HNF3β Expression Research Articles

Genetic and Epigenetic Modification of Rat Liver Progenitor Cells via HNF4α Transduction and 5' Azacytidine Treatment: An Integrated miRNA and mRNA Expression Profile Analysis.

Neonatal liver-derived rat epithelial cells (rLEC) from biliary origin are liver progenitor cells that acquire a hepatocyte-like phenotype upon sequential exposure to hepatogenic growth factors and cytokines. Undifferentiated rLEC express several liver-enriched transcription factors, including the hepatocyte nuclear factors (HNF) 3β and HNF6, but not the hepatic master regulator HNF4α. In this study, we first investigated the impact of the ectopic expression of HNF4α in rLEC on both mRNA and microRNA (miR) level by means of microarray technology. We found that HNF4α transduction did not induce major changes to the rLEC phenotype. However, we next investigated the influence of DNA methyl transferase (DNMT) inhibition on the phenotype of undifferentiated naïve rLEC by exposure to 5′ azacytidine (AZA), which was found to have a significant impact on rLEC gene expression. The transduction of HNF4α or AZA treatment resulted both in significantly downregulated C/EBPα expression levels, while the exposure of the cells to AZA had a significant effect on the expression of HNF3β. Computationally, dysregulated miRNAs were linked to target mRNAs using the microRNA Target Filter function of Ingenuity Pathway Analysis. We found that differentially regulated miRNA–mRNA target associations predict ectopic HNF4α expression in naïve rLEC to interfere with cell viability and cellular maturation (miR-19b-3p/NR4A2, miR30C-5p/P4HA2, miR328-3p/CD44) while it predicts AZA exposure to modulate epithelial/hepatic cell proliferation, apoptosis, cell cycle progression and the differentiation of stem cells (miR-18a-5p/ESR1, miR-503-5p/CCND1). Finally, our computational analysis predicts that the combination of HNF4α transduction with subsequent AZA treatment might cause changes in hepatic cell proliferation and maturation (miR-18a-5p/ESR1, miR-503-5p/CCND1, miR-328-3p/CD44) as well as the apoptosis (miR-16-5p/BCL2, miR-17-5p/BCL2, miR-34a-5p/BCL2 and miR-494-3p/HMOX1) of naïve rLEC.

Hepatocyte Nuclear Factor 3β Plays a Suppressive Role in Colorectal Cancer Progression

Background and Objective: Hepatocyte nuclear factor 3β (HNF3β) is a key transcription factor in the development of the gastrointestinal tract. However, only few studies have examined its' expression, function and potential clinical significance in colorectal cancer tumorigenesis and progression.Methods: HNF3β expression in colorectal cancer tissue samples of 174 patients was assessed by immunohistochemistry. The results were analyzed with respect to patients' clinicopathological characteristics and survival. Following the in vitro cell transfection, MTT, wound healing, and Transwell assays were used to test cell proliferation, migration, and invasion, respectively. Western blot was used to examine IL6, JAK1, and STAT3 protein expression. The potential for tumor formation was evaluated using a mouse xenograft model.Results: HNF3β expression was lower in colon cancer tissue compared to normal tissue and correlated with UICC clinical stage (P = 0.001), depth of invasion (P = 0.004), regional lymph node metastasis (P = 0.007), distant metastasis (P = 0.048), and poor survival (P < 0.001) in patients with colorectal cancer. Furthermore, HNF3β overexpression impeded proliferation, migration and invasion of SW480 cells via JAK-STAT3 signaling in vitro. Moreso, HNF3β overexpression showed a significant growth inhibition of subcutaneous xenograft tumors in vivo.Conclusions: The results show that HNF3β acts as a suppressor of colorectal cancer progression and decreased HNF3 β expression is closely related to the poor prognosis. Thus, HNF3β may be a potential molecular target for inhibition of colorectal cancer cells and development of new anti-tumor therapies.

Transcriptional Regulation of CYP2B6 Expression by Hepatocyte Nuclear Factor 3β in Human Liver Cells.

CYP2B6 plays an increasingly important role in xenobiotic metabolism and detoxification. The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) have been established as predominant regulators for the inductive expression of CYP2B6 gene in human liver. However, there are dramatic interindividual variabilities in CYP2B6 expression that cannot be fully explained by the CAR/PXR-based modulation alone. Here, we show that expression level of CYP2B6 was correlated with that of hepatocyte nuclear factor 3β (HNF3β) in human primary hepatocytes prepared from 35 liver donors. Utilizing recombinant virus-mediated overexpression or knockdown of HNF3β in HepG2 cells, as well as constructs containing serial deletion and site-directed mutation of HNF3β binding motifs in CYP2B6 luciferase reporter assays, we demonstrated that the presence or lack of HNF3β expression markedly correlated with CYP2B6 gene expression and its promoter activity. Novel enhancer modules of HNF3β located upstream of the CYP2B6 gene transcription start site were identified and functionally validated as key elements governing HNF3β-mediated CYP2B6 expression. Chromatin immunoprecipitation assays in human primary hepatocytes and surface plasmon resonance binding affinity experiments confirmed the essential role of these enhancers in the recruitment of HNF3β to the promoter of CYP2B6 gene. Overall, these findings indicate that HNF3β represents a new liver enriched transcription factor that is involved in the transcription of CYP2B6 gene and contributes to the large interindividual variations of CYP2B6 expression in human population.

Hepatitis B Virus X Protein Induces Hepatic Steatosis by Enhancing the Expression of Liver Fatty Acid Binding Protein.

Hepatitis B virus (HBV) has been implicated as a potential trigger of hepatic steatosis although molecular mechanisms involved in the pathogenesis of HBV-associated hepatic steatosis still remain elusive. Our prior work has revealed that the expression level of liver fatty acid binding protein 1 (FABP1), a key regulator of hepatic lipid metabolism, was elevated in HBV-producing hepatoma cells. In this study, the effects of HBV X protein (HBx) mediated FABP1 regulation on hepatic steatosis and the underlying mechanism were determined. mRNA and protein levels of FABP1 were measured by quantitative RT-PCR (qPCR) and Western blotting. HBx-mediated FABP1 regulation was evaluated by luciferase assay, coimmunoprecipitation, and chromatin immunoprecipitation. Hepatic lipid accumulation was measured by using Oil-Red-O staining and the triglyceride level. It was found that expression of FABP1 was increased in HBV-producing hepatoma cells, the sera of HBV-infected patients, and the sera and liver tissues of HBV-transgenic mice. Ectopic overexpression of HBx resulted in upregulation of FABP1 in HBx-expressing hepatoma cells, whereas HBx abolishment reduced FABP1 expression. Mechanistically, HBx activated the FABP1 promoter in an HNF3β-, C/EBPα-, and PPARα-dependent manner, in which HBx increased the gene expression of HNF3β and physically interacted with C/EBPα and PPARα. On the other hand, knockdown of FABP1 remarkably blocked lipid accumulation both in long-chain free fatty acids treated HBx-expressing HepG2 cells and in a high-fat diet-fed HBx-transgenic mice. Therefore, FABP1 is a key driver gene in HBx-induced hepatic lipid accumulation via regulation of HNF3β, C/EBPα, and PPARα. FABP1 may represent a novel target for treatment of HBV-associated hepatic steatosis. Accumulating evidence from epidemiological and experimental studies has indicated that chronic HBV infection is associated with hepatic steatosis. However, the molecular mechanism underlying HBV-induced pathogenesis of hepatic steatosis still remains to be elucidated. In this study, we found that expression of liver fatty acid binding protein (FABP1) was dramatically increased in the sera of HBV-infected patients and in both sera and liver tissues of HBV-transgenic mice. Forced expression of HBx led to FABP1 upregulation, whereas knockdown of FABP1 remarkably diminished lipid accumulation in both in vitro and in vivo models. It is possible that HBx promotes hepatic lipid accumulation through upregulating FABP1 in the development of HBV-induced nonalcoholic fatty liver disease. Therefore, inhibition of FABP1 might have therapeutic value in steatosis-associated chronic HBV infection.

MicroRNA-141 inhibits cell proliferation and invasion and promotes apoptosis by targeting hepatocyte nuclear factor-3β in hepatocellular carcinoma cells.

BackgroundHepatocyte nuclear factor-3β (HNF-3β) plays a critical role in hepatocyte differentiation and controls liver-specific gene expression during the development of hepatocellular carcinoma (HCC), but the molecular basis of this process has not been fully elucidated. microRNAs (miRNAs) are powerful, post-transcriptional regulators of gene expression. Whether miRNAs can impact the effects of HNF-3β in HCC is still unknown.MethodsHNF-3β and miR-141 expression levels were detected in HepG2 cells, using real-time quantitative RT-PCR (qRT-PCR). Luciferase reporter assays and Western blots were used to validate HNF-3β as a direct target gene of miR-141. Cell proliferation, invasion, and apoptosis were also examined to confirm whether miR-141 could impact on HNF-3β in HCC.ResultsIn this study, we found that HNF-3β protein levels were consistently upregulated in HCC clinical tissues compared with matched normal adjacent tissues. However, the mRNA levels of HNF-3β varied in random tissues, suggesting that a post-transcriptional mechanism was involved in its regulation. We used bioinformatic analyses to search for miRNAs that could potentially target HNF-3β, and identified specific targeting sites for miR-141 in the 3′-untranslated region (3′-UTR) of the HNF-3β gene. By overexpressing miR-141 in HepG2 cells, we experimentally validated that miR-141 directly regulated HNF-3β expression. Furthermore, the biological consequences of targeting HNF-3β by miR-141 were examined using cell proliferation, invasion and apoptosis assays in vitro. We demonstrated that the repression of HNF-3β by miR-141 suppressed the proliferation and invasion and promoted the apoptosis of HepG2 cells.ConclusionsmiR-141 functions as a tumor suppressor in HCC cells through the inhibition of HNF-3β translation.

Reciprocal regulation of microRNA-122 and c-Myc in hepatocellular cancer: Role of E2F1 and transcription factor dimerization partner 2

c-Myc is a well-known oncogene frequently up-regulated in different malignancies, whereas liver-specific microRNA (miR)-122, a bona fide tumor suppressor, is down-regulated in hepatocellular cancer (HCC). Here we explored the underlying mechanism of reciprocal regulation of these two genes. Real-time reverse-transcription polymerase chain reaction (RT-PCR) and northern blot analysis demonstrated reduced expression of the primary, precursor, and mature miR-122 in c-MYC-induced HCCs compared to the benign livers, indicating transcriptional suppression of miR-122 upon MYC overexpression. Indeed, chromatin immunoprecipitation (ChIP) assay showed significantly reduced association of RNA polymerase II and histone H3K9Ac, markers of active chromatin, with the miR-122 promoter in tumors relative to the c-MYC-uninduced livers, indicating transcriptional repression of miR-122 in c-MYC-overexpressing tumors. The ChIP assay also demonstrated a significant increase in c-Myc association with the miR-122 promoter region that harbors a conserved noncanonical c-Myc binding site in tumors compared to the livers. Ectopic expression and knockdown studies showed that c-Myc indeed suppresses expression of primary and mature miR-122 in hepatic cells. Additionally, Hnf-3β, a liver enriched transcription factor that activates miR-122 gene, was suppressed in c-MYC-induced tumors. Notably, miR-122 also repressed c-Myc transcription by targeting transcriptional activator E2f1 and coactivator Tfdp2, as evident from ectopic expression and knockdown studies and luciferase reporter assays in mouse and human hepatic cells. c-Myc represses miR-122 gene expression by associating with its promoter and by down-regulating Hnf-3β expression, whereas miR-122 indirectly inhibits c-Myc transcription by targeting Tfdp2 and E2f1. In essence, these results suggest a double-negative feedback loop between a tumor suppressor (miR-122) and an oncogene (c-Myc).

The Effect of Estrogen Compounds on Human Embryoid Bodies

Human embryonic stem cells are derived from the inner cell mass of preimplantation embryo at the blastocyst stage and their differentiation occurs through an intermediate step involving the formation of embryoid bodies (EBs), which are aggregates of embryonic stem cells. The EBs seem to be a powerful tool for investigating the development of embryos, as they can mimic the initial stages of embryonic development. In this study, we aimed to investigate the effect of estrogen compounds on the proliferation and differentiation of short-term and long-term cultured EBs in vitro. For this study, 10-day-old (short-term cultured) and 30-day-old (long-term cultured) EBs were subjected to estradiol (E2), estriol (E3), selective estrogen receptor modulator (raloxifene [RLX]), bisphenol A, and 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole for 7 days. To confirm the effects of estrogen treatment, ICI-182780 was added to the respective EBs for additional 7 days following estrogen treatment. Quantitative reverse transcription-polymerase chain reaction was performed to analyze the relative expression of differentiation marker genes representing the 3 germ layers. The expression of 7 marker genes, which included α-fetoprotein, hepatocyte nuclear factor (HNF)-3β, HNF-4α (endoderm), brachyury, cardiac actin ([cACT]; mesoderm), nestin (ectoderm), and Oct-4 (undifferentiated), was measured. Significantly, lower expression of HNF-4α in both short-term and long-term cultured EBs was observed after treatment of estrogen compounds compared to control. The expression of HNF-3β in short-term cultured EBs has been positively affected by E2, E3, and RLX. Regarding cACT, higher expression was observed after treatment of E2 (10(-7) mol/L) and E3 (10(-9) mol/L) in short-term cultured EBs, but opposite effects were demonstrated in long-term cultured EBs. The lower expressions of HNF-4α by E2 and RLX were negated by ICI-182780 treatment, although these findings were not statistically significant in E3-treated group. These findings suggest that estrogen compounds have effects on endodermal and mesodermal differentiation of human EBs.

Study of the expression levels of Hepatocyte nuclear factor 4 alpha and 3 beta in patients with different outcome of HBV infection

Hepatocyte nuclear factors 4 alpha (HNF4α) and 3 beta (HNF3β) are members of a group of liver-enriched transcription factors (LETFs) that play important roles in regulating the replication of hepatitis B virus (HBV) and liver inflammation. However, the relationship of the level of HNF4α and HNF3β with the severity of HBV-infected liver diseases is unclear. In this study, liver tissue samples from different types of HBV patients were collected, and HNF4α and HNF3β expression were detected by immunohistochemistry. The expression of HNF4α was significant higher in patients with severe hepatitis B(SHB) than those with chronic hepatitis B(CHB) and liver cirrhosis(LC) (both P < 0.05), but similar between patients with CHB and LC (P > 0.05). And the expression of HNF3β was similar among patients with CHB, LC and SHB (P > 0.05 for all pairwise comparison). This suggests that the expression level of HNF4α was different in patients with different outcome of HBV infection, high expression level of HNF4α may correlate with occurrence of SHB

The correlation of hepatocyte nuclear factor 4 alpha and 3 beta with hepatitis B virus replication in the liver of chronic hepatitis B patients

Hepatocyte nuclear factors 4 alpha (HNF4alpha) and 3 beta (HNF3beta) are members of a group of liver-enriched transcription factors (LETFs) that play important roles in regulating the replication of hepatitis B virus (HBV). Using cell culture and animal models, we showed that HNF4alpha supports HBV replication in nonhepatic cells and HNF3beta inhibits HBV replication. However, the expression of HNF4alpha and HNF3beta in the liver tissue of chronic HBV-infected patients and the relationship between the levels of HNF4alpha and HNF3beta and HBV replication are unclear. In this study, liver biopsy specimens from 86 chronic HBV-infected patients were collected. The expression levels of HNF4alpha, HNF3beta, hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) were detected by an immunohistochemical technique and the level of HBV DNA was checked by in situ hybridization with serial sections from liver biopsy tissue samples. We show here that samples with higher levels of HNF4alpha expression also have higher levels of HBsAg, HBcAg and HBV DNA. In contrast, in samples with higher levels of HNF3beta expression, levels of HBsAg, HBcAg and HBV DNA were lower. There was a positive correlation between HNF4alpha expression and HBV replication, and a negative correlation between HNF3beta expression and HBV replication, in the liver of chronic HBV-infected patients. This suggests that HNF4alpha and HNF3beta likely participate in HBV replication in patients with HBV infection, or that HBV replication may somehow influence the expression of HNF4alpha and HNF3beta in the liver.

SLC2A2 gene expression in kidney of diabetic rats is regulated by HNF-1α and HNF-3β

We hypothesize that, in kidney of diabetic rats, hepatocyte nuclear factors (HNF-1α and HNF-3β) play a critical role in the overexpression of solute carrier 2A2 (SLC2A2) gene. Diabetic rats submitted or not to rapid (up to 12 h) and short-term (1, 4 and 6 days) insulin treatment were investigated. Twofold increase in GLUT2 mRNA was observed in diabetic, accompanied by significant increases in HNF-1α and HNF-3β expression and binding activity. Additional 2-fold increase in GLUT2 mRNA and HNF-3β expression/activity was observed in 12-h insulin-treated rats. Six-day insulin treatment decreased GLUT2 mRNA and HNF-1α expression and activity to levels of non-diabetic rats, whereas HNF-3β decreased to levels of non-insulin-treated diabetic rats. Our results provide evidence for a link between the overexpression of SLC2A2 gene and the transcriptional activity of HNF-1α and HNF-3β in kidney of diabetic rats. Furthermore, recovery of SLC2A2 gene after 6-day insulin treatment also involves HNF-1α and HNF-3β activity.

15-Hydroxyprostaglandin Dehydrogenase is a Target of Hepatocyte Nuclear Factor 3β and a Tumor Suppressor in Lung Cancer

The forkhead transcription factor hepatocyte nuclear factor 3beta (HNF3beta) is essential in foregut development and the regulation of lung-specific genes. HNF3beta expression leads to growth arrest and apoptosis in lung cancer cells and HNF3beta is a candidate tumor suppressor in lung cancer. In a transcriptional profiling study using a conditional cell line system, we now identify 15-PGDH as one of the major genes induced by HNF3beta expression. 15-PGDH is a critical metabolic enzyme of proliferative prostaglandins, an antagonist to cyclooxygenase-2 and a tumor suppressor in colon cancer. We confirmed the regulation of 15-PGDH expression by HNF3beta in a number of systems and showed direct binding of HNF3beta to 15-PGDH promoter elements. Western blotting of lung cancer cell lines and immunohistochemical examination of human lung cancer tissues found loss of 15-PGDH expression in approximately 65% of lung cancers. Further studies using in vitro cell-based assays and in vivo xenograft tumorigenesis assays showed a lack of in vitro but significant in vivo tumor suppressor activity of 15-PGDH via an antiangiogenic mechanism analogous to its role in colon cancer. In summary, we identify 15-PGDH as a direct downstream effector of HNF3beta and show that 15-PGDH acts as a tumor suppressor in lung cancer.

Steroid hormones (17β-estradiol and hydrocortisone) upregulate hepatocyte nuclear factor (HNF)-3β and insulin-like growth factors I and II expression in the gonads of tilapia ( Oreochromis mossambicus) in vitro

Hepatocyte nuclear factors (HNF-1α, -1β and -3β) and insulin-like growth factors (IGF-I and -II), which are involved in liver-specific gene expression, metabolism, development and cell growth, have been found in the gonads of tilapia ( Oreochromis mossambicus). However, the functions of these factors and how they interact within the gonads of bony fish are not understood. In the present study, we provided experimental evidence that the expression of HNF-3β in the gonads of tilapia, but not HNF-1α and -1β, was affected in vitro by 17β-estradiol and hydrocortisone. Immunohistochemical staining confirmed that tilapia HNF-3β was mainly found in the nuclei of hepatocytes, the follicular granulosa cells of the ovaries, and the interstitial cells of the testes of adult tilapia. Further data were gathered at various steroid concentrations (0.1, 1, 10, 100, and 1000 nM) over various culture intervals (6, 12, 18, 24, 30, and 36 h) and subjected to semi-quantitative RT-PCR analysis. The expression of downstream genes (IGF-I and -II) followed the same temporal patterns as HNF-3β, albeit at decreased levels for 30 and 36 h culture intervals. Both hormones upregulated HNF-3β mRNA expression at concentrations of 0.1–10 nM, and reached optimal physiological concentrations for induction of IGFs at 1–10 nM. The identity of the PCR fragments was concurrently verified by sequencing and PCR-Southern hybridization. We inferred that HNF-3β and IGFs may play a regulatory role in tilapia gonads during oocyte maturation and spermatogenesis.

Induction of CYP2C12 expression in senescent male rats is well correlated to an increase of HNF3β expression, while the decline of CYP2C11 expression is unlikely due to a decrease of STAT5 activation

Ageing affects drugs metabolism influencing the therapeutic efficacy and safety of drugs. By using the experimental model of aged male rats, we investigated the influence of ageing on some CYP2C isoforms, the most important CYP450 sub-family in rats. The activity of the male specific CYP2C11 is decreased by 55% in senescent male rats. This correlates with a significant reduction of both protein content (80%) and mRNA (60%) indicating a demasculinization process. The expression of CYP2C12, a female specific isoform, is induced in senescent male rats indicating a feminization process. Neither the activity nor the expression of CYP2C6, a female predominant isoform, is modified in senescent male rats. Thereafter, certain putative GH mediators like some liver enriched transcription factors (LETFs) or STAT5b were investigated. The amount of HNF3β mRNA, a transcription factor involved in the up-regulation of CYP2C12, has been shown to increase by about three-fold in senescent male rats. With regard to STAT5b, which has been reported to be involved in the male specific regulation of CYP2C11, large amounts of phosphorylated STAT5 were observed in the liver of senescent male rats. These results indicate that while the induction of CYP2C12 during ageing could be due, at least partially, to the enhanced HNF3β expression, the decline of CYP2C11 is unlikely related to a decrease of STAT5 activation.

Dietary PUFA modulate the expression of proliferation and differentiation markers in Morris 3924A hepatoma cells

The effect of dietary polyunsaturated fatty acids on the expression of differentiation and proliferation markers in Morris 3924A hepatoma cells was investigated. ACT/I rats were conditioned 10 days with diets enriched with linoleic acid or α-linolenic acid before subcutaneous hepatoma cell transplantation. After 19 days from the inoculum, the mRNA levels of liver-enriched transcription factors and of their target genes were quantified. Both linoleic acid- and linolenic acid-enriched diets induced a decrease of β-actin, AFP, PCNA, c- myc and of hepatocyte nuclear factors HNF-1α and HNF-4α mRNA levels in tumor tissue whereas HNF-3β expression was induced by both dietary treatments. Only the α-linolenic acid-enriched diet was effective in reducing c- jun and increasing albumin mRNA levels. Since albumin is a C/EBPα target gene, C/EBPα gene transcription was evaluated at both protein and mRNA levels. It was found that α-linolenic acid-enriched diet did not enhance the C/EBPα mRNA content in hepatoma tissue while inducing C/EBPα protein expression with an isoform pattern similar to the hepatic phenotype. This evidence implies that α-linolenic acid or one of its metabolic products induce albumin synthesis in hepatoma cells by modulating C/EBPα gene expression at post-transcriptional level.

PKCλ regulates glucose-induced insulin secretion through modulation of gene expression in pancreatic β cells

Altered regulation of insulin secretion by glucose is characteristic of individuals with type 2 diabetes mellitus, although the mechanisms that underlie this change remain unclear. We have now generated mice that lack the λ isoform of PKC in pancreatic β cells (βPKCλ–/– mice) and show that these animals manifest impaired glucose tolerance and hypoinsulinemia. Furthermore, insulin secretion in response to high concentrations of glucose was impaired, whereas the basal rate of insulin release was increased, in islets isolated from βPKCλ–/– mice. Neither the β cell mass nor the islet insulin content of βPKCλ–/– mice differed from that of control mice, however. The abundance of mRNAs for Glut2 and HNF3β was reduced in islets of βPKCλ–/– mice, and the expression of genes regulated by HNF3β was also affected (that of Sur1 and Kir6.2 genes was reduced, whereas that of hexokinase 1 and hexokinase 2 genes was increased). Normalization of HNF3β expression by infection of islets from βPKCλ–/– mice with an adenoviral vector significantly reversed the defect in glucose-stimulated insulin secretion. These results indicate that PKCλ plays a prominent role in regulation of glucose-induced insulin secretion by modulating the expression of genes important for β cell function.

A transcriptional profiling study of CCAAT/enhancer binding protein targets identifies hepatocyte nuclear factor 3 beta as a novel tumor suppressor in lung cancer.

We showed previously that CCAAT/enhancer binding protein alpha (C/EBP alpha), a tissue-specific transcription factor, is a candidate tumor suppressor in lung cancer. In the present study, we have performed a transcriptional profiling study of C/EBP alpha target genes using an inducible cell line system. This study led to the identification of hepatocyte nuclear factor 3beta (HNF3 beta), a transcription factor known to play a role in airway differentiation, as a downstream target of C/EBP alpha. We found down-regulation of HNF3 beta expression in a large proportion of lung cancer cell lines examined and identified two novel mutants of HNF3 beta, as well as hypermethylation of the HNF3 beta promoter. We also developed a tetracycline-inducible cell line model to study the cellular consequences of HNF3 beta expression. Conditional expression of HNF3 beta led to significant growth reduction, proliferation arrest, apoptosis, and loss of clonogenic ability, suggesting additionally that HNF3 beta is a novel tumor suppressor in lung cancer. This is the first study to show genetic abnormalities of lung-specific differentiation pathways in the development of lung cancer.

Inhibition of mesodermal fate by Xenopus HNF3β/FoxA2

The winged-helix transcription factor HNF3β/FoxA2 is expressed in embryonic organizing centers of the gastrulating mouse, frog, fish, and chick. In the mouse, HNF3β is required for the formation of the mammalian node and notochord, and can induce ectopic floor plate formation when misexpressed in the developing neural tube; HNF3β expression in the extraembryonic endoderm is also necessary for the proper morphogenesis of the mammalian primitive streak. In the frog Xenopus laevis, several lines of evidence suggest that the related winged-helix factor Pintallavis functions as the ortholog of mammalian HNF3β in both axial mesoderm and neurectoderm; the role of Xenopus HNF3β itself, however, has not been clearly defined, and is the subject of this study. HNF3β is widely expressed in the vegetal pole but, as previously suggested, is excluded from the gastrula-stage mesoderm. We find that expression of an HNF3β-Engrailed repressor fusion protein induces ectopic axes and inhibits head formation in Xenopus embryos, while ectopic HNF3β inhibits mesoderm and anterior endoderm formation in explant assays and in vivo. Our studies suggest that HNF3β target genes function to limit the extent of mesoderm formation in the Xenopus gastrula, and point to related roles for Xenopus HNF3β and the extraembryonic component of mammalian HNF3β during vertebrate gastrulation.

Hepatocyte nuclear factor 3beta inhibits hepatitis B virus replication in vivo.

Hepatitis B virus (HBV) transgenic mice expressing rat hepatocyte nuclear factor 3beta (HNF3beta) were generated by breeding HBV transgenic mice with transgenic mice that constitutively overexpress the rat HNF3beta polypeptide in the liver. HBV 3.5-, 2.4- and 2.1-kb transcripts were reduced 2- to 4-fold in these mice relative to the HBV transgenic mouse controls. In contrast, the abundance of viral replication intermediates was profoundly reduced in HBV transgenic mice by overexpression of HNF3beta. This results, in part, from the preferential reduction in the level of the pregenomic 3.5-kb RNA relative to the precore 3.5-kb RNA. Therefore, it is apparent that increased expression of HNF3beta modestly reduces viral transcription and dramatically inhibits replication in vivo in the HBV transgenic mouse. This suggests that altering the activity of this transcription factor in vivo in chronic HBV carriers might be therapeutically beneficial.

Dual origin of the floor plate in the avian embryo.

Molecular analysis carried out on quail-chick chimeras, in which quail Hensen's node was substituted for its chick counterpart at the five- to six-somite stage (ss), showed that the floor plate of the avian neural tube is composed of distinct areas: (1) a median one (medial floor plate or MFP) derived from Hensen's node and characterised by the same gene expression pattern as the node cells (i.e. expression of HNF3beta and Shh to the exclusion of genes early expressed in the neural ectoderm such as CSox1); and (2) lateral regions that are differentiated from the neuralised ectoderm (CSox1 positive) and form the lateral floor plate (LFP). LFP cells are induced by the MFP to express HNF3beta transiently, Shh continuously and other floor-plate characteristic genes such as NETRIN: In contrast to MFP cells, LFP cells also express neural markers such as Nkx2.2 and Sim1. This pattern of avian floor-plate development presents some similarities to floor-plate formation in zebrafish embryos. We also demonstrate that, although MFP and LFP have different embryonic origins in normal development, one can experimentally obtain a complete floor plate in the neural epithelium by the inductive action of either a notochord or a MFP. The competence of the neuroepithelium to respond to notochord or MFP signals is restricted to a short time window, as only the posterior-most region of the neural plate of embryos younger than 15 ss is able to differentiate a complete floor plate comprising MFP and LFP. Moreover, MFP differentiation requires between 4 and 5 days of exposure to the inducing tissues. Under the same conditions LFP and SHH-producing cells only induce LFP-type cells. These results show that the capacity to induce a complete floor plate is restricted to node-derived tissues and probably involves a still unknown factor that is not SHH, the latter being able to induce only LFP characteristics in neuralised epithelium.

Zinc depletion reduced Egr-1 and HNF-3beta expression and apolipoprotein A-I promoter activity in Hep G2 cells.

We examined the influence of zinc status on expression of certain transcription factors involved in regulation of apolipoprotein A-I (apoAI) expression in human hepatoblastoma Hep G2 cells. A low zinc basal medium (zinc deficient, ZD) consisting of DMEM and 10% Chelex100-treated fetal bovine serum was used to deplete cellular zinc over one passage. Cells were also cultured for one passage in medium supplemented with 0.4 (ZD0.4), 4.0 (zinc normal, ZN), 16.0 (zinc adequate, ZA), or 32.0 microM zinc (zinc supplemented, ZS). Compared with ZN cells, cellular zinc levels were 43 and 31% lower in ZD and ZD0.4 cells but 70 and 146% higher in ZA and ZS cells, respectively. Supplementation of 0.4 microM zinc significantly increased DNA contents per plate, from 65% in ZD cells to 83% in ZD0.4 cells compared with ZN cells. Addition of >4 microM zinc in medium did not further increase DNA contents. The proportion of cells in G(1)/S and S phase was about fourfold higher and threefold lower, respectively, in ZD cells compared with ZN and other groups. Nuclear Egr-1 protein was markedly decreased in ZD and ZD0.4 cells. Moreover, hepatocyte nuclear factor (HNF)-3beta was severely degraded in ZD and ZD0.4 cells. In contrast, HNF-4alpha remained stable in all groups and was not significantly lower in ZD and ZD0.4 cells. Furthermore, downregulation of trans-acting factor Egr-1 and cleavage of HNF-3beta were associated with reduction of apoAI promoter activity in zinc-deficient Hep G2 cells. Thus zinc is critical in transcriptional regulation of apoAI gene expression in hepatocytes.