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.

Expression and activity of the calcitonin receptor family in a sample of primary human high-grade gliomas.

BACKGROUND: Glioblastoma (GBM) is the most common and aggressive type of primary brain cancer. With median survival of less than 15 months, identification and validation of new GBM therapeutic targets is of critical importance. RESULTS: In this study we tested expression and performed pharmacological characterization of the calcitonin receptor (CTR) as well as other members of the calcitonin family of receptors in high-grade glioma (HGG) cell lines derived from individual patient tumours, cultured in defined conditions. Previous immunohistochemical data demonstrated CTR expression in GBM biopsies and we were able to confirm CALCR (gene encoding CTR) expression. However, as assessed by cAMP accumulation assay, only one of the studied cell lines expressed functional CTR, while the other cell lines have functional CGRP (CLR/RAMP1) receptors. The only CTR-expressing cell line (SB2b) showed modest coupling to the cAMP pathway and no activation of other known CTR signaling pathways, including ERK1/2 and p38 MAP kinases, and Ca2+ mobilization, supportive of low cell surface receptor expression. Exome sequencing data failed to account for the discrepancy between functional data and expression on the cell lines that do not respond to calcitonin(s) with no deleterious non-synonymous polymorphisms detected, suggesting that other factors may be at play, such as alternative splicing or rapid constitutive receptor internalisation. CONCLUSIONS: This study shows that GPCR signaling can display significant variation depending on cellular system used, and effects seen in model recombinant cell lines or tumour cell lines are not always reproduced in a more physiologically relevant system and vice versa.

Targeting the CALCB/RAMP1 axis inhibits growth of Ewing sarcoma.

Ewing sarcoma (EwS) is an aggressive cancer characterized by chromosomal translocations generating fusions of the EWSR1 gene with ETS transcription factors (in 85% FLI1). EWSR1-FLI1 induces gene expression via binding to enhancer-like GGAA-microsatellites, whose activity correlates with the number of consecutive GGAA-repeats. Herein we investigate the role of the secretory neuropeptide CALCB (calcitonin-related polypeptide ß) in EwS, which signals via the CGRP (calcitonin gene-related peptide) receptor complex, containing RAMP1 (receptor activity modifying protein 1) as crucial part for receptor specificity. Analysis of 2678 gene expression microarrays comprising 50 tumor entities and 71 normal tissue types revealed that CALCB is specifically and highly overexpressed in EwS. Time-course knockdown experiments showed that CALCB expression is tightly linked to that of EWSR1-FLI1. Consistently, gene set enrichment analyses of genes whose expression in primary EwS is correlated to that of CALCB indicated that it is co-expressed with other EWSR1-FLI1 target genes and associated with signatures involved in stemness and proliferation. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) data for FLI1 and histone marks from EwS cell lines demonstrated that EWSR1-FLI1 binds to a GGAA-microsatellite close to CALCB, which exhibits characteristics of an active enhancer. Reporter assays confirmed the strong EWSR1-FLI1- and length-dependent enhancer activity of this GGAA-microsatellite. Mass spectrometric analyses of EwS cell culture supernatants demonstrated that CALCB is secreted by EwS cells. While short-term RNA interference-mediated CALCB knockdown had no effect on proliferation and clonogenic growth of EwS cells in vitro, its long-term knockdown decreased EwS growth in vitro and in vivo. Similarly, knockdown of RAMP1 reduced clonogenic/spheroidal growth and tumorigenicity, and small-molecule inhibitors directed against the RAMP1-comprising CGRP receptor reduced growth of EwS. Collectively, our findings suggest that CALCB is a direct EWSR1-FLI1 target and that targeting the CALCB/RAMP1 axis may offer a new therapeutic strategy for inhibition of EwS growth.

CGRP-CRLR/RAMP1 signal is important for stress-induced hematopoiesis.

Ecotropic viral integration site-1 (EVI1) has a critical role in normal and malignant hematopoiesis. Since we previously identified high expression of calcitonin receptor like receptor (CRLR) in acute myeloid leukemia (AML) with high EVI1 expression, we here characterized the function of CRLR in hematopoiesis. Since higher expression of CRLR and receptor activity modifying protein 1 (RAMP1) was identified in immature hematopoietic bone marrow (BM) cells, we focused on calcitonin gene-related peptide (CGRP), a specific ligand for the CRLR/RAMP1 complex. To elucidate the role of CGRP in hematopoiesis, Ramp1-deficient (Ramp1-/-) mice were used. The steady-state hematopoiesis was almost maintained in Ramp1-/- mice; however, the BM repopulation capacity of Ramp1-/- mice was significantly decreased, and the transplanted Ramp1-/- BM mononuclear cells had low proliferation capacity with enhanced reactive oxygen species (ROS) production and cell apoptosis. Thus, CGRP is important for maintaining hematopoiesis during temporal exposures with proliferative stress. Moreover, continuous CGRP exposure to mice for two weeks induced a reduction in the number of BM immature hematopoietic cells along with differentiated myeloid cells. Since CGRP is known to be increased under inflammatory conditions to regulate immune responses, hematopoietic exhaustion by continuous CGRP secretion under chronic inflammatory conditions is probably one of the important mechanisms of anti-inflammatory responses.

Molecular studies of CGRP and the CGRP family of peptides in the central nervous system.

BACKGROUND: Calcitonin gene-related peptide is an important target for migraine and other painful neurovascular conditions. Understanding the normal biological functions of calcitonin gene-related peptide is critical to understand the mechanisms of calcitonin gene-related peptide-blocking therapies as well as engineering improvements to these medications. Calcitonin gene-related peptide is closely related to other peptides in the calcitonin gene-related peptide family of peptides, including amylin. Relatedness in peptide sequence and in receptor biology makes it difficult to tease apart the contributions that each peptide and receptor makes to physiological processes and to disorders. SUMMARY: The focus of this review is the expression of calcitonin gene-related peptide, related peptides and their receptors in the central nervous system. Calcitonin gene-related peptide is expressed throughout the nervous system, whereas amylin and adrenomedullin have only limited expression at discrete sites in the brain. The components of two receptors that respond to calcitonin gene-related peptide, the calcitonin gene-related peptide receptor (calcitonin receptor-like receptor with receptor activity-modifying protein 1) and the AMY1 receptor (calcitonin receptor with receptor activity-modifying protein 1), are expressed throughout the nervous system. Understanding expression of the peptides and their receptors lays the foundation for more deeply understanding their physiology, pathophysiology and therapeutic use.

The influence of receptor activity-modifying protein-1 overexpression on angiogenesis in mouse brain capillary endothelial cells.

Receptor activity-modifying protein-1 (RAMP1) is highly expressed in the heart and vasculature, indicating that it might be related to the vascular system. However, the effects of RAMP1 on angiogenesis and the intrinsic mechanisms underlying this process remain unclear. Here, we verified that RAMP1 is a critical regulator of angiogenesis in a mouse brain capillary endothelial cell line (bEnd.3). We first constructed a RAMP1 overexpression lentiviral vector system and stably transfected bEnd.3 cells. We further showed that RAMP1 overexpression could lead to bEnd.3 migration and capillary tube formation in Matrigel without exogenous calcitonin gene-related peptide (CGRP) treatment. At the same time, RAMP1 overexpression had little effect on proliferation. More importantly, vascular endothelial growth factor (VEGF) and CGRP expression levels were not significantly higher in RAMP1-overexpressing cells than in control cells (P > 0.05), indicating that RAMP1 did not function through upregulating VEGF or CGRP expression in bEnd.3 cells. Strikingly, RAMP1 transfection increased adrenomedullin 2 (AM2) expression levels ( P < 0.05). Taken together, these data contribute to a better understanding of the molecular mechanisms of RAMP1 in angiogenesis.

RAMP1 in Kupffer cells is a critical regulator in immune-mediated hepatitis.

The significance of the relationship between the nervous and immune systems with respect to disease course is increasingly apparent. Immune cells in the liver and spleen are responsible for the development of acute liver injury, yet the regulatory mechanisms of the interactions remain elusive. Calcitonin gene-related peptide (CGRP), which is released from the sensory nervous system, regulates innate immune activation via receptor activity-modifying protein 1 (RAMP1), a subunit of the CGRP receptor. Here, we show that RAMP1 in Kupffer cells (KCs) plays a critical role in the etiology of immune-mediated hepatitis. RAMP1-deficient mice with concanavalin A (ConA)-mediated hepatitis, characterized by severe liver injury accompanied by infiltration of immune cells and increased secretion of pro-inflammatory cytokines by KCs and splenic T cells, showed poor survival. Removing KCs ameliorated liver damage, while depleting T cells or splenectomy led to partial amelioration. Adoptive transfer of splenic T cells from RAMP1-deficient mice led to a modest increase in liver injury. Co-culture of KCs with splenic T cells led to increased cytokine expression by both cells in a RAMP1-dependent manner. Thus, immune-mediated hepatitis develops via crosstalk between immune cells. RAMP1 in KCs is a key regulator of immune responses.

Expression of ASIC3 in the Trigeminal Nucleus Caudalis Plays a Role in a Rat Model of Recurrent Migraine.

Acid-sensing ion channel 3 (ASIC3) is abundant in the trigeminal nervous system and is most sensitive to a slight pH decrease. Recent studies have indicated that ASIC3 in the peripheral trigeminal ganglia is likely involved in the pathogenesis of migraine pain. However, it is unclear whether this receptor plays a role in recurrent migraine, namely, migraine chronicity. Here, we aimed to investigate the role of ASIC3 in an animal model of recurrent migraine (RM). In this study, we established a rat model of RM through repeated administration of inflammatory soup (IS) onto the dura. Then, we tested the mechanical pain thresholds of the face and hindpaws by von Frey filaments. qRT-PCR, Western blot and immunofluorescence labelling were used to detect the expression and localization of ASIC3 in the trigeminal nucleus caudalis (TNC). The protein levels of calcitonin gene-related peptide (CGRP), its receptor component receptor activity modifying protein 1 (RAMP1) and c-Fos were analysed following treatment with the ASIC3 inhibitor APETx2 and activator 2-guanidine-4-methylquinazoline (GMQ). We found decreased pain thresholds after repeated dural inflammatory stimulation, which suggested the establishment of an RM model. Based on this model, we observed elevated expression of ASIC3 in the TNC group compared to that in the Sham group. ASIC3 was primarily expressed in neurons but not in astrocytes of the TNC. Moreover, APETx2 attenuated tactile allodynia and significantly decreased the expression of c-Fos, CGRP and RAMP1, while GMQ aggravated these effects compared to those observed in the IS + vehicle group. These findings indicate a critical role of ASIC3 channels in the pathophysiology of RM, and ASIC3 might represent a potential therapeutic target to prevent the progression of migraine.