-
Marie-Dominique Ah Kioon,
Carine Asensio,
Hang-Korng Ea,
Frédéric Velard, Benjamin Uzan,
Sandrine Rullé,
Céline Bazille,
Caroline Marty,
Géraldine Falgarone,
Christelle Nguyen,
Corinne Collet,
Jean-Marie Launay,
Martine Cohen-Solal,
Frédéric Lioté
[show abstract]
[hide abstract]
ABSTRACT: Adrenomedullin(22-52) is a truncated peptide derived from adrenomedullin, a growth factor with antiapoptotic and immunoregulatory properties. It can act as an agonist or an antagonist depending on cell type. Its in vivo effects are unknown, but adrenomedullin(22-52) could possess immunomodulatory properties. This study was undertaken to evaluate the effect of adrenomedullin(22-52) in a mouse model of arthritis.
DBA/1 mice with collagen-induced arthritis (CIA) were treated with 1.2 μg/gm adrenomedullin(22-52) , adrenomedullin, or saline at arthritis onset. Bone mineral density was measured at the beginning of the experiment and when mice were killed. Mouse joints were processed for histologic analysis and protein studies, and spleens were examined for Treg cell expression. Cytokine expression was studied in mouse joint tissue and serum.
In mice with CIA, adrenomedullin and adrenomedullin(22-52) reduced clinical and histologic arthritis scores and shifted the pattern of articular and systemic cytokine expression from Th1 to Th2, as compared to untreated mice with CIA (controls). Tumor necrosis factor α, interleukin-6 (IL-6), and IL-17A levels were significantly decreased in the joints of mice with CIA treated with adrenomedullin or adrenomedullin(22-52) as compared to controls, whereas IL-4 and IL-10 levels were increased. Adrenomedullin(22-52) was more effective than adrenomedullin in modulating cytokine content and enhanced Treg cell function without changing Treg cell expression compared to controls. Adrenomedullin receptor binding and transcriptional adrenomedullin receptor expression were markedly increased in joints from controls, whereas adrenomedullin receptor binding was considerably decreased in treated animals. Mice with CIA treated with adrenomedullin or adrenomedullin(22-52) had considerably fewer apoptotic chondrocytes and diminished cartilage degradation. Adrenomedullin(22-52) completely prevented systemic bone loss by preserving osteoblastic activity, but without changes in osteoclastic activity.
Our findings indicate that adrenomedullin(22-52) , which has no vasoactive or tumor-inducing effects, is a potent antiinflammatory and bone-protective agent in this arthritis model.
Arthritis & Rheumatism 10/2011; 64(4):1069-81. · 7.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Rheumatoid arthritis (RA) is characterized by bone and cartilage invasion by fibroblast-like synoviocytes (FLSs). Adrenomedullin, a peptide with anabolic and antiapoptotic properties, is secreted by rheumatoid FLSs. Adrenomedullin also increases the expression of adhesion molecules in endothelial cells and keratinocytes. Here, we investigated whether adrenomedullin mediated FLS adhesion to extracellular matrix (ECM) proteins.
FLSs were isolated from synovial tissues from RA and osteoarthritis (OA) patients. Plates were coated overnight with the ECM proteins vitronectin, fibronectin, and type I collagen (Coll.I). Adrenomedullin was used as a soluble FLS ligand before plating. We tested interactions with the adrenomedullin receptor antagonist (22-52)adrenomedullin and with the protein kinase A (PKA) inhibitor H-89, and inhibition of co-receptor RAMP-2 by siRNA. Cell adhesion was measured by using color densitometry. Activation of α2 and β1 integrins was evaluated by fluorescent microscopy; integrin inhibition, by RGD peptides; and the talin-integrin interaction, by immunoprecipitation (IP).
Adrenomedullin specifically increased RA-FLS adhesion to vitronectin, fibronectin, and Coll.I; no such effect was found for OA-FLS adhesion. Basal or adrenomedullin-stimulated RA-FLS adhesion was inhibited by (22-52)adrenomedullin, H-89, and RAMP-2 siRNA. Adrenomedullin-stimulated adhesion was inhibited by RGD peptides, and associated with α2 and β1 integrin activation. This activation was shown with IP to be related to an integrin-talin interaction and was significantly decreased by (22-52)adrenomedullin.
Adrenomedullin-stimulated RA-FLS adhesion was specific for ECM proteins and mediated by α2 and β1 integrins. This effect of adrenomedullin was dependent on adrenomedullin receptors. These results support a new role for adrenomedullin in rheumatoid synovial fibroblast pathobiology.
Arthritis research & therapy 10/2010; 12(5):R190. · 4.27 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Wnt/beta-catenin signaling is critical for a variety of fundamental cellular processes. Here, we investigated the implication of the Wnt/beta-catenin signaling in the in vivo regulation of beta-cell growth and regeneration in normal and diabetic rats. To this aim, TCF7L2, the distal effector of the canonical Wnt pathway, was knocked down in groups of normal and diabetic rats by the use of specific antisense morpholino-oligonucleotides. In other groups of diabetic rats, the Wnt/beta-catenin pathway was activated by the inhibition of its negative regulator GSK-3beta. GSK-3beta was inactivated by either LiCl or anti-GSK-3beta oligonucleotides. The beta-cell mass was evaluated by morphometry. beta-cell proliferation was assessed in vivo and in vitro by BrdU incorporation method. In vivo beta-cell neogenesis was estimated by the evaluation of PDX1-positive ductal cells and GLUT2-positive ductal cells and the number of beta cells budding from the ducts. We showed that the in vivo disruption of the canonical Wnt pathway resulted in the alteration of normal and compensatory growth of beta-cells mainly through the inhibition of beta-cell proliferation. Conversely, activation of the Wnt pathway through the inhibition of GSK-3beta had a significant stimulatory effect on beta-cell regeneration in diabetic rats. In vitro, GSK-3beta inactivation resulted in the stimulation of beta-cell proliferation. This was mediated by the stabilization of beta-catenin and the induction of cyclin D. Taken together, our results demonstrate the involvement of the canonical Wnt signaling in the neonatal regulation of normal and regenerative growth of pancreatic beta-cells. Moreover, we provide evidence that activation of this pathway by pharmacological maneuvers can efficiently improve beta-cell regeneration in diabetic rats. These findings might have potential clinical applications in the regenerative therapy of diabetes.
AJP Endocrinology and Metabolism 11/2009; 298(2):E245-56. · 4.75 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Keratinocyte growth factor (KGF; palifermin) is a growth factor with a high degree of specificity for epithelial cells. KGF is an important effector of epithelial growth and tissue homeostasis in various organs including the pancreas. Here we investigated the intracellular signaling pathways involved in the mediation of pancreatic ductal cell proliferation and differentiation induced by exogenous KGF during beta-cell regeneration in diabetic rat.
In vitro and in vivo duct cell proliferation was measured by BrdU incorporation assay. The implication of MAPK-ERK1/2 in the mediation of KGF-induced cell proliferation was determined by inactivation of this pathway, using the pharmacological inhibitor or antisense morpholino-oligonucleotides against MEK1. In vivo KGF-induced duct cell differentiation was assessed by the immunolocalization of PDX1 and Glut2 in ductal cells and the implication of PI3K/AKT in this process was investigated. We showed that KGF exerted a potent mitogenic effect on ductal cells. Both in vitro and in vivo, its effect on cell proliferation was mediated through the activation of ERK1/2 as evidenced by the abolition of duct cell proliferation in the context of MEK/ERK inactivation. In vivo, KGF treatment triggered ductal cell differentiation as revealed by the expression of PDX1 and Glut2 in a subpopulation of ductal cells via a PI3K-dependent mechanism.
Here we show that KGF promotes beta-cell regeneration by stimulating duct cell proliferation in vivo. Moreover, we demonstrated for the first time that KGF directly induces the expression of PDX1 in some ductal cells thus inducing beta-cell neogenesis. We further explored the molecular mechanisms involved in these processes and showed that the effects of KGF on duct cell proliferation are mediated by the MEK-ERK1/2 pathway, while the KGF-induced cell differentiation is mediated by the PI3K/AKT pathway. These findings might have important implications for the in vivo induction of duct-to-beta cell neogenesis in patients with beta-cell deficiency.
PLoS ONE 02/2009; 4(3):e4734. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Journal of Cellular Physiology 05/2008; 215(1):122-8. · 3.87 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
The Journal of Immunology 06/2006; 176(9):5548-58. · 5.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Basic calcium phosphate (BCP) crystals, including hydroxyapatite, octacalcium phosphate (OCP) and carbonate-apatite, have been associated with severe osteoarthritis and several degenerative arthropathies. Most studies have considered the chondrocyte to be a bystander in the pathogenesis of calcium crystal deposition disease, assuming that synovial cell cytokines were the only triggers of chondrocyte activation. In the present study we identified direct activation of articular chondrocytes by OCP crystals, which are the BCP crystals with the greatest potential for inducing inflammation. OCP crystals induced nitric oxide (NO) production and inducible nitric oxide synthase (NOS) mRNA expression by isolated articular chondrocytes and cartilage fragments, in a dose-dependent manner and with variations over time. OCP crystals also induced IL-1beta mRNA expression. Using pharmacological and cytokine inhibitors, we observed that OCP crystals induced NO production and inducible NOS mRNA activation were regulated at both the transcriptional and the translational levels; were independent from IL-1beta gene activation; and involved p38 and c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways, as further confirmed by OCP crystal-induced p38 and JNK MAPK phosphorylation. Taken together, our data suggest that the transcriptional inducible NOS response to OCP crystals involved both the p38 and the JNK MAPK pathways, probably under the control of activator protein-1. NO, a major mediator of cartilage degradation, can be directly produced by BCP crystals in chondrocytes. Together with synovial activation, this direct mechanism may be important in the pathogenesis of destructive arthropathies triggered by microcrystals.
Arthritis research & therapy 02/2005; 7(5):R915-26. · 4.27 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Biochemical and Biophysical Research Communications 10/2004; 321(4):802-8. · 2.48 Impact Factor
