Calcitonin Receptor-like Receptor Research Articles

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, 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 NO 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.

The Anti-Calcitonin Gene-Related Peptide (Anti-CGRP) Antibody Fremanezumab Reduces Trigeminal Neurons Immunoreactive to CGRP and CGRP Receptor Components in Rats.

Treatment with the anti-CGRP antibody fremanezumab is successful in the prevention of chronic and frequent episodic migraine. In preclinical rat experiments, fremanezumab has been shown to reduce calcitonin gene-related peptide (CGRP) release from trigeminal tissues and aversive behaviour to noxious facial stimuli, which are characteristic pathophysiological changes accompanying severe primary headaches. To further decipher the effects of fremanezumab that underlie these antinociceptive effects in rats, immunohistochemistry and ELISA techniques were used to analyse the content and concentration of CGRP in the trigeminal ganglion, as well as the ratio of trigeminal ganglion neurons which are immunoreactive to CGRP and CGRP receptor components, 1-10 days after subcutaneous injection of fremanezumab (30 mg/kg) compared to an isotype control antibody. After fremanezumab treatment, the fraction of trigeminal ganglion neurons which were immunoreactive to CGRP and the CGRP receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) was significantly lowered compared to the control. The content and concentration of CGRP in trigeminal ganglia were not significantly changed. A long-lasting reduction in CGRP receptors expressed in trigeminal afferents may contribute to the attenuation of CGRP signalling and antinociceptive effects of monoclonal anti-CGRP antibodies in rats.

Abstract A020: Calcitonin receptor-like receptor is expressed in blood vessels in clear cell renal cell carcinoma and upregulated in endothelial cells co-cultured with tumor cells

Abstract Introduction. Clear cell renal cell carcinoma (ccRCC) is a highly vascularized and clinically aggressive kidney cancer, with high rates of metastasis and recurrence after cytoreductive nephrectomy. ccRCC can be treated with anti-angiogenic drugs, however drug resistance usually develops within one year and 5-year survival for metastatic cases is approximately 10%. Calcitonin receptor-like receptor (CLR) is a G-protein-coupled receptor (GPCR) expressed in endothelial cells (EC) in core- (intracellular) and terminally-glycosylated (cell surface-associated) forms. CLR and its three agonists play a role in angiogenesis. In ccRCC, CLR is upregulated in tumor vessels when compared to vessels in adjacent tissues. The aims of this study were to: (1) investigate which types of vessels (blood or lymphatic) in ccRCC tissues express CLR and (2) determine whether ccRCC tumor cells affect expression of this GPCR in EC. Methods. CLR expression was analyzed in sections of ccRCC tissues (primary tumors) from three patients by using immunohistochemistry and our rabbit anti-human CLR antibody LN1436, with controls performed using the antigen (21 amino-acid peptide). Distribution of CLR immunoreactivity was compared to pan-EC (cluster of differentiation 31, CD31) and lymphatic EC-specific (podoplanin, PDPN) marker expression patterns, revealed by using immunohistochemistry on serial sections with mouse monoclonal antibodies JC/70A and D2-40 respectively. Two ccRCC cell lines (786-O and KTCTL-140) were co-cultured with human dermal blood vessel EC (HDBEC) in vitro for 48 hours. Cell types were separated by using magnetic-activated cell sorting (MACS) with anti-CD31 antibody-conjugated microbeads. CLR expression in ccRCC and HDBEC before, during and after co-culture was analyzed by using immunoblotting and LN1436 antibody. Results. In ccRCC tissue sections, CLR immunoreactivity was detected predominantly in blood and not lymphatic vessels. Core- and terminally-glycosylated forms of CLR were expressed in HDBEC, and the expression levels of both were upregulated after co-culture with each of the two studied ccRCC cell lines. Conclusions. These data advance knowledge about CLR expression in ccRCC and suggest that tumor cells may be responsible for its upregulation in EC within the tumor microenvironment. Our findings warrant further investigation of the potential role of the expressed in tumor blood vessels GPCR CLR as a prognostic marker and/or a target for therapy in ccRCC. Citation Format: Matthew A. Morfitt, John Greenman, Anthony Maraveyas, Adrian L. Harris, Leonid L. Nikitenko. Calcitonin receptor-like receptor is expressed in blood vessels in clear cell renal cell carcinoma and upregulated in endothelial cells co-cultured with tumor cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A020.

Abstract B001: Calcitonin receptor-like receptor agonists induce p44/42 MAPK phosphorylation in VEGF-A-stimulated human blood endothelial cells after bevacizumab treatment

Abstract Introduction. Clear cell renal cell carcinoma (ccRCC) is a highly vascularized and clinically aggressive cancer. In the UK, there are around 9000 diagnoses and 3000 deaths annually. Targeted therapies that inhibit angiogenic regulators, such as bevacizumab (monoclonal antibody which inhibits vascular endothelial growth factor A, VEGF-A), have limited efficacy due to drug resistance. Expression of calcitonin receptor-like receptor (CLR) is upregulated in ccRCC and correlates with disease outcome. The expression of adrenomedullin (AM), peptide agonist of CLR and angiogenic factor, is upregulated in ccRCC. We hypothesized that the CLR signaling axis acts as an alternative pathway that enables resistance to drugs targeting the VEGF-A pathway in ccRCC. In the present study, we aimed to determine whether this signaling axis remains active in VEGF-A-stimulated and bevacizumab-treated human endothelial cells. Methods. Primary human dermal blood vessel endothelial cells (HDBEC; PromoCell) were characterized by immunofluorescence utilizing pan-endothelial (cluster of differentiation 31 and 144, CD31 and CD144, and von Willebrand factor, vWF) and lymphatic-specific (lymphatic vessel endothelial hyaluronan receptor 1, LYVE-1, and prospero homeobox 1, PROX1) markers and confocal microscopy. The activities of CLR agonists (AM; intermedin, IMD; calcitonin gene-related peptide, CGRP; 10−12 - 10−6 M at 10 min) and VEGF-A (50 ng/ml over 30-minute time course) were measured by analyzing phosphorylation of p44/42 mitogen-activated protein kinases (MAPK) using immunoblotting. HDBEC were pre-treated with bevacizumab or human IgG1 at 250 ng/ml and then stimulated with individual CLR agonists at 10−6 M for 10 minutes. Experiments were repeated at least three times. Data were analyzed using Shapiro-Wilk normality, unpaired statistics and appropriate multiple comparisons tests utilizing GraphPad Prism and p&amp;lt;0.05 interpreted as significant. Results. HDBEC were a pure population. AM, IMD and CGRP induced p44/42 MAPK phosphorylation at 10−6 M (p&amp;lt;0.05). For all three CLR agonists and VEGF-A, p44/42 MAPK phosphorylation peaked after 10 minutes of incubation (p&amp;lt;0.05). Bevacizumab treatment at 250 ng/ml was sufficient to inhibit VEGF-A-induced p44/42 MAPK phosphorylation (p&amp;lt;0.05). AM, IMD and CGRP were all able to induce phosphorylation of p44/42 MAPK after bevacizumab treatment (p&amp;lt;0.01). Conclusions. The findings that CLR agonists induce p44/42 MAPK phosphorylation in VEGF-A-stimulated HDBEC after bevacizumab treatment support our hypothesis. This work provides a foundation for investigating the role of the CLR signaling axis in mechanisms of ccRCC resistance to drugs targeting the VEGF-A pathway. Citation Format: Matthew A. Morfitt, John Greenman, Anthony Maraveyas, Leonid L. Nikitenko. Calcitonin receptor-like receptor agonists induce p44/42 MAPK phosphorylation in VEGF-A-stimulated human blood endothelial cells after bevacizumab treatment [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B001.

Reassessing the adrenomedullin scavenging function of ACKR3 in lymphatic endothelial cells.

Atypical chemokine receptor 3 (ACKR3) is a scavenger of the chemokines CXCL11 and CXCL12 and of several opioid peptides. Additional evidence indicates that ACKR3 binds two other non-chemokine ligands, namely the peptide hormone adrenomedullin (AM) and derivatives of the proadrenomedullin N-terminal 20 peptide (PAMP). AM exhibits multiple functions in the cardiovascular system and is essential for embryonic lymphangiogenesis in mice. Interestingly, AM-overexpressing and ACKR3-deficient mouse embryos both display lymphatic hyperplasia. Moreover, in vitro evidence suggested that lymphatic endothelial cells (LECs), which express ACKR3, scavenge AM and thereby reduce AM-induced lymphangiogenic responses. Together, these observations have led to the conclusion that ACKR3-mediated AM scavenging by LECs serves to prevent overshooting AM-induced lymphangiogenesis and lymphatic hyperplasia. Here, we further investigated AM scavenging by ACKR3 in HEK293 cells and in human primary dermal LECs obtained from three different sources in vitro. LECs efficiently bound and scavenged fluorescent CXCL12 or a CXCL11/12 chimeric chemokine in an ACKR3-dependent manner. Conversely, addition of AM induced LEC proliferation but AM internalization was found to be independent of ACKR3. Similarly, ectopic expression of ACKR3 in HEK293 cells did not result in AM internalization, but the latter was avidly induced upon co-transfecting HEK293 cells with the canonical AM receptors, consisting of calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying protein (RAMP)2 or RAMP3. Together, these findings indicate that ACKR3-dependent scavenging of AM by human LECs does not occur at ligand concentrations sufficient to trigger AM-induced responses mediated by canonical AM receptors.

46 Adrenomedullin: A Novel Peptide Hormone for Uterine Receptivity and Conceptus Elongation in Pigs?

Abstract Adrenomedullin (ADM) is an evolutionarily conserved multi-functional peptide hormone in mammalian species. The implantation sites of conceptuses (embryo/fetus and its extra-embryonic membranes) in pregnant Adm+/− female mice are abnormally spaced leading to fetal crowding, intrauterine growth restriction (IUGR) and increases in pregnancy loss. As litter-bearing species, pigs exhibit a high incidence of early embryonic death (30-40%) and naturally occurring IUGR (15-25%), and significant numbers of stillborn piglets (3-9%). Therefore, it is imperative that the roles of ADM in regulation of uterine receptivity, as well as growth and development of conceptus in pigs be established. This study determined abundances of ADM in uterine luminal fluid, and the patterns of expression of ADM and its receptor components in uteri from cyclic and pregnant gilts, as well as conceptuses during the peri-implantation period of pregnancy. ADM receptor components include: calcitonin receptor-like receptor (CALCRL; G protein-coupled receptor bound by ADM), receptor activity modifying protein (RAMP2; dimerized with CALCRL to form ADM1 receptor) and RAMP3 (dimerized with CALCRL to form ADM2 receptor with lower specificity to ADM) and atypical chemokine receptor 3 (ACKR3; a decoy receptor that serves as a cell-autonomous molecular rheostat to dampen ADM signaling). Total recoverable ADM was greater in the uterine fluid of pregnant compared with cyclic gilts between days 10 and 16 post-estrus and was from uterine luminal epithelial (LE) and conceptus trophectoderm (Tr) cells. Uterine expression of CALCRL, RAMP2, and ACKR3 were affected by day (P &amp;lt; 0.05), pregnant status (P &amp;lt; 0.01) and/or day x status (P &amp;lt; 0.05). Within porcine conceptuses, expression of CALCRL, RAMP2 and ACKR3 increased between days 10 and 16 of pregnancy. Using an established porcine trophectoderm (pTr1) cell line isolated from elongating porcine conceptuses recovered on day 2 post-breeding, it was determined that 10-7 M ADM stimulated proliferation of pTr1 cells (P &amp;lt; 0.05) at 48 h, and increased phosphorylated mechanistic target of rapamycin (p-MTOR) and 4E binding protein (p-4EBP1) by 6.1- and 4.9-fold (P &amp;lt; 0.0001), respectively. These novel results indicate a significant role for ADM in uterine receptivity for implantation and conceptus growth and development in pigs. They also provide a framework for future studies of ADM signaling to affect proliferation and migration of Tr cells, spacing of blastocysts, implantation and placentation in pigs. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2022-67015-36491 from the USDA National Institute of Food and Agriculture.

Clinical Potential of Adrenomedullin Signaling in the Cardiovascular System.

Numerous clinical studies have revealed the utility of circulating AM (adrenomedullin) or MR-proAM (mid-regional proAM 45-92) as an effective prognostic and diagnostic biomarker for a variety of cardiovascular-related pathophysiologies. Thus, there is strong supporting evidence encouraging the exploration of the AM-CLR (calcitonin receptor-like receptor) signaling pathway as a therapeutic target. This is further bolstered because several drugs targeting the shared CGRP (calcitonin gene-related peptide)-CLR pathway are already Food and Drug Administration-approved and on the market for the treatment of migraine. In this review, we summarize the AM-CLR signaling pathway and its modulatory mechanisms and provide an overview of the current understanding of the physiological and pathological roles of AM-CLR signaling and the yet untapped potentials of AM as a biomarker or therapeutic target in cardiac and vascular diseases and provide an outlook on the recently emerged strategies that may provide further boost to the possible clinical applications of AM signaling.