Two-thirds partial hepatectomy (PH) induces differentiated cells in the liver remnant to proliferate and regenerate to its original size. The proliferation-specific HNF-3/fork head homolog-11B protein (HFH-11B; also known as Trident and Win) is a family member of the winged helix/fork head transcription factors and in regenerating liver its expression is reactivated prior to hepatocyte entry into DNA replication (S phase). To examine whether HFH-11B regulates hepatocyte proliferation during liver regeneration, we used the ؊3-kb transthyretin (TTR) promoter to create transgenic mice that displayed ectopic hepatocyte expression of HFH-11B. Liver regeneration studies with the TTR-HFH-11B mice demonstrate that its premature expression resulted in an 8-h acceleration in the onset of hepatocyte DNA replication and mitosis. This liver regeneration phenotype is associated with protracted expression of cyclin D1 and C/EBP␤, which are involved in stimulating DNA replication and premature expression of M phase promoting cyclin B1 and cdc2. Consistent with the early hepatocyte entry into S phase, regenerating transgenic livers exhibited earlier expression of DNA repair genes (XRCC1, mHR21spA, and mHR23B). Furthermore, in nonregenerating transgenic livers, ectopic HFH-11B expression did not elicit abnormal hepatocyte proliferation, a finding consistent with the retention of the HFH-11B transgene protein in the cytoplasm. We found that nuclear translocation of the HFH-11B transgene protein requires mitogenic signalling induced by PH and that its premature availability in regenerating transgenic liver allowed nuclear translocation to occur 8 h earlier than in wild type.The mammalian liver is one of the few adult organs capable of completely regenerating itself in response to cellular injury from toxins, viral infections, or tissue removal (15,38,46). Liver regeneration after two-thirds partial hepatectomy (PH) represents a balance between hepatocyte proliferation and the maintenance of hepatocyte-specific gene expression required for liver homeostasis (22,46). A potent activation of hepatocyte immediate early transcription factors is observed during liver regeneration and includes c-Jun, c-Fos, c-Myc, NF-B, signal transducers, and activators of transcription 3 (stat3) and the CCAAT/enhancer protein ␤ (C/EBP␤) genes (7,9,25,46). Furthermore, maintenance of hepatocyte-specific gene transcription is coincident with sustained expression of hepatocyte nuclear factor genes (16,20,41). More recent genetic data demonstrated that the cytokine interleukin-6 (IL-6) plays an important role in establishing responsiveness of hepatocytes to growth factors which are released after liver injury (8,54). In a PH model of liver regeneration, homozygous null interleukin-6 (IL-6) or type 1 tumor necrosis factor receptor (TNFR-I) mice exhibited a 70% reduction in hepatocyte replication and this proliferation defect was eliminated by an intraperitoneal injection of IL-6 prior to surgery (8,43,54). This proliferation defect was accompanied by a failur...

To analyze the involvement of p53‐dependent transcriptional activation in normal development and in response to DNA damage in vivo, we created transgenic mice with a lacZ reporter gene under the control of a p53‐responsive promoter. Five independent strains showed similar patterns of transgene expression. In untreated animals, lacZ expression was limited to the developing nervous system of embryos and newborn mice and was strongly decreased in the adult brain. γ‐irradiation or adriamycin treatment induced lacZ expression in the majority of cells of early embryos and in the spleen, thymus and small intestine in adult mice. Transgene expression was p53 dependent and coincided with the sites of strong p53 accumulation. The lacZ‐expressing tissues and early embryos, unlike other adult tissues and late embryos, are characterized by high levels of p53 mRNA expression and respond to DNA damage by massive apoptotic cell death. Analysis of p53‐null mice showed that this apoptosis is p53 dependent. These data suggest that p53 activity, monitored by the reporter lacZ transgene, is the determinant of radiation and drug sensitivity in vivo and indicate the importance of tissue and stage specificity of p53 regulation at the level of mRNA expression.

FoxO transcription factors are important targets of insulin action. To better understand the role of FoxO proteins in the liver, we created transgenic mice expressing constitutively active FoxO1 in the liver using the ␣1-antitrypsin promoter. Fasting glucose levels are increased, and glucose tolerance is impaired in transgenic (TGN) versus wild type (WT) mice. Interestingly, fasting triglyceride and cholesterol levels are reduced despite hyperinsulinemia, and post-prandial changes in triglyceride levels are markedly suppressed in TGN versus WT mice. Activation of pro-lipogenic signaling pathways (atypical protein kinase C and protein kinase B) and the ability to suppress ␤-hydroxybutyrate levels are not impaired in TGN. In contrast, de novo lipogenesis measured with 3 H 2 O is suppressed by ϳ70% in the liver of TGN versus WT mice after refeeding. Gene-array studies reveal that the expression of genes involved in gluconeogenesis, glycerol transport, and amino acid catabolism is increased, whereas genes involved in glucose utilization by glycolysis, the pentose phosphate shunt, lipogenesis, and sterol synthesis pathways are suppressed in TGN versus WT. Studies with adenoviral vectors in isolated hepatocytes confirm that FoxO1 stimulates expression of gluconeogenic genes and suppresses expression of genes involved in glycolysis, the shunt pathway, and lipogenesis, including glucokinase and SREBP-1c. Together, these results indicate that FoxO proteins promote hepatic glucose production through multiple mechanisms and contribute to the regulation of other metabolic pathways important in the adaptation to fasting and feeding in the liver, including glycolysis, the pentose phosphate shunt, and lipogenic and sterol synthetic pathways.FoxO 2 transcription factors are important targets of insulin and growth factor action, and they contribute to the regulation of cell growth, differentiation, and metabolism (1-3). FoxO proteins form a subgroup within the family of Forkhead box (or Fox) transcription factors (4). Early studies indicated that Forkhead proteins interact with insulin response sequences (IRSs) in the promoter of the IGF-binding protein-1 (IGFBP-1) and the phosphoenolpyruvate carboxykinase (PEPCK) genes (5, 6) and that signaling through phosphatidylinositol 3Ј-kinase and protein kinase B (PKB) mediates IRS-dependent effects of insulin on gene expression (7). Genetic studies of Caenorhabditis elegans revealed that DAF-16, a FoxO transcription factor, is a major target of insulin-like signaling (8, 9). DAF-16 plays an important role in the adaptation to environmental stress, including nutrient restriction, and signaling through phosphatidylinositol 3Ј-kinase and PKB suppresses the function of DAF-16. Subsequent studies revealed that FoxO proteins contain highly conserved PKB phosphorylation sites (corresponding to Thr-24, Ser-256, and Ser-319 in human FoxO1) (10 -12) and that phosphorylation at these sites suppresses transactivation and promotes nuclear exclusion of FoxO proteins through multiple mechanisms (13)...

The winged helix transcription factor, hepatocyte nuclear factor-3␤ (HNF-3␤), mediates the hepatocytespecific transcription of numerous genes important for liver function. However, the in vivo role of HNF-3␤ in regulating these genes remains unknown because homozygous null HNF3␤ mouse embryos die in utero prior to liver formation. In order to examine the regulatory function of HNF-3␤, we created transgenic mice in which the ؊3-kb transthyretin promoter functions to increase hepatocyte expression of the rat HNF-3␤ protein.Postnatal transgenic mice exhibit growth retardation, depletion of hepatocyte glycogen storage, and elevated levels of bile acids in serum. The retarded growth phenotype is likely due to a 20-fold increase in hepatic expression of insulin-like growth factor binding protein 1 (IGFBP-1), which results in elevated levels in serum of IGFBP-1 and limits the biological availability of IGFs required for postnatal growth. The defects in glycogen storage and serum bile acids coincide with diminished postnatal expression of hepatocyte genes involved in gluconeogenesis (phosphoenolpyruvate carboxykinase and glycogen synthase) and sinusoidal bile acid uptake (Ntcp), respectively. These changes in gene transcription may result from the disruptive effect of HNF-3␤ on the hepatic expression of the endogenous mouse HNF-3␣,-3␤, -3␥, and -6 transcription factors. Furthermore, adult transgenic livers lack expression of the canalicular phospholipid transporter, mdr2, which is consistent with ultrastructure evidence of damage to transgenic hepatocytes and bile canaliculi. These transgenic studies represent the first in vivo demonstration that the HNF-3␤ transcriptional network regulates expression of hepatocyte-specific genes required for bile acid and glucose homeostasis, as well as postnatal growth.The liver performs essential functions in the body by uniquely expressing both hepatocyte-specific genes encoding plasma proteins and enzymes involved in the detoxification and in the homeostasis of glucose, cholesterol, and bile salts (4). Functional analysis of numerous hepatocyte-specific promoter and enhancer regions reveals that they are composed of multiple cis-acting DNA sequences that bind different families of hepatocyte nuclear factors (HNF) (reviewed in reference 4). These include the HNF-1, HNF-3, HNF-4, CCAAT/enhancer binding protein (C/EBP), HNF-6, and fetoprotein transcription factor families (4,29,15,52,53,57). Although none of these transcriptional regulatory proteins is entirely liver specific, the requirement for combinatorial protein interactions among them in order to achieve abundant transcriptional activity plays an important role in maintaining hepatocyte-specific gene expression.The HNF-3 proteins are members of an extensive family of transcription factors that share homology in the winged helix DNA binding domain and use a modified helix-turn-helix motif to bind DNA as a monomer (8, 37). To date, the winged helix family consists of over 50 members, which play important roles in the differentiat...

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DDB2 is an essential subunit of the damaged-DNA recognition factor DDB, which is involved in global genomic repair in human cells. Moreover, DDB2 is mutated in the repair-deficiency disease xeroderma pigmentosum (Group E). Expression of DDB2 in human cells is induced by P53, BRCA1 and by ionizing radiation. The DDB2 protein associates with transcriptional activator and coactivator proteins. In addition, DDB2 in conjunction with DDB1 associates with cullin 4A and the Cop9/signalosome. We generated a mouse strain deficient for DDB2 (DDB2À/À). Consistent with the human disease (XP-E), the DDB2À/À mice were susceptible to UV-induced skin carcinogenesis. We observed a significant difference in the initial rate of cyclobutane pyrimidine dimer (CPD)-removal from the skin following UV irradiation. Also, the DDB2-deficient mice exhibited a significantly reduced life span compared to their wild-type littermates. Moreover, unlike other XP-deficient mice, the DDB2-deficient mice developed spontaneous malignant tumors at a high rate between the ages of 20 and 25 months. The observations suggest that, in addition to DNA repair, the other interactions of DDB2 are significant in its tumor suppression function.

Background & Aims Over-expression of FoxM1 correlates with poor prognosis in hepatocellular carcinoma (HCC). Moreover, the Ras-signaling pathway is found to be ubiquitously activated in HCC through epigenetic silencing of the Ras-regulators. We investigated the roles of FoxM1 in Ras-driven HCC, and on HCC cells with stem-like features. Methods We employed a transgenic mouse model that expresses the oncogenic Ras in the liver. That strain was crossed with a strain that harbor floxed alleles of FoxM1 and the MxCre gene that allows conditional deletion of FoxM1. FoxM1 alleles were deleted after development of HCC, and the effects on the tumors were analyzed. Also, FoxM1-siRNA was used in human HCC cell lines to determine its role in the survival of the HCC cells with stem cell features. Results Ras-driven tumors over-express FoxM1. Deletion of FoxM1 inhibits HCC progression. There was increased accumulation of reactive oxygen species (ROS) in the FoxM1-deleted HCC cells. Moreover, FoxM1-deletion caused a disproportionate loss of the CD44+ and EpCAM+ HCC cells in the tumors. We show that FoxM1 directly activates expression of CD44 in human HCC cells. Moreover, the human HCC cells with stem cell features are addicted to FoxM1 for ROS-regulation and survival. Conclusion Our results provide genetic evidence for an essential role of FoxM1 in the progression of Ras-driven HCC. In addition, FoxM1 is required for the expression of CD44 in HCC cells. Moreover, FoxM1 plays a critical role in the survival of the HCC cells with stem cell features by regulating ROS.

JCV is a papovavirus which is widespread in the human population. The prototype Mad-1 variant of JCV induces a fatal demyelinating disease of the central nervous system (CNS) called Progressive Multifocal Leukoencephalopathy (PML) in immunosuppressed individuals. The unique tropism of JCV (Mad-1) to the CNS is attributed to the tissue-speci®c regulation of the viral early promoter which is responsible for the production of the viral regulatory protein, T-antigen. The archetype form of this virus, JCV(CY), which has been repeatedly isolated from the urine of PML and non-PML individuals, is distinct from JCV(Mad-1) in the structural organization of the regulatory sequence. To characterize the tissue speci®c expression of JCV(CY) and to investigate its potential in inducing disease, transgenic mice containing the early region of JCV(CY) were generated. Some of these mice between 9 ± 13 months of age exhibited signs of illness as manifested by paralysis of rear limbs, hunched posture, and poor grooming. Neuropathological examination indicated no sign of hypomyelination of the brain, but surprisingly, revealed the presence of primitive tumors originating from the cerebellum and the surrounding brain stem. The tumor masses also in®ltrated the surrounding tissue. Results from RNA and protein studies revealed a high level of Tantigen mRNA expression in hindbrains of clinically normal and aected transgenic mice. However, higher levels of T-antigen RNA and protein were detected in brains of the animals exhibiting severe illness. The close resemblance of JCV(CY) induced tumor in transgenic mice to the human medulloblastoma/primitive neuroectodermal tumor (PNETs) in location, histologic appearance, and expression of marker proteins strongly suggests the utility of this novel animal model for the study of human brain tumors.