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Abstract
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 49, Issue of December 5, pp. 49271–49278, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The a3 Isoform of the 100-kDa V-ATPase Subunit Is Highly but Differentially Expressed in Large (>10 Nuclei) and Small (<5 Nuclei) Osteoclasts* Received for publication, September 8, 2003, and in revised form, September 22, 2003 Published, JBC Papers in Press, September 22, 2003, DOI 10.1074/jbc.M309914200 Morris F. Manolson‡, Hesheng Yu, Weimin Chen, Yeqi Yao, Keying Li, Rita L. Lees, and Johan N. M. Heersche From the Faculty of Dentistry, University of Toronto, Toronto, Ontario M5G 1G6, Canada osteoclasts (i.e. 10 nuclei) are more likely to be in a resorptive Osteoclasts dissolve bone through acidification of an extracellular compartment by means of a multimeric state than small osteoclasts (i.e. 5 nuclei) (4) and are more vacuolar type H -ATPase (V-ATPase). In mammals, dependent on the vacuolar-type proton pumping ATPase there are four isoforms of the 100-kDa V-ATPase “a” (V-ATPase) to recover from an acid load (5). subunit. Mutations in the a3 isoform result in deficient V-ATPases are essential to osteoclastic bone resorption. Os- bone resorption and osteopetrosis, suggesting that a3 teoclasts demineralize the bone matrix through acidification of has a unique function in osteoclasts. It is thus surprising an extracellular resorption zone by actively pumping protons that several studies show a basal level of a3 expression across the plasma membrane (reviewed in Ref. 6). Transloca- in most tissues. To address this issue, we have compared tion of protons across the osteoclast plasma membrane is me- a3 expression in bone with expression in other tissues. diated by V-ATPases. V-ATPase-specific inhibitors reduce bone RNA blots revealed that the a3 isoform was expressed resorption in vitro (7–10) and in vivo (11), clearly demonstrat- highest in bone and confirmed its expression (in de- ing the essential role V-ATPases play in osteoclastic function creasing order) in liver, kidney, brain, lung, spleen, and and highlighting the potential of using V-ATPase inhibitors as muscle. In situ hybridization on bone tissue sections antiresorptive therapeutics. Unfortunately, V-ATPases are not revealed that the a3 isoform was highly expressed in just present on the osteoclast plasma membrane but are also on multinucleated osteoclasts but not in mononuclear stro- endomembrane organelles such as Golgi, lysosomes, endo- mal cells, whereas the a1 isoform was expressed in both somes, clathrin-coated vesicles, chromaffin granules, and syn- cell types at about the same level. We also found that a3 aptic vesicles (reviewed in Ref. 12). As V-ATPase activity is expression was greater in osteoclasts with 10 or more essential for basic housekeeping functions in almost all cell nuclei as compared with osteoclasts with five or fewer types, any therapeutic strategy to limit bone resorption nuclei. We hypothesize that these differences in a3 ex- through V-ATPase inhibition must be designed to specifically pression may be associated with previously demon- strated differences between large and small osteoclasts target the osteoclast plasma membrane V-ATPase. with reference to their resorptive activity. Isoforms of the V-ATPase “a” subunit may target or regu- late other V-ATPase subunits to distinct cellular locations. V-ATPases are composed of at least 11 subunits; seven are part Large osteoclasts predominate in diseases associated with of the hydrophilic catalytic core (V ), and the remaining four compose a hydrophobic domain (V ) spanning the membrane accelerated bone loss. Bone is a dynamic tissue. To maintain its structural integrity, bone is continually remodeled, first being bilayer. The hydrophobic domain contains one or two copies of the 100-kDa “a” subunit, the focus of this study. The mamma- resorbed by osteoclasts and then remade by osteoblasts. An increase in osteoclastic activity relative to osteoblastic activity lian “a” subunit has a hydrophilic 48-kDa N terminus and a hydrophobic 49-kDa C terminus containing up to nine puta- results in bone loss. Osteoclasts are multinucleated cells of hemopoietic origin. On average, osteoclasts in healthy bone tive transmembrane domains (13). In yeast, it has been shown contain between 3 and 10 nuclei. However, an increase in that the “a” subunit is essential for V-ATPase assembly (14) osteoclast size has been noted in Paget’s disease, end stage and activity (15, 16) and interacts with V subunits A and H renal disease, periodontal disease, and rheumatoid arthritis (17). We have cloned two isoforms of the “a” subunit in yeast (1–3). Interestingly, in vitro assays have shown that large (Vph1p, and Stv1p (14, 18)) and have demonstrated differential localization of V-ATPase complexes containing either isoform. Recent studies have shown that V-ATPases containing differ- * This work was supported by the Arthritis Society of Canada with ent “a” isoforms differ in their coupling efficiency and in vivo Operating Grant 99087 (to M. F. M.) and a fellowship (to H. Y.) and by dissociation between the V and V complexes (19). We hypoth- 1 o Canadian Institutes of Health Research Grant MT-15654 (to esize that isoforms of the “a” subunit may target or regulate J. N. M. H.). The costs of publication of this article were defrayed in other V-ATPase subunits to distinct cellular locations. part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section The “a3” V-ATPase subunit may be essential for but not 1734 solely to indicate this fact. unique to the osteoclast plasma membrane V-ATPase. Higher The nucleotide sequence(s) reported in this paper has been submitted TM to the GenBank /EBI Data Bank with accession number(s) AF393370, AF393371, and AF393372. ‡ To whom correspondence should be addressed: Faculty of Dentistry The abbreviations used are: V-ATPase, vacuolar proton-translocat- Research Institute, 124 Edward St., Rm. 429, Faculty of Dentistry, ing adenosine triphosphatase; GAPDH, glyceraldehyde-3-phosphate University of Toronto, Toronto, ON M5G 1G6, Canada. Tel.: 416- dehydrogenase; GST-sRANKL, glutathione S-transferase-soluble re- 979-4900 (ext. 4392); Fax: 416-979-4936; E-mail: m.manolson@ ceptor activator of NF-B ligand; TRAP, tartrate-resistant acid phos- utoronto.ca. phatase; RT, reverse transcriptase. This paper is available on line at http://www.jbc.org 49271 This is an Open Access article under the CC BY license. 49272 V-ATPase a3 Expression in Large and Small Osteoclasts perature for 10 min. Detached cells were washed away with culture eukaryotes have four isoforms of the “a” subunit, all of which medium, resulting in a 95% pure preparation of osteoclasts still at- appear to have differential expression patterns (20 –22). The tached to the culture dishes. a1, a2, and a3 isoforms have been shown to be expressed in Differentiation and Isolation of Large and Small Osteoclasts from the most tissues examined. However, the a1 isoform is most highly 6 RAW267.4 Cell Line—Approximately 5 10 third passage RAW267.4 expressed in brain and heart; a2 is most highly expressed in the cells were cultured in a 100-cm dish with 15 ml of Dulbecco’s modified acrosomal membrane within sperm (23) but also highly ex- Eagle’s medium, containing 10% fetal bovine serum, 20 units/ml peni- cillin, 20 g/ml streptomycin, 0.25 g/ml fungizone, and 200 ng/ml pressed in liver and kidney; and a3 is most highly expressed in recombinant glutathione S-transferase-soluble receptor activator of liver and heart (24, 25). The a4 isoform appears to be kidney- NF-B ligand (GST-sRANKL) in humidified air (37 °C and 5% CO ) specific (22, 26), and mutations within the human a4 isoform with medium changes at days 3, 5, and 7. For protein isolation, 10 result in distal tubular renal acidosis (27, 28). 2 100-cm dishes were plated as described above and visually inspected Evidence suggesting that a3 is essential to osteoclastic func- until at least 78% of the total nuclei were contained within small (5 tion first came from its initial identification as “OC-116Kda.” nuclei) osteoclasts (generally day 5) or within large (10 nuclei) oste- oclasts (generally day 8). Prior to protein isolation, osteoclasts were The OC-116Kda gene was cloned by a differential screening of further selected by gently washing the dishes with 3 10 ml of phos- a human osteoclastoma cDNA library, and its mRNA was re- phate-buffered saline using a 10-ml pipette. To extract protein, 8 ml of ported to be uniquely expressed in multinucleated giant cells TRIzol (Stratagene) was added per dish, and the isolation was per- within osteoclastoma tumors (29). In mice, a3 was induced formed according to the manufacturer’s instructions. during osteoclast differentiation, and the gene product was RT-PCR—95% pure preparations of osteoclasts (prepared as de- localized in the plasma membrane and cytoplasmic filamentous scribed) were immediately suspended in lysis buffer (mRNA Capture Kit; Roche Applied Science) and stored at 80 °C until further use. structures within osteoclasts (24). Disruption of the mouse a3 Once mRNA was extracted using the mRNA capture kit, RT-PCR was encoding gene, (referred to as either Atp6i (30) or MMUOC116 immediately performed using the RT-PCR Titan One Tube system (31)) resulted in severe osteopetrosis, whereas mutations in the (Roche Applied Science). The reverse transcription was performed at human a3 encoding gene (referred to as either TCIRG1 (32) or 50 °C for 30 min followed by 35 cycles of PCR (30 s at 94 °C, 30 s at OC116 (33)) resulted in a subset of autosomal recessive osteo- 55 °C, and 68 °C for 1 min). petroses (32) including infantile malignant osteopetrosis (33– Oligonucleotides for RT-PCR—To clone potential isoforms of the “a” subunit from rabbit osteoclasts, oligonucleotides were designed to encode 35). Despite this compelling genetic evidence suggesting that four evolutionarily conserved regions contained within the C terminus of a3 function is essential for and specific to osteoclastic bone the 100-kDa V-ATPase subunit nucleic acid sequences obtained from rats, resorption, expression studies suggest that a3 is ubiquitously mice, cows, and humans. Oligonucleotides were made 2– 8-fold redundant expressed and probably also has roles unrelated to osteoclastic to account for base pair differences found between species. Restriction function. RT-PCR revealed that a3 was expressed in all tissues sites for XbaI and SalI were encoded into the 5 ends of the sense and examined (36), whereas Northern blotting suggested that the antisense primers, respectively, to facilitate subcloning into pBluescript pBS-SK (Stratagene). Sequences (with base pairs in parentheses indicat- highest a3 expression was in the liver (24, 25). Bone tissue was ing regions of redundancy) of the four primers are as follows: M061 (sense not investigated in either of these studies. One alternatively strand), 5-agatctagaccttccc(c/g)tt(t/c)ctgtttgctgtgatg-3; MO62 (antisense spliced transcript of a3, lacking the first 217 amino acids off the strand), 5-gacgtcgacttcatctt(g/a)aaggagttgaggaa-3; M063 (sense strand), hydrophilic N terminus of a3, was identified as the T cell 5-agatctagattcctcaactcctt(c/t)aagatgaa-3; MO64 (antisense strand), membrane protein TIRC7 and was suggested to have a central gacgtcgacggtagga(c/g)gc(a/g)gtgttggaga(t/c)gcagcc-3. role in T cell activation in vitro and in vivo (37, 38). Plasmids, DNA Sequencing, and Sequence Analysis—For a house- keeping gene, a 292-bp fragment (from position 289 to 581 according to To address the disparate conclusions drawn from the genetic TM the GenBank accession number L23961) encoding rabbit GAPDH evidence and the expression patterns of a3, we decided to was used. The following plasmids were all obtained by performing directly compare a3 expression levels between bone and other RT-PCR with the indicated oligonucleotides on mRNA isolated from a tissues and the expression of a3 within the different bone cells. 95% pure preparation of osteoclasts obtained from the long bones of Finally, considering the differences between large and small New Zealand White rabbits. RT-PCR products were cut with XbaI and osteoclasts in resorptive activity and dependence on V-ATPase SalI (restriction sites added to the 5 end of the oligonucleotides) and cloned into XbaI and SalI of pBluescript pBS-SK. The inserts in the activity for intracellular pH regulation, we also explored plasmids described below were all commercially sequenced (York Uni- whether there is a correlation between a3 expression and os- versity Core Molecular Biology and DNA Sequencing Facility, Toronto, teoclast size. Ontario) to completion on both strands using the T3, T7, or custom TM synthesized oligonucleotides. Sequences deposited in GenBank are EXPERIMENTAL PROCEDURES the result of sequencing at least two independently obtained RT-PCR Materials—The murine macrophage cell line RAW267.4 was ob- products. Sequence analysis was performed using the Wisconsin Pack- tained from the American Type Culture Collection (ATCC catalog no. age, version 10.0 (Genetics Computer Group (GCG), Madison, WI). The TM TIB-71). Medium 199, -minimum essential medium, Dulbecco’s mod- following plasmids were obtained. pRab-a1-1 (MM642; GenBank ac- ified Eagle’s medium, antibiotics (penicillin G, gentamicin, and fungi- cession number AF393370) contains a 470-bp RT-PCR product result- zone) were obtained from Invitrogen; sterile fetal bovine serum was ing from oligonucleotides MO61 and MO62 (described above). It encodes obtained from MEDICORP Inc. Kits for mRNA purification and RT- part of the C terminus of the rabbit a1 isoform of the 100-kDa V-ATPase TM PCR were purchased from Roche Applied Science. Polyclonal antibodies subunit. pRab-a1–2 (MM644; GenBank accession number AF393370) to the V-ATPase a1 and a3 subunits were a kind gift from Dr. Beth Lee contains a 650-bp RT-PCR product resulting from oligonucleotides (Washington University School of Medicine), and monoclonal antibodies MO63 and MO64 (described above). It encodes the C terminus of the to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained rabbit a1 isoform of the 100-kDa V-ATPase subunit, and its sequence is TM from Abcam. Most other reagents were from either Sigma or Fisher. continuous with the 3 end of pRab-a1-1. pRab-a2 (MM656; GenBank Rabbit Osteoclast Isolation—Osteoclasts were obtained from 10-day- accession number AF393371) contains a 1060-bp RT-PCR product re- old New Zealand White rabbits as described in Ref. 39. Briefly, the long sulting from oligonucleotides MO61 and MO64 (described above). It bones (femurs, tibiae, humeri, and radii) were dissected out, adherent encodes part of the C terminus of the rabbit a2 isoform of the 100-kDa TM soft tissues were removed, and the cleaned shafts were cut longitudi- V-ATPase subunit. pRab-a3 (MM655; GenBank accession number nally. The interior surfaces were then curetted to release the bone cells, AF393372) contains a 950-bp RT-PCR product resulting from oligonu- followed by repeated pipetting to release additional cells attached to the cleotides MO61 and MO64 (described above). It encodes part of the C bone fragments. Cells were resuspended in -minimum essential me- terminus of the rabbit a3 isoform of the 100-kDa V-ATPase subunit. dium (pH 7.4) with 10% fetal calf serum and antibiotics (100 g/ml RNA Blot Hybridization—RNA was extracted from rabbit brain, penicillin G, 0.5 g/ml gentamicin, and 0.3 g/ml fungizone) and al- bone, kidney, liver, lung, muscle, and spleen tissue using TRIzol rea- lowed to attach overnight to culture dishes in humidified air (37 °C and gent (Invitrogen) following the manufacturer’s instructions. Approxi- 5% CO ). If further purification was required, cultures were incubated mately 15 g of RNA from each tissue was subjected to Northern blot the following day with 0.001% protease E, 0.01% EDTA at room tem- analysis performed essentially as described in Ref. 40 with the following V-ATPase a3 Expression in Large and Small Osteoclasts 49273 FIG.1. Relative mRNA expression of a3 in various rabbit tis- sues. A Northern blot was run with total RNA extracted from rabbit brain, bone, kidney, liver, lung, muscle, and spleen and probed with P-labeled cDNA encoding rabbit a3 (pRab-a3) and rabbit -actin. Quantification of the resulting autoradiogram is described under “Ex- perimental Procedures.” The a3 signal was first normalized to -actin and then presented relative to a3 expression in bone. FIG.2. In situ hybridization of paraffin sections of bone tissue reveals that a3 expression is mostly in osteoclasts. Three consec- exceptions. cDNA encoding a3 (pRab-a3) and -actin were labeled using utive 6-m sections of a 10-day-old rabbit femur were prepared as random hexamers and [ P]dCTP, with unincorporated nucleotides re- described under “Experimental Procedures” and stained for the oste- moved using an Amersham Biosciences NICK column. Prehybridization oclast-specific enzyme, TRAP, and probed with the digoxigenin-labeled (68 °C for 2 h) and hybridization (68 °C for 3 h) were performed using antisense (Antisense Probe) and sense (Sense Probe) cRNA encoding the Expresshyb hybridization solution (Clontech). After hybridization, the a3 subunit. blots were washed three times for 15 min each in 2 SSC, 0.1% SDS at 20 °C followed by two times for 20 min each in 0.1% SSC, 0.1% SDS at 61 °C. The resulting autoradiograms were scanned using a transpar- using the Image-Pro Plus 4.1 software (Media Cybernetics, L.P.) and ency adapter, and band intensities were quantified using AlphaEase subsequently confirmed by visual inspection, manually adding or sub- Image Analysis Software (Alpha Innotech Corp.). tracting grains that were missed or inappropriately labeled by the Quantification of Immunoblots—Immunoblots were performed ex- software package. Statistical analyses were performed using GraphPad actly as described in Ref. 16, with the resulting signal obtained using Instat 2.01 (Alias Software). the ECL detection system (Amersham Biosciences) and the FluorChem Imaging System (Alpha Innotech Corp.). To ensure that the chemilu- RESULTS minescent signals from the immunoblots were within the linear range, Deletions and mutations within the mouse and human a3 each protein sample was run on SDS-PAGE as a series of four serial genes result in an osteopetrotic phenotype (30 –33) despite the dilutions. Multiple exposure times were recorded by the CCD camera, and an exposure time was used for quantification only if the obtained fact that a3 expression has been reported in almost all tissues signal internally reflected the serial dilution of the sample. For each examined (36). To address these conflicting data, we set out to separate gel, the absolute value obtained for a1 and a3 at each of the directly compare the expression level of a3 in osteoclasts with four protein concentrations was divided by the absolute value obtained that in several other tissues. This study was initiated by first for GAPDH. GAPDH was used to normalize the signal between differ- determining which of the four known isoforms of the 100-kDa ent samples, since in situ hybridization revealed no difference in “a” subunit are expressed in rabbit osteoclasts. GAPDH gene expression between large and small osteoclasts (see Fig. 4E). The a1/GAPDH and a3/GAPDH ratios were then plotted against Bone Cells Express the a1, a2, and a3 Isoforms of the 100-kDa g of protein loaded per lane, and a linear regression line was fitted V-ATPase Subunit—To identify all V-ATPase a isoforms ex- using the method of least squares. The slope of this linear regression pressed in osteoclasts, we employed an RT-PCR strategy and line was then used to compare relative expression of a1 and a3 between designed two sense and two antisense oligonucleotides, each different experiments. encoding evolutionarily conserved regions found in all four of In Situ Hybridization—Femurs from 10-day-old rabbits were fixed in the a isoforms in several different species. These primers were 4% paraformaldehyde, decalcified using HCl (0.2 N HCl for 24 h), and embedded in paraffin using standard methods. Six-m sections were used to perform several RT-PCRs on mRNA extracted from a cut, placed onto silicon-treated glass slides and dried for 16 h at 40 °C. 95% pure preparation of osteoclasts isolated from the long Sections were dewaxed with two 10-min incubations in xylene and then bones of 10-day-old rabbits as described under “Experimental rehydrated as follows: 1 5 min in 100% EtOH, 1 5 min in 95% Procedures.” RT-PCR products were pooled and cloned into EtOH, 1 5 min in 70% EtOH, followed by two rinses in diethylpyro- sequencing vectors. Restriction mapping demonstrated that all carbonate-treated double-distilled H O. Prehybridization and hybrid- 60 of the resulting recombinant plasmids fell into only three ization conditions were as described in Ref. 41. Labeling and detection of the digoxigenin-labeled RNA probes were performed using the digoxi- groups (data not shown). Sequencing revealed that we had genin RNA labeling and detection kits from Roche Applied Science cloned three isoforms of the 100 V-ATPase “a” subunit (se- according to the manufacturer’s instructions. Tartrate-resistant acid TM quencing data have been deposited in the GenBank /EMBL phosphatase (TRAP) staining was performed exactly as described in Data Bank with accession numbers AF393370, AF393371, and Ref. 5. AF393372). These three rabbit isoforms have 91, 86, and 82% For in situ hybridization on cultured cells, freshly isolated osteoclast- amino acid identity, respectively, to the a1, a2, and a3 identi- containing cell suspensions (as described above) were plated on plastic chamber slides and cultured overnight. Cells were then fixed with 4% fied in mice (24, 25) and 85, 65, and 68% amino acid identity, paraformaldelyde, permeabilized with Triton X-100, hybridized with respectively, to the a1, a2, and a3 identified in chickens (43). the [ H]dCTP-labeled probes for 24 h at 42 °C, washed repeatedly, and The percentage similarity and the phylogenetic relationship finally dipped in emulsion and developed after 3 days of exposure as among these isoforms suggests that we have cloned the or- described in Ref. 42. tholog a1, a2, and a3 subunits from rabbits and that all three Digital images were taken at 400 using a Leitz microscope isoforms are expressed in a 95% pure osteoclast preparation. equipped with a SPOT RT digital camera (Spot Diagnostic Instruments, Inc.). For quantification, grains were first automatically highlighted Considering the numerous RT-PCRs performed and the num- 49274 V-ATPase a3 Expression in Large and Small Osteoclasts FIG.3. Quantification of in situ hybridization demonstrates that a3 is expressed primarily in osteoclasts, whereas a1 is expressed at similar levels in both multi- and mononuclear cells. A, cultured stromal cells from the long bones of new born rabbits were fixed, permeabilized, and probed with [ H]dCTP-labeled cDNA encoding a1 (a1, bottom panels), a3 (a3, top panels), and GAPDH (not shown) as described under “Experimental Procedures.” The a1 and a3 cDNA probes were equalized for size and radioactivity to enable comparisons between a1 and a3 expression in multi- and mononuclear cells. B, quantification of in situ hybridization was performed by counting individual grains in multinuclear (black bars) and mononuclear (white bars) cells using Image-Pro Plus 4.1 software. Results are normalized by expressing the number grains per cell as grains per nucleus to account for differences in cell size. Results are expressed as means S.E. for n number of cells. n 17 for a3-multinucleated cells, n 14 for a1-multinuclear cells, n 27 for a3-mononuclear cells, and n 29 for a1-mononuclear cells. *, p 0.001. ber of recombinant plasmids screened, the fact that we did not result in an osteopetrotic phenotype, we compared the expres- retrieve the fourth isoform of the 100-kDa subunit supports the sion levels of a3 in brain, kidney, liver, lung, muscle, and spleen view that the a4 isoform is specific to kidney. with that in bone. Northern analysis was performed using the The a3 Isoform of the 100-kDa Subunit Is Highly but Not a3 gene and, to account for differences in loading, -actin. With Exclusively Expressed in Osteoclasts—The pattern and magni- the a3 probe, we found a single intense band at 3.2 kb (data tude of a3 expression is still controversial. The original studies not shown). Quantification of the resulting autoradiographs using Northern blot analysis demonstrated that a3 was exclu- (Fig. 1) shows that whereas bone does have the highest level of sively found in osteoclastomas and not expressed in kidney, a3 expression, liver has only 10% less, with decreasing levels liver, skeletal muscle, or brain (29). Subsequent studies using seen in kidney, brain, lung, spleen, and muscle. However, bone RT-PCR revealed, however, that a3 was expressed in all tested is a complex tissue, and osteoclasts represent at most 1% of the human tissues (36), and Northern analysis by two different cells found within bone. If a3 mRNA expression were restricted groups demonstrated that the highest expression of a3 was in to osteoclasts, this result would imply that the a3 subunit is the liver (24, 25). In an attempt to understand why mutations highly expressed in osteoclasts. We therefore determined the and deletions of this apparently ubiquitously expressed gene expression of a3 in the various cell types within bone by per- V-ATPase a3 Expression in Large and Small Osteoclasts 49275 FIG.4. In situ hybridization reveals that a3 is differentially expressed in large and small osteoclasts. Cultured osteoclasts from newborn rabbit long bones were fixed, permeabilized, and probed with [ H]dCTP-labeled cDNA encoding rabbit a1, a3, and GAPDH as described under “Experimental Procedures.” The top panels show typical results for a3 expression in osteoclasts with 42 (A),2(B),3(C), and 4 (D) nuclei. Fig. 4E shows quantification of the in situ hybridization, which was performed by counting individual grains in large (10 nuclei, black bars) and small (5 nuclei, gray bars) cells using Image-Pro Plus 4.1 software. To account for the differences in size, the results are normalized by the number of nuclei per cell. The numbers reflect the mean taken from the images of 25 large and 25 small osteoclasts S.E. *, p 0.05. forming in situ hybridization (Fig. 2). Sequential bone slices pression in bone reflects predominately expression within were probed with a3 sense (right panel, negative control) and osteoclasts. antisense (middle panel) RNA, and osteoclasts were identified The a3 Isoform Is Differentially Expressed in Large (10 by TRAP staining (left panel). Fig. 2 reveals that a3 expression Nuclei) and Small (5 Nuclei) Osteoclasts—Osteoclasts are within bone is mostly limited to osteoclasts. heterogeneous, differing in size, shape, and resorptive activity. To quantify the results shown in Fig. 2, in situ hybridization Large osteoclasts (defined as having 10 nuclei (5)), are more was performed on isolated osteoclast-containing cell popula- likely to be found in diseases characterized by increased bone tions using H-labeled a1 and a3 cDNA probes. Fig. 3A shows resorption (1–3), more likely to be in a resorptive state (4), and that the a3 isoform was highly expressed in multinucleated are more dependent on V-ATPases to recover from an acid load cells but barely detectable in mononuclear cells. For quantifi- (5) than small osteoclasts (i.e. 5 nuclei). Considering the cation, the number of grains per cell was divided by the number differences in V-ATPase activity between large and small oste- of nuclei per cell to normalize counts and thereby account for oclasts, we addressed whether there was a concomitant differ- the variability in cell size. The results show that the a3 isoform ence in a3 expression, as suggested by visual inspection of the is expressed almost 30-fold higher in multinucleated cells com- in situ hybridization shown in Fig. 4. As before, defining small pared with mononuclear cells (Fig. 3B). In contrast, the a1 as 5 nuclei and large as 10 nuclei is arbitrary and made to isoform appears to be expressed at low but equal levels in both facilitate both the analysis of the data and comparison of the multi- and mononuclear cells, whereas the housekeeping gene, data with previous studies by creating two distinct nonoverlap- GAPDH, is expressed significantly higher in the mononuclear ping categories (4, 5). The number of nuclei per osteoclast, cells. These results thus support the view that the a3 ex- rather than surface area, was chosen to define cell size, since 49276 V-ATPase a3 Expression in Large and Small Osteoclasts FIG.5. Populations enriched in large or small osteoclasts can be ob- tained from RAW267.4 cells. A, RAW267.4 cells were cultured as de- scribed under “Experimental Procedures” with 200 ng/ml GST-sRANKL and media changes at days 3, 5, and 7. On days 5 (left panel)and8(right panel), plates were fixed and stained for TRAP. B, the num- ber of cells and the number of nuclei in each cell were counted for the following groups: TRAP-negative mononuclear cells (white), TRAP-positive cells containing 5 nuclei (gray), and TRAP-positive cells containing 10 nuclei (black). To account for differences in cell size, the histogram (B) is presented as the percentage of total nuclei in each group. cytoplasmic volume is known to correlate with the number of two populations reveal that the level of a1 and a3 translation in nuclei. Surface area is an unreliable measure of size, because large and small osteoclasts reflects the pattern of transcription osteoclasts in a migratory phase are spread out, whereas in a found in authentic osteoclasts by in situ hybridization (Fig. 6). stationary phase they assume a more rounded shape (44). The a3 expression was 2.86 0.79-fold (mean S.E., p 0.05) Quantification of these results shows that whereas there is no more in large osteoclasts than in small osteoclasts, whereas a1 difference in the expression of the housekeeping gene GAPDH, expression was similar (0.81 0.33-fold; mean S.E., p the expression of the a1 isoform in small osteoclasts is slightly 0.05) in large and small osteoclasts. but significantly higher than that in large osteoclasts. Con- These results lead us to speculate that the differences in a3 versely, a3 mRNA expression is 2.5-fold higher in large than in expression may be associated with the differences in the regu- small osteoclasts (Fig. 4E). Results comparing osteoclasts con- lation of the intracellular pH (5) and resorptive activity (4) taining 2–5, 6 –9, and 10 nuclei suggest a gradual increase in between large and small osteoclasts. Interestingly, 4% of the a3 expression with an increase in osteoclast size (data not small osteoclasts had a3 expression levels similar to large shown). osteoclasts (30 grains/nuclei). These numbers seem to corre- To test whether this observation was mirrored in a3 protein late with the observation that 5.6% of small osteoclasts are actively resorbing (4), which could further suggest that a3 expression, it was necessary to switch from using authentic rabbit osteoclasts to osteoclasts differentiated from the mouse expression is associated with the resorptive potential. macrophage cell line RAW267.4 since insufficient numbers of DISCUSSION osteoclasts can be isolated from rabbit long bones. RAW267.4 cells can differentiate into osteoclasts by culturing in media An ideal target for an antiresorptive therapeutic would be a containing sRANKL. By varying the concentration of GST- protein that is both uniquely expressed in osteoclasts and es- sRANKL, the number of media changes, and the culture period, sential to osteoclastic function. Even more advantageous would we obtained cultures in which at least 78% of the total nuclei be a therapeutic that could preferentially inhibit pathological within the culture dish were contained within small (5 nuclei) osteoclast activity resulting in bone loss while not affecting or large (10 nuclei) osteoclasts (Fig. 5). The number of oste- osteoclasts engaged in maintaining bone integrity. The most oclasts with 6 –9 nuclei was less than 5% for the two time points commonly used class of antiresorptive therapeutics, bisphos- selected (data not shown). There were variations in the number phonates, are effective not because their targets are unique to of days required to achieve the results shown in Fig. 5 (plus or osteoclasts or to osteoclast function but rather because bisphos- minus 1 day). Therefore, for all subsequent experiments, cul- phonates accumulate mostly in bone and are selectively taken tures were visually inspected on a daily basis, and protein was up by resorbing osteoclasts (45). This nonspecificity results in not extracted until enriched populations of large and small other tissues being affected that are exposed to higher concen- osteoclasts were obtained that were equal to or better than that trations of bisphosphonates, such as the gastrointestinal tract, shown in Fig. 5. Immunoblots of protein extracted from these resulting in side effects such as esophageal ulcers and gastro- V-ATPase a3 Expression in Large and Small Osteoclasts 49277 FIG.6. Immunoblots reveal that a3 is differentially expressed in large and small osteoclasts differentiated from RAW267.4 cells. A, protein ex- tracts from enriched populations of large (Large Osteoclasts) and small (Small Os- teoclasts) RAW-derived osteoclasts (as de- fined in the legend to Fig. 5) were run on SDS-PAGE as a series of four serial dilu- tions (5– 40 g), immunoblotted, and probed with antibodies specific to the V- ATPase a1 isoform (top panel), the a3 iso- form (bottom panel), and GAPDH (both panels). A shows typical results from a single experiment. B, immunoblots shown in A were quantified as described under “Experimental Procedures.” Considering that GAPDH mRNA expression in large and small osteoclasts was similar (Fig. 3B), both a1 and a3 expression were di- vided by GAPDH protein expression to normalize the signal within a single ex- periment. To compare between experi- ments, results from each blot were ex- pressed as a percentage difference in relative expression between large and small osteoclasts, with the relative ex- pression of small osteoclasts arbitrarily set at 1. The numbers reflect the mean of the relative expression S.E. from three separate experiments in which the popu- lations of large (black bars) and small (gray bars) osteoclasts were enriched to at least the same extent, if not better, than those shown in Fig. 5. *, p 0.05. intestinal infections (46, 47). The first reports describing the lack of a3 in all tissues except osteoclasts. Further evidence cloning and expression of the V-ATPase a3 subunit indicated that other isoforms may compensate for deficiencies, in partic- that this isoform was both unique to osteoclasts and essential ular for the “a” subunits, comes from mutational studies on the to osteoclast function (29), suggesting that a3 would make an yeast ortholog genes. excellent target for antiresorptive agents. Deletions and muta- Yeast have two isoforms of the V-ATPase “a” subunit, Vph1p tions within the mouse and human a3 gene result in an osteo- and Stv1p (14, 18). In wild type cells, Vph1p is the predomi- petrotic phenotype (30 –35), further supporting the notion that nantly expressed isoform and is localized to the yeast vacuole. a3 is both unique and essential for osteoclasts and hence an Disruption of the VPH1 gene eliminates vacuolar acidification excellent target for antiresorptive agents. However, expression and results in a phenotype similar to but not as severe as studies show a ubiquitous distribution (24, 25, 36), suggesting disruptions of other V-ATPase subunits encoded by single that a3 would not be a suitable therapeutic target. To address genes. This decrease in severity of the phenotype is most likely why mutations in a ubiquitously distributed protein result in due to the presence of the second isoform, Stv1p. Overexpres- sion of STV1 in vph1 strains results in mislocalization of osteopetrosis and to examine whether a3 is indeed a suitable target for an antiresorptive therapeutic, we have directly com- Stv1p to the yeast vacuole, restores vacuolar acidification, and eliminates all vph1 phenotypes, demonstrating that Stv1p pared a3 expression levels between bone and other tissues and compared the expression of a3 within the different bone cells. can functionally complement Vph1p. Disruption of STV1 does not result in any detectable phenotype, presumably because Similar to previous results, we found high levels of a3 mRNA expression in liver with decreasing amounts in kidney, brain, the constitutive high expression of Vph1p enables it to func- tionally complement for Stv1p absence. lung, spleen, and muscle, but the highest expression was found in mRNA extracted from bone (Fig. 1). Furthermore, in situ If a similar mechanism were operating in osteoclasts, dele- tions or mutations of one of the four mammalian V-ATPase “a” hybridization demonstrated that within bone, a3 expression was mostly in osteoclasts (Fig. 2). Considering that osteoclasts subunits could be partially compensated by the remaining three isoforms. A phenotype might only arise when very high represent less than 1% of bone cells, and assuming an even distribution of a3 in liver, these results would suggest that a3 expression levels of one particular isoform are required for a specific function. Considering the ubiquitous distribution of a3 expression in osteoclasts is 100-fold higher than in liver cells. This might explain why mutations and deletions within the a3 within mammalian cells, it is likely that this isoform is respon- sible for acidifying other compartments such as the lysosome gene result in an osteopetrotic phenotype. However, consider- ing the high levels of a3 expression in liver and its ubiquitous (47). Deletion of the a3 gene might not necessarily affect the acidification of other organelles if the other isoforms are able to distribution in other tissues, it is still surprising that other phenotypes do not result from a3 mutations. There is a prece- functionally compensate for its absence. Considering that we have shown that the expression levels of a1 and a3 are similar dent for osteoclasts being unique in this respect: Src / mice are also osteopetrotic due to deficient osteoclast function, in mononuclear cells (Fig. 3), it would seem possible that an a3 deficiency is compensated in tissues where other isoforms are whereas Src expression is also ubiquitous (46). A possible ex- planation may be that other isoforms can compensate for the normally expressed to a similar degree as a3. Since a3 expres- 49278 V-ATPase a3 Expression in Large and Small Osteoclasts 19. Kawasaki-Nishi, S., Nishi, T., and Forgac, M. (2001) J. Biol. Chem. 276, sion appears to be much higher than that of a1 in multinucle- 17941–17948 ated osteoclasts (Fig. 3), the levels of a1 (and possibly a2) may 20. Pujol, N., Bonnerot, C., Ewbank, J. J., Kohara, Y., and Thierry-Mieg, D. (2001) J. Biol. Chem. 276, 11913–11921 not be sufficient to compensate for the absence of a3, hence the 21. Oka, T., Toyomura, T., Honjo, K., Wada, Y., and Futai, M. (2001) J. Biol. Chem. osteopetrotic phenotype that results from mutations within a3. 276, 33079 –33085 Osteoclast size has been shown to increase in diseases char- 22. Oka, T., Murata, Y., Namba, M., Yoshimizu, T., Toyomura, T., Yamamoto, A., Sun-Wada, G. H., Hamasaki, N., Wada, Y., and Futai, M. (2001) J. Biol. acterized by increased bone resorption such as end stage renal Chem. 276, 40050 – 40054 disease (2), Paget’s disease (48), periodontal disease (3, 49), and 23. Sun-Wada, G. H., Imai-Senga, Y., Yamamoto, A., Murata, Y., Hirata, T., rheumatoid arthritis (50). We have previously shown that large Wada, Y., and Futai, M. (2002) J. Biol. Chem. 277, 18098 –18105 24. Toyomura, T., Oka, T., Yamaguchi, C., Wada, Y., and Futai, M. (2000) J. Biol. osteoclasts, as a population, are more active resorbers than Chem. 275, 8760 – 8765 small osteoclasts and that the reason for this is that the pro- 25. Nishi, T., and Forgac, M. (2000) J. Biol. Chem. 275, 6824 – 6830 26. Smith, A. N., Finberg, K. E., Wagner, C. A., Lifton, R. P., Devonald, M. A., Su, portion of large osteoclasts that are in a resorptive state is Y., and Karet, F. E. (2001) J. Biol. Chem. 276, 42382– 42388 larger (40%) than that of small osteoclasts (5.6%) (4). Here we 27. Smith, A. N., Skaug, J., Choate, K. A., Nayir, A., Bakkaloglu, A., Ozen, S., show that a3 mRNA (Fig. 4) and protein (Fig. 6) expression are Hulton, S. A., Sanjad, S. A., Al-Sabban, E. A., Lifton, R. P., Scherer, S. W., and Karet, F. E. (2000) Nat. Genet. 26, 71–75 higher in large compared with small osteoclasts, suggesting 28. Stover, E. H., Borthwick, K. J., Bavalia, C., Eady, N., Fritz, D. M., Rungroj, N., that a3 expression may be associated with the differences in Giersch, A. B., Morton, C. C., Axon, P. R., Akil, I., Al-Sabban, E. A., Baguley, D. M., Bianca, S., Bakkaloglu, A., Bircan, Z., Chauveau, D., the resorptive activity between large and small osteoclasts. We Clermont, M. J., Guala, A., Hulton, S. A., Kroes, H., Li Volti, G., Mir, S., also observed that 4% of small osteoclasts had a3 expression Mocan, H., Nayir, A., Ozen, S., Rodriguez Soriano, J., Sanjad, S. A., Tasic, levels similar to large osteoclasts. This correlates well with the V., Taylor, C. M., Topaloglu, R., Smith, A. N., and Karet, F. E. (2002) J. Med. Genet. 39, 796 – 803 observation that 5.6% of small osteoclasts are actively resorb- 29. Li, Y. P., Chen, W., and Stashenko, P. (1996) Biochem. Biophys. Res. Commun. ing (4), further suggesting that a3 expression is associated with 218, 813– 821 the resorptive potential. Considering these results, pharmaco- 30. Li, Y. P., Chen, W., Liang, Y., Li, E., and Stashenko, P. (1999) Nat. Genet. 23, 447– 451 logically targeting a3, one could preferentially inhibit the 31. Scimeca, J. C., Franchi, A., Trojani, C., Parrinello, H., Grosgeorge, J., Robert, larger and more active osteoclasts prevalent in pathological C., Jaillon, O., Poirier, C., Gaudray, P., and Carle, G. F. (2000) Bone 26, 207–213 bone loss. 32. Frattini, A., Orchard, P. J., Sobacchi, C., Giliani, S., Abinun, M., Mattsson, J. P., Keeling, D. J., Andersson, A. K., Wallbrandt, P., Zecca, L., Acknowledgments—We thank Dr. Beth Lee (Washington University Notarangelo, L. D., Vezzoni, P., and Villa, A. (2000) Nat. Genet. 25, 343–346 School of Medicine) for assistance with RAW cells and GST-sRANKL 33. 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Published: Dec 1, 2003