Effets cellulaires et voies de signalisation activés par le facteur anticoagulant, la protéine S, sur les cellules endothéliales (implication lors de l'angiogenèse)

L'angiogenèse est un processus physiologique qui correspond à la formation de nouveaux vaisseaux sanguins à partir d'un réseau vasculaire préexistant et est régulée par l'équilibre entre les facteurs endogènes pro- et anti-angiogéniques. La rupture de cet équilibre est associée à de nombreuses pathologies dont l'ischémie, la rétinopathie ou encore la progression tumorale. Etant donné que les cellules endothéliales, principal type cellulaire composant les vaisseaux sanguins expriment les récepteurs à activité tyrosine kinase du facteur anticoagulant, la protéine S, Tyro3, Axl et Mer et produisent de la protéine S, l'objectif de ce travail est d'étudier le rôle, de la protéine S dans l'angiogenèse. Dans la première partie de ce travail, nous avons montré in vivo que la protéine S inhibe l'angiogenèse induite par les facteurs pro-angiogéniques (VEGFA et FGF2). Parallèlement, nous avons observé in vitro une inhibition par la protéine S de la prolifération et de la migration des cellules endothéliales induites par le VEGFA. Cet effet est corrélé à l'inhibition par la protéine S des voies de signalisation des MAP-Kinases et de la phosphatidylinositol 3-kinase (PIK3) induites par le VEGFA. Nous avons ensuite mis en évidence, par l'utilisation d'inhibiteurs pharmacologiques et de petits ARNs interférents, que la protéine S inhibe, via l'activation du récepteur Mer et le recrutement de la protéine phosphatase SHP2, l'activation du VEGFR2, le principal récepteur du VEGFA. Dans la deuxième partie, nous avons montré de manière intéressante que le rôle joué par la protéine S lors de l'angiogenèse est plus complexe, puisqu'elle est capable d'activer directement la voie de signalisatioAngiogenesis is a physiological process that leads to new blood vessel formation and is regulated by a balance between pro-and anti-angiogenic endogenous factors. Disruption of this balance leads to many pathologies such as ischemia, retinopathies or tumor growth. Because endothelial cells, the main cellular type composing blood vessels, produce the anticoagulant factor, protein S and express its tyrosine kinase receptors Tyro3, Axl and Mer, we investigated the implication of protein S in angiogenesis. In the first part of this work, we demonstrated that protein S inhibits pro-angiogenic factors (VEGFA and FGF2)-induced angiogenesis in vivo. We also observed an inhibition of VEGFA-dependent endothelial cell proliferation and migration induced by protein S. These effects were correlated with protein S induced inhibition of VEGFA-dependent MAP-Kinases (Erk1, Erk2) and phosphatidylinositol 3-kinase (PIK3) activation. Furthermore, we demonstrated, using pharmacological inhibitors and small interfering RNAs, that protein S inhibits VEGFA-induced VEGFR-2 activation through Mer receptor activation and SHP2 protein phosphatase recruitment. In the second part, we demonstrated that, protein S on its own, is able to induce MAP-kinases pathway activation and endothelial cells proliferation. These cellular and molecular effects involved Mer receptor and SHP2 protein activation and required protein kinase SRC recruitment. Our results describe for the first time that protein S is an endogenous regulator for angiogenesis both in vitro and in vivo and may form the framework for the use of protein S as part of an anti-angiogenic treatment.POITIERS-SCD-Bib. électronique (861949901) / SudocSudocFranceF

Rôle du système urokinase/plasminogène et des métalloprotéases matricielles dans l'invasion des cellules du carcinome hépatocellulaire

PARIS7-Bibliothèque centrale (751132105) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Etude de l’implication de la Connexine 43 dans le processus d’invasion des glioblastomes humains

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Involvement of matrix metalloproteinase type-3 in hepatocyte growth factor-induced invasion of human hepatocellular carcinoma cells

International audienceIntra-hepatic invasion is a key feature of hepatocellular carcinoma (HCC) progression. We have shown that human liver myofibroblasts induce invasion of HCC cells through Matrigel, via the secretion of hepatocyte growth factor (HGF). In our study, we investigated the role of matrix metalloproteinases (MMP) in HGF-induced HCC cells invasion. Marimastat, a synthetic MMP inhibitor, dose-dependently decreased HGF-induced invasion of HepG2 cells with a maximum of 82.7 +/- 13.3% at 20 microM. TIMP-2, a natural inhibitor, decreased invasion up to 51.2 +/- 11.2% at 200 ng/ml. To determine the target for these inhibitors, we examined MMP expression using RT-PCR. MMPs 1, 7-9 and 10 were not expressed in HepG2 cells either in the absence or in the presence of HGF. MMP-2 and MMP-13 transcripts were detected in unstimulated cells but their expression was unchanged after exposition to HGF. MMP-3 transcripts were undetectable in unstimulated HepG2 cells. They became clearly expressed in HGF-stimulated cells, however, and this was confirmed by Northern blot. By Western blot, HGF dose-dependently stimulated the secretion of pro-MMP-3 in the culture medium. The role of MMP-3 in HGF-induced invasion was directly confirmed by using an antibody to MMP-3, that blocked invasion. Finally, RT-PCR demonstrated MMP-3 expression in 10/16 human HCCs tested, but not in normal liver. In conclusion, our data demonstrate that MMPs, most likely MMP-3, mediate HGF-induced invasion of HCC cells. The in vivo expression of MMP-3 in HCC suggests a role for this protease in HCC progression

Connexin43 mediates the invasive properties of human glioblastoma cells.

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Connexin43 increases the invasion capacity of human glioblastoma cells by inducing the formation of invadopodia.

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Neuropeptides of the VIP family inhibit glioblastoma cell invasion

International audienceVasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are neuropeptides acting through VPAC1, VPAC2 and PAC1 receptors (referred here as the VIP-receptor system). In the central nervous system, VIP and PACAP are involved in neurogenesis, cell differentiation and migration, suggesting that they could be implicated in the development of glioblastoma (GBM). The infiltrative nature of GBM remains a major problem for the therapy of these tumors. We previously demonstrated that the VIP-receptor system regulated cell migration of the human cell lines M059J and M059K, derived from a single human GBM. Here, we evaluated the involvement of the VIP-receptor system in GBM cell invasion. In Matrigel invasion assays, M059K cells that express more the VIP-receptor system than M059J cells were less invasive. Invasion assays performed in the presence of agonists, antagonists or anti-PACAP antibodies as well as experiments with transfected M059J cells overexpressing the VPAC1 receptor indicated that the more the VIP-receptor system was expressed and activated, the less the cells were able to invade. Western immunoblotting experiments revealed that the VIP-receptor system inactivated the signaling protein AKT. Invasion assays carried out in the presence of an AKT inhibitor demonstrated the involvement of this signaling kinase in the regulation of cell invasion by the VIP-receptor system in M059K cells. The inhibition by VIP of invasion and AKT was also observed in U87 cells. In conclusion, VIP and PACAP act as anti-invasive factors in different GBM cell lines, a function mediated by VPAC1 inhibition of AKT signaling in M059K cells

Connexins, important players in the dissemination of prostate cancer cells

International audienceOver the past 50years, increasing experimental evidences have established that connexins (Cxs) and gap junctional intercellular communication (GJIC) ensure an important role in both the onset and development of cancerous processes. In the present review, we focus on the impact of Cxs and GJIC during the development of prostate cancer (PCa), from the primary growth mainly localized in acinar glands and ducts to the distant metastasis mainly concentrated in bone. As observed in several other types of solid tumours, Cxs and especially Cx43 exhibit an ambivalent role with a tumour suppressor effect in the early stages and, conversely, a rather pro-tumoural profile for most of invasion and dissemination steps to secondary sites. We report here the current knowledge on the function of Cxs during PCa cells migration, cytoskeletal dynamics, proteinases activities and the cross talk with the surrounding stromal cells in the microenvironment of the tumour and the bones. In addition, we discuss the role of Cxs in the bone tropism even if the prostate model is rarely used to study the complete sequence of cancer dissemination compared to breast cancer or melanoma. Even if not yet fully understood, these recent findings on Cxs provide new insights into their molecular mechanisms associated with progression and bone targeted behaviour of PCa. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve

Techniques de carottages sur calcite souterraine. Enjeux et méthodes

International audienceSeveral analytical techniques are available to study underground calcite samples (U/Th, U/Pb dating, chemical and isotopic analyses, morphoscopy...). This leads to an increase of the number of samples taken from the caves. However, the conservation of the underground environment, a limitedlandscape impact and efficient documentation of the sampling are all elements that militate in favour of the development of adapted coring techniques. These techniques, presented here, cover three types of sampling: (i) diameter 4 to 10 mm, length 65/80 mm adapted to stalagmite bases; (ii) diameter 28 to 32 mm, length 0.3 to 1 m adapted to small floors and stalagmite bases; (iii) diameter 80 to 112 mm, length 0.3 to 2 m adapted to stalagmite floors and flowstones. A technical study group of the CNRS "Milieux Souterrains et Karsts" trade network offers here a synthesis of feedback and tests carried out in the field.The constraints of coring (protected cave), site accessibility (deep karst), extraction (conditioning) according to the type of sampling and their documentation are discussed. The final objective is to propose a methodological guide adapted to the different issues and situations encountered underground.Le développement de techniques analytiques permettant d'étudier des échantillons de calcite souterraine (datations U/Th, U/Pb, analyses chimiques et isotopiques, morphoscopie…) amène à multiplier les prélèvements d'échantillons sous terre. Cependant le souci de conservation du milieu souterrain, les enjeux d'un impact paysager limité, la nécessité d'une documentation efficace du prélèvement sont autant d'éléments qui militent pour le développement de techniques de carottage adaptées. Ces techniques, présentées ici, couvrent trois types d'échantillonnage : (i) diamètre 4 à 10 mm, longueur 65/80 mm adapté aux bases de stalagmites ; (ii) diamètre 28 à 32 mm, longueur 30 cm à 1 m adapté aux petits planchers et bases de stalagmites ; (iii) diamètre 80 à 112 mm, longueur 0,3 à 2 m adapté aux planchers et coulées stalagmitiques. Un groupe d'études techniques du réseau de métiers CNRS « Milieux Souterrains et Karsts » propose ici une synthèse des retours d'expérience et de tests menés sur le terrain. Les contraintes du carottage (grotte protégée), d'accessibilité au site (karst profond), d'extraction (conditionnement) en fonction du type d'échantillonnage et de leur documentation sont discutées. L'objectif final est de proposer un guide méthodologique adapté aux différents enjeux et situations rencontrées sous terre

Techniques de carottages sur calcite souterraine. Enjeux et méthodes

International audienceSeveral analytical techniques are available to study underground calcite samples (U/Th, U/Pb dating, chemical and isotopic analyses, morphoscopy...). This leads to an increase of the number of samples taken from the caves. However, the conservation of the underground environment, a limitedlandscape impact and efficient documentation of the sampling are all elements that militate in favour of the development of adapted coring techniques. These techniques, presented here, cover three types of sampling: (i) diameter 4 to 10 mm, length 65/80 mm adapted to stalagmite bases; (ii) diameter 28 to 32 mm, length 0.3 to 1 m adapted to small floors and stalagmite bases; (iii) diameter 80 to 112 mm, length 0.3 to 2 m adapted to stalagmite floors and flowstones. A technical study group of the CNRS "Milieux Souterrains et Karsts" trade network offers here a synthesis of feedback and tests carried out in the field.The constraints of coring (protected cave), site accessibility (deep karst), extraction (conditioning) according to the type of sampling and their documentation are discussed. The final objective is to propose a methodological guide adapted to the different issues and situations encountered underground.Le développement de techniques analytiques permettant d'étudier des échantillons de calcite souterraine (datations U/Th, U/Pb, analyses chimiques et isotopiques, morphoscopie…) amène à multiplier les prélèvements d'échantillons sous terre. Cependant le souci de conservation du milieu souterrain, les enjeux d'un impact paysager limité, la nécessité d'une documentation efficace du prélèvement sont autant d'éléments qui militent pour le développement de techniques de carottage adaptées. Ces techniques, présentées ici, couvrent trois types d'échantillonnage : (i) diamètre 4 à 10 mm, longueur 65/80 mm adapté aux bases de stalagmites ; (ii) diamètre 28 à 32 mm, longueur 30 cm à 1 m adapté aux petits planchers et bases de stalagmites ; (iii) diamètre 80 à 112 mm, longueur 0,3 à 2 m adapté aux planchers et coulées stalagmitiques. Un groupe d'études techniques du réseau de métiers CNRS « Milieux Souterrains et Karsts » propose ici une synthèse des retours d'expérience et de tests menés sur le terrain. Les contraintes du carottage (grotte protégée), d'accessibilité au site (karst profond), d'extraction (conditionnement) en fonction du type d'échantillonnage et de leur documentation sont discutées. L'objectif final est de proposer un guide méthodologique adapté aux différents enjeux et situations rencontrées sous terre