Pascale Bossard

Expression of COUP-TFII in metabolic tissues during development

In mammals, the COUP-TF-family consisting of two structurally related proteins, COUP-TFI and COUP-TFII belongs to the orphan member of the steroid/thyroid hormone receptor superfamily. In an attempt to gain insights into the role of COUP-TFII, we examined developmental expression pattern of the mouse COUP-TFII focusing our studies on endoderm-derived tissues, pancreas and liver in particular. Independent lines of transgenic mice expressing Escherichia coli beta-galactosidase driven by the COUP-TFII promoter were generated. Embryonic expression of the beta-gal protein at day 9 of gestation was detected in the notochord, the ventral neural tube and, interestingly, in the gut endoderm, a site where COUP-TFII has not been detected previously. Between 9.5 and 11.5 dpc, beta-gal expression pattern that was established earlier persisted and sections revealed a staining of the common atrial chamber of the heart. At 15.5 dpc, beta-gal activity was found in all endoderm-derived tissues. We found that COUP-TFII mRNA and protein were present in fetal and adult hepatocytes. Finally, COUP-TFII expression was detected in pancreas, as judged by co-expression of the beta-gal in some of the glucagon and PDX1 positive-cells at 12.5 dpc and co-expression with insulin positive-cells at 15.5 dpc. In adult pancreas, COUP-TFII protein was present in the endocrine islet cells.

COUP-TFII Controls Mouse Pancreatic β-Cell Mass through GLP-1-β-Catenin Signaling Pathways

Background The control of the functional pancreatic β-cell mass serves the key homeostatic function of releasing the right amount of insulin to keep blood sugar in the normal range. It is not fully understood though how β-cell mass is determined. Methodology/Principal Findings Conditional chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)-deficient mice were generated and crossed with mice expressing Cre under the control of pancreatic duodenal homeobox 1 (pdx1) gene promoter. Ablation of COUP-TFII in pancreas resulted in glucose intolerance. Beta-cell number was reduced at 1 day and 3 weeks postnatal. Together with a reduced number of insulin-containing cells in the ductal epithelium and normal β-cell proliferation and apoptosis, this suggests decreased β-cell differentiation in the neonatal period. By testing islets isolated from these mice and cultured β-cells with loss and gain of COUP-TFII function, we found that COUP-TFII induces the expression of the β-catenin gene and its target genes such as cyclin D1 and axin 2. Moreover, induction of these genes by glucagon-like peptide 1 (GLP-1) via β-catenin was impaired in absence of COUP-TFII. The expression of two other target genes of GLP-1 signaling, GLP-1R and PDX-1 was significantly lower in mutant islets compared to control islets, possibly contributing to reduced β-cell mass. Finally, we demonstrated that COUP-TFII expression was activated by the Wnt signaling-associated transcription factor TCF7L2 (T-cell factor 7-like 2) in human islets and rat β-cells providing a feedback loop. Conclusions/Significance Our findings show that COUP-TFII is a novel component of the GLP-1 signaling cascade that increases β-cell number during the neonatal period. COUP-TFII is required for GLP-1 activation of the β-catenin-dependent pathway and its expression is under the control of TCF7L2.

AXIN deficiency in human and mouse hepatocytes induces hepatocellular carcinoma in the absence of β-catenin activation

BACKGROUND & AIMS The Wnt/β-catenin pathway is the most frequently deregulated pathway in hepatocellular carcinoma (HCC). Inactivating mutations of the gene encoding AXIN1, a known negative regulator of the Wnt/β-catenin signaling pathway, are observed in about 10% of HCCs. Whole-genome studies usually place HCC with AXIN1 mutations and CTNNB1 mutations in the group of tumors with Wnt/β-catenin activated program. However, it has been shown that HCCs with activating CTNNB1 mutations form a group of HCCs, with a different histology, prognosis and genomic signature to those with inactivating biallelic AXIN1 mutations. We aimed to elucidate the relationship between CTNNB1 mutations, AXIN1 mutations and the activation level of the Wnt/β-catenin program. METHODS We evaluated two independent human HCC datasets for the expression of a 23-β-catenin target genes program. We modeled Axin1 loss of function tumorigenesis in two engineered mouse models and performed gene expression profiling. RESULTS Based on gene expression, we defined three levels of β-catenin program activation: strong, weak or no activation. While more than 80% CTNNB1-mutated tumors were found in the strong or in the weak activation program, most of the AXIN1-mutated tumors (>70%) were found in the subgroup with no activation. We validated this result by demonstrating that mice with a hepatocyte specific AXIN1 deletion developed HCC in the absence of β-catenin induction. We defined a 329-gene signature common in human and mouse AXIN1 mutated HCC that is highly enriched in Notch and YAP oncogenic signatures. CONCLUSIONS AXIN1-mutated HCCs occur independently of the Wnt/β-catenin pathway and involve Notch and YAP pathways. These pathways constitute potentially interesting targets for the treatment of HCC caused by AXIN1 mutations. LAY SUMMARY Liver cancer has a poor prognosis. Defining the molecular pathways involved is important for developing new therapeutic approaches. The Wnt/β-catenin pathway is the most frequently deregulated pathway in hepatocellular carcinoma (HCC). Mutations of AXIN1, a member of this pathway, represent about 10% of HCC mutations. Using both human HCC collections and engineered mouse models of liver cancers with AXIN1 mutation or deletion, we defined a common signature of liver tumors mutated for AXIN1 and demonstrate that these tumors occur independently of the activation of the Wnt/β-catenin pathway.

The Nutritional Induction of COUP-TFII Gene Expression in Ventromedial Hypothalamic Neurons Is Mediated by the Melanocortin Pathway

Background The nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an important coordinator of glucose homeostasis. We report, for the first time, a unique differential regulation of its expression by the nutritional status in the mouse hypothalamus compared to peripheral tissues. Methodology/Principal Findings Using hyperinsulinemic-euglycemic clamps and insulinopenic mice, we show that insulin upregulates its expression in the hypothalamus. Immunofluorescence studies demonstrate that COUP-TFII gene expression is restricted to a subpopulation of ventromedial hypothalamic neurons expressing the melanocortin receptor. In GT1-7 hypothalamic cells, the MC4-R agonist MTII leads to a dose dependant increase of COUP-TFII gene expression secondarily to a local increase in cAMP concentrations. Transfection experiments, using a COUP-TFII promoter containing a functional cAMP responsive element, suggest a direct transcriptional activation by cAMP. Finally, we show that the fed state or intracerebroventricular injections of MTII in mice induce an increased hypothalamic COUP-TFII expression associated with a decreased hepatic and pancreatic COUP-TFII expression. Conclusions/Significance These observations strongly suggest that hypothalamic COUP-TFII gene expression could be a central integrator of insulin and melanocortin signaling pathway within the ventromedial hypothalamus. COUP-TFII could play a crucial role in brain integration of circulating signal of hunger and satiety involved in energy balance regulation.

Un nouveau rôle de l’insuline dans la régulation du métabolisme glucido-lipidique hépatique

> Les facteurs de transcription sterol regulatory element binding protein (SREBP) sont des acteurs majeurs de la regulation du metabolisme lipidique puisqu’ils controlent l’expression des genes du metabolisme des acides gras, des triglycerides et du cholesterol [1]. Ces facteurs appartiennent a une famille composee de trois membres : SREBP-1a, SREBP-1c et SREBP2. L’isoforme SREBP-2 est codee par le gene SREBP-2 alors que SREBP-1a et 1c sont codees par le gene SREBP-1, par l’utilisation de deux promoteurs differents et par epissage alternatif. Les trois isoformes se distinguent par leur localisation tissulaire, leurs genes cibles et la regulation de leur activite transcriptionnelle (Tableau I) [2]. Les facteurs SREBP sont synthetises sous la forme de precurseurs inactifs ancres dans les membranes du reticulum endoplasmique. Le precurseur presente une structure tripartite similaire pour les trois isoformes comprenant un fragment aminoterminal correspondant au facteur de transcription, une boucle situee dans la lumiere du reticulum endoplasmique et un fragment carboxyterminal correspondant a un domaine de regulation. Le clivage proteolytique du precurseur SREBP permet la liberation de la partie transcriptionnellement active qui, une fois liberee, migre dans le noyau pour activer ses genes cibles [3]. Les travaux de Brown et Goldstein ont montre que la transcription et le clivage proteolytique des isoformes SREBP-1a et SREBP-2 etaient actives par la depletion en cholesterol [1]. Pour ces isoformes, la forme mature est liberee par un processus appele RIP (regulated intramembrane proteolysis) faisant intervenir la proteine SCAP (SREBP cleavage activating protein), les proteines Insig (insulin induced gene) et les proteases S1P et S2P [4]. La SCAP joue un double role : celui de cargo proteique puisqu’elle escorte le precurseur SREBP du reticulum endoplasmique vers le Golgi ou ce dernier sera clive par les deux proteases S1P et S2P, et celui de detecteur de la concentration intracellulaire de cholesterol (cholesterol sensor) puisqu’elle possede un domaine se liant au cholesterol. Ainsi, quand la concentration de cholesterol est faible, le complexe SCAP-SREBP migre du reticulum endoplasmique vers le Golgi ou le precurseur est clive par deux proteases. Au contraire, lorsque la concentration de cholesterol est elevee, le complexe SCAPSREBP est retenu dans les membranes du reticulum endoplasmique par les proteiNO UV EL LE S MEDECINE/SCIENCES 2005 ; 21 : 569-92

β-catenin-activated hepatocellular carcinomas are addicted to fatty acids

Objectives CTNNB1-mutated hepatocellular carcinomas (HCCs) constitute a major part of human HCC and are largely inaccessible to target therapy. Yet, little is known about the metabolic reprogramming induced by β-catenin oncogenic activation in the liver. We aimed to decipher such reprogramming and assess whether it may represent a new avenue for targeted therapy of CTNNB1-mutated HCC. Design We used mice with hepatocyte-specific oncogenic activation of β-catenin to evaluate metabolic reprogramming using metabolic fluxes on tumourous explants and primary hepatocytes. We assess the role of Pparα in knock-out mice and analysed the consequences of fatty acid oxidation (FAO) using etomoxir. We explored the expression of the FAO pathway in an annotated human HCC dataset. Results β-catenin-activated HCC were not glycolytic but intensively oxidised fatty acids. We found that Pparα is a β-catenin target involved in FAO metabolic reprograming. Deletion of Pparα was sufficient to block the initiation and progression of β-catenin-dependent HCC development. FAO was also enriched in human CTNNB1-mutated HCC, under the control of the transcription factor PPARα. Conclusions FAO induced by β-catenin oncogenic activation in the liver is the driving force of the β-catenin-induced HCC. Inhibiting FAO by genetic and pharmacological approaches blocks HCC development, showing that inhibition of FAO is a suitable therapeutic approach for CTNNB1-mutated HCC.

The role of chicken ovalbumin upstream promoter transcription factor II in the regulation of hepatic fatty acid oxidation and gluconeogenesis in newborn mice

Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an orphan nuclear receptor involved in the control of numerous functions in various organs (organogenesis, differentiation, metabolic homeostasis, etc.). The aim of the present work was to characterize the regulation and contribution of COUP-TFII in the control of hepatic fatty acid and glucose metabolisms in newborn mice. Our data show that postnatal increase in COUP-TFII mRNA levels is enhanced by glucagon (via cAMP) and PPARα. To characterize COUP-TFII function in the liver of suckling mice, we used a functional (dominant negative form; COUP-TFII-DN) and a genetic (shRNA) approach. Adenoviral COUP-TFII-DN injection induces a profound hypoglycemia due to the inhibition of gluconeogenesis and fatty acid oxidation secondarily to reduced PEPCK, Gl-6-Pase, CPT I, and mHMG-CoA synthase gene expression. Using the crossover plot technique, we show that gluconeogenesis is inhibited at two different levels: 1) pyruvate carboxylation and 2) trioses phosphate synthesis. This could result from a decreased availability in fatty acid oxidation arising cofactors such as acetyl-CoA and reduced equivalents. Similar results are observed using the shRNA approach. Indeed, when fatty acid oxidation is rescued in response to Wy-14643-induced PPARα target genes (CPT I and mHMG-CoA synthase), blood glucose is normalized in COUP-TFII-DN mice. In conclusion, this work demonstrates that postnatal increase in hepatic COUP-TFII gene expression is involved in the regulation of liver fatty acid oxidation, which in turn sustains an active hepatic gluconeogenesis that is essential to maintain an appropriate blood glucose level required for newborn mice survival.