Liver-enriched Transcription Factors Research Articles

Wnt/β-catenin signaling regulates amino acid metabolism through the suppression of CEBPA and FOXA1 in liver cancer cells

Deregulation of the Wnt/β-catenin pathway is associated with the development of human cancer including colorectal and liver cancer. Although we previously showed that histidine ammonia lyase (HAL) was transcriptionally reduced by the β-catenin/TCF complex in liver cancer cells, the mechanism(s) of its down-regulation by the complex remain to be clarified. In this study, we search for the transcription factor(s) regulating HAL, and identify CEBPA and FOXA1, two factors whose expression is suppressed by the knockdown of β-catenin or TCF7L2. In addition, RNA-seq analysis coupled with genome-wide mapping of CEBPA- and FOXA1-binding regions reveals that these two factors also increase the expression of arginase 1 (ARG1) that catalyzes the hydrolysis of arginine. Metabolome analysis discloses that activated Wnt signaling augments intracellular concentrations of histidine and arginine, and that the signal also increases the level of lactic acid suggesting the induction of the Warburg effect in liver cancer cells. Further analysis reveals that the levels of metabolites of the urea cycle and genes coding its related enzymes are also modulated by the Wnt signaling. These findings shed light on the altered cellular metabolism in the liver by the Wnt/β-catenin pathway through the suppression of liver-enriched transcription factors including CEBPA and FOXA1.

Abstract 7079: The Wnt/β-catenin signaling pathway regulates cellular metabolism through the suppression of HAL and ARG1 in liver cancer cells

Abstract Aberrant activation of the Wnt/β-catenin signaling pathway plays a crucial role in a wide range of human tumors including colorectal and liver cancer. The activated signaling causes nuclear accumulation of β-catenin, which leads to the elevated expression of target genes of TCF/LEF (T-cell factor/lymphoid enhancer factor) transcription factors through their interaction with nuclear β-catenin. Recent studies have identified a lot of genes induced by this pathway such as MYC, CCND1, AXIN2, and LGR5, and clarified their roles in tumorigenesis. However, the roles of the genes down-regulated by the pathway remain to be elucidated. Our previous study revealed that the histidine ammonia-lyase gene (HAL) encoding an enzyme involved in the catabolism of histidine was down-regulated by the signaling pathway in liver cancer. In addition, we identified a region that is involved in the Wnt-dependent regulation of HAL. To clarify the regulatory mechanism of HAL by the pathway, we searched for transcription factors that are associated with the regulatory region, and identified CEBPA and FOXA1, two liver-enriched transcription factors. It is of note that the expression of these factors was reduced by the TCF/β-catenin complex. Further investigation identified arginase 1 (ARG1) encoding an enzyme that catalyzes the hydrolysis of arginine as another target gene of CEBPA and FOXA1. Consistent with these data, inhibition of the Wnt signaling increased the expression of ARG1 as well as HAL in liver cancer cells. Furthermore, the up-regulation of HAL and ARG1 by the inhibition of this pathway decreased intracellular concentrations of histidine and arginine, suggesting that the Wnt signaling plays an important role in cellular metabolism through the regulation of the two transcription factors. These findings shed light on the role of the Wnt/β-catenin signaling pathway in the metabolism of amino acids in liver cancer cells. Citation Format: Saya Nakagawa, Kiyoshi Yamaguchi, Kiyoko Takane, Sho Tabata, Tsuneo Ikenoue, Yoichi Furukawa. The Wnt/β-catenin signaling pathway regulates cellular metabolism through the suppression of HAL and ARG1 in liver cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7079.

ZBTB7B is a permissive regulator of hepatocellular carcinoma initiation by repressing c-Jun expression and function

Hepatocarcinogenesis is a multi-step process. However, the regulators of hepatocellular carcinoma (HCC) initiation are understudied. Adult liver-specific gene expression was globally downregulated in HCC. We hypothesize that adult liver-specific genes, especially adult liver-enriched transcription factors may exert tumor-suppressive functions in HCC. In this study, we identify ZBTB7B, an adult liver-enriched transcription factor as a permissive regulator of HCC initiation. ZBTB7B is highly expressed in hepatocytes in adult livers, compared to fetal livers. To evaluate the functions of ZBTB7B in hepatocarcinogenesis, we performed hepatocyte-specific ZBTB7B knockout in hydrodynamic oncogene transfer-induced mouse liver cancer models. Hepatocyte-specific knockout of ZBTB7B promotes activated Akt and N-Ras-induced HCC development. Moreover, ZBTB7B deficiency sensitizes hepatocytes to a single oncogene Akt-induced oncogenic transformation and HCC initiation, which is otherwise incompetent in inducing HCC. ZBTB7B deficiency accelerates HCC initiation by down-regulating adult liver-specific gene expression and priming livers to a fetal-like state. The molecular mechanism underlying ZBTB7B functions in hepatocytes was investigated by integrated transcriptomic, phosphoproteomic, and chromatin immunoprecipitation-sequencing analyses. Integrative multi-omics analyses identify c-Jun as the core signaling node in ZBTB7B-deficient liver cancer initiation. c-Jun is a direct target of ZBTB7B essential to accelerated liver cancer initiation in ZBTB7B-deficient livers. Knockdown of c-Jun expression or dominant negative c-Jun expression delays HCC development in ZBTB7B-deficient livers. In addition, ZBTB7B competes with c-Jun for chromatin binding. Ectopic ZBTB7B expression attenuates the tumor-promoting functions of c-Jun. Expression of ZBTB7B signature, composed of 140 genes co-regulated by ZBTB7B and c-Jun, is significantly downregulated in early-stage HCCs compared to adjacent normal tissues, correlates to liver-specific gene expression, and is associated with good prognosis in human HCC. Thus, ZBTB7B functions as a permissive regulator of HCC initiation by directly regulating c-Jun expression and function.

Cell-Specific Regulation of Inflammatory Cytokines and Acute-Phase Proteins by the Glucocorticoid Receptor

Background. Literature and data mining found abnormal induction of chemokine (C-X-C motif) ligand 1 (CXCL1) and CXCL8 and down-regulation of CXCL2 in inflammatory liver diseases. This study was performed to understand the glucocorticoid receptor’s (GR’s) effects on chemokine and acute-phase protein expression in human liver, in settings of bacterial infection (modeled using LPS) or inflammation (modeled using TNFα). Methods. Primary human hepatocytes (PHH) were treated with combinations of tumor necrosis factor alpha (TNFα), lipopolysaccharide (LPS), and dexamethasone (DEX) for 24 h, following which chemokine mRNA and protein expression were analyzed using qPCR and enzyme-linked immunosorbent assay assays. Dual luciferase assays were performed on transfected cell lines. Mutant CXCL2 promoters were used in dual luciferase assays to identify specific regions of the CXCL2 promoter affected by GR, TNFα, or hepatocyte nuclear factor 4α (HNF4α, a liver-enriched transcription factor). Results. In PHH from donor 1, GR strongly inhibited LPS-induced CXCL1 and CXCL8 translation and transcription, whereas CXCL2 transcription tended to increase with DEX treatment. In PHH from donor 2, DEX treatment inhibited protein expression and secretion of CXCL1 and CXCL8 induced by TNFα and/or LPS, whereas CXCL2 upregulation was largely unaffected by DEX treatment. In nonliver HEK293T cells GR activity inhibited CXCL2 promoter activity. However, in liver-derived HEPG2 cells, GR induced CXCL2 promoter activity. A 407-base pair region upstream of CXCL2 promoter is necessary for full GR functionality in HEPG2 cells. TNFα synergized with HNF4α in inducing CXCL2 promoter activity in HEPG2 cells. Conclusions. GR’s effects on chemokine expression are cell-type specific and chemokine specific. GR down-regulated CXCL1 and CXCL8 in different cell types, whereas the specific activation of CXCL2 in hepatocytes and down-regulation of CXCL2 in nonhepatocytes by GR appears due to cell-specific utilization of CXCL2 promoter. By specifically increasing GR activity in the liver, we may normalize chemokine imbalances and prevent sepsis in inflammatory liver diseases.

Combinational Overexpression of <i>Foxa3</i> and <i>Hnf4a</i> Enhance the Proliferation and Prolong the Functional Maintenance of Primary Hepatocytes

In an in vitro culture system, primary hepatocytes usually display a low proliferation capacity, accompanied with a decrease of viability and a loss of hepatocyte-specific functions. Previous studies have demonstrated that the combination introductions of certain hepatocyte-specific transcription factors are able to convert fibroblasts into functional hepatocyte-like cells. However, such combinational usage of transcription factors in primary hepatocytes culture has not yet sufficiently studied. The forkhead box protein A3 (FoxA3) and hepatocyte nuclear factor 4α (Hnf4α) are liver-enriched transcription factors that play vital roles in the differentiation, and maintenance of hepatocytes. Thus, we simultaneously overexpressed the two genes, Foxa3 and Hnf4a, in rat hepatocytes and observed that the combinational augmentation of these two transcription factors have enhanced the proliferation and stabilized the hepatocyte-specific functions of primary hepatocytes over a long-term culture period.

Combinational Overexpression of Foxa3 and Hnf4α Enhance the Proliferation and Prolong the Functional Maintenance of Primary Hepatocytes

In an in vitro culture system, primary hepatocytes usually display a low proliferation capacity, accompanied with a decrease of viability and a loss of hepatocyte-specific functions. Previous studies have demonstrated that the combination introductions of certain hepatocyte-specific transcription factors are able to convert fibroblasts into functional hepatocyte-like cells. However, such combinational usage of transcription factors in primary hepatocytes culture has not yet sufficiently studied. The forkhead box protein A3 (FoxA3) and hepatocyte nuclear factor 4α (Hnf4α) are liver-enriched transcription factors that play vital roles in the differentiation, and maintenance of hepatocytes. Thus, we simultaneously overexpressed the two genes, Foxa3 and Hnf4α, in rat hepatocytes and observed that the combinational augmentation of these two transcription factors have enhanced the proliferation and stabilized the hepatocyte-specific functions of primary hepatocytes over a long-term culture period.

Transcriptional Inhibition of MicroRNA miR-122 by Small Molecules Reduces Hepatitis C Virus Replication in Liver Cells.

MicroRNAs (miRNAs) are noncoding RNA molecules of 22-24 nucleotides that are estimated to regulate thousands of genes in humans, and their dysregulation has been implicated in many diseases. MicroRNA-122 (miR-122) is the most abundant miRNA in the liver and has been linked to the development of hepatocellular carcinoma and hepatitis C virus (HCV) infection. Its role in these diseases renders miR-122 a potential target for small-molecule therapeutics. Here, we report the discovery of a new sulfonamide class of small-molecule miR-122 inhibitors from a high-throughput screen using a luciferase-based reporter assay. Structure-activity relationship (SAR) studies and secondary assays led to the development of potent and selective miR-122 inhibitors. Preliminary mechanism-of-action studies suggest a role in the promoter-specific transcriptional inhibition of miR-122 expression through direct binding to the liver-enriched transcription factor hepatocyte nuclear factor 4α. Importantly, the developed inhibitors significantly reduce HCV replication in human liver cells.

Unraveling Differential Transcriptomes and Cell Types in Zebrafish Larvae Intestine and Liver.

The zebrafish intestine and liver, as in other vertebrates, are derived from the endoderm. Great effort has been devoted to deciphering the molecular mechanisms controlling the specification and development of the zebrafish intestine and liver; however, genome-wide comparison of the transcriptomes between these two organs at the larval stage remains unexplored. There is a lack of extensive identification of feature genes marking specific cell types in the zebrafish intestine and liver at 5 days post-fertilization, when the larval fish starts food intake. In this report, through RNA sequencing and single-cell RNA sequencing of intestines and livers separately dissected from wild-type zebrafish larvae at 5 days post-fertilization, together with the experimental validation of 47 genes through RNA whole-mount in situ hybridization, we identified not only distinctive transcriptomes for the larval intestine and liver, but also a considerable number of feature genes for marking the intestinal bulb, mid-intestine and hindgut, and for marking hepatocytes and cholangiocytes. Meanwhile, we identified 135 intestine- and 97 liver-enriched transcription factor genes in zebrafish larvae at 5 days post-fertilization. Our findings provide rich molecular and cellular resources for studying cell patterning and specification during the early development of the zebrafish intestine and liver.

Mettl3-mediated mRNA m6A modification controls postnatal liver development by modulating the transcription factor Hnf4a

Hepatic specification and functional maturation are tightly controlled throughout development. N6-methyladenosine (m6A) is the most abundant RNA modification of eukaryotic mRNAs and is involved in various physiological and pathological processes. However, the function of m6A in liver development remains elusive. Here we dissect the role of Mettl3-mediated m6A modification in postnatal liver development and homeostasis. Knocking out Mettl3 perinatally with Alb-Cre (Mettl3 cKO) induces apoptosis and steatosis of hepatocytes, results in severe liver injury, and finally leads to postnatal lethality within 7 weeks. m6A-RIP sequencing and RNA-sequencing reveal that mRNAs of a series of crucial liver-enriched transcription factors are modified by m6A, including Hnf4a, a master regulator for hepatic parenchymal formation. Deleting Mettl3 reduces m6A modification on Hnf4a, decreases its transcript stability in an Igf2bp1-dependent manner, and down-regulates Hnf4a expression, while overexpressing Hnf4a with AAV8 alleviates the liver injury and prolongs the lifespan of Mettl3 cKO mice. However, knocking out Mettl3 in adults using Alb-CreERT2 does not affect liver homeostasis. Our study identifies a dynamic role of Mettl3-mediated RNA m6A modification in liver development.

HepG2-Based Designer Cells with Heat-Inducible Enhanced Liver Functions

Functional human hepatocytes have been a pivotal tool in pharmacological studies such as those investigating drug metabolism and hepatotoxicity. However, primary human hepatocytes are difficult to obtain in large quantities and may cause ethical problems, necessitating the development of a new cell source to replace human primary hepatocytes. We previously developed genetically modified murine hepatoma cell lines with inducible enhanced liver functions, in which eight liver-enriched transcription factor (LETF) genes were introduced into hepatoma cells as inducible transgene expression cassettes. Here, we establish a human hepatoma cell line with heat-inducible liver functions using HepG2 cells. The genetically modified hepatoma cells, designated HepG2/8F_HS, actively proliferated under normal culture conditions and, therefore, can be easily prepared in large quantities. When the expression of LETFs was induced by heat treatment at 43 °C for 30 min, cells ceased proliferation and demonstrated enhanced liver functions. Furthermore, three-dimensional spheroid cultures of HepG2/8F_HS cells showed a further increase in liver functions upon heat treatment. Comprehensive transcriptome analysis using DNA microarrays revealed that HepG2/8F_HS cells had enhanced overall expression of many liver function-related genes following heat treatment. HepG2/8F_HS cells could be useful as a new cell source for pharmacological studies and for constructing bioartificial liver systems.

Invivo partial cellular reprogramming enhances liver plasticity and regeneration.

Mammals have limited regenerative capacity, whereas some vertebrates, like fish and salamanders, are able to regenerate their organs efficiently. The regeneration in these species depends on cell dedifferentiation followed by proliferation. We generate a mouse model that enables the inducible expression of the four Yamanaka factors (Oct-3/4, Sox2, Klf4, and c-Myc, or 4F) specifically in hepatocytes. Transient invivo 4F expression induces partial reprogramming of adult hepatocytes to a progenitor state and concomitantly increases cell proliferation. This is indicated by reduced expression of differentiated hepatic-lineage markers, an increase in markers of proliferation and chromatin modifiers, global changes in DNA accessibility, and an acquisition of liver stem and progenitor cell markers. Functionally, short-term expression of 4F enhances liver regenerative capacity through topoisomerase2-mediated partial reprogramming. Our results reveal that liver-specific 4F expression invivo induces cellular plasticity and counteracts liver failure, suggesting that partial reprogramming may represent an avenue for enhancing tissue regeneration.

Royal jelly fatty acids downregulate ANGPTL8 expression through the decrease in HNF4α protein in human hepatoma HepG2 cells.

Royal jelly (RJ) intake has been reported to be effective for reducing serum lipids; however, the mechanism is not fully understood. Angiopoietin-like protein 8 (ANGPTL8), a secreted protein, plays a key role in lipid metabolism. In this study, we investigated the effects of specific fatty acids included in RJ (RJ fatty acids), such as 10-hydroxy-2-decenoic acid, 10-hydroxydecanoic acid, and sebacic acid (SA), on expression of ANGPTL8 in human hepatoma HepG2 cells. SA markedly reduced the expression of ANGPTL8. Reporter assay revealed that SA suppressed ANGPTL8 promoter activity. In addition, we identified a functional binding site of hepatocyte nuclear factor-4α (HNF4α), a liver-enriched transcription factor, in the ANGPTL8 promoter. SA reduced the levels of HNF4α protein and the binding of HNF4α to the ANGPTL8 promoter. Moreover, siRNA knockdown of HNF4α suppressed the expression of ANGTPL8 mRNA. Taken together, we conclude that SA downregulates ANGPTL8 expression via the decrease in HNF4α protein.

Liver progenitor cells may construct cysts having heterogeneous gene expression of liver-enriched transcription factors in mice with conditional knockout of the Hhex gene

The deletion of the Hhex (Hematopoietically expressed homeobox) gene causes agenesis of the liver and polycystic liver disease depending on its timing. The present study was undertaken to determine the role of the Hhex gene in not only signaling cascades to cyst and abnormal bile duct formation but also the liver progenitor contribution to cystic development. Liver-specific Hhex knockout mice (Alb-Cre/HhexloxP/loxP) in adult stages were used. Wild-type and conditional knockout (cKO) livers were immunohistologically compared for cell growth, and gene expression of liver functions, biliary markers and cystic markers. In Hhex cKO livers, cyst formation and dilated intrahepatic bile ducts were noted, which resembled the histology of the von Meyenburg complex. Ki67 immunohistochemistry showed that the growth activity in bile ducts and cysts of cKO livers was elevated compared with that of wild-type livers. There were far fewer liver progenitor cells or bile ductule cells around portal veins of cKO livers than in wild-type livers. Several liver-enriched transcription factors, including Foxa1 and Foxa2, were heterogeneously expressed in bile ducts and cysts of cKO livers whereas their expression in wild-type bile ducts was comparatively homogeneous. PC1 and PC2 immunohistochemistry revealed their up-regulation in cysts of cKO livers. These data indicate that Hhex is not only required for proper bile duct morphogenesis, but is also involved in cyst formation through promoted cell growth. Liver progenitor cells may form cysts. Unbalanced expression of liver-enriched transcription factors might be involved in cyst formation. Hhex cKO mice may be a good animal model for hepatic cystic diseases.

FACI Is a Novel CREB-H-Induced Protein That Inhibits Intestinal Lipid Absorption and Reverses Diet-Induced Obesity.

Background & AimsCREB-H is a key liver-enriched transcription factor governing lipid metabolism. Additional targets of CREB-H remain to be identified and characterized. Here, we identified a novel fasting- and CREB-H–induced (FACI) protein that inhibits intestinal lipid absorption and alleviates diet-induced obesity in mice.MethodsFACI was identified by reanalysis of existing transcriptomic data. Faci-/- mice were generated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)-mediated genome engineering. RNA sequencing was performed to identify differentially expressed genes in Faci-/- mice. Lipid accumulation in the villi was assessed by triglyceride measurement and Oil red O staining. In vitro fatty acid uptake assay was performed to verify in vivo findings.ResultsFACI expression was enriched in liver and intestine. FACI is a phospholipid-binding protein that localizes to plasma membrane and recycling endosomes. Hepatic transcription of Faci was regulated by not only CREB-H, but also nutrient-responsive transcription factors sterol regulatory element-binding protein 1 (SREBP1), hepatocyte nuclear factor 4α (HNF4α), peroxisome proliferator-activated receptor γ coactivator-1α (PGC1α), and CREB, as well as fasting-related cyclic adenosine monophosphate (cAMP) signaling. Genetic knockout of Faci in mice showed an increase in intestinal fat absorption. In accordance with this, Faci deficiency aggravated high-fat diet–induced obesity, hyperlipidemia, steatosis, and other obesity-related metabolic dysfunction in mice.ConclusionsFACI is a novel CREB-H–induced protein. Genetic disruption of Faci in mice showed its inhibitory effect on fat absorption and obesity. Our findings shed light on a new target of CREB-H implicated in lipid homeostasis.

Role of the liver-enriched transcription factor binding site mutation in the C promoter region of hepatitis B virus genome for HBx-enhanced hepatitis B virus replication

Objective: To construct a recombinant HBV replication-type plasmid with liver-enriched transcription factor binding site mutation at proximal of HBV C promoter in order to elucidate the role of HBx-enhanced HBV replication. Methods: Site-directed mutagenesis technology was used to construct a recombinant plasmid with liver-enriched transcription factor binding site mutation at proximal of HBV C promoter on the basis of wild-type HBV replicating plasmid and HBV replicating plasmid lacking HBx expression. Subsequently, plasmid transfection was carried out in HBV liver cancer cell replication model and mouse replication model, and HBV replication intermediates of cells and mouse liver tissue were extracted for detection. Results: Based on the HBV replicating plasmid, the HBV replicating plasmid with liver-enriched transcription factor binding site mutation at proximal of HBV C promoter was successfully constructed. HBx-enhanced HBV replication were detected in both the HBV liver cancer replication model and the mouse replication model. After mutating liver-enriched transcription factor binding site mutation at proximal of HBV C promoter, the effect of HBx on the enhancement of HBV replication was not significantly affected. Conclusion: HBx may not enhance HBV replication through liver-enriched transcription factor binding site mutation at proximal of HBV C promoter. The role of other liver-enriched transcription factor binding sites in HBx-enhanced HBV replication needs further study.

The Functional and Antiviral Activity of Interferon Alpha-Inducible IFI6 Against Hepatitis B Virus Replication and Gene Expression.

Hepatitis B virus is an enveloped DNA virus, that infects more than three hundred and sixty million people worldwide and leads to severe chronic liver diseases. Interferon-alpha inducible protein 6 (IFI6) is an IFN-stimulated gene (ISG) whose expression is highly regulated by the stimulation of type I IFN-alpha that restricts various kinds of virus infections by targeting different stages of the viral life cycle. This study aims to investigate the antiviral activity of IFI6 against HBV replication and gene expression. The IFI6 was highly induced by the stimulation of IFN-α in hepatoma cells. The overexpression of IFI6 inhibited while knockdown of IFI6 elevated replication and gene expression of HBV in HepG2 cells. Further study determined that IFI6 inhibited HBV replication by reducing EnhII/Cp of the HBV without affecting liver enriched transcription factors that have significant importance in regulating HBV enhancer activity. Furthermore, deletion mutation of EnhII/Cp and CHIP analysis revealed 100 bps (1715-1815 nt) putative sites involved in IFI6 mediated inhibition of HBV. Detailed analysis with EMSA demonstrated that 1715-1770 nt of EnhII/Cp was specifically involved in binding with IFI6 and restricted EnhII/Cp promoter activity. Moreover, IFI6 was localized mainly inside the nucleus to involve in the anti-HBV activity of IFI6. In vivo analysis based on the hydrodynamic injection of IFI6 expression plasmid along with HBV revealed significant inhibition of HBV DNA replication and gene expression. Overall, our results suggested a novel mechanism of IFI6 mediated HBV regulation that could develop potential therapeutics for efficient HBV infection treatment.

Development of a genetically modified hepatoma cell line with heat-inducible high liver function.

The online version of this article (10.1007/s10616-021-00457-4) contains supplementary material, which is available to authorized users.

Generation of Gene-Engineered Human Hepatoma Cells with Heat-Inducible Liver Functions

Hepatoma cells derived from liver carcinoma are a candidate cell source for bioartificial liver (BAL) systems due to their high proliferative capacity, although liver function of hepatoma cells is considerably low compared with primary hepatocytes. In our previous study, genetically engineered mouse hepatoma cells with inducible high liver function were established by transducing liver-enriched transcription factor (LETF) genes. In this study, we aimed to develop new gene-engineered human hepatoma cells, in which high liver functions are inducible by heat treatment. For this purpose, we constructed a gene expression system for eight LETF genes under control of tetracycline-dependent transactivator (tTA), and the system was introduced into the genome of HepG2-HSP cells, in which a tTA expression system induced by a heat-shock protein promoter with transcriptional amplification was introduced into HepG2 cells. Thus, the heat-inducible tTA promotes LETF genes to induce liver function. Upon the heat treatment of the cells (HepG2-HSP/8F) at 43°C for 30 min, liver functions such as albumin secretion and cytochrome P450 were significantly enhanced. The cells with heat-inducible liver function can be used as a new cell source for various hepatic studies including construction of BAL systems.

PXR Functionally Interacts with NF-κB and AP-1 to Downregulate the Inflammation-Induced Expression of Chemokine CXCL2 in Mice.

Pregnane X receptor (PXR) is a liver-enriched xenobiotic-responsive transcription factor. Although recent studies suggest that PXR shows anti-inflammatory effects by suppressing nuclear factor kappa B (NF-κB), the detailed mechanism remains unclear. In this study, we aimed to elucidate this mechanism. Mice were treated intraperitoneally with the PXR agonist pregnenolone 16α-carbonitrile (PCN) and/or carbon tetrachloride (CCl4). Liver injury was evaluated, and hepatic mRNA levels were determined via quantitative reverse transcription polymerase chain reaction. Reporter assays with wild-type and mutated mouse Cxcl2 promoter-containing reporter plasmids were conducted in 293T cells. Results showed that the hepatic expression of inflammation-related genes was upregulated in CCl4-treated mice, and PCN treatment repressed the induced expression of chemokine-encoding Ccl2 and Cxcl2 among the genes investigated. Consistently, PCN treatment suppressed the increased plasma transaminase activity and neutrophil infiltration in the liver. In reporter assays, tumor necrosis factor-α-induced Cxcl2 expression was suppressed by PXR. Although an NF-κB inhibitor or the mutation of an NF-κB-binding motif partly reduced PXR-dependent suppression, the mutation of both NF-κB and activator protein 1 (AP-1) sites abolished it. Consistently, AP-1-dependent gene transcription was suppressed by PXR with a construct containing AP-1 binding motifs. In conclusion, the present results suggest that PXR exerts anti-inflammatory effects by suppressing both NF-κB- and AP-1-dependent chemokine expression in mouse liver.

TRAIL inhibits HBV replication and expression by down-regulating liver-enriched transcription factors

Background and study aimsTo investigate the role of low-concentration TRAIL on HBV replication and expression. Material and methodsMTT assay was performed to determine the minimum concentrations of TRAIL protein in HepG2 cell apoptosis. HepG2 cells were transfected by HBV replication plasmid pHBV4.1. After the treatment with low concentration of TRAIL, the culture supernatant was collected to detect HBsAg and HBeAg by ELISA. Proteins were extracted from the resulted cells, followed by total RNA and HBV DNA intermediate replication. Southern Blot and Northern Blot were carried out to detect HBV RNA and HBV DNA replication intermediates, respectively. RT-PCR and Western Blot were carried out to detect gene and protein expressions for HNF4α, PPARα, and RXRα, respectively. Results50 ng/ml of TRAIL protein led to significant decline on the secretions of HBsAg and HBeAg. Expression levels of HBV RNA and HBV DNA replication intermediates were significantly decreased too. In addition, gene and protein expressions of HNF4α, PPARα and RXRα also dropped, especially for PPARα whose expressions significantly decreased. ConclusionTRAIL could inhibit HBV replication and expression by downregulating the expressions of liver-enriched transcription factors HNF4α, PPARα, and RXRα.