RESUMEN
Conditional deletion of the mouse Forkhead Box (Fox) m1b targeted allele in adult hepatocytes (Foxm1, previously called HFH-11B, Trident, Win, or MPP2) demonstrated that the Foxm1b transcription factor is essential for hepatocyte mitosis during liver regeneration. To determine the role of Foxm1b in liver development, we have generated Foxm1b -/- mice that deleted the Foxm1b exons encoding the winged helix DNA binding and transcriptional activation domains. Here, we show that all of the Foxm1b -/- embryos died in utero by 18.5 days postcoitum (dpc). Embryonic Foxm1b -/- livers displayed a 75% reduction in the number of hepatoblasts, resulting from diminished DNA replication and a failure to enter mitosis causing a polyploid phenotype. Reduced hepatoblast mitosis was associated with decreased protein levels of the Polo-like kinase 1 and Aurora B kinase, which phosphorylate regulatory proteins essential for orchestrating mitosis and cytokinesis. Diminished proliferation of Foxm1b -/- hepatoblasts contributed to abnormal liver development with significant reduction in the number of large hepatic veins compared to embryonic wild-type (WT) liver. Furthermore, embryonic Foxm1b -/- livers did not develop intrahepatic bile ducts, and these presumptive biliary hepatoblasts failed to express either biliary cytokeratins or nuclear levels of hepatocyte nuclear factor 1beta. These results suggest that Foxm1b is critical for hepatoblast precursor cells to differentiate toward biliary epithelial cell lineage. Finally, we used a hepatoblast-specific Cre recombinase transgene to mediate deletion of the Foxm1b fl/fl allele in the developing liver, and these embryos died in utero and exhibited diminished hepatoblast proliferation with similar abnormalities in liver morphogenesis, suggesting that the defect in liver development contributed to embryonic lethality.
Asunto(s)
Conductos Biliares Intrahepáticos/crecimiento & desarrollo , Hepatocitos/fisiología , Hígado/crecimiento & desarrollo , Mitosis , Morfogénesis , Factores de Transcripción/fisiología , Alelos , Animales , Aurora Quinasa B , Aurora Quinasas , Proteínas de Ciclo Celular , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead , Eliminación de Gen , Marcación de Gen , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Poliploidía , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Quinasa Tipo Polo 1RESUMEN
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. Here, we provide evidence that the Forkhead Box (Fox) m1b (Foxm1b or Foxm1) transcription factor is essential for the development of HCC. Conditionally deleted Foxm1b mouse hepatocytes fail to proliferate and are highly resistant to developing HCC in response to a Diethylnitrosamine (DEN)/Phenobarbital (PB) liver tumor-induction protocol. The mechanism of resistance to HCC development is associated with nuclear accumulation of the cell cycle inhibitor p27(Kip1) protein and reduced expression of the Cdk1-activator Cdc25B phosphatase. We showed that the Foxm1b transcription factor is a novel inhibitory target of the p19(ARF) tumor suppressor. Furthermore, we demonstrated that conditional overexpression of Foxm1b protein in osteosarcoma U2OS cells greatly enhances anchorage-independent growth of cell colonies on soft agar. A p19(ARF) 26-44 peptide containing nine D-Arg to enhance cellular uptake of the peptide was sufficient to significantly reduce both Foxm1b transcriptional activity and Foxm1b-induced growth of U2OS cell colonies on soft agar. These results suggest that this (D-Arg)(9)-p19(ARF) 26-44 peptide is a potential therapeutic inhibitor of Foxm1b function during cellular transformation. Our studies demonstrate that the Foxm1b transcription factor is required for proliferative expansion during tumor progression and constitutes a potential new target for therapy of human HCC tumors.
Asunto(s)
Carcinoma Hepatocelular/patología , Proteínas del Ojo , Lipoproteínas , Neoplasias Hepáticas Experimentales/patología , Proteínas del Tejido Nervioso , Factores de Transcripción/fisiología , Proteína p14ARF Supresora de Tumor/farmacología , Adenoma/genética , Adenoma/patología , Alquilantes/toxicidad , Animales , Apoptosis , Proteínas de Unión al Calcio/metabolismo , Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Ensayo de Unidades Formadoras de Colonias , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Proteínas de Unión al ADN/fisiología , Progresión de la Enfermedad , Antagonistas de Aminoácidos Excitadores/toxicidad , Femenino , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead , Genes p16 , Gutatión-S-Transferasa pi , Glutatión Transferasa/metabolismo , Hepatocitos/metabolismo , Hipocalcina , Humanos , Isoenzimas/metabolismo , Neoplasias Hepáticas Experimentales/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Osteosarcoma/metabolismo , Osteosarcoma/patología , Fragmentos de Péptidos/farmacología , Recoverina , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/metabolismo , Fosfatasas cdc25/metabolismoRESUMEN
The Forkhead Box (Fox) proteins are an extensive family of transcription factors that shares homology in the winged helix DNA binding domain. Liver regeneration studies with the -3 kb transthyretin (TTR) promoter-driven FoxM1B transgenic (TG) mice demonstrated that premature hepatocyte nuclear localization of the FoxM1B transgene protein at 16 h following partial hepatectomy (PHx) caused an 8-h acceleration in the onset of hepatocyte DNA replication (S-phase) and mitosis by stimulating earlier expression of cell cycle genes. Whether the FoxM1B transgene protein participates in immediate early events during liver regeneration remains to be determined. Here, we found that the FoxM1B transgene protein translocated to hepatocyte nuclei immediately following PHx, that its nuclear staining persisted for the first 6 h after surgery, and that this translocation was associated with an increase in size of regenerating TG hepatocytes. However, regenerating TTR-FoxM1B liver did not exhibit altered expression of proteins that have been implicated in mediating increased cell size, including serum-and-gucocorticoid-inducible protein kinase (SGK), protein kinase-B/Akt, the tumor suppresser gene PTEN (negative regulator of the PI3K/Akt pathway), c-Myc, or peroxisome proliferation. Moreover, we demonstrated that hepatocyte nuclear translocation of the FoxM1B transgene protein was rapidly induced during the hepatic acute phase response, which occurs during the immediate early stages of liver regeneration. Analysis of cDNA expression arrays identified a number of genes such as immediate early transcription factors (ID-3, Stat3, Nur77), matrix metalloproteinase-9 (MMP-9), and several glutathione S-transferase (GST) isoforms and stress response genes, whose expression is elevated in regenerating TTR-FoxM1B TG livers compared with regenerating wild-type (WT) liver. These liver regeneration studies demonstrate that hepatocyte nuclear translocation of the FoxM1B transgene protein was sustained for the first 6 h after PHx, and was associated with transient hypertrophy of regenerating TG hepatocytes and increased expression of genes that may enhance hepatocyte proliferation.
Asunto(s)
Tamaño de la Célula , Hepatocitos/citología , Hepatocitos/metabolismo , Regeneración Hepática/fisiología , Factores de Transcripción/metabolismo , Translocación Genética , Animales , Núcleo Celular , Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead , Perfilación de la Expresión Génica , Genes Inmediatos-Precoces/fisiología , Hepatectomía , Humanos , Ratones , Ratones Transgénicos , Mitosis , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Prealbúmina/genética , Ribonucleasa Pancreática/metabolismo , Fase S , Factores de Transcripción/genéticaRESUMEN
The Forkhead Box (Fox) proteins are an extensive family of transcription factors that shares homology in the winged helix DNA-binding domain and the members of which play essential roles in cellular proliferation, differentiation, and longevity. Reduced cellular proliferation during aging is associated with a progressive decline in both growth hormone (GH) secretion and Foxm1b expression. Liver regeneration studies with 12-month-old (old-aged) transgenic mice indicated that increased hepatocyte expression of Foxm1b alone is sufficient to restore hepatocyte proliferation to levels found in 2-month-old (young) regenerating liver. GH therapy in older people has been shown to cause an increase in cellular proliferation, but the transcription factors that mediated this stimulation in proliferation remain uncharacterized. In this study, we showed that human GH administration to old-aged Balb/c mice dramatically increased both expression of Foxm1b and regenerating hepatocyte proliferation. This increase in old-aged regenerating hepatocyte proliferation was associated with elevated protein expression of Cdc25A, Cdc25B, and cyclin B1, with reduced protein levels of cyclin-dependent kinase inhibitor p27(Kip1) (p27). GH treatment also was found to stimulate hepatocyte proliferation and expression of Foxm1b protein without partial hepatectomy (PHx). Furthermore, GH treatment of young Foxm1b -/- mice failed to restore regenerating hepatocyte DNA replication and mitosis caused by Foxm1b deficiency. These genetic studies provided strong evidence that the presence of Foxm1b is essential for GH to stimulate regenerating hepatocyte proliferation. In conclusion, our old-aged liver regeneration studies show that increased Foxm1b levels are essential for GH to stimulate hepatocyte proliferation, thus providing a mechanism for GH action in the elderly.
Asunto(s)
Envejecimiento/fisiología , Hepatocitos/efectos de los fármacos , Hormona de Crecimiento Humana/farmacología , Regeneración Hepática/efectos de los fármacos , Factores de Transcripción/fisiología , Animales , Proteínas de Ciclo Celular/análisis , Proteínas de Ciclo Celular/fisiología , División Celular/efectos de los fármacos , Ciclina B/fisiología , Ciclina B1 , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead , Hepatocitos/fisiología , Regeneración Hepática/fisiología , Ratones , Ratones Endogámicos BALB C , Fosforilación , Proteínas Supresoras de Tumor/análisis , Fosfatasas cdc25/análisis , Fosfatasas cdc25/fisiologíaRESUMEN
The Forkhead Box (Fox) proteins are an extensive family of transcription factors that shares homology in the winged helix DNA-binding domain and whose members play essential roles in cellular proliferation, differentiation, transformation, longevity, and metabolic homeostasis. Liver regeneration studies with transgenic mice demonstrated that FoxM1B regulates the onset of hepatocyte DNA replication and mitosis by stimulating expression of cell cycle genes. Here, we demonstrate that albumin-promoter-driven Cre recombinase-mediated hepatocyte-specific deletion of the Foxm1b Floxed (fl) targeted allele resulted in significant reduction in hepatocyte DNA replication and inhibition of mitosis after partial hepatectomy. Reduced DNA replication in regenerating Foxm1b(-/-) hepatocytes was associated with sustained increase in nuclear staining of the cyclin-dependent kinase (Cdk) inhibitor p21(Cip1) (p21) protein between 24 and 40 h after partial hepatectomy. Furthermore, increased nuclear p21 levels and reduced expression of Cdc25A phosphatase coincided with decreases in Cdk2 activation and hepatocyte progression into S-phase. Moreover, the significant reduction in hepatocyte mitosis was associated with diminished mRNA levels and nuclear expression of Cdc25B phosphatase and delayed accumulation of cyclin B1 protein, which is required for Cdk1 activation and entry into mitosis. Cotransfection studies demonstrate that FoxM1B protein directly activated transcription of the Cdc25B promoter region. Our present study shows that the mammalian Foxm1b transcription factor regulates expression of cell cycle proteins essential for hepatocyte entry into DNA replication and mitosis.
Asunto(s)
Quinasas CDC2-CDC28 , Replicación del ADN , Hepatocitos/metabolismo , Regeneración Hepática/fisiología , Mitosis , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Animales , Proteína Quinasa CDC2/fisiología , Proteínas de Ciclo Celular/metabolismo , Ciclina B/fisiología , Ciclina B1 , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/análisis , ADN Nucleotidiltransferasas/fisiología , Proteína Forkhead Box M1 , Factores de Transcripción Forkhead , Ratones , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/metabolismo , Recombinasas , Factores de Transcripción/genética , Fosfatasas cdc25/metabolismoRESUMEN
Recent liver regeneration studies indicate that maintaining hepatic Forkhead Box M1B (FoxM1B) expression in 12-month-old (old-aged) Transthyretin-FoxM1B transgenic mice increases hepatocyte proliferation and expression of cell cycle regulatory genes. Because these transgenic CD-1 mice maintain FoxM1B levels during the aging process, we conducted the current study to determine whether adenovirus delivery of the FoxM1B gene (AdFoxM1B) is sufficient to stimulate liver regeneration in old-aged Balb/c mice. Here we show that AdFoxM1B infection of old-aged mice caused a significant increase in FoxM1B expression, hepatocyte DNA replication, and mitosis following partial hepatectomy. This stimulation in hepatocyte S-phase progression was associated with diminished protein expression and perinuclear localization of cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) (p27) protein following partial hepatectomy. In contrast, old-aged mice infected with control virus displayed high hepatocyte levels of p27 protein, which had been localized to the nucleus prior to S-phase. Furthermore, we found that restoring FoxM1B expression did not influence p27 mRNA levels, and this new finding implicates FoxM1B in regulation of p27 protein levels. Likewise, AdFoxM1B-infected regenerating livers displayed elevated S-phase levels of Cdk2 kinase activity compared with old-aged mice infected with control virus. Furthermore, restoring FoxM1B expression in old-aged mice caused elevated levels of Cyclin B1, Cyclin B2, Cdc25B, Cdk1, and p55CDC mRNA as well as stimulating Cdc25B nuclear localization during liver regeneration, all of which are required for mitosis. These studies indicated that an acute delivery of the FoxM1B gene in old-aged mice is sufficient to re-establish proliferation of regenerating hepatocytes, suggesting that FoxM1B can be used for therapeutic intervention to alleviate the reduction in cellular proliferation observed in the elderly.