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1.
Mol Ther ; 32(4): 1125-1143, 2024 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-38311851

RESUMEN

The CTNNB1 gene, encoding ß-catenin, is frequently mutated in hepatocellular carcinoma (HCC, ∼30%) and in hepatoblastoma (HB, >80%), in which DLK1/DIO3 locus induction is correlated with CTNNB1 mutations. Here, we aim to decipher how sustained ß-catenin activation regulates DLK1/DIO3 locus expression and the role this locus plays in HB and HCC development in mouse models deleted for Apc (ApcΔhep) or Ctnnb1-exon 3 (ß-cateninΔExon3) and in human CTNNB1-mutated hepatic cancer cells. We identified an enhancer site bound by TCF-4/ß-catenin complexes in an open conformation upon sustained ß-catenin activation (DLK1-Wnt responsive element [WRE]) and increasing DLK1/DIO3 locus transcription in ß-catenin-mutated human HB and mouse models. DLK1-WRE editing by CRISPR-Cas9 approach impaired DLK1/DIO3 locus expression and slowed tumor growth in subcutaneous CTNNB1-mutated tumor cell grafts, ApcΔhep HB and ß-cateninΔExon3 HCC. Tumor growth inhibition resulted either from increased FADD expression and subsequent caspase-3 cleavage in the first case or from decreased expression of cell cycle actors regulated by FoxM1 in the others. Therefore, the DLK1/DIO3 locus is an essential determinant of FoxM1-dependent cell proliferation during ß-catenin-driven liver tumorigenesis. Targeting the DLK1-WRE enhancer to silence the DLK1/DIO3 locus might thus represent an interesting therapeutic strategy to restrict tumor growth in primary liver cancers with CTNNB1 mutations.


Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Humanos , Ratones , beta Catenina/genética , beta Catenina/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Carcinogénesis/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Cateninas/genética , Cateninas/metabolismo , Proliferación Celular/genética , Neoplasias Hepáticas/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Regulación hacia Arriba
2.
J Hepatol ; 77(2): 424-435, 2022 08.
Artículo en Inglés | MEDLINE | ID: mdl-35257829

RESUMEN

BACKGROUND & AIMS: One-third of hepatocellular carcinomas (HCCs) harbor mutations activating the ß-catenin pathway, predominantly via mutations in the CTNNB1 gene itself. Mouse models of Apc loss-of-function are widely used to mimic ß-catenin-dependent tumorigenesis. Given the low prevalence of APC mutations in human HCCs, we aimed to generate liver tumors through CTNNB1 exon 3 deletion (ßcatΔex3). We then compared ßcatΔex3 liver tumors with liver tumors generated via frameshift in exon 15 of Apc (Apcfs-ex15). METHODS: We used hepatocyte-specific and inducible mouse models generated through either a Cre-Lox or a CRISPR/Cas9 approach using adeno-associated virus vectors. Tumors generated by the Cre-Lox models were phenotypically analyzed using immunohistochemistry and were selected for transcriptomic analysis by RNA-sequencing (RNAseq). Mouse RNAseq data were compared to human RNAseq data (8 normal tissues, 48 HCCs, 9 hepatoblastomas) in an integrative analysis. Tumors generated via CRISPR were analyzed using DNA sequencing and immuno-histochemistry. RESULTS: Mice with CTNNB1 exon 3 deletion in hepatocytes developed liver tumors indistinguishable from Apcfs-ex15 liver tumors. Both Apcfs-ex15 and ßcatΔex3 mouse models induced growth of phenotypically distinct tumors (differentiated or undifferentiated). Integrative analysis of human and mouse tumors showed that differentiated mouse tumors cluster with well-differentiated human CTNNB1-mutated tumors. Conversely, undifferentiated mouse tumors cluster with human mesenchymal hepatoblastomas and harbor activated YAP signaling. CONCLUSION: Apcfs-ex15 and ßcatΔex3 mouse models both induce growth of tumors that are transcriptionally similar to either well-differentiated and ß-catenin-activated human HCCs or mesenchymal hepatoblastomas. LAY SUMMARY: New and easy-to-use transgenic mouse models of primary liver cancers have been generated, with mutations in the gene encoding beta-catenin, which are frequent in both adult and pediatric primary liver cancers. The mice develop both types of cancer, constituting a strong preclinical model.


Asunto(s)
Carcinoma Hepatocelular , Hepatoblastoma , Neoplasias Hepáticas , beta Catenina , Animales , Carcinoma Hepatocelular/patología , Hepatoblastoma/metabolismo , Hepatocitos/metabolismo , Humanos , Neoplasias Hepáticas/patología , Ratones , Ratones Transgénicos , Mutación , beta Catenina/genética
3.
Gastroenterology ; 157(3): 807-822, 2019 09.
Artículo en Inglés | MEDLINE | ID: mdl-31194980

RESUMEN

BACKGROUND & AIMS: In one-third of hepatocellular carcinomas (HCCs), cancer cells have mutations that activate ß-catenin pathway. These cells have alterations in glutamine, bile, and lipid metabolism. We investigated whether positron emission tomography (PET) imaging allows identification of altered metabolic pathways that might be targeted therapeutically. METHODS: We studied mice with activation of ß-catenin in liver (Apcko-liv mice) and male C57Bl/6 mice given injections of diethylnitrosamine, which each develop HCCs. Mice were fed a conventional or a methionine- and choline-deficient diet or a choline-deficient (CD) diet. Choline uptake and metabolism in HCCs were analyzed by micro-PET imaging of mice; livers were collected and analyzed by histologic, metabolomic, messenger RNA quantification, and RNA-sequencing analyses. Fifty-two patients with HCC underwent PET imaging with 18F-fluorodeoxyglucose, followed by 18F-fluorocholine tracer metabolites. Human HCC specimens were analyzed by immunohistochemistry, quantitative polymerase chain reaction, and DNA sequencing. We used hepatocytes and mouse tumor explants for studies of incorporation of radiolabeled choline into phospholipids and its contribution to DNA methylation. We analyzed HCC progression in mice fed a CD diet. RESULTS: Livers and tumors from Apcko-liv mice had increased uptake of dietary choline, which contributes to phospholipid formation and DNA methylation in hepatocytes. In patients and in mice, HCCs with activated ß-catenin were positive in 18F-fluorocholine PET, but not 18F-fluorodeoxyglucose PET, and they overexpressed the choline transporter organic cation transporter 3. The HCC cells from Apcko-liv mice incorporated radiolabeled methyl groups of choline into phospholipids and DNA. In Apcko-liv mice, the methionine- and choline-deficient diet reduced proliferation and DNA hypermethylation of hepatocytes and HCC cells, and the CD diet reduced long-term progression of tumors. CONCLUSIONS: In mice and humans, HCCs with mutations that activate ß-catenin are characterized by increased uptake of a fluorocholine tracer, but not 18F-fluorodeoxyglucose, revealed by PET. The increased uptake of choline by HCCs promotes phospholipid formation, DNA hypermethylation, and hepatocyte proliferation. In mice, the CD diet reverses these effects and promotes regression of HCCs that overexpress ß-catenin.


Asunto(s)
Carcinoma Hepatocelular/diagnóstico por imagen , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/diagnóstico por imagen , Neoplasias Hepáticas/genética , Mutación , Tomografía de Emisión de Positrones , beta Catenina/genética , Animales , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/patología , Proliferación Celular , Colina/administración & dosificación , Colina/análogos & derivados , Deficiencia de Colina/complicaciones , Metilación de ADN , Dietilnitrosamina , Modelos Animales de Enfermedad , Genes APC , Predisposición Genética a la Enfermedad , Humanos , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/patología , Masculino , Metionina/deficiencia , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Fosfolípidos/metabolismo , Valor Predictivo de las Pruebas , Radiofármacos/administración & dosificación , beta Catenina/metabolismo
4.
Liver Int ; 39(4): 727-739, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30721564

RESUMEN

BACKGROUND & AIMS: Loss of hepatocyte nuclear factor-4α (HNF4α), a critical factor driving liver development and differentiation, is frequently associated with hepatocellular carcinoma (HCC). Our recent data revealed that HNF4α level was decreased in mouse and human HCCs with activated ß-catenin signalling. In addition, increasing HNF4α level by miR-34a inhibition slowed tumour progression of ß-catenin-activated HCC in mice. METHODS: We generated a Hnf4aflox/flox/ Apcflox/flox /TTR-CreERT2 (Hnf4a/Apc∆Hep ) mouse line and evaluated the impact of Hnf4a disruption on HCC development and liver homoeostasis. RESULTS: There was no significant impact of Hnf4a disruption on tumour onset and progression in Apc∆Hep model. However, we observed an unexpected phenotype in 28% of Hnf4a∆Hep mice maintained in a conventional animal facility, which presented disorganized portal triads, characterized by stenosis of the portal vein and increased number and size of hepatic arteries and bile ducts. These abnormal portal structures resemble the human idiopathic non-cirrhotic portal hypertension syndrome. We correlated the presence of portal remodelling with a higher expression of protein and mRNA levels of TGFß and BMP7, a key regulator of the TGFß-dependent endothelial-to-mesenchymal transition. CONCLUSION: These data demonstrate that HNF4α does not play a major role during ß-catenin-driven HCC, thus revealing that the tumour suppressor role of HNF4α is far more complex and dependent probably on its temporal expression and tumour context. However, HNF4α loss in adult hepatocytes could induce abnormal portal structures resembling the human idiopathic non-cirrhotic portal hypertension syndrome, which may result from endothelial- and epithelial-to-mesenchymal transitions.


Asunto(s)
Factor Nuclear 4 del Hepatocito/metabolismo , Hepatocitos/metabolismo , Neoplasias Hepáticas/genética , beta Catenina/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Proteína Morfogenética Ósea 7/metabolismo , Carcinogénesis , Diferenciación Celular , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Factor Nuclear 4 del Hepatocito/genética , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Ratones , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo
5.
Differentiation ; 91(1-3): 42-9, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26856660

RESUMEN

Beta-catenin is known to play stage- and cell-specific functions during liver development. However, its role in development of bile ducts has not yet been addressed. Here we used stage-specific in vivo gain- and loss-of-function approaches, as well as lineage tracing experiments in the mouse, to first demonstrate that ß-catenin is dispensable for differentiation of liver precursor cells (hepatoblasts) to cholangiocyte precursors. Second, when ß-catenin was depleted in the latter, maturation of cholangiocytes, bile duct morphogenesis and differentiation of periportal hepatocytes from cholangiocyte precursors was normal. In contrast, stabilization of ß-catenin in cholangiocyte precursors perturbed duct development and cholangiocyte differentiation. We conclude that ß-catenin is dispensable for biliary development but that its activity must be kept within tight limits. Our work is expected to significantly impact on in vitro differentiation of stem cells to cholangiocytes for toxicology studies and disease modeling.


Asunto(s)
Conductos Biliares/crecimiento & desarrollo , Hígado/crecimiento & desarrollo , Morfogénesis/genética , beta Catenina/genética , Animales , Conductos Biliares/citología , Conductos Biliares/metabolismo , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Hepatocitos/citología , Hepatocitos/metabolismo , Hígado/metabolismo , Ratones
6.
Gut ; 65(6): 1024-34, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-25792709

RESUMEN

OBJECTIVE: Hepatocellular carcinoma (HCC) is the most prevalent primary tumour of the liver. About a third of these tumours presents activating mutations of the ß-catenin gene. The molecular pathogenesis of HCC has been elucidated, but mortality remains high, and new therapeutic approaches, including treatments based on microRNAs, are required. We aimed to identify candidate microRNAs, regulated by ß-catenin, potentially involved in liver tumorigenesis. DESIGN: We used a mouse model, in which ß-catenin signalling was overactivated exclusively in the liver by the tamoxifen-inducible and Cre-Lox-mediated inactivation of the Apc gene. This model develops tumours with properties similar to human HCC. RESULTS: We found that miR-34a was regulated by ß-catenin, and significantly induced by the overactivation of ß-catenin signalling in mouse tumours and in patients with HCC. An inhibitor of miR-34a (locked nucleic acid, LNA-34a) exerted antiproliferative activity in primary cultures of hepatocyte. This inhibition of proliferation was associated with a decrease in cyclin D1 levels, orchestrated principally by HNF-4α, a target of miR-34a considered to act as a tumour suppressor in the liver. In vivo, LNA-34a approximately halved progression rates for tumours displaying ß-catenin activation together with an activation of caspases 2 and 3. CONCLUSIONS: This work demonstrates the key oncogenic role of miR-34a in liver tumours with ß-catenin gene mutations. We suggest that patients diagnosed with HCC with ß-catenin mutations could be treated with an inhibitor of miR-34a. The potential value of this strategy lies in the modulation of the tumour suppressor HNF-4α, which targets cyclin D1, and the induction of a proapoptotic programme.


Asunto(s)
Ciclina D1/genética , Neoplasias Hepáticas Experimentales/genética , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Mutación , beta Catenina/genética , Animales , Carcinoma Hepatocelular/terapia , Humanos , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas Experimentales/terapia , Ratones
7.
Hepatology ; 59(6): 2344-57, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24214913

RESUMEN

UNLABELLED: ß-catenin signaling can be both a physiological and oncogenic pathway in the liver. It controls compartmentalized gene expression, allowing the liver to ensure its essential metabolic function. It is activated by mutations in 20%-40% of hepatocellular carcinomas (HCCs) with specific metabolic features. We decipher the molecular determinants of ß-catenin-dependent zonal transcription using mice with ß-catenin-activated or -inactivated hepatocytes, characterizing in vivo their chromatin occupancy by T-cell factor (Tcf)-4 and ß-catenin, transcriptome, and metabolome. We find that Tcf-4 DNA bindings depend on ß-catenin. Tcf-4/ß-catenin binds Wnt-responsive elements preferentially around ß-catenin-induced genes. In contrast, genes repressed by ß-catenin bind Tcf-4 on hepatocyte nuclear factor 4 (Hnf-4)-responsive elements. ß-Catenin, Tcf-4, and Hnf-4α interact, dictating ß-catenin transcription, which is antagonistic to that elicited by Hnf-4α. Finally, we find the drug/bile metabolism pathway to be the one most heavily targeted by ß-catenin, partly through xenobiotic nuclear receptors. CONCLUSIONS: ß-catenin patterns the zonal liver together with Tcf-4, Hnf-4α, and xenobiotic nuclear receptors. This network represses lipid metabolism and exacerbates glutamine, drug, and bile metabolism, mirroring HCCs with ß-catenin mutational activation.


Asunto(s)
Factor Nuclear 4 del Hepatocito/metabolismo , Hepatocitos/metabolismo , Neoplasias Hepáticas/etiología , Proteína 2 Similar al Factor de Transcripción 7/metabolismo , beta Catenina/metabolismo , Animales , Cromatina/metabolismo , Redes Reguladoras de Genes , Humanos , Metabolismo de los Lípidos , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Receptor Cross-Talk , beta Catenina/genética
8.
Dev Cell ; 10(6): 759-70, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16740478

RESUMEN

The molecular mechanisms by which liver genes are differentially expressed along a portocentral axis, allowing for metabolic zonation, are poorly understood. We provide here compelling evidence that the Wnt/beta-catenin pathway plays a key role in liver zonation. First, we show the complementary localization of activated beta-catenin in the perivenous area and the negative regulator Apc in periportal hepatocytes. We then analyzed the immediate consequences of either a liver-inducible Apc disruption or a blockade of Wnt signaling after infection with an adenovirus encoding Dkk1, and we show that Wnt/beta-catenin signaling inversely controls the perivenous and periportal genetic programs. Finally, we show that genes involved in the periportal urea cycle and the perivenous glutamine synthesis systems are critical targets of beta-catenin signaling, and that perturbations to ammonia metabolism are likely responsible for the death of mice with liver-targeted Apc loss. From our results, we propose that Apc is the liver "zonation-keeper" gene.


Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/genética , Genes APC , Genes Supresores de Tumor , Hígado/metabolismo , Adenoviridae/genética , Amoníaco/metabolismo , Animales , Regulación de la Expresión Génica , Vectores Genéticos , Hepatocitos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Hígado/citología , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Biológicos , Nitrógeno/metabolismo , Transducción de Señal , Urea/metabolismo , Proteínas Wnt/fisiología , beta Catenina/fisiología
9.
J Hepatol ; 55(1): 86-95, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21145869

RESUMEN

BACKGROUND & AIMS: ß-Catenin is an oncogene frequently mutated in hepatocellular carcinoma. In this study, we investigated target genes of ß-catenin signaling in hepatocyte proliferation. METHODS: We studied transgenic mice displaying either inactivation or activation of the ß-catenin pathway, focusing on analysis of liver proliferation due to aberrant ß-catenin activation, and on the regeneration process during which ß-catenin signaling is transiently activated. We localized in situ the various partners involved in proliferation or identified as targets of ß-catenin in these transgenic and regenerating livers. We also performed comparative transcriptome analyses, using microarrays. Finally, we extracted, from deep-sequencing data, both the DNA regulatory elements bound to the ß-catenin/Tcf nuclear complex and the expression levels of critical targets identified in microarrays. RESULTS: ß-Catenin activation during liver regeneration occurred during G1/S cell cycle progression and allowed zonal extension of the normal territory of active ß-catenin and panlobular proliferation. We found that ß-catenin controlled both cell-autonomous and non-cell-autonomous hepatocyte proliferation, through direct transcriptional and complex control of cyclin D1 gene expression and of the expression of a new target gene, Tgfα. CONCLUSIONS: We propose that ß-catenin controls panlobular hepatocyte proliferation partly by controlling, together with its Tcf4 nuclear partner, expression of the pro-proliferation cyclin D1 and Tgfα genes. This study constitutes a first step toward understanding the oncogenic properties of this prominent signaling pathway in the liver.


Asunto(s)
Genes bcl-1 , Hepatocitos/citología , Hepatocitos/metabolismo , Factor de Crecimiento Transformador alfa/genética , beta Catenina/metabolismo , Animales , Secuencia de Bases , Ciclo Celular , Proliferación Celular , ADN/genética , Perfilación de la Expresión Génica , Hígado/anatomía & histología , Hígado/metabolismo , Regeneración Hepática/genética , Regeneración Hepática/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos Biológicos , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de Señal , beta Catenina/deficiencia , beta Catenina/genética
10.
Proc Natl Acad Sci U S A ; 105(34): 12417-22, 2008 Aug 26.
Artículo en Inglés | MEDLINE | ID: mdl-18719115

RESUMEN

The H19 locus belongs to a cluster of imprinted genes that is linked to the human Beckwith-Wiedemann syndrome. The expression of H19 and its closely associated IGF2 gene is frequently deregulated in some human tumors, such as Wilms' tumors. In these cases, biallelic IGF2 expression and lack of expression of H19 are associated with hypermethylation of the imprinting center of this locus. These observations and others have suggested a potential tumor suppressor effect of the H19 locus. Some studies have also suggested that H19 is an oncogene, based on tissue culture systems. We show, using in vivo murine models of tumorigenesis, that the H19 locus controls the size of experimental teratocarcinomas, the number of polyps in the Apc murine model of colorectal cancer and the timing of appearance of SV40-induced hepatocarcinomas. The H19 locus thus clearly displays a tumor suppressor effect in mice.


Asunto(s)
Genes Supresores de Tumor/fisiología , ARN no Traducido/fisiología , Animales , Carcinoma Hepatocelular/patología , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Factor II del Crecimiento Similar a la Insulina , Ratones , Ratones Mutantes , Familia de Multigenes , ARN Largo no Codificante , ARN no Traducido/clasificación , Teratoma/patología
11.
Elife ; 92020 10 21.
Artículo en Inglés | MEDLINE | ID: mdl-33084574

RESUMEN

Erythropoietin (EPO) is a key regulator of erythropoiesis. The embryonic liver is the main site of erythropoietin synthesis, after which the kidney takes over. The adult liver retains the ability to express EPO, and we discovered here new players of this transcription, distinct from the classical hypoxia-inducible factor pathway. In mice, genetically invalidated in hepatocytes for the chromatin remodeler Arid1a, and for Apc, the major silencer of Wnt pathway, chromatin was more accessible and histone marks turned into active ones at the Epo downstream enhancer. Activating ß-catenin signaling increased binding of Tcf4/ß-catenin complex and upregulated its enhancer function. The loss of Arid1a together with ß-catenin signaling, resulted in cell-autonomous EPO transcription in mouse and human hepatocytes. In mice with Apc-Arid1a gene invalidations in single hepatocytes, Epo de novo synthesis led to its secretion, to splenic erythropoiesis and to dramatic erythrocytosis. Thus, we identified new hepatic EPO regulation mechanism stimulating erythropoiesis.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Eritropoyetina/metabolismo , Hepatocitos/metabolismo , Factores de Transcripción/metabolismo , beta Catenina/metabolismo , Adulto , Animales , Eritropoyesis , Femenino , Humanos , Hibridación in Situ , Masculino , Ratones , Vía de Señalización Wnt
12.
Hepatology ; 47(1): 247-58, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18038450

RESUMEN

UNLABELLED: During hepatogenesis, after the liver has budded out of the endoderm, the hepatoblasts quickly expand and differentiate into either hepatocytes or biliary cells, the latter of which arise only within the ductal plate surrounding the portal vein. Because the Wnt/beta-catenin pathway is involved in liver homeostasis and regeneration and in liver carcinogenesis, we investigated here a role for Wnt/beta-catenin signaling in the embryonic liver. A cyclization recombination (Cre)/locus of X-over P1 (loxP) strategy was chosen to perform adenomatous polyposis coli (Apc) invalidation in order to activate ectopic beta-catenin signaling in hepatoblasts; an appropriate transgenic model expressing the Cre recombinase was used. Phenotypic and immunolocalization studies, together with messenger RNA analyses, by microarray and real-time quantitative polymerase chain reaction approaches were performed on this model during normal hepatogenesis. The loss of Apc allowed beta-catenin activation in the hepatoblasts after the formation of the liver bud and led to embryonic lethality. In this model, the liver became hypoplastic, and hepatocyte differentiation failed, whereas beta-catenin-activated ducts developed and gave rise to fully differentiated bile ducts when transplanted into adult recipient livers. Microarray analyses suggested that beta-catenin plays a role in repressing the hepatocyte genetic program and remodeling the ductal plate. According to these data, in normal embryonic livers, beta-catenin was transiently activated in the nascent bile ducts. CONCLUSION: We demonstrated a key role for the Wnt/beta-catenin pathway in liver embryonic growth and in controlling the fate of hepatoblasts, preventing them from differentiating toward the hepatocyte lineage, and guiding them to biliary ductal morphogenesis.


Asunto(s)
Hígado/embriología , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Animales , Diferenciación Celular/fisiología , Pérdida del Embrión/fisiopatología , Hepatocitos/citología , Ratones , Morfogénesis/fisiología , Fenotipo , Transducción de Señal/fisiología
13.
Br J Nutr ; 101(6): 843-51, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18680626

RESUMEN

Ornithine aminotransferase (OAT) is a reversible enzyme expressed mainly in the liver, kidney and intestine. OAT controls the interconversion of ornithine into glutamate semi-aldehyde, and is therefore involved in the metabolism of arginine and glutamine which play a major role in N homeostasis. We hypothesised that OAT could be a limiting step in glutamine-arginine interconversion. To study the contribution of the OAT enzyme in amino acid metabolism, transgenic mice that specifically overexpress human OAT in the liver, kidneys and intestine were generated. The transgene expression was analysed by in situ hybridisation and real-time PCR. Tissue (liver, jejunum and kidney) OAT activity, and plasma and tissue (liver and jejunum) amino acid concentrations were measured. Transgenic male mice exhibited higher OAT activity in the liver (25 (sem 4) v. 11 (sem 1) nmol/min per microg protein for wild-type (WT) mice; P < 0.05) but there were no differences in kinetic parameters (i.e. Km and maximum rate of reaction (Vmax)) between WT and transgenic animals. OAT overexpression decreased plasma and liver ornithine concentrations but did not affect glutamine or arginine homeostasis. There was an inverse relationship between ornithine levels and OAT activity. We conclude that OAT overexpression has only limited metabolic effects, probably due to the reversible nature of the enzyme. Moreover, these metabolic modifications had no effect on phenotype.


Asunto(s)
Aminoácidos/metabolismo , Yeyuno/enzimología , Riñón/enzimología , Hígado/enzimología , Ornitina-Oxo-Ácido Transaminasa/metabolismo , Aminoácidos/análisis , Animales , Femenino , Expresión Génica , Homeostasis , Humanos , Inmunohistoquímica , Hibridación in Situ , Yeyuno/química , Hígado/química , Masculino , Ratones , Ratones Transgénicos , Ornitina-Oxo-Ácido Transaminasa/análisis , Ornitina-Oxo-Ácido Transaminasa/genética , Fenotipo , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Transgenes
14.
Future Oncol ; 4(5): 647-60, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18922122

RESUMEN

The Wnt/beta-catenin pathway is a key developmental pathway for which alterations have been described in various human cancers. The aberrant activation of this pathway is a major event in human hepatocellular carcinoma. Several laboratories have shown that the Wnt/beta-catenin pathway plays an essential role in all phases of liver development and maturation, and is required for the metabolic function of this organ. In this review, we summarize current knowledge regarding the role of the Wnt/beta-catenin pathway in hepatocellular carcinoma pathogenesis and liver biology, and the possibilities for developing new therapeutic interventions based on this knowledge.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Hígado/fisiología , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Carcinoma Hepatocelular/genética , Humanos , Neoplasias Hepáticas/genética , Proteínas Wnt/genética , beta Catenina/genética
15.
Cancer Res ; 66(4): 1949-55, 2006 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-16488993

RESUMEN

We analyzed the expression profiles of intestinal adenomas from a new murine familial adenomatous polyposis model (Apc(delta14/+)) using suppression subtractive hybridization to identify novel diagnostic markers of colorectal carcinogenesis. We identified 18 candidate genes having increased expression levels in the adenoma. Subsequent Northern blotting, real-time reverse transcription-PCR, and in situ hybridization analysis confirmed their induction in beta-catenin-activated epithelial cells of murine adenomas. We showed that most of the genes also have altered expression levels in human colonic adenomas and carcinomas. We focused on the IFITM genes that encode IFN-inducible transmembrane proteins. Serial analyses of gene expression levels revealed high levels of expression in early and late intestinal neoplasm in both mice and humans. Using a conditional mouse model of Apc inactivation and a human colon carcinoma cell line, we showed that IFITM gene expression is rapidly induced after activation of the beta-catenin signaling. Using a large-scale analysis of human tumors, we showed that IFITM gene expression is significantly up-regulated specifically in colorectal tumors and thus may be a useful diagnostic tool in these tumors.


Asunto(s)
Biomarcadores de Tumor/biosíntesis , Neoplasias Colorrectales/metabolismo , Proteínas de la Membrana/biosíntesis , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenoma/genética , Adenoma/metabolismo , Animales , Antígenos de Diferenciación , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Intestinales/genética , Neoplasias Intestinales/metabolismo , Masculino , Proteínas de la Membrana/genética , Ratones , Proteínas de Unión al ARN/biosíntesis , Proteínas de Unión al ARN/genética , Regulación hacia Arriba , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
16.
Diabetes ; 54(5): 1357-63, 2005 May.
Artículo en Inglés | MEDLINE | ID: mdl-15855320

RESUMEN

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) has been implicated in the control of blood glucose by its potent effect on expression and signaling of various nuclear receptors. To understand the role of COUP-TFII in glucose homeostasis, conditional COUP-TFII-deficient mice were generated and crossed with mice expressing Cre under the control of rat insulin II gene promoter, resulting in deletion of COUP-TFII in pancreatic beta-cells. Homozygous mutants died before birth for yet undetermined reasons. Heterozygous mice appeared healthy at birth and showed normal growth and fertility. When challenged intraperitoneally, the animals had glucose intolerance associated with reduced glucose-stimulated insulin secretion. Moreover, these heterozygous mice presented a mild increase in fasting and random-fed circulating insulin levels. In accordance, islets isolated from these animals exhibited higher insulin secretion in low glucose conditions and markedly decreased glucose-stimulated insulin secretion. Their pancreata presented normal microscopic architecture and insulin content up to 16 weeks of study. Altered insulin secretion was associated with peripheral insulin resistance in whole animals. It can be concluded that COUP-TFII is a new, important regulator of glucose homeostasis and insulin sensitivity.


Asunto(s)
Proteínas de Unión al ADN/fisiología , Glucosa/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/fisiología , Receptores de Esteroides/fisiología , Factores de Transcripción/fisiología , Animales , Glucemia/metabolismo , Factor de Transcripción COUP II , Factores de Transcripción COUP , Pollos , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Ácidos Grasos no Esterificados/sangre , Eliminación de Gen , Glucagón/sangre , Homeostasis , Insulina/sangre , Insulina/fisiología , Secreción de Insulina , Leptina/sangre , Lípidos/sangre , Ratones , Ratones Noqueados , Ratas , Receptores de Esteroides/deficiencia , Receptores de Esteroides/genética , Factores de Transcripción/deficiencia , Factores de Transcripción/genética
17.
Int J Exp Pathol ; 86(6): 397-413, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16309545

RESUMEN

Using several techniques, we have assessed morphological characteristics of a malignant thymic tumour in SV12 transgenic (Tg) mice expressing SV40 T and t antigens under control of an L-PK promoter. We describe the development of a carcinoma originating from thymic hyperplasia and followed by the formation of a benign tumour composed chiefly of medullary epithelial cells expressing the transgene and of lymphocytes, a pathology very rarely reported in mice. Our study of the SV12 Tg mice represents the first description of a model of a pure malignant thymic tumour associated with extensive angiogenesis maintained in numerous descendants. The formation of a large tumoral neovascular network, observed here, has never been described in human and/or experimental thymic tumours. Tumoral transformation and angiogenesis are demonstrated by immunolabelling with antibodies against various cytokeratins (CKs) of different molecular weights, vascular endothelial cell markers and VEGF/receptor-2 (Flk-1) present on the neovascular endothelial cells. Different points raised by the originality of this model are discussed. These include the medullary nature of the cells expressing the SV40 transgene and their relationship with the tumoral development. The subset of different molecular weight CK components and their modifications are also considered, as well as the presence of type IV epithelial cells, progenitors of medullary epithelial cells. Finally, the cell signals involved in angiogenesis and the possible action of an angiogenic factor, probably secreted by the tumoral cells themselves, are discussed.


Asunto(s)
Antígenos Transformadores de Poliomavirus/genética , Antígenos Virales de Tumores/genética , Neovascularización Patológica , Regiones Promotoras Genéticas , Piruvato Quinasa/genética , Neoplasias del Timo/patología , Animales , Antígenos Transformadores de Poliomavirus/metabolismo , Antígenos Virales de Tumores/metabolismo , Biomarcadores de Tumor/análisis , Transformación Celular Neoplásica , Femenino , Inmunohistoquímica/métodos , Masculino , Ratones , Ratones Endogámicos CBA , Ratones Transgénicos , Microscopía Inmunoelectrónica , Timo/patología , Neoplasias del Timo/genética
18.
Development ; 132(6): 1443-51, 2005 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-15716339

RESUMEN

Loss of Apc appears to be one of the major events initiating colorectal cancer. However, the first events responsible for this initiation process are not well defined and the ways in which different epithelial cell types respond to Apc loss are unknown. We used a conditional gene-ablation approach in transgenic mice expressing tamoxifen-dependent Cre recombinase all along the crypt-villus axis to analyze the immediate effects of Apc loss in the small intestinal epithelium, both in the stem-cell compartment and in postmitotic epithelial cells. Within 4 days, Apc loss induced a dramatic enlargement of the crypt compartment associated with intense cell proliferation, apoptosis and impairment of cell migration. This result confirms the gatekeeper role of Apc in the intestinal epithelium in vivo. Although Apc deletion activated beta-catenin signaling in the villi, we observed neither proliferation nor morphological change in this compartment. This highlights the dramatic difference in the responses of immature and differentiated epithelial cells to aberrant beta-catenin signaling. These distinct biological responses were confirmed by molecular analyses, revealing that Myc and cyclin D1, two canonical beta-catenin target genes, were induced in distinct compartments. We also showed that Apc is a crucial determinant of cell fate in the murine intestinal epithelium. Apc loss perturbs differentiation along the enterocyte, goblet and enteroendocrine lineages, and promotes commitment to the Paneth cell lineage through beta-catenin/Tcf4-mediated transcriptional control of specific markers of Paneth cells, the cryptdin/defensin genes.


Asunto(s)
Diferenciación Celular/fisiología , División Celular/fisiología , Genes APC , Intestinos/fisiología , Células de Paneth/fisiología , Animales , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/genética , Proteínas del Citoesqueleto/fisiología , Defensinas/genética , Defensinas/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/fisiología , Ratones , Ratones Noqueados , Células de Paneth/citología , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Transducción de Señal/fisiología , Transactivadores/fisiología , beta Catenina
19.
Dev Immunol ; 9(4): 223-31, 2002 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-15144019

RESUMEN

Bone marrow progenitors migrate to the thymus, where they proliferate and differentiate into immunologically competent T cells. In this report we show that mice transgenic for SV40 T and t antigens under the control of the L-pyruvate kinase promoter develop, in a first step, thymic hyperplasia of both thymocytes and epithelial cells. Morphological studies (histology, immunohistolabeling and electron microscopy) revealed modifications of the thymic microenvironment and gradual expansion of medullary epithelial cells in 1 month-old mice, taking over the cortical region. Then, a thymic carcinoma develops. Two-color labeling of frozen sections identified the transgene in medullary epithelial cells. Flow cytometry analysis demonstrated a marked increase in mature CD4+ and CD8+ thymocytes in adult mice (39 +/- 10 x 10(6) in transgenic mice and 12 +/- 5 x 10(6) in age-matched controls). Furthermore, thymocyte export was disturbed.


Asunto(s)
Antígenos Transformadores de Poliomavirus/inmunología , Células Epiteliales/fisiología , Virus 40 de los Simios/inmunología , Linfocitos T/fisiología , Neoplasias del Timo/fisiopatología , Animales , Antígenos Transformadores de Poliomavirus/genética , Diferenciación Celular/inmunología , Movimiento Celular/inmunología , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Células Epiteliales/ultraestructura , Femenino , Citometría de Flujo , Hiperplasia , Masculino , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Piruvato Quinasa/genética , Virus 40 de los Simios/genética , Linfocitos T/ultraestructura
20.
Hepatology ; 40(1): 167-76, 2004 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15239100

RESUMEN

To clarify molecular mechanisms underlying liver carcinogenesis induced by aberrant activation of Wnt pathway, we isolated the target genes of beta-catenin from mice exhibiting constitutive activated beta-catenin in the liver. Adenovirus-mediated expression of oncogenic beta-catenin was used to isolate early targets of beta-catenin in the liver. Suppression subtractive hybridization was used to identify the leukocyte cell-derived chemotaxin 2 (LECT2) gene as a direct target of beta-catenin. Northern blot and immunohistochemical analyses demonstrated that LECT2 expression is specifically induced in different mouse models that express activated beta-catenin in the liver. LECT2 expression was not activated in livers in which hepatocyte proliferation was induced by a beta-catenin-independent signal. We characterized by mutagenesis the LEF/TCF site, which is crucial for LECT2 activation by beta-catenin. We further characterized the chemotactic property of LECT2 for human neutrophils. Finally, we have shown an up-regulation of LECT2 in human liver tumors that expressed aberrant activation of beta-catenin signaling; these tumors constituted a subset of hepatocellular carcinomas (HCC) and most of the hepatoblastomas that were studied. In conclusion, our results show that LECT2, which encodes a protein with chemotactic properties for human neutrophils, is a direct target gene of Wnt/beta-catenin signaling in the liver. Since HCC develops mainly in patients with chronic hepatitis or cirrhosis induced by viral or inflammatory factors, understanding the role of LECT2 in liver carcinogenesis is of interest and may lead to new therapeutic perspectives.


Asunto(s)
Proteínas del Citoesqueleto/fisiología , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas del Tejido Nervioso , Transactivadores/fisiología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Línea Celular , Quimiotaxis , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Endogámicos , Ratones Transgénicos , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Hibridación de Ácido Nucleico/métodos , Transducción de Señal , Factores de Transcripción TCF , Factor de Transcripción 4 , Proteína 2 Similar al Factor de Transcripción 7 , Factores de Transcripción/metabolismo , Regulación hacia Arriba , beta Catenina
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