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 utilizing anti-human CLR antibody with label-free quantitative nano LC-MS/MS and quantitative in situ proximity ligation assay. By using this workflow, we identified 37 proteins interacting with endogenously expressed CLR amongst 4902 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 proximity ligation assay). 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.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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 with AML with FLT3-ITD or DNMT3A R882 single mutation. This study was designed to explore the specific role of Calcitonin Receptor Like (CALCRL) in AML with FLT3-ITD and DNMT3A R882 double mutations. MOLM13 cells were transduced with CRISPR knockout sgRNA constructs to establish the FTL3-ITD and DNMT3A-R882 double-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-C cells. 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 apoptosis and 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-ITD and DNMT3A-R882 double-mutated AML.

Pharmacological characterization of the relaxant effect induced by adrenomedullin in rat cavernosal smooth muscle

The aim of the present study was to determine the mechanisms underlying the relaxant effect of adrenomedullin (AM) in rat cavernosal smooth muscle (CSM) and the expression of AM system components in this tissue. Functional assays using standard muscle bath procedures were performed in CSM isolated from male Wistar rats. Protein and mRNA levels of pre-pro-AM, calcitonin receptor-like receptor (CRLR), and Subtypes 1, 2 and 3 of the receptor activity-modifying protein (RAMP) family were assessed by Western immunoblotting and quantitative real-time polymerase chain reaction, respectively. Nitrate and 6-keto-prostaglandin F1α (6-keto-PGF1α; a stable product of prostacyclin) levels were determined using commercially available kits. Protein and mRNA of AM, CRLR, and RAMP 1, -2, and -3 were detected in rat CSM. Immunohistochemical assays demonstrated that AM and CRLR were expressed in rat CSM. AM relaxed CSM strips in a concentration-dependent manner. AM22-52, a selective antagonist for AM receptors, reduced the relaxation induced by AM. Conversely, CGRP8-37, a selective antagonist for calcitonin gene-related peptide receptors, did not affect AM-induced relaxation. Preincubation of CSM strips with NG-nitro-L-arginine-methyl-ester (L-NAME, nitric oxide synthase inhibitor), 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, quanylyl cyclase inhibitor), Rp-8-Br-PET-cGMPS (cGMP-dependent protein kinase inhibitor), SC560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole, selective cyclooxygenase-1 inhibitor], and 4-aminopyridine (voltage-dependent K+ channel blocker) reduced AM-induced relaxation. On the other hand, 7-nitroindazole (selective neuronal nitric oxide synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), H89 (protein kinase A inhibitor), SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, adenylate cyclase inhibitor], glibenclamide (selective blocker of ATP-sensitive K+ channels), and apamin (Ca2+-activated channel blocker) did not affect AM-induced relaxation. AM increased nitrate levels and 6-keto-PGF1α in rat CSM. The major new contribution of this research is that it demonstrated expression of AM and its receptor in rat CSM. Moreover, we provided evidence that AM-induced relaxation in this tissue is mediated by AM receptors by a mechanism that involves the nitric oxide-cGMP pathway, a vasodilator prostanoid, and the opening of voltage-dependent K+ channels.

Effect and mechanism of intermedin in acute rat cardiac ischemic injury / 法医学杂志

OBJECTIVE@#To investigate the effect and potential mechanism of intermedin (IMD) in acute cardiac ischemic injury and to provide a new approach for exploring mechanism of sudden cardiac death.@*METHODS@#Seventy-two healthy male rats were randomly divided into 3 groups: control, ischemic and the IMD-treated group. The activity of lactate dehydrogenase (LDH), malondialdehyde (MDA) and superoxide dismutase (SOD) in heart blood were tested by enzyme chemistry method. The mRNA changes of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs) in cardiac were measured by real-time PCR analysis. Myocardial cyclic adenosine monophosphate (cAMP) content was determined by enzyme linked immunosorbent assay (ELISA). Apoptosis related factors Bcl-2 and Bax were detected by immunohistochemistry.@*RESULTS@#Comparing with the control group, LDH and MDA activity of ischemic group in heart blood increased and SOD activity decreased. The concentration of cAMP increased in ventricular muscle, Bcl-2 and Bax proteins expression ratio level decreased. The intravenation of IMD decreased the level of increased activity of LDH and MDA, and lessened the level of decreased activity of SOD. The mRNA expression of CRLR and RAMPs obviously increased in ventricular muscle.@*CONCLUSION@#The protective effect of IMD against myocardial ischemic injury could be caused by decreasing the oxidative stress of ischemia and inhibiting the myocardial apoptosis.

Changes of intermedin/adrenomedullin 2 and its receptors in the right ventricle of rats with chronic hypoxic pulmonary hypertension / 生理学报

The purpose of the present study was to explore the expression changes of intermedin/adrenomedullin 2 (IMD/ADM2), a novel small molecular bioactive peptide, and its receptors, calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMP1, RAMP2, RAMP3) in the right ventricle of rats with chronic hypoxia-induced pulmonary hypertension. Twenty male Sprague-Dawley rats were randomly divided into 4-week hypoxia group and normal control group (each n=10). The rats in hypoxia group were placed in an isobaric hypoxic chamber, in which O(2) content was maintained at 9%-11% by delivering N(2), and CO(2) content was maintained at <3% for 4 weeks (8 h/d, 6 d/week). The rats in the control group were housed in room air. The protein levels of IMD/ADM2 and adrenomedullin (ADM) in blood plasma and right ventricular tissue were measured by radioimmunoassay. The mRNA expressions of IMD/ADM2, ADM and their receptors CRLR, RAMP1, RAMP2, RAMP3 in right ventricular tissue were determined by reverse transcription-polymerase chain reaction (RT-PCR). The results showed that the ratio of right ventricle weight to left ventricle plus septum weight [RV/(LV+S)] and mean pulmonary arterial pressure (mPAP) were higher in hypoxia group than those in the control group (all P<0.01), suggesting that the rat model of pulmonary hypertension was successfully established. However, the mean carotid arterial pressure (mCAP) between the two groups had no significant difference. Compared with that in the control group, ADM contents in plasma and right ventricular tissue in hypoxia group increased by 1.26 and 1.68 folds (all P<0.01), respectively. Likewise, IMD/ADM2 contents in blood plasma and right ventricular tissue in hypoxia group increased by 0.90 and 1.19 folds (P<0.01), respectively, compared with that in the control group. The data of RT-PCR showed that mRNA levels of ADM, IMD/ADM2 and RAMP2 in hypoxia group increased by 155.1% (P<0.01), 80.9% (P<0.01) and 52.9% (P<0.05), respectively, compared with those in the control group. There were no significant differences in mRNA expressions of CRLR, RAMP1 and RAMP3 between the two groups (all P>0.05). Taken together, the results show that the level of IMD/ADM2 increases in the rats with chronic hypoxia-induced pulmonary hypertension.

Increased atria expression of receptor activity-modifying proteins in heart failure patients / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Receptor activity-modifying proteins (RAMPs) determine the ligand specificity of the calcitonin receptor-like receptor (CRLR); co-expression of RAMP1 and CRLR results in a calcitonin gene related peptide (CGRP) receptor, whereas the association of RAMP2 or RAMP3 with CRLR gives an adrenomedullin(ADM) receptor. As CGRP and ADM may play a beneficial role in heart failure, this study aimed at the question whether RAMPs mRNAs are changed in heart failure.</p><p><b>METHODS</b>Semi-quantitative reverse transcription-PCR (RT-PCR) was used to detect and quantify the mRNAs of RAMP1 and RAMP3 in the atria of heart failing patients.</p><p><b>RESULTS</b>It was found that the expressions of RAMP1, RAMP2 and RAMP3 mRNAs increased with the worsening of heart function, but the expressions of RAMP1 and RAMP2 mRNA decreased at level IV of heart failure.</p><p><b>CONCLUSION</b>The above results demonstrated in the atria of heart failure patients an up-regulation of CGRP receptor by an increase of RAMP1 in association with CRLR and an up-regulation of ADM receptor by an increase of RAMP2 expression in association with CRLR, thus suggesting that CGRP and ADM receptors be playing a functional role in compensating the chronic heart failure in human.</p>

The localization of adrenomedullin in rat kidney tissue and its inhibitory effect on the growth of cultured rat mesangial cells / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To observe the localization of adrenomedullin (AM) in rat kidney tissue and its inhibitory effect on the growth of cultured rat mesangial cells (MsC).</p><p><b>METHODS</b>A monoclonal antibody against AM developed by our laboratory was used to detect the localization of AM protein in rat kidney tissue by avidin-biotin complex immunohistochemistry. The expressions of AM and its receptor CRLR mRNA on cultured glomerular epithelial cells (GEC) and MsC were investigated by Northern blot assay, and the possible effect of AM secreted by GEC on MsC proliferation was observed using [3H]thymidine incorporation as an index.</p><p><b>RESULTS</b>A specific monoclonal antibody against AM was succesfully developed. AM was immunohistochemically localized mainly in glomeruli (GEC and endothelial cells), some cortical proximal tubules, medullary collecting duct cells, interstitial cells, vascular smooth muscle cells and endothelial cells. Northern blot assay showed that AM mRNA was expressed only on cultured GEC, but not on MsC, however, AM receptor CRLR mRNA was only expressed on MsC. GEC conditioned medium containing AM can inhibit MsC growth and AM receptor blocker CGRP8-37 may partially decreased this inhibitory effect.</p><p><b>CONCLUSION</b>AM produced by GEC inhibits the proliferation of MsC, which suggests that AM as an important regulator is involved in glomerular normal physiological functions and pathologic processes.</p>