Role of the epithelial sodium channel (ENaC) and its regulator CAP1/Prss8 in colon

Dans les cellules épithéliales sensibles à l'aldostérone, le canal sodique épithélial (ENaC) joue un rôle critique dans le contrôle de l'équilibre sodique, le volume sanguin, et la pression sanguine. Le rôle d'ENaC est bien caractérisé dans le rein et les poumons, cependant le rôle d'ENaC et son régulateur positif la protéase activatrice de canal 1 (CAP1 /Prss8) sur le transport sodique dans le côlon reste en grande partie inconnu. Nous avons étudié l'importance d'ENaC et de CAPMPrss8 dans le côlon. Les souris déficientes pour la sous- unité aENaC (souris ScnnlaKO) dans les cellules superficielles intestinales étaient viables et ne montraient pas de létalité embryonnaire ou postnatale. Sous diète normale (RS) ou pauvre en sodium (LS), la différence de potentiel rectale sensible à l'amiloride (APDamii) était drastiquement diminuée et son rythme circadien atténué. Sous diète normale (RS) ou diète riche en sodium (HS) ou fort chargement de potassium, le sodium et le potassium plasmatique et urinaire n'étaient pas significativement changé. Cependant, sous LS, les souris Senni aK0 perdaient des quantités significativement augmentées de sodium dans leurs fèces, accompagnées par de très hauts taux d'aldostérone plasmatique et une rétention urinaire en sodium augmentée. Les souris déficientes en CAPl/PmS (Prss8K0) dans les cellules superficielles intestinales étaient viables et ne montraient pas de létalité embryonnaire ou postnatale. Sous diètes RS et HS cependant, les souris Prss8KO montraient une diminution significative du APDamil dans l'après-midi, mais le rythme circadien était maintenu. Sous diète LS, la perte de sodium par les fèces était accompagnée par des niveaux d'aldostérone plasmatiques plus élevés. Par conséquent, nous avons identifié la protéase activatrice de canal CAP 1 IPrss8 comme un régulateur important d'ENaC dans le côlon in vivo. De plus, nous étudions l'importance d'ENaC et de CAPIIPrss8 dans les conditions pathologiques comme les maladies inflammatoires chroniques de l'intestin (MICI). Le résultat préliminaire out montre qu'une déficience d'Prss8 mènait à la détérioration de la colite induite par le DSS comparé aux modèles contrôles respectifs. En résumé, l'étude a montré que sous restriction de sel, l'absence d'ENaC dans Pépithélium de surface du côlon était compensée par 1'activation du système rénine-angiotensine- aldostérone (RAAS) dans le rein. Ceci a mené à un pseudohypoaldostéronisme de type I spécifique au côlon avec résistance aux minéralocorticoïdes sans signe d'altération de rétention de potassium. - In aldosterone-responsive epithelial cells of kidney and colon, the epithelial sodium channel (ENaC) plays a critical role in the control of sodium balance, blood volume, and blood pressure. The role of ENaC is well characterized in kidney and lung, whereas role of ENaC and its positive regulator channel-activating protease 1 (CAPl/PrasS) on sodium transport in colon is largely unknown. We have investigated the importance of ENaC and CAPI/Prss8 in colon for sodium and potassium balance. Mice lacking the aENaC subunit (Scnnla mice) in intestinal superficial cells were viable and did not show any fetal or perinatal lethality. Under regular (RS) or low salt (LS) diet, the amiloride sensitive rectal potential difference (APDamii) was drastically decreased and its circadian rhythm blunted. Under regular salt (RS) or high salt (HS) diets or under potassium loading, plasma and urinary sodium and potassium were not significantly changed. However, upon LS, the ScnnlaK0 mice lost significant amounts of sodium in their feces, accompanied by very high plasma aldosterone and increased urinary sodium retention. Mice lacking the CAPl/PrasS (Prss8K0) in intestinal superficial cells were viable and did not show any fetal or perinatal lethality. Upon RS and HS diets, however, Prss8K0 exhibited a significantly reduced APDamii in the afternoon, but its circadian rhythm was maintained. Upon LS diet, sodium loss through feces was accompanied by higher plasma aldosterone levels. Thus, we have identified the channel-activating protease CAPl/Prss8 as an important in vivo regulator of ENaC in colon. Furthermore, we are investigating the importance of ENaC and CAPI/Prss8 in pathological conditions like inflammatory bowel disease (IBD). Preliminary data showed that PmS-deficiency led to worsening of DSS-induced colitis as compared to their respective controls. Overall, the present study has shown that under salt restriction, the absence of ENaC in colonic surface epithelium was compensated by the activation of renin-angiotensin- aldosterone (RAAS) system in the kidney. This led to a colon specific pseudohypoaldosteroni sm type 1 with mineralocorticoid resistance without evidence of impaired potassium retention.

Crystallization and preliminary X-ray diffraction studies of Drosophila melanogaster Gαo-subunit of heterotrimeric G protein in complex with the RGS domain of CG5036.

Regulator of G-protein signalling (RGS) proteins negatively regulate heterotrimeric G-protein signalling through their conserved RGS domains. RGS domains act as GTPase-activating proteins, accelerating the GTP hydrolysis rate of the activated form of Gα-subunits. Although omnipresent in eukaryotes, RGS proteins have not been adequately analysed in non-mammalian organisms. The Drosophila melanogaster Gαo-subunit and the RGS domain of its interacting partner CG5036 have been overproduced and purified; the crystallization of the complex of the two proteins using PEG 4000 as a crystallizing agent and preliminary X-ray crystallographic analysis are reported. Diffraction data were collected to 2.0 Å resolution using a synchrotron-radiation source.

Differential insertion of GPI-anchored GFPs into lipid rafts of live cells.

Partitioning of proteins in cholesterol and sphingolipid enriched plasma membrane microdomains, called lipid rafts, is critical for many signal transduction and protein sorting events. Although raft partitioning of many signaling molecules remains to be determined, glycosylphosphatidyl-inositol (GPI)-anchored proteins possess high affinity for lipid rafts and are currently exploited as markers to investigate fundamental mechanisms in protein sorting and signal transduction events. In this study, we demonstrate that two recombinant GPI-anchored green fluorescent proteins (GFP-GPIs) that differ in their GPI signal sequence confer distinct localization in plasma membrane microdomains. GFP fused to the GPI signal of the decay accelerating factor GFP-GPI(DAF) partitioned exclusively in lipid rafts, whereas GFP fused to the GPI signal of TRAIL-R3, GFP-GPI(TRAIL-R3), associated only minimally with microdomains. In addition, we investigated the unique ability of purified GFP-GPIs to insert into membrane microdomains of primary lymphocytes. This cell surface painting allows rapid, stable, and functional association of the GPI-anchored proteins with the target cell plasma membrane. The distinct membrane localization of the two GFP-GPIs was observed irrespective of whether the GPI-anchored molecules were painted or transfected. Furthermore, we show that painted GFP-GPI(DAF) was totally dependent on the GPI anchor and that the membrane insertion was increased by the addition of raft-associated lipids such as cholesterol, sphingomyelin, and dipalmitoyl-phosphatidylethanolamine. Thus, this study provides evidence that different GPI signal sequences lead to distinct membrane microdomain localization and that painted GFP-GPI(DAF) serves as an excellent fluorescent marker for lipid rafts in live cells.

KRAS mutation testing for predicting response to anti-EGFR therapy for colorectal carcinoma: proposal for an European quality assurance program.

Novel therapeutic agents targeting the epidermal growth factor receptor (EGFR) have improved outcomes for patients with colorectal carcinoma. However, these therapies are effective only in a subset of patients. Activating mutations in the KRAS gene are found in 30-40% of colorectal tumors and are associated with poor response to anti-EGFR therapies. Thus, KRAS mutation status can predict which patient may or may not benefit from anti-EGFR therapy. Although many diagnostic tools have been developed for KRAS mutation analysis, validated methods and standardized testing procedures are lacking. This poses a challenge for the optimal use of anti-EGFR therapies in the management of colorectal carcinoma. Here we review the molecular basis of EGFR-targeted therapies and the resistance to treatment conferred by KRAS mutations. We also present guideline recommendations and a proposal for a European quality assurance program to help ensure accuracy and proficiency in KRAS mutation testing across the European Union.

Novel action of a wake-promoting neuropeptide in hippocampus : inhibition of nmda receptor function at excitatory synapses through orexin-a

One of the most intriguing functions of the brain is the ability to learn and memorize. The mechanism through which memory and learning are expressed requires the activation of NMDA receptors (NMDARs). These molecular entities are placed at the postsynaptic density of excitatory synapses and their function is tightly controlled by the actions of several modulators at the extracellular, intracellular and pore sites. A large part of the intracellular modulation comes from the action of G-protein coupled receptors (GPCRs). Through intracellular cascades typically involving kinases and phosphatases, GPCRs potentiate or inhibit NMDARs, controlling the conductive state but also the trafficking within the synapse. The GPCRs are involved in the modulation of a variety of brain functions. Many of them control cognition, memory and learning performance, therefore, their effects on NMDARs are extensively studied. The orexinergic system signals through GPCRs and it is well known for the regulation of waking, feeding, reward and autonomic functions. Moreover, it is involved in potentiating hippocampus-related cognitive tasks. Orexin receptors and fibers are present within the hippocampus, but whether these directly modulate hippocampal cells and synapses has not yet been determined. During my thesis, I studied orexinergic actions on excitatory synaptic transmission via whole-cell patch-clamp recordings in rat acute hippocampal slices. I observed that exogenously applied orexin-A (ox-A) exerted a strong inhibitory action on NMDAR-mediated synaptic potentials at mossy fiber (MF)-CA3 synapses, by postsynaptically activating orexin-2 receptors, a minor inhibition at Schaffer collateral-CAl synapses and did not affect other synapses with the CA3 area. Moreover, I demonstrated that the susceptibility of NMDARs to ox- A depends on the tone of endogenous orexin known to fluctuate during the day-night cycle. In fact, in slices prepared during the active period of the rats, when endogenous orexin levels are high, NMDAR-currents were not affected by exogenously applied ox-A. The inhibitory effect of ox-A was, however, reverted when interfering with the orexinergic system through intraperitoneal injections of almorexant, a dual orexin receptor antagonist, during the active phase prior to slice preparation. This thesis work suggests that the orexinergic system regulates NMDAR-dependent information flow through select hippocampal pathways depending on the time-of-day. The specific orexinergic modulation of NMDARs at MFs dampens the excitability of the hippocampal circuit and could impede the mechanisms related to memory formation, possibly also following extended periods of waking. -- La capacité d'apprentissage et de mémorisation est une des fonctions les plus intrigantes de notre cerveau. Il a été montré qu'elles requièrent l'activation des récepteurs NMDA (NMDARs). Ces entités moléculaires sont présentes au niveau de la densité post-synaptique des synapses excitatrices et leur fonction est étroitement contrôlée par l'action de nombreux modulateurs au niveau extracellulaire, intracellulaire et membranaire de ces récepteurs. Une grande partie de la modulation intracellulaire s'effectue via l'action de récepteurs couplés aux protéines G (GPCRs). Grace à leurs cascades intracellulaires typiquement impliquant des kinases et des phosphatases, les GPCRs favorisent l'activation ou l'inhibition des NMDARs, contrôlant ainsi leur perméabilité mais aussi leur mouvement à la synapse. Les GPCRs sont impliquées dans de nombreuses fonctions cérébrales telles que la cognition, la mémoire ainsi que la capacité d'apprentissage c'est pour cela que leurs effets sur les NMDARs sont très étudiés. Le système orexinergique fait intervenir ces GPCRs et est connu par son rôle dans la régulation de fonctions physiologiques telles que l'éveil, la prise alimentaire, la récompense ainsi que d'autres fonctions du système nerveux autonome. De plus, ce système est impliqué dans la régulation de tâches cognitives liées à l'hippocampe. Bien que les fibres et les récepteurs à l'orexine soient présents dans l'hippocampe, leur mécanisme d'action sur les cellules et les synapses de l'hippocampe n'a pas encore été élucidé. Durant ma thèse, je me suis intéressée aux effets de l'orexine sur la transmission synaptique excitatrice en utilisant la méthode d'enregistrement en patch-clamp en configuration cellule entière sur des tranches aiguës d'hippocampes de rats. J'ai observé que l'application exogène d'orexine A d'une part inhibe fortement les courants synaptiques dépendants de l'activation des NMDARs au niveau de la synapse entre les fibres moussues et CA3 via l'activation post-synaptique des orexine récepteurs 2 mais d'autre part n'inhibe que de façon mineure la synapse entre les collatérales de Schaffer et CAI et n'affecte pas les autres synapses impliquant CA3. J'ai également démontré que la sensibilité des NMDARs à l'orexine A dépend de sa concentration endogène qui fluctue durant le cycle éveil-sommeil. En effet, lorsque les coupes d'hippocampes sont préparées durant la période active de l'animal correspondant à un niveau endogène d'orexine élevé, l'application exogène d'orexine A n'a aucun effet sur les courants dépendants de l'activation des NMDARs. Cependant, l'injection dans le péritoine, durant la phase active de l'animal, d'un antagoniste des orexine récepteurs, l'almorexant, va supprimer l'effet inhibiteur de l'orexine A. Les résultats de ma thèse suggèrent donc que le système orexinergique module les informations véhiculées par les NMDARs via des voies de signalisation sélectives de l'hippocampe en fonction du moment de la journée. La modulation orexinergique des NMDARs au niveau des fibres moussues diminue ainsi l'excitabilité du circuit hippocampal et pourrait entraver les mécanismes liés à la formation de la mémoire, potentiellement après de longues périodes d'éveil.

Treatment implications of the emerging molecular classification system for melanoma.

Melanoma is an aggressive disease with few standard treatment options. The conventional classification system for this disease is based on histological growth patterns, with division into four subtypes: superficial spreading, lentigo maligna, nodular, and acral lentiginous. Major limitations of this classification system are absence of prognostic importance and little correlation with treatment outcomes. Recent preclinical and clinical findings support the notion that melanoma is not one malignant disorder but rather a family of distinct molecular diseases. Incorporation of genetic signatures into the conventional histopathological classification of melanoma has great implications for development of new and effective treatments. Genes of the mitogen-associated protein kinase (MAPK) pathway harbour alterations sometimes identified in people with melanoma. The mutation Val600Glu in the BRAF oncogene (designated BRAF(V600E)) has been associated with sensitivity in vitro and in vivo to agents that inhibit BRAF(V600E) or MEK (a kinase in the MAPK pathway). Melanomas arising from mucosal, acral, chronically sun-damaged surfaces sometimes have oncogenic mutations in KIT, against which several inhibitors have shown clinical efficacy. Some uveal melanomas have activating mutations in GNAQ and GNA11, rendering them potentially susceptible to MEK inhibition. These findings suggest that prospective genotyping of patients with melanoma should be used increasingly as we work to develop new and effective treatments for this disease.

Phytochemical investigation of alpine plants : the cottonsedges Eriophorum scheuchzeri Hoppe, E. angustifolium Honck. and E. latifolium Hoppe (CYPERACEAE)

Résumé: Alpine plants living at high altitudes undergo a series of climatic stress factors (chilling, enhanced UV radiation, short growing season, low nutriment supply...) which may influence their secondary compounds composition. Many publications showed in these last years that plants under stress conditions do synthesize a range of specific defence compounds (terpenes, flavonoids, coumarines...). A careful phytochemical investigation of those plants could therefore lead to the discovery of active molecules. Thus, for the biological and chemical screening, about 30 alpine plants have been collected above 2000 metres, in the alpine grass-lands. Eriophorum scheuchzeri Hoppe (Cyperaceae), not yet investigated phytochemically, revealed in its lipophilic and polar extracts the presence of various radical scavengers in a TLC autography with the DPPH (2,2-dipheny1-1- picrylhydrazyl) radical as spray reagent, as well as several antifungal compounds acitve against Cladosporium cucumerinum and Candida albi cans. The first part of this study consisted in the detection, isolation and characterization of the bioactive natural compounds present in the lipophilic extract of Eriophorum scheuchzeri. Among the eight isolated compounds, six were isoflavones. No isoflavones have been reported in the Cyperaceae family yet, nor in related families such as Poaceae or Juncaceae. Besides, isoflavones are generally rare in the plant kingdom and and they occur only in some families, such as Fabaceae, Rosaceae or Myristicaceae. In addition, out of these six isoflavones, three were new isoflavones. The known compounds were parvisoflavone A and B and cajanin which are already known isoflavones in the Fabaceae family. Two of the new isoflavones were particular, as they were C-methylated on the B-ring at the C-3' position. Methylated flavonoids are particularly rare in the plant kingdom. At present, no C-methylated isoflavones with methyl groups on the B-ring have ever been reported. The fourth new compound was a prenylated flavanone. Flavanones are also rare in the Cyperaceae family since they were found only in two genera (Cyperus and Schoenus). Finally, the widespread flavone tricin, characteristic of the Cyperaceae and Poaceae family has also been isolated. The second part of this study consisted in the characterization of the polar components present in the Me0H extract. In order to obtain mass and UV information about the secondary compounds present in the Eriophorum scheuchzeri methanolic extract, a LC-UV/DAD-APCl/MSn analysis has been performed as a first dereplication step. The UV/DAD spectra showed the presence of polyphenol compounds (phenylpropanoids and flavonoids). The LC-APCI/MSn analysis allowed the determination of the molecular weight of these compounds. Moreover, the fragmentation pattern of the [M+H]+ ions indicated presence of mono-, di- and tri-glycosides. LC-UV in combination with UV shift reagents added post-column was used in a second phase for the structural elucidation of the flavonoids. It allowed the positioning of the sugars on the aglycones. Finally, LC-NMR was used for a more detailed structural investigation of the compounds present in the crude MEOH extract. Thus, 10 fiavonoids have been totally or partially characterized by LC-UV-MS and LC-1H-RMN and UV-shift reagents added post column. However, the information obtained on-line was not always sufficient to allow a complete identification of all the compounds. Some of these compounds especially those with more than two sugar units attached to them, have been isolated in order to proceed to their complete characterization. Moreover, the Eriophorum scheuchzeri species was compared to two other species from the same genus. A LC-UV-ESI/MS analysis enabled a survey of the chemical composition of the DCM extracts of two related species E. angustifolium (Honck) and E. latifolium (Hoppe). The chromatograms of the three species showed some similarities in their flavonoid contents, especially by the recurrent presence of three compounds. The MEOH extracts of all three species have been compared by means of LC-UV-APCl/MS analyses. The chromatographic profile of all the three species showed even closer similarities than those found in the DCM extracts. E. angustifolium Honck. and E. latifolium species showed 7 compounds in common. Finally, the pure compounds obtained from the DCM (CH2Cl2) fraction were tested at different concentration, in order to evaluate their chemical and biological activities. All eight compounds showed an anti-scavenger activity against the DPPH radical, and four compounds showed antifungal activities against Cladosporium cucumerinum and Candida albicans. The pure compounds isolated from the MeOH extract were tested only for their biological activities as their antioxidant activity is already well documented in the literature. No compound showed a biological activity against Cladosporium cucumerinum and Candida albicans. Résumé: De nombreux travaux ont démontré ces dernières décennies que les plantes soumises à différents types de stress (basse température, UV, stress hydrique) synthétisent des composés secondaires (fiavonoides, coumarines, terpènes...) de protection et de défense. Les plantes d'altitude par exemple qui sont exposées à des conditions climatiques et environnementales difficiles, ont tendance à synthétiser des substances antioxydantes et antiradicalaires. Une investigation phytochirnique de ces plantes a conduit à la découverte de nouvelles molécules actives. Ainsi plusieurs plantes alpines ont été sélectionnées en fonction de leur habitat en vue de les soumettre aux tests biologiques (antifongiques) et chimiques (antiradicalaires) menés en routine dans notre laboratoire. Dans ce criblage biologique préliminaire, les extraits d'Eriophorum scheuchzeri Hoppe (Cyperaceae) ont réagi positivement aux différents tests. Il a donc été décidé d'entreprendre l'isolement des composés actifs. La première partie de ce travail a consisté à détecter, isoler et caractériser les composés naturels actifs présents dans l'extrait apolaire d' Eriophorum scheuchzeri. Parmi les huit composés isolés, quatre d'entre eux sont nouveaux. Un de ces produits est une flavanone et trois sont de nouvelles isoflavones, particulièrement intéressantes car elles possèdent des groupements C-méthylés au niveau du cycle B. Les flavonoides C-méthylés sont peu répandus dans le règne végétal et les rares exemples connus sont généralement C-méthylés sur le cycle A. Les quatre autres composés isolés n'ont jamais été décrits dans cette famille. Il s'agit d' isoflavones, les parvisoflavones A et B et la cajanine. Enfin, la flavone tricine, flavonoide caractéristique des Cyperaceae et des Poaceae a également été isolée. La deuxième partie de ce travail a consisté à caractériser les constituants polaires présents dans l'extrait methanolique. L'extrait a été analysé par chromatographie analytique couplée à différentes méthodes spectroscopiques (LC-UV-MS et LC-UV-1H RMN). De cette façon, douze flavonoides et un dérivé du phénylpropane, l'acide chlorogénique ont été identifiés. Les flavonoides tri-glycosylés ont dû être isolés afin de déterminer la nature et l'enchaînement des sucres. Finalement, l'espèce Eriophorum scheuchzeri a été comparée à deux autres espèces d' Eriophorum, soit E. angustifolium et E. latifolium. En conclusion, cette étude phytochimique a abouti à l'isolement de plusieurs nouvelles isoflavones aux activités antioxydantes et antifongiques ainsi qu'oestrogéniques.

The activity of the anti-apoptotic fragment generated by the caspase-3/p120 RasGAP stress-sensing module displays strict Akt isoform specificity.

The caspase-3/p120 RasGAP module acts as a stress sensor that promotes pro-survival or pro-death signaling depending on the intensity and the duration of the stressful stimuli. Partial cleavage of p120 RasGAP generates a fragment, called fragment N, which protects stressed cells by activating Akt signaling. Akt family members regulate many cellular processes including proliferation, inhibition of apoptosis and metabolism. These cellular processes are regulated by three distinct Akt isoforms: Akt1, Akt2 and Akt3. However, which of these isoforms are required for fragment N mediated protection have not been defined. In this study, we investigated the individual contribution of each isoform in fragment N-mediated cell protection against Fas ligand induced cell death. To this end, DLD1 and HCT116 isogenic cell lines lacking specific Akt isoforms were used. It was found that fragment N could activate Akt1 and Akt2 but that only the former could mediate the protective activity of the RasGAP-derived fragment. Even overexpression of Akt2 or Akt3 could not rescue the inability of fragment N to protect cells lacking Akt1. These results demonstrate a strict Akt isoform requirement for the anti-apoptotic activity of fragment N.

Adipose reduction in the activity of the repressor ICER is responsible for insulin resistance elicited by the transcriptional factors CREB in obesity

BACKGROUND AND AIMS: Sustained adipose activation of the transcriptional activators cAMP response binding proteins (CREB) in obesity leads to impaired expression of the glucose transporter GLUT4 and adiponectin (adipoq) in mice model of obesity. Diminution of GLUT4 and adipoq caused by CREB is indirect and relies on the increased repressive activity of the CREB target gene activating transcription factor 3 (ATF3). Specific inactivation of CREB in adipocytes decreases ATF3 production and improves whole-body insulin sensitivity of mice in the context of diet-induced obesity. Thus, elevation of CREB activity is a key mechanism responsible for adipocyte dysfunction and systemic insulin resistance. The inducible cAMP early repressor (ICER) is a negative regulator of the CREB activity. In fact, ICER antagonizes the CREB factor by competing for the regulation of similar target genes. The goal of the study was to investigate whether loss of ICER expression in adipocytes could be responsible for increased CREB activity in obesity. MATERIALS AND METHODS: Mice C57bl6 were fed with a high fat diet (HFD) for 12 weeks to increase body weight and generate insulin resistance. Biopsies of visceral adipose tissues (VAT) were prepared from human lean (BMI=24}0.5 Kg/m2) or obese subjects (BMI>35 Kg/m2). Total RNA and protein were prepared from white adipose tissues (WAT) of chow- or HFD-fed mice and VAT of lean and obese subjects. Activities of CREBs and ICER were monitored by electromobility shift assays (EMSA). The role of ICER on CREB activity was confirmed in 3T3-L1 adipocytes cells. Briefly after differentiation, the cells were electroporated with the plasmid coding for ICER cDNA. Gene expression was quantified by quantitative real-time PCR and western Blotting experiments. RESULTS: The expression of ICER is reduced in WAT of HFD-induced obese mice when compared to chow mice as measured by real-time PCR and EMSA. Similar result was found in human tissues. Reduction in ICER expression was associated with increased ATF3 expression and decreased adipoq and GLUT4 contents. Diminution in ICER levels was observed in adipocytes fraction whereas its expression was unchanged in stroma vascular fraction of WAT. Overexpression of ICER in 3T3-L1 adipocytes silenced the expression of ATF3, confirming the regulation of the factor by ICER. The expression of ICER is regulated by histone deacetylases activity (HDAC). Inhibition of HDACs in 3T3-L1 adipocytes cells using trichostatin inhibited the production of ICER. The whole activity of HDAC was reduced in WAT and VAT of obese mice and human obese subjects. CONCLUSION: Impaired adipose expression of ICER is responsible of increased CREB activity in adipocytes in obesity. This mechanism relies on reduction of the HDAC activity.

The role of endogenous and exogenous RasGAP-derived fragment N in protecting cardiomyocytes from peroxynitrite-induced apoptosis.

Peroxynitrite (PN) is a potent nitrating and oxidizing agent generated during various pathological situations affecting the heart. The negative effects of PN result, at least in part, from its ability to activate caspases and apoptosis. RasGAP is a ubiquitously expressed protein that is cleaved sequentially by caspase-3. At low caspase-3 activity, RasGAP is cleaved into an N-terminal fragment, called fragment N, that protects cells by activating the Ras/PI3K/Akt pathway. At high caspase-3 activity, fragment N is further cleaved and this abrogates its capacity to stimulate the antiapoptotic Akt kinase. Fragment N formation is crucial for the survival of cells exposed to a variety of stresses. Here we investigate the pattern of RasGAP cleavage upon PN stimulation and the capacity of fragment N to protect cardiomyocytes. PN did not lead to sequential cleavage of RasGAP. Indeed, PN did not allow accumulation of fragment N because it induced its rapid cleavage into smaller fragments. No situations were found in cells treated with PN in which the presence of fragment N was associated with survival. However, expression of a caspase-resistant form of fragment N in cardiomyocytes protected them from PN-induced apoptosis. Our results indicate that the antiapoptotic pathway activated by fragment N is effective at inhibiting PN-induced apoptosis (as seen when cardiomyocytes express a capase-3-resistant form of fragment N) but because fragment N is too transiently generated in response to PN, no survival response is effectively produced. This may explain the marked deleterious consequences of PN generation in various organs, including the heart.

Platforms for high-throughput screening of Wnt/Frizzled antagonists.

Signaling cascades initiated by Wnt lipoglycoproteins and their receptors of the Frizzled family regulate many aspects of animal development and physiology. Improper activation of this signaling promotes carcinogenic transformation and metastasis. Development of agents blocking the Wnt-Frizzled signaling is of prime interest for drug discovery. Despite certain progress no such agents are as yet brought to the market or even to clinical trials. One reason for these delays might be the use of suboptimal readout assays. In this article we overview existing and developing assay platforms to screen for Wnt-Frizzled antagonists. Among those, G protein-activating assays built on the emerging GPCR properties of Frizzleds are highlighted.

Fluorescence-activated cell sorting and cloning of bona fide CD8+ CTL with reversible MHC-peptide and antibody Fab' conjugates.

The isolation of subsets of Ag-specific T cells for in vitro and in vivo studies by FACS is compromised by the fact that the soluble MHC-peptide complexes and Abs used for staining, especially when combined, induce unwanted T cell activation and eventually apoptosis. This is especially a problem for CD8+ CTL, which are susceptible to activation-dependent cell death. In this study, we show that reversible MHC-peptide complexes (tetramers) can be prepared by conjugating MHC-peptide monomers with desthiobiotin (DTB; also called dethiobiotin) and multimerization by reaction with fluorescent streptavidin. While in the cold these reagents are stable and allow good staining, they rapidly dissociate in monomers at elevated temperatures, especially in the presence of free biotin. FACS cloning of Melan-A (MART-1)-specific CTL from a melanoma-infiltrated lymph node with reversible HLA-A2 Melan-A26-35 multimers yielded over two times more clones than when using the conventional biotin-containing multimers. CTL clones obtained by means of reversible multimers killed Melan-A-positive tumor cells more efficiently as compared with clones obtained with the stable multimers. Among the CTL obtained with the reversible multimers, but much less among those obtained with the stable multimers, a high proportion of clones exhibited high functional and physical avidity and died upon incubation with soluble MHC-peptide complexes. Finally, we show that Fab' of an anti-CD8 Ab can be converted in reversible DTB streptavidin conjugates the same way. These DTB reagents efficiently and reversibly stained murine and human CTL without affecting their viability.

The Oncogene ATF3 Is Potentiated by Cyclosporine A and Ultraviolet Light A.

Cutaneous squamous cell carcinoma (SCC) represents the most important cutaneous complication following organ transplantation. It develops mostly on sun-exposed areas. A recent study showed the role of activating transcription factor 3 (ATF3) in SCC development following treatment with calcineurin inhibitors. It has been reported that ATF3, which may act as an oncogene, is under negative calcineurin/nuclear factor of activated T cells (NFAT) control and is upregulated by calcineurin inhibitors. Still, these findings do not fully explain the preferential appearance of SCC on chronically sun-damaged skin. We analyzed the influence of UV radiation on ATF3 expression and its potential role in SCC development. We found that ATF3 is a specifically induced AP1 member in SCC of transplanted patients. Its expression was strongly potentiated by combination of cyclosporine A and UVA treatment. UVA induced ATF3 expression through reactive oxygen species-mediated nuclear factor erythroid 2-related factor 2 (NRF2) activation independently of calcineurin/NFAT inhibition. Activated NRF2 directly binds to ATF3 promoter, thus inducing its expression. These results demonstrate two mechanisms that independently induce and, when combined together, potentiate the expression of ATF3, which may then force SCC development. Taking into account the previously defined role of ATF3 in the SCC development, these findings may provide an explanation and a mechanism for the frequently observed burden on SCCs on sun-exposed areas of the skin in organ transplant recipients treated by calcineurin inhibitors.

Phenotypic and molecular characterization of the onset of neuropathy in rodent models of diabetes mellitus

Abstract : The principal focus of this work was to study the molecular changes leading to the development of diabetic peripheral neuropathy (DPN). DPN is the most common complication associated with both type I and II diabetes mellitus (DM). This pathology is the leading cause of non-traumatic amputations. Even though the pathological and morphological changes underlying DPN are relatively well described, the implicated molecular mechanisms remain poorly understood. The following two approaches were developed to study the development of DPN in a rodent model of DM type I. As a first approach, we studied the implication of lipid metabolism in DPN phenotype, concentrating on Sterol Response Element Binding Protein (SREBP)-lc which is the key regulator of storage lipid metabolism. We showed that SREBP-1c was expressed in peripheral nerves and that its expression profile followed the expression of genes involved in storage lipid metabolism. In addition, the expression of SREBP-1c in the endoneurium of peripheral nerves was dependant upon nutritional status and this expression was also perturbed in type I diabetes. In line with this, we showed that insulin elevated the expression of SREBP-1c in primary cultured Schwann cells by activating the SREBP-1c promoter. Taken together, these findings reveal that SREBP-1c expression in Schwann cells responds to metabolic stimuli including insulin and that this response is affected in type I diabetes mellitus. This suggests that disturbed SREBP-1c regulated lipid metabolism may contribute to the pathophysiology of DPN. As a second approach, we performed a comprehensive analysis of the molecular changes associated with DPN in the Akital~1~+ mouse which is a model of spontaneous early-onset type I diabetes mellitus. This mouse expresses a mutated non-functional isoform of insulin, leading to hypoinsulinemia and hyperglycaemia. To determine the onset of DPN, weight, blood glucose and motor nerve conduction velocity (MNCV) were measured in Akital+/+ mice during the first three months of life. A decrease in MNCV was evident akeady one week after the onset of hyperglycemia. To explore the molecular changes associated with the development of DPN in these mice, we performed gene expression profiling using sciatic nerve endoneurium and dorsal root ganglia (DRG) isolated from early diabetic male Akita+/+ mice and sex-matched littermate controls. No major transcriptional changes were detected either in the DRG or in the sciatic nerve endoneurium. This experiment indicates that the phenotypic changes observed during the development of DPN are not correlated with major transcriptional alterations, but mainly with alterations at the protein level. Résumé Lors ce travail, nous nous sommes intéressés aux changements moléculaires aboutissant aux neuropathies périphériques dues au diabète (NPD). Les NPD sont la complication la plus commune du diabète de type I et de type II. Cette pathologie est une cause majeure d'amputations. Même si les changements pathologiques et morphologiques associés aux NPD sont relativement bien décrits, les mécanismes moléculaires provoquant cette pathologie sont mal connus. Deux approches ont principalement été utilisées pour étudier le développement des NPD dans des modèles murins du diabète de type I. Nous avons d'abord étudié l'impact du métabolisme des lipides sur le développement des NPD en nous concentrant sur Sterol Response Element Binding Protein (SREBP)-1 c qui est un régulateur clé des lipides de stockage. Nous avons montré que SREBP-1 c est exprimé dans les nerfs périphériques et que son profil d'expression suit celui de gènes impliqués dans le métabolisme des lipides de stockage. De plus, l'expression de SREBP-1c dans l'endoneurium des nerfs périphériques est dépendante du statut nutritionnel et est dérégulée lors de diabète de type I. Nous avons également pu montrer que l'insuline augmente l'expression de SREBP-1c dans des cultures primaires de cellules de Schwann en activant le promoteur de SREBP-1c. Ses résultats démontrent que l'expression de SREBP-1c dans les cellules de Schwann est contrôlée par des stimuli métaboliques comme l'insuline et que cette réponse est affectée dans le cas d'un diabète de type I. Ces données suggèrent que la dérégulation de l'expression de SREBP-1c lors du diabète pourrait affecter le métabolisme des lipides et ainsi contribuer à la pathophysiologie des NPD. Comme seconde approche, nous avons réalisé une analyse globale des changements moléculaires associés au développement des NPD chez les souris Akita+/+, un modèle de diabète de type I. Cette souris exprime une forme mutée et non fonctionnelle de l'insuline provoquant une hypoinsulinémie et une hyperglycémie. Afin de déterminer le début du développement de la NPD, le poids, le niveau de glucose sanguin et la vitesse de conduction nerveuse (VCN) ont été mesurés durant les 3 premiers mois de vie. Une diminution de la VCN a été détectée une semaine seulement après le développement de l'hyperglycémie. Pour explorer les changements moléculaires associés avec le développement des NPD, nous avons réalisé un profil d'expression de l'endoneurium du nerf sciatique et des ganglions spinaux isolés à partir de souris Akital+/+ et de souris contrôles Akita+/+. Aucune altération transcriptionnelle majeure n'a été détectée dans nos échantillons. Cette expérience suggère que les changements phénotypiques observés durant le développement des NPD ne sont pas corrélés avec des changements importants au niveau transcriptionnel, mais plutôt avec des altérations au niveau protéique. Résumé : Lors ce travail, nous nous sommes intéressés aux changements moléculaires aboutissant aux neuropathies périphériques dues au diabète (NPD). Les NPD sont la complication la plus commune du diabète de type I et de type II. Cette pathologie est une cause majeure d'amputations. Même si les changements pathologiques et morphologiques associés aux NPD sont relativement bien décrits, les mécanismes moléculaires provoquant cette pathologie sont mal connus. Deux approches ont principalement été utilisées pour étudier le développement des NPD dans des modèles murins du diabète de type I. Nous avons d'abord étudié l'impact du métabolisme des lipides sur le développement des NPD en nous concentrant sur Sterol Response Element Binding Protein (SREBP)-1c qui est un régulateur clé des lipides de stockage. Nous avons montré que SREBP-1 c est exprimé dans les nerfs périphériques et que son profil d'expression suit celui de gènes impliqués dans le métabolisme des lipides de stockage. De plus, l'expression de SREBP-1c dans l'endoneurium des nerfs périphériques est dépendante du statut nutritionnel et est dérégulée lors de diabète de type I. Nous avons également pu montrer que l'insuline augmente l'expression de SREBP-1c dans des cultures primaires de cellules de Schwann en activant le promoteur de SREBP-1c. Ses résultats démontrent que l'expression de SREBP-1c dans les cellules de Schwann est contrôlée par des stimuli métaboliques comme l'insuline et que cette réponse est affectée dans le cas d'un diabète de type I. Ces données suggèrent que la dérégulation de l'expression de SREBP-1c lors du diabète pourrait affecter le métabolisme des lipides et ainsi contribuer à la pathophysiologie des NPD. Comme seconde approche, nous avons réalisé une analyse globale des changements moléculaires associés au développement des NPD chez les souris Akita~~Z~+, un modèle de diabète de type I. Cette souris exprime une forme mutée et non fonctionnelle de l'insuline provoquant une hypoinsulinémie et une hyperglycémie. Afin de déterminer le début du développement de la NPD, le poids, le niveau de glucose sanguin et la vitesse de conduction nerveuse (VCN) ont été mesurés durant les 3 premiers mois de vie. Une diminution de la VCN a été détectée une semaine seulement après le développement de l'hyperglycémie. Pour explorer les changements moléculaires associés avec le développement des NPD, nous avons réalisé un profil d'expression de l'endoneurium du nerf sciatique et des ganglions spinaux isolés à partir de souris Akital+/+ et de souris contrôles Akita+/+. Aucune altération transcriptionnelle majeure n'a été détectée dans nos échantillons. Cette expérience suggère que les changements phénotypiques observés durant le développement des NPD ne sont pas corrélés avec des changements importants au niveau transcriptionnel, mais plutôt avec des altérations au niveau protéique.

A WXW Motif Is Required for the Anticancer Activity of the TAT-RasGAP317-326 Peptide.

TAT-RasGAP317-326, a cell-permeable 10-amino acid-long peptide derived from the N2 fragment of p120 Ras GTPase-activating protein (RasGAP), sensitizes tumor cells to apoptosis induced by various anticancer therapies. This RasGAP-derived peptide, by targeting the deleted in liver cancer-1 (DLC1) tumor suppressor, also hampers cell migration and invasion by promoting cell adherence and by inhibiting cell movement. Here, we systematically investigated the role of each amino acid within the RasGAP317-326 sequence for the anticancer activities of TAT-RasGAP317-326. We report here that the first three amino acids of this sequence, tryptophan, methionine, and tryptophan (WMW), are necessary and sufficient to sensitize cancer cells to cisplatin-induced apoptosis and to reduce cell migration. The WMW motif was found to be critical for the binding of fragment N2 to DLC1. These results define the interaction mode between the active anticancer sequence of RasGAP and DLC1. This knowledge will facilitate the design of small molecules bearing the tumor-sensitizing and antimetastatic activities of TAT-RasGAP317-326.