Calcitonin Receptor Research Articles

A structural basis for amylin receptor phenotype.

Amylin receptors (AMYRs) are heterodimers of the calcitonin (CT) receptor (CTR) and one of three receptor activity-modifying proteins (RAMPs), AMY1R, AMY2R, and AMY3R. Selective AMYR agonists and dual AMYR/CTR agonists are being developed as obesity treatments; however, the molecular basis for peptide binding and selectivity is unknown. We determined the structure and dynamics of active AMYRs with amylin, AMY1R with salmon CT (sCT), AMY2R with sCT or human CT (hCT), and CTR with amylin, sCT, or hCT. The conformation of amylin-bound complexes was similar for all AMYRs, constrained by the RAMP, and an ordered midpeptide motif that we call the bypass motif. The CT-bound AMYR complexes were distinct, overlapping the CT-bound CTR complexes. Our findings indicate that activation of AMYRs by CT-based peptides is distinct from their activation by amylin-based peptides. This has important implications for the development of AMYR therapeutics.

Hydrogel-Mediated Release of TRPV1 Modulators to Fine Tune Osteoclastogenesis.

Bone defects, including bone loss due to increased osteoclast activity, have become a global health-related issue. Osteoclasts attach to the bone matrix and resorb the same, playing a vital role in bone remodeling. Ca2+ homeostasis plays a pivotal role in the differentiation and maturation of osteoclasts. In this work, we examined the role of TRPV1, a nonselective cation channel, in osteoclast function and differentiation. We demonstrate that endogenous TRPV1 is functional and causes Ca2+ influx upon activation with pharmacological activators [resiniferatoxin (RTX) and capsaicin] at nanomolar concentration, which enhances the generation of osteoclasts, whereas the TRPV1 inhibitor (5′-IRTX) reduces osteoclast differentiation. Activation of TRPV1 upregulates tartrate-resistant acid phosphatase activity and the expression of cathepsin K and calcitonin receptor genes, whereas TRPV1 inhibition reverses this effect. The slow release of capsaicin or RTX at a nanomolar concentration from a polysaccharide-based hydrogel enhances bone marrow macrophage (BMM) differentiation into osteoclasts whereas release of 5′-IRTX, an inhibitor of TRPV1, prevents macrophage fusion and osteoclast formation. We also characterize several subcellular parameters, including reactive oxygen (ROS) and nitrogen (RNS) species in the cytosol, mitochondrial, and lysosomal profiles in BMMs. ROS were found to be unaltered upon TRPV1 modulation. NO, however, had elevated levels upon RTX-mediated TRPV1 activation. Capsaicin altered mitochondrial membrane potential (ΔΨm) of BMMs but not 5′-IRTX. Channel modulation had no significant impact on cytosolic pH but significantly altered the pH of lysosomes, making these organelles less acidic. Since BMMs are precursors for osteoclasts, our findings of the cellular physiology of these cells may have broad implications in understanding the role of thermosensitive ion channels in bone formation and functions, and the TRPV1 modulator-releasing hydrogel may have application in bone tissue engineering and other biomedical sectors.

Lipidated Calcitonin Gene-Related Peptide (CGRP) Peptide Antagonists Retain CGRP Receptor Activity and Attenuate CGRP Action In Vivo.

Signaling through calcitonin gene-related peptide (CGRP) receptors is associated with pain, migraine, and energy expenditure. Small molecule and monoclonal antibody CGRP receptor antagonists that block endogenous CGRP action are in clinical use as anti-migraine therapies. By comparison, the potential utility of peptide antagonists has received less attention due to suboptimal pharmacokinetic properties. Lipidation is an established strategy to increase peptide half-life in vivo. This study aimed to explore the feasibility of developing lipidated CGRP peptide antagonists that retain receptor antagonist activity in vitro and attenuate endogenous CGRP action in vivo. CGRP peptide analogues based on the archetypal CGRP receptor antagonist, CGRP8-37, were palmitoylated at the N-terminus, position 24, and near the C-terminus at position 35. The antagonist activities of the lipidated peptide analogues were tested in vitro using transfected Cos-7 cells expressing either the human or mouse CGRP receptor, amylin subtype 1 (AMY1) receptor, adrenomedullin (AM) receptors, or calcitonin receptor. Antagonist activities were also evaluated in SK-N-MC cells that endogenously express the human CGRP receptor. Lipidated peptides were then tested for their ability to antagonize endogenous CGRP action in vivo using a capsaicin-induced dermal vasodilation (CIDV) model in C57/BL6J mice. All lipidated peptides except for the C-terminally modified analogue retained potent antagonist activity compared to CGRP8-37 towards the CGRP receptor. The lipidated peptides also retained, and sometimes gained, antagonist activities at AMY1, AM1 and AM2 receptors. Several lipidated peptides produced robust inhibition of CIDV in mice. This study demonstrates that selected lipidated peptide antagonists based on αCGRP8-37 retain potent antagonist activity at the CGRP receptor and are capable of inhibition of endogenous CGRP action in vivo. These findings suggest that lipidation can be applied to peptide antagonists, such as αCGRP8-37 and are a potential strategy for antagonizing CGRP action.

Piperlongumine Inhibits Titanium Particles-Induced Osteolysis, Osteoclast Formation, and RANKL-Induced Signaling Pathways.

Wear particle-induced aseptic loosening is the most common complication of total joint arthroplasty (TJA). Excessive osteoclast formation and bone resorptive activation have been considered to be responsible for extensive bone destruction and prosthesis failure. Therefore, identification of anti-osteoclastogenesis agents is a potential therapy strategy for the treatment of aseptic loosening and other osteoclast-related osteolysis diseases. In the present study, we reported, for the first time, that piperlongumine (PL), a key alkaloid compound from Piper longum fruits, could significantly suppress the formation and activation of osteoclasts. Furthermore, PL effectively decreased the mRNA expressions of osteoclastic marker genes such as tartrate-resistant acid phosphatase (TRAP), calcitonin receptor (CTR), and cathepsin K (CTSK). In addition, PL suppressed the receptor activator of nuclear factor-κB ligand (RANKL)-induced activations of MAPKs (ERK, JNK and p38) and NF-κB, which down-regulated the protein expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). Using a titanium (Ti) particle-induced calvarial osteolysis model, we demonstrated that PL could ameliorate Ti particle-induced bone loss in vivo. These data provide strong evidence that PL has the potential to treat osteoclast-related diseases including periprosthetic osteolysis (PPO) and aseptic loosening.

Roles of the Circadian Clock Mechanism in the Regulation of Daily Rhythms of Body Temperature

Body temperature of thermostatic animals does not stay constant but displays a regular circadian fluctuation, which has a role in maintaining homeostasis of sleep and metabolism as well as entraining the peripheral circadian clocks in the body. Following the discovery of clock genes that generate the circadian rhythm and the master clock structure in the brain and recent advances in infrared temperature imaging, there is a greater opportunity to investigate the mechanism underlying body temperature regulation, which currently remains unclear. In this review, we summarize our recent findings on a mechanism of body temperature regulation through a non-coding cis-element of the core clock gene Per2. The body temperature during siesta is controlled by the calcitonin receptors, which exhibit restricted expression in the suprachiasmatic nucleus, the locus of the central circadian clock in the brain. This review also refers to a recent machine learning-assisted thermography recording procedure, a technique that enables real-time simultaneous monitoring of circadian fluctuations in body temperature, locomotor activity, feeding, and drinking behaviors of animals. We will discuss the current challenges and open questions in understanding the molecular and circuit level mechanisms that give rise to circadian rhythms in body temperature.

Relayed signaling between mesenchymal progenitors and muscle stem cells ensures adaptive stem cell response to increased mechanical load.

Adaptation to mechanical load, leading to enhanced force and power output, is a characteristic feature of skeletal muscle. Formation of new myonuclei required for efficient muscle hypertrophy relies on prior activation and proliferation of muscle stem cells (MuSCs). However, the mechanisms controlling MuSC expansion under conditions of increased load are not fully understood. Here we demonstrate that interstitial mesenchymal progenitors respond to mechanical load and stimulate MuSC proliferation in a surgical mouse model of increased muscle load. Mechanistically, transcriptional activation of Yes-associated protein 1 (Yap1)/transcriptional coactivator with PDZ-binding motif (Taz) in mesenchymal progenitors results in local production of thrombospondin-1 (Thbs1), which, in turn, drives MuSC proliferation through CD47 signaling. Under homeostatic conditions, however, CD47 signaling is insufficient to promote MuSC proliferation and instead depends on prior downregulation of the Calcitonin receptor. Our results suggest that relayed signaling between mesenchymal progenitors and MuSCs through a Yap1/Taz-Thbs1-CD47 pathway is critical to establish the supply of MuSCs during muscle hypertrophy.

Long-acting amylin analogues for the management of obesity.

To summarize recent developments of long-acting amylin analogues for the treatment of obesity and to outline their mode of action. Amylin is a pancreatic hormone acting to control energy homeostasis and body weight. Activity at the calcitonin and amylin receptors in the area postrema seems to - at least partly - be responsible for these effects of amylin. Both preclinical and early-stage clinical studies investigating long-acting amylin receptor analogues demonstrate beneficial effects on body weight in obesity. Cagrilintide, a novel amylin analogue suitable for once-weekly administration, is in phase II clinical development and has shown promising body weight reducing effects alone and in combination with the glucagon-like peptide 1 receptor agonist semaglutide. Long-acting amylin analogues have emerged as a possible pharmacotherapy against obesity, but more studies are needed to support the utility and long-term effects of this strategy in relevant populations.

Accelerated Development With Increased Bone Mass and Skeletal Response to Loading Suggest Receptor Activity Modifying Protein-3 as a Bone Anabolic Target.

Knockout technologies provide insights into physiological roles of genes. Studies initiated into endocrinology of heteromeric G protein-coupled receptors included deletion of receptor activity modifying protein-3, an accessory protein that alters ligand selectivity of calcitonin and calcitonin-like receptors. Initially, deletion of Ramp3-/- appeared phenotypically silent, but it has emerged that mice have a high bone mass phenotype, and more subtle alterations to angiogenesis, amylin homeostasis, and a small proportion of the effects of adrenomedullin on cardiovascular and lymphatic systems. Here we explore in detail, effects of Ramp3-/- deletion on skeletal growth/development, bone mass and response of bone to mechanical loading mimicking exercise. Mouse pups lacking RAMP3 are healthy and viable, having accelerated development of the skeleton as assessed by degree of mineralisation of specific bones, and by microCT measurements. Specifically, we observed that neonates and young mice have increased bone volume and mineralisation in hindlimbs and vertebrae and increased thickness of bone trabeculae. These changes are associated with increased osteoblast numbers and bone apposition rate in Ramp3-/- mice, and increased cell proliferation in epiphyseal growth plates. Effects persist for some weeks after birth, but differences in gross bone mass between RAMP3 and WT mice lose significance in older animals although architectural differences persist. Responses of bones of 17-week old mice to mechanical loading that mimics effects of vigorous exercise is increased significantly in Ramp3-/- mice by 30% compared with WT control mice. Studies on cultured osteoblasts from Ramp3-/- mice indicate interactions between mRNA expression of RAMPs1 and 3, but not RAMP2 and 3. Our preliminary data shows that Ramp3-/- osteoblasts had increased expression β-catenin, a component of the canonical Wnt signalling pathway known to regulate skeletal homeostasis and mechanosensitivity. Given interactions of RAMPs with both calcitonin and calcitonin-like receptors to alter ligand selectivity, and with other GPCRs to change trafficking or ligand bias, it is not clear whether the bone phenotype of Ramp3-/- mice is due to alterations in signalling mediated by one or more GPCRS. However, as antagonists of RAMP-interacting receptors are growing in availability, there appears the likelihood that manipulation of the RAMP3 signalling system could provide anabolic effects therapeutically.

Mouse Microglial Calcitonin Receptor Knockout Impairs Hypothalamic Amylin Neuronal pSTAT3 Signaling but Lacks Major Metabolic Consequences.

Amylin and leptin synergistically interact in the arcuate nucleus of the hypothalamus (ARC) to control energy homeostasis. Our previous rodent studies suggested that amylin-induced interleukin-6 release from hypothalamic microglia may modulate leptin signaling in agouti-related peptide expressing neurons. To confirm the physiological relevance of this finding, the calcitonin receptor (CTR) subunit of the amylin receptor was selectively depleted in microglia by crossing tamoxifen (Tx) inducible Cx3cr1-CreERT2 mice with CTR-floxed mice. Unexpectedly, male mice with CTR-depleted microglia (KO) gained the least amount of weight of all groups regardless of diet. However, after correcting for the tamoxifen effect, there was no significant difference for body weight, fat mass or lean mass between genotypes. No alteration in glucose tolerance or insulin release was detected. However, male KO mice had a reduced respiratory quotient suggesting a preference for fat as a fuel when fed a high fat diet. Importantly, amylin-induced pSTAT3 was decreased in the ARC of KO mice but this was not reflected in a reduced anorectic response. On the other hand, KO mice seemed to be less responsive to leptin’s anorectic effect while displaying similar ARC pSTAT3 as Tx-control mice. Together, these data suggest that microglial amylin signaling is not a major player in the control of energy homeostasis in mice.

Calcitonin: current concepts and differential diagnosis.

Calcitonin (CT) is most effectively produced by the parafollicular cells of the thyroid gland. It acts through the calcitonin receptor (CTR), a seven-transmembrane class II G-protein-coupled receptor linked to multiple signal transduction pathways with its main secretagogues being calcium and gastrin. It is clinically used mostly in the diagnosis and follow-up of medullary thyroid carcinoma (MTC). Hypercalcitoninemia can be attributed to primary (e.g. CT-secreting tumor) or secondary (e.g. due to hypercalcemia) overproduction, underexcretion (e.g. renal insufficiency), drug reaction (e.g. β-blockers), or false-positive results. In clinical practice, elevated basal calcitonin (bCT) is indicative, but not pathognomonic, of MTC. Current literature leans toward an age as well as gender-specific cutoff approach. bCT >100 pg/ml has up to 100% positive prognostic value (PPV) for MTC, whereas bCT between 8 and 100 pg/ml for adult males and 6 and 80 pg/ml for adult females should be possibly further investigated with stimulation calcitonin (sCT) tests. Calcium is showing similar efficacy with pentagastrin (Pg) sCT; however, the real value of these provocative tests has been disputed given the availability of new, highly sensitive CT immunoassays. Anyhow, evidence concludes that sCT <2 times bCT may not be suggestive of MTC, in which case, thyroid in addition to whole body workup based on clinical evaluation is further warranted. Moreover, measurement of basal and stimulated procalcitonin has been proposed as an emerging concept in this clinical scenario. Measuring bCT levels in patients with thyroid nodules as a screening tool for MTC remains another controversial topic. It has been well established, though, that bCT levels raise the sensitivity of FNAB (Fine Needle Aspiration Biopsy) and correlate with disease progression both pre- and postoperatively in this situation. There have been numerous reports about extrathyroidal neoplasms that express CT. Pancreatic, laryngeal, and lung neuroendocrine neoplasms (NENs) are most frequently associated with hypercalcitoninemia, but CT production has also been described in various other neoplasms such as duodenal, esophageal, cutaneous, and paranasal NENs as well as prostate, colon, breast, and lung non-NENs. This review outlines the current biosynthetic and physiology concepts about CT and presents up-to-date information regarding the differential diagnosis of its elevation in various clinical situations.

The calcitonin receptor protects against bone loss and excessive inflammation in collagen antibody-induced arthritis

SummaryPharmacological application of teleost calcitonin (CT) has been shown to exert chondroprotective and anti-resorptive effects in patients with rheumatoid arthritis (RA). However, the role of endogenous CT that signals through the calcitonin receptor (CTR) remains elusive. Collagen II antibody-induced arthritis (CAIA) was stimulated in wild type (WT) and CTR-deficient (Calcr−/−) mice. Animals were monitored over 10 or 48 days. Joint inflammation, cartilage degradation, and bone erosions were assessed by clinical arthritis score, histology, histomorphometry, gene expression analysis, and μ-computed tomography. CAIA was accompanied by elevated systemic CT levels and CTR expression in the articular cartilage. Inflammation, cartilage degradation, and systemic bone loss were more pronounced in Calcr−/− CAIA mice. Expression of various pro-inflammatory, bone resorption, and catabolic cartilage markers were exclusively increased in Calcr−/− CAIA mice. Endogenous CT signaling through the mammalian CTR has the potential to protect against joint inflammation, cartilage degradation, and excessive bone remodeling in experimental RA.

Yukmijihwang-Tang Suppresses Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL)-Induced Osteoclast Differentiation and Prevents Ovariectomy (OVX)-Mediated Bone Loss.

Yukmijihwang-tang (YJ) has been used to treat diabetes mellitus, renal disorders, and cognitive impairment in traditional medicine. This study aimed to evaluate the anti-osteoporotic effect of YJ on ovariectomy (OVX)-induced bone loss in a rat and receptor activator of nuclear factor kappa-B ligand (RANKL)-mediated osteoclast differentiation in bone marrow macrophages (BMMs). YJ reduced the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) in an osteoclast/osteoblast co-culture system by regulating the ratio of RANKL/osteoprotegerin (OPG) by osteoblasts. Overall, YJ reduced TRAP-positive cell formation and TRAP activity and F-actin ring formation. Analysis of the underlying mechanisms indicated that YJ inhibited the activation of the nuclear factor of activated T cell cytoplasmic 1 (NFATc1) and c-Fos, resulting in the suppression of osteoclast differentiation-related genes such as TRAP, ATPase, H+ transporting, lysosomal 38 kDa, V0 subunit d2, osteoclast-associated receptor, osteoclast-stimulatory transmembrane protein, dendritic cell-specific transmembrane protein, matrix metalloproteinase-9, cathepsin K, and calcitonin receptor. YJ also inhibited the nuclear translocation of NFATc1. Additionally, YJ markedly inhibited RANKL-induced phosphorylation of signaling pathways activated in the early stages of osteoclast differentiation including the p38, JNK, ERK, and NF-κB. Consistent with these in vitro results, the YJ-administered group showed considerably attenuated bone loss in the OVX-mediated rat model. These results provide promising evidence for the potential novel therapeutic application of YJ for bone diseases such as osteoporosis.

Administration of High-Dose Methylprednisolone Worsens Bone Loss after Acute Spinal Cord Injury in Rats.

The administration of high-dose methylprednisolone (MP) for 24–48 h after traumatic spinal cord injury (SCI) has been shown to improve functional recovery. The known adverse effects of MP on skeletal muscle and the immune system, though, have raised clinically relevant safety concerns. However, the effect of MP administration on SCI-induced bone loss has not been evaluated to date. This study examined the adverse effects of high-dose MP administration on skeletal bone after acute SCI in rodents. Male rats underwent spinal cord transection at T3–T4, which was followed by an intravenous injection of MP and subsequent infusion of MP for 24 h. At 2 days, animals were euthanized and hindlimb bone samples were collected. MP significantly reduced bone mineral density (−6.7%) and induced deterioration of bone microstructure (trabecular bone volume/tissue volume, −18.4%; trabecular number, −19.4%) in the distal femur of SCI rats. MP significantly increased expression in the hindlimb bones of osteoclastic genes receptor activator of nuclear factor-κB ligand (RANKL; +402%), triiodothyronine receptor auxiliary protein (+32%), calcitonin receptor (+41%), and reduced osteoprotegerin/RANKL ratio (−72%) compared to those of SCI-vehicle animals. Collectively, 1 day of high-dose MP at a dose comparable to the dosing regimen prescribed to patients who qualify to receive this treatment approach with acute SCI increased loss of bone mass and integrity below the level of lesion than that of animals that had SCI alone, and was associated with further elevation in the expression of genes involved in pathways associated with osteoclastic bone resorption than that observed in SCI animals.

Biocompatible Customized 3D Bone Scaffolds Treated with CRFP, an Osteogenic Peptide.

Background: Currently used synthetic bone graft substitutes (BGS) are either too weak to bear the principal load or if metallic, they can support loading, but can lead to stress shielding and are unable to integrate fully. In this study, we developed biocompatible, 3D printed scaffolds derived from µCT images of the bone that can overcome these issues and support the growth of osteoblasts. Methods: Cylindrical scaffolds were fabricated with acrylonitrile butadiene styrene (ABS) and Stratasys® MED 610 (MED610) materials. The 3D-printed scaffolds were seeded with Mus musculus calvaria cells (MC3T3). After the cells attained confluence, osteogenesis was induced with and without the addition of calcitonin receptor fragment peptide (CRFP) and the bone matrix production was analyzed. Mechanical compression testing was carried out to measure compressive strength, stiffness, and elastic modulus. Results: For the ABS scaffolds, there was a 9.8% increase in compressive strength (p < 0.05) in the scaffolds with no pre-coating and the treatment with CRFP, compared to non-treated scaffolds. Similarly, MED610 scaffolds treated with CRFP showed an 11.9% (polylysine pre-coating) and a 20% (no pre-coating) increase (p < 0.01) in compressive strength compared to non-treated scaffolds. Conclusions: MED610 scaffolds are excellent BGS as they support osteoblast growth and show enhanced bone growth with enhanced compressive strength when augmented with CRFP.

Abstract 11079: Calcitonin Signaling Controls Disease-Induced Left Ventricular Remodeling

Introduction: We recently discovered that the well-known thyroid hormone calcitonin (CT) is produced by atrialcardiomyocytes (CMs) and regulates atrial fibrogenesis and arrhythmia susceptibility via CT receptors (CTRs) inatrial cardiofibroblasts (CFs). Whether the CT-CTR system regulates ventricular function and structure isunknown and was the object of this study. Method and Results: Human studies were performed on left ventricular (LV) samples from 27 patients who hadcardiac surgery (Oxford, UK; ethical approval 18/SC/0404). Animal studies used 8-10 week-old male control(CTR flx/flx ; n=150) and CTR -/- (n=131) mice subjected to transverse aortic constriction (TAC) and myocardialinfarction (MI), with selected analyses in wild type mice (n=5). Cardiac function was assessed byechocardiography at baseline, 3 and 6 weeks post-TAC, and 1 and 4 weeks post-MI.Expression profiling detected CTR and CT mRNA (qPCR; Fig. A-B) and protein (immunoblotting) in human LVtissue and mouse LV CMs and CFs, with a higher CT abundance in CMs. CTR protein was decreased in LV tissue from patients with hypertension vs non-hypertensive controls (Fig. C).CTR deletion enhanced atrial and LV fibrosis (Masson’s Trichrome; Fig. D-E), significantly exacerbated post-TAC LV ejection fraction reduction by 15.2% (Fig. F), and atrial (by 23.7%, Fig. G) and LV dilation (by 19.9%),while increasing induced-AF duration by 6-fold (Fig. H) and inducibility by 15% vs post-TAC controls.In CTR -/- mice, acute MI produced greater fibrosis in the LV risk-region (by 18%; Fig. I), ejection fractionreduction (by 14%, Fig. J) vs CTR flx/flx controls. Mortality, atrial dilation and AF-inducibility were similar inCTR -/- and control MI-mice. Conclusion: CTR deletion in mice exacerbates adverse LV remodeling and dysfunction in clinically-relevantdisease models, pointing towards an important protective role of CT-CTR signaling in ventricular myocardium.

Modulation of osteoclastogenesis through adrenomedullin receptors on osteoclast precursors: initiation of differentiation by asymmetric cell division

Adrenomedullin (ADM), a member of the calcitonin family of peptides, is a potent vasodilator and was shown to have the ability to modulate bone metabolism. We have previously found a unique cell surface antigen (Kat1 antigen) expressed in rat osteoclasts, which is involved in the functional regulation of the calcitonin receptor (CTR). Cross-linking of cell surface Kat1 antigen with anti-Kat1 antigen monoclonal antibody (mAbKat1) stimulated osteoclast formation only under conditions suppressed by calcitonin. Here, we found that ADM provoked a significant stimulation in osteoclastogenesis only in the presence of calcitonin; a similar biological effect was seen with mAbKat1 in the bone marrow culture system. This stimulatory effect on osteoclastogenesis mediated by ADM was abolished by the addition of mAbKat1. 125I-labeled rat ADM (125I-ADM)-binding experiments involving micro-autoradiographic studies demonstrated that mononuclear precursors of osteoclasts abundantly expressed ADM receptors, and the specific binding of 125I-ADM was markedly inhibited by the addition of mAbKat1, suggesting a close relationship between the Kat1 antigen and the functional ADM receptors expressed on cells in the osteoclast lineage. ADM receptors were also detected in the osteoclast progenitor cells in the late mitotic phase, in which only one daughter cell of the dividing cell express ADM receptors, suggesting the semiconservative cell division of the osteoclast progenitors in the initiation of osteoclastogenesis. Messenger RNAs for the receptor activity-modifying-protein 1 (RAMP1) and calcitonin receptor-like receptor (CRLR) were expressed in cells in the osteoclast lineage; however, the expression of RAMP2 or RAMP3 was not detected in these cells. It is suggested that the Kat1 antigen is involved in the functional ADM receptor distinct from the general ADM receptor, consisting of CRLR and RAMP2 or RAMP3. Modulation of osteoclastogenesis through functional ADM receptors abundantly expressed on mononuclear osteoclast precursors is supposed to be important in the fine regulation of osteoclast differentiation in a specific osteotrophic hormonal condition with a high level of calcitonin in blood.Adrenomedullin (ADM) significantly stimulates osteoclastogenesis only in the presence of calcitonin. Functional ADM receptor partly composed of osteoclast-specific cell surface Kat1 antigen was detected in cells in the osteoclast-lineage. Expression of ADM receptor was firstly detected in proliferating osteoclast progenitors performing asymmetric cell division and is then expressed in mononuclear osteoclast precursors. Specific regulation of ADM receptors expressed on mononuclear osteoclast precursors could provide an alternative way to modulate bone remodeling therapeutically.

Pharmacological characterisation of mouse calcitonin and calcitonin receptor-like receptors reveals differences compared with human receptors.

The calcitonin (CT) receptor family is complex, comprising two receptors (the CT receptor [CTR] and the CTR-like receptor [CLR]), three accessory proteins (RAMPs) and multiple endogenous peptides. This family contains several important drug targets, including CGRP, which is targeted by migraine therapeutics. The pharmacology of this receptor family is poorly characterised in species other than rats and humans. To facilitate understanding of translational and preclinical data, we need to know the receptor pharmacology of this family in mice. Plasmids encoding mouse CLR/CTR and RAMPs were transiently transfected into Cos-7 cells. cAMP production was measured in response to agonists in the absence or presence of antagonists. We report the first synthesis and characterisation of mouse adrenomedullin, adrenomedullin 2 and βCGRP and of mouse CTR without or with mouse RAMPs. Receptors containing m-CTR had subtly different pharmacology than human receptors; they were promiscuous in their pharmacology, both with and without RAMPs. Several peptides, including mouse αCGRP and mouse adrenomedullin 2, were potent agonists of the m-CTR:m-RAMP3 complex. Pharmacological profiles of receptors comprising m-CLR:m-RAMPs were generally similar to those of their human counterparts, albeit with reduced specificity. Mouse receptor pharmacology differed from that in humans, with mouse receptors displaying reduced discrimination between ligands. This creates challenges for interpreting which receptor may underlie an effect in preclinical models and thus translation of findings from mice to humans. It also highlights the need for new ligands to differentiate between these complexes. This article is part of a themed issue on Advances in Migraine and Headache Therapy (BJP 75th Anniversary).. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.3/issuetoc.

Methods to measure calcitonin receptor activity, up-regulated in cell stress, apoptosis and autophagy.

The expression of the calcitonin receptor (CT Receptor) is widespread throughout the life cycle of mammals and in many diseases, and in these contexts the functions of the common isoforms is largely unknown. The relatively recent development of anti-CT Receptor antibodies that bind separate epitopes on the CT a Receptor and CT b Receptor isoforms has advanced our knowledge and understanding of these events. CT Receptor at the protein level is upregulated in programmed cell death including apoptosis (as described in a previous publication) and autophagy, which is discussed in our upcoming, unpublished review. Incomplete data sets are cited in this review on the upregulation of CACLR (encoding CT Receptor) mRNA, in particular the insert-positive isoform (CT b Receptor), in response to cell stress. Cell stress is induced by growth in depleted foetal bovine serum (dFBS) or without FBS, both of which induce degrees of starvation and autophagy, or dFBS plus staurosporine, which induces apoptosis. Details of the methods deployed to generate these data are described here including measurement of the upregulation of CT b Receptor mRNA with qPCR and nanopore long range sequencing. An anti-CT Receptor antibody also known as CalRexin TM, which binds an epitope in the N-terminal domain, was conjugated to either fluorophore 568, which is accumulated into apoptotic cells as previously reported, or pHrodo Red, a pH dependent fluorescent dye, which is accumulated into autophagic and apoptotic cells. These conjugates are under development to image programmed cell death. The methods for conjugation and high content imaging on the Operetta platform are described. The high fluorescence intensity at low pH of CalRexin:pHrodo Red in both autophagic and apoptotic cells suggests localisation in autophago-lysosomes and lysosomes respectively. Overall, these observations and the methods that underpin them have contributed to our understanding of the widespread expression of CT Receptor isoforms.

Calcitonin receptor‐like (CALCRL) is a marker of stemness and an independent predictor of outcome in pediatric AML

AbstractWe have recently identified the G protein-coupled neuropeptide receptor calcitonin receptor‐like (CALCRL) as an independent prognostic biomarker and a therapeutic target in more than 1500 adult patients with acute myeloid leukemia (AML). Here, we confirmed CALCRL expression as a prognostic factor in a cohort of 284 pediatric patients with AML. High CALCRL expression was independently associated with event-free survival (hazard ratio [HR], 1.87; 95% confidence interval [CI], 1.36-2.57; P = .0001), overall survival (HR, 1.55; 95% CI, 1.06-2.27; P = .025), and cumulative incidence of relapse (HR, 2.10; 95% CI, 1.49-1.96; P < .0001) when adjusting for age, white blood cell count, and genetic risk. Despite its association with leukemia stem cell signatures, CALCRL expression remained associated with all end points when compared with the 17-gene leukemic stem cell score. The strong association of CALCRL expression with the risk of relapse also in the pediatric population supports its role as novel age-independent master regulator of relapse-initiating, drug-tolerant AML cells in humans.

Development of High Affinity Calcitonin Analog Fragments Targeting Extracellular Domains of Calcitonin Family Receptors.

The calcitonin and amylin receptors (CTR and AMY receptors) are the drug targets for osteoporosis and diabetes treatment, respectively. Salmon calcitonin (sCT) and pramlintide were developed as peptide drugs that activate these receptors. However, next-generation drugs with improved receptor binding profiles are desirable for more effective pharmacotherapy. The extracellular domain (ECD) of CTR was reported as the critical binding site for the C-terminal half of sCT. For the screening of high-affinity sCT analog fragments, purified CTR ECD was used for fluorescence polarization/anisotropy peptide binding assay. When three mutations (N26D, S29P, and P32HYP) were introduced to the sCT(22–32) fragment, sCT(22–32) affinity for the CTR ECD was increased by 21-fold. CTR was reported to form a complex with receptor activity-modifying protein (RAMP), and the CTR:RAMP complexes function as amylin receptors with increased binding for the peptide hormone amylin. All three types of functional AMY receptor ECDs were prepared and tested for the binding of the mutated sCT(22–32). Interestingly, the mutated sCT(22–32) also retained its high affinity for all three types of the AMY receptor ECDs. In summary, the mutated sCT(22–32) showing high affinity for CTR and AMY receptor ECDs could be considered for developing the next-generation peptide agonists.