Adrenomedullin is anti-apoptotic in osteoblasts through CGRP1 receptors and MEK-ERK pathway.

Adrenomedullin (ADM) has been shown to mediate multifunctional responses in cell culture and animal system such as regulation of growth and apoptosis. ADM stimulates the proliferation of osteoblasts in vitro and promotes bone growth in vivo. The ability of ADM to influence osteoblastic cell number through inhibition of apoptosis has not yet been studied. To address this question we have investigated its effect on the apoptosis of serum-deprived osteoblastic cells using mouse MC3T3-E1 cells which express both ADM and ADM receptors. Treatment with ADM significantly blunted apoptosis, evaluated by caspase-3 activity, DNA fragmentation quantification and annexin V-FITC labeling. This effect was abolished by the subtype-1 CGRP receptor antagonist, CGRP(8-37). Both ADM and its specific receptor antagonist, the (22-52) ADM fragment exhibited a similar anti-apoptotic effect. Thus, our data suggest that ADM exerts anti-apoptotic effects through CGRP1 receptors. This was substantiated by a similar protective effect of CGRP on MC3T3-E1 cells apoptosis. Accordingly, neutralization of endogenous ADM by a specific antibody enhanced apoptosis. Finally, the selective inhibitor of MAPK kinase (MEK), PD98059, abolished the apoptosis protective effect of ADM and prevented ADM activation of ERK1/2. These data show that ADM acts as a survival factor in osteoblastic cells via a CGRP1 receptor-MEK-ERK pathway, which provides further understanding on the physiological function of ADM in osteoblasts.

A critical role for adrenomedullin-calcitonin receptor-like receptor in regulating rheumatoid fibroblast-like synoviocyte apoptosis.

Rheumatoid arthritis (RA) is characterized by fibroblast-like synoviocyte (FLS) hyperplasia, which is partly ascribable to decreased apoptosis. In this study, we show that adrenomedullin (ADM), an antiapoptotic peptide, is constitutively secreted in larger amounts by FLS from joints with RA (RA-FLS) than with osteoarthritis (OA-FLS). ADM secretion was regulated by TNF-alpha. Peptidylglycine alpha-amidating monooxygenase, the ADM-processing enzyme, was expressed at the mRNA level by both RA-FLS and OA-FLS. Constituents of the ADM heterodimeric receptor calcitonin receptor-like receptor (CRLR)/receptor activity-modifying protein (RAMP)-2 were up-regulated at the mRNA and protein levels in cultured RA-FLS compared with OA-FLS. ADM induced rapid intracellular cAMP production in FLS and reduced caspase-3 activity, DNA fragmentation, and chromatin condensation in RA-FLS exposed to apoptotic conditions, indicating that CRLR/RAMP-2 was fully functional. ADM-induced cAMP production was less marked in OA-FLS than in RA-FLS, suggesting differences in receptor regulation and expression. ADM dose-dependently inhibited RA-FLS apoptosis, and this effect was reversed by the 22-52 ADM antagonist peptide. ADM inhibited RA-FLS apoptosis triggered by extrinsic and intrinsic pathways. Our data suggest that ADM may prevent or reduce RA-FLS apoptosis, via up-regulation of its functional receptor CRLR/RAMP-2. Regulation of ADM secretion and/or CRLR/RAMP-2 activation may constitute new treatment strategies for RA.

Post-transcriptional regulation of CRLR expression during hypoxia.

Adrenomedullin and CGRP are two potent vasodilator peptides, and their receptors are formed by heterodimerization of the CRLR and a RAMP molecule. Hypoxia is associated with many diseases of the cardiovascular system. It was recently shown that the human CRLR gene promoter contains an HIF-1alpha regulatory element, and that CRLR mRNA was increased by hypoxia in human endothelial cells. In the present work, we have assessed the effect of hypoxia on CRLR expression both in vivo and in vitro using two different experimental models. We have also investigated the effect of hypoxia on RAMP expression. (1) We analyzed the effects of a chronic hypobaric hypoxia on rat ventricle expression of RAMPs and CRLR. (2) Acute hypoxia was studied in human vascular smooth cells from coronary artery (CASMC) exposed for 6h to 2% O(2). RT-PCR was used to analyze the mRNA expression, and protein levels were determined by Western blotting. A sharp increase in HIF-1alpha protein levels was induced by hypoxia in CASMC, and 3.5-fold rise of the CRLR protein occurred after 1h of hypoxia in face of unchanged mRNA levels. The CRLR mRNA levels were only elevated later. A clear decrease of the CRLR protein level occurred after 3 and 6h of hypoxia. Thus, acute hypoxia in CASMC induced a rapid change of the CRLR protein amount independently of changes in the CRLR mRNA. This finding suggested a major post-transcriptional effect of hypoxia on CRLR expression in CASMC. RAMP2 and adrenomedullin mRNAs were increased after 4h, but no change was observed for RAMP1. Chronic hypoxia in rats enhanced both mRNA and protein levels of the three RAMPs and CRLR in right and left ventricles. Together, our in vivo and in vitro data suggested that hypoxia up-regulates both adrenomedullin and its receptor (CRLR/RAMP2) to enhance the signaling at the target cell.

[RAMPs and G protein coupled receptors]. / RAMP et récepteurs couplés aux protéines G.

RAMPs (receptor activity-modifying proteins) were discovered in 1998 as accessory proteins needed to the functionnal activity of CGRP (calcitonin gene-related peptide) receptors. Three RAMPs generated by three different genes are known in human, rat and mice. The coding sequences of such genes are described, but as yet, regulation sequences are unknown. RAMPs interact with GPCR (G protein-coupled receptors) of class II. In the case of the calcitonin/CGRP peptide family, RAMPs determine the functionnal specificity of the receptor, glycosylate and translocate the receptor to the cell surface. CGRP receptors are observed in presence of the RAMP1/calcitonin receptor-like receptor (CRLR), but the association of RAMP2 or RAMP3 with CRLR generates an adrenomedullin receptor. The calcitonin receptor (CTR) is translocated alone to the cell surface, but interactions of RAMPs with CTR forms amylin receptors. If RAMPs can interact with glucagon, parathyroid hormone and VIP/PACAP (vasoactive intestinal peptide/pituitary adenylate cyclase activating polypeptide (VPACR1)) receptors, the functionnal specificity of these receptors remains unaltered. However, the complex VPACR1/RAMP2 enhances specifically the phosphoinoside signaling pathway.

Dexamethasone decreases phospholipase C beta1 isozyme expression in human vascular smooth muscle cells.

The molecular characterization of the human PLC beta1 gene was just reported by Peruzzi et al. [Biochim. Biophys. Acta 1582 (2002) 46]. This prompted us to investigate the effects of dexamethasone on PLC beta1 expression in two types of human vascular smooth muscle cells--coronary artery smooth muscle cells (hCASMC) and aortic smooth muscle cells (hAoSMC), since glucocorticoids are known to affect the signaling pathways of Gprotein coupled receptors. Semi-quantitative RT-PCR was used to analyze mRNA expression and Western-blot for protein expression. Dexamethasone treatment in the two types of cells studied decreased (mRNA and protein) PLC beta1 isozyme expression. A rapid (2 h) fall in mRNA occurred in hCASMC after treatment, and hCASMC were more sensitive to dexamethasone (1 nM versus 100 nM) than hAoSMC. The major reduction (80%) was observed after 48 h of exposure in both VSMC. Treatment with mifeprisone, an antagonist of glucocorticoid receptors, blunted the dexamethasone effect on PLC beta1 mRNA and showed that this effect was mediated by glucocorticoids receptors.

Calcitonin gene-related peptide (CGRP) and CGRP receptor expression at the human implantation site.

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide produced by tissue-specific alternative splicing of the primary transcript of the calcitonin gene. The objectives of this study were: 1) to determine the expression of CGRP and its receptor at the human implantation site, and 2) to examine the possible in vitro effect of this neuropeptide on two major partners of implantation, decidual cells and extravillous cytotrophoblasts. Immunohistological analysis of first-trimester placental chorionic villi showed CGRP in decidual cells and glandular cells, but not in extravillous trophoblast cells. CGRP expression was confirmed in cultured decidual cells by Southern blot analysis and immunocytochemistry and by RIA in culture medium. Transcripts of calcitonin receptor-like receptor were detected by Southern blot analysis of RT-PCR amplicons from both decidual and extravillous trophoblast cells, whereas transcripts for the receptor activity-modifying protein 1 were detected in decidual cells only. In vitro, CGRP stimulated cAMP production but not nitric oxide (NO) release by cultured decidual cells; in contrast CGRP increased NO release but not cAMP production in cultured extravillous trophoblasts. The presence of NO synthase (endothelial and inducible) was confirmed by immunodetection in extravillous trophoblasts, both in situ and in vitro. This study points to a paracrine and autocrine effect of CGRP on decidual and extravillous trophoblast cells, two major actors in implantation.

Increased myocardial expression of RAMP1 and RAMP3 in rats with chronic heart failure.

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM) are potent vasodilators in humans and improved myocardial ischemia is observed after CGRP administration. Receptors for CGRP and ADM were already identified in heart. Receptor activity-modifying proteins (RAMPs) determine the ligand specificity of the calcitonin receptor-like receptor (CRLR); co-expression of RAMP1 and CRLR results in a CGRP receptor, whereas the association of RAMP2 or RAMP3 with CRLR gives an ADM receptor. As CGRP and ADM may play a beneficial role in heart failure, we investigated whether the CGRP and ADM receptors are upregulated in chronic heart failure. We have used semi-quantitative RT-PCR and Western-blot analysis to detect and quantify the mRNA and the protein of RAMP1 and RAMP3 in both atria and ventricles of failing hearts 6 months after aortic banding in rats. Our results showed for the first time an up-regulation of RAMP1 and RAMP3 mRNAs and proteins in this model of cardiac failure. No change was observed in mRNAs coding for CRLR, RAMP2, RDC1 (canine orphan receptor), and ADM. The present results suggested after congestive heart failure in adult rats, an up-regulation of the CGRP receptor (by an increase in RAMP1 that is associated with CRLR) in atria and ventricles and of ADM receptor (by increased RAMP3 expression that is associated with CRLR) in atria. These findings support a functional role for CGRP and ADM receptors to compensate the chronic heart failure in rats.