Calcitonin Receptor-like Receptor Research Articles

Upregulation of the neuropeptide receptor calcitonin receptor-like in the spinal cord via MLL2 in a mouse model of paclitaxel-induced peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a prevalent and severe side effect affecting cancer patients undergoing paclitaxel treatment. Growing evidence underscores the pivotal role of calcitonin-related peptide (CGRP) in the development of CIPN. Repeated administration of paclitaxel induces alterations in CGRP release from sensory neurons within the dorsal root ganglia (DRG). The density of the CGRP receptor is most prominent in the dorsal horn of the spinal cord, where it overlaps with the distribution of CGRP. However, the impact of chemotherapy treatment on expression of the CGRP receptor in the spinal cord remains unclear, as well as the potential therapeutic benefits of a CGRP receptor antagonist in an animal model of CIPN. Using a mouse model of paclitaxel-induced mechanical hypersensitivity, we show upregulation of Calcitonin receptor-like receptor (Calcrl) mRNA expression in the spinal cord, an event that occurred in association with upregulation of the H3K4 methyltransferase MLL2. This effect of repeated paclitaxel administration was also linked to an increase in the recruitment of MLL2, thereby enhancing levels of the active mark H3K4me2 at the Calcrl promoter. Furthermore, administration of the CGRP receptor antagonist BIBN4096 mitigated mechanical and cold hypersensitivity in paclitaxel-treated mice. Together, these observations suggest the CGRP receptor in the spinal cord as a potential target for reducing paclitaxel-induced neuropathic pain in animal models.

TNBS colitis induces architectural changes and alpha-synuclein overexpression in mouse distal colon: A morphological study

Alpha-synuclein (α-syn) is widely expressed in presynaptic neuron terminals, and its structural alterations play an important role in the pathogenesis of Parkinson’s disease (PD). Aggregated α-syn has been found in brain, in the peripheral nerves of the enteric nervous system (ENS) and in the intestinal neuroendocrine cells during synucleinopathies and inflammatory bowel disorders. In the present study, we evaluated the histomorphological features of murine colon with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, a common model of colitis. Thereafter, we investigated the expression of α-syn, Toll-like receptor 4 (TLR4), choline acetyltransferase (ChAT), vasoactive intestinal peptide (VIP), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), and calcitonin-like receptor (CALCR). Finally, we investigated the presence of phosphorylated α-syn (pS129 α-syn) aggregates and their relationship with inflammatory cells. Colon from TNBS mice showed an increase in inflammatory cells infiltrate and significative changes in the architecture of the intestinal mucosa. α-Syn expression was significantly higher in inflamed colon. VIP was increased in both the mucosa and muscularis externa of TNBS mice, while TH, CGRP, and CALCR were significantly reduced in TNBS mice. Amyloid aggregates of pS129 α-syn were detectable in the ENS, as in the macrophages around the glands of the mucosa correlating with the markers of inflammation. This study describes — for the first time — the altered expression of α-syn and the occurrence of amyloid α-syn aggregates in the inflammatory cells under colitis, supporting the critical role of bowel inflammation in synucleinopathies and the involvement of α-syn in IBD.

Crosstalk between bone metastatic cancer cells and sensory nerves in bone metastatic progression.

Although the role of peripheral nerves in cancer progression has been appreciated, little is known regarding cancer/sensory nerve crosstalk and its contribution to bone metastasis and associated pain. In this study, we revealed that the cancer/sensory nerve crosstalk plays a crucial role in bone metastatic progression. We found that (i) periosteal sensory nerves expressing calcitonin gene-related peptide (CGRP) are enriched in mice with bone metastasis; (ii) cancer patients with bone metastasis have elevated CGRP serum levels; (iii) bone metastatic patient tumor samples express elevated calcitonin receptor-like receptor (CRLR, a CGRP receptor component); (iv) higher CRLR levels in cancer patients are negatively correlated with recurrence-free survival; (v) CGRP induces cancer cell proliferation through the CRLR/p38/HSP27 pathway; and (vi) blocking sensory neuron-derived CGRP reduces cancer cell proliferation in vitro and bone metastatic progression in vivo. This suggests that CGRP-expressing sensory nerves are involved in bone metastatic progression and that the CGRP/CRLR axis may serve as a potential therapeutic target for bone metastasis.

Sex differences in expression of CGRP family of receptors and ligands in the rat trigeminal system

BackgroundCalcitonin gene-related peptide (CGRP) is part of the calcitonin peptide family, which includes calcitonin (CT), amylin (AMY), and adrenomedullin (ADM). CGRP and its receptor are highly present in the trigeminovascular system (TVS). Recent research suggests that other members of the calcitonin family could be feasible therapeutic targets in the treatment of migraine. The present study aims to elucidate the distribution of ADM, AMY, CT, and their receptors in the rat TVS, and to explore potential sex differences in their expression.MethodsTrigeminal ganglia (TG) were dissected from male and female adult rats. Protein and gene expression were assessed through immunohistochemistry and RT-qPCR. Additionally, the dura mater was isolated for further investigation of protein expression and fiber localization using immunohistochemistry.ResultsQuantitative gene expression analysis revealed the presence of all genes in male and female TGs, except for calcitonin receptor (CTR). Notably, CGRP mRNA levels in TG were several folds higher than those of other genes. The receptor activity-modifying protein-1 (RAMP1) mRNA levels were significantly higher in female compared to male.No AMY or CT immunoreactivity was observed in the TVS. In contrast, immunoreactivity for ADM, CGRP, RAMP1, CTR, and calcitonin-like receptor (CLR) were observed in the cytoplasm of TG neurons. Immunoreactive Aδ-fibers storing RAMP1, ADM and CLR were also identified. RAMP2 and RAMP3 were expressed in nucleus of TG neurons and in satellite glial cells. Furthermore, RAMP1 and CLR were co-localized with CASPR in the nodes of Ranvier located in Aδ-fibers.ConclusionsThis study provides valuable insights into the distribution of the CGRP family of peptides and their receptors in the TVS. CGRP mRNA levels in the TG were markedly higher than those of other genes, demonstrating the key role of CGRP. The co-localization of CLR and RAMP1 on Aδ-fibers with CASPR suggests a potential role for this receptor in modulating trigeminal nerve function and neuronal excitability, with implications for migraine pathophysiology. Additionally, RAMP1 mRNA levels were significantly higher in female TG compared to males, indicating sex-specific differences in gene expression. These findings underscore the need for further research into the functional significance of gender-related variations.

Loss of Calcitonin Gene Related Receptor component protein (RCP) in nervous system can bias "gepant" antagonism.

Research in calcitonin gene-related peptide (CGRP) has primarily focused on ligand- receptor interactions at the calcitonin-like receptor (CLR) and receptor activity-modifying unit 1 (RAMP1) subunits. However, the role of receptor component protein (RCP), which mediates signaling via the Gα-stimulatory pathway, is less understood. A novel tamoxifen-inducible mouse model, nestinRCP (-/-), was generated to study loss of RCP in CGRP signaling in the nervous system, and behavioral changes to motion-induced nausea and postural sway were studied after systemic injections of CGRP or CGRP co-delivered with migraine drugs. Findings from this study suggest the loss of CGRP-RCP can bias "gepant" antagonists like olcegepant, and may promote development of therapies to inhibit the RCP-CLR interactions.

Extracellular bimolecular fluorescence complementation for investigating membrane protein dimerization: a proof of concept using class B GPCRs.

Bimolecular fluorescence complementation (BiFC) methodology uses split fluorescent proteins to detect interactions between proteins in living cells. To date, BiFC has been used to investigate receptor dimerization by splitting the fluorescent protein between the intracellular portions of different receptor components. We reasoned that attaching these split proteins to the extracellular N-terminus instead may improve the flexibility of this methodology and reduce the likelihood of impaired intracellular signal transduction. As a proof-of-concept, we used receptors for calcitonin gene-related peptide, which comprise heterodimers of either the calcitonin or calcitonin receptor-like receptor in complex with an accessory protein (receptor activity-modifying protein 1). We created fusion constructs in which split mVenus fragments were attached to either the C-termini or N-termini of receptor subunits. The resulting constructs were transfected into Cos7 and HEK293S cells, where we measured cAMP production in response to ligand stimulation, cell surface expression of receptor complexes, and BiFC fluorescence. Additionally, we investigated ligand-dependent internalization in HEK293S cells. We found N-terminal fusions were better tolerated with regards to cAMP signaling and receptor internalization. N-terminal fusions also allowed reconstitution of functional fluorescent mVenus proteins; however, fluorescence yields were lower than with C-terminal fusion. Our results suggest that BiFC methodologies can be applied to the receptor N-terminus, thereby increasing the flexibility of this approach, and enabling further insights into receptor dimerization.

Quantitative proteomics reveals CLR interactome in primary human cells

The G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) mediates essential functions in several cell types and is implicated in cardiovascular pathologies, skin diseases, migraine and cancer. To date, the network of proteins interacting with CLR (“CLR interactome”) in primary cells, where this GPCR is expressed at endogenous (physiologically relevant) levels, remains unknown. To address this knowledge gap, we established a novel integrative methodological workflow/approach for conducting a comprehensive/proteome-wide analysis of Homo sapiens CLR interactome. We used primary human dermal lymphatic endothelial cells and combined immunoprecipitation (IP) utilising anti-human CLR antibody with label-free quantitative nano-liquid chromatography-tandem mass spectrometry (nano LC-MS/MS) and quantitative in situ proximity ligation assay (PLA). By using this workflow, we identified 37 proteins interacting with endogenously expressed CLR amongst 4,902 detected members of the cellular proteome (by quantitative nano LC-MS/MS) and revealed direct interactions of two kinases and two transporters with this GPCR (by in situ PLA). All identified interactors have not been previously reported as members of CLR interactome. Our approach and findings uncover the hitherto unrecognized compositional complexity of the interactome of endogenously expressed CLR and contribute to fundamental understanding of the biology of this GPCR. Collectively, our study provides a first-of-its-kind integrative methodological approach and datasets as valuable resources and robust platform/springboard for advancing the discovery and comprehensive characterization of physiologically relevant CLR interactome at a proteome-wide level in a range of cell types and diseases in future studies.

Expression of Calcitonin Gene-Related Peptide and Calcitonin Receptor-like Receptor in Colorectal Adenocarcinoma.

Colorectal cancer is one of the most widespread types of cancer that still causes many deaths worldwide. The development of new diagnostic and prognostic markers, as well as new therapeutic methods, is necessary. The calcitonin gene-related peptide (CGRP) neuropeptide alongside its receptor calcitonin receptor-like receptor (CRLR) could represent future biomarkers and a potential therapeutic target. Increased levels of CGRP have been demonstrated in thyroid, prostate, lung, and breast cancers and may also have a role in colorectal cancer. At the tumor level, it acts through different mechanisms, such as the angiogenesis, migration, and proliferation of tumor cells. The aim of this study was to measure the level of CGRP in colorectal cancer patients' serum by enzyme-linked immunosorbent assay (ELISA) and determine the level of CGRP and CRLR at the tumor level after histopathological (HP) and immunohistochemical (IHC) analysis, and then to correlate them with the TNM stage and with different tumoral characteristics. A total of 54 patients with newly diagnosed colorectal adenocarcinoma were evaluated. We showed that serum levels of CGRP, as well as CGRP and CRLR tumor level expression, correlate with the TNM stage, with local tumor extension, the presence of lymph node metastasis, and distant metastasis, and also with the tumor differentiation degree. CGRP is present in colorectal cancer from the incipient TNM stage, with levels increasing with the stage, and can be used as a diagnostic and prognostic marker and may also represent a potentially new therapeutic target.

Coronary Artery Disease Risk Variant Dampens the Expression of CALCRL by Reducing HSF Binding to Shear Stress Responsive Enhancer in Endothelial Cells In Vitro.

CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary artery disease. In this study, we functionally characterized the noncoding regulatory elements carrying coronary artery disease that risks single-nucleotide polymorphisms and studied their role in the regulation of CALCRL expression in endothelial cells. To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene CALCRL, we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq [transposase-accessible chromatin with sequencing], chromatin immunoprecipitation assay-quantitative polymerase chain reaction, and electromobility shift assay) analyses, alongside luciferase reporter assays, and targeted gene and enhancer perturbations (siRNA and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) in human aortic endothelial cells. We demonstrate that the regulatory element harboring rs880890 exhibits high enhancer activity and shows significant allelic bias. The A allele was favored over the G allele, particularly under shear stress conditions, mediated through alterations in the HSF1 (heat shock factor 1) motif and binding. CRISPR deletion of rs880890 enhancer resulted in downregulation of CALCRL expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and CALCRL expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow. CALCRL knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial nitric oxide synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production. Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates CALCRL expression. A better understanding of CALCRL gene regulation and the role of single-nucleotide polymorphisms in the modulation of CALCRL expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses.

Cryo-EM Structure of the Human Amylin 1 Receptor in Complex with CGRP and Gs Protein.

Inhibition of calcitonin gene-related peptide (CGRP) or its cognate CGRP receptor (CGRPR) has arisen as a major breakthrough in the treatment of migraine. However, a second CGRP-responsive receptor exists, the amylin (Amy) 1 receptor (AMY1R), yet its involvement in the pathology of migraine is poorly understood. AMY1R and CGRPR are heterodimers consisting of receptor activity-modifying protein 1 (RAMP1) with the calcitonin receptor (CTR) and the calcitonin receptor-like receptor (CLR), respectively. Here, we present the structure of AMY1R in complex with CGRP and Gs protein and compare it with the reported structures of the AMY1R complex with rat amylin (rAmy) and the CGRPR in complex with CGRP. Despite similar protein backbones observed within the receptors and the N- and C-termini of the two peptides bound to the AMY1R complexes, they have distinct organization in the peptide midregions (the bypass motif) that is correlated with differences in the dynamics of the respective receptor extracellular domains. Moreover, divergent conformations of extracellular loop (ECL) 3, intracellular loop (ICL) 2, and ICL3 within the CTR and CLR protomers are evident when comparing the CGRP bound to the CGRPR and AMY1R, which influences the binding mode of CGRP. However, the conserved interactions made by the C-terminus of CGRP to the CGRPR and AMY1R are likely to account for cross-reactivity of nonpeptide CGRPR antagonists observed at AMY1R, which also extends to other clinically used CGRPR blockers, including antibodies.

Functional Analysis and Tissue-Specific Expression of Calcitonin and CGRP with RAMP-Modulated Receptors CTR and CLR in Chickens.

Calcitonin (CT) and calcitonin gene-related peptide (CGRP) are critical regulators of calcium balance and have extensive implications for vertebrate physiological processes. This study explores the CT and CGRP signaling systems in chickens through cloning and characterization of the chicken calcitonin receptor (CTR) and calcitonin receptor-like receptor (CLR), together with three receptor activity-modifying proteins (RAMPs). We illuminated the functional roles for chickens between the receptors examined alone and in RAMP-associated complexes using luciferase reporter assays. Chicken CTRs and CLRs stimulated the cAMP/PKA and MAPK/ERK signaling pathways, signifying their functional receptor status, with CT showing appreciable ligand activity at nanomolar concentrations across receptor combinations. Notably, it is revealed that chicken CLR can act as a functional receptor for CT without or with RAMPs. Furthermore, we uncovered a tissue-specific expression profile for CT, CGRP, CTR, CLR, and RAMPs in chickens, indicating the different physiological roles across various tissues. In conclusion, our data establish a clear molecular basis to reveal information on CT, CGRP, CTR, CLR, and RAMPs in chickens and contribute to understanding the conserved or divergent functions of this family in vertebrates.

Activation of CGRP receptor-mediated signaling promotes tendon-bone healing.

Calcitonin gene-related peptide (CGRP), an osteopromotive neurotransmitter with a short half-life, shows increase while calcitonin receptor-like (CALCRL) level is decreased at the early stage in bone fractures. Therefore, the activation of CALCRL-mediated signaling may be more critical to promote the tendon-bone healing. We found CGRP enhanced osteogenic differentiation of BMSCs through PKA/CREB/JUNB pathway, contributing to improved sonic hedgehog (SHH) expression, which was verified at the tendon-bone interface (TBI) in the mice with Calcrl overexpression. The osteoblast-derived SHH and slit guidance ligand 3 were reported to favor nerve regeneration and type H (CD31hiEMCNhi) vessel formation, respectively. Encouragingly, the activation or inactivation of CALCRL-mediated signaling significantly increased or decreased intensity of type H vessel and nerve fiber at the TBI, respectively. Simultaneously, improved gait characteristics and biomechanical performance were observed in the Calcrl overexpression group. Together, the gene therapy targeting CGRP receptor may be a therapeutic strategy in sports medicine.

Structural basis of distinct signaling kinetics of calcitonin-like receptor peptide ligands

Structural basis of distinct signaling kinetics of calcitonin-like receptor peptide ligands

Graphene as a potential treatment for acute migraine: A computational study

Here, we study the graphene nanosheets with various functional groups including –COOH, -NH2, –COOH-NH2, -OH, and O interacting with calcitonin receptor-like receptor (CLR) in combination with receptor activity-modifying protein (RAMP1). This study uses molecular dynamics (MD) simulations to study the interaction between CLR/RAMP1 and graphene-family planar nanosheets: Gr, Gr-COOH, Gr-NH2, Gr-OH, Gr-O, and Gr-COOH&-NH2. The Ubrogepant was used to validate the results and comparison of the Gr-family performance. The MD simulation data revealed that Gr, Gr-OH, and Gr-COOH interacted with CLR/RAMP1 more intensely, due to changes in electrostatic interactions. Furthermore, the binding affinity of the nanosheets, as well as Ubrogepant toward the protein, have been calculated using docking. Docking analysis with both CLR fragment and CLR/RAMP1revealed similar trends in the interactions where Gr-OH has the strongest affinity in binding to the neuropeptide. The DFT calculations demonstrated a discernible surface polarity hierarchy among the functionalized graphene materials, revealing Gr-O and Gr-OH with the highest surface polarity, followed by Gr-COOH and Gr-NH2, respectively, consistent with our findings. Altogether, our study shows that three candidates; Gr-OH> Gr-COOH> pristine Gr can inhibit CGRPR binding by disrupting the protein’s structure. Significantly, the Gr-family outperformed Ubrogepant as an indicator of its potential anti-migraine applications. The results of this study provide detailed and reliable theoretical guidance for using the graphene family in treating migraine and can assist in the further development of new graphene-based materials.

CALCRL knockdown suppresses cancer stemness and chemoresistance in acute myeloid leukemia with FLT3-ITD and DNM3TA-R882 double mutations.

Acute myeloid leukemia (AML)patients with FLT3 internal tandem duplication (FLT3-ITD)and DNA methyltransferase 3A (DNMT3A)R882 double mutations had a worse prognosis compared withAML with FLT3-ITDor DNMT3A R882 single mutation. This study was designed to explore the specific role of Calcitonin Receptor Like (CALCRL)in AML with FLT3-ITDand DNMT3A R882 double mutations. MOLM13 cells were transduced with CRISPR knockout sgRNA constructs to establish the FTL3-ITDand DNMT3A-R882double-mutated AML cell model. Quantitative real-time PCR and Western blot assay were carried out to examine corresponding gene and protein expression. Methylation of CALCRL promoter was measured by methylation-specific PCR (MSP).Cell viability, colony formation, flow cytometry, and sphere formation assays were conducted to determine cell proliferation, apoptosis, and stemness. MOLM13 cells were exposed to stepwise increasing concentrations of cytarabine (Ara-C) to generate MOLM13/Ara-Ccells. An in vivo AML animal model was established, and the tumor volume and weight were recorded. TUNEL assay was adopted to examine cell apoptosis in tumor tissues. DNMT3A-R882 mutation upregulated the expression of CALCRL while downregulated the DNA methylation level of CALCRL in MOLM13 cells. CALCRL knockdown greatly inhibited cell proliferation, promoted apoptosisand repressed cell stemness, accompanied with the downregulated Oct4, SOX2, and Nanog in DNMT3A-R882-mutated MOLM13 cells and MOLM13/Ara-C cells. Furthermore, CALCRL knockdown restricted tumor growth and the chemoresistance of AML in vivo, as well as inducing cell apoptosis in tumor tissues. Together, these data reveal that CALCRL is a vital regulator of leukemia cell survival and resistance to chemotherapy, suggesting CALCRL as a promising therapeutic target for the treatment of FTL3-ITDand DNMT3A-R882 double-mutated AML.

Receptor activity-modifying proteins of adrenomedullin (RAMP2/3): Roles in the pathogenesis of ARDS.

Acute respiratory distress syndrome (ARDS) is a life-threatening lung condition characterized by widespread inflammation and pulmonary edema. Adrenomedullin (AM), a bioactive peptide with various functions, is expected to be applied in treating ARDS. Its functions are regulated primarily by two receptor activity-modifying proteins, RAMP2 and RAMP3, which bind to the AM receptor calcitonin receptor-like receptor (CLR). However, the roles of RAMP2 and RAMP3 in ARDS remain unclear. We generated a mouse model of ARDS via intratracheal administration of lipopolysaccharide (LPS), and analyzed the pathophysiological significance of RAMP2 and RAMP3. RAMP2 expression declined with LPS administration, whereas RAMP3 expression increased at low doses and decreased at high doses of LPS. After LPS administration, drug-inducible vascular endothelial cell-specific RAMP2 knockout mice (DI-E-RAMP2−/−) showed reduced survival, increased lung weight, and had more apoptotic cells in the lungs. DI-E-RAMP2−/− mice exhibited reduced expression of Epac1 (which regulates vascular endothelial cell barrier function), while RAMP3 was upregulated in compensation. In contrast, after LPS administration, RAMP3−/− mice showed no significant changes in survival, lung weight, or lung pathology, although they exhibited significant downregulation of iNOS, TNF-α, and NLRP3 during the later stages of inflammation. Based on transcriptomic analysis, RAMP2 contributed more to the circulation-regulating effects of AM, whereas RAMP3 contributed more to its inflammation-regulating effects. These findings indicate that, while both RAMP2 and RAMP3 participate in ARDS pathogenesis, their functions differ distinctly. Further elucidation of the pathophysiological significance and functional differences between RAMP2 and RAMP3 is critical for the future therapeutic application of AM in ARDS.

CALCRL induces resistance to daunorubicin in acute myeloid leukemia cells through upregulation of XRCC5/TYK2/JAK1 pathway.

Chemotherapy is the main treatment option for acute myeloid leukemia (AML), but acquired resistance of leukemic cells to chemotherapeutic agents often leads to difficulties in AML treatment and disease relapse. High calcitonin receptor-like (CALCRL) expression is closely associated with poorer prognosis in AML patients. Therefore, this study was performed by performing CALCRL overexpression constructs in AML cell lines HL-60 and Molm-13 with low CALCRL expression. The results showed that overexpression of CALCRL in HL-60 and Molm-13 could confer resistance properties to AML cells and reduce the DNA damage and cell cycle G0/G1 phase blocking effects caused by daunorubicin (DNR) and others. Overexpression of CALCRL also reduced DNR-induced apoptosis. Mechanistically, the Cancer Clinical Research Database analyzed a significant positive correlation between XRCC5 and CALCRL in AML patients. Therefore, the combination of RT-PCR and Western blot studies further confirmed that the expression levels of XRCC5 and PDK1 genes and proteins were significantly upregulated after overexpression of CALCRL. In contrast, the phosphorylation levels of AKT/PKCε protein, a downstream pathway of XRCC5/PDK1, were significantly upregulated. In the response study, transfection of overexpressed CALCRL cells with XRCC5 siRNA significantly upregulated the drug sensitivity of AML to DNR. The expression levels of PDK1 protein and AKT/PKCε phosphorylated protein in the downstream pathway were inhibited considerably, and the expression of apoptosis-related proteins Bax and cleaved caspase-3 were upregulated. Animal experiments showed that the inhibitory effect of DNR on the growth of HL-60 cells and the number of bone marrow invasions were significantly reversed after overexpression of CALCRL in nude mice. However, infection of XCRR5 shRNA lentivirus in HL-60 cells with CALCRL overexpression attenuated the effect of CALCRL overexpression and upregulated the expression of apoptosis-related proteins induced by DNR. This study provides a preliminary explanation for the relationship between high CALCRL expression and poor prognosis of chemotherapy in AML patients. It offers a more experimental basis for DNR combined with molecular targets for precise treatment in subsequent studies.

RAMP1 as a novel prognostic biomarker in pan-cancer and osteosarcoma.

Receptor activity modifying protein 1 (RAMP1) facilitates the localization of the calcitonin-like receptor (CLR) to the plasma membrane, but its role in osteosarcoma (OS) remains unclear. We evaluated the RAMP1 expression and prognostic value across different cancers, studying tumor immune infiltration. The prognostic value was analyzed using the GSE39058 and TARGET datasets. Differential gene expression was evaluated. a protein-protein interaction network was constructed, and gene set enrichment analysis was performed. The function of RAMP1 in the tumor microenvironment was analyzed, and its expression in OS cell lines was validated using quantitative real-time PCR. High RAMP1 expression correlated with poor prognosis relative to low RAMP1 expression (p < 0.05). Low RAMP1 expression correlated with an abundance of CD4+ memory-activated T cells. whereas a high expression level correlated with a high proportion of gamma-delta T cells (γδ T cells). Differentially expressed genes from TARGET was enriched in olfactory transduction pathways (normalized enrichment scores [NES] = 1.6998, p < 0.0001). RAMP1 expression negatively correlated with CD44 expression but positively correlated with TNFSF9 expression. The RAMP1 gene is substantially expressed in OS cells compared to the normal osteoblast cell line hFOB1.19. Thus, RAMP1 may be a prognostic biomarker and potential therapeutic target in OS.

Pharmacological characterisation of erenumab, Aimovig, at two calcitonin gene-related peptide responsive receptors.

Calcitonin gene-related peptide (CGRP) is involved in migraine pathophysiology. CGRP can signal through two receptors. The canonical CGRP receptor comprises the calcitonin receptor-like receptor and receptor activity-modifying protein 1 (RAMP1); the AMY1 receptor comprises the calcitonin receptor with RAMP1. Drugs that reduce CGRP activity, such as receptor antagonists, are approved for the treatment and prevention of migraine. Despite being designed to target the canonical CGRP receptor, emerging evidence suggests that these antagonists, including erenumab (a monoclonal antibody antagonist) can also antagonise the AMY1 receptor. However, it is difficult to estimate its selectivity because direct comparisons between receptors under matched conditions have not been made. We therefore characterised erenumab at both CGRP-responsive receptors with multiple ligands, including αCGRP and βCGRP. Erenumab antagonism was quantified through IC50 and pKB experiments, measuring cAMP production. We used SK-N-MC cells which endogenously express the human CGRP receptor, and HEK293S and Cos7 cells transiently transfected to express either human CGRP or AMY1 receptors. Erenumab antagonised both the CGRP and AMY1 receptors with an ~20-120-fold preference for the CGRP receptor, depending on the cells, agonist, analytical approach and/or assay format. Erenumab antagonised both forms of CGRP equally, and appeared to act as a competitive reversible antagonist at both receptors. Despite being designed to target the CGRP receptor, erenumab can antagonise the AMY1 receptor. Its ability to antagonise CGRP activity at both receptors may be useful in better understanding the clinical profile of erenumab.

Multi-biological functions of intermedin in diseases.

Intermedin (IMD) is a member of the calcitonin gene-related peptide (CGRP)/calcitonin (CT) superfamily, and it is expressed extensively throughout the body. The typical receptors for IMD are complexes composed of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein (RAMP), which leads to a biased activation towards Gαs. As a diagnostic and prognostic biomarker, IMD regulates the initiation and metastasis of multiple tumors. Additionally, IMD functions as a proangiogenic factor that can restrain excessive vascular budding and facilitate the expansion of blood vessel lumen, ultimately resulting in the fusion of blood vessels. IMD has protective roles in various diseases, including ischemia-reperfusion injury, metabolic disease, cardiovascular diseases and inflammatory diseases. This review systematically elucidates IMD's expression, structure, related receptors and signal pathway, as well as its comprehensive functions in the context of acute kidney injury, obesity, diabetes, heart failure and sepsis. However, the precise formation process of IMD short peptides in vivo and their downstream signaling pathway have not been fully elucidated yet. Further in-depth studies are need to translate IMD research into clinical applications.