Extracellular Calcium (Ca2+o)-Sensing Receptor in a Mouse Monocyte-Macrophage Cell Line (J774):...

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Extracellular Calcium (Ca 21 o )-Sensing Receptor in a Mouse Monocyte-Macrophage Cell Line (J774): Potential Mediator of the Actions of Ca 21 o on the Function of J774 Cells TORU YAMAGUCHI, OLGA KIFOR, NAIBEDYA CHATTOPADHYAY, MEI BAI, and EDWARD M. BROWN ABSTRACT The calcium-sensing receptor (CaR) is a G protein– coupled receptor that plays key roles in extracellular calcium ion (Ca 21 o ) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites of osteoclastic bone resorption during bone remodeling and may play a role in the “reversal” phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca 21 o that could provide a signal for bone marrow mononuclear cells in the vicinity, leading us to investigate whether such mononuclear cells express the CaR. In this study, we used the mouse J774 cell line, which exhibits a pure monocyte-macrophage phenotype. Both immunocytochemistry and Western blot analysis, using polyclonal antisera specific for the CaR, detected CaR protein in J774 cells. The use of reverse transcriptase-polymerase chain reaction with CaR-specific primers, including a set of intron-spanning primers, followed by nucleotide sequencing of the amplified products, also identified CaR transcripts in J774 cells. Exposure of J774 cells to high Ca 21 o (2.8 mM or more) or the polycationic CaR agonist, neomycin (100 mM), stimulated both chemotaxis and DNA synthesis in J774 cells. Therefore, taken together, our data strongly suggest that the monocyte-macrophage cell line, J774, possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. (J Bone Miner Res 1998;13:1390–1397) INTRODUCTION W E PREVIOUSLY CLONED highly homologous Ca 21 o -sens- ing receptors (CaRs) from bovine and human para- thyroid gland, (1,2) rat kidney, (3) and thyroid C-cells, (4) which activate phospholipase C (PLC) and raise the levels of inositol trisphosphate (IP 3 ) and the cytosolic calcium con- centration. The physiological relevance of the CaR has been documented in humans by showing that inactivating and activating mutations in the human CaR gene cause inherited hyper- and hypocalcemic disorders, respectively, rendering affected family members inappropriately “resis- tant” or “sensitive” to the usual effects of Ca 21 o on para- thyroid and renal functions. (5,6) Bone, like parathyroid gland and kidney, is thought to be involved in systemic mineral ion homeostasis, and thus it is possible that the CaR may also play some role within the skeleton by sensing local changes in Ca 21 o caused by bone remodeling. Since substantial release of calcium ions takes place dur- ing bone resorption, (7) and marrow cells are likely to be exposed to high local calcium concentrations, the CaR could possibly modulate bone and marrow physiology by sensing such changes in Ca 21 o . Of all the cell types present in bone marrow, macrophage-like mononuclear cells are particularly good candidates for sensing local changes in Ca 21 o because they appear at sites of osteoclastic bone resorption at the end of the resorptive phase of bone re- modeling, (8,9) during which local increases in Ca 21 o within Endocrine-Hypertension Division, Department of Medicine, Brigham and Women’s Hospital, and Harvard Medical School, Boston, Massachusetts, U.S.A. JOURNAL OF BONE AND MINERAL RESEARCH Volume 13, Number 9, 1998 Blackwell Science, Inc. © 1998 American Society for Bone and Mineral Research 1390

Transcript of Extracellular Calcium (Ca2+o)-Sensing Receptor in a Mouse Monocyte-Macrophage Cell Line (J774):...

Extracellular Calcium (Ca21o)-Sensing Receptor in a

Mouse Monocyte-Macrophage Cell Line (J774):Potential Mediator of the Actions of Ca21

oon the Function of J774 Cells

TORU YAMAGUCHI, OLGA KIFOR, NAIBEDYA CHATTOPADHYAY, MEI BAI, andEDWARD M. BROWN

ABSTRACT

The calcium-sensing receptor (CaR) is a G protein–coupled receptor that plays key roles in extracellular calciumion (Ca21

o) homeostasis in parathyroid gland and kidney. Macrophage-like mononuclear cells appear at sites ofosteoclastic bone resorption during bone remodeling and may play a role in the “reversal” phase followingosteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases inCa21

o that could provide a signal for bone marrow mononuclear cells in the vicinity, leading us to investigatewhether such mononuclear cells express the CaR. In this study, we used the mouse J774 cell line, which exhibitsa pure monocyte-macrophage phenotype. Both immunocytochemistry and Western blot analysis, using polyclonalantisera specific for the CaR, detected CaR protein in J774 cells. The use of reverse transcriptase-polymerase chainreaction with CaR-specific primers, including a set of intron-spanning primers, followed by nucleotide sequencingof the amplified products, also identified CaR transcripts in J774 cells. Exposure of J774 cells to high Ca21

o

(2.8 mM or more) or the polycationic CaR agonist, neomycin (100 mM), stimulated both chemotaxis and DNAsynthesis in J774 cells. Therefore, taken together, our data strongly suggest that the monocyte-macrophage cellline, J774, possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney.(J Bone Miner Res 1998;13:1390–1397)

INTRODUCTION

WE PREVIOUSLY CLONED highly homologous Ca21o-sens-

ing receptors (CaRs) from bovine and human para-thyroid gland,(1,2) rat kidney,(3) and thyroid C-cells,(4) whichactivate phospholipase C (PLC) and raise the levels ofinositol trisphosphate (IP3) and the cytosolic calcium con-centration. The physiological relevance of the CaR hasbeen documented in humans by showing that inactivatingand activating mutations in the human CaR gene causeinherited hyper- and hypocalcemic disorders, respectively,rendering affected family members inappropriately “resis-tant” or “sensitive” to the usual effects of Ca21

o on para-thyroid and renal functions.(5,6) Bone, like parathyroid

gland and kidney, is thought to be involved in systemicmineral ion homeostasis, and thus it is possible that theCaR may also play some role within the skeleton by sensinglocal changes in Ca21

o caused by bone remodeling.Since substantial release of calcium ions takes place dur-

ing bone resorption,(7) and marrow cells are likely to beexposed to high local calcium concentrations, the CaRcould possibly modulate bone and marrow physiology bysensing such changes in Ca21

o. Of all the cell types presentin bone marrow, macrophage-like mononuclear cells areparticularly good candidates for sensing local changes inCa21

o because they appear at sites of osteoclastic boneresorption at the end of the resorptive phase of bone re-modeling,(8,9) during which local increases in Ca21

o within

Endocrine-Hypertension Division, Department of Medicine, Brigham and Women’s Hospital, and Harvard Medical School, Boston,Massachusetts, U.S.A.

JOURNAL OF BONE AND MINERAL RESEARCHVolume 13, Number 9, 1998Blackwell Science, Inc.© 1998 American Society for Bone and Mineral Research

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the immediate vicinity of osteoclasts are known to reachlevels as high as 40 mM.(7) This high level of Ca21

o couldprovide these cells with a signal that modulates their sub-sequent physiological responses. In fact, some studies haveshown that high Ca21

o induces chemotaxis of mononuclearcells(10) and that conditioned medium from monocytes cul-tured at high Ca21

o stimulates DNA synthesis and alkalinephosphatase (ALP) activity in osteoblastic MC3T3-E1cells(10,11) and inhibits the formation of osteoclast-like cellsfrom their precursors.(12) These findings suggest that mono-nuclear cells might play a key role in the reversal phase ofbone remodeling by controlling osteoblast and osteoclastfunctions through their capacity to sense Ca21

o. However,the precise molecular mechanism(s) by which these cellsdetect changes in Ca21

o remains unknown.Macrophage-like mononuclear cells are also known to

have the capacity to differentiate into mature functionalosteoclasts under specific culture conditions.(13) Severalstudies showed that high Ca21

o induces rapid increases inthe cytosolic Ca21 concentration in osteoclasts, therebycausing a marked cell retraction followed by a profoundinhibition of bone resorption.(14–17) Thus, Ca21

o-sensingmechanisms seem to be intimately related to the entireprocess of the differentiation from mononuclear cells tomature osteoclasts.

In a previous study using immunohistochemistry withCaR-specific antisera, we showed the expression of thisreceptor in bone marrow mononuclear cells as well as inALP-positive, putative osteoblast precursors, erythroid pre-cursors, and megakaryocytes.(18) These findings suggestedthat the CaR might be involved in the Ca21

o-sensing mech-anism of these bone marrow–derived cells. In this study, tofocus on the role of the CaR in bone marrow mononuclearcells, we used the mouse J774 cell line, which expressesseveral characteristics of the monocyte-macrophage lin-eage, including the capacities to perform phagocytosis andundergo antigen-stimulated proliferation.(19,20)

MATERIALS AND METHODS

Materials

All routine culture media were obtained from GIBCOBRL (Grand Island, NY, U.S.A.). Neomycin sulfate andanhydrous calcium chloride (CaCl2) were purchased fromSigma Chemical Co. (St. Louis, MO, U.S.A.), and [3H]methylthymidine was from DuPont-NEN (Boston, MA,U.S.A).

Cell culture

J774 cells were obtained from the American Type Cul-ture Collection (ATCC, Rockville, MD, U.S.A.). J774 cellswere grown in Dulbecco’s modified essential medium(DMEM; Ca21 1.8 mM, Mg21 0.4 mM) supplemented with10% fetal bovine serum (Hyclone, Logan, UT, U.S.A.) and1% penicillin/streptomycin in 5% CO2 at 37°C. The me-dium was changed twice weekly, and the cells were subcul-tured into 25-cm2 culture flasks by detaching them gentlywith a cell scraper after reaching subconfluency. For mor-

phological evaluation, J774 cells were plated onto 12-mmcircular glass coverslips in 24-well (2.0-cm2) plates. After24 h of culture, the medium was discarded, and each cov-erslip with adherent cells was washed once with phosphate-buffered saline (PBS), fixed with 4% formaldehyde in PBSfor 5 minutes, and washed with PBS once again. Each cov-erslip was stored at 4°C until assessment for the presence ofthe CaR as described below.

Immunocytochemistry for CaR in J774 cells

A CaR-specific polyclonal antiserum (LSN), generouslyprovided by Drs. A. Spiegel and P. Goldsmith (NIDDK,National Institutes of Health, Bethesda, MD, U.S.A.), wasemployed for performing immunocytochemistry in thisstudy. The antiserum was raised against a peptide corre-sponding to amino acids 174–193 of the human CaR, whichresides within the predicted amino-terminal extracellulardomain of the CaR. J774 cells, fixed as described above,were treated with DAKO Protein Block Serum-Free Solu-tion (DAKO Corp., Carpenteria, CA, U.S.A.) for 1 h andthen incubated overnight at 4°C with primary antiserum(anti-CaR antiserum LSN) at a concentration of 5 mg/ml inblocking solution (DAKO Corp.). Negative controls werecarried out by incubation with the anti-CaR antiserum pre-absorbed with 10 mg/ml of the synthetic CaR peptideagainst which it was raised. After washing the cells threetimes with 0.5% bovine serum albumin in PBS for 10 min-utes, ALP-coupled, goat anti-rabbit immunoglobulin G (1:200; GIBCO BRL) was added and incubated for 1 h atroom temperature. The cells were then washed with PBSthree times for 10 minutes each, and the color reaction wasdeveloped for 10–20 minutes using a solution consisting of44 ml of Nitroblue Tetrazolium Chloride (NBT, 75 mg/ml)and 33 ml of 5-Bromo-4-chloro-3-indolylphosphate p-Tolu-idine Salt (BCIP, 50 mg/ml) in 10 ml of 0.1 M Tris-HCl(pH 9.5), 0.1 M NaCl, 50 mM MgCl2, and 1 mg/ml levami-sole, which was included for suppression of endogenouscellular ALP activity. The color reaction was stopped bywashing twice in the above solution without NBT or BCIPand then twice in water.

Western blot analysis of CaR in J774 cells

A CaR-specific polyclonal antiserum (4637) was gener-ously provided by Drs. Forrest Fuller and Karen Krapcho ofNPS Pharmaceuticals, Inc. (Salt Lake City, UT, U.S.A.).This antiserum was raised against a peptide (FF-7) corre-sponding to amino acids 345–359 of the bovine CaR, whichresides within the predicted amino-terminal extracellulardomain of the CaR. The antiserum was subjected to furtherpurification using an affinity column conjugated with theFF-7 peptide, and the affinity-purified antiserum was usedfor Western blot analysis.

Monolayers of J774 cells in 75 cm2 culture flasks wererinsed with 1 mM EDTA in PBS and lysed with 1.0 ml of alysis solution (1% sodium dodecyl sulfate [SDS], 10 mMTris-HCl, pH 7.4) heated to 65°C. The cells were scrapedfrom the flasks, transferred to a microcentrifuge tube, andheated for an additional 5 minutes at 65°C. The viscosity of

CaR AND MOUSE CELL LINE, J774 1391

the sample was reduced by brief sonication, and insolublematerial was removed by centrifugation for 5 minutes. Theresultant whole cell lysate in the supernatant was stored at–20°C until Western blot analysis was carried out.

Aliquots of 150 mg of protein were dissolved in SDS-Laemmli gel loading buffer containing 100 mM dithiothre-itol, incubated at 37°C for 15 minutes, and resolved elec-trophoretically on 6.5% SDS-polyacrylamide gels. Proteinswere electrophoretically transferred to nitrocellulose at240 mA for 40 minutes in transfer buffer containing 19 mMTris-HCl, 150 mM glycine, 0.015% SDS, and 20% metha-nol. The blots were then blocked with 1% bovine serumalbumin in PBS containing 0.25% Triton X-100 (blockingsolution) for 2 h and incubated with the affinity-purifiedantiserum (4637) or with peptide-blocked antiserum (thesame amount of antiserum preincubated at room tempera-ture for 60 minutes with twice the amount [w/w] of FF-7peptide) at a concentration of 1 mg/ml in the blocking so-lution overnight at 4°C. The blots were washed three timeswith PBS containing 0.25% Triton X-100 (washing solution)at room temperature for 10 minutes each. The blots werefurther incubated with a 1:2000 dilution of horseradishperoxidase–coupled, goat anti-rabbit immunoglobulin G(Sigma Chemical Co.) in the blocking solution for 1 h atroom temperature. The blots were then washed three timeswith the washing solution at room temperature for 40 min-utes each, and specific protein bands were detected using anenhanced chemiluminescence system (Amersham, Arling-ton Heights, IL, U.S.A.).

Polymerase chain reaction amplification of CaR inJ774 cells

Total RNA was prepared from J774 cells with the TRIzolReagent (GIBCO BRL). One microgram of total RNA wasused for the synthesis of single-stranded cDNA (cDNAsynthesis kit, GIBCO BRL). The resultant first-strandedcDNA was used for the polymerase chain reaction (PCR)procedure. PCR was performed at a final concentration of20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.8 mM MgCl2,0.2 mM dNTP, 0.4 mM of forward primer, 0.4 mM of re-verse primer, and 1 ml of ELONGASE enzyme mix(GIBCO BRL). Two different sets of primer pairs wereused: 59-ATGGTTTGGCTACTGTTTGG-39, sense; 59-CAGAGCCTTGGAGACGGTGT-39, antisense, designedfrom the partially cloned extracellular domain of the mouseAtT-20 cell CaR(21); 59-AGAAGTTCCGAGAG-GAAGCC-39, sense; 59-ACCTGTTGCCACCTTCTTCG-39, antisense, designed from the extracellular domain of therat kidney CaR (RaKCaR).(3) The first primer pair wasdesigned to span one intron of the CaR gene, to distinguishproducts amplified from cDNA and genomic DNA. In or-der to perform “hot start” PCR, the enzyme was addedduring the initial 3-minute denaturation and was followedby 35 cycles of amplification (30 s denaturation at 94°C, 30 sannealing at 47°C, and 1 minute extension at 72°C). Thereaction was completed with an additional 10 minute incu-bation at 72°C to allow completion of extension. PCR prod-ucts were fractionated on 1.2% agarose gels. The presence

of 319 and 480 nucleotide-long amplified products, respec-tively, were indicative of positive PCR reactions.

Cloning and sequencing of CaR reverse transcriptasePCR products

Reverse transcriptase (RT)-PCR products were ligatedinto the pCR 2.1 vector of the TA cloning kit (Invitrogen,Carlsbad, CA, U.S.A.) by incubation overnight at 14°C.Competent cells were transformed according to the manu-facturer’s instructions and placed on ampicillin-containingagar in the presence of X-gal. Transformed cells were iden-tified after overnight growth at 37°C as white colonies. Thewhite colonies were used to inoculate into LB media, andplasmid DNA was extracted, digested with EcoRI and frac-tionated by agarose gel electrophoresis. DNA from positiveclones was further purified using the Qiagen Plasmid kit(Qiagen Inc., Chatsworth, CA, U.S.A.) and sequenced bi-directionally using M13 forward and M13 reverse primerswith an automated sequencer (AB377, Applied Biosystems,Foster City, CA, U.S.A.) in the Automatic Sequencing andGenotyping Faculty, Brigham and Women’s Hospital (Bos-ton, MA, U.S.A.), using dideoxy terminator Taq technol-ogy.

Chemotaxis assay of J774 cells

Chemotaxis was evaluated using a Neuroprobe BW200Sblindwell chamber (Neuroprobe, Gaithersburg, MD,U.S.A.) as previously described.(22) Various concentrationsof CaCl2 or 100 mM neomycin sulfate in serum-free DMEMwere loaded in the lower chamber, which is separated fromthe upper well by a 5 mm membrane with 5 mm pores. J774cells (1 3 105 cells/ml) were washed twice, suspended inserum-free DMEM, and added to the upper chamber. Aftera 4 h incubation at 37°C, cells on the upper surface of themembrane that had not migrated were scraped from themembrane, and cells that had migrated to the opposite sideof the membrane were fixed with methanol and stained withGiemsa. The cells that had migrated to the lower surface ofthe filter were counted in six high-power fields (3400) usinga light microscope.

DNA synthesis in J774 cells

We assessed DNA synthesis in J774 cells using [3H]thy-midine incorporation. J774 cells were seeded in 24-well(2.0 cm2) plates at a density of 1000 cells/well in 500 ml ofDMEM containing 10% fetal bovine serum as well as var-ious concentrations of CaCl2 or 100 mM neomycin sulfate.After a 72 h incubation at 37°C, cells were pulsed with[3H]thymidine (1 mCi/well). The incubation was terminatedafter overnight incubation by removal of the medium andaddition of 5% trichloroacetic acid. Cell layers werescraped and transferred to microcentrifuge tubes. Aftercentrifugation at 15,000g and removal of the supernatant,the precipitate was washed with 75% ethanol and desic-cated at room temperature. The residual pellet was dis-solved in 20 mM NaOH and 1% SDS, and a scintillation

1392 YAMAGUCHI ET AL.

cocktail was added. Samples were counted in a liquid scin-tillation counter.

Statistics

Results are expressed as the mean 6 SEM. Statisticalevaluation for differences between groups was done usingone-way analysis of variance followed by Fisher’s projectedleast significant difference test. For all statistical tests, val-ues of p , 0.05 were considered significant.

RESULTS

Immunoreactivity of CaR protein in J774 cells usingCaR-specific antiserum

To clarify the expression of the CaR by cells of themonocyte-macrophage lineage, we investigated the pres-ence of the CaR in the mouse J774 cell line, because itpossesses a pure monocyte-macrophage phenotype. Immu-nocytochemistry of J774 cells with a CaR-specific polyclonalantiserum (LSN) revealed strong CaR staining (Fig. 1A).

There was some heterogeneity in staining intensity betweenthe cells, with monocyte-like round-shaped cells stainingstronger than cells with flatter and/or more elongatedmorphologies. Multinucleated J774 cells were also stronglystained (data not shown). The staining was eliminated bypreincubating the primary antiserum with the peptideagainst which it was raised (Fig. 1B). We stained the cellswith another CaR-specific antiserum (4637) and obtainedsimilar results (data not shown).

We also performed Western analysis on proteins isolatedfrom J774 cells (Fig. 2). A band at ;150–160 kDa, whichwas stained in the presence of specific antiserum (4637) butmarkedly diminished following preabsorption of the anti-serum with its specific peptide, was of a size consistent withthat of the intact glycosylated CaR (lane 1). Bands at lowermolecular weights (;110, 105, 90, and 75 kDa) were alsodetected in the J774 cell extract. These bands may representdegradation products of the CaR protein generated by theintrinsic proteases of J774 cells. The specificity of the anti-CaR antiserum used for Western blot analysis in this studyis apparent from the marked reduction in the intensities ofthe bands in the lane incubated with peptide-preabsorbedanti-CaR antiserum (Fig. 2, lane 2).

Analysis of CaR mRNA in J774 cells by RT-PCR

RT-PCR with two sets of CaR-specific primers (Fig. 3),one of which was intron-spanning, amplified two productsof the expected sizes, 319 bp (lane 1) and 480 bp (lane 3),for CaR-derived products. No products were observedwhen the RT was omitted during synthesis of cDNA (lane2 and 4, respectively). DNA sequence analysis of the twoPCR products revealed 100% and 95% sequence identitieswith the mouse AtT-20 cell CaR sequence (data not shown)and the RaKCaR sequence (Fig. 4), respectively. The480 bp PCR product showed one amino acid difference

FIG. 1. Immunocytochemistry of J774 cells carried out asdescribed in the Materials and Methods using a CaR-spe-cific antibody (LSN). Immunocytochemistry of J774 cellsrevealed strong CaR staining (A), which was eliminated bypreincubating the primary antibody with the peptide againstwhich it was raised (B). The photomicrographs were takenat a magnification of 3630.

FIG. 2. Western analysis of membrane preparations fromJ774 cells performed as described in the Materials andMethods. A band at ;150–160 kDa that was stained in thepresence of specific antibody (4637) was consistent with theintact glycosylated CaR (lane 1). Bands at lower molecularweights may be degradation products of the CaR proteingenerated by intrinsic proteases of J774 cells, because thespecificity of the labeling by the antibody used in this studyto detect the CaR was confirmed by the marked reductionin the intensities of these bands in the lane incubated withpeptide-preabsorbed CaR-antibody (lane 2).

CaR AND MOUSE CELL LINE, J774 1393

from the rat sequence at position 132. These results showedthat the PCR products corresponded to CaR sequences,indicating the presence of bona fide CaR transcripts inthese cells.

Chemotactic activity of J774 cells toward high Ca21o

or neomycin sulfate

A chemotaxis assay was performed to determine thecapacity of J774 cells to migrate toward CaR agonists. Asshown in Fig. 5, elevated levels of Ca21

o induced a concen-tration-dependent chemotactic response of J774 cells overcontrol values at 1.8 mM Ca21

o (p , 0.05), which appearedto reach a plateau at 4.8 mM Ca21

o. Neomycin sulfate(100 mM) also induced a significant chemotactic responseover the control (p , 0.05). Using the Zigmond-Hirschcheckerboard analysis,(23) the Ca21

o-induced cell move-ment was found to be directed chemotaxis but not to berandom chemokinesis (data not shown).

DNA synthesis in J774 cells stimulated by high Ca21o

or neomycin sulfate

We found that treatment of J774 cells with increasinglevels of Ca21

o resulted in a dose-dependent stimulation ofDNA synthesis over control values at 1.8 mM Ca21

o

(p , 0.05) as assessed by [3H]thymidine incorporation,which appeared to reach a plateau at 4.8 mM Ca21

o

(Fig. 6). This pattern was similar to that of the chemotacticresponse presented in Fig. 5. Neomycin sulfate (100 mM)also induced a significant stimulation of DNA synthesisover the control (p , 0.05).

DISCUSSION

In a previous study, we examined low-density mononu-clear cells in primary cultures of mouse bone marrow andshowed that these cells exhibited CaR-immunoreactivity byimmunocytochemistry, especially following incubation withhigh Ca21

o (4.8 mM). They also demonstrated a predomi-

nance of the monocyte-macrophage phenotype as indicatedby the colocalization of nonspecific esterase (NSE) activitywith CaR-immunoreactivity.(18) Since these monocyte-mac-rophage–like cells were taken directly from bone marrowand may be similar to those appearing at sites of osteoclas-tic bone resorption during the reversal phase of bone re-modeling in vivo, this finding suggested that the CaR mightbe expressed by these cells in vivo. However, we observedthat these preparations were slightly contaminated withother kinds of cells, including ALP-positive, putative osteo-blast-like cells (,10%). Thus, such marrow-derived prepa-rations might be not ideal for investigating the existence ofthe CaR in monocytes-macrophages using RT-PCR, whichrequires a pure preparation of cells for definitive identifi-cation of the cell type expressing the relevant transcript. Tocircumvent this problem, we used mouse J774 cells possess-ing a pure monocyte-macrophage phenotype(19,20) in thisstudy. J774 cells showed clear expression of CaR protein byimmunocytochemistry as well as by Western blot analysis,which revealed a specific band at a molecular weight con-sistent with that of the intact, glycosylated CaR (;150–160 kDa). In addition, RT-PCR performed on total RNAfrom J774 cells followed by sequence analysis of the PCRproducts indicated the presence of bona fide CaR tran-scripts in these cells. Thus, the present study shows that acell line of the monocyte-macrophage lineage expressesboth CaR protein and mRNA.

Although J774 cells are not the same as the mononuclearcells present at sites of bone remodeling, recent evidencesuggests that monocytes-macrophages, regardless of theirsite of origin, can exhibit properties reflecting the functionsof mononuclear cells localized at the resorptive site in bonemarrow. Pacifici et al. demonstrated that interleukin-1(IL-1) activity released from human peripheral bloodmonocytes reflects the bone formation rate in osteoporoticpatients(24) and that bone matrix constituents stimulateIL-1 release from human blood mononuclear cells.(25) J774cells are also known to be functionally similar to mononu-clear cells in vivo, with the secretion of IL-1 and othercytokines upon stimulation.(26,27)

In this study, exposure of J774 cells to the polycationicCaR agonists, high Ca21

o or neomycin, stimulated bothchemotaxis and DNA synthesis in the cells. In the micro-environment where bone remodeling takes place, macro-phage-like mononuclear cells are known to migrate to re-sorption pits at the end of the osteoclastic resorption phase.During the phase of active osteoclastic resorption, the re-sorptive site on the bone surface is sealed by the osteoclastplasma membrane, and the local Ca21

o in this compartmentcan rise to as high as 40 mM.(7) Thus, it is possible that thishigh level of Ca21

o could raise Ca21o in the surrounding

bone/bone marrow microenvironment after the release ofcalcium from beneath an actively resorbing osteoclast fol-lowing its migration to another site or degeneration andcould provide a signal for mononuclear cells to migrate intothe resorption pit. Our results suggest that mononuclearcells could detect such changes in Ca21

o, potentially via theCaR, thereby causing their migration and proliferation inresponse to this signal. In accordance with our findings,stimulation of chemotaxis by high Ca21

o has also been

FIG. 3. Identification of CaR transcripts in J774 cellsusing RT-PCR with two sets of CaR-specific primers, per-formed as described in the Materials and Methods. Twoproducts were amplified from reverse transcribed RNAisolated from J774 cells, which were of the expected sizes,319 bp (lane 1) and 480 bp (lane 3), for CaR-derived prod-ucts. The PCR reactions without RT showed no products(lane 2 and 4, respectively).

1394 YAMAGUCHI ET AL.

detected in human peripheral monocytes,(10) although inone study expression of the CaR could not be detected byRT-PCR in human peripheral blood monocytes.(28) Therole of the CaR in the control of cell proliferation has notbeen clear until recently, when the receptor has been con-clusively shown to be involved in the stimulation of cellproliferation by CaR agonists. Mailland et al.(29) reportedthat CaR agonists stimulate the proliferation of CCL39hamster fibroblasts transfected with the CaR, and Hoffet al.(30) reported that transfection of NIH-3T3 cells with ahuman CaR cDNA harboring an activating mutation in-duced neoplastic transformation and cell proliferation.These findings therefore are consistent with the hypothesisthat the CaR mediates the stimulation of the proliferationof J774 cells by high Ca21

o and neomycin, although addi-tional studies will be needed to document further causalrelationships between expression of the CaR and the con-trol of chemotaxis and cell proliferation by high Ca21

o andother CaR agonists in this cell type.

Monocytes-macrophages are known to secrete variouscytokines, including IL-1, IL-6, and tumor necrosis factor,upon stimulation, such as following the development ofestrogen deficiency in the immediate postmenopausal peri-od.(31) Recently, Ca21

o was shown to induce the secretion

FIG. 5. Chemotactic activity of J774 cells toward highCa21

o or neomycin sulfate. The number of J774 cells thatmigrated to the side of the membrane to which CaCl2 orneomycin sulfate had been added 4 h previously wascounted as described in the Materials and Methods. Eachbar expresses the mean 6 SEM for six determinations.*p , 0.05 compared with cells exposed to 1.8 mM Ca21

o.

FIG. 4. Nucleotide and deducedamino acid sequences of the 480 bp CaRRT-PCR fragment. DNA sequence anal-ysis of the 480 bp PCR product amplifiedfrom reverse-transcribed RNA isolatedfrom mouse J774 cells revealed 95% se-quence identity with the nucleotide se-quence of the RaKCaR cDNA. ThePCR product showed one amino aciddifference at position 132. The nucleo-tide sequence of the 319 bp PCR prod-uct was 100% identical with the corre-sponding region of the mouse AtT-20cell CaR sequence reported previous-ly(21) (data not shown).

CaR AND MOUSE CELL LINE, J774 1395

of these cytokines from human peripheral blood mononu-clear cells,(28,32) suggesting that a putative Ca21

o-sensingmechanism is involved in this process. Since these cytokinescan modulate the function of osteoclasts and osteoblastsand are thought to be intimately involved in the pathogen-esis of postmenopausal osteoporosis,(31) it is possible thatthe CaR could affect bone turnover by inducing secretion ofthese cytokines from mononuclear cells after sensingchanges in Ca21

o. In fact, some studies have shown thatconditioned medium from human peripheral blood mono-cytes pretreated with Ca21

o had an inhibitory action onosteoclast formation(12) and stimulated DNA synthesis andALP activity in osteoblastic MC3T3-E1 cells.(10,11)

Monocytes-macrophages are also known to have the ca-pacity to fuse with one another and to differentiate intomature functional osteoclasts under specific culture condi-tions.(13) Hence, our results suggest that mature osteoclastscould potentially incorporate the CaR from their precursorsand express it in the cell surface. In fact, Zaidi et al. showedthat various divalent cations, including elevated levels ofCa21

o, inhibit bone resorption by isolated rat oste-oclasts,(33) although the pharmacological profile for theinhibitory actions of these cations differed somewhatfrom that reported for the CaR. Further study will beneeded to elucidate whether the CaR is actually involvedin these processes.

Although the CaR was first cloned from parathyroid andkidney,(1–3) more recent data have suggested the presenceof this receptor in additional tissues, such as brain, intes-tine, and skin.(34) In this study, we also show that mono-cytes-macrophages, one of the hematopoietic cell lineages,express both CaR protein and mRNA. Our results suggestthat this receptor may be involved in physiological re-sponses of these cells, such as chemotaxis and proliferationafter Ca21

o stimulation. These events are observed duringthe reversal phase of bone remodeling in vivo, suggestingthat the CaR could potentially play a key role in bone cell

functions within the bone/bone marrow microenviron-ment.

ACKNOWLEDGMENTS

The authors gratefully acknowledge generous grant sup-port from the following sources: The Mochida MemorialFoundation Grant for Medical and Pharmaceutical Re-search (to T.Y.), The Yamanouchi Foundation Grant forResearch on Metabolic Disorders (to T.Y.), NPS Pharma-ceuticals, Inc. (to E.M.B.), The St. Giles Foundation (toE.M.B.), USPHS (DK41415, DK48330, DK52005) (toE.M.B.), and the National Space Bioscience Research In-stitute (NSBRI) (to E.M.B.).

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Address reprint requests toToru Yamaguchi, M.D.

Endocrine-Hypertension DivisionDepartment of Medicine

Brigham and Women’s Hospital221 Longwood Avenue

Boston, MA 02115 U.S.A.

Received in original form November 26, 1997; in revised formMarch 17, 1998; accepted May 11, 1998.

CaR AND MOUSE CELL LINE, J774 1397