Roles of hepatocyte nuclear factors in hepatitis B virus infection.

Approximately 350 million people are estimated to be persistently infected with hepatitis B virus (HBV) worldwide. HBV maintains persistent infection by employing covalently closed circular DNA (cccDNA), a template for all HBV RNAs. Chronic hepatitis B (CHB) patients are currently treated with nucleos(t)ide analogs such as lamivudine, adefovir, entecavir, and tenofovir. However, these treatments rarely cure CHB because they are unable to inhibit cccDNA transcription and inhibit only a late stage in the HBV life cycle (the reverse transcription step in the nucleocapsid). Therefore, an understanding of the factors regulating cccDNA transcription is required to stop this process. Among numerous factors, hepatocyte nuclear factors (HNFs) play the most important roles in cccDNA transcription, especially in the generation of viral genomic RNA, a template for HBV replication. Therefore, proper control of HNF function could lead to the inhibition of HBV replication. In this review, we summarize and discuss the current understanding of the roles of HNFs in the HBV life cycle and the upstream factors that regulate HNFs. This knowledge will enable the identification of new therapeutic targets to cure CHB.

Pregnane X receptor, constitutive androstane receptor and hepatocyte nuclear factors as emerging players in cancer precision medicine.

Great research effort has been focused on elucidating the contribution of host genetic variability on pharmacological outcomes in cancer. Nuclear receptors have emerged as mediators between environmental stimuli and drug pharmacokinetics and pharmacodynamics. The pregnane X receptor, constitutive androstane receptor and hepatocyte nuclear factors have been reported to regulate transcription of genes that encode drug metabolizing enzymes and transporters. Altered nuclear receptor expression has been shown to affect the metabolism and pharmacological profile of traditional chemotherapeutics and targeted agents. Accordingly, polymorphic variants in these genes have been studied as pharmacogenetic markers of outcome variability. This review summarizes the state of knowledge about the roles played by pregnane X receptor, constitutive androstane receptor and hepatocyte nuclear factor expression and genetics as predictive markers of anticancer drug toxicity and efficacy, which can improve cancer precision medicine.

Hepatocyte nuclear factor-4α interacts with other hepatocyte nuclear factors in regulating transthyretin gene expression.

Transthyretin is a negative acute phase protein whose serum level decreases during the acute phase response. Transthyretin gene expression in the liver is regulated at the transcriptional level, and is controlled by hepatocyte nuclear factor (HNF)-4α and other HNFs. The site-directed mutagenesis of HNF-4, HNF-1, HNF-3 and HNF-6 binding sites in the transthyretin proximal promoter dramatically decreases transthyretin promoter activity. Interestingly, the mutation of the HNF-4 binding site not only abolishes the response to HNF-4α, but also reduces significantly the response to other HNFs. However, mutation of the HNF-4 binding site merely affects the specific binding of HNF-4α, but not other HNFs, suggesting that an intact HNF-4 binding site not only provides a platform for specific interaction with HNF-4α, but also facilitates the interaction of HNF-4α with other HNFs. In a cytokine-induced acute phase response cell culture model, we observed a significant reduction in the binding of HNF-4α, HNF-1α, HNF-3ß and HNF-6α to the transthyretin promoter, which correlates with a decrease in transthyretin expression after injury. These findings provide new insights into the mechanism of the negative transcriptional regulation of the transthyretin gene after injury caused by a decrease in the binding of HNFs and a modulation in their coordinated interactions.

Synergistic effect of radiation and interleukin-6 on hepatitis B virus reactivation in liver through STAT3 signaling pathway.

PURPOSE: Hepatitis B virus (HBV) reactivation can occur after radiotherapy (RT) for hepatobiliary malignancies. Our previous in vitro culture study identified interleukin-6 (IL-6) as the main bystander mediator of RT-induced HBV replication. We attempted to examine the molecular mechanism in HBV-transgenic mice. METHODS AND MATERIALS: HBV transgenic mice were treated with whole liver RT (4 Gy daily for 5 days) with or without administration of IL-6 (400 ng twice daily for 15 days). The serum level of HBV DNA was measured using real-time polymerase chain reaction, and the IL-6 concentration was measured using enzyme-linked immunosorbent assay. The intensity of immunostaining with antibodies to HBV core protein and phosphorylated signal transducer and activator of transcription (STAT)3 in the mouse liver was qualitatively analyzed. HepG2.2.15 cells (a human hepatoblastoma cell line that persistently produces HBV DNA) were used to investigate the molecular role of IL-6 plus RT in HBV reactivation. RESULTS: HBV reactivation was induced in vivo with IL-6 plus RT (5.58-fold) compared with RT alone (1.31-fold, p = .005), IL-6 alone (1.31-fold, p = .005), or sham treatment (1.22-fold, p = .004). HBV core protein staining confirmed augmentation of intrahepatic HBV replication. IL-6 plus RT-induced HBV DNA replication in HepG2.2.15 cells was suppressed by the STAT3 inhibitor AG490 and by transfection with dominant-negative STAT3 plasmid. Phosphorylated STAT3 staining was strongest in liver tissue from mice treated with IL-6 plus RT. The mobility shift assay demonstrated that reactivation was mediated through the interaction of phosphorylated STAT3/hepatocyte nuclear factor-3 complex with HBV enhancer 1. CONCLUSION: RT to the liver and longer sustained IL-6 induced HBV reactivation through the STAT3 signal transduction pathway.

[On the role of liver-enriched transcription factors in regulating HBV transcription and replication].

OBJECTIVE: To investigate the effects of various liver-enriched transcription factors in regulating HBV transcription and replication, and to explore their potential roles in HBV hepatotropism. METHODS: The replication-competent HBV recombinant plasmid pHBV4.1 plus different liver-enriched transcription factor (HNF1, HNF3, HNF4, HNF6, C/EBP and RXRa/PPARa) expression plasmids were co-transfected into nonhepatic cell lines (NIH3T3, HeLa, 293T, SW1353, CV-1 and COS1). The transcription levels of 3.5 kb, 2.4/2.1 kb and 0.7 kb HBV RNA were analyzed by Northern blot hybridization, and the level of HBV DNA replication intermediates was detected by Southern blot hybridization analysis. RESULTS: In the absence of co-transfected liver enriched transcription factor expression vectors, the 3.5 kb HBV RNA is not transcribed and HBV DNA replication is not detected after transfecting of NIH 3T3 cells with pHBV4.1. Expression of the liver-enriched transcription factor HNF4 or RXRalpha/PPARalpha, stimulates the transcription of 3.5 kb HBV RNA and the replication of HBV DNA. In contrast, expression of HNF1, HNF3, HNF6 and C/EBP does not stimulate the transcription of 3.5 kb HBV RNA and therefore does not activate viral replication. HNF4 and RXRalpha/PPARalpha were also shown to activate the transcription of 3.5 kb HBV RNA and viral replication in divers cell types including HeLa, 293T, SW1353, CV-1 and COS1 cells. Mutation of the proximal nucleocapsid HNF4 binding site results in a greatly decreased level of HNF4 or RXRalpha/PPARalpha dependent HBV replication. CONCLUSION: This study demonstrated that the liver-enriched transcription factors HNF4 and RXRa/PPARa can support HBV transcription and replication in nonhepatic cells, indicating that liver-specific gene transcription is one of the determinants of HBV hepatotropism.

Regulatory mechanisms controlling human E-cadherin gene expression.

In cancer cells, loss of E-cadherin gene expression caused dysfunction of the cell-cell junction system, triggering cancer invasion and metastasis. Therefore, E-cadherin is an important tumor-suppressor gene. To understand how E-cadherin gene expression is regulated in cancer cells, we have used E-cadherin-positive and -negative expressing cells to find out the possible up- or down regulating transcription factors in human E-cadherin regulatory sequences. Functional analysis of human E-cadherin regulatory sequences constructs indicated that AML1, Sp1, and p300 may play important roles in promoting E-cadherin expression. In addition, we found there are four HNF3-binding sites in human E-cadherin regulatory sequences. The exogenous HNF3 can enhance the E-cadherin promoter activity in metastatic breast cancer cells and the metastatic breast cancer cells stably transfected with HNF3 showed re-expression of E-cadherin. The HNF3 stable transfectants changed from mesenchymal-like into epithelial morphology. The transwell assays showed the re-expressed E-cadherin reduced cell motility of metastatic breast cancer cells. These results suggested HNF3 may play important roles in the upregulation of the E-cadherin promoter, with the consequent re-expression of E-cadherin, thus reducing the metastatic potential of breast cancer cells. These findings suggested HNF3 plays important roles in the upregulation of the E-cadherin gene and may be able to reduce the motility of metastatic breast cancer cells.