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Endocrinology Vol. 138, No. 2 521-529
Copyright © 1997 by The Endocrine Society

Articles

Regulation by Calcitonin and Glucocorticoids of Calcitonin Receptor Gene Expression in Mouse Osteoclasts1

Seiki Wada, Nobuyuki Udagawa2, Takuhiko Akatsu, Naokazu Nagata, T. John Martin and David M. Findlay3

St. Vincent’s Institute of Medical Research (S.W.. N.U., T.J.M., D.M.F.), Fitzroy, Victoria, Australia; and the Third Department of Internal Medicine, National Defense Medical College (S.W., T.A., N.N.), Namiki, Tokorozawa, Saitama, Japan

Address all correspondence and requests for reprints to: Seiki Wada, M.D., Ph.D., Third Department of Internal Medicine, National Defense Medical College, 3–2 Namiki, Tokorozawa, Saitama 359, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously studied regulation of the calcitonin (CT) receptor (CTR) by glucocorticoid (GC) and CT in cultures of mature mouse osteoclast-like cells (OCLs). The present studies were designed to examine the interaction of CT and GC in regulation of the CTR in osteoclasts and the molecular mechanisms involved. Treatment of OCLs with 10-7 M dexamethasone (Dex) increased the CTR number in a time-dependent manner, whereas treatment with 10-9 M salmon CT (sCT) reduced CTR number; neither treatment changed receptor affinity. Dex pretreatment somewhat antagonized the CT-induced reduction in [125I]sCT specific binding. Dex increased, and sCT pretreatment decreased, the sCT-responsive adenylate cyclase activity in parallel with the change in receptor binding. Dex treatment resulted in an increase in CTR messenger RNA (mRNA) levels, as assessed by reverse transcription-PCR, indicating that the increased CTR number was mediated by de novo CTR synthesis. This effect was specific to GCs and was not reproduced by mineralocorticoids or sex steroids. Treatment with sCT resulted in a rapid and profound reduction in CTR mRNA expression, and this reductions was somewhat delayed by Dex pretreatment. OCLs were treated with 5,6-dichloro-1ß-D-ribofuranosyl benzimidazole to enable estimation of the mRNA decay rates in the absence of ongoing transcription. The stability of CTR mRNA was similar to the control value in Dex-treated OCLs, suggesting that the effect of Dex may be due to changes in transcriptional activity. Interestingly, transcriptional inhibition by 5,6-dichloro-1ß-D-ribofuranosyl benzimidazole abolished the ability of CT to reduce CTR mRNA levels, suggesting that CT may act by increasing the rate of CTR mRNA decay, and that this effect requires ongoing transcription. The 3'-untranslated region of the mouse CTR mRNA contains four copies of the AUUUA motif, as well as other A/U-rich sequences, which have been shown to determine the stability of other mRNA transcripts. The stability results were consistent with the results of the nuclear transcript run-on assay, which indicated that treatment with Dex enhanced the rate of transcription, whereas CT had no effect. These results show that GC and CT influence CTR expression by distinct mechanisms and provide the basis for identification of the cellular factors involved.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MOST important recognized action of the 32-amino acid peptide hormone calcitonin (CT) is inhibition of osteoclastic bone resorption. However, when CT is used repeatedly in vivo and in vitro, resistance to its action develops, a phenomenon termed escape (1, 2). It has been reported that glucocorticoids (GCs) protect against CT escape when used in the treatment of malignancy-associated hypercalcemia (3, 4). Although there are several possible explanations for this effect of GCs, there is some evidence that GCs act directly on bone cells to modulate the effect of CT (5, 6). In a previous report, we showed that GCs enhance the CT responsiveness of adenylate cyclase of OCLs in parallel with up-regulation of CT receptor (CTR) in mature mouse osteoclast-like cells (OCLs) prepared in an in vitro mouse coculture system (7). The recent cloning of CTR complementary DNA (cDNA) from several species (8, 9, 10, 11) has enabled study of the regulation of CTR messenger RNA (mRNA) expression in osteoclasts (12, 13, 14). Thus, we found that CT treatment induced down-regulation of the CTR in OCLs through a prolonged decrease in CTR mRNA expression, with a parallel reduction in CT responsiveness (12). This homologous CTR down-regulation has also been shown in human osteoclast-like cells of giant cell tumor origin (15). We have also shown that the protein kinase A (PKA) pathway is central to the process of CT-induced homologous CTR down-regulation of OCLs (16). The likely relevance of this work to an understanding of osteoclast biology was our finding that this down-regulation was induced, not only by pharmacological CT concentrations, but also by concentrations of CT down to the physiological range (16).

The molecular mechanisms of CTR gene regulation remained to be clarified. The CTR belongs to the seven-transmembrane G protein-coupled receptor superfamily. Regulation of another member of this receptor class, the ß2-adrenergic receptor (ß2AR) is subject to ligand-induced down-regulation, which is mediated by reduction of the receptor mRNA levels (17). It has been found that this mRNA down-regulation is due to destabilization of the ß2AR mRNA rather than a decrease in the rate of transcription of the ß2AR gene (17). There are now many examples of regulated mRNA species, for example several cytokine mRNAs, in which the rate of degradation of the mRNA plays an important role in regulating steady state mRNA levels (18). It now appears that analogous mechanisms of mRNA destabilization apply to the ß2AR mRNA, with cognate adenylate/uridylate (A/U)-rich sequences in the 3'-untranslated region (3'UTR) involved in the regulation of mRNA stability and turnover (19, 20, 21).

In the present study, we examined the molecular and cellular mechanisms of CTR regulation by GCs and CT in OCLs. We were interested to explore the interaction of these agents with respect to CTR expression. The results show that GCs enhance CTR gene expression in OCLs predominantly through increased transcription rate of the gene, whereas CT induces posttranscriptional destabilization of the receptor mRNA. GC treatment of OCL cultures somewhat slow the CT-induced down-regulation of CTR and CTR mRNA. These findings provide the basis for identification of the cellular factors that mediate the effects of these agents in cells of the osteoclast lineage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and materials
Newborn C57/BL6J mice and 6- to 9-week-old male C57/BL6J mice were purchased from Australia Animal Resource (Mt. Waverley, Australia). Animal handling and treatment were performed according to protocols provided by the National Health and Medical Research Council of Australia. Approval for the procedures described herein was granted by St. Vincent’s Hospital animal ethics committee. Salmon CT (sCT) and 1{alpha},25-dihydroxyvitamin D3 [1,25-(OH)2D3] were supplied by Bachem (Torrance, CA) and Hoffman La Roche (Nutley, NJ), respectively. Fast Garnet GBC, AS-BI phosphate, dexamethasone (Dex), prednisolone, triamcinolone, aldosterone, dehydroepiandrosterone, progesterone, 17ß-estradiol, SDS, and isobutylmethylxanthine (IBMX) were purchased from Sigma Chemical Co. (St. Louis, MO). Collagen gel solutions (cell matrix, type 1-A) were purchased from Nitta Gelatin Co. (Osaka, Japan). Bacterial collagenase was purchased from Boehringer Mannheim (Mannheim, Germany). 5,6-Dichloro-1-ß-D-ribofuranosyl benzimidazole (DRB) was obtained from Calbiochem-Novabiochem (La Jolla, CA). The cAMP assay kit was purchased from Yamasa Shoyu (Chiba, Japan).

Cell culture
OCLs were prepared as previously described (22, 23). Briefly, primary osteoblastic cells prepared from newborn mouse calvaria (5 x 105 cells/dish) and bone marrow cells from male mice (5 x 106 cells/dish) were cocultured on dishes coated with collagen gel, as previously described (22), in {alpha}MEM containing 10% FBS in the presence of 10-8 M 1,25-(OH)2D3 for 7–8 days. At the end of the culture, dishes were treated with collagenase (0.2%), and intercellular attachment was gently disrupted by syringing in 21-gauge needles. Equal aliquots of the cells were inoculated, and cells were allowed to settle for 3 h before further experiments in medium without added 1,25-(OH)2D3. In these cultures, multinuclear OCLs were abundant, occupying 40–60% of the whole cell population, and mononuclear OCLs were ~20% of the total OCL nuclei.

[125I]sCT binding experiments
sCT was iodinated by a modified chloramine-T method, achieving a specific activity of ~160 mCi/mg. OCLs (1000–3000 multinuclear OCL/well) in 24-well trays were incubated for 1 h with sCT (10-9 M) 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 36 h with or without Dex; cells previously treated with Dex were resupplemented with Dex (10-7 M). The CTR-binding capacity of OCLs was determined by incubation with [125I]sCT (~20,000 cpm/500 µl; ~4 x 10-11 M) in {alpha}MEM containing 0.1% BSA at 4 C for 4 h. Nonspecific binding was assessed by coincubation with 10-7 M sCT. After the incubation, cells were washed twice with cold PBS and dissolved in 0.5 N NaOH, and cell-bound radioactivity was measured. Scatchard analysis of the binding data was calculated from competitive binding studies with a fixed amount of [125I]sCT and increasing amounts of unlabeled sCT.

Measurement of cAMP production
Equal aliquots of OCLs (500–1000 multinuclear OCLs/well) in 24-well trays were incubated with sCT (10-9 M) for 1 h with or without Dex (10-7 M); Dex was added 12 h before the addition of sCT. After removal of the medium, OCLs were washed with PBS twice before the addition of fresh growth medium containing 10% FBS. Cells were further incubated for 24 h with or without Dex; cells previously treated with Dex were resupplemented with Dex (10-7 M). Twenty-four hours after sCT removal, the media were discarded, and OCLs were treated with sCT (10-9 M) in {alpha}MEM containing 0.1% BSA and 1 mM IBMX for 20 min at 37 C. cAMP was extracted by sonication for 10 sec in 0.1 N HCl and was measured by RIA as described previously (24).

RNA extraction
Cells on collagen gels on days 7–8 were treated with collagenase, and equal aliquots of the cells were inoculated into 10-cm dishes (multinucleated OCL number was ~10,000/dish). For the experiments to determine the time course of the effects of Dex, OCLs were allowed to settle for 2 h before the addition of Dex (10-7 M) and were further incubated for various time intervals before extraction of total RNA by the guanidine thiocyanate-phenol chloroform method (25).

For experiments to measure CTR mRNA stability, OCLs were treated with or without sCT (10-9 M) for 1 h in the presence or absence of Dex; Dex was added 12 h before the addition of sCT. The cells were then washed with PBS, and the media were replaced with fresh growth media containing DRB (25 µg/ml) and 10% FBS. In the Dex-incubated cultures, fresh Dex (10-7 M) was added to the replaced medium. Cells were further incubated for various time intervals, and total RNA was extracted.

PCR amplification of reverse transcribed mRNA
First strand cDNA was synthesized from 2.5 µg total RNA by incubation for 1 h at 42 C using AMV reverse transcriptase (Promega, Madison, WI) with random hexanucleotides (Promega), as reported previously (12, 16, 26). From this reaction mixture, 2.5 µl of 25 µl were subjected to PCR to amplify sequences of the CTR or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA as specified below. The reaction mixture contained 50 pmol of each oligonucleotide primer; 25 mM deoxy (d)-ATP, dGTP, dCTP, and dTTP (Pharmacia, Uppsala, Sweden); 2 µl of a 10-fold concentrated reaction buffer (Boehringer Mannheim, Indianapolis, IN); 1 U Taq DNA polymerase (Boehringer Mannheim); and sterile distilled water and was overlaid with paraffin oil. Amplification was performed in a Perkin-Elmer DNA Thermal Cycler 480 (Norwalk, CT), 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, or annealing at 55 C for GAPDH. Preliminary experiments were performed to ensure that the number of cycles employed was within the exponential phase of the amplification curve, as shown previously (12). PCR products were resolved on a 2% (wt/vol) agarose gel, and the specificity of the reaction was confirmed by Southern transfer to nylon filter (Hybond-N membrane, Amersham, Arlington Heights, IL) and hybridization with 32P-labeled internal oligonucleotide probes. The signals were quantitated using a Molecular Dynamics PhosphorImager (Sunnyvale, CA).

Oligonucleotides used for this study were purchased from Bresatec (Thebarton, Australia). The oligonucleotides for mouse (m) CTR were: mCTR2, 5'-CAAGGCACGGACAATGTTGAGAAG-3' (3'-primer complementary to nucleotides 1564–1586); and mCTR1, 5'-TTTCAAGAACCTTAGCTGCCAGAG-3' (5'-primer complementary to nucleotides 1023–1046) (8). The products were verified with the internal sense strand oligonucleotide, 5'-AAGCACATGTTCCTTACTTA-3' (mCTR3, complementary to nucleotides 1046–1079), 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 mouse GAPDH (27). Oligonucleotides for GAPDH were: 5'-primer, 5'-CATGGAGAAGGCTGGGGCTC-3' (GAPDH4); and 3'-primer, 5'-AACGGATACATTGGGGGTAG-3' (GAPDH5). PCR products were verified with a 32P-labeled internal sense oligonucleotide, 5'-GCTGTGGGCAAGGTCATCCC-3' (GAPDH1), as we previously reported.

Nuclear run-on assay
OCLs on collagen gels (~20 10-cm diameter dishes) on days 7–8 were treated with collagenase, and equal aliquots of the cells were inoculated into 175-cm2 flasks (the number of multinuclear OCL was ~500,000/flask). OCLs were allowed to settle for 2 h in {alpha}MEM containing 10% FBS before the addition of Dex (10-7 M) and sCT (10-9 M), and cells were further incubated for 3 h with these agents. After washing twice with ice-cold PBS, cell nuclei were isolated using Nonidet P-40 lysis buffer. The transcription rate was determined using the nuclear run-on technique, as described previously (28, 29). Complementary cDNA probes for CTR (rat C1a isoforms; 10 µg cDNA including vector sequence), GAPDH (10 µg cDNA including vector sequence), and vector itself (10 µg) were immobilized onto nylon filters (Hybond-N membrane, Amersham). Newly synthesized transcripts incorporated with [32P]UTP were hybridized with the cDNAs for 36 h at 65 C in Tris-EDTA-SDS-NaCl solution and washed with suitable stringency. Filters were exposed to x-ray films or a PhosphorImager was used to quantitate the signals.

Statistical analysis
Experimental data were analyzed by one-way ANOVA, and individual comparisons from control were performed by Dunnett’s multiple comparison test. For the data shown in Figs. 1Go and 3Go, individual comparisons between pairs were performed by Fisher’s protected least significant difference (Fisher’s protected least significant difference) procedure.



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  		Figure 1. Effect of Dex and/or sCT treatment on [125I]sCT binding. OCLs 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 up to 36 h with or without Dex. Cells previously treated with Dex were resupplemented with Dex (10-7 M). Twelve, 24, or 36 h after removal of sCT, OCLs were washed with PBS, and the CTR-binding capacity was determined by incubating with [125I]sCT at 4 C for 4 h. Cell-bound radioactivity was measured after dissolving the cells with 0.5 N NaOH. A, Twelve hours after sCT removal; B, 24 h after sCT removal; C, 36 h after sCT removal. Cont, Not treated with Dex or sCT; Dex, treated with Dex alone; sCT, treated with sCT alone; Dex+sCT, treated with Dex and sCT. Each point represents the mean ± SD for four wells. a, P < 0.01 vs. control; b, P < 0.01 vs. Dex alone. Nonspecific binding in these studies was ~10% of the total binding. This experiment is representative of three separate experiments in which similar results were obtained.

 


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  		Figure 3. sCT-stimulated cAMP responses in OCLs pretreated with sCT and/or Dex. OCLs 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, OCLs 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 hours 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. a, P < 0.01 vs. control OCLs stimulated by sCT. V, Basal cAMP production of control OCLs stimulated with vehicle; sCT, cAMP production of OCLs stimulated with sCT; Control, not pretreated with Dex or 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.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of Dex and sCT treatment on CTR in OCLs
We have studied the effects of GCs and CTs in mature mouse OCLs, where GCs up-regulated and CTs down-regulated the CTR of OCLs. Here, we examined the interaction of GCs and CT with respect to regulation of CTR in OCLs. OCLs 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, OCLs 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, or 36 h after sCT removal, binding of [125I]sCT to cells was measured as described in Materials and Methods. OCLs treated with Dex showed a ~2-fold increase in [125I]sCT specific binding compared with control cells from 12–36 h (Fig. 1Go). In OCLs treated solely with sCT 10-9 M, specific binding decreased to ~50-20% 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 delayed. At 12 h, specific binding primarily reflected the enhancement due to Dex treatment, whereas measurement 36 h after sCT removal in cells cotreated with Dex and sCT showed that specific binding subsequently decreased compared with that in control cells.

To investigate more precisely the action of Dex and sCT on cell surface CTR concentration and receptor affinity in OCLs, we performed competitive binding experiments on OCL cultures of each treatment. OCLs were treated with Dex and/or sCT using the same experimental protocol described above. Twenty-four hours after sCT removal, cells were submitted to competitive binding, as described in Materials and Methods. The results (Fig. 2Go) showed a 53% decrease in receptor number in sCT-treated cells, as indicated by the x-intercept in the Scatchard plot, and a 80% increase in Dex-treated cultures, respectively. There were minimal effects on receptor Kd, as indicated by the slope of the lines of best fit in the Scatchard plot, in either case. Treatment with Dex and sCT together resulted in essentially no change in receptor number and a minimal change in receptor Kd. None of the treatments was associated with alteration in the number of tartrate-resistant acid phosphatase-positive cells in the OCL cultures, as we have shown previously (7, 12).



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  		Figure 2. Effect of Dex and/or sCT treatment on [125I]sCT binding sites and affinity in OCLs. OCLs 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 24 h. Cells previously treated with Dex were resupplemented with Dex (10-7 M). Twenty-four hours after removal of sCT, OCLs were washed with PBS, and cells were subjected to competitive binding experiments with a fixed amount of [125I]sCT and increasing amounts of unlabeled sCT. The data were examined by Scatchard analysis. Each point represents the mean values for four wells. Open circle, Untreated (control) cells [solid line; Kd = 0.39 nM; maximum binding (Bmax) = 2.0 x 106 sites/mononuclear cell]; closed circle, sCT-treated cells (Kd = 0.35 nM; Bmax = 0.95 x 106); open square, Dex-treated cells (Kd = 0.41 nM; Bmax = 3.6 x 106); closed square, Dex- and sCT-treated cells (dotted line; Kd = 0.40 nM; Bmax = 1.9 x 106). This experiment is representative of three separate experiments in which similar results were obtained.

 
Effects of Dex and sCT pretreatment on sCT-stimulated cAMP production in OCLs
We next examined the effects of Dex and sCT pretreatment on sCT-responsive adenylate cyclase activity to determine whether the effects observed on binding capacity would relate to the biological response of adenylate cyclase activity in OCLs. OCLs 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, OCLs 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 hours later, cells were examined for the adenylate cyclase response to sCT (10-9 M) for 20 min at 37 C. As shown in Fig. 3Go, 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 ~2-fold; preincubation with sCT (10-9 M) for 1 h decreased the response to ~30% of that in control cells 24 h after sCT removal. When OCLs 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.

Effects of Dex and sCT treatment on CTR mRNA expression in OCLs
To study the effects of Dex and sCT treatment on CTR mRNA expression, we applied a semiquantitative RT-PCR procedure, as previously described (16, 26). 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 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 effects of Dex treatment on CTR mRNA expression, OCLs 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 around 12 h of exposure, after which the increased levels of CTR mRNA expression gradually decreased up to 48 h of exposure (Fig. 4Go).



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  		Figure 4. Effect of Dex on CTR mRNA expression in OCLs. OCLs were treated with Dex (10-7 M) for various periods of time. Total RNA was extracted from 0–48 h after the addition of Dex. 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 are shown above as the ratio of CTR/GAPDH; the values were compared with those of control OCLs at 0 h (the value of 1.0). C, Control OCLs (open circles); D, Dex-treated OCLs (closed circles). This experiment is representative of three separate experiments in which similar results were obtained.

 
sCT treatment of OCLs was recently found to result in a sustained decrease in CTR mRNA levels (12, 30). Thus, we next examined the effect of cotreatment with Dex and sCT on CTR mRNA expression in OCLs. OCLs 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, OCLs 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 0–48 h after sCT removal. Treatment with Dex enhanced CTR mRNA expression in OCLs, whereas sCT treatment for 1 h resulted in a subsequent decrease in CTR mRNA levels in both control and Dex-treated cultures (Fig. 5Go). However, in the Dex-treated cells, the decrease in CTR mRNA levels was somewhat delayed compared with that in control cells. 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 even by 48 h after sCT removal.



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  		Figure 5. Effects of sCT and/or Dex treatment on CTR mRNA expression in OCLs. OCLs 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, OCLs 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 OCLs (the value of 1.0). Control, Control OCLs not treated with Dex or 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.

 
We have shown previously that up-regulation of CTR by Dex was specific for GCs and was not reproduced by mineralocorticoids or sex steroids (7). We then examined whether increased CTR mRNA expression by Dex is specific for GCs. Enhancement of CTR mRNA by Dex was reproduced by other GCs, triamcinolone and prednisolone (not shown), but not by other mineralocorticoids or sex steroids (aldosterone, progesterone, 17ß-estrogen, and dehydroepiandrosterone; Fig. 6Go).



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  		Figure 6. Effects of various steroid hormone and sCT on CTR mRNA expression in OCLs. OCLs were treated with triamcinolone (10-7 M), sCT (10-9 M), progesterone (10-7 M), dehydroepiandrosterone (DHEA; 10-7 M), aldosterone (10-7 M), and 17ß-estradiol (10-7 M) for 24 h. The 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 OCLs (the value of 1.0). This experiment is representative of three separate experiments in which similar results were obtained.

 
Effects of Dex and sCT treatment on CTR mRNA stability in OCLs
To investigate the mechanism of regulation of CTR gene expression in the above experiments, the half-life of mRNA was examined using the RNA polymerase II inhibitor DRB, which inhibits ongoing transcription. OCLs 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 sCT, cells were washed thoroughly with PBS. The media were replaced with fresh growth media containing 10% FBS, at which time DRB (25 µg/ml) was added to the culture media. Cells previously treated with Dex were resupplemented with Dex, and total RNA was extracted at various time intervals for RT-PCR analysis. These experiments yielded the following information. Firstly, the calculated half-life of CTR mRNA was ~13.5 h in Dex-treated OCLs compared with ~14 h in control cells (Fig. 7BGo). Based on these data, it appears that Dex treatment did not enhance CTR mRNA by increasing its stability. Secondly, although previously and in the present experiments we have shown that sCT treatment of OCLs resulted in a rapid decrease in CTR mRNA levels, when DRB was added to sCT-treated cultures, the inhibitory effect of sCT on CTR mRNA expression was abolished, and the calculated t1/2 was the same as that in non-sCT-pretreated cells. This was also true for OCLs treated with sCT in the presence of Dex. Thirdly, as shown in Fig. 7CGo, direct comparison of cells treated or untreated with DRB underscores the protective effect of DRB on CTR mRNA. In the absence of DRB, treatment with sCT markedly reduced CTR mRNA expression in OCLs, in the presence or absence of Dex, 12 h after the removal of sCT. In contrast, there was no difference between control and sCT-treated cells or between Dex-treated and Dex- and sCT-treated cells in the presence of DRB.



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  		Figure 7. Effect of Dex and sCT on CTR mRNA stability in OCLs. OCLs 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 1-h treatment with sCT, cells were washed with PBS, and the media was replaced with fresh growth media, at which time DRB (25 µg/ml) was added to the culture. Cells previously treated with Dex were resupplemented with Dex (10-7 M), and total RNA was extracted at various time intervals. RNA was reverse transcribed and subjected to PCR using specific primers. Products were verified with specific internal oligonucleotides as described in Materials and Methods. The autoradiograph signals (A) were quantitated and are shown in B. The intensities of the signals were compared with the control at time zero (the value of 1.0) and are displayed as a semilog plot on the y-axis. C shows the results for 12 h samples, with or without DRB, expressed as the ratio of CTR/GAPDH signals. The values were compared with those of control OCLs treated with neither Dex nor sCT (the value of 1.0). A: Cont, OCLs not treated with Dex or sCT; Dex, OCLs treated with Dex; sCT, OCLs treated with sCT; Dex+sCT, OCLs treated with Dex and sCT. DRB(+), OCLs treated with DRB; DRB(-), OCLs not treated with DRB. B: Open circle, OCLs not treated with sCT or Dex (dotted line); closed square, OCLs treated with sCT; closed circle, OCLs treated with Dex; open square (dotted line), OCLs treated Dex and sCT. This experiment is representative of three separate experiments in which similar results were obtained.

 
Effects of Dex and sCT treatment on the transcriptional rate of CTR gene in OCLs
To further test the mechanism of regulation of CTR mRNA levels by Dex (10-7 M) and sCT (10-9 M), the transcription rate of the gene was examined by nuclear run-on assay. OCLs were inoculated into large flasks (the number of multinuclear OCL was ~500,000/flask). OCLs were allowed to settle for 2 h in {alpha}MEM containing 10% FBS before the addition of Dex and sCT. The cell nuclei were isolated after 3-h exposure to Dex and sCT, and then the newly transcribed RNA was quantitated with CTR and GAPDH cDNAs as described in Materials and Methods. There was an increase in the transcription rate of the CTR gene after Dex treatment (Fig. 8Go), although the increased magnitude caused by Dex treatment was variable (6.75 ± 3.86-fold; n = 4; P < 0.01 vs. control). Treatment with sCT, on the other hand, did not inhibit the transcription rate of the CTR gene (1.30 ± 0.50-fold; n = 4; not significantly different from control). There was no specific hybridization of the newly transcribed RNA to the vector sequence.



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  		Figure 8. Effect of Dex and sCT treatment on transcriptional activity of CTR gene in OCLs. OCLs (~500,000 MNC/flask) were treated with sCT (10-9 M) and Dex (10-7 M) for 3 h in {alpha}MEM containing 10% FBS. The cell nuclei were isolated, and the newly transcribed RNA was quantitated with CTR and GAPDH cDNAs 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 OCLs (the value of 1.0). Cont, Control OCLs not treated with Dex or sCT; Dex, Dex-treated OCLs; sCT, sCT-treated OCLs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown here that GCs enhanced, and CT attenuated, the [125I]sCT specific binding capacity in mouse OCLs. Both effects were mediated by modulation of CTR mRNA expression in OCLs. The effect of Dex on CTR mRNA expression was time dependent and evident by 6 h, and the maximum enhancement of gene expression was observed around 12 h of exposure. This effect was specific to GCs and was not reproduced by mineralocorticoids or sex steroids, consistent with the results of our previous binding studies (7). The interaction of Dex and CT, with respect to regulation of OCL CTR expression, was studied by pretreating cultures with Dex. Under those circumstances, Dex pretreatment delayed the down-regulation of [125I]sCT binding in OCLs induced by sCT; however, even in the presence of GC, treatment with sCT for 1 h eventually resulted in a reduced [125I]sCT-binding capacity compared with the control value. Scatchard plots indicated that neither agent, alone or in combination, changed the affinity of CT binding. Dex and sCT pretreatment changed the sCT-responsive adenylate cyclase activity in a manner qualitatively similar to the change in receptor binding, suggesting that these changes were primarily mediated by changes in CTR expression, rather than other components of the signal transduction pathway. Consistent with this, treatment with sCT also resulted in a marked reduction of CTR mRNA expression, whereas Dex treatment increased expression. In OCLs cotreated with Dex and sCT, the rate of reduction of mRNA levels was delayed considerably compared with that in cells treated with sCT alone.

The results presented here provide some insight into the mechanisms of regulation of the mouse OCL CTR by CT and GC. Treatment of cultures with CT for 1 h resulted in a subsequent rapid and profound decrease in CTR mRNA, as assessed by RT-PCR, so that by 12 h after CT was removed from the cultures, CTR mRNA was reduced to very low levels. This is consistent with our previous experiments performed according to the same protocol, in which the half-time of CTR mRNA reduction in CT-treated OCLs was ~6 h; that in control cultures was ~24-36 h (12). In addition, we have previously shown that CTR mRNA levels can be reduced in OCLs by ligand-independent activation of PKA and that the CT-induced down-regulation of mRNA was cAMP mediated (16). The current data show that the CT-induced reduction of mRNA could not be accounted for by decreased transcription of the CTR gene, according to nuclear run-on assay, suggesting that CT treatment led to a destabilization of the CTR mRNA. However, when transcription was inhibited using DRB, CTR mRNA decayed at the same rate in CT-treated cells as in control cells.

Taken together, these findings are remarkably similar to the regulation of the ß2-AR seen in several cell systems (19, 20, 21). Steady state levels of ß2AR protein and mRNA have been shown to decline in cells stimulated with ß-adrenergic agonists (17), and PKA activation was essential for the agonist-promoted down-regulation of receptor mRNA levels (31). Also consistent with the results for the OCL CTR was that destabilization of the ß2AR mRNA, rather than a reduced rate of transcription, appears to be primarily responsible for the ligand-mediated receptor regulation.

It is now well established that many mRNAs are regulated at the level of stability. Notable examples include the mRNA of various cytokines, for example, granulocyte-macrophage colony-stimulating factor (32) and interleukin-3 (33), and the oncogenes c-myc (34) and c-fos (35, 36). Cognate sequences in the 3'UTR of mRNA have been shown to function as signals for rapid mRNA degradation. These include A/U-rich elements (AREs), in which the core pentamer AUUUA (19, 20, 37) or the larger UUAUUUA(U/A)(U/A) (38) have been variously implicated as influencing mRNA degradation. In the case of the ß2AR, AREs in the 3'UTR have been shown to bind a number of cytosolic proteins in the size range 30–40 kDa (19, 20, 21). Similarly, in several systems, ARE-binding proteins of 30–45 kDa have been recently characterized (39, 40, 41). Although the role of these mRNA-binding proteins is not well established, by analogy with in vitro decay of c-myc mRNA (42), it is speculated that binding contributes to accelerated mRNA degradation (21). Moreover, agonist treatment of ß2AR-bearing cells resulted in an increase in the levels of certain of these proteins, suggesting that the receptor and mRNA-binding proteins are inversely regulated. Indications that A/U-rich motifs might be more widely important in regulating the stability of mRNAs encoding G protein-coupled receptors were, firstly, that the 3'UTRs of a number of these receptor mRNAs contain one or more AUUUA pentamers (20). Secondly, the hypothesis that the presence of this motif might be predictive of a regulated mRNA was tested using the thrombin receptor mRNA, which contains five copies of AUUUA (20). Evidence was obtained for agonist- and cAMP-mediated posttranscriptional regulation of the thrombin receptor mRNA, and moreover, it was observed that the ß2AR mRNA-binding protein recognized and bound to the thrombin receptor mRNA (20). A possible relationship between 3'UTR sequences in the CTR mRNA and binding of cytosolic proteins remains to be tested. However, it is interesting that the mouse and rat CTR mRNAs contain four AUUUA motifs in the 3'UTR, as well as other A/U-rich domains, including AUUUC and a large number of poly-U regions (Table 1Go). We, therefore, propose that CT binding might reduce CTR mRNA levels in OCLs by a mechanism involving increased levels or activation of A/U-rich mRNA-binding proteins. That these mechanisms are cell specific is indicated by experiments in nonosteoclastic cells (12, 43), in which the rat CTR underwent homologous down-regulation, not mediated by PKA and not involving modulation of CTR mRNA levels (43). The loss of CT-induced effects on mRNA stability in the presence of DRB is consistent with results in other systems in which mRNA regulation is achieved by destabilization. For example, destabilization of human tissue factor mRNA was not seen in the presence of actinomycin D, indicating that the destabilization factor(s) required ongoing transcription for activity (35, 44, 45).


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  		Table 1. Uridine richness in the 3'-UTR region of the various cloned CTRs

 
Treatment of OCL cultures with Dex resulted in enhanced CTR mRNA levels, which peaked by 12 h of Dex treatment. Dex treatment resulted in a consistent increase in CTR gene transcription, although of variable magnitude, as determined by nuclear run-on. According to mRNA stability in the presence of DRB, Dex did not greatly influence the turnover of CTR mRNA. If anything, Dex treatment slightly decreased the stability of CTR mRNA, and it thus appears that Dex does not enhance CTR mRNA by an effect on stability. However, as discussed above, interpretation of mRNA turnover studies in the presence of transcriptional inhibitors might be complicated by concurrent effects on levels of mRNA-destabilizing proteins. Interestingly, in ß2AR-bearing DDT1-MF2 cells, Dex concurrently up-regulated ß2AR mRNA and down-regulated ß2AR mRNA-binding protein (19), which recognizes A/U-rich elements in the 3'UTR. Dex treatment of OCL cultures considerably delayed the reduction by CT of CTR mRNA levels, although eventually (by 24 h) mRNA levels reflected the action of CT. This suggests that the action of CT to destabilize the CTR mRNA predominates over increased transcription by GC.

It remains to be determined whether the effects of Dex on OCL CTR expression are direct or indirect via osteoblasts or other stromal cells in the cocultures. It is possible, for example, that the effect is mediated by a cytokine(s) produced and secreted by these other cell types (46). We have recently shown, for example, that GCs up-regulated the interleukin-6 receptor in osteoblasts, which, in turn, promoted osteoclast differentiation in the presence of interleukin-6 (47). With respect to the present study, there was no difference in the number of multinucleated osteoclast-like cells between control and GC-treated cultures. Future isolation of the promoter region of the mouse CTR gene will greatly assist investigation of its regulation in various cell types.

In conclusion, the results obtained in this study suggest that GCs play an important role to modulate the regulation of CTR gene expression in OCLs. As GCs showed modulation of CT-induced homologous CTR down-regulation, it is possible that these results might have clinical implications. This study also shows that the CT-induced decrease in CTR mRNA in OCLs is mediated by increased CTR mRNA destabilization. Future studies will focus on the cellular factors responsible.


   Acknowledgments
 
We acknowledge helpful discussions and suggestion with Drs. Matthew T. Gillespie and Kathy Traianedes.


   Footnotes
 
1 This work was supported by grants from the National Health and Medical Research Council of Australia. Back

2 C. R. Roper Research Fellow, University of Melbourne (Melbourne, Australia). Back

3 Senior Research Fellow of National Health and Medical Research Council of Australia. Present address: Department of Orthopedic Surgery and Trauma, Royal Adelaide Hospital, Adelaide, South Australia 5001, Australia. Back

Received August 27, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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