Neogambogic Acid Suppresses Receptor Activator of Nuclear Factor κB Ligand (RANKL)-Induced Osteoclastogenesis by Inhibiting the JNK and NF-κB Pathways in Mouse Bone Marrow-Derived Monocyte/Macrophages.

BACKGROUND Neogambogic acid (NGA) is used in traditional Chinese medicine. The aim of this study was to investigate the effects of NGA on gene signaling pathways involved in osteoclastogenesis in mouse bone marrow-derived monocyte/macrophages (BMMs) and on bone resorption in vitro. MATERIAL AND METHODS Primary mouse BMMs were cultured with increasing concentrations of NGA. Real-time polymerase chain reaction was used to study the expression of mRNAs corresponding to gene products specific to receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation, including tartrate-resistant acid phosphatase (TRAP), calcitonin receptor (CTR), cathepsin K (CTSK), and nuclear factor of activated T cells c1 (NFATc1). A cell counting kit-8 assay was used to evaluate cell proliferation. Western blotting and confocal immunofluorescence microscopy were used to investigate the signaling pathways. A bone resorption model was used to quantify bone resorption. RESULTS An NGA dose of ≤0.4 µg/ml had no significant effect on the proliferation of mouse BMMs in vitro (P>0.05); concentrations of between 0.1-0.4 µg/ml significantly inhibited RANKL-induced osteoclastogenesis (P<0.01) in a dose-dependent manner. Compared with the control group, NGA significantly reduced RANKL-induced bone resorption in vitro (P <0.01), and downregulated the expression of osteoclast-related mRNAs of TRAP, CTR, CTSK, and NFATc1. NGA suppressed the activation of JNK but not the p38 signaling pathway and significantly reduced NF-κB p65 phosphorylation and the nuclear transport of NF-κB molecules, which inhibited NFATc1 expression. CONCLUSIONS NGA suppressed RANKL-induced osteoclastogenesis by inhibiting the JNK and NF-κB pathways in mouse BMMs in vitro and reduced osteoclastic bone resorption.

Pharmacologic Characterization of AMG 334, a Potent and Selective Human Monoclonal Antibody against the Calcitonin Gene-Related Peptide Receptor.

Therapeutic agents that block the calcitonin gene-related peptide (CGRP) signaling pathway are a highly anticipated and promising new drug class for migraine therapy, especially after reports that small-molecule CGRP-receptor antagonists are efficacious for both acute migraine treatment and migraine prevention. Using XenoMouse technology, we successfully generated AMG 334, a fully human monoclonal antibody against the CGRP receptor. Here we show that AMG 334 competes with [(125)I]-CGRP binding to the human CGRP receptor, with a Ki of 0.02 nM. AMG 334 fully inhibited CGRP-stimulated cAMP production with an IC50 of 2.3 nM in cell-based functional assays (human CGRP receptor) and was 5000-fold more selective for the CGRP receptor than other human calcitonin family receptors, including adrenomedullin, calcitonin, and amylin receptors. The potency of AMG 334 at the cynomolgus monkey (cyno) CGRP receptor was similar to that at the human receptor, with an IC50 of 5.7 nM, but its potency at dog, rabbit, and rat receptors was significantly reduced (>5000-fold). Therefore, in vivo target coverage of AMG 334 was assessed in cynos using the capsaicin-induced increase in dermal blood flow model. AMG 334 dose-dependently prevented capsaicin-induced increases in dermal blood flow on days 2 and 4 postdosing. These results indicate AMG 334 is a potent, selective, full antagonist of the CGRP receptor and show in vivo dose-dependent target coverage in cynos. AMG 334 is currently in clinical development for the prevention of migraine.

Mevalonates restore zoledronic acid-induced osteoclastogenesis inhibition.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is likely to be caused by continuous imperfection of bone healing after surgical treatments in patients with long-term administration of nitrogen-containing bisphosphonates (NBPs). NBPs inhibit osteoclastic bone resorption by impairing the mevalonic acid sterol pathway in osteoclasts. Thus, we hypothesized that exogenous mevalonic acid metabolites restore the inhibitory effects of NBPs on osteoclastogenesis and bone remodeling. To clarify the effects of mevalonic acid metabolites, especially geranylgeranyl pyrophosphate (GGPP) and geranylgeranyl transferase substrate geranylgeranyl acid (GGOH), we examined the effects of zoledronic acid with or without GGOH or GGPP on osteoclast differentiation, multinucleation, and bone mineral deposition in tooth-extracted sockets. Zoledronic acid decreased the number of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells derived from mouse osteoclast precursors treated with receptor activator of nuclear factor-κB ligand and macrophage colony-stimulating factor. Zoledronic acid simultaneously suppressed not only the expressions of osteoclastic differentiation-related molecules such as TRAP, cathepsin K, calcitonin receptor, and vacuolar H-ATPase but also those of multinucleation-related molecules such as dendrocyte-expressed 7 transmembrane proteins and osteoclast stimulatory transmembrane protein. Treatment with GGOH or GGPP, but not farnesyl acid, restored the zoledronic acid-inhibited number of TRAP-positive multinuclear cells together with the expressions of these molecules. Although intraperitoneal administration of zoledronic acid and lipopolysaccharide into mice appeared to induce BRONJ-like lesions with empty bone lacunae and decreased mineral deposition in tooth-extracted socket, both GGOH and GGPP partially restored the inhibitory effects on zoledronic acid-related mineral deposition. These results suggest the potential of mevalonic acid metabolites as therapeutic agents for BRONJ.

Long-term dose- and time-dependent effects of endodontic sealers in human in vitro osteoclastogenesis.

INTRODUCTION: This study evaluates the concentration and time-dependent effects of endodontic sealers' extracts (AH Plus [Dentsply DeTrey, Konstanz, Germany], GuttaFlow [Roeko, Colténe/Whaledent, Germany], Tubliseal [Kerr/Sybron, Romulus, MI], Sealapex [Kerr/Sybron, Romulus, MI], and RealSeal [SybronEndo, Orange, CA]) in the differentiation and function of both unstimulated and stimulated osteoclast precursors, simulating, respectively, immature/undifferentiated precursors and cells undergoing osteoclastogenesis. METHODS: The sealers were mixed according to the manufacturers' instructions, freshly extracted with culture medium (1.3 cm(2)/mL, 24 hours, 37°C, 5% CO2/air), and diluted (1:20, 1:100, 1:500, and 1:2500). Human peripheral blood mononuclear cells were used as osteoclast precursor cells. After overnight attachment, peripheral blood mononuclear cell cultures were exposed to the sealers' extracts during 21 days in the absence (unstimulated) or presence (stimulated) of recombinant macrophage colony-stimulating factor and receptor for the activation of nuclear factor-κB ligand. Cultures performed in the absence of the extracts were used as the control. Cultures were characterized for osteoclastic differentiation and function. RESULTS: Extracts caused mostly inhibitory effects on osteoclastic cells, both in unstimulated and stimulated conditions, which were reflected by a decrease in tartrate-resistant acid phosphatase activity, the presence of actin rings, vitronectin and calcitonin receptors, the calcium phosphate resorbing ability, and the expression of osteoclastic genes. Also, the extracts induced alterations in the relative contribution of some intracellular signaling pathways involved in osteoclastogenic events. The sealers differed in the dose- and time-dependent profile. An adaptive cell response was noticed for the inhibitory effects after long-term exposure. CONCLUSIONS: Endodontic sealers affect the osteoclastic differentiation and activity, which is followed by an adaptive cell response. Our results suggest that the deleterious effect in the bone periapical tissues observed with the root canal sealers might involve, at least partially, a direct effect on the osteoclastic cells.

Structure-activity relationships for α-calcitonin gene-related peptide.

UNLABELLED: Calcitonin gene-related peptide (CGRP) is a member of the calcitonin (CT) family of peptides. It is a widely distributed neuropeptide implicated in conditions such as neurogenic inflammation. With other members of the CT family, it shares an N-terminal disulphide-bonded ring which is essential for biological activity, an area of potential α-helix, and a C-terminal amide. CGRP binds to the calcitonin receptor-like receptor (CLR) in complex with receptor activity-modifying protein 1 (RAMP1), a member of the family B (or secretin-like) GPCRs. It can also activate other CLR or calcitonin-receptor/RAMP complexes. This 37 amino acid peptide comprises the N-terminal ring that is required for receptor activation (residues 1-7); an α-helix (residues 8-18), a region incorporating a ß-bend (residues 19-26) and the C-terminal portion (residues 27-37), that is characterized by bends between residues 28-30 and 33-34. A few residues have been identified that seem to make major contributions to receptor binding and activation, with a larger number contributing either to minor interactions (which collectively may be significant), or to maintaining the conformation of the bound peptide. It is not clear if CGRP follows the pattern of other family B GPCRs in binding largely as an α-helix. LINKED ARTICLES: This article is part of a themed section on Neuropeptides. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.170.issue-7.

Interactive effect of fluoride burden with calcitonin receptor gene polymorphisms on the risk of F bone injury.

PURPOSE: This study aims to examine the interactive effect of fluoride burden with calcitonin receptor (CTR) gene polymorphisms on the risk of fluoride (F) bone injury and provide the basis for determination of F bone injury risk factors. METHODS: In this case-control study, a total of 119 cases and 126 controls were enrolled from 2 aluminum plants in Hubei province. F burden (UF) was measured by F ion-selective electrode method. The CTR gene polymorphisms were determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Logistic regression analysis was used to estimate multivariate-adjusted odds ratios, 95% confidence intervals (CI). RESULTS: The odds of developing F bone injury for participants in the moderate F burden group versus the mild F burden group were 4.1 (95% CI: 1.9, 8.7); the heavy F burden group versus the mild F burden group were 14.1 (95% CI: 6.5, 30.6). The odds of developing F bone injury for participants with the TC & TT genotypes versus the CC genotype were 2.6 (95% CI: 1.4, 4.7). The interactions between TC & TT genotypes and moderate, heavy F burden were significant (OR = 14.4; OR = 40.3). CONCLUSION: The interactive effect of F burden and CTR genotype was significant, which increased the F bone injury risk.

Oral salmon calcitonin--pharmacology in osteoporosis.

IMPORTANCE OF THE FIELD: Osteoporosis is a slow progressive disease with develops over decades, and where intervention is needed for an extended number of years. This highlights the need for safe intervention possibilities, which have sustained beneficial effects post-treatment. AREAS COVERED IN THIS REVIEW: Articles on salmon calcitonin appearing on Pubmed from 1960 until today, with focus on a newly developed oral formulation showing increased exposure and efficacy compared with nasal formulation is reviewed. The second half focuses on long-term phenomena, such as bone quality and resolution effects. The final part discusses potential additional benefits of salmon calcitonin. WHAT THE READER WILL GAIN: Insight into the clinical development of an orally formulated peptide, as well as a detailed understanding of why this approach could revive salmon calcitonin as a treatment for osteoporosis. TAKE HOME MESSAGE: The oral formulation of salmon calcitonin provides additional benefits and increased efficacy on bone based on Phase I and II clinical trials data, as compared with the nasal formulation. Hence, the results on the ongoing Phase III fracture trial are awaited with great interest.

Effect of the calcitonin gene-related peptide (CGRP) receptor antagonist telcagepant in human cranial arteries.

INTRODUCTION: Calcitonin gene-related peptide (CGRP) is a neuronal messenger in intracranial sensory nerves and is considered to play a significant role in migraine pathophysiology. MATERIALS AND METHODS: We investigated the effect of the CGRP receptor antagonist, telcagepant, on CGRP-induced cranial vasodilatation in human isolated cerebral and middle meningeal arteries. We also studied the expression of the CGRP receptor components in cranial arteries with immunocytochemistry. Concentration response curves to αCGRP were performed in human isolated cerebral and middle meningeal arteries in the absence or presence of telcagepant. Arterial slices were stained for RAMP1, CLR and actin in a double immunofluorescence staining. RESULTS: In both arteries, we found that: (i) telcagepant was devoid of any contractile or relaxant effects per se; (ii) pretreatment with telcagepant antagonised the αCGRP-induced relaxation in a competitive manner; and (iii) immunohistochemistry revealed expression and co-localisation of CLR and RAMP1 in the smooth muscle cells in the media layer of both arteries. CONCLUSIONS: Our findings provide morphological and functional data on the presence of CGRP receptors in cerebral and meningeal arteries, which illustrates a possible site of action of telcagepant in the treatment of migraine.

Amylin(1-8) is devoid of anabolic activity in bone.

Amylin(1-8), a cyclic peptide consisting of the eight N-terminal amino acids of the 37-amino acid peptide amylin, has been shown to induce proliferation of primary osteoblasts and to induce bone formation in healthy male mice, whereas no data on efficacy in bone disease-related models have been reported. Therefore, we evaluated any effects of amylin(1-8) in ovariectomized rats with established osteopenia, a model for postmenopausal osteoporosis. At doses up to 100 nmol/kg/day, a dose highly effective in healthy mice, amylin(1-8) was unable to increase bone mineral density in ovariectomized rats during an 8-week treatment period. Histomorphometric analysis of the tibia indicated that amylin(1-8) did not change bone histomorphometric parameters. In an attempt to verify any potential biological effects of amylin(1-8), we investigated the efficacy of this peptide in various in vitro assays. Experiments designed to confirm previously published results on the proliferative effects of amylin(1-8) on primary osteoblasts failed to show any response. Amylin(1-8) was able to partially displace (125)I-rat amylin(1-37) from amylin receptors composed of the calcitonin receptor and RAMP1, indicating specific interaction of the peptide with the amylin binding site. However, in vitro efficacy assays with amylin(1-8) in calcitonin receptor-RAMP-positive HEK293T and MCF7 cells failed to reveal any agonist activity of amylin(1-8), whereas amylin(1-37) showed the expected agonist activity. In conclusion, our results indicate that amylin(1-8) does not show agonist activity on amylin receptors, does not affect osteoblast proliferation, and is devoid of anabolic activity in bone.

Intracoronary intermedin 1-47 augments cardiac perfusion and function in anesthetized pigs: role of calcitonin receptors and beta-adrenoreceptor-mediated nitric oxide release.

Systemic intermedin (IMD)1-47 administration has been reported to result in vasodilation and marked hypotension through calcitonin-related receptor complexes. However, its effects on the coronary circulation and the heart have not been examined in vivo. The present study was therefore planned to determine the primary in vivo effect of IMD1-47 on coronary blood flow and cardiac function and the involvement of the autonomic nervous system and nitric oxide (NO). In 35 anesthetized pigs, IMD1-47, infused into the left anterior descending coronary artery at doses of 87.2 pmol/min, at constant heart rate and arterial blood pressure, augmented coronary blood flow and cardiac function. These responses were graded in a further five pigs by increasing the infused dose of IMD1-47 between 0.81 and 204.1 pmol/min. In the 35 pigs, the blockade of cholinergic receptors (intravenous atropine, 5 pigs), alpha-adrenoceptors (intravenous phentolamine, 5 pigs), and beta1-adrenoceptors (intravenous atenolol, 5 pigs) did not abolish the cardiac response to IMD1-47, the effects of which were prevented by blockade of beta2-adrenoceptors (intravenous butoxamine, 5 pigs), NO synthase (intracoronary N(omega)-nitro-l-arginine methyl ester, 5 pigs), and calcitonin-related receptors (intracoronary CGRP8-37/AM22-52, 10 pigs). In porcine coronary endothelial cells, IMD1-47 induced the phosphorylation of endothelial NO synthase and NO production through cAMP signaling leading to ERK, Akt, and p38 activation, which was prevented by the inhibition of beta2-adrenoceptors, calcitonin-related receptor complexes, and K+ channels. In conclusion, IMD1-47 primarily augmented coronary blood flow and cardiac function through the involvement of calcitonin-related receptor complexes and beta2-adrenoreceptor-mediated NO release. The intracellular signaling involved cAMP-dependent activation of kinases and the opening of K+ channels.

Increased expression in calcitonin-like receptor induced by aldosterone in cerebral arteries from spontaneously hypertensive rats does not correlate with functional role of CGRP receptor.

OBJECTIVE: To analyze the effect of aldosterone on the expression of calcitonin gene-related peptide (CGRP) receptor components, calcitonin-like receptor (CL receptor) and receptor activity modifying protein 1 (RAMP1), as well as the effect of this mineralocorticoid on CGRP-mediated vasodilation in middle cerebral arteries from Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). RESULTS: CGRP 0.1 nM-0.1 microM induced a concentration-dependent relaxation that was nitric oxide independent and higher in SHR middle cerebral arteries. CL receptor and RAMP1 expression were similar in both strains. The relaxation to CGRP was not modified by aldosterone 1 microM in either strain, although aldosterone 1 microM increased expression of CL receptor without modifying RAMP1 in segments from SHR rats. CONCLUSIONS: CGRP elicits greater vasodilation in middle cerebral arteries from SHR than WKY rats, that is nitric oxide independent, and by mechanism independent of CGRP receptor components expression. Although aldosterone increases the expression of CL receptor in SHR, it does not alter vasodilation to CGRP, since RAMP1 expression is not increased. These results indicate that the increase in CL receptor, without an increase in RAMP1, does not correlate with changes in functional role of the CGRP receptor.

Sensitization of calcitonin gene-related peptide receptors by receptor activity-modifying protein-1 in the trigeminal ganglion.

The neuropeptide calcitonin gene-related peptide (CGRP) from the trigeminal ganglion has been established as a key player in the pathogenesis of migraine. In this study, we provide evidence that the responsiveness of neuronal CGRP receptors is strongly enhanced in vitro and in vivo by expression of human receptor activity-modifying protein-1 (hRAMP1), an obligatory subunit of the CGRP receptor. We first demonstrated that activation of CGRP receptors on cultured trigeminal ganglion neurons increased endogenous CGRP mRNA levels and promoter activity. The promoter activation was cAMP dependent and blocked by the antagonist BIBN4096BS [1-piperidinecarboxamide, N-[2-[[5-amino-l-[[4-(4-pyridinyl)-l-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)], a new antimigraine drug. Gene transfer using an adenoviral hRAMP1 expression vector increased the maximal production of cAMP by 1.8 +/- 0.2-fold and decreased the EC50 to 2.3 +/- 0.8 nM from 9.0 +/- 5.9 nM and 15.6 +/- 5.2 nM in uninfected and control-infected cultures, respectively. To establish whether RAMP1 is limiting in vivo as indicated from the culture studies, a transgenic mouse expressing hRAMP1 in the nervous system was generated. After CGRP injection into the whiskerpad, the hRAMP1 transgenic mice displayed 2.2 +/- 0.2-fold greater plasma extravasation, which is a measure of neurogenic inflammation. These results demonstrate that RAMP1 is functionally rate limiting for CGRP receptor activity in the trigeminal ganglion, which raises the possibility that elevated RAMP1 might sensitize some individuals to CGRP actions in migraine.

Calcitonin induces apoptosis resistance in prostate cancer cell lines against cytotoxic drugs via the Akt/survivin pathway.

The expression of calcitonin (CT) and CT-receptor (CTR) mRNA in primary prostate tumors increase with tumor progression. Since advanced prostate tumors display chemoresistance, we tested a hypothesis that CT increases apoptosis resistance of prostate cells against cytotoxic drugs. We examined the effect of CT on etoposide-induced apoptosis in PC-3M, LNCaP and NRP-152 cell lines. The cytoprotective actions of CT were then tested on paclitaxel-, dexamethasone- and selenite-induced apoptosis. We also examined cytotoxic actions of these drugs in CTR-silenced PC-3M cells. Since the role of Akt and inhibitors of apoptosis proteins (IAPs) in chemoresistance of advanced prostate cancers has been established, we tested the effect of CT on phospho-Akt and survivin levels in PC-3M cells. Finally, the cytoprotective effect of CT on PC-3M cells was tested in the presence of PI3K inhibitors such as LY 294002 and wortmannin. Acutely added CT significantly attenuated apoptosis of PC cell lines in response to etoposide, dexamethasone and selenite treatment, but could not reduce paclitaxel-induced apoptosis. CT potently stimulated phospho-Akt and survivin synthesis in PC-3M cells in a sustained manner, and LY 294002 attenuated CT-induced survivin synthesis as well as apoptosis resistance. These results suggest that CT induces chemoresistance to etoposide, dexamethasone and selenite but not to paclitaxel in prostate cells. Cytoprotective action of CT is mediated by CTR-induced activation of Akt-survivin pathway. Since CT/CTR expression in prostate cancers increases with tumor progression, the suppression of "CT System" may enhance the effectiveness of chemotherapy.

Insights into interactions between the alpha-helical region of the salmon calcitonin antagonists and the human calcitonin receptor using photoaffinity labeling.

Fish-like calcitonins (CTs), such as salmon CT (sCT), are widely used clinically in the treatment of bone-related disorders; however, the molecular basis for CT binding to its receptor, a class II G protein-coupled receptor, is not well defined. In this study we have used photoaffinity labeling to identify proximity sites between CT and its receptor. Two analogues of the antagonist sCT(8-32) containing a single photolabile p-benzoyl-l-phenylalanine (Bpa) residue in position 8 or 19 were used. Both analogues retained high affinity for the CT receptor and potently inhibited agonist-induced cAMP production. The [Bpa(19)]sCT(8-32) analogue cross-linked to the receptor at or near the equivalent cross-linking site of the full-length peptide, within the fragment Cys(134)-Lys(141) (within the amino terminus of the receptor, adjacent to transmembrane 1) (Pham, V., Wade, J. D., Purdue, B. W., and Sexton, P. M. (2004) J. Biol. Chem. 279, 6720-6729). In contrast, proteolytic mapping and mutational analysis identified Met(49) as the cross-linking site for [Bpa(8)]sCT(8-32). This site differed from the previously identified cross-linking site of the agonist [Bpa(8)]human CT (Dong, M., Pinon, D. I., Cox, R. F., and Miller, L. J. (2004) J. Biol. Chem. 279, 31177-31182) and may provide evidence for conformational differences between interaction with active and inactive state receptors. Molecular modeling suggests that the difference in cross-linking between the two Bpa(8) analogues can be accounted for by a relatively small change in peptide orientation. The model was also consistent with cooperative interaction between the receptor amino terminus and the receptor core.

A key role for transmembrane prolines in calcitonin receptor-like receptor agonist binding and signalling: implications for family B G-protein-coupled receptors.

Calcitonin receptor like-receptor is a family B G-protein coupled receptor (GPCR). It requires receptor activity modifying protein (RAMP) 1 to give a calcitonin gene-related peptide (CGRP) receptor. Little is known of how members of this receptor family function. Proline residues often form important kinks in alpha-helices. Therefore, all proline residues within the transmembrane helices of the receptor (Pro241, Pro244 in helix 4, Pro275 in helix 5, Pro321 and Pro331 in helix 6) were mutated to alanine. Pro241, Pro275, and Pro321 are highly conserved throughout all family B GPCRs. The binding of CGRP and its ability to stimulate cAMP production were investigated in mutant and wild-type receptors after transient transfection into COS-7 cells with RAMP1. The P321A mutation significantly decreased the pEC(50) for CGRP and reduced its affinity but did not change cell-surface expression. Antagonist binding [CGRP(8-37) and 1-piperidinecarboxamide, N-[2-[[5amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl) (BIBN4096BS)] was little altered by the mutation. Adrenomedullin-mediated signaling was disrupted when P321A was coexpressed with RAMP1, RAMP2, or RAMP3. The P331A mutant produced a moderate reduction in CGRP binding and receptor activation. Mutation of the other residues had no effect on receptor function. Thus, Pro321 and Pro331 are required for agonist binding and receptor activation. Modeling suggested that Pro321 induces a bend in helix 6, bringing its C terminus near that of helix 3, as seen in many family A GPCRs. This is abolished in P321A. P321A-I325P, predicted to restore this conformation, showed wild-type activation. Modeling can also rationalize the effects of transmembrane proline mutants previously reported for another family B GPCR, the VPAC(1) receptor.

Changes in the expression of calcitonin receptor-like receptor, receptor activity-modifying protein (RAMP) 1, RAMP2, and RAMP3 in rat uterus during pregnancy, labor, and by steroid hormone treatments.

Calcitonin gene-related peptide (CGRP) and its related peptide, adrenomedullin (AM), are potent smooth muscle relaxants in a variety of tissues. The CGRP has been reported to play an important role in maintaining uterine relaxation during pregnancy. We have previously reported that CGRP-induced uterine relaxation was gestationally regulated. Calcitonin receptor-like receptor (CRLR), a seven-domain transmembrane protein functions as CGRP-A receptor, in association with receptor activity-modifying protein (RAMP) 1, a single-domain transmembrane protein, whereas CRLR and RAMP2 or RAMP3 constitute a receptor for AM. In the present investigation, we examined the mRNA expression of CRLR, RAMP1, RAMP2, and RAMP3 in rat uterus (n = 8) by reverse transcriptional analysis and polymerase chain reaction to assess the changes in the expression of CGRP-A- and AM-receptor components during pregnancy and labor and by steroid hormone treatments in adult ovariectomized rats. The changes in mRNA are expressed relative to the 18S mRNA in the uterus of rats at various stages: nonpregnant, pregnant on Day 18, spontaneous labor at term, Day 2 postpartum, and in pregnant rats on treatment with RU486. Ovariectomized rats treated for 3 days twice daily s.c. with estradiol-17beta (2.5 microg/injection), progesterone (2 mg/injection), and the combination of estradiol-17beta and progesterone (same doses as above) were also examined for the expression of various receptor components. Results showed that mRNA expression of the receptor components was significantly higher (P < 0.001 for CRLR, P < 0.01 for RAMP1, P < 0.05 for RAMP2, and P < 0.01 for RAMP3) in pregnant compared to nonpregnant rats. Except for RAMP3, expression of all the other three genes decreased significantly (P < 0.05) during labor. A progesterone antagonist, RU486 significantly decreased (P < 0.01 for CRLR, P < 0.05 for RAMP1, RAMP2, and RAMP3) all the receptor components during pregnancy. In adult ovariectomized rats, progesterone caused significant increases in CRLR (P < 0.001), RAMP1 (P < 0.05), and RAMP2 (P < 0.01). Levels of RAMP3 were unaffected by the progesterone treatment. Estradiol-17beta treatment decreased all of the four receptor components significantly (P < 0.01 for CRLR, P < 0.05 for RAMP1, RAMP2, and RAMP3). Our results demonstrate that both CGRP and AM may play a role in uterine quiescence during pregnancy and that their receptor components are regulated by the steroid hormones.

Quantitation in receptor pharmacology.

The activity of drugs on receptors can be visualized in appropriate systems. However, for comparisons of activity to be made, these effects must be quantified. The common currency for this quantitation is the dose-response curve. Thus, the shape, location (along the concentration axis), and maximal asymptote of dose-response curves are used to quantify drug activity and comparisons to mathematical models of receptors are made to describe mechanism of action. This review cites examples of where these quantitative procedure yield information beyond what is readily apparent through observation of the data and thus support the use of quantitative methods to maximize the information gained from experiments.

Calcitonin receptor regulation and responsiveness to calcitonin 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 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 [125I]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.

Calcitonin.

The peptide calcitonin (CT) was initially discovered in 1962 as a novel hypocalcemic hormone. This hypocalcemic response was principally due to a potent inhibitory action of CT on osteoclast mediated bone resorption and it is this action which underlies its widespread clinical use for the treatment of bone disorders, including Paget's disease, osteoporosis and hypercalcemia of malignancy. In this article we review the basic physiology of CT action, structure-function studies on CT peptides, cloning of CT receptors and the identification of isoforms of the receptor derived from alterative splicing of the receptor mRNA. We also review the state of understanding on CT receptor mediated signaling and receptor regulation, along with developing concepts of how CT peptides interact with the receptor, including how the receptors may interact with receptor activity modifying proteins to produce novel phenotypes. Finally, current therapeutic use is reviewed, and the potential for expanded use that may come with advances in delivery of peptides or CT mimetics.

Calcitonin receptor-mediated growth suppression of HEK-293 cells is accompanied by induction of p21WAF1/CIP1 and G2/M arrest.

We investigated the mechanisms by which calcitonin (CT) suppresses cellular proliferation, using HEK-293 cells stably transfected with either the rat C1a CT receptor (CTR) or the insert-negative form of the human CTR. CT treatment of clonal cell lines expressing either receptor type, but not untransfected HEK-293 cells, strongly suppressed cell growth in a concentration-dependent manner. The reduction in cell growth with CT treatment could not be attributed to cellular necrosis or apoptotic cell death, the latter assessed by both DNA fragmentation analysis and caspase 3 (CPP-32) assay. Growth inhibition was associated with an accumulation of cells in the G2 phase of the cell cycle. CT treatment of the human and rat CTR-expressing cell lines resulted in a rapid and sustained induction of mRNA encoding the cyclin-dependent kinase inhibitor, p21WAF1/CIP1, increased levels of which were maintained at least 48 h after initiation of treatment. Western blot analysis showed a rapid corresponding increase in p21WAF1/CIP1 protein, whereas protein levels of another member of the cyclin-dependent kinase inhibitor family, p27kip1, were unchanged. In parallel with the induction of p21, CT treatment reduced levels of p53 mRNA and protein. CT treatment resulted in a specific cell cycle block in G2, which was associated with inhibition of Cdc2/cyclin B kinase activity as measured by histone H1 phosphorylation. There was no evidence for p21 association with this complex despite the inhibition of Cdc2 activity. Evidence that p21 induction was causative of cell growth suppression was obtained from p21 antisense oligonucleotide experiments. Treatment with a p21 antisense oligonucleotide blocked induction of p21 expression and significantly reduced the CT-mediated growth inhibition. These observations suggest that p21 is required for the G2 arrest in response to CT, but argue against a direct role of p21 in the inhibition of Cdc2 activity. These studies suggest a novel regulation of cell cycle progression by CT and will provide a basis for detailed examination of the molecular mechanisms involved.