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Endocrinology Vol. 141, No. 10 3774-3782
Copyright © 2000 by The Endocrine Society

ARTICLES

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

Atsuyoshi Samura, Seiki Wada, Satoru Suda, Makoto Iitaka and Sigehiro Katayama

The Fourth Department of Internal Medicine, Saitama Medical School, Saitama 350-0495 Japan

Address all correspondence and requests for reprints to: Seiki Wada, M.D., Ph.D., The 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 short exposure to calcitonin (CT) resulted in prolonged reduction of CT binding by inhibiting de novo CT receptor (CTR) synthesis. Additionally, CT-treated OCs demonstrated resistance to CT rechallenge on the inhibitory effect of CT in osteoclastic bone resorption. There is, however, scant information on CT effects on human osteoclasts. In this study, we examined the features of CTR down-regulation and its recovery after short exposure to CT of human OCs. OCs were prepared by treatment of peripheral blood mononuclear cells in vitro with osteoclast differentiation factor and macrophage colony-stimulating factor. Treatment of OCs with salmon CT (sCT) and human CT (hCT) resulted in a dose-dependent reduction in [125I]sCT binding capacity. Continued receptor occupancy by ligand was excluded by using a glycine-acid washing procedure. Treatment with sCT reduced CTR messenger RNA expression, suggesting that CTR down-regulation is, at least partly, attributable to an inhibition of de novo CTR synthesis. To investigate the intracellular signal transduction pathways that mediate these effects, we examined the effects of activation of the protein kinase (PK)A, PKC, and Ca2+-calmodulin-dependent kinases. Treatment with PKC activators mimicked CT, whereas neither activation of PKA nor elevation of intracellular Ca2+ did so. We further investigated the intracellular signaling pathways responsible for the inhibitory effects of CT on bone resorption, which showed that treatment with PKC activators reproduced the effects of CT. These data suggest that the PKC pathway plays an important role in homologous CTR down-regulation, as well as inhibition of bone-resorbing activity by CT, in human OCs. Short exposure of OCs to CT (10-9 M, 1 h) reduced [125l]sCT-specific binding for a prolonged period, as we have shown previously in mouse OCs. The reduced specific binding, CTR messenger RNA levels, and CT-sensitive adenylate cyclase responsiveness returned to the control levels by 96 h after removal of CT. These results strongly support the notion that escape from CT inhibition of osteoclastic bone resorption in humans is attributable to the development of resistance by OCs to CT. This study also showed that even short exposure to CT induced prolonged desensitization to CT rechallenge, although the OCs eventually regained responsiveness to sCT rechallenge.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CALCITONIN (CT) directly inhibits osteoclastic bone resorption and is 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 eventually causes a loss of its inhibitory effects on osteoclastic bone resorption. We and other investigators have studied the mechanism of this escape phenomenon, mostly using mouse and rat osteoclasts (4, 5, 6). The results indicated that escape or desensitization to CT was closely associated with the down-regulation of the CT receptor (CTR). This down-regulation was caused not only by internalization of the receptor (5) but also by reduced cell surface receptor expression through inhibition of de novo CTR synthesis (6, 7). The intracellular signaling responsible for the process of CT-induced homologous CTR down-regulation in mouse osteoclast-like cells (OCs) was activation of the protein kinase (PK)A pathway through cell surface CTR (8). There is, however, scant information on CTR regulation and the mechanism of homologous desensitization to CT, in cells of osteoclast lineage in 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 (9, 10) had proposed a membrane-bound factor that would be expressed on osteoblasts/stromal cells in response to the stimulators of bone resorption and that induces osteoclastogenesis by signaling to osteoclast progenitors. Recently, several groups have succeeded in the molecular cloning of osteoclast differentiation factor (ODF), which mediates an essential signal for osteoclast differentiation from its precursor cells (11). ODF was also found identical to tumor necrosis factor-related activation-induced cytokine (TRANCE) (12) and receptor activator of nuclear factor-{kappa}B ligand (RANKL) (13). Using soluble (s) RANKL and macrophage colony-stimulating factor (M-CSF), Matsuzaki et al. (14) recently developed a new method to prepare human OCs from peripheral blood mononuclear cells, which enables study of the mechanisms that mediate the biological responses to CT in cells of human osteoclast lineage. In this manuscript, we describe the use of human OCs prepared in vitro, to study the regulation of CTR, information which is also important for the optimal use of CT therapy. We report the intriguing finding that the signaling pathway responsible for CTR down-regulation in human OCs is different from that in mouse OCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant human sRANKL was purchased from PEPRO TECH EC Ltd. (London, UK). Recombinant human M-CSF was a kind gift from Morinaga Milk Ind. Co. Ltd. (Tokyo, Japan). Salmon CT (sCT), human CT (hCT), forskolin (FSK), dibutyryl cAMP (dbcAMP), calcium ionophore A23187, isobutylmethylxanthine (IBMX), trypsin-EDTA, {alpha}-MEM, and Histopaque 1077 were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate (PDD), and phorbol monoacetate (PA) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). cAMP assay kits were purchased from Yamasa Shoyu Co. (Chiba, Japan).

Preparation and culture of human OCs
Human OCs were prepared using a slight modification of the method reported by Matsuzaki et al. (14). Briefly, peripheral blood was collected from healthy normal volunteers in syringes containing 1,000 U/ml of 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 7–14 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 (100 ng/ml) and human M-CSF (200 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 CT or other agents for the times indicated. [125I]sCT with a specific activity of approximately 600 mCi/mg was purchased from Amersham Pharmacia Biotech Ltd. (Buckinghamshire, UK). OCs were incubated with CT or other agents in {alpha}MEM containing 10% FCS. Just before the binding experiments, cells were rinsed with acidified buffer (0.15 N NaCl/0.05 M glycine, pH 2.5, for 1 min) to remove CT, which binds to cell surface receptors, and rinsed thoroughly with cold PBS. 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 PBS and dissolved in 0.5 N NaOH, and cell-bound radioactivity was measured. EC50 was calculated from competitive binding studies with a fixed amount of [125I]sCT and increasing amounts of unlabeled sCT.

TRAP staining of the cells was as described previously (15). 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 positive for TRAP was counted.

RNA extraction
Equal aliquots of OCs (~3.0 x 104 multinuclear OCs/dish) were treated with CT or other agents for the time indicated. After washing cells with cold PBS, total RNA was extracted by the acid guanidinium-phenol-chloroform method, as reported previously (16).

PCR amplification of reverse transcribed messenger RNA (mRNA)
The RT of RNA to complementary DNA and the subsequent amplification were performed as described previously (6). 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 MuLV reverse transcriptase, 2.5 µM random hexamers, 1.0 U/µl ribonuclease inhibitor, and 1.0 mM deoxynucleotide triphosphate 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 10x 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 Ltd.) 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 hCTR1, 5'-GCAATGCTTTCACTCCTGAGAAAC-3' (5'-primer); and hCTR2, 5'-CAGTAAACACAGCCACGACAATGAG-3' (3'-primer). The products were verified with the internal sense strand oligonucleotide, 5'-GTTGAAGTAGTACCCAATGGA-3' (hCTR3) 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'.

Bone resorption assay
Bone resorption (pit formation on dentine slices) was assayed as reported previously (17). On days 7–8, OCs in 60-mm dishes (~3.0 x 104 multinuclear OCs/dish) were treated with 0.25% trypsin-EDTA for 10 min. OCs adherent to culture dishes were gently removed by sterile scrapers (Costar, Cambridge, MA). Cells were rinsed with PBS, and equal numbers of the cells (~1000 multinuclear OCs/dentine slice) were resuspended onto dentine slices (4-mm diameter; ~200-µm thick) in {alpha}MEM containing 10% FCS, sRANKL (100 ng/ml), and M-CSF (200 ng/ml) with CT or other agents, as specified. After incubation for 48 h, dentine slices were sonicated to remove the adherent cells in 1.0 N NH4OH. Slices were stained with Mayer’s Hematoxylin solution (Wako Pure Chemical Industries Ltd.) for 2 min, rinsed with distilled water, and then air-dried. Resorption pits visualized by this staining were identified by light microscopy. The total pit areas were measured in four randomly selected areas of each dentine slice using an image analysis system (NIH image version 1.61).

Measurement of cAMP production
OCs (~3.0 x 103 multinuclear OCs/well) were treated with CT or other agents in {alpha}MEM containing 0.1% BSA and 1.0 mM IBMX for 20 min at 37 C. Cellular cAMP production was measured by RIA using a Yamasa RIA kit. Details of this assay were described in a previous report (18).

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


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of CT treatment on [125I]sCT binding in human OCs
In mouse OCs, reduced sensitivity to CT was closely associated with homologous down-regulation of the CTR (6); and in human giant cell tumors, treatment with CT reduced CTR mRNA expression (7). To examine more closely the effects of CT in normal human OCs, cells were treated with various concentrations of sCT and hCT, and then receptor binding capacity was measured. To determine total cell surface CTR, receptor-bound CT was removed by washing cells with isotonic acidified buffer, which we have previously shown removes CT from CTR in mouse OCs without affecting cellular viability (8, 19). This method was originally reported for epidermal growth factor binding studies by Haigler et al. (20). In human OCs, we also found that acid washing can effectively remove CT from its binding sites (Table 1Go) without reducing OCs viability: short treatment (<5 min) with acidified buffer at 4 C did not affect subsequent TRAP activity of OCs examined by cytochemical analysis. Acid washing, after treatment with 10-9 M sCT for 24 h at 37 C, regenerated approximately 67% of the control [125I]sCT-binding capacity, indicating that approximately 33% of cell surface CTR was internalized after sCT treatment (Table 1Go). After treatment with sCT (10-13–10-8 M) and hCT (10-10–10-6 M) for 24 h, cells were rinsed with acidified buffer, and receptor binding capacity for [125I]sCT was measured. As shown in Fig. 1Go, [125I]sCT-specific binding was reduced by exposure of cells to CT, dose dependently. The ED50 values for the effect of CT on CTR down-regulation, calculable from the experiments shown in Fig. 1Go, were 7.5 x 10-11 M for sCT and 3.8 x 10-8 M for hCT. To study more precisely the action of CT treatment on CTR, we examined the binding of [125I]sCT and evaluated the binding capacity with a fixed amount of [125I]sCT and increasing amounts of unlabeled sCT. Cells were treated for 24 h with 10-9 M sCT, rinsed with acidified buffer and PBS thoroughly. OCs were then submitted to competitive binding studies. EC50 values for control and sCT-treated cells were 1.42 x 10-9 M and 2.16 x 10-9 M, respectively (Fig. 2Go). Cell numbers, both mono- and multinuclear cells, were the same in sCT-treated and control groups (data not shown).


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  		Table 1. Effects of acid washing on [125I]sCT-specific binding capacity after sCT treatment

 


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  		Figure 1. Effect of CT treatment on [125I]sCT-specific binding in human OCs. OCs were incubated with sCT and hCT (sCT, 10-13–10-8 M; hCT, 10-10–10-6 M), for 24 h, in {alpha}MEM containing 10% FCS. After removal of the media, cells were rinsed with acidified buffer and PBS, as described in Materials and Methods. Specific binding of [125I]sCT was measured. Open circles, hCT-treated cells; closed circles, sCT-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. Effect of sCT (10-9 M) treatment on [125I]sCT binding and its EC50 in human OCs. OCs were treated ,with or without sCT (10-9 M), for 24 h, in {alpha}MEM containing 10% FCS. After removal of the media, cells were rinsed with acidified buffer and PBS. Binding 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. Open circles, Untreated (control) cells (EC50 = 1.42 x 10-9 M); closed circles, sCT-treated cells (EC50 = 2.16 x 10-9 M). This experiment is representative of three separate experiments in which similar results were obtained.

 
Effects of CT treatment on CTR mRNA expression in human OCs
To examine the action of CT on CTR mRNA expression, we applied a semiquantitative RT-PCR procedure. For the PCR, between 15–35 (the actual cycle number we used for PCR in the following experiments was 32) cycles of amplification were optimal for semiquantitative analysis of mRNA expression for CTR, and between 10–25 (the actual cycle number we used for PCR in the following experiments was 20) cycles were optimal for GAPDH. The amplification was in the exponential phase over these ranges. OCs were treated with sCT for the indicated times, and RNA was extracted from the cells. OCs treated with sCT (10-9 M) for up to 48 h showed a marked decrease of CTR mRNA expression in a time-dependent fashion (Fig. 3Go). Significant reduction of mRNA expression was observed 12 h after treatment with CT, with no recovery of mRNA expression observed over the experimental period.



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  		Figure 3. Effect of sCT on CTR mRNA expression in human OCs. OCs were prepared as described in Materials and Methods (~3.0 x 104 OCs/dish). OCs were treated with sCT (10-9 M) for 4–48 h, and total RNA was extracted. RNA was reverse transcribed and subjected to PCR in 32 cycles for CTR mRNA amplification and 20 cycles for GAPDH amplification, using specific primers, as described in Materials and Methods. PCR products were transferred to a nylon filter and hybridized with 32P-labeled hCTR3, an internal sense oligonucleotide specific to human CTR sequence, and 32P-labeled GAPDH1, specific to human GAPDH sequence. The intensities of autoradiograph signals were quantitated and are shown below as the ratio of CTR/GAPDH compared with control (the value of 1.0). Each point represents the mean ± SD for four PCR products. *, P < 0.01 vs. control cells. This figure is representative of four separate experiments; similar results were obtained in the other three experiments. Cont, Control.

 
Effects of CT and activators of PKA, PKC, and Ca2+-calmodulin-dependent kinase on [125I]sCT-specific binding and CTR mRNA expression in human OCs
Experiments with cloned human (21), rat (22), and porcine (23, 24) CTR have shown that these receptors are capable of coupling to at least two distinct G protein-mediated intracellular signaling pathways, to elevate intracellular Ca2+ and activate PKC, and to elevate intracellular cAMP and activate PKA-mediated pathways. To investigate the intracellular signal transduction pathways that mediate homologous CTR down-regulation and the associated decrease of CTR mRNA expression in human OCs, we examined the effects of activation of PKA, PKC, and Ca2+-calmodulin dependent kinases. The active phorbol esters (PMA and PDD) directly activate PKC (25), whereas the inactive analog PA is incapable of activating the kinase in these cells. To examine the role of the PKA pathway, we used FSK, a direct activator of the catalytic unit of adenylate cyclase (26) and the cAMP analog, dbcAMP. The calcium ionophore (A23187) can elevate intracellular Ca2+ and lead to activation of Ca2+-calmodulin-dependent signaling. As shown in Fig. 4Go, treatment with PMA (10-7 M) and PDD (10-7 M) for 24 h markedly reduced [125I]sCT-specific binding, which mimics the effects of CT. In contrast, treatment of OCs for 24 h with PA (10-7 M), FSK (10-5 M), dbcAMP (10-4 M), or A23187 (10-7 M) had no significant effect on [125I]sCT binding capacity in human OCs. Treatment with these agents in culture for up to 24 h did not affect the number of OCs (data not shown).



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  		Figure 4. Effects of activators of PKC, PKA, and Ca2+-calmodulin-dependent kinase on [125l]sCT-specific binding in human OCs. OCs were treated with sCT (10-9 M), active phorbol esters (PMA, 10-7 M; PDD, 10-7 M), inactive phorbol ester (PA, 10-7 M), FSK (10-5 M), dbcAMP (10-4 M), and calcium ionophore (A23187, 10-7 M) in growth media for 24 h. After removal of the media, cells were thoroughly rinsed with acidified buffer and PBS, as described in Materials and Methods. Specific binding of [125I]sCT was measured as described. Each point represents the mean ± SD for four wells. *, P < 0.01 vs. control cells. Nonspecific binding in these studies was approximately 10% of the total binding. This figure is representative of four separate experiments, and similar results were obtained in the other three experiments.

 
To study the action of CT and activators of PKA, PKC, and Ca2+-calmodulin-dependent kinase on CTR mRNA expression in OCs, we again applied a semiquantitative RT-PCR procedure, as described above. OCs were treated with PMA (10-7 M), PDD (10-7 M), FSK (10-5 M), dbcAMP (10-4 M), and A23187 (10-7 M) for 24 h before extraction of total RNA. Similar to the results obtained in the binding studies, treatment with activators of PKC (PMA and PDD) resulted in a marked decrease of CTR mRNA expression. On the other hand, neither activation of PKA (FSK and dbcAMP) nor Ca2+-calmodulin-dependent kinase (A23187) significantly affected CTR mRNA expression (Fig. 5Go).



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  		Figure 5. Effects of activators of PKC, PKA, and Ca2+-calmodulin-dependent kinase on CTR mRNA expression in human OCs. OCs were prepared as described in Materials and Methods (the number of multinuclear OCs was ~3.0 x 104/dish). OCs were treated with sCT (10-9 M), PMA (10-7 M), PDD (10-7 M), FSK (10-5 M), dbcAMP (10-4 M), and A23187 (10-7 M), for 24 h, in {alpha}MEM containing 10% FCS. After washing with PBS, total RNA was extracted. RNA was reverse transcribed and subjected to 32 cycles of PCR for CTR mRNA amplification and 20 cycles for GAPDH amplification using specific primers, as described in Materials and Methods. PCR products were transferred to a nylon filter and hybridized with 32P-labeled hCTR3, an internal sense oligonucleotide specific to the CTR sequence, and 32P-labeled GAPDH1, specific to the GAPDH sequence. Signals were quantitated and are shown in the lower panel as the ratio of CTR/GAPDH PCR products compared with control (the value of 1.0). Each point represents the mean ± SD for four PCR products. *, P < 0.01 vs. control cells. This figure is representative of three separate experiments in which similar results were obtained.

 
Effects of activation of PKA, PKC, and Ca2+-calmodulin-dependent kinase on bone-resorbing activity (pit formation) in human OCs
In mouse OCs, Suzuki et al. (27) clearly showed that the effect of CT to inhibit bone resorption was predominantly mediated by PKA pathways, whereas the cellular signaling responsible for the process in humans has not been fully investigated. We therefore designed experiments to clarify the intracellular signaling pathways involved in the inhibition of osteoclastic resorption by CT. When the OCs were removed by trypsin from the dishes and then recultured, OCs were still positive for TRAP staining, but they only showed a very minimal resorptive activity in the absence of sRANKL and M-CSF. In this study, therefore, mature OCs were subcultured in growth media containing sRANKL (100 ng/ml) and M-CSF (200 ng/ml), for 48 h, in the absence or presence of sCT (10-9 M), PMA (10-7 M), PDD (10-7 M), FSK (10-5 M), dbcAMP (10-4 M), and A23187 (10-7 M) (~1000 multinuclear OCs/dentine slice). Subsequently the resorbed areas of bone were quantitated, as described in Materials and Methods. The number of cells was assessed by TRAP staining and was not changed by these treatments. Treatment of OCs for 48 h with PKA activators (FSK and dbcAMP) or with A23187 did not inhibit osteoclastic bone-resorbing activity. In contrast, treatment with active phorbols (PMA and PDD) for 48 h inhibited the bone-resorbing function of OCs (Fig. 6Go).



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  		Figure 6. Effects of activators of PKA, PKC, and Ca2+-calmodulin-dependent kinase on pit formation of human OCs. OCs on plastic dishes were treated with 0.25% trypsin-EDTA for 10 min and gently removed with scrapers, and equal numbers of OCs were settled onto dentine slices (~1000 multinuclear OCs/dentine slice). After settlement, the OCs were cultured with sCT (10-9 M), PMA (10-7 M), PDD (10-7 M), FSK (10-5 M), dbcAMP (10-4 M), and A23187 (10-7 M) in growth media containing 10% FCS, sRANKL (100 ng/ml), and M-CSF (200 ng/ml) for 48 h. The area of bone resorbed was quantitated as described in Materials and Methods. Each point represents the mean ± SD of four random fields. *, P < 0.01 vs. control cells. This figure is representative of three separate experiments in which similar results were obtained. Bar, 200 µm.

 
Recovery of [125l]sCT binding capacity and CTR mRNA expression after preincubation of sCT in human OCs
As reported previously (6, 17, 28), even brief treatment with CT affects osteoclast activity for a prolonged period. Using a mouse coculture system, it was reported that the recovery of CTR mRNA and CT binding was not observed until 72 h after short CT treatment (28). However, there is scant information on when and how osteoclasts regain their responsiveness to CT. In this study, we applied a method of coincubation with sRANKL(100 ng/ml) and M-CSF (200 ng/ml), which not only generates OCs from peripheral blood mononuclear cells but also elongates the survival of mature OCs (29). This procedure enables investigation of biochemical features of OCs in culture for prolonged periods. When the OCs were treated with sCT (10-9 M) for 1 h and then rinsed, binding capacity for [125I] sCT was decreased, as shown in Fig. 7Go. The sCT-treated OCs, however, regained binding capacity toward the level of control cells by 96 h after sCT removal. Under the conditions of the experiments reported here, CT treatment had no statistically significant effect on the number of mononuclear or multinuclear OCs. Furthermore, nucleus number per multinucleated cells did not differ significantly between treated and control cells (Fig. 7Go).



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  		Figure 7. Effect of short treatment with sCT (10-9 M, 1 h) on [125l]sCT binding in human OCs. OCs were treated with sCT (10-9 M) for 1 h. After removal of the media, cells were thoroughly rinsed by PBS and incubated up to 7 days in growth media containing 10% FCS, sRANKL (100 ng/ml), and M-CSF (200 ng/ml). Specific binding of [125l]sCT was measured as described in Materials and Methods. Each point represents the mean ± SD for four wells. The values were compared with untreated cells and are shown as percent of control. *, P < 0.01 vs. control (non-sCT treated) cells. Nonspecific binding in these studies was approximately 10% of the total binding. The numbers of mononuclear and multinuclear TRAP-positive cells, along with nucleus number per multinuclear OCs, were shown below. There was no significant difference between the control and sCT-treated cells. Similar results were obtained in the other three experiments.

 
Effects of CT on CTR mRNA expression were also evaluated by short exposure to sCT in the OCs. OCs were treated with sCT (10-9 M, 1 h), rinsed thoroughly with PBS, and further cultured in the growth media containing sRANKL and M-CSF. The total RNA was extracted, at each time point, as described in Materials and Methods. Reduced expression of CTR mRNA was observed around 24–48 h after removal sCT, but the expression of the mRNA returned toward control levels by 72 h after removal of sCT (Fig. 8Go).



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  		Figure 8. Effect of short treatment with sCT (10-9 M, 1 h) on CTR mRNA expression in human OCs. OCs were prepared as described in Materials and Methods (~3.0 x 104 multinuclear OCs/dish). OCs were treated with sCT (10-9 M) for 1 h. After removal of the media, cells were thoroughly rinsed with PBS and further incubated up to 7 days with fresh growth media containing 10% FCS, sRANKL (100 ng/ml), and M-CSF (200 ng/ml). Total RNA was extracted at each time point. RNA was reverse transcribed and subjected to 32 cycles of PCR for CTR mRNA amplification and 20 cycles for GAPDH amplification using specific primers, as described in Materials and Methods. PCR products were transferred to a nylon filter and hybridized with 32P-labeled hCTR3, an internal sense oligonucleotide specific to hCTR sequence, and 32P-labeled GAPDH1, specific to GAPDH sequence. The intensities of autoradiograph signals were quantitated and are shown below as the ratio of CTR/GAPDH compared with control cells (the value of 1.0). Each point represents the mean ± SD for four PCR products. *, P < 0.01 vs. control cells. This figure is representative of four separate experiments; similar results were obtained in the other three experiments.

 
cAMP responses to CT rechallenge in human OCs
To investigate the recovery process and sensitivity to CT rechallenge in the OCs, we measured cAMP production in CT-pretreated OCs. OCs were pretreated with sCT (10-9 M) for 1 h, rinsed thoroughly with PBS, and cultured with fresh growth media containing sRANKL and M-CSF. Basal and sCT (10-9 M)-stimulated cAMP production was measured on days 0, 1, 4, and 7 after removal of sCT. In control cells, treatment with CT in OCs increased adenylate cyclase response, as shown in Fig. 9Go. Twenty-four hours after the removal of sCT, OCs showed desensitization to a rechallenge with sCT. Ninety-six hours after the removal of sCT, responsiveness to CT returned. However, full responsiveness of sCT-pretreated OCs to the rechallenge with CT required longer than 96 h and was seen at 7 days after removal of sCT (Fig. 9Go).



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  		Figure 9. Effect of preincubation with sCT (10-9 M, 1 h) on sCT-responsive cAMP production in human OCs. OCs were preincubated with sCT (10-9 M) for 1 h. After removal of the media, cells were thoroughly rinsed with PBS and incubated up to 7 days. At the times indicated, OCs were rechallenged with sCT (10-9 M), for 20 min, in the presence of IBMX. Basal and sCT (10-9 M)-stimulated cAMP production was measured. cAMP production was assayed, by RIA, as described in Materials and Methods. Each point represents the mean ± SD for four wells. *, P < 0.05; **, P < 0.01 vs. control (nonpreincubated) cells treated with sCT. This figure is representative of three separate experiments, all of which yielded similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study confirms our earlier work, where we found that treatment of OCs with CT induced prolonged homologous CTR down-regulation, decline in CTR mRNA expression, and desensitization of CT-stimulated adenylate cyclase activity (6). Concentrations of sCT or hCT, which did not acutely reduce CTR binding capacity, could nevertheless induce homologous down-regulation of the CTR. In the present work, we applied an acid pH washing procedure to exclude the possibility that continuous treatment with CT for 24 h simply occupied cell surface CTR rather than decreasing their concentration. Along with reduced CTR mRNA expression, a decreased receptor number after acid washing clearly indicated that the decreased de novo CTR synthesis is responsible for the homologous CTR down-regulation in human OCs. The mechanisms of homologous down-regulation of peptide hormone receptors have been studied in various cells and tissues. In general, the number of functional receptors expressed on the cell surface is regulated by several mechanisms, including modulation of receptor expression, partitioning of functional receptors between the cell surface and internal pools (sequestration), and degradation of receptor protein. Experiments to explore the molecular basis of the CTR gene expression in osteoclasts suggested that CT down-regulate CTR by inhibiting transcription of CTR gene expression (30) and/or by reducing stability of CTR mRNA (31). The effect of CT on CTR mRNA stability 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 (32). Multiple copies of the AUUUA motif have been identified in the 3' untranslated regions of CTR gene in various species (31), including the human (33).

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 (19, 34, 35). Similar results have been observed in rat and mouse osteoclasts (4, 6), suggesting that the basic relationships of ligand-receptor interactions of CT are conserved among cell types and different species. The results obtained from the studies of cells transfected with clonal CTR showed that CTRs couple to effector systems that lead to activation of PKA, PKC, and pathways mediated by elevation of intracellular Ca2+ (21, 22, 23, 24). Although transfected studies are beneficial to understanding basic receptor biology, e.g. receptor-ligand recognition and subsequent induction of intracellular events, there is a potential limitation that they may not always reflect all aspects of receptor biology, such as physiological receptor regulation, as they occur in target cells. 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-resorbing activity (36), although some of the important aspects would be conserved irrespective of the cell types (37).

In the present study, treatment of OCs with activators of PKC mimicked the effect of CT on CTR down-regulation and CTR mRNA expression, whereas neither activation of PKA nor elevation of intracellular Ca2+ did so. These data suggest that the PKC pathway is primarily involved in homologous down-regulation of the CTR in human OCs. The effect of CT on inhibition of bone resorption was also found to be mediated predominantly through a PKC-dependent signaling pathway in the cells (Fig. 6Go). In rat osteoclasts, the inhibitory effect of CT was shown to be mediated by both PKA- and PKC-mediated signaling (36, 38): activation of the PKC pathway induced rapid osteoclast retraction, and that of the PKA pathway inhibited subsequent osteoclast motility. In mouse OCs, it was shown, however, that CT inhibition of F-actin ring formation and bone resorption was mediated by PKA rather than PKC-mediated signaling (27), results which were also consistent with our work on the intracellular signaling responsible for homologous CTR regulation in mouse OCs (8). The results obtained in human OCs were contrary to those studies: the action of CT on human OCs seem to occur predominantly through activation of a PKC-dependent cascade, leading to homologous CTR down-regulation and inhibition of bone resorption. It has been demonstrated that peptide hormones act on target tissues in cell- and species-specific ways in various experimental models. In brain tissue, CT apparently causes either no change, or an inhibition, of adenylate cyclase activity (39). Nevertheless, CTRs cloned from brain are capable of coupling to stimulatory GTP-binding protein when they are expressed in other cell types (22). In hepatocytes, CT prevented CCl4-induced oxyradical formation and cellular damage by a mechanism involving activation of PKC (40). In LLC-PK1 pig kidney cells, intracellular signaling varied in a cell cycle-dependent manner (41). It was shown recently that CT induced Shc phosphorylation and activation of Erk1/2, a serine/threonine PK in human embryonic kidney cells transfected with C1a isoform of the rabbit CTR (42). CT also phosphorylated human enhancer of filamentation 1 (HEF1), a p130Cas-like docking protein, which is involved in the modulation of cell attachment and cytoskeletal function (37). These signaling networks were found to be the downstream target of PKC and an elevation of intracellular Ca2+ but were largely independent of PKA (37, 42).

CT has been used to treat malignancy-associated hypercalcemia, Paget’s disease of bone and osteoporosis (1, 2, 3). Usage of CT in osteoporotic patients varies among the protocols: daily CT, given intranasally (43, 44) or im (45, 46), has shown its efficacy for treatment, whereas the other studies showed that im injection of CT only once or twice a week could achieve significant improvement of bone mineral density (47). It has been shown, however, that the effects of CT were diminished by repetitive administration, which has been shown in earlier clinical studies (48), as well as in vitro studies (49). Although CT potently inhibits osteoclastic bone-resorbing activity, OCs exposed to CT showed resistance or refractoriness to the rechallenge with CT, which was parallel to the homologous CTR down-regulation (6). There are also some reports showing that the generation of neutralizing antibodies to CT may relate to the refractoriness to the rechallenge with CT (50), although the contribution of antibodies to the so-called escape phenomenon remains to be fully elucidated. Because it had been difficult to culture osteoclasts for a long period of time, recovery of cell surface CTR expression and changes of sensitivity to CT rechallenge have not been examined in osteoclasts. Jimi et al. (29) recently found that treatment with sRANKL and M-CSF could maintain viability of OCs and elongate survival of the cells. In the presence of sRANKL and M-CSF, the OCs pretreated with CT were cultured up to 14 days, to examine biochemical features of CTR. When human OCs were treated with sCT (10-9 M) for 1 h and then rinsed, binding capacity for [125I] sCT was immediately reduced, but the binding capacity gradually returned toward the level of control cells by 96 h after sCT removal (Fig. 7Go). The recovery was also confirmed at the level of CTR mRNA expression and CT-sensitive adenylate cyclase responses. These results indicated that OCs pretreated with CT could regain responsiveness to CT rechallenge eventually. Intermittent administration of CT might be effective to attenuate desensitization to CT, which occurred in CT target tissues.

In conclusion, this study shows that the intracellular signaling pathway responsible for homologous regulation of CTR and inhibition of osteoclastic bone resorption in human OCs is the PKC pathway. Our results also suggest that, in human OCs, even short exposure to CT induced prolonged desensitization to CT rechallenge, which recovers gradually. This may suggest that intermittent administration of CT would be effective for the treatment of osteoporosis, resulting in reduced desensitization in CT target cells.


   Acknowledgments
 
The authors are grateful for excellent technical assistance by Ms. Keiko Nagatani and for scientific advice by Drs. Shigemitsu Yasuda and Shinji Kitahama.


   Footnotes
 
1 This work was supported, in part, by Grant No. 10770558 from the Ministry of Education, 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 (Grant No. 23566) from Japan Health Sciences Foundation, and Casio Science Promotion Foundation. Back

Received March 20, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Doftos LJ 1993 Calcitonin. In: Favus MJ (ed) Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, ed 2. Raven Press, New York, pp 70–76
  2. Martin TJ 1999 Calcitonin, an update. Bone 24:63S–65S
  3. Sexton PM, Findlay DM, Martin TJ 1999 Calcitonin. Curr Med Chem 6:1069–1093
  4. Nicholson GC, Moseley JM, Yates AJ, Martin TJ 1987 Control of cyclic adenosine 3',5'-monophosphate production in osteoclasts: calcitonin-induced persistent activation and homologous desensitization of adenylate cyclase. Endocrinology 120:1902–1908[Abstract]
  5. Ikegame M, Ejiri S, Ozawa H 1994 Histochemical and autoradiographic studies on elcatonin internalization and intracellular movement in osteoclasts. J Bone Miner Res 9:25–37[Medline]
  6. Wada S, Martin TJ, Findlay DM 1995 Homologous regulation of the calcitonin receptor in mouse osteoclast-like cells and human breast cancer T47D cells. Endocrinology 136:2611–2621[Abstract]
  7. Takahashi S, Goldring S, Katz M, Hilsenbeck S, Williams R, Roodman GD 1995 Down-regulation of calcitonin receptor mRNA expression by calcitonin during human osteoclast-like cell differentiation. J Clin Invest 95:167–171
  8. Wada S, Udagawa N, Nagata N, Martin TJ, Findlay DM 1996 Physiological levels of calcitonin regulate the mouse osteoclast calcitonin receptor by a protein kinase A-mediated mechanism. Endocrinology 137:312–320[Abstract]
  9. Suda T, Takahashi N, Martin TJ 1992 Modulation of osteoclast differentiation. Endocr Rev 13:66–80[CrossRef][Medline]
  10. Takahashi N, Udagawa N, Suda T 1999 A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function. Biochem Biophys Res Commun 256:449–455[CrossRef][Medline]
  11. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Higashio K, Udagawa N, Takahashi N, Suda T 1998 Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 95:3597–3602[Abstract/Free Full Text]
  12. Wong BR, Rho J, Arron J, Robinson E, Orlinick J, Chao M, Kalachikov S, Cayani E, Bartlett III FS, Frankel WN, Lee SY, Choi Y 1997 TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells. J Biol Chem 272:25190–25194[Abstract/Free Full Text]
  13. Anderson DM, Maraskovsky E, Billingsley WL, Dougall WC, Tometsko ME, Roux ER, Teepe MC, DuBose RF, Cosman D, Galibert L 1997 A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function. Nature 390:175–179[CrossRef][Medline]
  14. Matsuzaki K, Udagawa N, Takahashi N, Yamaguchi K, Yasuda H, Shima N, Morinaga T, Toyama Y, Yabe Y, Higashio K, Suda T 1998 Osteoclast differentiation factor (ODF) induces osteoclast-like cell formation in human peripheral blood mononuclear cell cultures. Biochem Biophys Res Commun 246:199–204[CrossRef][Medline]
  15. Wada S, Akatsu T, Tamura T, Takahashi N, Suda T, Nagata N 1994 Glucocorticoid regulation of calcitonin receptor in mouse osteoclast-like multinucleated cells. J Bone Miner Res 9:1705–1712[Medline]
  16. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  17. Wada S, Udagawa N, Nagata N, Martin TJ, Findlay DM 1996 Calcitonin receptor down-regulation relates to calcitonin resistance in mature mouse osteoclasts. Endocrinology 137:1042–1048[Abstract]
  18. Wada S, Yasutomo Y, Kosano H, Kugai N, Nagata N 1991 The effect of PGF2{alpha} on parathyroid hormone-stimulated cyclic AMP production in mouse osteoblastic cell, MC3T3E1. Biochim Biophys Acta 1074:182–188[Medline]
  19. Findlay DM, Martin TJ 1984 Relationship between internalization and calcitonin-induced receptor loss in T 47D cells. Endocrinology 115:78–83[Abstract]
  20. Haigler HT, Maxfield FR, Willingham MC, Pastan I 1980 Dansylcadaverine inhibits internalization of 125I-epidermal growth factor in BALB 3T3 cells. J Biol Chem 255:1239–1241[Abstract/Free Full Text]
  21. Gorn AH, Lin HY, Yamin M, Auron PE, Flannery MR, Tapp DR, Manning CA, Lodish HF, Krane SM, Goldring SR 1992 Cloning, characterization, and expression of a human calcitonin receptor from an ovarian carcinoma cell line. J Clin Invest 90:1726–1735
  22. Houssami S, Findlay DM, Brady CL, Myers DE, Martin TJ, Sexton PM 1994 Isoforms of the rat calcitonin receptor: consequences for ligand binding and signal transduction. Endocrinology 135:183–190[Abstract]
  23. Chabre O, Conklin BR, Lin HY, Lodish HF, Wilson E, Ives HE, Catanzariti L, Hemmings BA, Bourne HR 1992 A recombinant calcitonin receptor independently stimulates 3',5'-cyclic adenosine monophosphate and Ca2+/inositol phosphate signaling pathways. Mol Endocrinol 6:551–556[Abstract]
  24. Findlay DM, Houssami S, Lin HY, Myers DE, Brady CL, Darcy PK, Ikeda K, Martin TJ, Sexton PM 1994 Truncation of the porcine calcitonin receptor cytoplasmic tail inhibits internalization and signal transduction but increases receptor affinity. Mol Endocrinol 8:1691–1700[Abstract]
  25. Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, Nishizuka Y 1982 Direct activation of calcium-activated, phospholipid-dependent protein kinase by tumor-promoting phorbol esters. J Biol Chem 257:7847–7851[Abstract/Free Full Text]
  26. Seamon KB, Padgett W, Daly JW 1981 Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc Natl Acad Sci USA 78:3363–3367[Abstract/Free Full Text]
  27. Suzuki H, Nakamura I, Takahashi N, Ikuhara T, Matsuzaki K, Isogai Y, Hori M, Suda T 1996 Calcitonin-induced changes in the cytoskeleton are mediated by a signal pathway associated with protein kinase A in osteoclasts. Endocrinology 137:4685–4690[Abstract]
  28. Rakopoulos M, Ikegame M, Findlay DM, Martin TJ, Moseley JM 1995 Short treatment of osteoclasts in bone marrow culture with calcitonin causes prolonged suppression of calcitonin receptor mRNA. Bone 17:447–453[Medline]
  29. Jimi E, Akiyama S, Tsurukai T, Okahashi N, Kobayashi K, Udagawa N, Nishihara T, Takahashi N, Suda T 1999 Osteoclast differentiation factor acts as a multifunctional regulator in murine osteoclast differentiation and function. J Immunol 163:434–442[Abstract/Free Full Text]
  30. Inoue D, Shih C, Galson DL, Goldring SR, Horne WC, Baron R 1999 Calcitonin-dependent down-regulation of the mouse C1a calcitonin receptor in cells of the osteoclast lineage involves a transcriptional mechanism. Endocrinology 140:1060–1068[Abstract/Free Full Text]
  31. Wada S, Udagawa N, Akatsu T, Nagata N, Martin TJ, Findlay DM 1997 Regulation by calcitonin and glucocorticoids of calcitonin receptor gene expression in mouse osteoclasts. Endocrinology 138:521–529[Abstract/Free Full Text]
  32. Pende A, Tremmel KD, DeMaria CT, Blaxall BC, Minobe WA, Sherman JA, Bisognano JD, Bristow MR, Brewer G, Port J 1996 Regulation of the mRNA-binding protein AUF1 by activation of the beta-adrenergic receptor signal transduction pathway. J Biol Chem 271:8493–8501[Abstract/Free Full Text]
  33. Kuestner RE, Elrod RD, Grant FJ, Hagen FS, Kuijper JL, Matthewes SL, O’Hara PJ, Sheppard PO, Stroop SD, Thompson DL, Whitmore TE, Findlay DM, Houssami S, Sexton PM, Moore EE 1994 Cloning and characterization of an abundant subtype of the human calcitonin receptor. Mol Pharmacol 46:246–255[Abstract]
  34. Lamp SJ, Findlay DM, Moseley JM, Martin TJ 1981 Calcitonin induction of a persistent activated state of adenylate cyclase in human breast cancer cells (T 47D). J Biol Chem 256:12269–12274[Free Full Text]
  35. Findlay DM, Martin TJ 1986 Kinetics of calcitonin receptor internalization in lung cancer (BEN) and osteogenic sarcoma (UMR 106–06) cells. J Bone Miner Res 1:277–283[Medline]
  36. Zaidi M, Datta HK, Moonga BS, MacIntyre I 1990 Evidence that the action of calcitonin on rat osteoclasts is mediated by two G proteins acting via separate post-receptor pathways. J Endocrinol 126:473–481[Abstract]
  37. Zhang Z, Hernandez-Lagunas L, Horne WC, Baron R 1999 Cytoskeleton-dependent tyrosine phosphorylation of the p130(Cas) family member HEF1 downstream of the G protein-coupled calcitonin receptor. Calcitonin induces the association of HEF1, paxillin, and focal adhesion kinase. J Biol Chem 274:25093–25098[Abstract/Free Full Text]
  38. Su Y, Chakraborty M, Nathanson MH, Baron R 1992 Differential effects of the 3',5'-cyclic adenosine monophosphate and protein kinase C pathways on the response of isolated rat osteoclasts to calcitonin. Endocrinology 131:1497–1502[Abstract]
  39. Yamin M, Gorn AH, Flannery MR, Jenkins NA, Gilbert DJ, Copeland NG, Tapp DR, Krane SM, Goldring SR 1994 Cloning and characterization of a mouse brain calcitonin receptor complementary deoxyribonucleic acid and mapping of the calcitonin receptor gene. Endocrinology 135:2635–2643[Abstract]
  40. Chen S, Morimoto S, Tamatani M, Fukuo K, Nakahashi T, Nishibe A, Jiang B, Ogihara T 1996 Calcitonin prevents CCl4-induced hydroperoxide generation and cytotoxicity possibly through C1b receptor in rat hepatocytes. Biochem Biophys Res Commun 218:865–871[CrossRef][Medline]
  41. Chakraborty M, Chatterjee D, Kellokumpu S, Rasmussen H, Baron R 1991 Cell cycle-dependent coupling of the calcitonin receptor to different G proteins. Science 251:1078–1082[Abstract/Free Full Text]
  42. Chen Y, Shyu JF, Santhanagopal A, Inoue D, David JP, Dixon SJ, Horne WC, Baron R 1998 The calcitonin receptor stimulates Shc tyrosine phosphorylation and Erk1/2 activation. Involvement of Gi, protein kinase C, and calcium. J Biol Chem 273:19809–19816[Abstract/Free Full Text]
  43. Overgaard K, Riis BJ, Christiansen C, Podenphant J, Johansen JS 1989 Nasal calcitonin for treatment of established osteoporosis. Clin Endocrinol (Oxf) 30:435–442[Medline]
  44. Ellerington MC, Hillard TC, Whitcroft SI, Marsh MS, Lees B, Banks LM, Whitehead MI, Stevenson JC 1996 Intranasal salmon calcitonin for the prevention and treatment of postmenopausal osteoporosis. Calcif Tissue Int 59:6–11[CrossRef][Medline]
  45. Pontiroli AE, Pajetta E, Scaglia L, Rubinacci A, Resmini G, Arrigoni M, Pozza G 1994 Analgesic effect of intranasal and intramuscular salmon calcitonin in post-menopausal osteoporosis: a double-blind, double-placebo study. Aging (Milano) 6:459–463[Medline]
  46. Rico H, Revilla M, Hernandez ER, Villa LF, Alvarez de Buergo M 1995 Total and regional bone mineral content and fracture rate in postmenopausal osteoporosis treated with salmon calcitonin: a prospective study. Calcif Tissue Int 56:181–185[CrossRef][Medline]
  47. Orimo H, Morii H, Inoue T, Tamamoto K, Minaguchi H, Ishii Y, Murota K, Fujimaki E, Watanabe R, Harata S, Honjo H, Fujita T 1996 Effect of elcatonin on involutional osteoporosis. J Bone Miner Metab 14:73–78
  48. Silva OL, Becker KL 1973 Salmon calcitonin in the treatment of hypercalcemia. Arch Intern Med 132:337–339[CrossRef][Medline]
  49. Wener JA, Gorton SJ, Raisz LG 1972 Escape from inhibition or resorption in cultures of fetal bone treated with calcitonin and parathyroid hormone. Endocrinology 90:752–759[Medline]
  50. Hosking DJ, Denton LB, Cadge B, Martin TJ 1979 Functional significance of antibody formation after long-term calcitonin therapy. Clin Endocrinol (Oxf) 10:243–252[Medline]



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