Signal transduction pathways mediating the effect of adrenomedullin on osteoblast survival.

Adrenomedullin (ADM) plays an important role in the regulation of osteoblastic cells through both a proliferative and an anti-apoptotic effects. The present study investigated mechanisms involved in the effect of ADM on survival. We report that ADM can act in osteoblasts both through a non-transcriptional action, by phosphorylation of different kinases and components, and through a transcriptional effect by activation of CREB. So, we observed by Western blot analysis, modifications in the downstream targets of ERK, the pro-apoptotic protein Bad, which is inactivated by increase in Ser155 phosphorylation, and the transcription factor CREB, which is activated by phosphorylation at Ser133. CREB activation was confirmed by a CRE-dependent gene transcription assay and an immunocytochemical study. This increase in CREB phosphorylation could lead to its enhanced transcriptional activity, as indicated by the induced expression of the proliferation marker, PCNA. Moreover, ADM could also activate the tyrosine kinase Src and the PI3-Kinase, both of which are implicated in survival. The use of specific pharmacological inhibitors allowed to establish that ADM could activate a signaling cascade involving Src, MEK, ERK, p90RSK, and that the effect of ADM, in particular on the CREB protein, greatly depends on the regulatory control of interfering signaling pathways. Moreover, as Wnt signaling plays an important role in the control of osteoblast apoptosis, we explored a major component of this pathway, protein GSK3ß. ADM-induced inactivation of GSK3ß by phosphorylation at Ser9, highly suggests that ADM could also exert its survival effect in osteoblast via components of the Wnt pathway.

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.

RAMPs and CRLR expressions in osteoblastic cells after dexamethasone treatment.

Adrenomedullin (ADM) is a potent stimulator of osteoblastic activity and promotes bone growth in vivo. ADM receptors are formed by heterodimerization of the CRLR and a RAMP2 or RAMP3 molecule. Since glucocorticoid responsive elements were recently identified in the human CRLR promoter and that glucocorticoids exert a major action in bones, we investigated the acute effect of dexamethasone (Dex) treatment on ADM receptor components in osteoblastic cell types: the MC3T3-E1 cells and calvaria-derived osteoblastic cells. Changes in expression of CRLR and RAMPs molecules were evaluated at mRNA levels using RT-PCR and at protein levels by Western blot analysis. We found that Dex increased expression of RAMP1 and RAMP2 mRNA in a time-dependent but dose-independent manner, while RAMP3 was unchanged. In contrast, Dex decreased the CRLR mRNA expression and these changes were reflected at protein levels. We suggest that Dex, in osteoblastic cells, altered ADM receptor by inhibition of CRLR expression and consequently could impair the ADM anabolic effect on bone. Our findings could explain in part, the detrimental side effects observed at bone level during glucocorticoid therapy.

Sequencing of a calcitonin receptor-like receptor in salmon Oncorhynchus gorbuscha. Functional studies using the human receptor activity-modifying proteins.

The calcitonin gene-related peptide (CGRP) receptor and adrenomedullin (ADM) receptor are generated by the concomitant expression of a calcitonin receptor-like receptor (CL receptor) and a specific receptor activity-modifying protein (RAMP) in mammals. We have identified the sequence encoding the salmon CL receptor (sCL receptor) and studied its function after co-expression with the human RAMPs in Cos-7 cells. The potential open-reading frame encoded a 465-amino-acid protein which is 72% identical to the human CL receptor and 85.8% identical to the flounder CL receptor. Function was assessed by measuring the cyclic adenosine monophosphate (cAMP) produced by Cos-7 cells transiently transfected with recombinant vectors for the sCL receptor and human RAMP. Co-expression of the CL receptor and RAMP1, formed a CGRP receptor, as in mammals. This CGRP receptor responded to selective analogs as a type 1 CGRP receptor. Cells co-expressing the CL receptor and RAMP2 did not produce increased cAMP in response to human ADM. Cells co-expressing the CL receptor and RAMP3, produced such a response, as in mammals, indicating that the human ADM molecule is not the cause of the previous unresponsiveness. We suggest that the human RAMP2 molecule does not interact with the sCL receptor because of major differences in the sequences of the salmon CL receptor and the mammalian CL receptor. The availability of this receptor must allow to further study their structural basis. This identification of a non-mammalian CL receptor, and characterization of its function, give insight in the evolution of the CL receptor molecule.