Alcohol consumption causes liver diseases, designated as Alcoholic Liver Disease (ALD). Because alcohol is detoxified by alcohol dehydrogenase (ADH), a major ethanol metabolism system, the development of ALD was initially believed to be due to malnutrition caused by alcohol metabolism in liver. The discovery of the microsomal ethanol oxidizing system (MEOS) changed this dogma. Cytochrome P450 enzymes (CYP) constitute the major components of MEOS. Cytochrome P450 2E1 (CYP2E1) in MEOS is one of the major ROS generators in liver and is considered to be contributive to ALD. Our labs have been studying the relationship between CYP2E1 and ALD for many years. Recently, we found that human CYP2A6 and its mouse analog CYP2A5 are also induced by alcohol. In mice, the alcohol induction of CYP2A5 is CYP2E1-dependent. Unlike CYP2E1, CYP2A5 protects against the development of ALD. The relationship of CYP2E1, CYP2A5, and ALD is a major focus of this review.

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Au cours des processus de croissance folliculaire et d'ovulation, les cellules stéroïdogéniques, y compris les cellules de granulosa (CG), jouent un rôle crucial dans la maturation et la libération de l'ovocyte. Notre laboratoire a identifié et caractérisé pour la première fois tribbles pseudokinase 2 (TRIB2) dans les CG de follicules ovariens. Des études ont démontré que TRIB2 joue un rôle dans la coordination de la mitose et la morphogenèse chez la drosophile alors que chez la souris, son expression dans les cellules du cumulus serait liée à la maturation ovocytaire. Cependant, le rôle exact de TRIB2 dans la fonction des CG et ses effets sur les voies de signalisation impliquées dans la croissance folliculaire restait à être défini. La présente étude de doctorat avait donc pour but d’étudier la fonction et les partenaires de liaison de TRIB2 dans les CG de follicules ovariens bovins. À l’aide d’un modèle d’étude in vivo consistant en des CG obtenues à partir de follicules à différents stades de développement, nous avons démontré une régulation à la baisse de TRIB2 par l’hormone lutéinisante (LH) et la gonadotrophine chorionique humaine (hCG) aussi bien au niveau du messager que de la protéine. De plus, les analyses utilisant un modèle in vitro de CG en culture ont montré que la FSH stimule l'expression de TRIB2 tandis que l'inhibition de TRIB2 via CRISPR/Cas9 entraîne une réduction significative de la prolifération des CG (P<0,05). Les analyses Western blot ont montré une augmentation des niveaux de phosphorylation d’ERK1/2 (MAPK3/1) et p38MAPK (MAPK14) suite à la surexpression de TRIB2. Ces résultats suggèrent un rôle de TRIB2 dans la croissance folliculaire et la modulation des voies MAPK. Avec l’approche double hybride chez la levure, nous avons identifié CALM1, INHBA, INPPL1, NT5E, SCD, SDHB et RAB14 comme partenaires de liaison de TRIB2. Les analyses RT-PCR ont montré que ces partenaires sont régulés différemment au cours du développement folliculaire et les manipulations de l’expression de TRIB2 (inhibition ou surexpression) résulte en une régulation différente (augmentation ou diminution) de l’expression des partenaires dans les CG. Ces résultats suggèrent un rôle de TRIB2 dans la régulation de cibles effectrices en lien avec la fonction des CG et le développement folliculaire. Enfin, un modèle de CG provenant de vaches en période post-partum a été utilisé pour mieux comprendre le contrôle de l’activité des CG. Des analyses complémentaires avec ce modèle ont révélé une réduction significative de TRIB2 chez les vaches ayant un taux élevé de BHB (>1.4mmol/L) comparé à celles ayant un faible taux de BHB (<1.2mmol/L). Cette réduction était concomitante à une augmentation d’interleukines pro-inflammatoires et une réduction d’interleukines anti-inflammatoires dans les CG. L’ensemble de ces résultats supporte un rôle de TRIB2 dans la modulation de la signalisation MAPK dans les CG, apporte une preuve solide que TRIB2 pourrait agir comme un régulateur de la prolifération et de la fonction des CG et de l’expression de gènes cibles, et suggère que TRIB2 pourrait affecter la stéroïdogenèse au cours du développement folliculaire et lors de la période post-partum. / During the processes of follicular growth and ovulation, steroidogenic cells, including granulosa cells, play a crucial role in the maturation and release of the oocyte. Our lab identified and characterized for the first time tribble pseudokinase 2 (TRIB2) in GC of ovarian follicles. Previous studies have shown that TRIB2 plays a role in the coordination of mitosis and morphogenesis in Drosophila while in mice its expression in cumulus cells is linked to oocyte maturation. However, the exact mechanism of action of TRIB2 as well as its function and effects on signaling pathways in GC during follicular growth remained to be defined. This study aimed therefore to further investigate TRIB2 function and identify its binding partners in GC. TRIB2 inhibition and overexpression experiments were conducted using, respectively, CRISPR/Cas9 technology and the pQE1 system. Using an in vivo model consisting of GC obtained from follicles at different stages of development, we demonstrated a downregulation of TRIB2 by the endogenous luteinizing hormone (LH) and human Chorionic Gonadotropin (hCG) at both the messenger and protein levels. In addition, analyzes using an in vitro model of cultured GC, we showed that FSH stimulates the expression of TRIB2 while inhibition of TRIB2 via CRISPR-Cas9 resulted in a significant reduction in GC proliferation (P<0.05). Western blot analyzes showed an increase in the phosphorylation levels of ERK1/2 (MAPK3/1) and p38MAPK (MAPK14) following TRIB2 overexpression. These results suggested a role of TRIB2 in follicular growth and modulation of MAPK pathways. In the second part of the thesis, we identified CALM1, INHBA, INPPL1, NT5E, SCD, SDHB and RAB14 as binding partners of TRIB2 in GC using the yeast two-hybrid approach. RT-qPCR analyzes showed that all of these partners are present in the dominant follicles but are differently regulated during follicular development. Moreover, TRIB2 manipulation (inhibition or overexpression) results in a different regulation (up- or down-regulation) of these partners expression in GC. These results suggest a role of TRIB2 in the regulation of effector targets genes related to follicular development and GC activity. In the third part of the thesis, a GC model from postpartum cows was also used to better understand the control of GC activity. Further analyses using this model revealed a significant decrease of TRIB2 in cows with high level of BHB (>1.4 mmol/L) as compared to those with a low BHB levels (<1.2 mmol/L). This reduction of TRIB2 was concomitant with an increase in pro-inflammatory interleukins and a reduction in anti-inflammatory interleukins in GC. Overall, these results support a role of TRIB2 in the modulation of MAPK signaling in GC, provide strong evidence that TRIB2 could act as a regulator of GC proliferation and function as well as expression of target genes in GC, and suggests that TRIB2 might affect steroidogenesis during follicular development and during the post-partum period.

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Kolorektale Karzinome (CRC) zeigen eine heterogene Ätiologie. Die Progression prämaligner Vorläufer zu CRC unterscheidet (U) sich in Morphologie, molekularen Veränderungen und Interaktion mit der Tumorumgebung. CRC weisen oft onkogene Mutationen in KRAS und BRAF auf. Diese steigern die MAPK Signalwegaktivität (Mpa). Obwohl sie im selben Signalweg wirken, sind KRAS und BRAF auf die CRC-Entitäten U verteilt. Dabei ist KRAS häufiger im sogenannten konventionellen und BRAF im serratierten Weg zu CRC mutiert. In dieser Studie nutzte ich murine intestinale Organoide (iO), die induzierbare (Ind) KRAS oder BRAF Onkogene exprimieren. Große U zwischen KRAS und BRAF zeigten sich sowohl in Signaltransduktion (ST) als auch im Phänotyp. Phosphoprotein-, ERK-Reporter-, scRNA-Seq und EM-Analysen ergaben eine starke Mpa durch BRAF, die zu hoher Expression von MAPK-Zielgenen und Verlust der epithelialen Integrität führte. iO nach KRAS-Ind blieben intakt, korrelierend mit moderater, zelltypspezifischer (ZS) Mpa in sekretorischen und undifferenzierten Zellen. Die meisten Enterozyten waren Mpa-negativ. ERK-Reporter zeigten: Das ZS Muster der Mpa ist nicht nur gegenüber KRAS, sondern auch dem Entzug von Wachstumsfaktoren stabil. Dies spricht für eine intrinsische, robuste Regulierung der Mpa. BRAF-Ind Mpa setzte die ZS Regulierung der MAPK außer Kraft und schädigte das Gewebe, im Einklang mit einer oberen Grenze tolerabler Mpa. Die ZS Mpa wurde in CRC-Zelllinien bestätigt, deren Mpa durch KRAS aber nicht BRAF U ausfiel. Ferner, nutzte ich iO mit bCatenin+KRAS-Ind, um den konventionellen Weg zu CRC zu modellieren. Die Kombination führte zu synergistischen Effekten, die sich in EGFR-unabhängigem Wachstum und der Aufhebung der ZS Mpa-Blockade äußerten, die durch eine Verschiebung der Differenzierung zu mehr Progenitorzellen bewirkt wurde. Zusammenfassend konnte ich U in der Mpa durch KRAS oder BRAF im Darmepithel feststellen, was dazu beiträgt, deren Rollen in der CRC-Genese zu bestimmen. / Colorectal cancer (CRC) is a disease with heterogeneous etiology. Premalignant lesions follow distinct routes of progression to carcinoma reflected by differences in morphology, molecular alterations and the tumor environment. Mutant KRAS and BRAF are frequent, leading to MAPK pathway activation (Mpa), which is relevant for CRC therapy. Despite acting in the same pathway, mutant KRAS and BRAF segregate to different entities, as KRAS is more frequent in the conventional- and BRAF being specific for the serrated route to CRC. I used murine intestinal organoids (iO) expressing inducible oncogenic KRAS or BRAF to study the impact of oncogenes in primary cells. I found marked differences in signal transduction and phenotype. Phospho-protein, ERK-reporter, scRNA-seq and EM data showed strong Mpa upon BRAF induction followed by ERK-target gene expression leading to tissue disruption. In contrast, KRAS left the tissue intact resulting in less and cell type-dependent Mpa limited to secretory cells, a subset of late-stage enterocytes and undifferentiated crypt cells. Most enterocytes were irresponsive to KRAS. The pattern of Mpa was robust towards KRAS or growth factor depletion arguing in favor of intrinsic, resilient MAPK regulation. In iO, BRAF-induced Mpa could break this cell type-specific regulation, indicating an upper limit of tolerable Mpa. I validated these findings in CRC cell lines that differed in Mpa in response to oncogenic KRAS but not BRAF. Finally, I used iO expressing an inducible form of stabilized bCatenin in combination with KRAS to mimic events frequently found in the conventional pathway to CRC. Expression of KRAS and bCatenin synergized in driving EGFR independent growth and breaking the villus-specific block of Mpa by altering differentiation towards progenitor cell types. In summary, this study emphasizes differences between Mpa induced by oncogenic KRAS or BRAF which helps clarifying their nature in different etiological routes to CRC genesis.

Dans ce travail, nous démontrons que STIM1, un senseur calcique activé par la déplétion du Ca2+ intracellulaire du réticulum endoplasmique, est indispensable pour la signalisation calcique et la préférence oro-sensorielle du gras. Nous observons que l'acide linoléique (LA), en activant les phospholipases A2 via CD36, produit de l'acide arachidonique (AA) et de la lyso-phosphatidylcholine (lyso-PC). Cette activation déclenche un influx calcique dans les cellules CD36-positives, et induit la production du facteur CIF (Ca2+ Influx Factor). CIF, AA et lyso-PC exercent différentes actions sur l'ouverture des canaux SOC (Stored Operated Calcium Channel) constitués de protéines Orai et contrôlés par STIM1. Par ailleurs, les souris au phénotype Stim1-/- perdent la préférence spontanée pour les lipides et la libération de la sérotonine à partir des cellules gustatives dans le milieu extracellulaire chez les animaux sauvages. Nous demontrons aussi que la signalisation calcique médiée via CD36 est doublement modulée lors de l'obésité. L'augmentation de la [Ca2+]i dans les cellules gustatives observée chez le Psammomys obesus, un modèle d'obésité nutritionelle, est fortement diminuée chez les souris rendues obèses par un regime hyperlipidique. Nous avons constaté également que l'interaction de LA avec le CD36 induit l'activation des MAP Kinases de la voie MEK1/2/ERK1/2/Elk-1 qui est non seulement à l'origine de l'activation des aires cérébrales telles que le NTS, le noyau arqué, l'hippocampe mais aussi indispensable pour la préférence spontanée pour les lipides alimentaires. Nos résultats suggèrent pour la prémière fois, que la voie ERK1/2 des MAPK et la signalisation calcique lipidique controlée par STIM1 sont impliquées dans la perception oro-gustative des lipides

Nitric oxide (NO) is an important biological molecule within the body, which over production of this molecule in response to different stimulations can cause various inflammatory diseases. Over production of this molecule is caused by the induction of the inducible nitric oxide synthase (iNOS) enzyme. This enzyme uses L-arginine as a substrate and therefore the presence and transport of this amino acid into the cells can be a key factor in regulating NO over production. Different signalling mechanisms have been implicated in the regulation of this pathway and one of which involves the Mitogen Activated Protein Kinases (MAPK). This family of proteins respond to inflammatory conditions and may mediate effects induced by inflammatory mediators. Of the MAPKs, the role of the c-Jun-N-terminal kinase (JNK) pathway in the induction of iNOS is still controversial. JNK and its downstream target, the transcription factor Activator Protein-1 (AP-1), have shown contradictory effects on iNOS induction leading to controversies over their role in regulating iNOS expression in different cell systems or with various stimuli. The studies described in this thesis have determined the role of JNK/AP-1 on iNOS expression, NO production, L-arginine uptake and also on the transporters responsible for L-arginine transport into the cells. The studies were carried out in two different cell types: rat aortic smooth muscle cells (RASMCs) and J774 macrophages which are both critically associated with the over production of NO in vascular inflammatory disease states. The first approach was to block the expression of the inducible L-arginine-NO pathway using SP600125 and JNK Inhibitor VIII which are both pharmacological inhibitors of JNK. The results from these studies showed that the pharmacological intervention was without effect in RASMCs, but inhibited iNOS, NO and L-arginine transport in J774 macrophages. In contrast, the molecular approach employed using two dominant negative constructs of AP-1 (TAM-67 and a-Fos) revealed a different profile of effects in RASMCs, where a-Fos caused an induction in iNOS and NO while TAM-67 had an inhibitory effect on iNOS, NO, L-arginine transport and CAT-2B mRNA expression. The latter was unaffected in RASMCs but suppressed in J774 macrophages by SP600125. Examination of JNK isoforms expression showed the presence of JNK1 and 2 in both cell systems. Moreover, stimulation with LPS/IFN- or LPS alone resulted in JNK phosphorylation which did not reveal any difference between smooth muscle cells and macrophages. In contrast, expression and activation of AP-1 subunits revealed differences between the two cell systems. Activation of cells with LPS and IFN- (RASMCs) or LPS alone (J774 macrophages) resulted in changes in the activated status of the different AP-1 subunit which was different for the two cell systems. In both cell types c-Jun, JunD and Fra-1 were increased and in macrophages, FosB activity was also enhanced. Inhibition of JNK with SP600125 caused down-regulation in c-Jun in both cell types. Interestingly this down-regulation was in parallel with increases in the subunits JunB, JunD, c-Fos and Fra-1 in RASMCs or JunB and Fra-1 in J774 macrophages. Since, SP600125 was able to exert inhibitory effects in the latter cell type but not in RASMCs, it is possible that the compensatory up-regulation of certain AP-1 subunits in the smooth muscle cells may compensate for c-Jun inhibition thereby preventing suppression of iNOS expression. This notion clearly needs to be confirmed but it is potentially likely that hetero-dimers formed between JunB, JunD, c-Fos and Fra-1 could sustain gene transcription in the absence of c-Jun. The precise dimer required has not been addressed but unlikely to exclusively involve JunB and Fra-1 as these are up-regulated in macrophages but did not sustain iNOS, NO or induced L-arginine transport in the presence of SP600125. To further support the argument above, the dominant negatives caused varied effects on the activation of the different subunits. a-Fos down-regulated c-Jun, c-Fos, FosB, Fra-1 whereas TAM-67 reduced c-Jun and c-Fos but marginally induced Fra-1 activity. Associated with these changes was an up-regulation of iNOS-NO by a-Fos and inhibition by TAM-67. Taken together, the data proposes a complex mechanism(s) that regulate the expression of the inducible L-arginine-NO pathway in different cell systems and the complexity may reflect diverse intracellular changes that may be different in each cell type and not always be apparent using one experimental approach especially where this is pharmacological. Moreover, these findings strongly suggest exercising caution when interpreting pure pharmacological findings in cell-based systems particularly where these are inconsistent or contradictory.

El gen humano 11&#61538;-HSD2 es un modelo para investigar la contribución de los efectos de los receptores de esteroides en células de cáncer de mama. El análisis del promotor mostró que la región distal está implicada en la mayor parte de la activación dependiente de hormona. En respuesta a hormona, STAT5A se recluta a la región distal y PR a las regiones distal y proximal del promotor. El reclutamiento de PR se debe a dos mecanismos diferentes, la unión directa de PR a la región proximal, y la implicación vía JAK/STAT en el reclutamiento a la región distal. La inducción del gen 11&#61538;-HSD2 por hormonas disminuye parcialmente por inhibidores de MAPK y PI3K/Akt y totalmente por inhibidores de JAK/STAT. Así, los efectos citoplasmáticos del PR están implicados en la inducción del gen progesterona. La forma activa de la ARN-polimerasa II es reclutada por la inducción con hormonas a la región distal del promotor 11&#61538;-HSD2 y la región distal tiene respuesta a hormonas por sí misma, indicando que la inducción del gen por hormonas empieza antes del sitio de inicio de transcripción descrito previamente. / The human 11&#61538;-HSD2 gene is a model to investigate the contribution of steroid hormone receptors effects on a progesterone responsive promoter in breast cancer cells. Deletion analysis of the 11&#61538;-HSD2 promoter showed that the distal region is involved in most of the hormone-dependent activation. ChIP showed hormone-dependent STAT5A-recruitment to the distal region and PR-recruitment to the distal and proximal promoter regions. Results suggest two different mechanisms of hormone-induced PR-recruitment, since cells stably expressing PR containing a mutated DNA-binding domain have affected hormone-dependent PR-recruitment to proximal promoter, and JAK/STAT pathway inhibition blocks PR-recruitment to distal promoter. Hormone-stimulated 11&#61538;-HSD2 gene-expression was partially decreased by MAPK and PI3K/AKT pathway inhibitors and totally blocked by JAK/STAT pathways inhibitors, indicating that cytoplasmic PR effects involvement in progestin-induced 11&#61538;-HSD2 expression. Importantly, upon hormone induction active RNA-polymerase II is recruited from the 11&#61538;-HSD2 distal promoter region and the distal minimal promoter has hormone-responsiveness by itself, suggesting that progesterone-dependent 11&#61538;-HSD2 expression starts upstream the previously characterized transcription start site.