Calcitonin Receptor mRNA Expression Research Articles

Appropriate clinical usage of calcitonin escape phenomenon and intermittent v.s. daily administration of calcitonin

Since Calcitonin (CT) inhibits osteoclastic bone resorption, it has been widely used to treat metabolic bone disorders, such as Paget's disease of bone, malignancy-associated hypercalcemia, and osteoporosis. It is recognized, however, that continuous treatment with CT causes a loss of its inhibitory effects on bone resorption. We and other investigators have studied the mechanism and the results indicated that desensitization to CT was closely associated with the down regulation of the CT receptor (CTR). This down regulation was due not only to internalization of the receptor but also to reduced cell surface receptor concentration through inhibition of de novo CTR synthesis. An essential signal for osteoclast differentiation from its precursor cells, which was termed as ODF, was also found identical to tumor necrosis factor (TNF) related activation induced cytokine (TRANCE) and receptor activator of nuclear factor-kappa B ligand (RANKL). Using soluble RANKL and macrophage colony stimulating factor, we recently studied the mechanisms of the biological responses to CT in cells of human osteoclast lineage. The signaling pathway responsible for CTR down regulation in human osteoclasts is different from that observed in mouse osteoclasts: the activation of protein kinase A pathway is primarily responsible for CTR regulation in mouse osteoclasts, while the activation of protein kinase C was predominant in humans. Treatment with CT reduced concentration of cellular surface CTR and CTR mRNA expression also in human osteoclasts. The reduced specific binding, CTR mRNA levels and CT-sensitive adenylate cyclase responsiveness returned to the control levels by 96h after removal of CT. These results may suggest that intermittent administration of CT would be effective for the treatment of osteoporosis, resulting in reduced desensitization in CT target cells.

Calcitonin is a prostate epithelium-derived growth stimulatory peptide

Locally secreted growth factors and neuropeptides may play an important role in sustaining the growth of hormone-independent prostate cancer. Our previous studies have shown that calcitonin-like immunoreactive peptide (CTI) is secreted by primary prostate cells in culture, and its secretion from malignant prostate cells is significantly higher than benign cells. Exogenously added calcitonin (CT) induces DNA synthesis in serum-starved prostate cancer LNCaP and PC-3M cells. Present studies extended these findings by cloning cDNAs for CT and CT receptor (CT-R) from prostate cancer cells and studying the expression of CT and CT-R mRNA in prostate cancer cell lines and primary prostate tumor specimens. The results have shown that PC-3 cells expressed CT, and not CT-R, mRNA, whereas CT-R, but not CT, mRNA was expressed by LNCaP cells. Conditioned media from PC-3 cells induced DNA synthesis of LNCaP cells, and this mitogenic response was abolished by anti-CT serum. Highly aggressive PC-3M cells co-expressed CT and CT-R mRNAs. CT also induced a twofold increase in DNA synthesis of primary prostate cells and anti-CT serum caused a 56% decline. In-situ hybridization histochemistry of archival prostate specimens has selectively localized CT and CT-R mRNA in basal epithelium of benign and low grade PC specimens, and these mRNAs were not detected in either luminal epithelium or stroma. In contrast, CT and CT-R mRNA were detected throughout the luminal epithelium of moderate and high-grade PC specimens. Most epithelial cells of low and moderately differentiated tumors expressed either CT or CT-R mRNA, suggesting that CT may serve as a paracrine factor. In contrast, CT and CT-R mRNAs were co-expressed by most tumor cells in advanced PC specimens. The cells expressing CT-R mRNA in primary tumors also co-expressed PCNA. These results, when combined with mitogenic actions of CT on primary prostate cells as well as PC cell lines, strongly support the role for CT in sustaining the growth of cancer cells.

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

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

Regulation of calcitonin receptor by glucocorticoid in human osteoclast-like cells prepared in vitro using receptor activator of nuclear factor-kappaB ligand and macrophage colony-stimulating factor.

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

Acid pH increases carbonic anhydrase II and calcitonin receptor mRNA expression in mature osteoclasts.

Numerous resorptive stimuli have been shown to enhance osteoclast differentiation, increasing osteoclast numbers and accelerating bone resorption. Currently, there is much less understanding of regulation of mature osteoclast activity. Indeed, there is presently only minimal evidence of changes in gene expression as a mechanism for altering bone resorption. We investigate here, in the mature osteoclast, regulation of 2 genes-carbonic anhydrase II (CAII) and calcitonin receptor (CTR) in response to acidosis, which is known to increase bone resorption. We studied the effect of acid pH on CAII and CTR mRNA expression in mature osteoclasts raised in coculture of ST-2 and primary marrow cells. On day 6 of culture, stromal cells were removed with collagenase, the remaining osteoclasts were incubated overnight, and then exposed to varying pH. RT-PCR was performed on total RNA using primers for CAII, CTR, or glyceraldehyde dehydrogenase phosphate (GAP). Expression of CTR mRNA was increased 2.14 +/- 0.41 and 2.56 +/- 0.45 (P < 0.05)-fold by a 4-hour exposure to pH 6.75 and 6.5, respectively. CAII mRNA was similarly increased 2.18 +/- 0.42 and 2.63 +/- 0.48 (P < 0.05)-fold by pH 6.75 and 6.5, respectively. Increased expression of CAII and CTR mRNA was seen by 2 hours and maximally by 4 hours. Increased expression of CTR and CAII mRNA was not explained by increases in osteoclast numbers: pH 7.4-100 +/- 3.7, 6.75-133 +/- 8.3, 6.5-124 +/- 7.8. These results demonstrate upregulation of two osteoclast genes in response to acidosis, illustrating the ability of the mature osteoclast to respond to resorptive signals with increased functional gene expression.

Localization of calcitonin receptor mRNA in the mouse brain: coexistence with serotonin transporter mRNA

To elucidate the sites of and mechanisms of analgesic effect of centrally injected calcitonin, we examined expression of calcitonin receptor mRNA in the mouse brain by in situ hybridization techniques. Calcitonin receptor mRNA was expressed in various brain regions, including the preoptic area, dorsomedial hypothalamic nucleus, lateral hypothalamic area, periaqueductal gray, dorsal raphe nucleus, locus coeruleus, lateral parabrachial nucleus, gigantocellular reticular nucleus alpha part, lateral paragigantocellular nucleus, raphe magnus nucleus and solitary tract nucleus, which are known to play important roles in pain modulation. In addition, a double in situ hybridization technique demonstrated the intense expression of calcitonin receptor mRNA on serotonergic neurons in some raphe nuclei and the lateral paragigantocellular nucleus, suggesting the involvement of central serotonergic pathways in analgesic effect of calcitonin.

Enhanced expression of osteocalcin mRNA in human osteoarthritic trabecular bone of the proximal femur is associated with decreased expression of interleukin-6 and interleukin-11 mRNA.

Few studies have investigated the factors or mechanisms that may lead to structural changes in OA bone. This study examines the in vivo expression of messenger RNA encoding the osteoclastogenic cytokines interleukin-6 (IL-6) and interleukin-11 (IL-11), together with the osteoblastic marker osteocalcin (OCN) and the calcitonin receptor (CTR), which in bone is exclusively expressed by osteoclasts. Total RNA was isolated from intertrochanteric trabecular bone from OA patients, and from controls taken at autopsy. The patterns of mRNA expression of IL-6, IL-11, OCN, and CTR were examined using reverse-transcription polymerase chain reaction (RT-PCR) by determining the relative ratios of the amplified products with respect to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both IL-6 and IL-11 mRNA were significantly less abundant in OA than in the control group. Expression of IL-11 mRNA decreased significantly with age for both groups. OCN mRNA expression was significantly more abundant in OA, and there was no significant difference for CTR mRNA between the two groups. For both OCN and CTR in OA, expression increased significantly with increasing age. These differences in expression between the OA and control groups are consistent with an hypothesis that biochemical and genetic factors in bone can contribute or perhaps underlie the degenerative joint changes seen in OA.

Calcitonin responsiveness and receptor expression in porcine and murine osteoclasts: a comparative study

The presence of the calcitonin (CT) receptor is a distinguishing characteristic of osteoclasts; however, species variability exists with respect to functional responsiveness to CT. In the present study, CT responsiveness and temporal expression of the CT receptor in differentiating cultures of porcine osteoclasts was examined and compared to murine osteoclasts. In vitro porcine osteoclast differentiation was evaluated using bone marrow cultures from neonatal pigs. Murine osteoclast differentiation was studied using cocultures of murine bone marrow and BALC cells, a calvarial-derived cell line. In the presence of 1,25 (OH)2D3, a time-dependent increase in osteoclast differentiation was observed in porcine and murine cultures. Salmon CT (sCT) and porcine CT (pCT) inhibited 1,25 (OH)2D3-stimulated porcine osteoclast differentiation at 10−8 and 10−7 mol/L (60% with 10−7 mol/L sCT and 85% inhibition with 10−7 mol/L pCT). Treatment of murine cocultures with sCT (10−17–10−7 mol/L) resulted in a concentration-dependent decrease in osteoclast differentiation with a maximal inhibition of 70%. Osteoclast differentiation was inhibited in a concentration-dependent manner by recombinant human transforming growth factor-beta1 (rhTGF-β1) in both species. The effects of CT on resorption lacunae formation were determined by culturing in vitro generated porcine or murine osteoclasts on bovine cortical bone slices for 18 h in the presence or absence of CT. With both porcine and murine osteoclasts, a concentration-dependent decrease in resorption lacunae formation was observed between 10−13 and 10−7 mol/L sCT with the highest concentrations completely abolishing resorption. However, pCT only inhibited porcine osteoclastic resorption at 10−7 mol/L. CT receptor messenger ribonucleic acid (mRNA) expression was determined at different time points during in vitro osteoclast differentiation. In porcine cultures, expression of CT receptor mRNA correlated with the presence of osteoclasts. In murine cocultures, mRNA for the CT receptor was observed at each time point examined and was independent of the presence of multinucleated osteoclasts. Thus, porcine and murine differentiating osteoclast cultures express CT receptor mRNA; however, receptor expression correlates with osteoclast formation only in the porcine cultures. In summary, porcine and murine osteoclasts express CT receptor mRNA and functional responsiveness to CT. These findings suggest that the effects of sCT on osteoclast resorption are similar in murine and porcine cells, but that sCT is a less potent inhibitor of porcine than murine osteoclast differentiation.

Calcitonin receptor mRNA expression in TT cells: Effect of dexamethasone

Among the four isoforms of the calcitonin receptor (CTR) described in humans, two differ by the presence of h-CTR1 or absence of h-CTR2 of 16 amino acids in the first intracellular loop. Both receptors are biologically active. The TT cell line derived from a human medullary carcinoma of the thyroid is characterized by the secretion of large amounts of calcitonin. We have recently shown that this cell line expresses h-CTR2. In the present work we have studied the expression of CTR during TT cell proliferation and used dexamethasone to modify calcitonin expression in order to establish if an autocrine regulation involving calcitonin and its receptor was functional in the TT cells. The expression of this receptor and of calcitonin during TT cell proliferation was studied by reverse transcriptase-polymerase chain reaction (RT-PCR). Dexamethasone, a potent inhibitor of TT cell proliferation, levels (day 6 of culture) specifically increased receptor levels from day 8 onwards. CT peptide and CT mRNA levels decreased or were similar during experimental time. CTR regulation by glucocorticoids is suggested in TT cells. Autocrine regulation of CTR is also suggested by relation between CT mRNA levels and CTR mRNA.

Osteoclasts from human giant cell tumors of bone lack estrogen receptors.

Although estrogen is important in human skeletal homeostasis, the major target cell in bone is unknown. Estrogen receptors (ER) have been demonstrated in osteoblasts and bone marrow stromal cells, but their presence in osteoclasts remains controversial because completely pure preparations have not been available. We have examined expression of ER-alpha and ER-beta messenger RNA (mRNA) by RT-PCR in samples from human giant cell tumor of bone (GCT), including: whole tumor, cultured mononuclear cells, and a pure osteoclast population obtained by microisolation. Whole tumor expressed both ER-alpha and calcitonin receptor (CTR) mRNA and apparently lower levels of ER-beta mRNA. Passaged cultures of tumor mononuclear stromal cells also expressed ER-alpha and low ER-beta but not CTR mRNA. In pure preparations of microisolated osteoclasts, expression of ER-alpha or ER-beta mRNA was not detected, whereas expression of CTR mRNA was readily identified. Microisolated GCT mononuclear cells expressed ER-alpha, but no detectable CTR mRNA. Fluorescence in situ hybridization (FISH) using an ER-alpha riboprobe demonstrated strong signal in the mononuclear cells but multinucleated osteoclasts showed no detectable signal. In contrast, CTR mRNA was detected in multinucleated osteoclasts but not in stromal-like tumor cells by FISH. 17Beta-estradiol consistently showed no effect on bone resorbing activity of osteoclasts from GCT cultured on cortical bone, although calcitonin was a potent inhibitor. These findings indicate that significant expression of ER does not occur in osteoclasts derived from human GCT and suggest that estrogen effects are mediated by other cells of the bone environment.

Calcitonin receptor mRNA is expressed in human medullary thyroid carcinoma.

We recently reported the presence of a truncated form (h-CTR2) of the human calcitonin receptor (CTR) in TT cells, a cell line derived from medullary thyroid carcinoma (MTC). This form (h-CTR2), characterized by the absence of 16 amino acids in the first intracellular domain, was also detected in two cases of MTC. In the present study we determined the expression of CTR mRNA in a larger sample, representative of the different clinical forms of MTC, and in normal thyroid. h-CTR2 was expressed in all MTC specimens (both sporadic and familial) and in the normal thyroid samples. The expression of the receptor mRNA was higher in MTC compared with normal thyroid. Moreover, CT and CTR mRNA levels were modified significantly during proliferation. This result suggests that CT may be involved in proliferation of MTC via autocrine/paracrine regulation. Calcitonin secretion by MTC may play a role in the development and spread of these tumors.

Regulation by calcitonin and glucocorticoids of calcitonin receptor gene expression in mouse osteoclasts.

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-1 beta-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-1 beta-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.

The role of gp130-mediated signals in osteoclast development: regulation of interleukin 11 production by osteoblasts and distribution of its receptor in bone marrow cultures.

Interleukin (IL)-11 is a multifunctional cytokine whose role in osteoclast development has not been fully elucidated. We examined IL-11 production by primary osteoblasts and the effects of rat monoclonal anti-mouse glycoprotein 130 (gp130) antibody on osteoclast formation, using a coculture of mouse osteoblasts and bone marrow cells. IL-1, TNF alpha, PGE2, parathyroid hormone (PTH) and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) similarly induced production of IL-11 by osteoblasts, but IL-6, IL-4, and TGF beta did not. Primary osteoblasts constitutively expressed mRNAs for both IL-11 receptor (IL-11R alpha) and gp130. Osteotropic factors did not modulate IL-11R alpha mRNA at 24 h, but steady-state gp130 mRNA expression in osteoblasts was upregulated by 1 alpha,25(OH)2D3, PTH, or IL-1. In cocultures, the formation of multinucleated osteoclast-like cells (OCLs) in response to IL-11, or IL-6 together with its soluble IL-6 receptor was dose-dependently inhibited by rat monoclonal anti-mouse gp130 antibody. Furthermore, adding anti-gp130 antibody abolished OCL formation induced by IL-1, and partially inhibited OCL formation induced by PGE2, PTH, or 1 alpha,25(OH)2D3. During osteoclast formation in marrow cultures, a sequential relationship existed between the expression of calcitonin receptor mRNA and IL-11R alpha mRNA. Osteoblasts as well as OCLs expressed transcripts for IL-11R alpha, as indicated by RT-PCR analysis and in situ hybridization. These results suggest a central role of gp130-coupled cytokines, especially IL-11, in osteoclast development. Since osteoblasts and mature osteoclasts expressed IL-11R alpha mRNA, both bone-forming and bone-resorbing cells are potential targets of IL-11.

Calcitonin receptor mrna expression in the human prostate

Objectives A subpopulation of prostate neuroendocrine (NE) cells contain calcitonin (CT). It has been postulated that CT-producing cells in the prostate account for the high CT level in the semen, and may be involved in the regulation of other epithelial cells via a paracrine mechanism. The presence of CT binding sites in the plasma membrane fraction of prostate tissue has been demonstrated by radioligand binding assay. In the present study, we investigated the CT receptor gene expression in the human prostate, a key component of the autocrine/paracrine loop in the CT functional pathway. Methods Reverse transcription polymerase chain reaction (RT-PCR) was carried out to evaluate the CT receptor mRNA expression in normal prostate tissue. Subsequent DNA sequencing was used to verify RT-PCR amplified products and to determine the isoform of the receptor. To define the location of the CT receptor expression, nonradioactive in situ hybridization was performed with a digoxigenin-labeled probe complementary to the coding region of the CT receptor mRNA. A polyclonal antibody against CT was used to reveal the CT-secreting cells in the prostate. Results CT receptor mRNA expression was detected in the prostate tissue. Further analysis of the DNA sequence showed that CT receptor expressed in the prostate was the isoform without a 16-amino acid insert in the first intracellular domain. In situ hybridization revealed that CT receptor was present in the prostate NE cells. Immunocytochemical staining of mirror image sections showed that some CT-secreting cells also expressed CT receptor. Conclusions CT receptor expression in the prostate, a key component in the CT functional pathway, is located in subsets of dispersed NE cells (CT secreting and CT nonsecreting), which indicates that prostate CT may play an important role in the autocrine/paracrine regulation of the prostate NE system.

Expression of the calcitonin receptor in bone marrow cell cultures and in bone: a specific marker of the differentiated osteoclast that is regulated by calcitonin.

We studied the temporal sequence of osteoclast (OC) differentiation from precursor cells in murine marrow cultures. Two markers of the OC phenotype, calcitonin (CT) receptor (CTR) and tartrate resistant acid phosphatase (TRAP), were assessed. Marrow cells from C57BL/6 mice were cultured for 3, 5, 7, and 9 days with or without 1,25-(OH)2vitamin D3 (10(-8) M). In controls only small numbers of osteoclastic multinucleated cells 9MNCs) formed per well (< 15 per well). In contrast, 1,25-(OH)2D3 strongly stimulated MNC formation (> 80 per well on day 7). Messenger RNA (mRNA) for TRAP was detectable by reverse transcription-polymerase chain reaction amplification in both control and 1,25-(OH)2D3 treated groups at all times. However, TRAP mRNA was detectable in MNCs by the less sensitive in situ hybridization only on days 5, 7, and 9 and only in 1,25-(OH)2D3 treated cells. In control cultures, CTR mRNA was present on day 3 only in nonadherent cells and was not present in adherent cells (where MNCs formed) at any time point. In 1,25-(OH)2D3 treated cultures CTR mRNA was detectable in nonadherent cells on day 3 and in adherent cells on day 5 and thereafter. Peak levels of CTR mRNA were seen in adherent cells on day 7 (15-fold more than day 5 and 4-fold more than day 9). CT (10(-7) M) treatment of 7 day cultures, which had been stimulated to express the osteoclastic phenotype, caused a marked decrease in CTR mRNA expression at 24 h. There was no effect of CT treatment on CTR mRNA expression at 3 h or on TRAP mRNA expression at 3 or 24 h. In neonatal mouse calvaria cultures, CTR mRNA expression was constitutively present and was markedly decreased by 48 h of CT treatment. Similarly, bone resorption in these cultures was inhibited at 24 h by CT treatment, but at 48 and 72 h there was escape from the inhibitory effects of CT on resorption. In the marrow cultures, MNCs were greater than 98% positive for [125I]-salmon calcitonin (sCT) binding and this binding was completely competed away by excess cold sCT (10(-7) M). All primary isolated osteoclasts from 1- to 3-day-old mouse long bones exhibited [125I]-sCT binding and TRAP activity and were strongly positive for CTR and TRAP mRNA by in situ hybridization. Both MNCs that formed in bone marrow cultures and isolated primary osteoclasts formed resorption pits on bone slices.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of two human skeletal calcitonin receptor isoforms cloned from a giant cell tumor of bone. The first intracellular domain modulates ligand binding and signal transduction.

Two distinct calcitonin (CT) receptor (CTR)-encoding cDNAs (designated GC-2 and GC-10) were cloned and characterized from giant cell tumor of bone (GCT). Both GC-2 and GC-10 differ structurally from the human ovarian cell CTR (o-hCTR) that we cloned previously, but differ from each other only by the presence (GC-10) or absence (GC-2) of a predicted 16-amino acid insert in the putative first intracellular domain. Expression of all three CTR isoforms in COS cells demonstrated that GC-2 has a lower binding affinity for salmon (s) CT (Kd approximately 15 nM) than GC-10 or o-hCTR (Kd approximately 1.5 nM). Maximal stimulatory concentrations of CT resulted in a mean accumulation of cAMP in GC-2 transfected cells that was greater than eight times higher than in cells transfected with GC-10 after normalizing for the number of receptor-expressing cells. The marked difference in maximal cAMP response was also apparent after normalizing for receptor number. GC-2 also demonstrated a more potent ligand-mediated cAMP response compared with GC-10 for both human (h) and sCT (the EC50 values for GC-2 were approximately 0.2 nM for sCT and approximately 2 nM for hCT; EC50 values for GC-10 were approximately 6 nM for sCT and approximately 25 nM for hCT). Reverse transcriptase PCR of GCT RNA indicated that GC-2 transcripts are more abundant than those encoding for GC-10. In situ hybridization on GCT tissue sections demonstrated CTR mRNA expression in osteoclast-like cells. We localized the human CTR gene to chromosome 7 in band q22. The distinct functional characteristics of GC-2 and GC-10, which differ in structure only in the first intracellular domain, indicate that the first intracellular domain of the CTR plays a previously unidentified role in modulating ligand binding and signal transduction via the G protein/adenylate cyclase system.

Downregulation of calcitonin receptor mRNA expression by calcitonin during human osteoclast-like cell differentiation.

Calcitonin inhibits both osteoclast formation and bone resorption, and is a primary treatment for patients with hypercalcemia and increased bone turnover. However, the clinical utility of calcitonin is limited because patients become refractory to calcitonin after several days (the calcitonin "escape phenomenon"). The molecular basis for calcitonin "escape" is unclear. To determine the regulatory mechanisms controlling calcitonin receptor (CTR) expression in osteoclasts and their precursors, we treated immature mononuclear precursors for human osteoclast-like multinucleated cells (MNC) formed in vitro with 1,25-(OH)2D3, to induce their differentiation to committed mononuclear precursors, and mature multinucleated osteoclasts, and used reverse transcriptase (RT)-PCR to assess expression of CTR mRNA in both committed mononuclear precursors and MNC. The PCR fragment produced was cloned and sequenced to confirm that it was derived from CTR mRNA. CTR mRNA expression was detected in mononuclear MNC precursors after 7 d of 1,25-(OH)2D3 treatment. It was also present in osteoclast-like MNC and highly purified giant cells from osteoclastomas, but not in monocytes or macrophage polykaryons formed in vitro. Calcitonin markedly decreased CTR but not actin mRNA expression in giant cells and MNC after 12 h, and removal of calcitonin restored CTR mRNA expression. Similarly, calcitonin decreased calcitonin-induced adenylate cyclase activity. These data suggest: (a) downregulation of CTR gene expression by calcitonin may in part explain the calcitonin "escape phenomenon"; and (b) expression of CTR mRNA occurs in mononuclear osteoclast precursors within 7 d after exposure to 1,25-(OH)2D3.