ILC2-derived CGRP triggers acute inflammation and nociceptive responses in bacterial cystitis.

Calcitonin gene-related peptide (CGRP), a neuropeptide involved in nociceptor neuronal function, plays a critical role in mediating neuroinflammation and pain. In this study, we find that bladder group 2 innate lymphoid cells (ILC2s) function as primary producers of CGRP in the early phase of bacterial cystitis, contributing to increased inflammation, altered voiding behavior, and heightened pelvic allodynia. Furthermore, we demonstrate that interleukin (IL)-33, a cytokine secreted by urothelial cells, upregulates CGRP production by ILC2s in the bladder during uropathogenic Escherichia coli (UPEC) infection. Moreover, our research reveals that monocytes expressing high levels of receptor activity-modifying protein 1 (RAMP1), a CGRP receptor, mediate the pro-inflammatory effects of CGRP-producing ILC2s. In summary, our results underscore the significance of the immune cell-derived neuropeptides in the pathology of UPEC infection, suggesting a promising therapeutic approach targeting the IL-33-ILC2-CGRP axis for managing lower urinary tract symptoms in bacterial cystitis.

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.

Differential expression of components of the CGRP-receptor family in human coronary and human middle meningeal arteries: functional implications.

BACKGROUND: Different responses in human coronary arteries (HCA) and human middle meningeal arteries (HMMA) were observed for some of the novel CGRP receptor antagonists, the gepants, for inhibiting CGRP-induced relaxation. These differences could be explained by the presence of different receptor populations in the two vascular beds. Here, we aim to elucidate which receptors are involved in the relaxation to calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2) in HCA and HMMA. METHODS: RNA was isolated from homogenized human arteries (23 HCAs; 12 F, 11 M, age 50 ± 3 years and 26 HMMAs; 14 F, 12 M, age 51 ± 3 years) and qPCR was performed for different receptor subunits. Additionally, relaxation responses to CGRP, AM or AM2 of the human arteries were quantified using a Mulvany myograph system, in the presence or absence of the adrenomedullin 1 receptor antagonist AM22-52 and/or olcegepant. RESULTS: Calcitonin-like receptor (CLR) mRNA was expressed equally in both vascular beds, while calcitonin receptor (CTR) and receptor activity-modifying protein 3 (RAMP3) expression was low and could not be detected in all samples. RAMP1 expression was similar in HCA and HMMA, while RAMP2 expression was higher in HMMA. Moreover, receptor component protein (RCP) expression was higher in HMMA than in HCA. Functional experiments showed that olcegepant inhibits relaxation to all three agonists in both vascular beds. In HCA, antagonist AM22-52 did not inhibit relaxation to any of the agonists, while a trend for blocking relaxation to AM and AM2 could be observed in HMMA. CONCLUSION: Based on the combined results from receptor subunit mRNA expression and the functional responses in both vascular tissues, relaxation of HCA is mainly mediated via the canonical CGRP receptor (CLR-RAMP1), while relaxation of HMMA can be mediated via both the canonical CGRP receptor and the adrenomedullin 1 receptor (CLR-RAMP2). Future research should investigate whether RAMP2 predominance over RAMP1 in the meningeal vasculature results in altered migraine susceptibility or in a different response to anti-migraine medication in these patients. Moreover, the exact role of RCP in CGRP receptor signalling should be elucidated in future research.

RAMP1 Signaling Mitigates Acute Lung Injury by Distinctively Regulating Alveolar and Monocyte-Derived Macrophages.

Acute respiratory distress syndrome (ARDS) is a life-threatening lung injury that induces cytokine hypersecretion. Receptor activity-modifying protein (RAMP) 1, a subunit of the calcitonin gene-related peptide (CGRP) receptor, regulates the production of cytokines. This study examined the role of RAMP1 signaling during lipopolysaccharide (LPS)-induced acute lung injury (ALI). LPS administration to wild-type (WT) mice depleted alveolar macrophages (AMs) and recruited monocyte-derived macrophages (MDMs) and neutrophils. RAMP1-deficient (RAMP1-/-) mice exhibited higher lung injury scores, cytokine levels, and cytokine-producing neutrophil infiltration. RAMP1-deficient AMs produced more cytokines in response to LPS than WT AMs. Adoptive transfer of RAMP1-deficient AMs to RAMP1-/- mice increased cytokine levels and neutrophil accumulation compared to the transfer of WT AMs. RAMP1-/- mice had reduced MDM recruitment and lower pro-inflammatory and reparative macrophage profiles. Cultured bone marrow (BM)-derived RAMP1-deficient macrophages stimulated with LPS showed decreased expression of pro-inflammatory and pro-repairing genes. CGRP administration to WT mice reduced cytokine production and neutrophil accumulation. These findings indicate that RAMP1 signaling mitigates LPS-induced ALI by inactivating AMs and promoting inflammatory and repair activities of MDMs. Targeting RAMP1 signaling presents a potential therapeutic approach for the treatment of ARDS.

Signalling of the neuropeptide calcitonin gene-related peptide (CGRP) through RAMP1 promotes liver fibrosis via TGFß1/Smad2 and YAP pathways.

The liver is innervated by primary sensory nerve fibres releasing the neuropeptide calcitonin gene-related peptide (CGRP). Elevated plasma levels of CGRP have been found in patients with liver fibrosis or cirrhosis. We hypothesised that signalling of CGRP and its receptors might regulate liver fibrosis and propose a novel potential target for the treatment. In this study, hepatic expression of CGRP and its receptor component, the receptor activity-modifying protein 1 (RAMP1), was dramatically increased in diseased livers of patients. In a murine liver fibrosis model, deficiency of RAMP1 resulted in attenuated fibrogenesis characterized by less collagen deposition and decreased activity of hepatic stellate cells (HSC). Mechanistically, activity of the TGFß1 signalling core component Smad2 was severely impaired in the absence of RAMP1, and Yes-associated protein (YAP) activity was found to be diminished in RAMP1-deficient liver parenchyma. In vitro, stimulation of the HSC line LX-2 cells with CGRP induces TGFß1 production and downstream signalling as well as HSC activation documented by increased α-SMA expression and collagen synthesis. We further demonstrate in LX-2 cells that CGRP promotes YAP activation and its nuclear translocation subsequent to TGFß1/Smad2 signals. These data support a promotive effect of CGRP signalling in liver fibrosis via stimulation of TGFß1/Smad2 and YAP activity.

Lack of RAMP1 Signaling Suppresses Liver Regeneration and Angiogenesis Following Partial Hepatectomy in Mice.

BACKGROUND/AIM: The liver effectively restores both size and function following partial hepatectomy (PHx). Angiogenesis is crucial for the repair and regeneration of liver tissue post-PHx. Calcitonin gene-related peptide (CGRP) released from sensory nerves and its receptor-receptor activity-modifying protein 1 (RAMP1) are involved in angiogenesis. This study aimed to assess the role of RAMP1 signaling in angiogenesis during liver regeneration following PHx. MATERIALS AND METHODS: RAMP1 deficient (RAMP1-/-) and wild-type (WT) mice were subjected to PHx. RESULTS: RAMP1-/- mice demonstrated delayed liver regeneration, indicated by lower liver-to-body weight ratios compared to WT mice. This was associated with lower levels of Ki67+ hepatocytes and hepatic trophic growth factors. Additionally, RAMP1-/- mice exhibited lower levels of endothelial cell markers, including CD31, compared to WT mice. This reduction was associated with reduced levels of vascular endothelial growth factor (VEGF)-C, VEGF-D, and VEGF receptor 3 (VEGFR3). In WT mice with PHx, the administration of a VEGFR3 inhibitor reduced the liver-to-body weight ratio, Ki67+ hepatocytes, and VEGF-C/VEGFR3 expression levels in the liver compared to those in the vehicle-treated group. CONCLUSION: The deletion of RAMP1 signaling suppresses liver regeneration and angiogenesis through VEGFR3. Specific activation of RAMP1 signaling may represent a potential therapeutic strategy for liver regeneration following PHx.

Sensory nerves drive migration of dental pulp stem cells via the CGRP-Ramp1 axis in pulp repair.

Dental pulp stem cells (DPSCs) are responsible for maintaining pulp structure and function after pulp injury. DPSCs migrate directionally to the injury site before differentiating into odontoblast-like cells, which is a prerequisite and a determinant in pulp repair. Increasing evidence suggests that sensory neuron-stem cell crosstalk is critical for maintaining normal physiological functions, and sensory nerves influence stem cells mainly by neuropeptides. However, the role of sensory nerves on DPSC behaviors after pulp injury is largely unexplored. Here, we find that sensory nerves released significant amounts of calcitonin gene-related peptide (CGRP) near the injury site, acting directly on DPSCs via receptor activity modifying protein 1 (RAMP1) to promote collective migration of DPSCs to the injury site, and ultimately promoting pulp repair. Specifically, sensory denervation leads to poor pulp repair and ectopic mineralization, in parallel with that DPSCs failed to be recruited to the injury site. Furthermore, in vitro evidence shows that sensory nerve-deficient microenvironment suppressed DPSC migration prominently among all related behaviors. Mechanistically, the CGRP-Ramp1 axis between sensory neurons and DPSCs was screened by single-cell RNA-seq analysis and immunohistochemical studies confirmed that the expression of CGRP rather than Ramp1 increases substantially near the damaged site. We further demonstrated that CGRP released by sensory nerves binds the receptor Ramp1 on DPSCs to facilitate cell collective migration by an indirect co-culture system using conditioned medium from trigeminal neurons, CGRP recombinant protein and antagonists BIBN4096. The treatment with exogenous CGRP promoted the recruitment of DPSCs, and ultimately enhanced the quality of pulp repair. Targeting the sensory nerve could therefore provide a new strategy for stem cell-based pulp repair and regeneration.

The neuroimmune CGRP-RAMP1 axis tunes cutaneous adaptive immunity to the microbiota.

The somatosensory nervous system surveils external stimuli at barrier tissues, regulating innate immune cells under infection and inflammation. The roles of sensory neurons in controlling the adaptive immune system, and more specifically immunity to the microbiota, however, remain elusive. Here, we identified a mechanism for direct neuroimmune communication between commensal-specific T lymphocytes and somatosensory neurons mediated by the neuropeptide calcitonin gene-related peptide (CGRP) in the skin. Intravital imaging revealed that commensal-specific T cells are in close proximity to cutaneous nerve fibers in vivo. Correspondingly, we observed upregulation of the receptor for the neuropeptide CGRP, RAMP1, in CD8+ T lymphocytes induced by skin commensal colonization. The neuroimmune CGRP-RAMP1 signaling axis functions in commensal-specific T cells to constrain Type 17 responses and moderate the activation status of microbiota-reactive lymphocytes at homeostasis. As such, modulation of neuroimmune CGRP-RAMP1 signaling in commensal-specific T cells shapes the overall activation status of the skin epithelium, thereby impacting the outcome of responses to insults such as wounding. The ability of somatosensory neurons to control adaptive immunity to the microbiota via the CGRP-RAMP1 axis underscores the various layers of regulation and multisystem coordination required for optimal microbiota-reactive T cell functions under steady state and pathology.

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.

Receptor activity-modifying protein 1 regulates mouse skin fibroblast proliferation via the Gαi3-PKA-CREB-YAP axis.

BACKGROUND: Skin innervation is crucial for normal wound healing. However, the relationship between nerve receptors and wound healing and the intrinsic mechanism remains to be further identified. In this study, we investigated the role of a calcitonin gene-related peptide (CGRP) receptor component, receptor activity-modifying protein 1 (RAMP1), in mouse skin fibroblast (MSF) proliferation. METHODS: In vivo, Western blotting and immunohistochemical (IHC) staining of mouse skin wounds tissue was used to detect changes in RAMP1 expression. In vitro, RAMP1 was overexpressed in MSF cell lines by infection with Tet-On-Flag-RAMP1 lentivirus and doxycycline (DOX) induction. An IncuCyte S3 Live-Cell Analysis System was used to assess and compare the proliferation rate differences between different treatment groups. Total protein and subcellular extraction Western blot analysis, quantitative real-time-polymerase chain reaction (qPCR) analysis, and immunofluorescence (IF) staining analysis were conducted to detect signalling molecule expression and/or distribution. The CUT & RUN assay and dual-luciferase reporter assay were applied to measure protein-DNA interactions. RESULTS: RAMP1 expression levels were altered during skin wound healing in mice. RAMP1 overexpression promoted MSF proliferation. Mechanistically, total Yes-associated protein (YAP) and nuclear YAP protein expression was increased in RAMP1-overexpressing MSFs. RAMP1 overexpression increased inhibitory guanine nucleotide-binding protein (G protein) α subunit 3 (Gαi3) expression and activated downstream protein kinase A (PKA), and both elevated the expression of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) and activated it, promoting the transcription of YAP, elevating the total YAP level and promoting MSF proliferation. CONCLUSIONS: Based on these data, we report, for the first time, that changes in the total RAMP1 levels during wound healing and RAMP1 overexpression alone can promote MSF proliferation via the Gαi3-PKA-CREB-YAP axis, a finding critical for understanding RAMP1 function, suggesting that this pathway is an attractive and accurate nerve target for skin wound treatment. Video Abstract.

Schwann cell endosome CGRP signals elicit periorbital mechanical allodynia in mice.

Efficacy of monoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor (calcitonin receptor-like receptor/receptor activity modifying protein-1, CLR/RAMP1) implicates peripherally-released CGRP in migraine pain. However, the site and mechanism of CGRP-evoked peripheral pain remain unclear. By cell-selective RAMP1 gene deletion, we reveal that CGRP released from mouse cutaneous trigeminal fibers targets CLR/RAMP1 on surrounding Schwann cells to evoke periorbital mechanical allodynia. CLR/RAMP1 activation in human and mouse Schwann cells generates long-lasting signals from endosomes that evoke cAMP-dependent formation of NO. NO, by gating Schwann cell transient receptor potential ankyrin 1 (TRPA1), releases ROS, which in a feed-forward manner sustain allodynia via nociceptor TRPA1. When encapsulated into nanoparticles that release cargo in acidified endosomes, a CLR/RAMP1 antagonist provides superior inhibition of CGRP signaling and allodynia in mice. Our data suggest that the CGRP-mediated neuronal/Schwann cell pathway mediates allodynia associated with neurogenic inflammation, contributing to the algesic action of CGRP in mice.

Regulation of Cutaneous Immunity In Vivo by Calcitonin Gene-Related Peptide Signaling through Endothelial Cells.

Calcitonin gene-related peptide (CGRP) can bias the outcome of Ag presentation to responsive T cells in vitro away from Th1-type immunity and toward the Th2 and Th17 poles through actions on endothelial cells (ECs). To test the in vivo significance of this observation, we engineered a mouse lacking functional CGRP receptors on ECs (EC receptor activity modifying protein 1 [RAMP1] knockout mice). On percutaneous immunization to 1-fluoro-2,4-dinitrobenzene, stimulated CD4+ T cells from draining lymph nodes showed significantly reduced IL-17A expression with significantly increased IFN-γ, IL-4, and IL-22 expression at the protein and mRNA levels compared with control mice. Retinoic acid receptor-related orphan receptor γ t mRNA was significantly reduced, while mRNAs for T-box expressed in T cells and GATA binding protein 3 were significantly increased. In addition, EC RAMP1 knockout mice had significantly reduced contact hypersensitivity responses, and systemic administration of a CGRP receptor antagonist similarly inhibited contact hypersensitivity in wild-type mice. These observations provide compelling evidence that CGRP is a key regulator of cutaneous immunity through effects on ECs and suggest a novel pathway for potential therapeutic manipulation.

Activation of Calcitonin Gene-Related Peptide and Adrenomedullin Receptors by PEGylated Adrenomedullin.

Adrenomedullin (AM) improves colitis in animal models and patients with inflammatory bowel disease. We have developed a PEGylated AM derivative (PEG-AM) for clinical application because AM has a short half-life in the blood. However, modification by addition of polyethylene glycol (PEG) may compromise the function of the original peptide. In this paper, we examined the time course of cAMP accumulation induced by 5 and 60 kDa PEG-AM and compared the activation of calcitonin gene-related peptide (CGRP), AM1 and AM2 receptors by AM, 5 and 60 kDa PEG-AM. We also evaluated the effects of antagonists on the action of 5 and 60 kDa PEG-AM. PEG-AM stimulated cAMP production induced by these receptors; the increase in cAMP levels resulting from application of PEG-AM peaked at 15 min. Moreover, PEG-AM activity was antagonized by CGRP (8-37) or AM (22-52) (antagonists of CGRP and AM receptors, respectively) and the maximal response was not suppressed. These findings indicate that the effects of PEG-AM are similar to those of native AM.

[Study on the correlation between prognosis of patients with chronic hepatitis B under interferon treatment and polymorphism of both calcitonin gene related peptide and receptor activity modifying protein 1].

Objective: To analyze the association of two single-nucleotide polymorphisms (SNP) [Calcitonin gene related peptide (CGRP) rs155209 and receptor activity modifying protein 1 (RAMP1) rs3754701] and the prognosis of chronic hepatitis B patients who were under interferon therapy. Methods: A total of 317 patients and their anticoagulant blood samples were collected in this study. The SNPs in the CGRP and region RAMP1 were genotyped using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Logistic regression method was used to assess the results from different phenotypic outcomes between cases and controls, after adjusted for sex and age in co-dominant, dominant and recessive genetic models. Results: Data from this study clearly demonstrated the relevance of CGRP rs155209 and RAMP1 rs3754701 with DNA response and ALT response. RAMP1 rs3754701T was strongly associated with both DNA response and ALT response (OR=2.277, 95%CI: 1.386-3.741, P=0.001; OR=1.694, 95%CI: 1.073-2.675, P=0.024). However, CGRP rs155209C was less prone to DNA response and ALT response (OR=0.150, 95%CI: 0.083-0.271, P<0.001; OR=0.583, 95%CI: 0.367-0.925, P=0.022). Conclusions: Results from our study suggested that both RAMP1 rs3754701 and CGRP rs155209 were associated with the prognosis of patients under interferon therapy in Han population, from the northern parts of China while RAMP1 rs3754701T was a protective factor for both ALT response and DNA response, but CGRP rs155209C carriers were less prone to DNA and ALT responses.

CGRP in rat mesenteric artery and vein - receptor expression, CGRP presence and potential roles.

CGRP is a potent dilator of arteries and despite rich perivascular CGRP immunoreactivity in both arteries and veins the role of CGRP in veins remains unknown. The aim of the current study was to compare perivascular CGRP immunoreactivity and expression of CGRP receptor mRNA and CGRP receptor immunoreactivity in rat mesenteric arteries and veins. Furthermore, potential vasomotor effects of CGRP were explored in veins. Immunohistochemical studies reproduced rich perivascular CGRP innervation in arteries and in veins. Further, the presence of mRNA encoding the CGRP receptor subunits, CLR and RAMP1, were demonstrated in both arteries and veins using qPCR. Before comparing the vasoactive effects of CGRP in arteries and veins, we aimed to identify an experimental setting where vasomotor responses could be detected. Therefore, a length-tension study was performed in artery and vein segments. Whereas the arteries showed the characteristic monophasic curve with an IC/IC100 value of 0.9, surprisingly the veins showed a biphasic response with two corresponding IC/IC100 values of 0.7 and 0.9, respectively. There was no significant difference between fresh and cultured vasculature segments. To investigate whether a potential tension-dependent CGRP-induced dilation of veins caused the decline between the two IC/IC100 peaks, a second study was performed, with the CGRP receptor antagonist, BIBN4096BS (olcegepant) and the sensory nerve secretagogue, capsaicin. No significant vascular role of endogenous perivascular CGRP in mesenteric veins could be concluded, and a potential role of the rich perivascular CGRP and CGRP receptor abundancy in veins remains unknown.

RAMP1 signaling in immune cells regulates inflammation-associated lymphangiogenesis.

Calcitonin gene-related peptide (CGRP) regulates inflammation via signaling through receptor activity-modifying protein (RAMP) 1. Here, we investigated the role of RAMP1 signaling in growth of lymphatic vessels during inflammation. Lymphangiogenesis in the diaphragm of RAMP1-deficient (-/-) mice or their wild-type (WT) counterparts was induced by repeated intraperitoneal injection of lipopolysaccharide (LPS). Compared with WT mice, LPS-induced lymphangiogenesis in RAMP1-/- mice was suppressed. This was accompanied by the reduced expression of vascular endothelial growth factor (VEGF)-C and VEGF-D. The number of CD4+ cells in diaphragm tissue from WT mice was greater than RAMP1-/- mice. Removing CD4+ cells attenuated lymphangiogenesis and expression of VEGF-C and VEGF-D. CD4+ cells isolated from RAMP1-/- mice exhibited reduced expression of VEGF-C and VEGF-D. The number of CD11b+ cells from RAMP1-/- mice was higher than WT mice and was associated with the upregulated expression of genes related to pro-inflammatory macrophage phenotype and downregulation of reparative macrophage phenotype-related expression. When fluorescein isothiocyanate (FITC)-dextran was injected into the peritoneal cavity, the amount of residual FITC-dextran in WT mice was lower than that in RAMP1-/- mice. The present results suggest that RAMP1 signaling in immune cells plays a critical role in inflammation-related lymphangiogenesis; therefore, it represents a novel target for controlling lymphangiogenesis.

RAMP1 and RAMP3 Differentially Control Amylin's Effects on Food Intake, Glucose and Energy Balance in Male and Female Mice.

Amylin is a pancreatic peptide, which acts as a key controller of food intake and energy balance and predominately binds to three receptors (AMY 1-3). AMY 1-3 are composed of a calcitonin core receptor (CTR) and associated receptor-activity modifying proteins (RAMPs) 1-3. Using RAMP1, RAMP3 and RAMP1/3 global KO mice, this study aimed to determine whether the absence of one or two RAMP subunits affects food intake, glucose homeostasis and metabolism. Of all the RAMP-deficient mice, only high-fat diet fed RAMP1/3 KO mice had increased body weight. Chow-fed RAMP3 KO and high-fat diet fed 1/3 KO male mice were glucose intolerant. Fat depots were increased in RAMP1 KO male mice. No difference in energy expenditure was observed but the respiratory exchange ratio (RER) was elevated in RAMP1/3 KO. RAMP1 and 1/3 KO male mice displayed an increase in intermeal interval (IMI) and meal duration, whereas IMI was decreased in RAMP3 KO male and female mice. WT and RAMP1, RAMP3, and RAMP1/3 KO male and female littermates were then assessed for their food intake response to an acute intraperitoneal injection of amylin or its receptor agonist, salmon calcitonin (sCT). RAMP1/3 KO were insensitive to both, while RAMP3 KO were responsive to sCT only and RAMP1 KO to amylin only. While female mice generally weighed less than male mice, only RAMP1 KO showed a clear sex difference in meal pattern and food intake tests. Lastly, a decrease in CTR fibers did not consistently correlate with a decrease in amylin- induced c-Fos expression in the area postrema (AP). Ultimately, the results from this study provide evidence for a role of RAMP1 in mediation of fat utilization and a role for RAMP3 in glucose homeostasis and amylin's anorectic effect.

Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase.

We developed a novel and convenient method for rapidly identifying CRISPR/Cas9-based genome-edited biallelic knockout (KO) cells/individuals carrying insertions or deletions of a few nucleotides (indels) by performing PCR on genomic DNA samples under stringent conditions and low MgCl2 concentrations. The biallelic KO samples can be judged as 'negative' under these conditions. The sense primer corresponds to the sequence recognised by guide RNA and subsequently cleaved by Cas9 immediately upstream of a target gene's proto-spacer adjacent motif (PAM), and the reverse primer corresponds to the sequence ~200 bp downstream from the PAM. PCR performed using this primer set under standard MgCl2 concentrations (1.5-2.5 mM) should generate PCR products derived from both mutated and unedited alleles, whereas PCR performed using lower MgCl2 concentrations (0.8-2 mM) should yield products derived from unedited alleles. This enables high-throughput screening of biallelic mutants among cells/embryos having ≥1 indels at a region within 5 bp upstream of the PAM (where more than 94% of indels are known to appear). We performed proof-of-principle analyses of this novel approach using genome-edited Et1, Tyr, Ramp1, Ramp3, and Rosa26 mouse samples carrying various types of indels, and demonstrate that this new technique allows rapid identification of biallelic KO mutants among samples carrying various types of indels and mosaic mutations with 100% accuracy. We name this system detection of biallelic KO mutants harbouring indels using PCR (Bindel-PCR).

Development of chimeric and bifunctional antagonists for CLR/RAMP receptors.

CGRP, adrenomedullin (ADM), and adrenomedullin 2 (ADM2) family peptides are important neuropeptides and hormones for the regulation of neurotransmission, vasotone, cardiovascular morphogenesis, vascular integrity, and feto‒placental development. These peptides signal through CLR/RAMP1, 2 and 3 receptor complexes. CLR/RAMP1, or CGRP receptor, antagonists have been developed for the treatment of migraine headache and osteoarthritis pain; whereas CLR/RAMP2, or ADM receptor, antagonists are being developed for the treatment of tumor growth/metastasis. Based on the finding that an acylated chimeric ADM/ADM2 analog potently stimulates CLR/RAMP1 and 2 signaling, we hypothesized that the binding domain of this analog could have potent inhibitory activity on CLR/RAMP receptors. Consistent with this hypothesis, we showed that acylated truncated ADM/ADM2 analogs of 27-31 residues exhibit potent antagonistic activity toward CLR/RAMP1 and 2. On the other hand, nonacylated analogs have minimal activity. Further truncation at the junctional region of these chimeric analogs led to the generation of CLR/RAMP1-selective antagonists. A 17-amino-acid analog (Antagonist 2-4) showed 100-fold selectivity for CLR/RAMP1 and was >100-fold more potent than the classic CGRP receptor antagonist CGRP8-37. In addition, we showed (1) a lysine residue in the Antagonist 2-4 is important for enhancing the antagonistic activity, (2) an analog consisted of an ADM sequence motif and a 12-amino-acid binding domain of CGRP exhibits potent CLR/RAMP1-inhibitory activity, and (3) a chimeric analog consisted of a somatostatin analog and an ADM antagonist exhibits dual activities on somatostatin and CLR/RAMP receptors. Because the blockage of CLR/RAMP signaling prevents migraine pain and suppresses tumor growth/metastasis, further studies of these analogs, which presumably have better access to the tumor microenvironment and nerve endings at the trigeminal ganglion and synovial joints as compared to antibody-based therapies, may lead to the development of better anti-CGRP therapy and alternative antiangiogenesis therapy. Likewise, the use of bifunctional somatostatin-ADM antagonist analogs could be a promising strategy for the treatment of high-grade neuroendocrine tumors by targeting an antiangiogenesis agent to the neuroendocrine tumor microenvironment.

The neuropeptide receptor subunit RAMP1 constrains the innate immune response during acute pancreatitis in mice.

OBJECTIVES: The importance of the Calcitonin-gene-related-peptide-pathway (CGRP) as neuronal modulator of innate immune responses in mice has been previously demonstrated. The CGRP-receptor is composed of two subunits: the receptor-activity-modifying-protein-1 (RAMP1) and the calcitonin-receptor-like-receptor (CLR). CGRP can influence immune cells and their capacity of producing inflammatory cytokines. Using a RAMP1 knockout-mouse (RAMP1-/-) we examined the role of the CGRP-receptor in the acute-phase of cerulein-induced pancreatitis. METHODS: Hourly cerulein-injections for a period of 8 h in RAMP1-/- and wild-type mice were performed. To compare severity and extent of inflammation in RAMP1-/- and wild-type mice, histological analyses were done and cytokine levels were assessed using qRT-PCR 8 h, 24 h, 2 days, and 7 days post-cerulein-treatment. Furthermore, serum activities of LDH and lipase were determined. RESULTS: After 8 h RAMP1-/- mice showed a higher pancreas-to-body-weight-ratio, increased tissue edema and immune cell infiltration with higher amount of F4/80-positive cells as compared to wild-type mice. Overall infiltration of immune cells at 24 h was increased in RAMP1-/- mice and composed predominantly of MPO-positive neutrophils. In addition, after 24 h RAMP1-/- mice presented a higher pancreas-to-body-weight-ratio, higher expression of Ccl3, Il6, and Il1b and increased number of cleaved caspase 3 positive cells. Serum lipase correlated with the extent of tissue damage in RAMP1-/- compared to wild-type mice 24 h post-cerulein treatment. CONCLUSION: Mice lacking RAMP1 showed increased inflammation, tissue edema, and pancreas injury particularly in the early phase of acute pancreatitis. This study highlights the essential role of CGRP for dampening the innate immune response in acute pancreatitis.