Éditeur(s) : HAL CCSD Public Library of Science
Résumé : International audience
Ro 31-8220 is a potent protein kinase C (PKC) inhibitor belonging to the chemical class of bisindolylmaleimides (BIMs). Various PKC-independent effects of Ro 31-8220 have however been demonstrated, including inhibition of the ATP-binding cassette drug transporter breast cancer resistance protein. In the present study, we reported that the BIM also blocks activity of the solute carrier organic cation transporter (OCT) 1, involved in uptake of marketed drugs in the liver, in a PKC-independent manner. Ro 31-8220, in contrast to other pan-PKC inhibitors such as staurosporine and chelerythrine, was thus shown to cis-inhibit uptake of the reference OCT1 substrate tetraethylammonium in OCT1-transfected HEK293 cells in a concentration-dependent manner (IC50 = 0.18 μM) and without altering membrane expression of OCT1. This blockage of OCT1 was also observed in human hepatic HepaRG cells that constitutionally express OCT1. It likely occurred through a mixed mechanism of inhibition. Ro 31-8220 additionally trans-inhibited TEA uptake in OCT1-transfected HEK293 cells, which likely discards a transport of Ro 31-8220 by OCT1. Besides Ro 31-8220, 7 additional BIMs, including the PKC inhibitor LY 333531, inhibited OCT1 activity, whereas 4 other BIMs were without effect. In silico analysis of structure-activity relationships next revealed that various molecular descriptors, especially 3D-WHIM descriptors related to total size, correspond to key physico-chemical parameters for inhibition of OCT1 activity by BIMs. In addition to activity of OCT1, Ro 31-8220 inhibited those of other organic cation transporters such as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K, whereas, by contrast, it stimulated that of OCT2. Taken together, these data extend the nature of cellular off-targets of the BIM Ro 31-8220 to OCT1 and other organic cation transporters, which has likely to be kept in mind when using Ro 31-8220 and other BIMs as PKC inhibitors in experimental or clinical studies
ISSN: 1932-6203

hal-01255837
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01255837
DOI : 10.1371/journal.pone.0144667
PUBMED : 26657401
PUBMEDCENTRAL : PMC4675551

Éditeur(s) : HAL CCSD Cambridge University Press
Résumé : International audience
The liver is the most important link between the circadian system and metabolism. As a food-entrainable oscillator, the hepatic clock needs to be entrained by food-related signals. The objective of the present study was to investigate the possible role of ghrelin (an orexigenic peptide mainly synthesized in the gastrointestinal tract) as an endogenous synchronizer of the liver oscillator in teleosts. To achieve this aim, we first examined the presence of ghrelin receptors in the liver of goldfish. Then, the ghrelin regulation of clock gene expression in the goldfish liver was studied. Finally, the possible involvement of the phospholipase C/ protein kinase C (PLC/ PKC) and adenylate cyclase/protein kinase A (AC/PKA) intracellular signalling pathways was investigated. Ghrelin receptor transcripts, ghs-r1a, are present in the majority of goldfish hepatic cells. Ghrelin induced the mRNA expression of the positive (gbmal1a, gclock1a) and negative (gper genes) elements of the main loop of the molecular clock machinery, as well as grev-erba (auxiliary loop) in cultured liver. These effects were blocked, at least in part, by a ghrelin antagonist. Incubation of liver with a PLC inhibitor (U73122), a PKC activator (phorbol 12-myristate 13-acetate) and a PKC inhibitor (chelerythrine chloride) demonstrated that the PLC/ PKC pathway mediates such ghrelin actions. Experiments with an AC activator (forskolin) and a PKA inhibitor (H89) showed that grev-erba regulation could be due to activation of PKA. Taken together, the present results show for the first time in vertebrates a direct action of ghrelin on hepatic clock genes and support a role for this hormone as a temporal messenger in the entrainment of liver circadian functions.
Journal of Experimental Biology

hal-01519254
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01519254
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01519254/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01519254/file/Ghrelin%20induces%20clock%20gene%20expression.pdf
DOI : 10.1242/jeb.144253

Éditeur(s) : HAL CCSD Nature Publishing Group
Résumé : International audience
Necroptosis is a programmed cell death pathway that has been shown to be of central pathophysiological relevance in multiple disorders (hepatitis, brain and cardiac ischemia, pancreatitis, viral infection and inflammatory diseases). Necroptosis is driven by two serine threonine kinases, RIPK1 (Receptor Interacting Protein Kinase 1) and RIPK3, and a pseudo-kinase MLKL (Mixed Lineage Kinase domain-Like) associated in a multi-protein complex called necrosome. In order to find new inhibitors for use in human therapy, a chemical library containing highly diverse chemical structures was screened using a cell-based assay. The compound 6E11, a natural product derivative, was characterized as a positive hit. Interestingly, this flavanone compound: inhibits necroptosis induced by death receptors ligands TNF-α (Tumor Necrosis Factor) or TRAIL (TNF-Related Apoptosis-Inducing Ligand); is an extremely selective inhibitor, among kinases, of human RIPK1 enzymatic activity with a nM Kd; has a non-ATP competitive mode of action and a novel putative binding site; is weakly cytotoxic towards human primary blood leukocytes or retinal pigment epithelial cells at effective concentrations; protects human aortic endothelial cells (HAEC) from cold hypoxia/reoxygenation injury more effectively than necrostatin-1 (Nec-1) and Nec-1s. Altogether, these data demonstrate that 6E11 is a novel potent small molecular inhibitor of RIPK1-driven necroptosis.
ISSN: 2045-2322

hal-01616456
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01616456
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01616456/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01616456/file/s41598-017-12788-4.pdf
DOI : 10.1038/s41598-017-12788-4

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Hepatic drug transporters are now recognized as major actors of hepatobiliary elimination of drugs. Characterization of their regulatory pathways is therefore an important issue. In this context, the present study was designed to analyze the potential regulation of human hepatic transporter expression by protein kinase C (PKC) activation. Treatment by the reference PKC activator phorbol 12-myristate 13-acetate (PMA) for 48 h was shown to decrease mRNA expression of various sinusoidal transporters, including OATP1B1, OATP2B1, NTCP, OCT1 and MRP3, but to increase that of OATP1B3, whereas mRNA expression of canalicular transporters was transiently enhanced (MDR1), decreased (BSEP and MRP2) or unchanged (BCRP) in human hepatoma HepaRG cells. The profile of hepatic transporter mRNA expression changes in PMA-treated HepaRG cells was correlated to that found in PMA-exposed primary human hepatocytes and was similarly observed in response to the PKC-activating marketed drug ingenol mebutate. It was associated with concomitant repression of OATP1B1 and OATP2B1 protein expression and reduction of OATP, OCT1, NTCP and MRP2 activity. The use of chemical PKC inhibitors further suggested a contribution of novel PKCs isoforms to PMA-mediated regulations of transporter mRNA expression. PMA was finally shown to cause epithelial-mesenchymal transition (EMT) in HepaRG cells and exposure to various additional EMT inducers, i.e., hepatocyte growth factor, tumor growth factor-β1 or the HNF4α inhibitor ≡I6015, led to transporter expression alterations highly correlated to those triggered by PMA. Taken together, these data highlight PKC-dependent regulation of human hepatic drug transporter expression, which may be closely linked to EMT triggered by PKC activation
ISSN: 0006-2952

hal-01216205
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01216205
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01216205/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01216205/file/Protein%20kinase%20C-dependent%20regulation.pdf
DOI : 10.1016/j.bcp.2015.10.007

Éditeur(s) : HAL CCSD
Résumé : International audience
Necroptosis is a regulated form of cell death involved in several disease models including in particular liver diseases. Receptor-interacting protein kinases, RIPK1 and RIPK3, are the main serine/threonine kinases driving this cell death pathway. We screened a noncommercial, kinase-focused chemical library which allowed us to identify Sibiriline as a new inhibitor of necroptosis induced by tumor necrosis factor (TNF) in Fas-associated protein with death domain (FADD)-deficient Jurkat cells. Moreover, Sib inhibits necroptotic cell death induced by various death ligands in human or mouse cells while not protecting from caspase-dependent apoptosis. By using competition binding assay and recombinant kinase assays, we demonstrated that Sib is a rather specific competitive RIPK1 inhibitor. Molecular docking analysis shows that Sib is trapped closed to human RIPK1 adenosine triphosphate-binding site in a relatively hydrophobic pocket locking RIPK1 in an inactive conformation. In agreement with its RIPK1 inhibitory property, Sib inhibits both TNF-induced RIPK1-dependent necroptosis and RIPK1-dependent apoptosis. Finally, Sib protects mice from concanavalin A-induced hepatitis. These results reveal the small-molecule Sib as a new RIPK1 inhibitor potentially of interest for the treatment of immune-dependent hepatitis. © 2017 Federation of European Biochemical Societies
ISSN: 1742464X

hal-01616420
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01616420
DOI : 10.1111/febs.14176
PUBMED : 28715128

Éditeur(s) : HAL CCSD MDPI
Résumé : International audience
Drug transporters are now recognized as major actors in pharmacokinetics, involved notably in drug-drug interactions and drug adverse effects. Factors that govern their activity, localization and expression are therefore important to consider. In the present review, the implications of protein kinases C (PKCs) in transporter regulations are summarized and discussed. Both solute carrier (SLC) and ATP-binding cassette (ABC) drug transporters can be regulated by PKCs-related signaling pathways. PKCs thus target activity, membrane localization and/or expression level of major influx and efflux drug transporters, in various normal and pathological types of cells and tissues, often in a PKC isoform-specific manner. PKCs are notably implicated in membrane insertion of bile acid transporters in liver and, in this way, are thought to contribute to cholestatic or choleretic effects of endogenous compounds or drugs. The exact clinical relevance of PKCs-related regulation of drug transporters in terms of drug resistance, pharmacokinetics, drug-drug interactions and drug toxicity remains however to be precisely determined. This issue is likely important to consider in the context of the development of new drugs targeting PKCs-mediated signaling pathways, for treating notably cancers, diabetes or psychiatric disorders.
ISSN: 1422-0067

hal-01516024
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01516024
DOI : 10.3390/ijms18040764
PUBMED : 28375174

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Aryl hydrocarbon receptor-dependent genomic effects of environmental polycyclic aromatic hydrocarbons (PAHs) have been shown to be modulated by non-genomic protein kinase C (PKC)-related pathways. The present study was designed to determine whether PKC activation may also impair estrogenic genomic response triggered by PAHs. Treatment by the PKC activator phorbol 12-myristate 13-acetate (PMA) was found to markedly and differentially impair the up-regulation of estrogenic markers triggered by the estrogenic PAH benzanthracene (BZA) in cultured human mammary cells; BZA-mediated mRNA up-regulation of pS2 and amphiregulin was thus increased, whereas that of progesterone receptor and CXCL12 was repressed. BZA/PMA cotreatment however failed to alter BZA-mediated increase of activity of a luciferase gene reporter construct driven by an estrogen response element, thus discarding any global effect of PMA toward BZA-triggered estrogen receptor activation. Various chemicals inhibiting PKCs or extracellular signal-regulated kinase (ERK) as well as the knock-down of PKCδ expression counteracted the PMA-mediated increase of pS2 mRNA up-regulation triggered by BZA, demonstrating that it was dependent on PKCs, including PKCδ isoform, and ERKs. This non-genomic modulation of estrogenic effects of PAHs by PKC activation may have to be considered when considering the deleterious effects of these environmental contaminants towards the endocrine system.
ISSN: 0887-2333

inserm-00872862
http://www.hal.inserm.fr/inserm-00872862
DOI : 10.1016/j.tiv.2012.05.006
PUBMED : 22643241

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Formaldehyde (FA) is a volatile organic compound (VOC), considered as a major indoor air pollutant and suspected to favor the development of inflammatory lung diseases. The present study was aimed at identifying cytokines/chemokines targeted by FA in human lung cells. This VOC was demonstrated to up-regulate interleukin-11 (IL-11) mRNA and secretion levels in a dose-dependent manner in cultured lung epithelial BEAS-2B cells. It concomitantly induced mRNA expression of transforming growth factor (TGF)-β1, a fibrogenic marker regulated by IL-11. FA was also found to trigger an early phosphorylation of p38 and extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinases (MAPKs) in BEAS-2B cells, whose inhibition by ERK and p38 MAPK chemical inhibitors (U0126 and SB203580, respectively) counteracted FA-mediated induction of IL-11. In addition, FA increased phosphorylation of cAMP response element binding protein (CREB) and the use of small-interfering RNA targeting CREB demonstrated that this transcription factor was required for the up-regulation of IL-11 by FA. Implication of protein kinase C (PKC) in FA-induced IL-11 expression was moreover demonstrated by using RO-31-8220, a PKC inhibitor. We finally showed using SB203580 and RO-31-8220 that phosphorylation of CREB and CREB-promoter activity induced by FA are under the control of both p38 MAPK and PKC. Taken together, the results showed that FA uses different pathways to induce IL-11 expression in lung BEAS-2B cells. IL-11, well-known to contribute to lung inflammatory diseases, appears thus as a molecular target of FA, which could be involved in putative deleterious inflammatory and fibrogenic pulmonary effects of this VOC.
ISSN: 0300-483X

hal-00682486
https://hal.archives-ouvertes.fr/hal-00682486
DOI : 10.1016/j.tox.2011.11.011
PUBMED : 22138297

Éditeur(s) : HAL CCSD Springer Verlag
Résumé : International audience
Interleukin-33 (IL-33), a cytokine belonging to the IL-1 family, is crucially involved in inflammatory pathologies including liver injury and linked to various modes of cell death. However, a link between IL-33 and necroptosis or programmed necrosis in liver pathology remains elusive. We aimed to investigate the regulation of IL-33 during necroptosis-associated liver injury. The possible regulation of IL-33 during liver injury by receptor-interacting protein kinase 1 (RIPK1) and poly(ADP-ribose) polymerase 1 (PARP-1) was investigated in mice in vivo and in hepatic stellate cells in vitro. The liver immunohistopathology, flow cytometry, serum transaminase measurement, ELISA, and qPCR-based cytokine measurement were carried out. By using a chemical approach, we showed that pretreatment of mice with Necrostatin-1 (Nec-1) (inhibitor of RIPK1) and/or PJ34 (inhibitor of PARP-1) significantly protected mice against concanavalin A (ConA) liver injury (aspartate amino-transferase (AST)/alanine amino-transferase (ALT)) associated with down-regulated hepatocyte-specific IL-33 expression. In contrast, the expression level of most systemic cytokines (except for IL-6) or activation of liver immune cells was not altered by chemical inhibitors rather an increased infiltration of neutrophils in the liver. During polyinosine-polycytidylic acid (Poly(I:C))-induced acute hepatitis, liver injury and hepatocyte-specific IL-33 expression was also inhibited by PJ34 without any protective effect of PJ34 in CCl4-induced liver injury. Moreover, PJ34 down-regulated the protein expression of IL-33 in activated hepatic stellate cells by cocktail of cytokines or staurosporine in vitro. In conclusion, we evidenced that the Nec-1/PJ34 is a potent inhibitor of liver injury and Nec-1/PJ34 down-regulated hepatocyte-specific IL-33 expression in the liver in vivo or in hepatic stellate cells in vitro, suggesting IL-33 as a possible readout of necroptosis-involved liver pathologies. Necroptosis inhibitors can protect mice against liver injury induced by ConA or Poly(I:C). IL-33 expression in liver injury in vivo is inhibited by PJ34. IL-33 expression in hepatic stellate cells in vitro is inhibited by PJ34. Hepatocyte-specific IL-33 expression is down-regulated by Nec-1/PJ34 during hepatitis. IL-33 is a new marker of necroptosis-associated liver injuries.
ISSN: 0946-2716

hal-01134319
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01134319
DOI : 10.1007/s00109-015-1270-6
PUBMED : 25747661

Éditeur(s) : HAL CCSD
Résumé : Croton urucurana Baillon (Euphorbiaceae), plant species popularly known in Brazil as “sangra-d’água”, is commonly found in Mato Grosso do Sul State (midwestern Brazil), where its latex is popularly used to treat several types of cancer. Despite the ethnopharmacological significance of the species, no phytochemical or cytotoxic studies of its latex have been published. In this context, the aim of the present study was to assess the cytotoxic activity of crude extracts, fractions and isolated compound from the latex of C. urucurana and to investigate the effects of crourorb A1 on the viability, apoptosis, cell cycle and cell migration of human hepatocarcinoma cell lines (Huh-7). Also, we have evaluated the viability of Huh-7 cells treated with crourorb A1 in 3D collagen gels. The cytotoxicity was detected using the sulforhodamine B (SRB) assay against six human cancer cells lines and one nontumor NIH/3T3 cell line (murine fibroblast). Studies on the mechanism of cell death of crourorb A1 were conducted in different test systems in vitro. The bioactive ethyl acetate phase obtained from the latex of C. urucurana afforded a novel cyclic peptide, [1−9-NαC]-crourorb A1, that proved active against NCIADR/ RES (ovary, multidrug-resistance phenotype) cells with the same potency as doxorubicin (positive control) and inactive up to the highest concentration tested against nontumor NIH/3T3 cells (GI50 = > 250 μg/mL). Crourorb A1 treatment decreases the cell viability of Huh-7 at dose and time dependent manner associated with induction of apoptosis by caspase 3/7 activation and increasing expression of pro-apoptotic proteins: Bak, Bid, Bax, Puma, Bim, and Bad and clived-caspase3 protein. The JNK/MAP pathway is involved in this induced cell death. Crourorb A1 effect is also associated with G2/M phase cell cycle arrest. In parallel, we observed that crourorb A1 treatment resulted in an increase of cyclin-dependent kinase (CDK1), cyclin B1 and cyclin D1 expressions. A significant reduction of Huh-7 cells migration by crourorb A1 can be assessed and we also show that cells grown on 3D collagen gels exhibited increased resistance to crourorb A1 when compared to 2D cell culture.
Croton urucurana Baillon (Euphorbiaceae), est une plante très connue et utilisée en médecine traditionnelle au Brésil, la «sangra-d’água» retrouvée dans l'État du Mato Grosso do Sul (région Centre-Ouest du Brésil). Son latex est abondamment utilisé en médecine traditionnelle dans le traitement de plusieurs types de cancer. En dépit de son importance ethnopharmacologique, on ne trouve aucune étude sur sa composition chimique ou sur sa potentielle activité anti-cancéreuse. L’objectif de ce travail a été d'évaluer l'activité cytotoxique des extraits totaux, fractions d’extraits et de molécules purifiées, isolées du latex de C. urucurana. Nous avons étudié plus particulièrement les effets du crourorb A1 sur la viabilité, l'apoptose, le cycle cellulaire et la migration de cellules issues de carcinome hépatocellulaire humaine (Huh-7). Nous avons également évalué l’effet du crourorb A1sur la viabilité des cellules Huh-7 cultivées dans les gels de collagène en 3D. L' activité cytotoxique ont été réalisées par le test à la sulforhodamine B sur six lignées cellulaire cancéreuses et sur une lignée cellulaire non cancéreuse de fibroblastes murins, les cellules NIH/3T3. L'étude phytochimique de la fraction ‘acétate d'éthyle’ du latex de C. urucurana bioguidé a permis d’obtenir un nouveau cyclopeptide que nous avons nommé [1-9-NαC]-crourorb A1. Le crourorb présente une cytotoxicité puissante sur la lignée cellulaire cancéreuse NCI-ADR/RES (ovaire, le phénotype de multirésistance) à un niveau équivalent à celle de la doxorubicine (témoin positif). Ce composé est de plus pratiquement inactif (GI50 = >250 μg/mL) sur la lignée non cancéreuse NIH/3T3. L'évaluation de la cytotoxicité in vitro du crourorb A1 (test wst1) sur les cellules Huh-7 a démontré que la viabilité des cellules est inhibée de manière dose et temps-dépendant. Nous avons mesuré une forte activation des caspases 3 et 7 et une nette augmentation de l’expression des protéines pro-apoptotiques (Bak, Bid, Bax, Puma, Bim et Bad) ainsi que de la caspase 3 clivée par le crourorb A1. Nous avons également déterminé que la voie de signalisation JNK-MAPkinase semble nécessaire à l’induction de la mort cellulaire des cellules Huh-7 traitées au crourorb A1. Le crourob A1 induit un arrêt du cycle cellulaire en phase G2/M. En parallèle, les expressions de la cycline-dépendante kinase CDK1, des cyclines B1 et D1 sont augmentées après traitement au crourorb A1. De plus, le crourorb A1 a un effet inhibiteur significatif de la migration des cellules Huh-7, en présence ou absence de mitomycine C. Nous avons également confirmé, l’effet inhibiteur du crourob A1 sur la viabilité des cellules Huh7 cultivées en 3D dans les gels de collagène 1 et démontré que les cellules cultivées en 3D semblent plus résistantes au crourorb A1 par rapport aux cultures en monocouche 2D.
Croton urucurana Baillon (Euphorbiaceae) é uma espécie arbórea conhecida popularmente no Brasil como “sangra d´água”, comumente encontrada no estado de Mato Grosso do Sul, onde seu látex tem sido utilizado na medicina popular no tratamento de vários tipos de câncer. Apesar de sua importância etnofarmacológica, não há relatos de estudos relacionados à sua composição química ou atividades anticâncer na literatura. Nesse contexto, o objetivo do presente estudo foi avaliar a atividade citotóxica de extratos, fases, frações e da substância isolada do látex de C. ururucurana e investigar os mecanismos celulares e moleculares in vitro responsáveis pela citotoxicidade da substância isolada considerando seu potencial apoptótico e influência sobre a progressão do ciclo celular em uma linhagem de hepatocarcinoma celular humano (Huh-7), bem como, os efeitos desta substância sobre a migração e viabilidade de células Huh-7 cultivadas em geis de colágeno em 3D. O ensaio de citotoxicidade in vitro foi realizado com o corante sulforrodamina B (SRB) em seis linhagens de células neoplásicas humanas e uma linhagem de células não neoplásicas NIH/3T3 (fibloblasto murinho). Os estudos de mecanismos de ação com crourorb A1 foram conduzidos em diferentes sistemas-teste in vitro. O estudo fitoquímico biomonitorado da fase bioativa acetato de etila do látex de C. urucurana resultou no isolamento de um novo ciclopeptídeo denominado [19-NαC]-crourorb A1, que apresentou potente atividade citotóxica frente à linhagem de células neoplásicas NCI-ADR/RES (ovário com fenótipo de resistência a múltiplos fármacos), com a mesma potência que a doxorrubicina (controle positivo), e foi inativa até a maior concentração testada (GI50 = >250 μg/mL) frente à linhagem de células não neoplásicas 3T3. A exposição de células Huh-7 ao crourorb A1 resultou em uma diminuição da viabilidade celular de maneira dose e tempo-dependentes. Nós observamos um aumento da atividade de caspases 3 e 7 e um aumento da expressão de proteínas próapoptóticas (Bak, Bid, Bax, Puma, Bim e Bad) e caspase 3 clivada após tratamento com crourorb A1. Além disso, observamos que a via de sinalização celular MAP/JNK foi necessária para induzir a morte celular em células Huh-7 tratadas com crourorb A1. Os efeitos de crourorb A1 foram também associados com o acúmulo de células na fase G2/M do ciclo celular. Em paralelo, as expressões da cinase dependente da ciclina (CDK1), ciclina B1 e ciclina D1 foram aumentadas após tratamento com crourorb A1. Foi demonstrado também o envolvimento de crourorb A1 na redução da migração de células Huh-7 cultivadas na presença e ausência de mitomicina C. A avaliação da viabilidade de células Huh-7 cultivadas em geis de colágeno em 3D apontou uma maior resistência destas células ao crourorb A1 quando comparadas com a cultura de células em 2D.
https://tel.archives-ouvertes.fr/tel-01478286

NNT : 2016REN1B010
tel-01478286
https://tel.archives-ouvertes.fr/tel-01478286
https://tel.archives-ouvertes.fr/tel-01478286/document
https://tel.archives-ouvertes.fr/tel-01478286/file/DE-MATOS-CANDIDO-BACANI-Priscila.pdf

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Background & Aims: The severity of liver diseases is exacerbated by the death of hepatocytes, which can be induced by the sensing of pathogen associated molecular patterns (PAMPs) derived from the gut microbiota. The molecular mechanisms regulating these cell death pathways are poorly documented. In this study, we investigated the role of the receptor interacting protein kinase 1 (RIPK1), a protein known to regulate cell fate decisions, in the death of hepatocytes using two in vivo models of PAMP-induced hepatitis. Methods: Hepatitis was induced in mice by independent injections of two different bacterial PAMPs: lipopolysaccharide (LPS) and unmethylated CpG oligodeoxynucleotide (CpG-DNA) motifs. The role of RIPK1 was evaluated by using mice specifically lacking RIPK1 in liver parenchymal cells (Ripk1(LPC-KO)). Administration of liposome-encapsulated clodronate served to investigate the role of Kupffer cells in the establishment of the disease. Etanercept, a tumor necrosis factor (TNF)-decoy receptor, was used to study the contribution of TNF-alpha during LPS-mediated liver injury. Results: Whereas RIPK1 deficiency in liver parenchymal cells did not trigger basal hepatolysis, it greatly sensitized hepatocytes to apoptosis and liver damage following a single injection of LPS or CpG-DNA. Importantly, hepatocyte death was prevented by previous macrophage depletion or by TNF inhibition. Conclusions: Our data highlight the pivotal function of RIPK1 in maintaining liver homeostasis in conditions of macrophage-induced TNF burst in response to PAMPs sensing. Lay summary: Excessive death of hepatocytes is a characteristic of liver injury. A new programmed cell death pathway has been described involving upstream death ligands such as TNF and downstream kinases such as RIPK1. Here, we show that in the presence of LPS liver induced hepatic injury was due to secretion of TNF by liver macrophages, and that RIPK1 acts as a powerful protector of hepatocyte death. This newly identified pathway in the liver may be helpful in the management of patients to predict their risk of developing acute liver failure. (C) 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
ISSN: 0168-8278

hal-01532060
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01532060
DOI : 10.1016/j.jhep.2017.01.005
PUBMED : 28088582

Éditeur(s) : HAL CCSD
Résumé : Anti-Müllerian hormone (AMH) is a member of the TGF-ß superfamily. AMH is well known for its role in Müllerian duct regression in male fetuses. Postnatally, AMH is secreted by granulosa cells (GCs) of small growing follicles (preantral and small antral). However, despite the increasing interest of ovarian AMH in clinics, little is known on its mechanism of action and its role in female reproductive tract. My PhD project focuses on the identification of AMH function in the female reproductive tract.AMH signals through a type II transmembrane serine/threonine kinase receptor (AMHR-II) which forms a complex with a type I serine/threonine kinase receptor (ActR-IA, BMPR-IA, BMPR-IB). The type II receptor phosphorylates serine and threonine residues of type I receptor. Once activated, the type I receptor phosphorylates the receptor-regulated Smads (R-Smad1/5/8) which interact with a common partner Smad4. The Smad complex accumulates into the nucleus and regulates target gene expression. This canonical signalling pathway is regulated at different levels, in particular by co-receptors which amplify or antagonize TGF-ß family members action. The type I receptors and R-Smads involved in AMH effects on post-natal GCs remain unknown. In addition, to date, no co-receptor has been found for AMH. To define the involvement of the different type I receptors, we used siRNA technology to inactivate Acvr1, Bmpr1a and Bmpr1b in GC. In parallele, we analysed GC extracted from conditional mutant mice for Acvr1 and Bmpr1a. We found that BMPR-IA is the most important type I receptor for AMH to transduce its signal in GC. A Smad-Gal4/UAS-luciferase reporter gene technology allowed us to show that Smad1 and 5 are involved in AMH signaling pathway. Recently, new BMPs coreceptors were found, RGMs for Repulsive Guidance Molecules. There are three RGMs : RGMa, b and c. Because AMH shares with BMPs its type I receptors and R-Smad proteins, we hypothesized that they also share the same co-receptors, the RGM. We showed that RGMb was the only one expressed in GC and after siRNA transfection we demonstrated that this coreceptor is not essential for AMH to transduce its signal.To date, only few AMH target genes have been identified. Aromatase (Cyp19a1) and LH receptor (Lhcgr) are down-regulated by AMH in rat and porcine GCs. We used micro-array technology (Affymetrix) by comparing Wt and knockout immature ovaries to find new AMH target genes. This experiment evidenced that Ovgp1 and Kcnj2 are two new potential AMH target genes in the ovary.The last part of my project was to define a potential role of AMH in murine uterus. Only one study showed that AMHR-II is expressed in the mouse myometrium. We showed that Amh gene is slightly expressed in uterus but the results are not confirmed at the protein level. Using PCR-array, we found a lot of differentially expressed genes between Wt and Amh KO uterus. Therefore, AMH could regulate uterine function through the modulation of different genes located in the myometrium.
L’hormone anti-Müllerienne (AMH) est un membre de la famille TGF-β impliquée dans la différenciation du tractus reproductif mâle. Elle est aussi exprimée par les cellules de la granulosa de l’ovaire adulte. Cependant, son rôle physiologique chez la femelle n’a pas encore été entièrement établi. Mon projet de thèse a pour objectif d’élucider le(s) rôle(s) de l’AMH dans le tractus reproductif femelle. L’AMH transduit ses effets par l’intermédiaire de deux récepteurs transmembranaires sérine/thréonine kinase : un récepteur de type II qui lui est spécifique (AMHR-II) et un récepteur de type I (ActR-IA, BMPR-IA, BMPR-IB) qu’elle partage avec les BMPs. Après fixation de l’hormone sur le récepteur de type II, celui-ci recrute et phosphoryle le récepteur de type I. Ce dernier phosphoryle à son tour les Smads spécifiques (Smad1, 5 et 8) qui s’associent à la Smad commune, Smad4. L’ensemble transloque dans le noyau et en association avec des facteurs de transcription régule les gènes cibles de l’hormone. L'utilisation de souris KO conditionnelles pour les récepteurs Acvr1 et Bmpr1a et d'une technique de siRNA dirigés contre chacun des trois récepteurs de type I a permis de mettre en évidence que le récepteur BMPR-IA est un acteur essentiel de la voie de signalisation de l'AMH dans les cellules de la granulosa. Pour déterminer la ou les Smad(s) impliquées, une technique de gènes rapporteurs, Smad-Gal4/UAS-luciférase, a été utilisée. Nous avons pu montrer que les Smad1 et 5 sont importantes pour la transduction du signal de l'AMH dans les cellules de la granulosa. Récemment des corécepteurs aux BMPs, les Repulsive Guidance Molecule (RGMs), ont été mis en évidence. L’AMH partageant sa voie de signalisation avec les BMPs, nous avons cherché à déterminer si ces corécepteurs pouvaient également intervenir dans la voie de signalisation de l’AMH. Il existe 3 types de RGMs: RGMa, RGMb et RGMc. Nous avons montré en q-PCR que seul RGMb est exprimé dans les cellules de la granulosa alors que les 3 RGMs sont exprimés dans l’ovaire. L'utilisation de siRNA dirigés contre RGMb a permis de montrer que ce récepteur n'est pas nécessaire à la transduction du signal de l'AMH. Actuellement, seuls deux gènes cibles de l’AMH sont connus dans les cellules de la granulosa : l’aromatase et le récepteur LH. Nous avons réalisé des analyses de puces à ADN (ou micro-array) pour décrire de nouveaux gènes cibles de l'AMH. L'analyse des puces a permis de décrire de nouveaux gènes régulés par cette hormone tels qu’Ovgp1 ou Kcnj2. La dernière partie de mon projet visait à déterminer un rôle potentiel de l'AMH dans l'utérus. En effet, le récepteur de cette hormone est exprimé dans le myomètre utérin de souris permettant de supposer qu’elle peut agir sur cet organe. Nous avons pu mettre en évidence une expression faible du gène de l’Amh dans l’utérus. En revanche, l’expression et la localisation de la protéine restent encore à définir. Une expérience de PCR-array a permis de montrer que de nombreux gènes sont différentiellement exprimés entre l’utérus Wt et l’utérus KO Amh. Ceci indique que l’AMH jouerait un rôle sur la régulation de la fonction utérine qu’elle soit exprimée ou non dans cet organe.
https://tel.archives-ouvertes.fr/tel-00989662

Droits : info:eu-repo/semantics/OpenAccess
NNT : 2014PA11T014
tel-00989662
https://tel.archives-ouvertes.fr/tel-00989662
https://tel.archives-ouvertes.fr/tel-00989662/document
https://tel.archives-ouvertes.fr/tel-00989662/file/VA_SEDES_LAURIANE_03042014.pdf

Éditeur(s) : HAL CCSD BMJ Publishing Group
Résumé : International audience
BACKGROUND: Mutations in the leucine-rich-repeat kinase 2 (LRRK2) gene have been identified in families with autosomal dominant Parkinson's disease (ADPD), the most common of which is the p.G2019S substitution that has been found at varying frequencies worldwide. Because of the size of the LRRK2 gene, few studies have analysed the entire gene in large series of ADPD families. METHODS: We performed extensive mutation analyses of all 51 coding exons of the LRRK2 gene in index cases from 226 Parkinson's disease families compatible with autosomal dominant inheritance, mostly from France (n = 182) and North Africa (n = 14). RESULTS: We found 79 sequence variants, 29 of which were novel. Eight potentially or proven pathogenic mutations were found in 22 probands (9.7%). There were four novel amino acid substitutions that are potentially pathogenic (p.S52F, p.N363S, p.I810V, p.R1325Q) and two novel variants, p.H1216R and p.T1410M, that are probably not causative. The common p.G2019S mutation was identified in 13 probands (5.8%) including six from North Africa (43%). The known heterozygous p.R1441H and p.I1371V mutations were found in two probands each, and the p.E334K variant was identified in one single patient. Most potentially or proven pathogenic mutations were located in the functional domains of the Lrrk2 protein. CONCLUSION: This study leads us to conclude that LRRK2 mutations are a common cause of autosomal dominant Parkinson's disease in Europe and North Africa.
ISSN: 0022-2593

inserm-00529272
http://www.hal.inserm.fr/inserm-00529272
DOI : 10.1136/jmg.2008.062612
PUBMED : 19357115

Éditeur(s) : HAL CCSD BioMed Central
Résumé : International audience
Background: The Hippo pathway regulates growth rate and organ size in fly and mouse, notably through control of cell proliferation. Molecular interactions at the heart of this pathway are known to have originated in the unicellular ancestry of metazoans. They notably involve a cascade of phosphorylations triggered by the kinase Hippo, with sub‑ sequent nuclear to cytoplasmic shift of Yorkie localisation, preventing its binding to the transcription factor Scalloped, thereby silencing proliferation genes. There are few comparative expression data of Hippo pathway genes in non‑ model animal species and notably none in non‑bilaterian phyla.Results: All core Hippo pathway genes could be retrieved from the ctenophore Pleurobrachia pileus and the hydro‑ zoan cnidarian Clytia hemisphaerica, with the important exception of Yorkie in ctenophore. Expression study of the Hippo, Salvador and Scalloped genes in tentacle " cellular conveyor belts " of these two organisms revealed striking differences. In P. pileus, their transcripts were detected in areas where undifferentiated progenitors intensely prolifer‑ ate and where expression of cyclins B and D was also seen. In C. hemisphaerica, these three genes and Yorkie are expressed not only in the proliferating but also in the differentiation zone of the tentacle bulb and in mature tentacle cells. However, using an antibody designed against the C. hemiphaerica Yorkie protein, we show in two distinct cell lineages of the medusa that Yorkie localisation is predominantly nuclear in areas of active cell proliferation and mainly cytoplasmic elsewhere.Conclusions: This is the first evidence of nucleocytoplasmic Yorkie shift in association with the arrest of cell prolifera‑ tion in a cnidarian, strongly evoking the cell division‑promoting role of this protein and its inhibition by the activated Hippo pathway in bilaterian models. Our results furthermore highlight important differences in terms of deployment and regulation of Hippo pathway genes between cnidarians and ctenophores.
ISSN: 2041-9139

Droits : http://creativecommons.org/licenses/by/
hal-01286578
http://hal.upmc.fr/hal-01286578
http://hal.upmc.fr/hal-01286578/document
http://hal.upmc.fr/hal-01286578/file/comparative.pdf
DOI : 10.1186/s13227-016-0041-y

Éditeur(s) : HAL CCSD Royal Society
Résumé : International audience
Maternal Embryonic Leucine zipper Kinase (MELK) was recently shown to be involved in cell division of Xenopus embryo epithelial cells. The cytokinetic furrow of these cells ingresses asymmetrically and is developmentally regulated. Two subpopulations of xMELK, the mMELK (for "mitotic" xMELK) and iMELK ("interphase" xMELK), which differ in their spatial and temporal regulation, are detected in Xenopus embryo. How cells regulate these two xMELK populations is unknown. In this study we show that, in epithelial cells, xMELK is present at a higher concentration at the apical junctional complex, in contrast to mesenchyme-like cells, which have uniform distribution of cortical MELK. Interestingly, mMELK and iMELK also differ by their requirements towards cell-cell contacts to establish their proper cortical localization both in epithelial and mesenchyme-like cells. Receptor for Activated protein Kinase C (RACK1), which we identified as an xMELK partner, co-localizes with xMELK at the tight junction. Moreover, a truncated RACK1 construct interferes with iMELK localization at cell-cell contacts. Collectively, our results suggest that iMELK and RACK1 are present in the same complex and that RACK1 is involved in the specific recruitment of iMELK at the apical junctional complex in epithelial cells of Xenopus embryos.
ISSN: 2046-6390

hal-00874053
https://hal.archives-ouvertes.fr/hal-00874053
https://hal.archives-ouvertes.fr/hal-00874053/document
https://hal.archives-ouvertes.fr/hal-00874053/file/Cell-cycle_dependent-accepted.pdf
DOI : 10.1242/bio.20136080

Éditeur(s) : HAL CCSD
Résumé : International audience
Viral replication in the liver is generally detected by cellular endosomal Toll-like receptors (TLRs) and cytosolic helicase sensors that trigger antiviral inflammatory responses. Recent evidence suggests that surface TLR2 may also contribute to viral detection through recognition of viral coat proteins but its role in the outcome of acute viral infection remains elusive. In this study, we examined in vivo the role of TLR2 in acute infections induced by the highly hepatotrophic mouse hepatitis virus (MHV) type 3 and weakly hepatotrophic MHV-A59 serotype. To address this, C57BL/6 (wild-type; WT) and TLR2 knockout (KO) groups of mice were intraperitoneally infected with MHV3 or MHV-A59. MHV3 infection provoked a fulminant hepatitis in WT mice, characterized by early mortality and high alanine and aspartate transaminase levels, histopathological lesions and viral replication whereas infection of TLR2 KO mice was markedly less severe. MHV-A59 provoked a comparable mild and subclinical hepatitis in WT and TLR2 KO mice. MHV3-induced fulminant hepatitis in WT mice correlated with higher hepatic expression of interferon-β, interleukin-6, tumour necrosis factor-α, CXCL1, CCL2, CXCL10 and alarmin (interleukin-33) than in MHV-A59-infected WT mice and in MHV3-infected TLR2 KO mice. Intrahepatic recruited neutrophils, natural killer cells, natural killer T cells or macrophages rapidly decreased in MHV3-infected WT mice whereas they were sustained in MHV-A59-infected WT mice and MHV3-infected TLR2 KO. MHV3 in vitro infection of macrophagic cells induced rapid and higher viral replication and/or interleukin-6 induction in comparison to MHV-A59, and depended on viral activation of TLR2 and p38 mitogen-activated protein kinase. Taken together, these results support a new aggravating inflammatory role for TLR2 in MHV3-induced acute fulminant hepatitis. © 2016 John Wiley & Sons Ltd
Immunology

Droits : info:eu-repo/semantics/OpenAccess
hal-01373946
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01373946
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01373946/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01373946/file/Toll-like%20receptor%20%28tlr%29-2%20exacerbates%20murine%20acute%20viral%20hepatitis.pdf
DOI : 10.1111/imm.12627

Éditeur(s) : HAL CCSD Elsevier
Résumé : Thanks to Dr M. Blanchard-Desce (UMR CNRS 5255, University of Bordeaux, France), who kindly provided us with fluorescent dye RS19 and the PIXEL, MRIc, flow cytometry and H2P2 platforms (SFR Biosit, UMS CNRS 3480/US INSERM 018, Rennes).
International audience
[1-9-NαC]-crourorb A1 is a cyclic peptide isolated from Croton urucurana Baillon latex, found in midwestern Brazil, that has been shown to exert cytotoxic effects against a panel of cancer cell lines. However, the underlying mechanisms responsible for the crourorb A1-induced cytotoxicity in cancer cells remain unknown. In this study, the effects of crourorb A1 on the viability, apoptosis, cell cycle and migration of Huh-7 (human hepatocarcinoma) cells were investigated. We evaluated the viability of Huh-7 cells treated with crourorb A1 in 2D and 3D collagen cultures and found that cells in 3D culture exhibited increased resistance to crourorb A1 compared to cells in 2D culture (IC50: 62μg/ml versus 35.75μg/ml). Crourorb A1 treatment decreases the viability of Huh-7 cells in a dose- and time-dependent manner and is associated with the induction of apoptosis, in the absence of necrotic cells, through the activation of caspase-3/7 and increased expression of the pro-apoptotic proteins Bak, Bid, Bax, Puma, Bim, and Bad. The effects of crourorb A1 are also associated with G2/M phase cell cycle arrest and increases in cyclin-dependent kinase (CDK1) and cyclin B1 expression. A significant reduction in Huh-7 cell migration induced by crourorb A1 was also observed in the presence of mitomycin C. Finally, we showed that the JNK/MAP pathway, but not ERK signaling, is involved in crourorb A1-induced hepatocarcinoma cell mortality.
ISSN: 0378-4274

hal-01526429
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01526429
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01526429/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01526429/file/de%20Matos%20C%C3%A2ndido-Bacani%20et%20al.%20-%20%5B1%E2%80%939-N%CE%B1C%5D-crourorb%20A1%20isolated%20from%20Croton%20urucura.pdf
DOI : 10.1016/j.toxlet.2017.03.020