Physiopathologie du coeur embryonnaire soumis à l'anoxie-réoxygénation: rôle protecteur du NO et des canaux KATP

RESUME Introduction : Dans le coeur adulte, l'ischémie et la reperfusion entraînent des perturbations électriques, mécaniques, biochimiques et structurales qui peuvent causer des dommages réversibles ou irréversibles selon la sévérité de l'ischémie. Malgré les récents progrès en cardiologie et en chirurgie foetales, la connaissance des mécanismes impliqués dans la réponse du myocarde embryonnaire à un stress hypoxique transitoire demeure lacunaire. Le but de ce travail a donc été de caractériser les effets chrono-, dromo- et inotropes de l'anoxie et de la réoxygénation sur un modèle de coeur embryonnaire isolé. D'autre part, les effets du monoxyde d'azote (NO) et de la modulation des canaux KATP mitochondriaux (mito KATP) sur la récupération fonctionnelle postanoxique ont été étudiés. La production myocardique de radicaux d'oxygène (ROS) et l'activité de MAP Kinases (ERK et JNK) impliquées dans la signalisation cellulaire ont également été déterminées. Méthodes : Des coeurs d'embryons de poulet âgés de 4 jours battant spontanément ont été placés dans une chambre de culture puis soumis à une anoxie de 30 min suivie d'une réoxygénation de 60 min. L'activité électrique (ECG), les contractions de l'oreillette, du ventricule et du conotroncus (détectées par photométrie), la production de ROS (mesure de la fluorescence du DCFH) et l'activité kinase de ERK et JNK dans le ventricule ont été déterminées au cours de l'anoxie et de la réoxygénation. Les coeurs ont été traités avec un bloqueur des NO synthases (L-NAME), un donneur de NO (DETA-NONOate), un activateur (diazoxide) ou un inhibiteur (5-HD) des canaux mitoKATP un inhibiteur non-spécifique des PKC (chélérythrine) ou un piégeur de ROS (MPG). Résultats : L'anoxie et la réoxygénation entraînaient des arythmies (essentiellement d'origine auriculaire) semblables à celles observées chez l'adulte, des troubles de la conduction (blocs auriculo-ventriculaires de 1er, 2ème et 3ème degré) et un ralentissement marqué du couplage excitation-contraction (E-C) ventriculaire. En plus de ces arythmies, la réoxygénation déclenchait le phénomène de Wenckelbach, de rares échappements ventriculaires et une sidération myocardique. Aucune fibrillation, conduction rétrograde ou activité ectopique n'ont été observées. Le NO exogène améliorait la récupération postanoxique du couplage E-C ventriculaire alors que L'inhibition des NOS la ralentissait. L'activation des canaux mito KATP augmentait la production mitochondriale de ROS à la réoxygénation et accélérait la récupération de la conduction (intervalle PR) et du couplage E-C ventriculaire. La protection de ce couplage était abolie par le MPG, la chélérythrine ou le L-NAME. Les fonctions électrique et contractile de tous les coeurs récupéraient après 30-40 min de réoxygénation. L'activité de ERK et de JNK n'était pas modifiée par L'anoxie, mais doublait et quadruplait, respectivement, après 30 min de réoxygénation. Seule l'activité de JNK était diminuée (-60%) par l'activation des canaux mitoKATP. Cet effet inhibiteur était partiellement abolit par le 5-HD. Conclusion: Dans le coeur immature, le couplage E-C ventriculaire semble être un paramètre particulièrement sensible aux conditions d'oxygénation. Sa récupération postanoxique est améliorée par l'ouverture des canaux mitoKATP via une signalisation impliquant les ROS Ies PKC et le NO. Une réduction de l'activité de JNK semble également participer à cette protection. Nos résultats suggèrent que les mitochondries jouent un rôle central dans la modulation des voies de signalisation cellulaire, en particulier lorsque les conditions métaboliques deviennent défavorables. Le coeur embryonnaire isolé représente donc un modèle expérimental utile pour mieux comprendre les mécanismes associés à une hypoxie in utero et pour améliorer les stratégies thérapeutiques en cardiologie et chirurgie foetales. ABSTRACT Physiopathology of the anoxic-reoxygenated embryonic heart: Protective role of NO and KATP channel Aim: In the adult heart, the electrical, mechanical, biochemical and structural disturbances induced by ischemia and reperfusion lead to reversible or irreversible damages depending on the severity and duration of ischemia. In spite of recent advances in fetal cardiology and surgery, little is known regarding the cellular mechanisms involved in hypoxia-induced dysfunction in the developing heart. The aim of this study was to precisely characterize the chrono-, dromo- and inotropic disturbances associated with anoxia-reoxygenation in an embryonic heart model. Furthermore, the roles that nitric oxide (NO), reactive oxygen species (ROS), mitochondrial KATP, (mito KATP) channel and MAP Kinases could play in the stressed developing heart have been investigated. Methods: Embryonic chick hearts (4-day-old) were isolated and submitted in vitro to 30 min anoxia followed by 60 min reoxygenation. Electrical (ECG) and contractile activities of atria, ventricle and conotruncus (photometric detection), ROS production (DCFH fluorescence) and ERK and JNK activity were determined in the ventricle throughout anoxia-reoxygenation. Hearts were treated with NO synthase inhibitor (L-NAME), NO donor (DETA-NONOate), mitoKATP channel opener (diazoxide) or blocket (5-HD), PKC inhibitor (chelerythrine) and ROS scavenger (MPG). Results: Anoxia and reoxygenation provoked arrhythxnias (mainly originating from atrial region), troubles of conduction (st, 2nd, and 3rd degree atrio-ventricular blocks) and disturbances of excitation-contraction (E-C) coupling. In addition to these types of arrhythmias, reoxygenation triggered Wenckebach phenomenon and rare ventricular escape beats. No fibrillations, no ventricular ectopic beats and no electromechanical dissociation were observed. Myocardial stunning was observed during the first 30 min of reoxygenation. All hearts fully recovered their electrical and mechanical functions after 30-40 min of reoxygenation. Exogenous NO improved while NOS inhibition delayed E-C coupling recovery. Mito KATP, channel opening increased reoxygenation-induced ROS production and improved E-C coupling and conduction (PR) recovery. MPG, chelerythrine or L-NAME reversed this effect. Reoxygenation increased ERK and JNK activities land 4-fold, respectively, while anoxia had no effect. MitoKATP channel opening abolished the reoxygenation-induced activation of JNK but had no effect on ERK activity. This inhibitory effect was partly reversed by mitoKATP channel blocker but not by MPG. Conclusion: In the developing heart, ventricular E-C coupling was found to be specially sensitive to hypoxia-reoxygenation and its postanoxic recovery was improved by mitoKATP channel activation via a ROS-, PKC- and NO-dependent pathway. JNK inhibition appears to be involved in this protection. Thus, mitochondria can play a pivotal role in the cellular signalling pathways, notably under critical metabolic conditions. The model of isolated embryonic heart appears to be useful to better understand the mechanisms underlying the myocardial dysfunction induced by an in utero hypoxia and to improve therapeutic strategies in fetal cardiology and surgery.

Interaction of Leukocyte Elastase Inhibitor/L-DNase II with BCL-2 and BAX

Leukocyte Elastase Inhibitor (LEI, also called serpin B1) is a protein involved in apoptosis among other physiological processes. We have previously shown that upon cleavage by its cognate protease, LEI is transformed into L-DNase II, a protein with a pro-apoptotic activity. The caspase independent apoptotic pathway, in which L-DNase II is the final effector, interacts with other pro-apoptotic molecules like Poly-ADP-Ribose polymerase (PARP) or Apoptosis Inducing Factor (AIF). The screening of LEI/L-DNase II interactions showed a possible interaction with several members of the BCL-2 family of proteins which are known to have a central role in the regulation of caspase dependent cell death. In this study, we investigated the regulation of LEI/L-DNase II pathway by two members of this family of proteins: BAX and BCL-2, which have opposite effects on cell survival. We show that, in both BHK and HeLa cells, LEI/L-DNase II can interact with BCL-2 and BAX in apoptotic and non-apoptotic conditions. These proteins which are usually thought to be anti-apoptotic and pro-apoptotic respectively, both inhibit the L-DNase II pro-apoptotic activity. These results give further insight in the regulation of caspase-independent pathways and highlight the involvement of the intracellular environment of a given protein in the determinism of its function. They also add a link between caspase-dependent and independent pathways of apoptosis.

Study of drug tolerance mechanisms and of the calcineurin pathway in the opportunistic pathogen "Candida albicans"

ABSTRACT :Azole antifungal drugs possess fungistatic activity in Candida albicans making this human pathogen tolerant to these agents. The conversion of azoles into fungicidal agents is of interest since their fungistatic properties increase the ability of C. albicans to develop drug resistance. In C. albicans, the phosphatase calcineurin (calcineurin) is essential for antifungal drug tolerance. Up to now, the only known target of calcineurin is Crzl, which is a transcription factor (TF) involved in responses to ionic stress. Thus, most of the components of the calcineurin signaling remain to be identified in C. albicans.In this work, the calcineurin pathway was investigated in order to i) characterize the role of calcineurin in the biology of C. albicans, ii) identify putative targets of calcineurin and iii) characterize the phenomenon of tolerance to antifungal drugs. Towards these aims, four different approaches were used.First, using C. albicans microarrays, an attempt was made to identify a set of calcineurindependent genes (CDGs). Since CDGs were highly dependent upon the external stimulus used to activate calcineurin (Ca2+ or terbinafine), this stimulus bias was bypassed by the construction of strains expressing a truncated autoactive form of calcineurin (Cmp1tr) in a doxycyclinedependent manner. The characterization of Cmpltr was undertaken and results showed that it mimicked awild-type activated calcineurin for all tested phenotypes (i.e. Cnbl-dependence, inhibition by FK506, phosphatase 2B activity, ability to dephosphorylate Crzl and to regulate Crz1-and calcineurin-dependent genes, role in antifungal drug tolerance and susceptibility, role in colony formation on Spider agar). Cmp1tr was therefore considered as a valid tool to study the calcineurin signaling pathway. In silico analysis of CDGs allowed the identification of i) a significant overlap between CDGs and genes regulated by the Cyrl signalíng pathway, ii) putative interactions between calcineurin activation and cell wall reorganization and phospholipid transport, iii) a putative interactión between calcineurin and the regulation of translation and iv) a putative relation between calcineurin and proteasome regulation. Further in silico analyses of the promoters of Crz1-independent CDGs were performed to identify TFs (other than Crz1) that were likely to regulate CDGs and therefore to be a direct target of calcineurin. The analyses revealed that Rpn4 and Mnl1 were TFs likely to be regulated by calcineurin.Second, in order to better characterize azole tolerance, an attempt was made to i) confirm the role of Hsp90 in fluconazole tolerance with a doxycycline-dependent Hsp90 expression system and ii) assess its calcineurin-dependence. Hsp90 was found to be significantly involved in fluconazole tolerance. However, results were not in agreement with the hypothesis that Hsp90 mediates fluconazole tolerance by the only downstream effector calcineurin. Rather Hsp90 is interacting with numerous components for fluconazole tolerance.Third, a collection of C. albicans TFs mutants were screened for loss of tolerance to terbinafine and fluconazole in order to identify TFs involved in antifungal drug tolerance. Out of the 265 TFs mutants screened, only the upc2Δ/Δ mutant showed a loss of fluconazole and terbinafine tolerance. Interestingly, no relation between Upc2 and calcineurin activity was found. These results suggested that the tolerance to antifungal drugs must not be only considered as a calcineurin-dependent phenomenon in C. albicans.Fourth, using FRCS analyses, an attempt was made to identify putative signs of programmed cell death (PCD) in calcineurin mutant cells upon loss of tolerance to terbinafine. A high proportion of cells died from both RO5-dependent (which is a sign of PCD) and ROS-independent (which is a sign of loss of homeostasis) processes in the calcineurin mutant. While these results suggest that calcineurin represses both loss of homeostasis and PCD, the role of calcineurin in PCD is still an open question.In conclusion, this work allowed i) the identification of several putative calcineurin targets, ii) the discovery of several links between calcineurin and signaling pathways and important biological processes and iii) the identification of novel components of calcineurin-independent mechanisms that participate in tolerance to antifungal drugs in C. albicans.RÉSUME :Les azoles sont des antifongiques qui présentent une activité fongistatique contre Candida albicans et rendent cette levure tolérante à ces agents. La conversion des azoles en agents fongicides est d'intérêts car leurs propriétés fongistatiques favorisent le développement de résistance aux drogues chez C. albicans. La calcineurine (calcineurin) est une phosphatase essentielle pour la tolérance aux antifongiques chez C. albicans. La seule cible connue de la calcineurin est Crz1, un facteur de transcription (FT) impliqué dans la réponse aux stress ionique. Ainsi, la plupart des constituants de la voie de signalisation de la calcineurin restent encore à être identifiés chez C. albicans.Dans ce travail de thèse, la voie de signalisation de la calcineurin a été étudiée de sorte à i) caractériser le rôle de la calcineurin dans la biologie de C. albicans, ii) identifier de nouvelles cibles de la calcineurin et iii) caractériser le phénomène de tolérance aux antifongiques. A ce propos, quatre approches ont été entreprises.Premièrement, des puces à ADN de C. albicans ont été utilisées afin d'identifier les gènes dépendants de la calcineurin (GDCs). Les GDCs étant étroitement dépendants du stimulus utilisé pour activer la calcineurin, le biais «stimulus» a été évité via la construction d'une souche exprimant une forme tronquée et autoactive de la calcineurin (Cmp1tr), en présence de doxycycline. La caractérisation de Cmp1tr a été entreprise et les résultats ont montré qu'elle mimait une calcineurin sauvage et activée pour la plupart des phénotypes testés (i.e. dépendance à Cnb1, inhibition par le FK506, activité phosphatase 2B, déphosphorylation de Crz1 et régulation de gènes dépendant de la calcineurin, rôle dans la tolérance et la susceptibilité aux antifongiques, rôle dans la formation des colonies sur milieu Spider). Cmp1tr a donc été considéré comme un outil pertinent pour l'étude de la voie de signalisation de la calcineurin. Les analyses in silico des GDCs ont permis l'identification i) d'un chevauchement entre les GDCs èt les gènes régulés par la voie de signalisation de Cyrl, ii) d'une interaction entre la calcineurin et la réorganisation de la paroi cellulaire ainsi que le transport des phospholipides, iii) d'une interaction entre calcineurin et la régulation de la traduction et iv) une relation entre la calcineurin et la régulation du protéasome. De plus, une analyse in silico des promoteurs des GDCs avec une régulation indépendante de Crz1 a permis d'identifier deux FTs qui pourraient être des cibles directes de la calcineurin, Rpn4 et Mnll.Deuxièmement, afin de caractériser la tolérance aux azoles, il a été entrepris i) de confirmer le rôle de Hsp90 dans la tolérance au fluconazole en utilisant un système d'expression dépendant de la doxycycline et ii) de caractériser sa dépendance à la calcineurin. Hsp90 a été montré impliqué dans la tolérance aux azoles. Cependant, les résultats n'ont pas corroboré une hypothèse expliquant le rôle d'Hsp90 dans la tolérance aux antifongiques par son unique. interaction avec la calcineurin. Il a été proposé que le rôle d'Hsp90 dans la tolérance aux antifongiques soit dû à ces multiples interactions avec le protéome de C. albicans plutôt que par son interaction avec un partenaire unique.Troisièmement, une collection de mutant pour des FTs de C. albicans a été criblée pour une perte de tolérance au fluconazole ou à la terbinafine, de sorte à identifier les FTs impliqués dans la tolérance aux antifongiques. Sur les 265 FTs passés au crible, seul le mutant upc2Δ/Δ a montré une perte de tolérance au fluconazole et à la terbinafine. Aucune relation n'a été trouvée entre la calcineurin et l'activité d'Upc2. Ces résultats suggèrent que la perte de tolérance aux antifongiques ne doit pas être considérée comme un phénomène exclusivement lié à la voie de signalisation de la calcineurin.Quatrièmement, en utilisant la cytométrie de flux, la présence de signes de mort cellulaire programmée (MCP) a été recherchée lors de la perte de tolérance du mutant calcineurin incubé avec de la terbinafine. Une grande proportion de cellules mortes incluant ou non une production de ROS (un signe de MCP) a été détectée dans le mutant calcineurin. Ces résultats préliminaires suggèrent que la calcineurin réprime autant la perte d'homéostasie qu'elle régule l'entrée en MCP. Cependant d'autres analyses sont nécessaires pour démontrer clairement le rôle de la calcineurin dans la régulation de la MCP.En conclusion, ce travail de thèse a permis i) l'identification de plusieurs cibles possibles de la calcineurine, ii) la découverte de plusieurs interactions entre la calcineurine et d'autres voies de signalisation et processus biologiques importants et iii) de démontrer la présence de voies indépendantes de la calcineurine impliquées dans la tolérance aux antifongiques chez C. albicans.

Jasmonate biochemical pathway.

Plants possess a family of potent fatty acid-derived wound-response and developmental regulators: the jasmonates. These compounds are derived from the tri-unsaturated fatty acids alpha-linolenic acid (18:3) and, in plants such as Arabidopsis thaliana and tomato, 7(Z)-, 10(Z)-, and 13(Z)-hexadecatrienoic acid (16:3). The lipoxygenase-catalyzed addition of molecular oxygen to alpha-linolenic acid initiates jasmonate synthesis by providing a 13-hydroperoxide substrate for formation of an unstable allene oxide by allene oxide synthase (AOS). This allene oxide then undergoes enzyme-guided cyclization to produce 12-oxophytodienoic acid (OPDA). These first steps take place in plastids, but further OPDA metabolism occurs in peroxisomes. OPDA has several fates, including esterification into plastid lipids and transformation into the 12-carbon prohormone jasmonic acid (JA). JA is itself a substrate for further diverse modifications, including the production of jasmonoyl-isoleucine (JA-Ile), which is a major biologically active jasmonate among a growing number of jasmonate derivatives. Each new jasmonate family member that is discovered provides another key to understanding the fine control of gene expression in immune responses; in the initiation and maintenance of long-distance signal transfer in response to wounding; in the regulation of fertility; and in the turnover, inactivation, and sequestration of jasmonates, among other processes.

Leishmania major metacaspase can replace yeast metacaspase in programmed cell death and has arginine-specific cysteine peptidase activity.

The human protozoan parasite Leishmania major has been shown to exhibit several morphological and biochemical features characteristic of a cell death program when differentiating into infectious stages and under a variety of stress conditions. Although some caspase-like peptidase activity has been reported in dying parasites, no caspase gene is present in the genome. However, a single metacaspase gene is present in L. major whose encoded protein harbors the predicted secondary structure and the catalytic dyad histidine/cysteine described for caspases and other metacaspases identified in plants and yeast. The Saccharomyces cerevisiae metacaspase YCA1 has been implicated in the death of aging cells, cells defective in some biological functions, and cells exposed to different environmental stresses. In this study, we describe the functional heterologous complementation of a S. cerevisiae yca1 null mutant with the L. major metacaspase (LmjMCA) in cell death induced by oxidative stress. We show that LmjMCA is involved in yeast cell death, similar to YCA1, and that this function depends on its catalytic activity. LmjMCA was found to be auto-processed as occurs for caspases, however LmjMCA did not exhibit any activity with caspase substrates. In contrast and similarly to Arabidopsis thaliana metacaspases, LmjMCA was active towards substrates with arginine in the P1 position, with the activity being abolished following H147A and C202A catalytic site mutations. These results suggest that metacaspases are members of a family of peptidases with a role in cell death conserved in evolution notwithstanding possible differences in their catalytic activity.

Fas-associated death domain protein (FADD) and caspase-8 mediate up-regulation of c-Fos by Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via a FLICE inhibitory protein (FLIP)-regulated pathway.

Fas, a death domain-containing member of the tumor necrosis factor receptor family and its ligand FasL have been predominantly studied with respect to their capability to induce cell death. However, a few studies indicate a proliferation-inducing signaling activity of these molecules too. We describe here a novel signaling pathway of FasL and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that triggers transcriptional activation of the proto-oncogene c-fos, a typical target gene of mitogenic pathways. FasL- and TRAIL-mediated up-regulation of c-Fos was completely dependent on the presence of Fas-associated death domain protein (FADD) and caspase-8, but caspase activity seemed to be dispensable as a pan inhibitor of caspases had no inhibitory effect. Upon overexpression of the long splice form of cellular FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein (cFLIP) in Jurkat cells, FasL- and TRAIL-induced up-regulation of c-Fos was almost completely blocked. The short splice form of FLIP, however, showed a rather stimulatory effect on c-Fos induction. Together these data demonstrate the existence of a death receptor-induced, FADD- and caspase-8-dependent pathway leading to c-Fos induction that is inhibited by the long splice form FLIP-L.

Constant p53 pathway inactivation in a large series of soft tissue sarcomas with complex genetics.

Alterations of the p53 pathway are among the most frequent aberrations observed in human cancers. We have performed an exhaustive analysis of TP53, p14, p15, and p16 status in a large series of 143 soft tissue sarcomas, rare tumors accounting for around 1% of all adult cancers, with complex genetics. For this purpose, we performed genomic studies, combining sequencing, copy number assessment, and expression analyses. TP53 mutations and deletions are more frequent in leiomyosarcomas than in undifferentiated pleomorphic sarcomas. Moreover, 50% of leiomyosarcomas present TP53 biallelic inactivation, whereas most undifferentiated pleomorphic sarcomas retain one wild-type TP53 allele (87.2%). The spectrum of mutations between these two groups of sarcomas is different, particularly with a higher rate of complex mutations in undifferentiated pleomorphic sarcomas. Most tumors without TP53 alteration exhibit a deletion of p14 and/or lack of mRNA expression, suggesting that p14 loss could be an alternative genotype for direct TP53 inactivation. Nevertheless, the fact that even in tumors altered for TP53, we could not detect p14 protein suggests that other p14 functions, independent of p53, could be implicated in sarcoma oncogenesis. In addition, both p15 and p16 are frequently codeleted or transcriptionally co-inhibited with p14, essentially in tumors with two wild-type TP53 alleles. Conversely, in TP53-altered tumors, p15 and p16 are well expressed, a feature not incompatible with an oncogenic process.

Identification, using a siRNA screen approach, and molecular characterization of a new Fas pathway modulator: STK11/

Abstract : Apoptosis is an evolutionarily conserved cellular suicide mechanism that can be triggered by activation of various pathways, such as the Fas-Pathway. Upon stimulation by its specific ligand (FasL), present at the surface of Cytotoxic Τ lymphocytes, the death receptor Fas initiates a signaling cascade culminating in the activation of cellular caspases, leading thus to cell death of the target cell (e.g. transformed cell). Dysregulation of apoptosis in general, and of Fas pathway in particular, was shown to contribute to pathogenesis of cancers and many human diseases. Even though, during the last decades the molecular mechanisms of apoptosis have been widely studied, it is important to better understand the mechanisms leading to apoptosis, to improve our understanding of pathological processes, and generate more subtle apoptosis-modulating therapies to fight cancer and other diseases. In order to identify new components of the Fas signaling pathway, a screen based on the mechanism of RNA interference was undertaken. After a first and a second manual whole-kinome screen, we identified several strong positive hits that showed a protection against Fas ligand-induced apoptosis with distinct siRNAs, notably STK11, an interesting tumor suppressor mutated in several sporadic and inherited cancers. The STK11 functional characterization reveals that this kinase represents an apically acting general pro-apoptotic modulator of the extrinsic pathway (FasL, TRAIL, TNF-induced apoptosis), but not of the intrinsic apoptotic pathway. The STK11 action on the Fas pathway was shown to be dependent on its kinase activity, but independent of AMPK, a well-characterized STK11 downstream substrate. Furthermore, STK11 was shown to interact with caspase-8, a major mediator of the extrinsic pathway, and modulate its activity through an unclear mechanism that may involve an STK11-dependant caspase-8 phosphorylation. This modification may allow a proper caspase-8 polyubiquitination and activation in p62 sequestosmes aggregates, but may also increase the activation of caspase-8 at the DISC level. In addition, we observed that STK11 modulate not only the apoptotic pathway induced by Fas engagement, but also FasL-induced JNK and NF- KB, sustaining an upstream role of this kinase in the pathway. In conclusion, our report reveals that STK11 is an important pro-apoptotic modulator of the Fas pathway in particular, and extrinsic pathway in general. Our finding could explain, at least partially, why inactivating mutations of the kinase leads to cancer, by allowing resistance to apoptosis and accordingly evasion of immune surveillance. Résumé : L'apoptose est un mécanisme de suicide cellulaire, conservé dans diverses espèces, et qui au niveau moléculaire est déclenché par différentes voies de signalisation, comme par exemple lors de l'activation du récepteur Fas. La liaison du ligand FasL au récepteur de la mort Fas, induit une cascade de signalisation qui conduit à l'activation des caspases. Les lymphocytes Τ cytotoxiques peuvent utiliser la voie Fas pour induire la mort et se débarrasser de cellules dangereuses pour le reste de l'organisme, tel que les cellules transformées. La dysrégulation de l'apoptose en général, et de la voie Fas en particulier, peut contribuer à diverses maladies telles que le cancer. Même si ces dernières décennies, les mécanismes moléculaires conduisant à l'apoptose ont été extensivement étudiés, il reste néanmoins important de mieux comprendre le phénomène d'apoptose, pour améliorer notre compréhension des processus pathologiques, mais surtout dans le but de développer de nouvelles thérapies ciblant l'apoptose contre le cancer et d'autres pathologies. Pour identifier de nouveau constituants de la voie Fas, un criblage génétique basé sur l'interférence à l'ARN a été entrepris. Après un premier et un deuxième criblage d'une librairie du kinome, nous avons identifié différentes protéines qui pourraient jouer un rôle positif dans la voie Fas, et en particulier la protéine suppresseur de tumeur STK11, qui est fréquemment mutée dans divers cancers sporadiques et héréditaires. La caractérisation fonctionnelle de STK11 a révélé que cette kinase était un modulateur apical de la voie extrinsèque de l'apoptose en général (Fas, TNF, TRAIL), mais pas de la voie intrinsèque. L'action de STK11 sur la voie Fas est dépendante de sa fonction kinase, mais indépendante de l'AMPK, un substrat bien caractérisé de STK11. De plus, STK11 interagît avec la caspase-8, un constituant majeur de la voie Fas, et module son activité, par un mécanisme encore peu clair qui pourrait impliquer une phosphorylation de la caspase-8 par STK11. Cette modification pourrait permettre une activation optimale de la caspase-8 en jouant un rôle dans le processus de polyubiquitination de la caspase-8, phénomène qui semble être important pour l'activation de la caspase-8 dans des agrégats protéiques avec p62, mais qui pourrait aussi augmenter son activation au niveau du DISC. Finalement, nous avons observé que STK11 modulait non seulement la voie apoptotique déclenchée par l'activation de Fas, mais aussi les voies non-apoptotiques de Fas, comme JNK et NF-KB. En conclusion notre étude, révèle que STK11 est un important modulateur pro- apoptotique de la voie Fas, et de la voie extrinsèque en général. Cette découverte pourrait expliquer, du moins partiellement, pourquoi les mutations inactivatrices de STK11 conduisent au cancer, par une augmentation de la résistance à l'apoptose et donc par l'évasion de la surveillance immunitaire.

Association of NTRK3 and its interaction with NGF suggest an altered cross-regulation of the neurotrophin signaling pathway in eating disorders

Eating disorders (EDs) are complex psychiatric diseases that include anorexia nervosa and bulimia nervosa, and have higher than 50% heritability. Previous studies have found association of BDNF and NTRK2 to ED, while animal models suggest that other neurotrophin genes might also be involved in eating behavior. We have performed a family-based association study with 151 TagSNPs covering 10 neurotrophin signaling genes: NGFB, BDNF, NTRK1, NGFR/p75, NTF4/5, NTRK2, NTF3, NTRK3, CNTF and CNTFR in 371 ED trios of Spanish, French and German origin. Besides several nominal associations, we found a strong significant association after correcting for multiple testing (P = 1.04 × 10−4) between ED and rs7180942, located in the NTRK3 gene, which followed an overdominant model of inheritance. Interestingly, HapMap unrelated individuals carrying the rs7180942 risk genotypes for ED showed higher levels of expression of NTRK3 in lymphoblastoid cell lines. Furthermore, higher expression of the orthologous murine Ntrk3 gene was also detected in the hypothalamus of the anx/anx mouse model of anorexia. Finally, variants in NGFB gene appear to modify the risk conferred by the NTRK3 rs7180942 risk genotypes (P = 4.0 × 10−5) showing a synergistic epistatic interaction. The reported data, in addition to the previous reported findings for BDNF and NTRK2, point neurotrophin signaling genes as key regulators of eating behavior and their altered cross-regulation as susceptibility factors for EDs.

Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule.

Cell death is achieved by two fundamentally different mechanisms: apoptosis and necrosis. Apoptosis is dependent on caspase activation, whereas the caspase-independent necrotic signaling pathway remains largely uncharacterized. We show here that Fas kills activated primary T cells efficiently in the absence of active caspases, which results in necrotic morphological changes and late mitochondrial damage but no cytochrome c release. This Fas ligand-induced caspase-independent death is absent in T cells that are deficient in either Fas-associated death domain (FADD) or receptor-interacting protein (RIP). RIP is also required for necrotic death induced by tumor necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL). In contrast to its role in nuclear factor kappa B activation, RIP requires its own kinase activity for death signaling. Thus, Fas, TRAIL and TNF receptors can initiate cell death by two alternative pathways, one relying on caspase-8 and the other dependent on the kinase RIP.

Regulation of the NaCI cotransporter by the SGK1-Nedd4-2 pathway

SummaryRegulation of renal Na+ transport is essential for controlling blood pressure, as well as Na+ and K+ homeostasis. Aldosterone stimulates Na+ reabsorption in the aldosterone-sensitive distal nephron (ASDN), via the Na+-CI" cotransporter (NCC) in the distal convoluted tubule (DCT), and the epithelial Na+ channel (ENaC) in the late DCT, connecting tubule and collecting duct. Importantly, aldosterone increases NCC protein expression by an unknown post-translational mechanism. The ubiquitin-protein ligase Nedd4-2 is expressed along the ASDN and regulates ENaC: under aldosterone induction, the serum/glucocorticoid-regulated kinase SGK1 phosphorylates Nedd4-2 on S328, thus preventing the Nedd4-2/ENaC interaction, ubiquitylation and degradation of the channel. Here, we present evidence that Nedd4-2 regulates NCC. In transfected HEK293 cells, Nedd4-2 co-immunoprecipitates with NCC and stimulates NCC ubiquitylation at the cell surface. In Xenopus laevis oocytes, co- expression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreases NCC activity and surface expression. This inhibition is prevented by SGK1 in a kinase-dependent manner. Moreover, we show that NCC expression is up-regulated in inducible renal tubule-specific Nedd4-2 knockout mice and in mDCT15 cells silenced for Nedd4-2. On the other hand, in inducible renal tubule-specific SGK1 knockout mice, NCC expression is down-regulated.Interestingly, in contrast to ENaC, Nedd4-2-mediated NCC inhibition is independent of a PY motif in NCC. Moreover, whereas single mutations of Nedd4-2 S328 or S222 to alanine do not interfere with SGK1 action, the double mutation enhances Nedd4-2 activity and abolishes SGK1-dependent inhibition. These results indicate that NCC expression and activity is controlled by a regulatory pathway involving SGK1 and Nedd4-2, and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.RésuméLa régulation du transport de sodium est cruciale dans le maintien de la pression artérielle. L'aldostérone stimule la réabsorption de Na+ dans la partie du néphron sensible à l'aldostérone (ASDN), via le co-transporteur Na+-CI" (NCC) au niveau du tubule contourné distale et via le canal à sodium (Epithelial Na+ Channel ; ENaC) dans la deuxième partie du tubule contourné distale, dans le tube connecteur et le tube collecteur. L'aldostérone augmente l'expression de NCC au niveau protéique par un mécanisme non élucidé. La protéine ubiquitine ligase Nedd4-2 est exprimée tout le long du néphron sensible à l'aldostérone. ENaC est connu pour être régulé par Nedd4-2. Suite à une stimulation par l'aldostérone, la kinase Ser/Thr SGK1 phosphoryle Nedd4-2, ce qui empêche l'interaction entre Nedd4-2 et ENaC. Dans des cellules HEK293 transfectées, nous avons montré que Nedd4-2 interagit avec le co-transporteur NCC et stimule l'ubiquitylation de NCC à la surface. Nous avons montré dans les oocytes de Xenopus laevis que l'expression de NCC avec Nedd4-2 diminue l'activité du co-transporteur. Cette diminution n'est pas observée lorsqu'on exprime NCC avec le mutant inactif de Nedd4-2. Cette inhibition de NCC est contrée par SGK1. L'effet de SGK1 sur NCC dépend de son activité kinase. Nous avons montré dans des souris knock-out pour Nedd4-2, dans le néphron et de manière inductible, que l'expression de NCC est augmentée. Nous avons également montré que la suppression de la protéine Nedd4-2 dans les cellules mDCT15 provoque l'augmentation de NCC. Au contraire dans les souris knock-out pour la kinase SGK1, dans le néphron et de manière inductible, nous observons une diminution de la protéine NCC. Contrairement à ce qui a été montré pour le canal ENaC l'inhibition de NCC par Nedd4-2 est indépendante des motifs PY. De plus, La mutation des sérines 328 ou 222 sur Nedd4-2 en alanine n'interfère pas avec l'action de SGK1 pour prévenir l'inhibition. Par contre, la double mutation, les sérines 222 et 328 mutées en alanine, augmente l'action de Nedd4-2 sur l'activité de NCC et prévient l'effet de SGK1. Ces résultats montrent que l'expression et l'activité de NCC sont contrôlées par une voie de régulation impliquant Nedd4-2-SGK1 et nous fournissent une explication pour l'augmentation de NCC observé après une induction avec l'aldostérone.Résumé large publicOn estime que des millions de personnes seraient hypertendues. L'hypertension artérielle est responsable d'environ 8 millions de décès par ans dans le monde. L'hypertension est responsable de la moitié environs des accidents cardiaques, mais aussi des accidents vasculaires cérébraux. Il est très important de comprendre les mécanismes qui se trouvent derrière cette pathologie.Le co-transporteur NCC joue un grand rôle dans le maintien de la balance sodique. Il a été montré que des perturbations dans l'expression de NCC pouvaient engendrer de l'hypertension.Le co-transporteur NCC est exprimé dans la partie distale du néphron, l'unité fonctionnelle du rein. Plusieurs études ont montrées que NCC était sous le contrôle de l'hormone aldostérone.Le travail de cette thèse consiste à étudier les mécanismes impliqués dans la régulation de NCC. On a ainsi pu montrer que NCC interagit avec la protéine ubiquitine ligase Nedd4-2. La protéine Nedd4-2 diminue l'expression de NCC à la surface cellulaire et aussi son activité Nous avons également montré que la kinase SGK1 pouvait prévenir l'interaction entre Nedd4-2 et NCC par phosphorylation de Nedd4-2. Nous avons montré dans des souris deletée pour Nedd4-2, dans le néphron, que l'expression de NCC est augmentée. Nous avons également montré que la suppression de la protéine Nedd4-2 dans les cellules mDCT15 provoque l'augmentation de NCC. Au contraire, dans les souris deletée pour la kinase SGK1, dans le néphron, nous observons une diminution de la protéine NCC. La connaissance des processus impliqués dans la régulation du co-transporteur NCC pourrait amener au développement de nouveau médicaments pour soigner l'hypertension.

Anorectal Malformations: Finding the Pathway out of the Labyrinth.

Anorectal malformations (ARMs) are a complex group of congenital anomalies involving the distal anus and rectum, as well as the urinary and genital tracts in a significant number of cases. Most ARMs result from abnormal development of the urorectal septum in early fetal life. In most cases, the anus is not perforated and the distal enteric component ends blindly (atresia) or as a fistula into the urinary tract, genital tract, or perineum. ARMs are also present in a great number of syndromes and associations of congenital anomalies. The classification of ARMs is mainly based on the position of the rectal pouch relative to the puborectal sling, the presence or absence of fistulas, and the types and locations of the fistulas. All of this information is crucial in determining the most appropriate surgical approach for each case. Imaging studies play a key role in evaluation and classification of ARMs. In neonates, clinical and radiologic examinations in the first 3 days of life help determine the type of ARM and the need for early colostomy. In older children, preoperative pelvic magnetic resonance imaging is the most efficient diagnostic method for evaluating the size, morphology, and grade of development of the sphincteric musculature.

Retinal ischemia-induced apoptosis is associated with alteration in Bax and Bcl-x(L) expression rather than modifications in Bak and Bcl-2.

PURPOSE: Apoptosis is known to play a key role in cell death after retinal ischemia. However, little is known about the kinetics of the signaling pathways involved and their contribution to this process. The aim of this study was to determine whether changes in the expression of molecules in the mitochondrial apoptotic pathway might explain the progression of retinal damage following ischemia/reperfusion. METHODS: Retinal ischemia was induced by elevating intraocular pressure in the vitreous cavity to 150 mmHg for a period of 60 min. At time 0, 3 h (early phase), and 24 h (late phase) after reperfusion, the retinas were harvested and modifications in the expression of Bax, Bak, Bcl-2, and Bcl-x(L) as well as caspase-3 and -7, were examined by qPCR and, in some cases, by western blot. RESULTS: qPCR analysis performed at the early phase after ischemia revealed a time dependent decrease in Bax, Bak, and Bcl-x(L) and no alteration in Bcl-2 mRNA expression in response to retinal ischemia. At the protein level, proapoptotic Bax and Bak were not modulated while Bcl-2 and Bcl-x(L) were significantly upregulated. At this stage, the Bax per Bcl-2 and Bax:Bcl-x(L) ratios were not modified. At the late phase of recovery, Bax and Bcl-x(L) mRNAs were downregulated while Bak was increased. Increased Bax:Bcl-2 and Bax:Bcl-x(L) ratios at both the mRNA and protein levels were observed 24 h after the ischemic insult. Analysis of caspases associated with mitochondria-mediated apoptosis revealed a specific increase in the expression of caspase-3 in the ischemic retinas 24 h after reperfusion, and a decrease in the expression of caspase-7. CONCLUSIONS: This study revealed that Bcl-2-related family members were differently regulated in the early and late phases after an ischemic insult. We showed that the Bax:Bcl-2 and Bax:Bcl-x(L) balances were not affected in the initial phases, but the Bax:Bcl-x(L) ratio shifted toward apoptosis during the late phase of recovery. This shift was reinforced by caspase-3 upregulation.

Rhumatologie. Une nouvelle option thérapeutique dans la prise en charge de l'inflammation chronique en rhumatologie: les inhibiteurs des janus kinases [A new therapeutical option for chronic inflammation in rheumatology: janus kinases inhibitors (JAK)].

In the last 15 years, the therapeutical options for the treatment of chronic inflammatory diseases in rheumatology have increased a lot. Nevertheless, some patients do not respond or respond partially to the current therapies--including to the biologics therapy. Tofacitinib (Xeljanz) is now on the Swiss market. It inhibits the JAK pathway. Tofacitinib--as monotherapy or with methotrexate--improves the control of rheumatoid arthritis (RA). In a comparative study, tofacitinib was as effective as adalimumab. Further, tofacitinib reduced structural damages in RA and is considered as an alternative, in case of non-response, to anti-TNF and probably to other biologics therapy. The side effects are upper respiratory tract and opportunist infections and tuberculosis. Blood count, lipids, kidney function, liver tests, CK and blood pressure have to be monitored.

The inositol Inpp5k 5-phosphatase affects osmoregulation through the vasopressin-aquaporin 2 pathway in the collecting system.

Inositol Inpp5k (or Pps, SKIP) is a member of the inositol polyphosphate 5-phosphatases family with a poorly characterized function in vivo. In this study, we explored the function of this inositol 5-phosphatase in mice and cells overexpressing the 42-kDa mouse Inpp5k protein. Inpp5k transgenic mice present defects in water metabolism characterized by a reduced plasma osmolality at baseline, a delayed urinary water excretion following a water load, and an increased acute response to vasopressin. These defects are associated with the expression of the Inpp5k transgene in renal collecting ducts and with alterations in the arginine vasopressin/aquaporin-2 signalling pathway in this tubular segment. Analysis in a mouse collecting duct mCCD cell line revealed that Inpp5k overexpression leads to increased expression of the arginine vasopressin receptor type 2 and increased cAMP response to arginine vasopressin, providing a basis for increased aquaporin-2 expression and plasma membrane localization with increased osmotically induced water transport. Altogether, our results indicate that Inpp5k 5-phosphatase is important for the control of the arginine vasopressin/aquaporin-2 signalling pathway and water transport in kidney collecting ducts.