ChREBP has emerged as one of the key controllers of hepatic lipogenesis. While the function of ChREBP has been extensively investigated, mechanisms underlying its transcriptional regulation remain largely unknown. We located a conserved POU-binding site within mammalian ChREBP promoters, and demonstrated that the POU homeodomain protein Oct-1 binds to this site in the human HepG2 cell line. Oct-1 transfection significantly repressed ChREBP promoter activity 50-75%. Conversely, knockdown of Oct-1 expression with shRNA significantly increased ChREBP expression levels. Furthermore, insulin treatment resulted in a two-fold activation of ChREBP promoter activity, and stimulated endogenous ChREBP expression. We found that the stimulatory effect of insulin on the ChREBP promoter is at least partially dependent on the presence of the POU-binding site, and that insulin treatment reduced Oct-1 expression. Our observations identify Oct-1 as a transcriptional repressor of ChREBP, and suggest that insulin stimulates ChREBP expression via attenuating the repressive effect of Oct-1.

ChREBP has emerged as one of the key controllers of hepatic lipogenesis. While the function of ChREBP has been extensively investigated, mechanisms underlying its transcriptional regulation remain largely unknown. We located a conserved POU-binding site within mammalian ChREBP promoters, and demonstrated that the POU homeodomain protein Oct-1 binds to this site in the human HepG2 cell line. Oct-1 transfection significantly repressed ChREBP promoter activity 50-75%. Conversely, knockdown of Oct-1 expression with shRNA significantly increased ChREBP expression levels. Furthermore, insulin treatment resulted in a two-fold activation of ChREBP promoter activity, and stimulated endogenous ChREBP expression. We found that the stimulatory effect of insulin on the ChREBP promoter is at least partially dependent on the presence of the POU-binding site, and that insulin treatment reduced Oct-1 expression. Our observations identify Oct-1 as a transcriptional repressor of ChREBP, and suggest that insulin stimulates ChREBP expression via attenuating the repressive effect of Oct-1.

C-reactive protein (CRP), a plasma protein of the innate immune system, is produced by hepatocytes. A critical regulatory region (-42 to -57) on the CRP promoter contains binding site for the IL-6-activated transcription factor C/EBPβ. The IL-1β-activated transcription factor NF-κB binds to a κB site located nearby (-63 to -74). The κB site overlaps an octamer motif (-59 to -66) which is the binding site for the constitutively active transcription factor Oct-1. Oct-1 is known to function both as a transcriptional repressor and as an activator depending upon the promoter context. Also, Oct-1 can regulate gene expression either by binding directly to the promoter or by interacting with other transcription factors bound to the promoter. The aim of this study was to investigate the functions of Oct-1 in regulating CRP expression. In luciferase transactivation assays, overexpressed Oct-1 inhibited (IL-6 + IL-1β)-induced CRP expression in Hep3B cells. Deletion of the Oct-1 site from the promoter drastically reduced the cytokine response because the κB site was altered as a consequence of deleting the Oct-1 site. Surprisingly, overexpressed Oct-1 inhibited the residual (IL-6 + IL-1β)-induced CRP expression through the promoter lacking the Oct-1 site. Similarly, deletion of the Oct-1 site reduced the induction of CRP expression in response to overexpressed C/EBPβ, and overexpressed Oct-1 inhibited C/EBPβ-induced CRP expression through the promoter lacking the Oct-1 site. We conclude that Oct-1 acts as a transcriptional repressor of CRP expression and it does so by occupying its cognate site on the promoter and also via other transcription factors by an as yet undefined mechanism.

Les anthracyclines, comme la doxorubicine (DOX) ou la daunorubicine (DNR), sont utilisées dans le traitement d’une grande variété de cancers allant des lymphomes, au cancer du sein, en passant par certaines leucémies. Encore aujourd’hui, beaucoup pensent que les anthracyclines entrent dans les cellules par diffusion passive, toutefois, la plupart de ces mêmes personnes sont d’accord pour dire que la p-glycoprotéine est responsable d’exporter ces molécules hors de la cellule. Mais pourquoi une molécule aurait besoin d’un transporteur pour sortir de la cellule, et pas pour y entrer ? Qu’est-ce qui ferait que la diffusion passive fonctionnerait dans un sens, mais pas dans l’autre, d’autant que l’entrée des anthracyclines dans les cellules est très rapide ? Nous pensons qu’il existe bel et bien un transporteur responsable de faire passer les anthracyclines du milieu extracellulaire au cytoplasme, et nous voulons développer un modèle de levure qui permettrait de déterminer si une protéine, un transporteur, issue d’un autre organisme eucaryote est en mesure de transporter la DOX à l’intérieur de la cellule. Pour ce faire, nous avons rassemblé un groupe de mutants présentant une déficience dans l’absorption d’autres molécules chargées positivement telles que la bléomycine ou le NaD1 et avons déterminé le taux d’absorption de DOX de chacun de ces mutants. Les simples mutants sam3Δ ou dur3Δ n’ont montré qu’une faible réduction de l’absorption de DOX, voire, aucune, par rapport à la souche parentale. Si le double mutant sam3Δdur3Δ a montré une réduction relativement importante de l’absorption de DOX, c’est le mutant agp2Δ qui présentait la plus grande réduction d’absorption de DOX, ainsi qu’une résistance notable à son effet létal. Nous avons utilisé, par la suite, ce mutant pour exprimer, à l’aide d’un vecteur d’expression, une protéine du ver Caenorhabditis elegans, OCT-1 (CeOCT-1). Les résultats ont montré que cette protéine était en mesure de restaurer l’absorption de DOX, compromise chez le mutant agp2Δ ainsi que d’augmenter la sensibilité de la souche parentale à son effet létal, lorsqu’exprimée chez celle-ci. Cela suggère que CeOCT-1 est un transporteur fonctionnel de DOX et contredit également le dogme selon lequel les anthracyclines entrent dans les cellules par diffusion passive. / Anthracyclines such as doxorubicin (DOX) or daunorubicin (DNR) are used for treatment of a wide variety of cancers from lymphomas to breast cancer going through some leukemia. Still today, several people think that anthracyclines enter cells through passive diffusion, but these same people believe that the p-glycoprotein is involved in efflux of anthracyclines. We think that there is a transporter responsible for uptake of anthracyclines by cells and we want to develop a yeast model that would allow us to determine if protein from another eukaryote organism could be able to transport DOX into yeast cells. In order to do that, we gathered a group of mutants that are already known to be resistant to other positively charged molecules such as bleomycin and NaD1 and determined their ability to take up DOX. The simple mutants sam3Δ or dur3Δ showed no or slight decrease of DOX compared to parent. If the double mutant sam3Δdur3Δ showed rather significant uptake decrease, it is the agp2Δ that showed the grater decrease in DOX uptake as a resistance to its lethal effect. We used that mutant to express a protein from the worm Caenorhabditis elegans, OCT-1 (CeOCT-1). The results showed that this protein was able restore back the uptake of DOX that was compromised in agp2Δ and was also able to increase the sensitivity of the parent to its lethal effect. This leads us to believe that CeOCT-1 is functional transporter for DOX and goes against the dogma wherein anthracyclines enter the cells through passive diffusion.

Human C-reactive protein (CRP) is the prototypic acute phase protein whose serum concentration increases rapidly during inflammation. CRP is also associated with atherosclerosis; it is deposited at lesion sites where it may interact with modified lipoproteins. There are 2 major questions regarding CRP: 1. How is the serum concentration of CRP regulated? 2. What are the functions of CRP in atherosclerosis? Our first aim was to determine the role of the constitutively expressed transcription factor Oct-1 in regulating CRP gene expression. We found that Oct-1 overexpression inhibited (IL-6+IL-1β)- induced CRP gene expression; maximal inhibition required the binding of Oct-1 to an octamer motif at (-59 to -66) on the CRP promoter. Oct-1 overexpression inhibited both (IL-6+IL-1β)- induced and C/EBPβ-induced CRP gene expression even when the Oct-1 site was deleted. These findings suggest that Oct-1 is a repressor of CRP gene expression that acts via binding to its cognate site on the CRP promoter as well as through indirect interactions with other promoterbound transcription factors. Our second aim was to investigate the interaction of CRP with oxidized low density lipoprotein (ox-LDL). Acidic pH, a hallmark of atherosclerotic lesions, reversibly alters CRP structure and exposes a hidden binding site that enables CRP to bind ox-LDL. Using site-directed mutagenesis we constructed a CRP mutant (E42Q) that showed significant binding to ox-LDL at physiological pH. E42Q CRP required a less acidic pH for maximal binding and bound ox-LDL more efficiently than wild type CRP at any pH. We then examined if reactive oxygen species also induced CRP – ox-LDL interaction. H2O2-treated CRP bound ox-LDL at physiological pH. Like acidic pH, H2O2-treatment induced only a local structural change exposing the ox-LDL binding site. E42Q and H2O2-modified CRP are tools to study the function of CRP in animal models of atherosclerosis, which may not have an inflammatory environment sufficient to modify CRP and induce binding to atherogenic ox-LDL. We conclude that Oct-1 is one of the critical regulators of CRP gene expression, and that CRP can be modified in vitro to convert it into an atherogenic LDL-binding molecule.

Une démarche de type génie de la réaction chimique est appliquée à l'hydroformylation modèle d'oct-1-ène par des complexes lipophobes du rhodium préparés à partir de Rh(CO)2(acac) en phase liquide ionique ([Bmim][PF6]) ou en phase liquide ionique supportée sur silice. La réaction étant contrôlée par la concentration des réactifs dans la phase liquide ionique catalytique, une première étape a consisté à mesurer ces concentrations tant pour les deux gaz (H2 et CO) que pour l'oct-1-ène à différentes températures et pressions. Diverses méthodes de mesures sont utilisées pour la solubilité de l'oléfine : thermogravimétrie et chromatographie gazeuse après extraction multiple d’espace de tête, en présence de solvant (décane) et du produit de la réaction (nonanal). Le transfert gaz-liquide, qui peut conditionner la vitesse de réaction dans ces milieux visqueux, est également mesuré par une technique dynamique de variation de pression, en liquide ionique pur et en mélange biphasique liquide ionique-phase organique, dans un réacteur autoclave à autoaspiration de gaz par arbre creux. Une corrélation générale est proposée montrant une forte influence de la vitesse d'agitation.Une étude cinétique est réalisée en conditions de transferts non limitants en gaz-liquide organique-liquide ionique avec la TPPTS comme ligand. Les comportements habituels de l’hydroformylation  en  phase organique ou en phase aqueuse sont retrouvés : ordre voisin de 1 pour H2, inhibition par CO à forte concentration, énergie d'activation élevée. Si le turnover est convenable (70 h-1), le rapport n/iso est par contre très bas ce qui n'est pas en faveur de ce système catalytique. Quelques résultats permettent aussi une première analyse de la catalyse biphasique avec le ligand sulfoxantphos et de la catalyse en phase liquide ionique supportée sur silice avec la TPPTS. / A chemical reaction engineering approach is applied to the hydroformylation of 1-octene using lipophobic complexes of rhodium prepared from Rh(CO)2(acac) in ionic liquid phase ([Bmim] [PF6]) or in the ionic liquid phase supported on silica. As the reaction is controlled by the concentration of the reagents in the catalytic ionic liquid phase, the concentrations of both gases (H2 and CO) and also of 1-octene are measured at various temperatures and pressures as an initial step. Different methods are used for the measurement of the olefin solubility inside the ionic liquid: thermogravimetry and multiple headspace chromatography, in the presence of solvent (decane) and reaction product (nonanal). The gas-liquid mass transfer, which can be a rate controlling step in these viscous media, is also measured by a dynamic technique of pressure variation, both in case of pure ionic liquid and biphasic mixture of ionic liquid and organic phase, in an autoclave reactor with self induced stirrer. A general correlation is proposed showing the strong influence of the agitation speed. A kinetic study is realized in no gas–liquid nor organic–ionic liquid mass transfer limiting conditions (chemical regime) with TPPTS as ligand. The usual hydroformylation behaviour is observed, as already found in organic phase or in aqueous phase: order close to 1 for H2, inhibition by CO at large concentration, and high activation energy. If the turnover frequency is suitable (70 h-1), the n/iso ratio is very low which is not favourable to this catalytic system. Some experimental results also allow a first analysis of biphasic catalysis with sulfoxantphos ligand and of ionic liquid phase supported catalysis with TPPTS ligand.