole determinant for binding to NHERF-1, either in the kidney
or in the osteoclast. Using rat kidney homogenates, we found that when
the PDZ-binding motif was deleted, NHERF-1 was still recovered,
although the recovery was far less than that with the WT Npt2a protein,
consistent with our recent demonstration that the COOH-terminal TRL
motif of Npt2a is not the sole determinant of apical membrane
expression in polarized opossum kidney (OK) cells (13). An
upstream proline-arginine (P578R579) sequence appeared to play an additional role in membrane expression of the Npt2a
protein (13). Our affinity-precipitation studies using the
GST-Npt2a-WT and GST-Npt2a-
TRL fusion proteins contain the upstream
PR sequence, which may explain the finding that Npt2a lacking the
COOH-terminal TRL motif can still bind NHERF-1. It is of interest,
however, that nearly identical findings have been found using similar
Npt2a fusion proteins in affinity-precipitation assays in a renal
epithelial cell line, suggesting that NHERF-1 and Npt2a may bind at
more than one site (Lederer ED and Weinman EJ, unpublished
observations). Moreover, a similar paradigm may also characterize the
interaction between NHERF-1 and NHE3 (Weinman EJ, unpublished
observations). The nature of the putative alternate site or sites of
interaction between NHERF-1 and Npt2a remains to be determined.
We have shown that Npt2a associates indirectly with ezrin in the
osteoclast, a finding that may explain the indirect association with
the actin cytoskeleton. In the kidney, NHERF-1 is predominantly found
in brush-border membranes, with a minor fraction in the basolateral
membrane and in the cytosol (30). These separate pools of
NHERF-1 have been hypothesized to shuttle between the different
membrane domains to regulate ion transport. NHERF-1 associates with an
NH2-terminal ezrin/radixin/moesin domain (ERMAD), conserved
in proteins that belong to the ERM family of actin-binding proteins,
serving as a membrane-cytoskeleton bridge; several studies have
suggested that various transporters are linked to the actin cytoskeleton through the NHERF-1-ERM complex (26). This
association between NHERF and the ERM complex may determine the
membrane sorting of Npt2a in the osteoclast. We found some discernible
differences in the sorting of the Npt2a-WT and the Npt2a-TRL
proteins in the osteoclast, as seen from our immunofluorescence
studies. In actively resorbing osteoclasts, multiple actin rings were
seen to form, which delineated areas of resorptive activity. There was
a redistribution of the Npt2a (WT and
TRL) protein, from the plasma
membrane to these actin rings. However, in nonresorbing osteoclasts
(plated on glass), our immunofluorescence data indicate that the plasma
membrane localization of the Npt2a-WT protein is more intense than that
of Npt2a-
TRL. This observation is consistent with findings in
opossum kidney (OK) cells, where the TRL-truncated Npt2a cotransporter
was still partially expressed at the apical membrane, although part of
it was detected inside the cell (13). Indeed, an upstream
sequence (i.e., P578R579) is also involved in
apical expression of the cotransporters (13). Mutation of this PR motif, or removal of all downstream residues, resulted in
almost a total loss of apical membrane expression of Npt2a in OK cells.
It was suggested that there was an "unmasking" of this internal PR
motif after removal of several amino acid residues upstream of the
COOH-terminal TRL motif. The question remains whether the internal PR
sequence functions as a sorting signal or as an anchoring determinant
for Npt2a in the osteoclast or the kidney. Future studies, using either
adenoviral or retroviral vectors for high-efficiency gene transfer,
will permit a more in-depth investigation of the activity and sorting
behavior of Npt2a-WT and the Npt2a-
TRL proteins. Furthermore, the
application of pulse chase and surface biotinylation strategies will
facilitate the study of Npt2a half-life and membrane sorting in
osteoclasts and kidney derived from NHERF-1 null mice
(25).
We have previously reported that the hypophosphatemia in young
Npt2a/
mice is associated with skeletal abnormalities
and a mild osteoclast defect (11). The absence of a more
flagrant osteopetrotic phenotype in Npt2a
/
mice
suggested that other Pi transport mechanism(s) in the
osteoclast compensate(s) for the deficiency of Npt2a. We have
hypothesized that the type III family of Na/Pi
cotransporters may adapt to the lack of the Npt2a protein in
osteoclasts (11). In the present study we demonstrate that PiT-1, a member of the type III family of Na/Pi
cotransporters, is expressed in the murine osteoclast. This was
accomplished by RT-PCR of RNA isolated from osteoclasts in culture
and PCR-based screening of an osteoclast cDNA library. Moreover,
our preliminary data show that PiT-1 in the osteoclast is probably
identical to that found in the kidney.
We have also demonstrated that PiT-1 is localized to the basolateral
membrane of the polarized osteoclast and that its distribution in the
osteoclast is similar to what we previously reported for Npt2a
(10). Moreover, the colocalization of PiT-1 with actin in
the polarized osteoclast is consistent with the plasma membrane localization of PiT-1. It is of interest that the receptor for amphotropic murine leukemia viruses, PiT-2, has been reported to
associate with actin in CHO cells (24). The colocalization of PiT-2 with actin in these cells can be modified by variations in
concentration of extracellular Pi (24). On the
basis of these findings, it was postulated that changes in the actin
cytoskeleton (such as formation of stress fibers) may be responsible
for conformational changes in PiT-2 that in turn determine its activity
as a Pi transporter. Although PiT-1 does not contain any
recognizable PDZ-binding motifs that may mediate an indirect
association with actin, the activity of PiT-1 in the osteoclast in
response to ambient Pi levels may be linked to changes in
the osteoclast actin network that occur during attachment to bone
matrix and subsequent resorption. Future studies are necessary to
assess the relative contribution of PiT-1 to osteoclast function in
both WT and Npt2a/
mice.
![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-44706 and a Designated Research Initiatives Fund Grant from the University of Maryland, Baltimore, MD, to A. Gupta, National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-46292 to M. A. Chellaiah, and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-55881 and a Department of Veterans Affairs Research Service grant to E. J. Weinman. H. S. Tenenhouse and H. Murer were supported by grants from the Canadian Institutes for Health Research (FRN 44355) and the Swiss National Science Foundation (31.46523 and 31.6539), respectively.
![]() |
FOOTNOTES |
---|
* M. A. Khadeer and Z. Tang contributed equally to this work.
Address for reprint requests and other correspondence: A. Gupta, Dept. of Oral and Craniofacial Biological Sciences, Univ. of Maryland, 666 West Baltimore St., Baltimore, MD 21201 (E-mail: ang001{at}dental.umaryland.edu).
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.
First published February 26, 2003;10.1152/ajpcell.00580.2002
Received 12 December 2002; accepted in final form 21 February 2003.
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