Mouse Osteoblastic Cell Line (MC3T3-E1) Expresses Extracellular Calcium (Ca2+o)–Sensing Receptor and Its Agonists Stimulate Chemotaxis and Proliferation of MC3T3-E1 Cells

Authors

  • Toru Yamaguchi,

    Corresponding author
    1. Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachussetts, U.S.A.
    • Address reprint requests to: Toru Yamaguchi, M.D., Endocrine-Hypertension Division, Brigham and Women's Hospital, 221 Longwood Avenue, Boston, MA 02115 U.S.A.
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  • Naibedya Chattopadhyay,

    1. Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachussetts, U.S.A.
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  • Olga Kifor,

    1. Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachussetts, U.S.A.
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  • Robert R. Butters JR.,

    1. Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachussetts, U.S.A.
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  • Toshitsugu Sugimoto,

    1. Third Division, Department of Medicine, Kobe Universeity School of Medicine, Kobe, Japan
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  • Edward M. Brown

    1. Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachussetts, U.S.A.
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Abstract

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the “reversal” phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.

INTRODUCTION

BONE FORMATION DURING remodeling of the skeleton is initiated by the migration of preosteoblasts to sites of osteoclastic bone resorption during the “reversal” phase that precedes the laying down of new bone. These cells subsequently differentiate into mature osteoblasts and eventually deposit and mineralize osteoid protein.1 Bone resorption induces local increases in the extracellular calcium concentration (Ca2+o) within the immediate vicinity of osteoclasts that are known to reach levels as high as 40 mM.2 The latter could therefore provide preosteoblasts with a signal that modulates their subsequent physiological responses, such as migration and proliferation. In fact, some studies have shown that high Ca2+o induces chemotaxis3,4 and DNA synthesis5,6 of the mouse osteoblastic clonal cell line MC3T3-E1,7 which differentiates from preosteoblasts to mature osteoblasts as a function of time in culture.8–10

Although the mechanism by which MC3T3-E1 cells respond to changes in Ca2+o is unclear, one possibility is that this occurs via the Ca2+o-sensing receptor (CaR), which has recently been cloned from bovine and human parathyroid gland,11,12 rat kidney,13 and thyroid C cells.14 The physiological relevance of the CaR has been documented in humans by showing that inactivating and activating mutations of the CaR gene cause inherited hyper- and hypocalcemic disorders,15,16 respectively, rendering affected family members inappropriately “resistant” or “sensitive,” respectively, to the usual effects of Ca2+o on parathyroid and renal functions. The CaR binds its cationic ligands, such as Ca2+, gadolinium (Gd3+), and neomycin, with EC50 values of 3 mM, 20 μM, and 60 μM, respectively. These agonists produce a G protein-dependent activation of phospholipase C (PLC), leading to elevations in the levels of inositol trisphosphate (IP3) and the cytosolic calcium concentration.11 Indeed, Quarles et al. showed that these three agonists stimulate DNA synthesis in MC3T3-E1 cells with EC50 values close to those described above,6 suggesting that these cells have a Ca2+o-sensing mechanism functionally similar to the CaR. However, they failed to detect expression of the CaR by Northern analysis or reverse transcription-polymerase chain reaction (RT-PCR) in MC3T3-E1 cells,17 and thus the precise molecular mechanism(s) by which these osteoblastic cells detect changes in Ca2+o still remains unknown.

In a previous study, using immunohistochemistry with CaR-specific antisera, we showed expression of this receptor in diverse cell types in human bone marrow, including alkaline phosphatase (ALP)-positive, putative osteoblast precursors, nonspecific esterase-positive mononuclear cells, erythroid precursors, and megakaryocytes.18 These findings suggested that the CaR might be involved in the Ca2+o-sensing mechanism of these bone marrow-derived cells. In this study, we used MC3T3-E1 cells as a model of osteoblasts in bone marrow and further examined the presence and role of the CaR in these cells. We demonstrate clear expression of the CaR in MC3T3-E1 cells as assessed by immunocytochemical staining and Western blot analysis using an anti-CaR antiserum as well as Northern analysis with a CaR-specific probe and RT-PCR with CaR-specific primers. We also confirm that CaR agonists stimulate both chemotaxis and DNA synthesis in the CaR-expressing MC3T3-E1 cells examined in this study. These results show that both CaR protein and mRNA are expressed in the MC3T3-E1 cell line; in addition, they suggest that the receptor could potentially play a pivotal role in regulating the function of osteoblasts present within the marrow by sensing local changes in Ca2+o related to bone remodeling.

MATERIALS AND METHODS

Materials

All routine culture media were obtained from GIBCO BRL (Grand Island, NY, U.S.A.). Neomycin sulfate and anhydrous calcium chloride (CaCl2) were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.), and Gd3+ (III) chloride hexahydrate was from Aldrich Chemical Co. (Milwaukee, WI, U.S.A.). [3H]methylthymidine was purchased from DuPont New England Nuclear (Boston, MA, U.S.A.).

Cell culture

MC3T3-E1 cells, established as an osteoblastic cell line from normal mouse calvaria,7 were the generous gift from Dr. H. Kodama (Ohu University, Koriyama, Japan). A different batch of MC3T3-E1 cells was provided by NPS Pharmaceuticals, Inc. (Salt Lake City, UT, U.S.A.). MC3T3-E1 cells were grown in alpha-modified minimal essential medium (α-MEM; Ca2+, 1.8 mM; Mg2+, 0.81 mM; H2PO4, 1.0 mM) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, U.S.A.) and 1% penicillin/streptomycin in 5% CO2 at 37°C. The medium was changed twice weekly, and the cells were subcultured into 25 cm2 culture flasks by detaching them gently with a cell scraper after reaching subconfluency. For morphological evaluation, MC3T3-E1 cells were plated onto 12-mm circular glass coverslips in 24-well (2.0 cm2) plates. After 24 h of culture, the medium was discarded, and each coverslip with adherent cells was washed once with phosphate-buffered saline (PBS), fixed with 4% formaldehyde in PBS for 5 minutes, and washed with PBS once again. Each coverslip was stored at 4°C until assessment for the presence of the CaR as described below.

Immunocytochemistry for CaR in MC3T3-E1 cells

A CaR-specific polyclonal antiserum (4637) was generously provided by Drs. Forrest Fuller and Karen Krapcho of NPS Pharmaceuticals, Inc. This antiserum was raised against a peptide (FF-7) corresponding to amino acids 345–359 of the bovine CaR, which resides within the predicted amino-terminal extracellular domain of the CaR. The antiserum was subjected to further purification using an affinity column conjugated with the FF-7 peptide, and the affinity-purified antiserum was used for immunocytochemistry and Western blot analysis as described below. As described previously,18 while there is some similarity in the region of the metabotropic glutamate receptors corresponding to the FF-7 peptide (40–53%), the 4637 antiserum and several other anti-CaR antisera that we have employed in other studies (M. Bai et al., manuscript in preparation) failed to react on Western analysis with cellular protein isolated from HEK293 cells transfected with the metabotropic glutamate receptor (mGluR1) cDNA. In contrast, these proteins were readily detected on Western blot using an anti-mGluR antiserum. Fixed MC3T3-E1 cells were treated with peroxidase blocking reagent (DAKO Corp., Carpenteria, CA, U.S.A.) for 10 minutes to inhibit endogenous peroxidases, followed by treatment with protein block serum-free solution (DAKO Corp.) for 1 h and then incubated overnight at 4°C with the affinity-purified, anti-CaR antiserum at a concentration of 5 μg/ml in blocking solution. Negative controls were carried out by incubating the cells with affinity-purified antiserum 4637 that had been preabsorbed with 10 μg/ml of the FF-7 peptide. After washing the cells three times with 0.5% bovine serum albumin (BSA) in PBS for 10 minutes each, peroxidase-coupled, goat anti-rabbit immunoglobulin G (IgG, 1:200; Sigma Chemical Co.) was added and incubated for 1 h at room temperature. The cells were then washed with PBS three times for 10 minutes each, and the color reaction was developed using the DAKO AEC substrate system (DAKO Corp.) for about 10 minutes. The color reaction was stopped by washing three times in water.

Western analysis of CaR in MC3T3-E1 cells

Monolayers of MC3T3-E1 cells in 75 cm2 culture flasks that had been cultured for 5, 13, or 20 days were rinsed twice with 1 mM EDTA in PBS and lysed with 1.0 ml of a lysis solution (1% sodium dodecyl sulfate [SDS], 10 mM Tris-HCl, pH 7.4) heated to 65°C. The cells were scraped from the flasks, transferred to microcentrifuge tubes, and heated for an additional 5 minutes at 65°C. The viscosity of the sample was reduced by brief sonication, and insoluble material was removed by centrifugation for 5 minutes. The resultant whole cell lysate in the supernatant was stored at –20°C until Western blot analysis was carried out.

Aliquots of 150 μg of protein were dissolved in SDS-Laemmli gel loading buffer containing 100 mM dithiothreitol, incubated at 37°C for 15 minutes, and resolved electrophoretically on 6.5% SDS-polyacrylamide gels. Proteins were electrophoretically transferred to nitrocellulose at 240 mA for 40 minutes in transfer buffer containing 19 mM Tris-HCl, 150 mM glycine, 0.015% SDS, and 20% methanol. The blots were blocked for 2 h with 1% BSA in PBS containing 0.25% Triton X-100 (blocking solution) and then incubated overnight at 4°C with the affinity-purified antiserum (4637) or with peptide-blocked antiserum (the same amount of antiserum preincubated at room temperature for 60 minutes with twice the amount of FF-7 peptide) at a concentration of 1 μg/ml in the blocking solution. The blots were washed three times with PBS containing 0.25% Triton X-100 (washing solution) at room temperature for 10 minutes each. The blots were further incubated with a 1:2000 dilution of horseradish peroxidase-coupled, goat anti-rabbit IgG (Sigma Chemical Co.) in the blocking solution for 1 h at room temperature. The blots were then washed three times with the washing solution at room temperature for 40 minutes each, and specific protein bands were detected using an enhanced chemiluminescence system (Amersham, Arlington Heights, IL, U.S.A.).

Western analysis of the mGluR in MC3T3-E1 cells

Western analysis of mGluR1 in MC3T3-E1 cells was performed using an anti-mGluR1 antiserum (Pharmingen, San Diego, CA, U.S.A.) and horseradish peroxidase-coupled, goat anti-mouse IgG (Sigma Chemical Co.) essentially as described above for Western blots carried out with the anti-CaR antiserum.

Detection of CaR transcripts by Northern blot analysis

For the purpose of determining the sizes of the CaR transcripts in MC3T3-E1 cells, Northern blot analysis was employed on aliquots of 5 μg of poly(A+) RNA obtained using oligo-dT cellulose chromatography of total RNA. Rat kidney poly(A+)-enriched RNA, 5.0 μg, was used as positive control employing the same cRNA probe in a different blot. RNA samples were denatured and electrophoresed in 2.2 M formaldehyde-1% agarose gels along with an 0.24–9.5 kb RNA ladder (GIBCO BRL) and transferred overnight to nylon membranes (Duralon; Stratagene, La Jolla, CA, U.S.A.). A 577 bp XhoI-SacI fragment corresponding to nucleotides 721–1298 of the RaKCaR cDNA was subcloned into the pBluescript(SK+) vector. The plasmid was then linearized with BglII, and a32P-labeled riboprobe was synthesized with the MAXIscript T3 kit (Pharmacia Biotech, Piscataway, NJ, U.S.A.) using T3 polymerase and32P-UTP. Nylon membranes were prehybridized for 2 h at 52°C in a solution consisting of 50% formamide, 4× Denhardt's solution (50× Denhardt's = 5 g of Ficoll, 5 g of polyvinylpyrrolidone, and 5 g of BSA), 5× SSPE (20× SSPE = 2.98 M NaCl and 0.02 M EDTA in 0.2 M phosphate buffer, pH 7.0), 0.5% SDS, 10% dextran sulfate, 250 μg/ml yeast tRNA, and 200 μg/ml calf thymus DNA. Labeled cRNA probe (2 × 106 cpm/ml) was then added, and the membranes were hybridized overnight at 52°C for the MC3T3-E1 sample and at 65°C for kidney RNA sample. Washing was carried out for 20 minutes at moderate stringency (0.3× SSC [20× SSC = 3 M NaCl and 0.3 M Na3-citrate · 2H2O], 0.5% SDS at 55°C) for the RNA from MC3T3-E1 cells, while for mouse kidney RNA, the blot was washed at 65°C for 20 minutes. Moderate stringency washing was employed for MC3T3-E1 RNA samples because of the relatively low level of expression of CaR transcripts in these cells and because of the use of a heterologous probe (e.g., derived from the rat kidney CaR). In control studies comparing the use of moderate versus high stringency washes for Northern blot analysis of RNA from kidney, although there was some increase in the intensity of the bands after washing at lower stringency, no additional bands were observed. Membranes were then exposed to X-ray film (Kodak XAR-5; Eastman Kodak, Rochester, NY, U.S.A.) for 4 days at –70°C.

PCR amplification of CaR in MC3T3-E1 cells

Total RNA was prepared from MC3T3-E1 cells using the TRIzol Reagent (GIBCO BRL). One microgram of total RNA was used for the synthesis of single-stranded cDNA (cDNA synthesis kit; GIBCO BRL). The resultant first-stranded cDNA was used for the PCR procedure. PCR was performed at a final concentration of 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.8 mM MgCl2, 0.2 mM dNTP, 0.4 μM of forward primer, 0.4 mM of reverse primer and 1 μl of ELONGASE enzyme mix (a Taq/Pyrococcus species GB-D DNA polymerase mixture; GIBCO BRL). The primer sequences were: 5′-ATGGTTTGGCTACTGTTTGG-3′, sense; 5′-CAGAGCCTTGGAGACGGTGT-3′, antisense, which encode nucleotides 40–59 and 339–358, respectively, of the partially cloned extracellular domain of the mouse AtT-20 pituitary cell CaR.19 This primer set was designed to span one intron of the CaR gene, to distinguish products amplified from cDNA and genomic DNA. To perform “hot start” PCR, the enzyme was added during the initial 3-minute denaturation and was followed by 35 cycles of amplification (30-s denaturation at 94°C, 30-s annealing at 47°C and 1-minute extension at 72°C). The reaction was completed with an additional 10-minute incubation at 72°C to allow completion of extension. PCR products were fractionated on 1.2% agarose gels. The presence of a 319 nucleotide-long amplified product was indicative of a positive PCR reaction. The PCR product in the reaction mixture was purified using the QIAquick PCR purification kit (Qiagen, Santa Clarita, CA, U.S.A.) and subjected to direct, bidirectional sequencing employing the same primer pairs used for PCR by means of an automated sequencer (AB377; Applied Biosystems, Foster City, CA, U.S.A.) in the DNA Sequence Faculty of the University of Maine (Orono, ME, U.S.A.), using dideoxy terminator Taq technology.

Chemotaxis assay of MC3T3-E1 cells

Chemotaxis was evaluated using a blindwell chamber (BW200S; Neuro Probe Inc., Gaithersburg, MD, U.S.A.) as previously described.3,20 CaCl2, 1.8 or 4.8 mM, 300 μM neomycin sulfate, or 100 mM GdCl3 · 6H2O in serum-free Dulbecco's MEM were loaded in the lower chamber, which is separated from the upper well by a 5 mm membrane with 5-μm pores. MC3T3-E1 cells (1 × 105 cells/ml) were dissociated with trypsin-EDTA solution (GIBCO BRL), washed twice, suspended in serum-free α-MEM, and added to the upper chamber. After a 12-h incubation at 37°C, cells on the upper surface of the membrane that had not migrated were scraped from the membrane, and cells that had migrated to the opposite side of the membrane were fixed with methanol and stained with Giemsa. The cells that had migrated to the lower surface of the filter were counted in six high-power fields (×400) using a light microscope. For the purpose of comparison between multiple assays, the data were normalized as the fold increase in cellular chemotaxis relative to that of the control.

DNA synthesis in MC3T3-E1 cells

We assessed DNA synthesis in MC3T3-E1 cells using [3H]thymidine incorporation. MC3T3-E1 cells were dissociated by gentle scraping and repeated pipeting and seeded in 24-well (2.0 cm2) plates at a density of 1000 cells/well in 500 μl of α-MEM containing 10% fetal bovine serum as well as various concentrations of CaCl2, neomycin sulfate, or GdCl3 · 6H2O as indicated in Fig. 5. After a 48 h incubation at 37°C, cells were pulsed with [3H]thymidine (1 μCi/well). Incubations were terminated after overnight incubation by removal of the medium and addition of 5% trichloroacetic acid. Cells were scraped and transferred to microcentrifuge tubes. After centrifugation at 15,000g and removal of the supernatant, the precipitate was washed with 75% ethanol and desiccated at room temperature. The residual pellet was dissolved in 20 mM NaOH and 1% SDS, and a scintillation cocktail was added. Samples were counted in a liquid scintillation counter.

Figure FIG. 5.

(A) Chemotactic activity of MC3T3-E1 cells toward high Ca2+o, neomycin sulfate or Gd3+o. The number of MC3T3-E1 cells that migrated to the side of the membrane to which CaCl2, neomycin sulfate, or GdCl3·6H2O had been added 12 h previously were counted as described in the Materials and Methods. Each bar expresses the mean ± SEM for six determinations. *p < 0.05 compared with cells exposed to 1.8 mM Ca2+o. (B) Stimulation of DNA synthesis in MC3T3-E1 cells by high Ca2+o, neomycin sulfate, or Gd3+o. MC3T3-E1 cells were treated with high Ca2+o, neomycin sulfate, or Gd3+o for 3 days. Values are expressed as percentage of control (cells treated with 1.8 mM Ca2+o). Each bar represents the mean ± SEM for six determinations. *p < 0.05 compared with cells treated with 1.8 mM Ca2+o.

Statistics

Results are expressed as the mean ± SEM. Statistical evaluation for differences between groups was done using one-way analysis of variance followed by Fisher's protected least significant difference. For all statistical tests, values of p < 0.05 were considered significant.

RESULTS

Immunoreactivity of CaR protein in MC3T3-E1 cells using CaR-specific antiserum

To clarify whether the CaR is expressed by osteoblasts, we investigated the presence of the receptor in the mouse clonal osteoblastic cell line, MC3T3-E1. Immunocytochemistry of MC3T3-E1 cells with a CaR-specific polyclonal antiserum revealed strong CaR staining (Fig. 1A), which was eliminated by preincubating the primary antiserum with the peptide against which it was raised (Fig. 1B).

Figure FIG. 1.

Immunocytochemistry of MC3T3-E1 cells carried out as described in the Materials and Methods using a CaR-specific antiserum (4637). Immunocytochemistry revealed strong CaR staining (A), which was eliminated by preincubating the primary antiserum with the peptide against which it was raised (B). The photomicrographs were taken at a magnification of ×1000.

We also performed Western analysis of CaR on proteins isolated from MC3T3-E1 cells. Bands were stained in proteins obtained from the two different batches of the cells in the presence of specific antiserum (Fig. 2, lanes 1 and 2). The molecular weight of the band at ∼150–160 kDa in each lane was of a size consistent with those of the intact, glycosylated human CaR (140–200 kDa),21 bovine parathyroid CaR (∼130–150 kDa),22 and mouse kidney CaR (∼140 kDa).23 The specificity of the anti-CaR antiserum used in this study was confirmed by the abolition of the bands following preabsorption of the anti-CaR antiserum with the peptide against which it was raised (Fig. 2, lanes 3 and 4). No immunoreactivity for mGluR1 was observed in proteins extracted from MC3T3-E1 cells by Western analysis using an anti-mGluR1 antiserum (Fig. 2, lanes 5 and 6).

Figure FIG. 2.

Western analysis of whole cell lysates from MC3T3-E1 cells. Western analyses of CaR and mGluR1 were each performed on the two different batches of the MC3T3-E1 cells as described in the Materials and Methods. Bands at ∼150–160 kDa that were stained in the presence of specific antiserum were consistent with the intact glycosylated CaR21–23 (lanes 1 and 2). The specificity of the labeling by the antiserum used in this study to detect the CaR was confirmed by abolition of the bands in extracts of cells incubated with peptide-preabsorbed CaR-antiserum (lanes 3 and 4). No mGluR1 immunoreactivity was observed in the cells using the anti-mGluR1 antiserum (lanes 5 and 6). The cells cultured for 5, 13, or 20 days (lanes 7–9, respectively) showed immunoreactivity for the CaR over the entire range of culture periods.

When MC3T3-E1 cells were cultured up to 20 days, they began to generate osmiophilic nodular regions with strong ALP staining (data not shown), confirming their capacity to differentiate to mature osteoblasts as reported previously.8 Western analysis using anti-CaR antiserum on proteins isolated from the cells cultured for 5, 13, or 20 days (lanes 7–9, respectively) showed that the cells expressed CaR protein over the entire range of culture periods during which osteoblastic differentiation took place.

Detection of CaR mRNA in MC3T3-E1 cells by Northern blot analysis and RT-PCR

In Fig. 3, lane 1 shows Northern blot analysis performed using a riboprobe derived from the rat kidney CaR on poly(A+) RNA isolated from MC3T3-E1 cells, which revealed three transcripts with sizes of 9.5, 4.5, and 1.5 kb; these sizes were consistent with those of the mouse CaR transcripts from AtT-20 pituitary cell line reported previously.19 However, a 7.5 kb band observed in AtT-20 cells was not visible in MC3T3-E1 cells even after exposing the blot for 24 h in a phosphorimager cassette. Northern blot analysis of mouse kidney revealed a predominant CaR mRNA transcript of 7.5 kb and two minor transcripts of 9.5 kb and 4.1 kb (Fig. 3, lane 2).

Figure FIG. 3.

Northern blot analysis of CaR transcripts in MC3T3-E1 cells (lane 1) and mouse kidney (lane 2) performed as described in the Materials and Methods. Arrows show the sizes of the CaR transcripts.

RT-PCR with mouse CaR-specific primers, which were intron-spanning to preclude amplification of a similar sized product from any contaminating genomic DNA, amplified a product of the expected size, 319 bp for a CaR-derived product (Fig. 4, lane 1). No products were observed when the RT was omitted during synthesis of cDNA (Fig. 4, lane 2). The 319 bp PCR product was also amplified from cDNA prepared from a different batch of MC3T3-E1 cells (Fig. 4, lane 3). No products were detected in the control PCR reaction without RT (Fig. 4, lane 4). DNA sequence analysis of the 319 bp PCR products revealed a 100% sequence identity with the mouse AtT-20 cell CaR sequence reported previously19 (data not shown). These results show that the RT-PCR products corresponded to an authentic CaR sequence, indicating the presence of bona fide CaR transcripts in these cells.

Figure FIG. 4.

Identification of CaR transcripts in MC3T3-E1 cells using RT-PCR with intron-spanning mouse CaR-specific primers, performed as described in the Materials and Methods. A product was amplified from reverse transcribed RNA isolated from MC3T3-E1 cells, which was of the expected size, 319 bp (lane 1) for a CaR-derived product. The PCR reaction without RT showed no products (lane 2). A 319-bp PCR product was also amplified from cDNA isolated from the different batch of MC3T3-E1 cells (lane 3), while no PCR product was apparent in the RT-minus control reaction (lane 4). DNA sequence analysis of the 319 bp PCR products revealed a 100% sequence identity with the mouse AtT-20 cell CaR sequence reported previously19 (data not shown).

Chemotactic activity of MC3T3-E1 cells toward high Ca2+o, neomycin sulfate, or Gd3+o

A chemotaxis assay was performed to determine the capacity of MC3T3-E1 cells to migrate toward CaR agonists. As shown in Fig. 5A, 4.8 mM Ca2+o induced a chemotactic response of MC3T3-E1 cells over control values at 1.8 mM Ca2+o (p < 0.05). Both neomycin sulfate (300 μM) and Gd3+o (100 μM) also induced significant chemotactic responses over the control (p < 0.05).

DNA synthesis of MC3T3-E1 cells stimulated by high Ca2+o, neomycin sulfate, or Gd3+o

We found that treatment of MC3T3-E1 cells with increasing levels of Ca2+o up to 4.8 mM resulted in a dose-dependent stimulation of DNA synthesis over control values at 1.8 mM Ca2+o (p < 0.05) (Fig. 5B). Neomycin sulfate (100 and 300 μM) and Gd3+o (25 and 100 μM) also induced significant stimulations of DNA synthesis over the control (p < 0.05).

DISCUSSION

In a previous study, we examined the expression of the CaR in primary cultures of human bone marrow and showed that low-density mononuclear cells isolated from whole human bone marrow, which are putatively enriched in marrow progenitor cells, including bone cell precursors, expressed the CaR by Northern analysis, RT-PCR, and immunocytochemistry.18 About one third of these adherent, CaR-immunoreactive cells were also positive for ALP, suggesting the expression of the CaR in putative osteoblast precursors or osteoblasts. Since these osteoblast-like cells were taken directly from bone marrow and may be similar to those present in marrow in vivo, this finding suggested that the CaR might be expressed by preosteoblasts or osteoblasts in vivo. However, we observed that these preparations also contained other types of cells, including nonspecific esterase-positive, monocyte-macrophage-like cells. Thus, such marrow-derived preparations might not be ideal for addressing specifically the existence and role of the CaR in osteoblasts. To circumvent this problem, we used mouse MC3T3-E1 cells possessing an osteoblastic phenotype in this study.7 Our results showed that MC3T3-E1 cells clearly expressed CaR protein by immunocytochemistry (i.e., Fig. 1) as well as by Western blot analysis (e.g., Fig. 2), which revealed a specific band at a molecular weight consistent with that of the intact, glycosylated CaR (∼150–160 kDa).21–23 In addition, both Northern analysis performed on poly(A+) RNA from MC3T3-E1 cells and RT-PCR performed on total RNA from these cells followed by sequence analysis of the PCR products indicated the presence of bona fide CaR transcripts (Figs. 3 and 4). Thus, the present study shows that this osteoblast cell line expresses both CaR protein and mRNA.

It is known that MC3T3-E1 cells exhibit properties of osteoprogenitor cells and preosteoblasts in their actively growing stage; following growth arrest, they differentiate to develop markers of mature osteoblasts, including the expression of high levels of ALP and the capacity to form mineralized bone matrix.8–10 Thus, this cell line seems to be a useful model for examining the process of osteoblastic development in vitro. In this study, MC3T3-E1 cells expressed the CaR by Western blot analysis over the entire range of culture periods examined (5, 13, and 20 days) (Fig. 2), which may provide an in vitro model of the developmental stages from preosteoblast to mature osteoblast.9,10 This result suggests that MC3T3-E1 cells (and, perhaps, osteoblastic cells in vivo) express the CaR throughout their differentiation.

Osteoblasts are known to play a crucial role in the formation phase of bone remodeling, by laying down the structural components of bone (matrix and mineral) and secreting various cytokines and growth factors that influence both bone formation and resorption.24 Bone formation is initiated by the migration of preosteoblasts into resorption pits at the end of osteoclastic bone resorption.1 Substantial amounts of Ca2+o are released from mineralized bone matrix during osteoclastic resorption,2 raising the level of Ca2+o in the vicinity of resorption sites. It is possible that the CaR senses these high levels of Ca2+o, thereby providing a signal for preosteoblasts that induces their migration into sites where new bone formation is required. In fact, we previously showed that high Ca2+o induced both a chemotactic response and DNA synthesis of MC3T3-E1 cells,3,5 and Quarles et al. showed that high Ca2+o and other CaR agonists, such as Gd3+o and neomycin, stimulated DNA synthesis in MC3T3-E1 cells.6 These findings suggest that a calcium-sensing mechanism is present in these osteoblastic cells and is involved in their migration and proliferation. In this study, we confirmed these previous observations and further showed that the CaR agonists, Gd3+ and neomycin, as well as high Ca2+o per se could induce chemotaxis of MC3T3-E1 cells (Fig. 5). Although Quarles et al. failed to detect CaR expression by either RT-PCR or Northern analysis in MC3T3-E1 cells,17 we have documented using multiple detection methods that MC3T3-E1 cells express both CaR protein and mRNA that are similar if not identical to those in the parathyroid gland and kidney (Figs. 1, 2, 3, 4), suggesting that the CaR might also potentially be involved in the migration and proliferation of MC3T3-E1 cells.

Although there are other reports failing to show expression of the CaR in human peripheral blood monocytes25 and human osteoblast-like SAOS-2 cells,26 these results were based on the single finding of negative results of RT-PCR using CaR-specific primers, but not on methods detecting CaR protein expression, such as immunocytochemistry and Western analysis. We found that detection of CaR transcripts by RT-PCR or Northern blot analysis in MC3T3-E1 cells was, in general, more difficult than identification of receptor protein using immunocytochemistry and Western analysis. Therefore, we believe that negative results obtained using the former two methods require more cautious interpretation. Since the complete DNA sequence of the mouse CaR has not been determined, Quarles et al. performed RT-PCR of mouse CaR with primers designed from human CaR. They failed to amplify CaR-derived products at an annealing temperature of 60°C in MC3T3-E1 cells.17 However, this temperature might be too high for effective priming of the mouse CaR in MC3T3-E1 cells in view of the species mismatch between primers and their DNA template as well as the probable lower expression of CaR mRNA in the osteoblastic cells relative to those in the parathyroid and kidney. Indeed, we also failed to amplify any PCR products even with mouse CaR-derived primers in MC3T3-E1 cells when annealing temperatures above 50°C were used and found that a temperature of 47°C was optimal for successful amplification of the CaR-derived product by PCR. Moreover, we found that ordinary Taq DNA polymerase was relatively ineffective for amplifying CaR mRNA from MC3T3-E1 cells, and that only “hot start” PCR using a mixture of Taq/Pyrococcus species GB-D DNA polymerases, which have proofreading activity, resulted in successful amplification. Therefore, RT-PCR of the mouse CaR may require careful selection of the experimental conditions that are ideal for PCR. In this study, the possibility of amplification of CaR from genomic DNA was totally eliminated because the primer pair was designed to span one intron of the CaR gene, the primer pair amplified a product of the size expected for a CaR-derived product, and no PCR product was amplified without RT.

Similarly, we only successfully detected CaR transcripts in MC3T3-E1 cells using Northern blot analysis under moderately stringent hybridization and washing conditions, utilizing a rat CaR-specific cRNA probe and 5 μg of poly(A+) RNA instead of the cDNA probe and 2 μg of poly(A+) RNA that were employed by Quarles et al.17 The principal bands observed in MC3T3-E1 cells were similar in size to two minor bands observed on Northern analysis of mouse kidney performed on poly(A+) under high stringency conditions. We employed two different stringencies with MC3T3-E1 cells and mouse kidney because of the use of a heterologous (e.g., rat) probe and since the relative abundance of the CaR transcripts in MC3T3-E1 cells are much lower than in mouse kidney. The use of high stringency conditions for hybridization and washing would have greatly reduced the signal intensity observed in the MC3T3-E1 cells relative to that in kidney. The 9.5 and 4.5 kb transcripts present in MC3T3-E1 cells are of sizes similar to transcripts expressed in both mouse kidney and murine AtT-20 cells.19 However, MC3T3-E1 cells did not express any of the 7.5 kb transcript observed in AtT-20 cells19 or mouse kidney, even after exposing the blot for 24 h using phosphorimager analysis. There are previous instances where the relative ratios of the abundance of CaR transcripts varies from organ to organ.27 Because 4.5 kb transcript encodes the entire functional CaR protein, the significance of the larger 9.5 and 7.5 kb transcripts remain uncertain. However, the possibility of organ/cell type specific, post-transcriptional regulation of CaR expression as a result of variations in the stabilities of the various CaR transcripts cannot be ruled out.

Another explanation for the difference between our results and those of Quarles et al. could be variations in the clones of MC3T3-E1 cells that were employed in the two studies, and that some clones show little or no expression of the CaR. However, this explanation appears less likely because we have confirmed the existence of the CaR in the two different batches of MC3T3-E1 cells, both of which have the capacity to differentiate to mature osteoblasts after prolonged culture, as assessed by Western blot analysis and RT-PCR using intron-spanning primers.

The CaR is known to share amino acid sequence homology and topological similarity with the mGluRs,28 which are G protein-coupled receptors present in the central nervous system that respond to glutamate, the major excitatory neurotransmitter in the brain. Thus, it was important to consider the possibility of our anti-CaR antiserum cross-reacting with mGluRs in MC3T3-E1 cells. We compared the amino acid sequences of the CaR-derived FF-7 peptide, to which the antiserum, 4637, that was used in this study was raised, and the corresponding regions of the mGluRs, and found that they shared 40–53% homologies to one another. These values may well not represent sufficient degrees of similarity to produce cross-reactivity of our anti-CaR antiserum with the mGluRs. We failed to find any evidence in the published literature describing the expression of mGluRs in MC3T3-E1 cells or other osteoblast-like cells. Moreover, Western analysis in this study showed that an anti-mGluR1 antiserum failed to detect mGluR protein in MC3T3-E1 cells. Moreover, we have observed that HEK293 cells transfected with the cDNA for mGluR1 were negative on Western analysis using anti-CaR antiserum, 4637, despite being positive on Western analysis using an anti-mGluR1 antiserum18 (M. Bai and E. M. Brown, manuscript in preparation), indicating that it is unlikely that the anti-CaR antiserum could detect a mGluR even if the cells expressed it. Thus, we consider it unlikely that the protein in MC3T3-E1 cells recognized by our anti-CaR antiserum represents mGluR(s) instead of CaR.

Recently, Hinson et al. have reported that nucleotide sequences of putative CaR-related receptors (Casr-rs) were identified in mouse genomic libraries by PCR.29 The deduced protein sequence of one of these putative receptors (Casr-rs1) was 63% similar and 40% identical to the CaR over the available transmembrane region. However, as with the mGluRs, these levels of identity in predicted protein sequences may well not be large enough to result in cross-reactivity of our anti-CaR antiserum with these related protein. Moreover, although this CaR-related nucleotide sequence was initially identified in MC3T3-E1 cells by RT-PCR and was used as a probe to screen mouse genomic libraries to identify other related sequences, it could not be identified in subsequent analyses of mouse tissues, including MC3T3-E1 cells, by RT-PCR.29 Additional studies are necessary to determine whether these CaR-related nucleotide sequences are actually expressed as mature proteins in MC3T3-E1 cells using specific antisera raised to their predicted protein sequences.

The role of the CaR in the control of cellular proliferation has not been clear until recently, when the receptor has been conclusively shown to be involved in the stimulation of cell proliferation by CaR agonists. Mailland et al. reported that CaR agonists stimulate the proliferation of CCL39 hamster fibroblasts transfected with the CaR,30 and Hoff et al. reported that transfection of NIH-3T3 cells with a human CaR cDNA harboring an activating mutation induced cellular transformation and proliferation.31 In addition, McNail et al. have recently demonstrated that rat-1 fibroblasts express an endogenous CaR and that high Ca2+o stimulates the proliferation of these cells through a proliferative pathway involving CaR-mediated activation of SRC kinase and ERK1.32 Moreover, several recent studies have clarified the signaling mechanisms underlying Ca2+o-stimulated chemotaxis and proliferation of MC3T3-E1 cells. Godwin and Soltoff demonstrated that inhibition of the activation of either G protein or PLC blocked nearly all of the chemotaxis in MC3T3-E1 cells, whereas the inhibition of protein kinase C (PKC) or phosphoinositide-3-kinase did not.4 These results suggested that Ca2+o-stimulated chemotaxis of this cell line is linked to the activation of G protein and PLC. Quarles et al. showed that a variety of polyvalent cations, including Al3+, Gd3+, Ca2+ and neomycin, stimulated DNA synthesis in MC3T3-E1 cells through a mechanism coupled to the activation of G protein and PKC.6 Since the CaR activates PLC in a G protein-dependent manner, thereby raising the levels of IP3 and the cytosolic calcium concentration and activating PKC,11,33 these findings do not contradict the hypothesis that the CaR is involved in the proliferation and chemotaxis of MC3T3-E1 cells induced by high Ca2+o and other CaR agonists. However, one study reported that neither Gd3+o nor Ca2+o increased either inositol phosphate formation or the cytosolic calcium concentration in MC3T3-E1 cells.34 Thus, additional studies are needed to document further causal relationships between expression of the CaR, its signal transduction pathways and the control of chemotaxis and cell proliferation by CaR agonists in this osteoblastic cell line.

Although the CaR was first cloned from the parathyroid and kidney,11–13 bone is also intimately involved in systemic calcium ion homeostasis.35 In this study, using MC3T3-E1 cells as a model, we show that osteoblasts, which play an important role in bone remodeling within the skeleton, express both CaR protein and mRNA. Our results suggest that this receptor may be involved in important physiological responses of these cells, such as chemotaxis and proliferation after stimulation by Ca2+o. These events are observed at the beginning of the bone formation phase of skeletal remodeling in vivo, suggesting that the CaR could potentially play a key role in the functions of bone cells within the bone/bone marrow microenvironment.

Acknowledgements

The authors gratefully acknowledge generous grant support from the following sources: The Mochida Memorial Foundation Grant for Medical and Pharmaceutical Research (to T.Y.), The Yamanouchi Foundation Grant for Research on Metabolic Disorders (to T.Y.), NPS Pharmaceuticals, Inc. (to E.M.B.), The St. Giles Foundation (to E.M.B.), USPHS Grants DK-41415, DK-48330, and DK-52005 (to E.M.B.), and the National Space Bioscience Research Institute (NSBRI) (to E.M.B.).

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