No rim, a maior parte da carga de glicose filtrada é reabsorvida na porção inicial do túbulo proximal, no qual são co-expressos os transportadores: SGLT2 e GLUT2. No diabetes mellitus ocorre aumento no fluxo transepitelial de glicose, o que decorre de aumento na expressão desses transportadores, e pode ser revertido pelo tratamento com insulina. Os fatores transcricionais HNF1A, HNF4A e FOXA2 são descritos como potenciais reguladores do gene Slc2a2. A proteína AKT medeia efeitos da insulina, e é capaz de ativar fatores transcricionais. O objetivo deste estudo foi investigar em linhagem celular IRPTC, o efeito da alta concentração de glicose e da insulina sobre a expressão de Slc2a2/GLUT2 e Slc5a2/SGLT2, assim como a participação da AKT e dos fatores transcricionais. Observamos que a alta concentração de glicose aumentou a expressão do Slc2a2/GLUT2 e a atividade de ligação dos fatores transcricionais HNF4A e FOXA2 na região promotora do gene Slc2a2, por mecanismo mediado pela AKT. A insulina reverteu o efeito sobre o Slc2a2, porém não alterou o conteúdo de GLUT2. / Glucose filtrated load is reabsorbed in renal proximal tubule by the coordinate action of the glucose transporters SGLT2 and GLUT2. In diabetes, renal glucose reabsorption increases; that involves overexpression of the glucose transporters, and is reversed by insulin therapy. The transcription factors HNF1A, HNF4A and FOXA2 have been proposed as modulators of Slc2a2 gene expression. The AKT protein is an important mediator of insulin action, and has been able to activate transcription factors. The present study investigates in immortalized rat proximal tubule cells the effects of high glucose and insulin concentrations upon the Slc2a2/GLUT2 and Slc5a2/SGLT2 expression, as well as the participation of AKT, HNF1A, HNF4A and FOXA2. On the other hand, 25 mM glucose increased the expression of Slc2a2GLUT2, which was accompanied by increased HNF4A and FOXA2 binding in the Slc2a2 promoter, in an AKT-mediated way. Insulin reversed the Slc2a2 mRNA regulation, but did not alter GLUT2 content.

No rim, a maior parte da carga de glicose filtrada é reabsorvida na porção inicial do túbulo proximal, no qual são co-expressos os transportadores: SGLT2 e GLUT2. No diabetes mellitus ocorre aumento no fluxo transepitelial de glicose, o que decorre de aumento na expressão desses transportadores, e pode ser revertido pelo tratamento com insulina. Os fatores transcricionais HNF1A, HNF4A e FOXA2 são descritos como potenciais reguladores do gene Slc2a2. A proteína AKT medeia efeitos da insulina, e é capaz de ativar fatores transcricionais. O objetivo deste estudo foi investigar em linhagem celular IRPTC, o efeito da alta concentração de glicose e da insulina sobre a expressão de Slc2a2/GLUT2 e Slc5a2/SGLT2, assim como a participação da AKT e dos fatores transcricionais. Observamos que a alta concentração de glicose aumentou a expressão do Slc2a2/GLUT2 e a atividade de ligação dos fatores transcricionais HNF4A e FOXA2 na região promotora do gene Slc2a2, por mecanismo mediado pela AKT. A insulina reverteu o efeito sobre o Slc2a2, porém não alterou o conteúdo de GLUT2. / Glucose filtrated load is reabsorbed in renal proximal tubule by the coordinate action of the glucose transporters SGLT2 and GLUT2. In diabetes, renal glucose reabsorption increases; that involves overexpression of the glucose transporters, and is reversed by insulin therapy. The transcription factors HNF1A, HNF4A and FOXA2 have been proposed as modulators of Slc2a2 gene expression. The AKT protein is an important mediator of insulin action, and has been able to activate transcription factors. The present study investigates in immortalized rat proximal tubule cells the effects of high glucose and insulin concentrations upon the Slc2a2/GLUT2 and Slc5a2/SGLT2 expression, as well as the participation of AKT, HNF1A, HNF4A and FOXA2. On the other hand, 25 mM glucose increased the expression of Slc2a2GLUT2, which was accompanied by increased HNF4A and FOXA2 binding in the Slc2a2 promoter, in an AKT-mediated way. Insulin reversed the Slc2a2 mRNA regulation, but did not alter GLUT2 content.

Gastrulation is one of the most critical events of embryogenesis, generating the three primary germ layers (definitive endoderm, mesoderm, and ectoderm) and establishing the embryonic body plan. The definitive endoderm, which generates the lungs, liver, pancreas, and digestive tract, has become a tissue of particular interest in recent years. Understanding definitive endoderm formation and patterning will greatly aid progress in the in vitro differentiation of embryonic stem cells to definitive endoderm for use in treatment of diseases such as diabetes and hepatitis as an alternative for whole organ replacement. Gene targeting studies have demonstrated a critical role for the Nodal signaling pathway and the forkhead transcription factors Foxh1 and Foxa2 in specification of a group of cells referred to as the anterior primitive streak (APS). However, the transcriptional targets of Foxh1 and/or Foxa2 other than Nodal that regulate specification of this group of cells are currently unknown. Fate mapping and lineage tracing experiments have shown the APS to be the source of the definitive endoderm. However, many questions regarding specification and patterning of the definitive endoderm remain. The study of this tissue has been hampered by the lack of genetic markers specific for the definitive endoderm as many of the current markers, including Cerl, Foxa2, and Sox17, are also expressed in the visceral endoderm, an extraembryonic tissue. To further investigate the role of Foxh1 in specification of the anterior primitive streak and to address the lack of genetic markers for the definitive endoderm we performed expression profiling on post-implantation mouse embryos using Affymetrix™ GeneChips®. From this analysis we identified and characterized a novel marker of the mouse definitive endoderm. Examination of this, and other, novel endoderm markers in Foxh1 and Foxa2 deficient mouse embryos revealed that contrary to current models of definitive endoderm formation, we find some definitive endoderm is formed in these mutants. Specifically, specification of the midgut and hindgut definitive endoderm is largely unaffected, while foregut formation is severely affected. These results suggest that the formation of the midgut and hindgut definitive endoderm populations is independent of the anterior primitive streak and separate from the foregut definitive endoderm. This represents a major insight into the mechanisms regulating endoderm formation and patterning.

Gastrulation is one of the most critical events of embryogenesis, generating the three primary germ layers (definitive endoderm, mesoderm, and ectoderm) and establishing the embryonic body plan. The definitive endoderm, which generates the lungs, liver, pancreas, and digestive tract, has become a tissue of particular interest in recent years. Understanding definitive endoderm formation and patterning will greatly aid progress in the in vitro differentiation of embryonic stem cells to definitive endoderm for use in treatment of diseases such as diabetes and hepatitis as an alternative for whole organ replacement. Gene targeting studies have demonstrated a critical role for the Nodal signaling pathway and the forkhead transcription factors Foxh1 and Foxa2 in specification of a group of cells referred to as the anterior primitive streak (APS). However, the transcriptional targets of Foxh1 and/or Foxa2 other than Nodal that regulate specification of this group of cells are currently unknown. Fate mapping and lineage tracing experiments have shown the APS to be the source of the definitive endoderm. However, many questions regarding specification and patterning of the definitive endoderm remain. The study of this tissue has been hampered by the lack of genetic markers specific for the definitive endoderm as many of the current markers, including Cerl, Foxa2, and Sox17, are also expressed in the visceral endoderm, an extraembryonic tissue. To further investigate the role of Foxh1 in specification of the anterior primitive streak and to address the lack of genetic markers for the definitive endoderm we performed expression profiling on post-implantation mouse embryos using Affymetrix™ GeneChips®. From this analysis we identified and characterized a novel marker of the mouse definitive endoderm. Examination of this, and other, novel endoderm markers in Foxh1 and Foxa2 deficient mouse embryos revealed that contrary to current models of definitive endoderm formation, we find some definitive endoderm is formed in these mutants. Specifically, specification of the midgut and hindgut definitive endoderm is largely unaffected, while foregut formation is severely affected. These results suggest that the formation of the midgut and hindgut definitive endoderm populations is independent of the anterior primitive streak and separate from the foregut definitive endoderm. This represents a major insight into the mechanisms regulating endoderm formation and patterning.

Gastrulation is one of the most critical events of embryogenesis, generating the three primary germ layers (definitive endoderm, mesoderm, and ectoderm) and establishing the embryonic body plan. The definitive endoderm, which generates the lungs, liver, pancreas, and digestive tract, has become a tissue of particular interest in recent years. Understanding definitive endoderm formation and patterning will greatly aid progress in the in vitro differentiation of embryonic stem cells to definitive endoderm for use in treatment of diseases such as diabetes and hepatitis as an alternative for whole organ replacement. Gene targeting studies have demonstrated a critical role for the Nodal signaling pathway and the forkhead transcription factors Foxh1 and Foxa2 in specification of a group of cells referred to as the anterior primitive streak (APS). However, the transcriptional targets of Foxh1 and/or Foxa2 other than Nodal that regulate specification of this group of cells are currently unknown. Fate mapping and lineage tracing experiments have shown the APS to be the source of the definitive endoderm. However, many questions regarding specification and patterning of the definitive endoderm remain. The study of this tissue has been hampered by the lack of genetic markers specific for the definitive endoderm as many of the current markers, including Cerl, Foxa2, and Sox17, are also expressed in the visceral endoderm, an extraembryonic tissue. To further investigate the role of Foxh1 in specification of the anterior primitive streak and to address the lack of genetic markers for the definitive endoderm we performed expression profiling on post-implantation mouse embryos using Affymetrix™ GeneChips®. From this analysis we identified and characterized a novel marker of the mouse definitive endoderm. Examination of this, and other, novel endoderm markers in Foxh1 and Foxa2 deficient mouse embryos revealed that contrary to current models of definitive endoderm formation, we find some definitive endoderm is formed in these mutants. Specifically, specification of the midgut and hindgut definitive endoderm is largely unaffected, while foregut formation is severely affected. These results suggest that the formation of the midgut and hindgut definitive endoderm populations is independent of the anterior primitive streak and separate from the foregut definitive endoderm. This represents a major insight into the mechanisms regulating endoderm formation and patterning. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

No description available.

The mammalian skin provides a necessary barrier between the organism and the environment, defending against loss of water and solutes, preventing the invasion of pathogens as well as protecting against chemical and physical assault. Claudin (Cldn)-based Tight Junctions (TJs) are the main functional part of the skin barrier. In particular, Cldn6 through its cytoplasmic tail has been shown to be important for barrier function. In other to further investigate the role of the Cldn6 tail in TJ-function, we developed Cldn6 mouse mutants carrying varying truncations of the Cldn6 tail. Both of these mice present with epidermal differentiation perturbations and delayed barrier function that is repaired later in life. These studies support the importance of the tail portion of the Cldn molecules in epidermal differentiation and barrier function. In addition, both of these mouse models are useful for the study of barrier function in preterm infants and in aging, with the hope of developing novel therapeutics for the alleviation of barrier dysfunction. Cdx is a family of homeodomain (HD) transcription factors (TFs) essential for many key developmental processes. In particular, Cdx2 is important for the establishment and maintenance of posterior identity in the developing endoderm. In spite of this, only a few Cdx targets in the developing endoderm have been discovered. In addition, the interplay between Cdx and its targets within the endoderm is poorly understood. In this study, we show that the forkhead box transcription factor, Foxa2 is a Cdx2 target. We also show that Foxa2 and Cdx2 physically and genetically interact to regulate a subset of genes that are implicated in endodermal development. These studies help to further our understanding of endoderm biology with the goal of developing new strategies to diagnose and treat diseases associated with defective endoderm development.

Le foie est un organe clef dans la régulation du métabolisme énergétique de l’organisme. La superfamille des récepteurs nucléaires y joue un rôle primordial de senseur de l’environnement métabolique. Parmi ces récepteurs nucléaires, le récepteur des acides biliaires FXR participe aux mécanismes de régulation de l’activité du foie à travers son action sur les métabolismes des acides biliaires, des glucides et des lipides. FXR est devenu ainsi une cible thérapeutique potentielle dans le traitement de nombreuses maladies impliquant un désordre métabolique comme les cholestases, le diabète de type 2 ou la stéatohépatite non-alcoolique. Malgré des résultats prometteurs dans le traitement de la stéatohépatite non-alcoolique, le traitement de patients avec un agoniste de FXR, le INT747, semble augmenter la concentration plasmatique du LDL-Cholestérol et diminue la concentration du HDL-Cholestérol suggérant un risque accru de développement d’athérosclérose. Ces effets sur le profil lipidique sont le frein majeur du développement clinique de ses agonistes. Les mécanismes moléculaires impliqués dans la régulation par FXR de nombreuses voies comme le métabolisme des lipides et du cholestérol sont peu explorés et peu compris. Compte-tenu de ces informations, il est d’autant plus intéressant d’approfondir les connaissances de ces mécanismes et d’identifier des facteurs ou de nouveaux partenaires capables de moduler l’activité transcriptionnelle de FXR plus spécifiquement dans le cadre du contrôle du métabolisme des lipides et du cholestérol. L’un des facteurs de transcription connu comme régulateur majeur de ces voies métaboliques dans le foie est le facteur de transcription de la famille forkhead FOXA2. Ce facteur de transcription, dont l’activité est dépendante des conditions physiologiques, est activé par le glucagon et inhibé par l’insuline. De plus, c’est également un régulateur du métabolisme des acides biliaires, du cholestérol et des lipides.L’objectif de cette thèse a été d’étudier l’interaction entre les voies de signalisation de FXR et de FOXA2 dans différentes lignées cellulaires d’hépatocytes humains ou murins et dans le foie. Nous avons établi que FOXA2 et FXR sont colocalisés sur la chromatine des cellules HepG2 et dans le foie de souris à proximité de gènes impliqués dans la régulation du métabolisme des lipides et du cholestérol. Ces zones de cofixation de FXR et de FOXA2 présentent très peu de motifs de fixation de FOXA2 suggérant l’implication d’autres motifs de fixation non connus ou un mécanisme de type « tethering ». Nous avons montré que la fixation de FOXA2 à ces zones de cofixation avec FXR est augmentée par l’activation de FXR par son agoniste, le GW4064, évoquant une potentielle interaction entre ces deux facteurs. Nous avons démontré que ces deux facteurs interagissaient physiquement et que FOXA2 est un répresseur de l’activité transcriptionnelle de FXR à travers l’utilisation de différentes approches et modèles cellulaires. Finalement, dans les hépatocytes primaires de souris, FOXA2 est impliqué dans la répression de l’activité transcriptionnelle de FXR par le glucagon sur le gène Shp.L’ensemble de ce travail met en évidence pour la première fois la répression de l’activité de FXR par le facteur de transcription caractéristique du jeûne FOXA2 à travers un mécanisme moléculaire suggérant une transrépression de type «tethering». Ces résultats présentent un mécanisme inédit par lequel l’activité de FXR peut être modulée par le statut nutritionnel de façon gène-spécifique. / The liver is a key regulator of whole-body energy metabolism. The nuclear receptor super-family plays a leading role in the metabolic sensing of the liver. Among the nuclear receptors, the bile acid nuclear receptor FXR contribute to the modulation of liver activity in particular through the regulation of bile acid, lipids and glucose homeostasis. Consequently, FXR became a potential therapeutic target for many diseases implicated metabolic disorder such as cholestasis, type 2 diabete or Non-Alcoholic Steatohepatitis (NASH). Despite promising results especially on NASH, patient treatment with FXR agonist the INT747 seems to increase LDL-Cholesterol plasma concentrations together with a decreased concentration of HDL-Cholesterol suggesting a higher risk to develop atherosclerosis. These effects on plasma lipid profile are the major break against the development of agonists in clinics. Giving the poor understanding and knowledge of the molecular mechanisms which govern FXR regulation of activity on various signaling pathways, it is of major interest to find new partners and regulators of FXR and especially on lipid and cholesterol homeostasis. One of the transcription factor known to be active in the control of these signaling pathways in the liver is the forkhead box transcription factor FOXA2. This transcription factor whose activity is dependent of physiological conditions is activated by glucagon and inhibited by insulin. In addition, this factor is known to regulate bile acid, cholesterol and lipid metabolism, functions very close from FXR activities in the liver.The objective of this PhD was to study the interaction between FXR and FOXA2 signaling pathways in different hepatic cells lines from human or mouse origin and in the liver. We established that FOXA2 and FXR are colocalised in HepG2 cells and liver chromatin near genes implicated in the lipid and cholesterol metabolism. These FXR/FOXA2 cobinding zones present few consensus FOXA2 response elements suggesting the implication of non consensus binding motifs or a “tethering” mechanism. We show that FOXA2 binding to FXR/FOXA2 cobinding zones is increased when FXR is activated and/or more present in the chromatin evoking a potential interaction between these two factors. We demonstrate that FXR and FOXA2 interact physically and that FOXA2 is a repressor of FXR transcriptional activity using different approaches and cellular models. Finally, we show that FOXA2 is implicated in glucagon-induced repression of FXR transcriptional activity on Shp gene.To conclude, our results show for the first time that the fasting key regulator of lipid and cholesterol homeostasis FOXA2 is a repressor of FXR transcriptional activity through a plausible mechanism involving “tethering” process. This work gives a novel mechanism by which FXR activity can be modified by nutritional status in a gene-specific manner.

We have previously shown that the arcuate organization of cell fates within the ventral midbrain critically depends upon the morphogen, Sonic Hedgehog (SHH), which is secreted from a signaling center located along the ventral midline, called the floor plate (FP). Thus, it is ultimately the specification of the FP that is responsible for the patterning and specification of ventral midbrain cell fates. Interestingly, we have found that the chick midbrain FP can be divided into medial (MFP) and lateral (LFP) regions on the basis of gene expression, mode of induction and function. Overexpression of SHH alone is sufficient to recapitulate the entire pattern of ventral cell fates, although remarkably it cannot induce MFP, consistent with the observation that the MFP is refractory to any perturbations of HH signaling. In contrast, overexpression of the winged-helix transcription factor FOXA2/HNF3[beta]robustly induced the MFP fate throughout ventral midbrain while blocking its activity resulted in the absence of the MFP. Thus, by analyzing the differences between SHH and FOXA2 blockade and overexpression, we were able to attribute functions to each the LFP and the MFP. Notably, we observed that FOXA2 overexpression caused a bending of the midbrain neurepithelium that resembled the endogenous median hinge-point observed during neurulation. Additionally, FOXA2 misexpression led to a robust induction of DA progenitors and neurons that was never observed after SHH expression alone. In contrast, we found that all other ventral cell types required HH signaling directly, at a distance and early on in the development of the midbrain when its tissue size is relatively small. Additionally, HH blockade resulted in increased cell-scatter of the arcuate territories and in the disruption of the regional boundaries between the ventral midbrain and adjacent tissue. Thus, we bring new insight into the mechanism by which midbrain FP is specified and ascribe functional roles to its subregions. We propose that while the MFP regulates the production of dopaminergic progenitors and the changes in cellshape required for bending and shaping the neural tube, the LFP appears to be largely responsible for cell survival and the formation of a spatially coherent pattern of midbrain cell fates. / text

No description available.