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Endocrinology Vol. 142, No. 4 1471-1478
Copyright © 2001 by The Endocrine Society

ARTICLES

Regulation of Calcitonin Receptor by Glucocorticoid in Human Osteoclast-Like Cells Prepared in Vitro Using Receptor Activator of Nuclear Factor-{kappa}B Ligand and Macrophage Colony-Stimulating Factor1

Seiki Wada, Shigemitsu Yasuda, Tsutomu Nagai, Tomoya Maeda, Shinji Kitahama, Satoru Suda, David M. Findlay, Makoto Iitaka and Shigehiro Katayama

Fourth Department of Internal Medicine, Saitama Medical School (S.W., S.Y., T.N., T.M., S.Ki., S.S., M.I., S.Ka.), Saitama 350-0495, Japan; and Department of Orthopedics and Trauma, University of Adelaide (D.M.F.), Adelaide, South Australia 5001, Australia

Address all correspondence and requests for reprints to: Seiki Wada, M.D., Fourth Department of Internal Medicine, Saitama Medical School, 38 Morohongo Moroyama-cho, Iruma-gun, Saitama 350-0495, Japan. E-mail: wadas{at}saitama-med.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Using mouse osteoclast-like cells (OCs), we have shown that treatment with glucocorticoids (GCs) resulted in an increase in calcitonin (CT) binding by enhancing CT receptor (CTR) gene transcription. Additionally, treatment with GCs demonstrated increased sensitivity to CT. There is, however, scant information on the effects of GC or CTR regulation by GCs in human osteoclasts. In this study we examined CTR regulation by GCs and the effects of GCs and CT together in human OCs. OCs were prepared by treatment of peripheral blood mononuclear cells in vitro with soluble receptor activator of nuclear factor-{kappa}B ligand and macrophage colony-stimulating factor. Treatment of mature OCs with dexamethasone (Dex) resulted in a dose- and time-dependent increase in [125I]salmon CT (sCT) binding capacity. Treatment with Dex enhanced CTR messenger RNA (mRNA) expression, suggesting that CTR up-regulation is at least partly due to an increase in de novo CTR synthesis. Triamcinolone and prednisolone reproduced the Dex effect on [125I]sCT-specific binding and CTR mRNA expression, but 17ß-estradiol, progesterone, dehydroepiandrosterone, and aldosterone did not. A Scatchard plot analysis showed that Dex enhanced CTR number with a minimal change in the affinity to sCT. Autoradiographic studies using [125I]sCT showed that Dex enhanced the CTR density on individual multinuclear OCs. Up-regulation of [125I]sCT-specific binding and CTR mRNA expression was seen even in the presence of sCT, but the enhancement diminished subsequently at later times (36–48 h after sCT removal), which was consistent with our previous observation in mouse OCs. This suggests that GCs and CTs act on CTR expression differently, consistent with our previous work using mouse OCs, in which we found that GCs increased transcription of CTR gene expression, whereas CT reduced CTR mRNA stability. The results obtained in this study show that GC increased CTR expression and sensitivity to CT in cells of the human osteoclast lineage and provide the basis for understanding the beneficial effects of combination treatment with GCs and CTs in malignancy-associated hypercalcemia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AS CALCITONIN (CT) inhibits osteoclastic bone resorption, it has been widely used to treat metabolic bone disorders, such as Paget’s disease of bone, malignancy-associated hypercalcemia, and osteoporosis (1, 2, 3). It is recognized, however, that continuous treatment with CT causes a loss of its inhibitory effects on bone resorption. We and other investigators have studied the cellular mechanism for this, mostly using mouse and rat osteoclast-like cells (4, 5, 6). The results indicated that desensitization to CT was closely associated with down-regulation of the CT receptor (CTR). This down-regulation was due not only to internalization of the receptor (5), but also to reduced cell surface receptor concentration through inhibition of de novo CTR synthesis (6, 7). There is, however, scant information on CTR regulation or the mechanism of homologous desensitization to CT in cells of osteoclast lineage in human and other species, because of the difficulties in obtaining sufficient cells for biochemical studies.

The generation of human osteoclasts in vitro has been a requirement to evaluate osteoclast pathophysiology in bone and calcium metabolism. Suda and co-workers proposed a factor that would be expressed on osteoblasts/stromal cells in response to the stimulators of osteoclastogenesis to osteoclast progenitors (8). Several groups have recently succeeded in the cloning of osteoclast differentiation factor, which mediates an essential signal for osteoclast differentiation from its precursor cells (9, 10). Osteoclast differentiation factor was also found to be identical to tumor necrosis factor-related activation-induced cytokine (11) and receptor activator of nuclear factor-{kappa}B ligand (RANKL) (12). Using soluble (s) RANKL and macrophage colony-stimulating factor (M-CSF), Matsuzaki et al. (13) developed a new method to prepare human OCs from peripheral blood mononuclear cells, which enables us to study the mechanisms of the biological responses to CT and other agents in cells of human osteoclast lineage. Using this system, we recently found that the signaling pathway responsible for CTR down-regulation in human OCs is different from that observed in mouse OCs; activation of the PKA pathway is primarily responsible for CTR regulation in mouse OCs (14), whereas activation of PKC was predominant in human OCs (15).

Glucocorticoids (GCs), alone or in combination with CTs, have been used to treat hypercalcemia due to malignancy. It has been postulated that treatment with GCs impairs osteoclastic bone resorption by inhibiting OC survival (16), reduces transcription of some genes related to osteoclast-generating factors, e.g. PTH-related peptide (17), and occasionally reduces tumor mass (18). However, the beneficial effect of GCs in the treatment of hypercalcemia has been appreciated when they were combined with CT (19, 20). The mechanism of the synergistic action has been studied in mouse OCs. The results indicated that GCs up-regulate CTR through increased de novo CTR synthesis (21, 22). It was also found recently that treatment with GCs increased gene transcription of CTR in mouse OCs (22); however, there are only a few studies on the effects of GCs in osteoclasts, in particular GC regulation of the CTR in human OCs.

In this report we describe the effect of GCs on human OCs prepared in vitro to study more precisely the mechanism of CTR regulation, information that is important for the optimal use of CT and GC therapy for the treatment of malignancy-associated hypercalcemia.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant human sRANKL was purchased from PeproTech Ltd. (London, UK). Recombinant human M-CSF was a gift from Morinaga Milk Industries Co. Ltd. (Tokyo, Japan). Salmon CT (sCT), dexamethasone (Dex), prednisolone, triamcinolone, 17ß-estradiol, progesterone, dehydroepiandrosterone (DHEA), aldosterone, isobutylmethylxanthine (IBMX), trypsin-EDTA, {alpha}MEM, and Histopaque 1077 were purchased from Sigma-Aldrich Corp. (St. Louis, MO). cAMP assay kits were purchased from Yamasa Shoyu Co. (Chiba, Japan).

Preparation and culture of human osteoclast-like cells (OCs)
Human OCs were prepared using a slight modification of the method reported by Matsuzaki et al. (13). Briefly, peripheral blood was collected from healthy normal volunteer in syringes containing 1000 U/ml preservative-free heparin. Informed consent was obtained before blood aspiration. Mononuclear cells were isolated by centrifugation over Histopaque 1077 density gradients and resuspended at approximately 5.0 x 106 cells/ml in {alpha}MEM supplemented with 10% FCS. The cells were then cultured for about 14 days in 24-well plates (~3.0 x 106 cells/well) or 60-mm culture dishes (2.5 x 107 cells/dish) in the presence of human sRANKL (50 ng/ml) and human M-CSF (100 ng/ml). Culture media were replenished with fresh media every 3–4 days. Cells were used for experiments when mature multinuclear cells were predominant in the cultures.

[125I]sCT binding experiments and tartrate-resistant acid phosphatase (TRAP) staining
Equal aliquots of OCs (~3.0 x 103 multinuclear OCs/well) were treated with Dex or other agents for the times indicated. [125I]sCT (SA, ~600 mCi/mg) was purchased from Amersham Pharmacia Biotech (Aylesbury, UK). OCs were incubated with Dex or other agents in {alpha}MEM containing 10% FCS. A solution of 0.1% ethanol and 0.01 N acetic acid was used as vehicle for steroids and CT, respectively. These solutions alone did not significantly modify the binding capacity of [125I]sCT. Cells were then incubated with [125I]sCT (~20,000 cpm/400 µl; ~2 x 10-11 M) at 4 C for 4 h. Nonspecific binding was assessed by coincubation with 10-6 M sCT. At the end of the incubation, cells were rinsed with cold phosphate-buffered saline (PBS) and dissolved in 0.5 N NaOH, and cell-bound radioactivity was measured. Scatchard analysis of the binding data from competitive binding studies was performed with a fixed amount of [125I]sCT and increasing amounts of unlabeled sCT.

The cells were stained by TRAP as described previously (6). In brief, cells were incubated for 20 min in 50 mM acetic acid buffer (pH 5.0) containing sodium tartrate dihydrate (50 mM), naphthol AS-MX phosphate (0.1 mg/ml), and Fast Red Violet LB salt (0.6 mg/ml). After washing with distilled water and drying, the number of cells staining positively for TRAP was counted.

Autoradiography of [125I]sCT binding
After a 3-h settlement in 4- or 8-well chamber slides (Nunc, Inc., Naperville, IL) or plastic coverslips (Nunc, Inc.) in 24-well plates, OCs were treated with dexamethasone for the times indicated (0–48 h). After removal of the media, cells were washed twice with PBS and then used for binding experiments with 10-9 M [125I]sCT in {alpha}MEM containing 0.1% BSA for 1 h at 37 C. After TRAP staining, the cells were subjected to autoradiography. Autoradiographic studies have been detailed previously (6). In brief, the slides were dipped into NR-M2 nuclear emulsion (Konica Corp., Tokyo, Japan) and exposed for 7–10 days at 4 C in the dark, then developed with Phenisol x-ray developer (Ilford, Mobberley, UK) for 4 min at 20 C, fixed with Hypam rapid fixer (Ilford) for 5 min, and washed in tap water. To determine CTR density on control or Dex-treated OCs, the number of silver grains was counted on 20 randomly selected multinuclear OCs in each treatment group by superimposing a grid over the cells. The number of silver grains is expressed per 100 µm2 with background grain density subtracted.

RNA extraction
Equal aliquots of OCs (~1.0 x 104 multinuclear OCs/dish) were treated with Dex or other agents for the times indicated. A solution of 0.1% ethanol and 0.01 N acetic acid, was used as vehicle for steroids and CT, respectively. These solutions alone did not significantly modify the expression of CTR messenger RNA (mRNA). After washing cells with cold PBS, total RNA was extracted by the acid guanidinium-phenol-chloroform method, as reported previously (15).

PCR amplification of reverse transcribed mRNA
The RT of RNA to complementary DNA and the subsequent amplification were performed as described previously (15). First strand complementary DNA was synthesized from 1.0 µg total RNA by incubation for 1 h at 42 C with 2.5 U/µl murine leukemia virus reverse transcriptase, 2.5 µM random hexamers, 1.0 U/µl ribonuclease inhibitor, and 1.0 mM deoxy-NTP mix. From this reaction mixture, 1 µl of 20 µl was submitted to PCR to amplify the sequence of the CTR mRNA specified below and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the OCs using GeneAmp RNA PCR kit components (PE Applied Biosystems, Foster City, CA). The reaction mixture, containing 100 pmol of each primer, 1.5 µl 25 mM MgCl2 solution, 2.5 µl 10 x reaction buffer II, 5.0 U AmpliTaq DNA polymerase, and sterile distilled water, was overlaid with 50 µl paraffin oil. Amplification was performed by TAKARA DNA Thermal cycler (TAKARA, Biotechnology, Kusatsu, Japan), with cycles of denaturation at 94 C for 1 min, annealing at 60 C for 1 min, and extension at 72 C for 1 min for CTR and GAPDH. Preliminary experiments were performed to ensure that the number of cycles employed was within the exponential phase of the amplification curve. PCR products were resolved on a 1.0% (wt/vol) agarose gel, and the specificity of the reaction was confirmed by Southern transfer to nylon filter (Hybond+-N membrane, Amersham Pharmacia Biotech) and hybridization with 32P-labeled internal oligonucleotide probes. The signals were quantitated using an image analysis system (NIH image version 1.61).

Oligonucleotides used for this study were synthesized by Amersham Pharmacia Biotech). The oligonucleotides for human CTR were: human CTR1, 5'-GCAATGCTTTCACTCCTGAGAAAC-3' (5'-primer); and human CTR2, 5'-CAGTAAACACAGCCACGACAATGAG-3' (3'-primer). The products were verified with the internal sense strand oligonucleotide, 5'-GTTGAAGTAGTACCCAATGGA-3' (human CTR3), by Southern hybridization. To ensure equal starting quantities of DNA for the experiments and to allow semiquantitation of the PCR products representing CTR, reverse transcribed RNA samples were also amplified using oligonucleotide primers specific for human GAPDH sequence. Oligonucleotides for GAPDH were: GAPDH3, 5'-CACTGACACGTTGGCAGTGG-3' (3'-primer); and GAPDH4, 5'-CATGGAGAAGGCTGGGGCTC-3' (5'-primer). PCR products were verified with a 32P-labeled internal sense oligonucleotide GAPDH1, 5'-GCTGTGGGCAAGGTCATCCC-3'.

Measurement of cAMP production
OCs (~3.0 x 103 multinuclear OCs/well) were pretreated with Dex and/or sCT and then challenged with sCT or other agents in {alpha}MEM containing 0.1% BSA and 1.0 mM IBMX for 20 min at 37 C. A solution of 0.1% ethanol and 0.01 N acetic acid was used as vehicle for Dex and CT, respectively. These solutions alone did not significantly modify CT-sensitive cAMP production. Cellular cAMP production was measured by RIA using a Yamasa RIA kit. Details of this assay were described in a previous report (21).

Statistical analysis
Experimental data were analyzed using one-way ANOVA with a post-hoc Bonferroni-Dunn test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of Dex treatment on [125I]sCT binding in human OCs
We have studied the effects of GCs and CTs in mature mouse OCs, where GCs up-regulated and CTs down-regulated the CTR of OCs (21, 22). Here, we examined the action of GCs on CTR in human OCs, assessing modulation of the specific binding capacity of [125I]sCT. When the OCs were treated with Dex, [125I]sCT-specific binding was increased in a dose- and time-dependent manner (Fig. 1Go). This effect was evident at 10-9 M, and the maximum response was observed at 10-7 M. In the time-course experiment, the increased binding capacity was found more than 12 h after addition of Dex, and the effect was observed for up to 48 h (Fig. 1BGo). We have shown previously that up-regulation of CTR by Dex was specific for GCs and was not reproduced by mineralocorticoids or sex steroids in mouse OCs (21). We therefore examined whether increased [125I]sCT-specific binding by Dex is also specific for GCs in humans. Enhancement of [125I]sCT-specific binding after Dex treatment was only reproduced by the GCs, triamcinolone and prednisolone, not by mineralocorticoids or sex steroids (17ß-estrogen, progesterone, DHEA, and aldosterone; Fig. 2Go).



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  		Figure 1. Effect of Dex treatment on [125I]sCT-specific binding in human OCs. A, Dose response of [125I]sCT-specific binding in human OCs after 24-h treatment with Dex. B, Time course of the effect of Dex (10-7 M) on [125I]sCT-specific binding in human OCs. OCs were incubated with Dex at the dose (A) and time (B) indicated in {alpha}MEM containing 10% FCS. After removal of the media, cells were washed with PBS, and specific binding of [125I]sCT was measured as described in Materials and Methods. B: {circ}, Control cells; •, Dex-treated cells. Each point represents the mean ± SD for four wells. Nonspecific binding in these studies was approximately 10% of the total binding. This figure is representative of three separate experiments in which similar results were obtained.

 


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  		Figure 2. Effects of various steroids on sCT-specific binding. OCs were treated without (control) or with triamcinolone (10-7 M), prednisolone (10-7 M), 17ß-estradiol (10-7 M), progesterone (10-7 M), DHEA (10-7 M), and aldosterone (10-7 M) for 24 h. After treatment, cells were washed with PBS, and specific binding of [125I]sCT was measured. Each point represents the mean ± SD for four wells. Nonspecific binding in these studies was approximately 10% of the total binding. This figure is representative of three separate experiments in which similar results were obtained.

 
To investigate more precisely the action of Dex on the cell surface CTR concentration and receptor affinity in OCs, we evaluated the effects of Dex through competitive binding experiments. Twenty-four hours after treatment with Dex (10-7 M), OCs were submitted to competitive binding with fixed amounts of [125I]sCT (~20,000 cpm/400 ml; 2 x 10-11 M) and increasing amounts of unlabeled sCT, as described in Materials and Methods. The results (Fig. 3Go) showed a 2.4-fold increase in Dex-treated cultures in receptor number, as indicated by the x-intercept in the Scatchard plot. There were minimal effects on receptor Kd, as indicated by the slope of the lines of best fit in the Scatchard plot. Treatment with Dex for 24 h did not alter either the number of TRAP-positive cells or the mean nucleus number in the OC cultures. Using autoradiographic techniques, we further examined whether the increase in CTR number was accurately reflected by the increase in [125I]sCT-specific binding capacity in each human OC. The results (Table 1Go) demonstrated that Dex enhanced the density of the grains on TRAP-positive multinuclear and mononuclear OCs, but not on other types of cells. Cells not positively stained by TRAP staining (e.g. fibroblastic spindle-shaped cells) showed low levels of silver grain density (20 ± 10/100 µm2; n = 10), similar to background grain density (23 ± 8/100 µm2; n = 10).



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  		Figure 3. Effect of Dex (10-7 M) treatment on [125I]sCT binding and its IC50 in human OCs. OCs were treated with or without Dex (10-7 M) for 24 h in {alpha}MEM containing 10% FCS. After removal of the media, cells were washed with PBS. Binding capacity was then examined by incubating cells for 4 h at 4 C with a fixed concentration of [125I]sCT and varying concentrations of unlabeled sCT. A representative experiment was shown in this figure, and each point is the mean of four values. A, Competitive binding curve; B, Scatchard analysis of the binding data. {circ}, Untreated (control) cells (binding capacity, 16.9 x 106/multinuclear cells; Kd = 1.6 nM); •, Dex-treated cells (binding capacity, 32.5 x 106/multinuclear cells; Kd = 1.5 nM). This experiment is representative of three separate experiments in which similar results were obtained.

 

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  		Table 1. Effect of treatment with Dex (10-7 M) on the number of silver grains in human OCs, using [125I] sCT autoradiography

 
Using mouse OCs, we previously reported that treatment with CT induces homologous down-regulation of its receptor in both mouse (6) and human (15) OCs. Although pretreatment of mouse OCs with GCs increased CTR expression, the effects of GCs were attenuated when the cells were also exposed to CT. To examine the interaction of GCs and CT with respect to regulation of CTR in human OCs, OCs were treated with sCT (10-9 M) for 1 h in the presence or absence of Dex (10-7 M); Dex was added 12 h before the addition of sCT. After sCT removal, OCs were washed twice with PBS, and the media were replaced with fresh growth media. Cells previously treated with Dex were resupplemented with Dex (10-7 M). Twelve, 24, 36, or 48 h after sCT removal, binding of [125I]sCT to cells was measured, as described in Materials and Methods. OCs treated with Dex showed an approximately 2-fold increase in [125I]sCT-specific binding, compared with control cells, from 12–48 h (Fig. 4Go). In OCs treated solely with sCT 10-9 M, specific binding decreased to approximately 20–50% of that in control cells at 24 and 36 h after sCT removal. When the cells were treated with Dex and sCT together, the CT-induced CTR loss was somewhat delayed. At 12 h, the specific binding primarily reflected the enhancement due to Dex treatment, whereas measurement at 36 and 48 h after sCT removal in cells cotreated with Dex and sCT showed that specific binding subsequently decreased, compared with that in control cells, even in the continuous presence of Dex.



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  		Figure 4. Effect of Dex and/or sCT on [125I]sCT-specific binding in human OCs. OCs were prepared as described in Materials and Methods (~3.0 x 104 OCs/dish). Cells were treated with sCT (10-9 M) for 1 h in the presence or absence of Dex (10-7 M); Dex was added 12 h before the addition of sCT. After removal of the media, cells were washed twice with PBS, and the media were replaced with fresh growth media containing 10% FCS. Cells were further incubated for up to 48 h with or without Dex. Cells previously treated with Dex were resupplemented with Dex (10-7 M). After removal of the media, cells were washed with PBS as described in Materials and Methods. Specific binding of [125I]sCT was measured. Cont, Control OCs treated with neither Dex nor sCT; Dex, treated with Dex alone; sCT, treated with sCT alone; Dex+sCT, treated with Dex and sCT. This experiment is representative of three separate experiments in which similar results were obtained.

 
Effect of Dex treatment on CTR mRNA expression in human OCs
To study the effect of Dex on CTR mRNA expression in human OCs, we used semiquantitative RT-PCR, as previously described (6, 15). For the PCR, between 15–35 (actual cycle number we used for PCR in the following experiments, 30) cycles of amplification were optimal for semiquantitative analysis of mRNA expression for CTR, and between 10–25 (actual cycle number we used for PCR in the following experiments, 20) cycles were optimal for GAPDH. The amplification was in the exponential phase over these ranges. The PCR products were resolved and were authenticated by Southern blot analysis with 32P-labeled internal sense oligonucleotides, as described in Materials and Methods. The CTR mRNA levels were compared with those of GAPDH as an internal control.

To study the time course of the effect of Dex treatment on CTR mRNA expression, OCs were treated with Dex (10-7 M), and total RNA was extracted at various times from 0–48 h after the addition of Dex. Treatment with Dex resulted in an increase in CTR mRNA expression by 6 h after addition. The maximum increase was observed after about 24-h exposure, after which the increased levels of CTR mRNA expression gradually returned to control levels after 48-h exposure (Fig. 5).

We have also shown previously that up-regulation of CTR mRNA by Dex was specific for GCs in mouse OCs and was not reproduced by mineralocorticoids or sex steroids (22). We therefore examined whether increased CTR mRNA expression by Dex is specific for GCs in human OCs. Indeed, enhancement of CTR mRNA by Dex was reproduced by other GCs, triamcinolone and prednisolone, but not by mineralocorticoids or sex steroids (Fig. 6Go). The effects of prednisolone and triamcinolone on the enhanced expression of CTR mRNA did not differ significantly from those of Dex when they were examined in the same experiments (data not shown).



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  		Figure 6. Effects of various steroids on CTR mRNA expression in OCs. OCs were treated with prednisolone (10-7 M), triamcinolone (10-7 M), progesterone (10-7 M), DHEA (10-7 M), aldosterone (10-7 M), and 17ß-estradiol (10-7 M) for 24 h. Total RNA was extracted and then reverse transcribed. The products were amplified by PCR using specific internal oligonucleotides. The PCR products were transferred to a nylon filter and hybridized with 32P-labeled internal sense oligonucleotide specific for CTR and GAPDH sequence as described in Materials and Methods. The intensities of autoradiograph signals were quantitated and are shown below as the ratio of CTR/GAPDH; the values were compared with that of control OCs (the value of 1.0). Each bar represents the mean ± SD of three samples. This experiment is representative of three separate experiments in which similar results were obtained.

 
To determine the effects of Dex and sCT together on CTR mRNA expression, OCs were treated with sCT (10-9 M) for 1 h in the presence or absence of Dex (10-7 M); Dex was added 12 h before the addition of sCT. After sCT removal, OCs were washed twice with PBS, and the media were replaced with fresh growth media. Cells previously treated with Dex were resupplemented with Dex (10-7 M). Total RNA was extracted at 0–48 h after sCT removal. Treatment with Dex enhanced CTR mRNA expression in OCs, whereas sCT treatment for 1 h resulted in a subsequent decrease in CTR mRNA levels in both control and Dex-treated cultures (Fig. 7Go). In both cell groups, treatment with sCT eventually resulted in a profound decrease in CTR mRNA levels, which did not return to the control levels by 48 h after sCT removal.



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  		Figure 7. Effects of sCT and/or Dex treatment on CTR mRNA expression in OCs. OCs were treated with sCT (10-9 M) for 1 h in the presence or absence of Dex (10-7 M); Dex was added 12 h before the addition of sCT. After removal of the media, cells were washed twice with PBS, and the media were replaced with fresh growth media containing 10% FBS. Cells were further incubated for up to 48 h with or without Dex. Cells previously treated with Dex were resupplemented with Dex (10-7 M). After removal of sCT, OCs were washed with PBS and total RNA was extracted. RNA was reverse transcribed and subjected to PCR using specific primers. The PCR products were transferred to a nylon filter and hybridized with 32P-labeled internal sense oligonucleotide specific for CTR and GAPDH sequence as described in Materials and Methods. The intensities of autoradiograph signals were quantitated and are shown below as the ratio of CTR/GAPDH; the values were compared with those of control OCs at each time (value of 1.0). Control, Control OCs treated with neither Dex nor sCT; sCT, treated with sCT alone; Dex, treated with Dex alone; Dex+sCT, treated with Dex and sCT. This experiment is representative of three separate experiments in which similar results were obtained.

 
Effect of Dex pretreatment on sCT-stimulated cAMP production in OCs
To determine whether the effects of GC observed on binding capacity and CTR mRNA expression would relate to the biological response of adenylate cyclase activity in OCs, we examined the effects of Dex on sCT-responsive adenylate cyclase activity. OCs were treated with sCT (10-9 M) for 1 h in the presence or absence of Dex (10-7 M); Dex was added 12 h before the addition of sCT. After sCT removal, OCs were washed twice with PBS, and the media were replaced with fresh growth media. Cells previously treated with Dex were treated with Dex. Twenty-four, 36, or 48 h after sCT removal, cells were examined for the adenylate cyclase response to subsequent exposure to sCT (10-9 M) for 20 min at 37 C in the presence of IBMX. As shown in Fig. 8Go, both Dex and sCT pretreatment modulated sCT-responsive adenylate cyclase in a manner qualitatively similar to the change in receptor binding. Pretreatment with Dex (10-7 M) increased the maximum sCT-responsive adenylate cyclase activity about 2-fold; treatment with sCT (10-9 M) for 1 h, on the other hand, diminished the response to about 15% of that in control cells at 24 h after sCT removal. When OCs were pretreated with Dex and sCT together in this protocol, the cAMP response to sCT was approximately the same as that in control cells not exposed to these agents. At 36 and 48 h after sCT removal, a diminished response was observed, even in the presence of Dex, compared with that in control cells (neither treated with Dex nor sCT).



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  		Figure 8. sCT-stimulated cAMP responses in human OCs pretreated with sCT and/or Dex. OCs were treated with sCT (10-9 M) for 1 h in the presence or absence of Dex (10-7 M): Dex was added 12 h before the addition of sCT. After sCT removal, OCs were washed twice with PBS, and the media were replaced with fresh growth media. Cells previously treated with Dex were resupplemented with Dex (10-7 M). Twenty-four (A), 36 (B), and 48 (C) h later, cells were examined for the adenylate cyclase response to sCT (10-9 M) for 20 min in the presence of IBMX. cAMP was assayed as described in Materials and Methods. Each bar shows the mean ± SD for four wells. *, P < 0.01 vs. control OCs stimulated by sCT. V, Basal cAMP production of control OCs stimulated with vehicle; sCT, cAMP production of OCs stimulated with sCT; Control, pretreated with neither Dex nor sCT; Dex, pretreated with Dex alone; sCT, pretreated with sCT alone; Dex+sCT, pretreated with Dex and sCT. This experiment is representative of three separate experiments in which similar results were obtained.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study confirms our earlier work (21, 22), where we found that treatment of OCs with GCs induced CTR up-regulation, caused a rise in CTR mRNA expression, and enhanced responsiveness of CT-stimulated adenylate cyclase activity. GCs have been shown to regulate some peptide hormone actions at the level of hormone synthesis and secretion or at the level of hormone receptors. ß2-Adrenergic receptors in smooth muscle cells (23) and PTH/PTH-related peptide receptors in ROS 17/2.8 osteosarcoma cells (24) have been reported to be up-regulated by GCs. Such regulation has been reported to occur at the level of gene transcription, but recently it was shown that GCs act on mRNA stability of certain target genes (25). Up-regulation by GCs on peptide hormone actions has not been appreciated in clinical application, with the apparent exception of treatment of hypercalcemia, in which the potentiation of the hypocalcemic effect of CT by GCs has been described. The combination of GCs and CT produced a rapid decline in the serum calcium concentration (19) and an increase in the effectiveness of CT to decrease the serum calcium level in hypercalcemic patients (20).

The mechanism of CTR regulation has been studied in clonal cells, in which evidence was consistent with rapid ligand-induced internalization of the CT-CTR complex and persistent activation of adenylate cyclase by CT treatment (26, 27, 28). Similar results have been observed in rat and mouse osteoclasts (4, 6), suggesting that the basic mechanism of CTR regulation is conserved among cell types and different species. The results obtained from the recent studies of cells transfected with CTR showed that CTRs couple to multiple intracellular effector systems (29, 30, 31). Although these transfected studies are useful to identify basic receptor biology, there is a potential limitation that they do not always reflect all aspects of receptor biology, such as physiological receptor regulation, as they occur in target cells (6). Osteoclasts, the main target cells of CT in bone, respond to CT very differently from the model cell systems. CT induces retraction of osteoclasts and inhibits osteoclastic bone resorption (32), although some of the important aspects of this were found to be conserved regardless of the cell type (33). We previously studied the effects of GCs and CTs using mouse osteoclasts prepared in vitro, where we found that GCs up-regulate CTR through increased de novo CTR synthesis and CTs down-regulate CTR by inhibiting de novo CTR synthesis along with internalization of the ligand-receptor complex (6). Treatment with Dex was found to increase the transcription rate of CTR gene in mouse OCs (22). Interestingly, however, when human OCs were studied with respect to homologous down-regulation, it was found that the mechanism of CTR regulation by CTs in human OCs was somewhat different from that observed in mouse OCs. The basic features of the CTR regulation, i.e. ligand receptor recognition, subsequent internalization, and decreased concentration of CTR on cell surface, were essentially the same in both species. However, the intracellular pathway responsible for CTR down-regulation differed between the two species; the homologous down-regulation was predominantly through the activation of PKC in human OCs (15), whereas activation of protein kinase A was important in mice (14). This difference prompted the present study in human OCs to investigate the basic mechanism of the beneficial interaction of GCs and CT seen in treatment for hypercalcemia (19, 20).

When the OCs were treated with Dex, [125I]sCT-specific binding was increased in a dose- and time-dependent manner. Enhancement of [125I]sCT-specific binding and CTR mRNA expression by Dex was only reproduced by GCs, suggesting that the effects of Dex were specific for GCs. Scatchard analysis showed a 2.4-fold increase in Dex-treated cultures in receptor number, as indicated by the x-intercept in the Scatchard plot. Using autoradiographic techniques, it was shown that the apparent increase in CTR number was reflected in the increased [125I]sCT-specific binding capacity in individual human OCs. The life span of mature osteoclast is limited, and in the present experiments the number of TRAP-positive osteoclasts was reduced after the peak of cell maturation, which probably accounted for the reduced sCT-induced cAMP levels in each of the treatment groups. Despite this, the effects of Dex to enhance CT-sensitive adenylate cyclase were still present for up to 48 h after the addition of Dex. Although this effect could be possible through the involvement of intracellular components other than surface CTR expression, for example, G proteins and catalytic subunits, it seemed most likely that the effects of GCs were predominantly through up-regulation of CTR, as preliminary data showed that GCs did not affect the influence of forskolin (a direct activator of catalytic subunit) or cholera toxin (a Gs activator) on adenylate cyclase activity (data not shown). It is of great interest to clarify the molecular mechanism of interaction between GCs and CTs on CTR regulation. As we have previously described (6, 14, 15, 21, 22), the mechanism of CTR regulation is specific for particular target cells. Experiments to explore the molecular basis of the CTR gene expression in mouse OCs suggested that GCs up-regulate CTR by increasing transcription of CTR gene (22). Although CTs induced a rapid and profound decrease in CTR mRNA expression in mouse OCs, it was not possible to show an effect of CT on CTR gene transcription in mouse OCs (22). Recently, however, Inoue et al. showed that CT may reduce CTR gene transcription, using an RNA protection assay (34). Studies on mRNA turnover in the presence of transcriptional inhibitors suggest that the action of CT to destabilize the CTR mRNA predominates over increased transcription by GC (22). The mRNA stability observed in mouse OCs required ongoing transcription, suggesting the involvement of a labile protein mRNA-degrading factor. The AUUUA motifs as well as other A/U-rich sequences have been shown to determine the stability of other mRNA transcripts through binding with multiple proteins in this region (35). Multiple copies of the AUUUA motif have been identified in the 3'-untranslated regions of the CTR gene in various species (22), including the human (29). In the present study treatment with GCs increased the concentration of cell surface CTR; however, the effects of GCs were attenuated when the cells were exposed to sCT in OCs. Further experiments are required to determine the molecular mechanisms responsible for these opposing actions of GC and CT on CTR in human OCs.

It remains to be determined whether the effects of Dex on OC CTR expression are direct or indirect via osteoblasts or other stromal cells in the cultures. It is possible, for example, that the effect is mediated by a cytokine(s) produced in response to Dex treatment. We have previously shown that GCs up-regulated the interleukin-6 receptor in osteoblasts, which, in turn, promoted osteoclast differentiation in the presence of interleukin-6 (36). With respect to the present study, there was no difference in the number of multi- and mononuclear TRAP-positive cells between control and GC-treated cultures. Future isolation of the promoter region of the CTR gene will greatly assist investigation of its regulation in various cell types.

In conclusion, this study shows that treatment with GCs increased the concentration of cell surface CTR and CTR mRNA expression in human OCs. Our results may at least partially explain the beneficial interaction of GCs and CTs observed clinically.



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  		Figure 5. Effect of Dex on CTR mRNA expression in OCs. OCs were prepared as described in Materials and Methods (~3.0 x 104 OCs/dish). OCs were treated with Dex (10-7 M) for various periods of time. After removal of the media, OCs were washed (0–48 h) with PBS, and then total RNA was extracted. RNA was reverse transcribed and subjected to PCR using specific primers as described in Materials and Methods. Products were verified with specific internal oligonucleotides. The intensities of autoradiograph signals were quantitated and were shown below as the ratio of CTR/GAPDH; the values were compared with those of control OCs at 0 h (the value of 1.0). C, Control OCs ({circ}); D, Dex-treated OCs (•). This experiment is representative of three separate experiments in which similar results were obtained.

 

   Acknowledgments
 
The authors are grateful for excellent technical assistance of Ms. Keiko Nagatani and the scientific advice of Drs. Akinobu Minagawa and Satomi Fujimaki.


   Footnotes
 
1 This work was supported in part by the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan, Research on Health Sciences Focusing on Drug Innovation (no. 73001) from the Japan Health Sciences Foundation and the Casio Science Promotion Foundation. Back

Received September 6, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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