Itch receptor MRGPRX4 interacts with the receptor activity-modifying proteins.

Cholestatic itch is a severe and debilitating symptom in liver diseases with limited treatment options. The class A G protein-coupled receptor (GPCR) Mas-related GPCR subtype X4 (MRGPRX4) has been identified as a receptor for bile acids, which are potential cholestatic pruritogens. An increasing number of GPCRs have been shown to interact with receptor activity-modifying proteins (RAMPs), which can modulate different aspects of GPCR biology. Using a combination of multiplexed immunoassay and proximity ligation assay, we show that MRGPRX4 interacts with RAMPs. The interaction of MRGPRX4 with RAMP2, but not RAMP1 or 3, causes attenuation of basal and agonist-dependent signaling, which correlates with a decrease of MRGPRX4 cell surface expression as measured using a quantitative NanoBRET pulse-chase assay. Finally, we use AlphaFold Multimer to predict the structure of the MRGPRX4-RAMP2 complex. The discovery that RAMP2 regulates MRGPRX4 may have direct implications for future drug development for cholestatic itch.

RAMP3 determines rapid recycling of atypical chemokine receptor-3 for guided angiogenesis.

Receptor-activity-modifying proteins (RAMPs) are single transmembrane-spanning proteins which serve as molecular chaperones and allosteric modulators of G-protein-coupled receptors (GPCRs) and their signaling pathways. Although RAMPs have been previously studied in the context of their effects on Family B GPCRs, the coevolution of RAMPs with many GPCR families suggests an expanded repertoire of potential interactions. Using bioluminescence resonance energy transfer-based and cell-surface expression approaches, we comprehensively screen for RAMP interactions within the chemokine receptor family and identify robust interactions between RAMPs and nearly all chemokine receptors. Most notably, we identify robust RAMP interaction with atypical chemokine receptors (ACKRs), which function to establish chemotactic gradients for directed cell migration. Specifically, RAMP3 association with atypical chemokine receptor 3 (ACKR3) diminishes adrenomedullin (AM) ligand availability without changing G-protein coupling. Instead, RAMP3 is required for the rapid recycling of ACKR3 to the plasma membrane through Rab4-positive vesicles following either AM or SDF-1/CXCL12 binding, thereby enabling formation of dynamic spatiotemporal chemotactic gradients. Consequently, genetic deletion of either ACKR3 or RAMP3 in mice abolishes directed cell migration of retinal angiogenesis. Thus, RAMP association with chemokine receptor family members represents a molecular interaction to control receptor signaling and trafficking properties.

A selective role for receptor activity-modifying proteins in subchronic action of the amylin selective receptor agonist NN1213 compared with salmon calcitonin on body weight and food intake in male mice.

The role of receptor activity-modifying proteins (RAMPs) in modulating the pharmacological effects of an amylin receptor selective agonist (NN1213) or the dual amylin-calcitonin receptor agonist (DACRA), salmon calcitonin (sCT), was tested in three RAMP KO mouse models, RAMP1, RAMP3 and RAMP1/3 KO. Male wild-type (WT) and knockout (KO) littermate mice were fed a 45% high-fat diet for 20 weeks prior to the 3-week treatment period. A decrease in body weight after NN1213 was observed in all WT mice, whereas sCT had no effect. The absence of RAMP1 had no significant effect on NN1213 efficacy, and sCT was still inactive. However, the absence of RAMP3 impeded NN1213 efficacy but improved sCT efficacy. Similar results were observed in RAMP1/3 KO suggesting that the amylin receptor 3 (AMY3 = CTR + RAMP3) is necessary for NN1213's maximal action on body weight and food intake and that the lack of AMY3 allowed sCT to be active. These results suggest that the chronic use of DACRA such as sCT can have unfavourable effect on body weight loss in mice (which differs from the situation in rats), whereas the use of the amylin receptor selective agonist does not. AMY3 seems to play a crucial role in modulating the action of these two compounds, but in opposite directions. The assessment of a long-term effect of amylin and DACRA in different rodent models is necessary to understand potential physiological beneficial and unfavourable effects on weight loss before its transition to clinical trials.

IAPP-driven metabolic reprogramming induces regression of p53-deficient tumours in vivo.

TP53 is commonly altered in human cancer, and Tp53 reactivation suppresses tumours in vivo in mice (TP53 and Tp53 are also known as p53). This strategy has proven difficult to implement therapeutically, and here we examine an alternative strategy by manipulating the p53 family members, Tp63 and Tp73 (also known as p63 and p73, respectively). The acidic transactivation-domain-bearing (TA) isoforms of p63 and p73 structurally and functionally resemble p53, whereas the ΔN isoforms (lacking the acidic transactivation domain) of p63 and p73 are frequently overexpressed in cancer and act primarily in a dominant-negative fashion against p53, TAp63 and TAp73 to inhibit their tumour-suppressive functions. The p53 family interacts extensively in cellular processes that promote tumour suppression, such as apoptosis and autophagy, thus a clear understanding of this interplay in cancer is needed to treat tumours with alterations in the p53 pathway. Here we show that deletion of the ΔN isoforms of p63 or p73 leads to metabolic reprogramming and regression of p53-deficient tumours through upregulation of IAPP, the gene that encodes amylin, a 37-amino-acid peptide co-secreted with insulin by the ß cells of the pancreas. We found that IAPP is causally involved in this tumour regression and that amylin functions through the calcitonin receptor (CalcR) and receptor activity modifying protein 3 (RAMP3) to inhibit glycolysis and induce reactive oxygen species and apoptosis. Pramlintide, a synthetic analogue of amylin that is currently used to treat type 1 and type 2 diabetes, caused rapid tumour regression in p53-deficient thymic lymphomas, representing a novel strategy to target p53-deficient cancers.

Adrenomedullin 2 and 5 activate the calcitonin receptor-like receptor (clr) - Receptor activity-modifying protein 3 (ramp3) receptor complex in Xenopus tropicalis.

The adrenomedullin (AM) family is involved in diverse biological functions, including cardiovascular regulation and body fluid homeostasis, in multiple vertebrate lineages. The AM family consists of AM1, AM2, and AM5 in tetrapods, and the receptor for mammalian AMs has been identified as the complex of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 2 (RAMP2) or RAMP3. However, the receptors for AM in amphibians have not been identified. In this study, we identified the cDNAs encoding calcrl (clr), ramp2, and ramp3 receptor components from the western clawed frog (Xenopus tropicalis). Messenger RNAs of amphibian clr and ramp2 were highly expressed in the heart, whereas that of ramp3 was highly expressed in the whole blood. In HEK293T cells expressing clr-ramp2, cAMP response element luciferase (CRE-Luc) reporter activity was activated by am1. In HEK293T cells expressing clr-ramp3, CRE-Luc reporter activity was increased by the treatment with am2 at the lowest dose, but with am5 and am1 at higher dose. Our results provided new insights into the roles of AM family peptides through CLR-RAMP receptor complexes in the tetrapods.

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.

Structure-function analyses reveal a triple ß-turn receptor-bound conformation of adrenomedullin 2/intermedin and enable peptide antagonist design.

The cardioprotective vasodilator peptide adrenomedullin 2/intermedin (AM2/IMD) and the related adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) signal through three heterodimeric receptors comprising the calcitonin receptor-like class B G protein-coupled receptor (CLR) and a variable receptor activity-modifying protein (RAMP1, -2, or -3) that determines ligand selectivity. The CGRP receptor (RAMP1:CLR) favors CGRP binding, whereas the AM1 (RAMP2:CLR) and AM2 (RAMP3:CLR) receptors favor AM binding. How AM2/IMD binds the receptors and how RAMPs modulate its binding is unknown. Here, we show that AM2/IMD binds the three purified RAMP-CLR extracellular domain (ECD) complexes with a selectivity profile that is distinct from those of CGRP and AM. AM2/IMD bound all three ECD complexes but preferred the CGRP and AM2 receptor complexes. A 2.05 Å resolution crystal structure of an AM2/IMD antagonist fragment-bound RAMP1-CLR ECD complex revealed that AM2/IMD binds the complex through a unique triple ß-turn conformation that was confirmed by peptide and receptor mutagenesis. Comparisons of the receptor-bound conformations of AM2/IMD, AM, and a high-affinity CGRP analog revealed differences that may have implications for biased signaling. Guided by the structure, enhanced-affinity AM2/IMD antagonist variants were developed, including one that discriminates the AM1 and AM2 receptors with ∼40-fold difference in affinities and one stabilized by an intramolecular disulfide bond. These results reveal differences in how the three peptides engage the receptors, inform development of AM2/IMD-based pharmacological tools and therapeutics, and provide insights into RAMP modulation of receptor pharmacology.

Probing the Mechanism of Receptor Activity-Modifying Protein Modulation of GPCR Ligand Selectivity through Rational Design of Potent Adrenomedullin and Calcitonin Gene-Related Peptide Antagonists.

Binding of the vasodilator peptides adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) to the class B G protein-coupled receptor calcitonin receptor-like receptor (CLR) is modulated by receptor activity-modifying proteins (RAMPs). RAMP1 favors CGRP, whereas RAMP2 and RAMP3 favor AM. Crystal structures of peptide-bound RAMP1/2-CLR extracellular domain (ECD) heterodimers suggested RAMPs alter ligand preference through direct peptide contacts and allosteric modulation of CLR. Here, we probed this dual mechanism through rational structure-guided design of AM and CGRP antagonist variants. Variants were characterized for binding to purified RAMP1/2-CLR ECD and for antagonism of the full-length CGRP (RAMP1:CLR), AM1 (RAMP2:CLR), and AM2 (RAMP3:CLR) receptors. Short nanomolar affinity AM(37-52) and CGRP(27-37) variants were obtained through substitutions including AM S45W/Q50W and CGRP K35W/A36S designed to stabilize their ß-turn. K46L and Y52F substitutions designed to exploit RAMP allosteric effects and direct peptide contacts, respectively, yielded AM variants with selectivity for the CGRP receptor over the AM1 receptor. AM(37-52) S45W/K46L/Q50W/Y52F exhibited nanomolar potency at the CGRP receptor and micromolar potency at AM1 A 2.8-Å resolution crystal structure of this variant bound to the RAMP1-CLR ECD confirmed that it bound as designed. CGRP(27-37) N31D/S34P/K35W/A36S exhibited potency and selectivity comparable to the traditional antagonist CGRP(8-37). Giving this variant the ability to contact RAMP2 through the F37Y substitution increased affinity for AM1, but it still preferred the CGRP receptor. These potent peptide antagonists with altered selectivity inform the development of AM/CGRP-based pharmacological tools and support the hypothesis that RAMPs alter CLR ligand selectivity through allosteric effects and direct peptide contacts.

RAMP3 is a prognostic indicator of liver cancer and might reduce the adverse effect of TP53 mutation on survival.

AIM: To assess the prognostic value of RAMP3 expression in terms of overall survival (OS) and recurrence-free survival (RFS) in hepatocellular carcinoma (HCC) patients. MATERIALS & METHODS: Immunochemistry staining was performed to detect RAMP3 expression. Data in the Cancer Genome Atlas-Liver Hepatocellular Cancer were used for secondary analysis. RESULTS: RAMP3 expression was significantly downregulated in HCC tissues than in normal liver tissues. Increased RAMP3 expression was an independent prognostic factor of favorable OS (hazard ratio [HR]: 0.772, 95% CI: 0.689-0.864; p < 0.001) and RFS (HR = 0.719, 95% CI: 0.633-0.817; p < 0.001). High RAMP3 expression was associated with significantly better RFS in both TP53 mutant and wildtype groups. CONCLUSION: High RAMP3 RNA expression is an independent prognostic factor of favorable OS and RFS in patients with HCC.

Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties.

Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.

Effect of Valsartan on Cerebellar Adrenomedullin System Dysregulation During Hypertension.

Adrenomedullin (AM) and its receptors components, calcitonin-receptor-like receptor (CRLR), and receptor activity-modifying protein (RAMP1, RAMP2, and RAMP3) are expressed in cerebellum. Cerebellar AM, AM binding sites and receptor components are altered during hypertension, suggesting a role for cerebellar AM in blood pressure regulation. Thus, we assessed the effect of valsartan, on AM and its receptor components expression in the cerebellar vermis of Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Additionally, we evaluated AM action on superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activity, and thiobarbituric acid reactive substances (TBARS) production in cerebellar vermis. Animals were treated with valsartan or vehicle for 11 days. Rats were sacrificed by decapitation; cerebellar vermis was dissected; and AM, CRLR, RAMP1, RAMP2, and RAMP3 expression was quantified by Western blot analysis. CAT, SOD, and GPx activity was determined spectrophotometrically and blood pressure by non-invasive plethysmography. We demonstrate that AM and RAMP2 expression was lower in cerebellum of SHR rats, while CRLR, RAMP1, and RAMP3 expression was higher than those of WKY rats. AM reduced cerebellar CAT, SOD, GPx activities, and TBARS production in WKY rats, but not in SHR rats. Valsartan reduced blood pressure and reversed the altered expression of AM and its receptors components, as well the loss of AM capacity to reduce antioxidant enzyme activity and TBARS production in SHR rats. These findings demonstrate that valsartan is able to reverse the dysregulation of cerebellar adrenomedullinergic system; and they suggest that altered AM system in the cerebellum could represent the primary abnormality leading to hypertension.

Intermedin1-53 attenuates vascular calcification in rats with chronic kidney disease by upregulation of α-Klotho.

Deficiency in α-Klotho is involved in the pathogenesis of vascular calcification. Since intermedin (IMD)1-53 (a calcitonin/calcitonin gene-related peptide) protects against vascular calcification, we studied whether IMD1-53 inhibits vascular calcification by upregulating α-Klotho. A rat model of chronic kidney disease (CKD) with vascular calcification induced by the 5/6 nephrectomy plus vitamin D3 was used for study. The aortas of rats with CKD showed reduced IMD content but an increase of its receptor, calcitonin receptor-like receptor, and its receptor modifier, receptor activity-modifying protein 3. IMD1-53 treatment reduced vascular calcification. The expression of α-Klotho was greatly decreased in the aortas of rats with CKD but increased in the aortas of IMD1-53-treated rats with CKD. In vitro, IMD1-53 increased α-Klotho protein level in calcified vascular smooth muscle cells. α-Klotho knockdown blocked the inhibitory effect of IMD1-53 on vascular smooth muscle cell calcification and their transformation into osteoblast-like cells. The effect of IMD1-53 to upregulate α-Klotho and inhibit vascular smooth muscle cell calcification was abolished by knockdown of its receptor or its modifier protein, or treatment with the protein kinase A inhibitor H89. Thus, IMD1-53 may attenuate vascular calcification by upregulating α-Klotho via the calcitonin receptor/modifying protein complex and protein kinase A signaling.

Calcitonin Gene-Related Peptide Rescues Proximity Associations of Its Receptor Components, Calcitonin Receptor-Like Receptor and Receptor Activity-Modifying Protein 1, in Rat Uterine Artery Smooth Muscle Cells Exposed to Tumor Necrosis Factor Alpha.

Calcitonin gene-related peptide (CALCB), adrenomedullin (ADM), and ADM2/intermedin play critical roles in vascular adaptation during pregnancy through calcitonin receptor-like receptor (CALCRL) and receptor activity-modifying proteins (RAMPs). This study was designed to assess the predominant RAMP that associates with CALCRL to form a functional receptor in the rat uterine artery smooth muscle (RUASM). We also determined if these receptor component associations are decreased by tumor necrosis factor (TNF) alpha and if CALCB, ADM, or ADM2 can rescue CALCRL/RAMP associations. Using proximity ligation assay in RUASM cells, this study shows that CALCRL predominantly associates with RAMP1 forming a CALCB receptor, and minimally with RAMP2 and RAMP3 that confer specificity for ADM and ADM2. However, knockdown of RAMP1 mRNA increases the interaction between CALCRL and RAMP3 without affecting the association of CALCRL and RAMP2. Furthermore, CALCB, ADM, and ADM2 have no effects on the associations of CALCRL with any of the RAMPs in RUASM cells. Interestingly, CALCB reverses the TNFalpha-induced decreases in CALCRL/RAMP1 associations. Furthermore, CALCB increases ERK1/2 phosphorylation in a time-dependent manner in RUASM, and the protective effect of CALCB on TNFalpha-induced inhibition of CALCRL/RAMP1 associations was significantly blocked in presence of ERK inhibitor (PD98059). In conclusion, this study demonstrates that CALCRL predominantly associates with RAMP1 forming a CALCB-specific receptor complex in RUASM cells, which is dissociated by TNFalpha. Rescue of TNFalpha-induced dissociation of CALCRL/RAMP1 complex by CALCB in RUASM cells suggests a potential use of CALCB in developing therapeutic strategies for pregnancy-related complications that are vulnerable to abnormal levels of TNFalpha, such as fetal growth restriction and preeclampsia.

Involvement of Receptor Activity-Modifying Protein 3 (RAMP3) in the Vascular Actions of Adrenomedullin in Rat Mesenteric Artery Smooth Muscle Cells.

CALCB, ADM, and ADM2 are potent vasodilators that share a seven-transmembrane GPCR, calcitonin receptor-like receptor (CALCRL), whose ligand specificity is dictated by the presence of one of the three receptor activity-modifying proteins (RAMPs). We assessed the relative pharmacologic potency of these peptides in mesenteric artery smooth muscle cells (VSMCs) and the specific RAMP that mediates the effect of ADM in VSMCs. VSMCs, with or without RAMP knockdown, were treated with CALCB, ADM, or ADM2 in the presence or absence of their antagonists, CALCB8-37, ADM22-52, and ADM217-47, respectively, to assess the relative effect of peptides on cAMP production and their pharmacologic potency. Proximity ligation assay was used to assess the specific RAMP that associates with CALCRL to mediate the actions of ADM in VSMCs. All three peptides induced cAMP generation in VSMCs and the order of their potency is CALCB > ADM > ADM2. Effects of CALCB were blocked by CALCB8-37, ADM effects were blocked by CALCB8-37 and ADM217-47 but not ADM22-52, and ADM2 effects were blocked by all three antagonists. Knockdown of RAMP2 was ineffective, whereas knockdown of RAMP3 inhibited ADM-induced cAMP production in VSMCs, suggesting involvement of RAMP3 with CALCRL to mediate ADM effects. Absence of both RAMP2 and RAMP3 further increased CALCB-induced cAMP synthesis compared to control (P < 0.05). ADM increased CALCRL and RAMP3 association and RAMP3 knockdown inhibited the interaction of ADM with CALCRL.

Ligand-induced internalization, recycling, and resensitization of adrenomedullin receptors depend not on CLR or RAMP alone but on the receptor complex as a whole.

Adrenomedullins (AM) is a multifaceted distinct subfamily of peptides that belongs to the calcitonin gene-related peptide (CGRP) superfamily. These peptides exert their functional activities via associations of calcitonin receptor-like receptors (CLRs) and receptor activity-modifying proteins (RAMPs) RAMP2 and RAMP3. Recent studies established that RAMPs and CLRs can modify biochemical properties such as trafficking and glycosylation of each other. However there is very little or no understanding regarding how RAMP or CLR influence ligand-induced events of AM-receptor complex. In this study, using pufferfish homologs of CLR (mfCLR1-3) and RAMP (mfRAMP2 and mfRAMP3), we revealed that all combinations of CLR and RAMP quickly underwent ligand-induced internalization; however, their recycling rates were different as follows: mfCLR1-mfRAMP3>mfCLR2-mfRAMP3>mfCLR3-mfRAMP3. Functional receptor assay confirmed that the recycled receptors were resensitized on the plasma membrane. In contrast, a negligible amount of mfCLR1-mfRAMP2 was recycled and reconstituted. Immunocytochemistry results indicated that the lower recovery rate of mfCLR3-mfRAMP3 and mfCLR1-mfRAMP2 was correlated with higher proportion of lysosomal localization of these receptor complexes compared to the other combinations. Collectively our results indicate, for the first time, that the ligand-induced internalization, recycling, and reconstitution properties of RAMP-CLR receptor complexes depend on the receptor-complex as a whole, and not on individual CLR or RAMP alone.

Intermedin is upregulated and attenuates renal fibrosis by inhibition of oxidative stress in rats with unilateral ureteral obstruction.

AIM: Transforming growth factor-ß1 (TGF-ß1) plays a pivotal role in the progression of renal fibrosis. Reactive oxygen species mediate profibrotic action of TGF-ß1. Intermedin (IMD) has been shown to inhibit oxidative stress, but its role in renal fibrosis remains unclear. Here, we investigated the effects of IMD on renal fibrosis in a rat model of unilateral ureteral obstruction (UUO). METHODS: The expression of IMD and its receptors, calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP1/2/3), in the obstructed kidney was detected by real-time polymerase chain reaction (PCR), western blotting and immunohistochemistry. To evaluate the effects of IMD on renal fibrosis, we locally overexpressed exogenous IMD in the obstructed kidney using an ultrasound-microbubble-mediated delivery system. Renal fibrosis was determined by Masson trichrome staining. The expression of TGF-ß1, connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA) and fibronectin was measured. Smad2/3 activation and macrophage infiltration were evaluated. We also studied oxidative stress by measuring superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. RESULTS: mRNA and protein expression of IMD increased after UUO. CRLR, RAMP1, RAMP2 and RAMP3 were also induced by ureteral obstruction. IMD overexpression remarkably attenuated UUO-induced tubular injury and blunted fibrotic response as shown by decreased interstitial collagen deposition and downregulation of fibronectin. Macrophage infiltration, α-SMA and CTGF upregulation caused by UUO were all relieved by IMD, whereas TGF-ß1 upregulation and Smad2/3 activation were not affected. Meanwhile, we noted increased oxidative stress in obstruction, which was also attenuated by IMD gene delivery. CONCLUSIONS: Our results indicate that IMD is upregulated after UUO. IMD plays a protective role in renal fibrosis via its antioxidant effects.

Functional differentiation of RAMP2 and RAMP3 in their regulation of the vascular system.

Adrenomedullin (AM) is a vasoactive peptide that possesses various bioactivities. AM receptors are dimers consisting of CLR with one of two accessory proteins, RAMP2 or RAMP3. The functional difference between CLR/RAMP2 and CLR/RAMP3 and the relationship between the two receptors remain unclear. To address these issues, we generated RAMP2 and RAMP3 knockout (-/-) mice and have been studying their physiological activities in the vascular system. AM-/- and RAMP2-/- mice die in utero due to blood vessel abnormalities, which is indicative of their essential roles in vascular development. In contrast, RAMP3-/- mice were born normally without any major abnormalities. In adult RAMP3-/- mice, postnatal angiogenesis was normal, but lymphangiography using indocyanine green (ICG) showed delayed drainage of subcutaneous lymphatic vessels. Moreover, chyle transport by intestinal lymphatics was delayed in RAMP3-/- mice, which also showed more severe interstitial edema than wild-type mice in a tail lymphedema model, with characteristic dilatation of lymphatic capillaries and accumulation of inflammatory cells. In scratch-wound assays, migration of isolated RAMP3-/- lymphatic endothelial cells was delayed as compared to wild-type cells, and AM administration failed to enhance the re-endothelialization. The delay in re-endothelialization was due to a primary migration defect rather than a decrease in proliferation. These results suggest that RAMP3 regulates drainage through lymphatic vessels, and that the AM-RAMP3 system could be a novel therapeutic target for controlling postoperative lymphedema.

A hepatocellular carcinoma 5-gene score associated with survival of patients after liver resection.

BACKGROUND & AIMS: Due to the phenotypic and molecular diversity of hepatocellular carcinomas (HCC), it is a challenge to determine a patient's prognosis. We aimed to identify new prognostic markers of patients with HCC treated by liver resection. METHODS: We collected 314 HCC samples from patients at Bordeaux (1998-2007) and Créteil (2003-2007) hospitals in France. We analyzed the gene expression patterns of the tumors and compared expression patterns with patient survival times. Using the coefficient and regression formula of the multivariate Cox model, we identified a "5-gene score" associated with survival times. This molecular score was then validated in 2 groups of patients from Europe and the United States (n = 213) and China (n = 221). RESULTS: The 5-gene score, based on combined expression level of HN1, RAN, RAMP3, KRT19, and TAF9, was associated with disease-specific survival times of 189 patients with resected HCC in Bordeaux (hazard ratio = 3.5; 95% confidence interval: 1.9-6.6; P < .0001). The association between the 5-gene score and disease-specific survival was validated in an independent cohort of 125 patients in Créteil (hazard ratio = 2.3; 95% confidence interval: 1.1-4.9; P < .0001). The 5-gene score more accurately predicted patient outcomes than gene expression signatures reported previously. In multivariate analyses, the 5-gene score was associated with disease-specific survival, independent of other clinical and pathology feature of HCC. Disease-specific survival was also predicted by combining data on microvascular invasion, the Barcelona Clinic Liver Cancer classification system, and the 5-gene score in a nomogram. The prognostic accuracy of the 5-gene score was further validated in European and US patients with hepatitis C, cirrhosis, and HCC (overall survival P = .002) and in Asian patients with HCC with hepatitis B (overall survival, P = .02). Combining the 5-gene score with the expression pattern of 186 genes in corresponding cirrhotic tissues increased its prognostic accuracy. CONCLUSIONS: The molecular 5-gene score is associated with outcomes of patients with HCC treated by resection in different clinical settings worldwide. This new biomarker should be tested in clinical trials to stratify patients in therapeutic decisions.

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.

Temporo-spatial expression of adrenomedullin and its receptors in the bovine placenta.

BACKGROUND: Adrenomedullin (AM) is a potent vasodilator peptide and is also involved in various physiological activities. In humans and rodents, AM is found in the uteroplacental unit and may be responsible for fetal development and maintenance of placental function. This study investigated 1) the mRNA expression patterns of AM and its receptor components (calcitonin receptor-like receptor (CRLR), receptor activity modifying protein (RAMP) 2 and RAMP3) during pregnancy and 2) mRNA and protein localization of AM, CRLR and RAMPs in the bovine placentome. METHODS: For real-time quantitative RT-PCR, bovine uteroplacental tissues were collected from Day 25, 60, 100, 150, 200 and 250 of gestation and separated into uterine caruncle (CAR), intercaruncular endometrium (ICAR), extra-embryonic membranes on Day 25 and cotyledonary villous after Day 60 (EEM-COT) and intercotyledonary chorion (ICOT). In situ hybridization and immunohistochemistry was performed to investigate the cellular localization of mRNA and protein of AM, CRLR, RAMP2 and RAMP3 in the placentome on Day 56, 150 and 230 of gestation and interplacentomal tissues on Day 56 of gestation. RESULTS: AM mRNA was highly expressed on Day 200 in EEM-COT, CAR and ICAR. CRLR mRNA was highly expressed on Day 60 in all portions. RAMP2 mRNA was also highly expressed on Day 60 in ICOT and ICAR. In EEM-COT, mRNA expression of CRLR and RAMP2 decreased from Day 150 to 250. RAMP3 mRNA was highly expressed on Day 150 in EEM-COT, ICOT and ICAR. A distinct AM mRNA and protein signal were only found in trophoblast binucleate cells (BNCs), whereas those of CRLR, RAMP2 and RAMP3 were detected in cotyledonary villous and caruncular epithelial cells. In interplacentomal tissues, AM was detected in BNCs of fetal membrane and a small part of luminal epithelium, endothelial lineage of blood vessels and glandular epithelium of the endometrium. Distinct signals of CRLR, RAMP2 and RAMP3 were found in trophoblast cells, luminal epithelium, stroma under the epithelium, endothelial lineage of blood vessels and glandular epithelium. CONCLUSIONS: Our results indicate that the AM system in the bovine uteroplacental unit may be activated at placentation and transition from the mid to late gestation period. Locally produced AM in the BNCs may play a crucial role in regulation of placental vascular and cellular functions during pregnancy.