Hepatocyte Nuclear Factor 4α Research Articles

HNF4α is required for Tkfc promoter activation by ChREBP.

Triokinase/FMN cyclase (Tkfc) is involved in fructose metabolism and is responsible for the phosphorylation of glyceraldehyde to glyceraldehyde-3-phosphate. In this study, we show that refeeding induced hepatic expression of Tkfc in mice. Luciferase reporter gene assays using the Tkfc promoter revealed the existence of two hepatocyte nuclear factor 4α (HNF4α)-responsive elements (HNF4RE1 and HNF4RE2) and one carbohydrate-responsive element-binding protein (ChREBP)-responsive element (ChoRE1). Deletion and mutation of HNF4RE1 and HNF4RE2 or ChoRE1 abolished HNF4α and ChREBP responsiveness, respectively. HNF4α and ChREBP synergistically stimulated Tkfc promoter activity. ChoRE1 mutation attenuated but maintained HNF4α responsiveness, whereas HNF4RE1 and HNF4RE2 mutations abolished ChREBP responsiveness. Moreover, Tkfc promoter activity stimulation by ChREBP was attenuated upon HNF4α knockdown. Furthermore, Tkfc expression was decreased in livers of ChREBP-/- and liver-specific HNF4-/- (Hnf4αΔHep) mice. Altogether, our data indicate that Tkfc is a target gene of ChREBP and HNF4α, and Tkfc promoter activity stimulation by ChREBP requires HNF4α.

Tripartite motif-containing protein 21 is involved in IFN-γ-induced suppression of hepatitis B virus by regulating hepatocyte nuclear factors.

The antiviral role of the tripartite motif-containing (TRIM) protein family , a member of the E3-ubiquitin ligase family, has recently been actively studied. Hepatitis B virus (HBV) infection is a major contributor to liver diseases; however, the host factors regulated by cytokine-inducible TRIM21 to suppress HBV remain unclear. In this study, we showed the antiviral efficacy of TRIM21 against HBV in hepatoma cell lines, primary human hepatocytes isolated from patient liver tissues, and mouse model. Using TRIM21 knock-out cells, we confirmed that the antiviral effects of interferon-gamma, which suppress HBV replication, are diminished when TRIM21 is deficient. Northern blot analysis confirmed a reduction of HBV RNA levels by TRIM21. Using Luciferase reporter assay, we also discovered that TRIM21 decreases the activity of HBV enhancers, which play a crucial role in covalently closed circular DNA transcription. The participation of the RING domain and PRY-SPRY domain in the anti-HBV effect of TRIM21 was demonstrated through experiments using deletion mutants. We identified a novel interaction between TRIM21 and hepatocyte nuclear factor 4α (HNF4α) through co-immunoprecipitation assay. More specifically, ubiquitination assay revealed that TRIM21 promotes ubiquitin-mediated proteasomal degradation of HNF4α. HNF1α transcription is down-regulated as a result of the degradation of HNF4α, an activator for the HNF1α promoter. Therefore, the reduction of key HBV enhancer activators, HNF4α and HNF1α, by TRIM21 resulted in a decline in HBV transcription, ultimately leading to the inhibition of HBV replication.IMPORTANCEDespite extensive research efforts, a definitive cure for chronic hepatitis B remains elusive, emphasizing the persistent importance of this viral infection as a substantial public health concern. Although the risks associated with hepatitis B virus (HBV) infection are well known, host factors capable of suppressing HBV are largely uncharacterized. This study elucidates that tripartite motif-containing protein 21 (TRIM21) suppresses HBV transcription and consequently inhibits HBV replication by downregulating the hepatocyte nuclear factors, which are host factors associated with the HBV enhancers. Our findings demonstrate a novel anti-HBV mechanism of TRIM21 in interferon-gamma-induced anti-HBV activity. These findings may contribute to new strategies to block HBV.

Novel role of MHC class II transactivator in hepatitis B virus replication and viral counteraction.

The major histocompatibility class II (MHC II) transactivator, known as CIITA, is induced by Interferon gamma (IFN-γ) and plays a well-established role in regulating the expression of class II MHC molecules in antigen-presenting cells. Primary human hepatocytes (PHH) were isolated via therapeutic hepatectomy from two donors who tested negative for hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis D virus (HDV). The hepatocellular carcinoma (HCC) cell lines HepG2 and Huh7 were used for the mechanistic study, and HBV infection was performed in HepG2-NTCP cells. HBV DNA replication intermediates and secreted antigen levels were measured using Southern blotting and ELISA, respectively. We identified a non-canonical function of CIITA in the inhibition of hepatitis B virus (HBV) replication in both HCC cells and patient-derived PHH. Notably, in vivo experiments demonstrated that HBV DNA and secreted antigen levels were significantly decreased in mice injected with the CIITA construct. Mechanistically, CIITA inhibited HBV transcription and replication by suppressing the activity of HBV-specific enhancers/promoters. Indeed, CIITA exerts antiviral activity in hepatocytes through ERK1/2-mediated down-regulation of the expression of hepatocyte nuclear factor 1α (HNF1α) and HNF4α, which are essential factors for virus replication. In addition, silencing of CIITA significantly abolished the IFN-γ-mediated anti-HBV activity, suggesting that CIITA mediates the anti-HBV activity of IFN-γ to some extent. HBV X protein (HBx) counteracts the antiviral activity of CIITA via direct binding and impairing its function. Our findings reveal a novel antiviral mechanism of CIITA that involves the modulation of the ERK pathway to restrict HBV transcription. Additionally, our results suggest the possibility of a new immune avoidance mechanism involving HBx.

KRT81 and HNF1A expression in pancreatic ductal adenocarcinoma: investigation of predictive and prognostic value of immunohistochemistry-based subtyping.

Even after decades of research, pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal disease and responses to conventional treatments remain mostly poor. Subclassification of PDAC into distinct biological subtypes has been proposed by various groups to further improve patient outcome and reduce unnecessary side effects. Recently, an immunohistochemistry (IHC)-based subtyping method using cytokeratin-81 (KRT81) and hepatocyte nuclear factor 1A (HNF1A) could recapitulate some of the previously established molecular subtyping methods, while providing significant prognostic and, to a limited degree, also predictive information. We refined the KRT81/HNF1A subtyping method to classify PDAC into three distinct biological subtypes. The prognostic value of the IHC-based method was investigated in two primary resected cohorts, which include 269 and 286 patients, respectively. In the second cohort, we also assessed the predictive effect for response to erlotinib + gemcitabine. In both PDAC cohorts, the new HNF1A-positive subtype was associated with the best survival, the KRT81-positive subtype with the worst, and the double-negative with an intermediate survival (p < 0.001 and p < 0.001, respectively) in univariate and multivariate analyses. In the second cohort (CONKO-005), the IHC-based subtype was additionally found to have a potential predictive value for the erlotinib-based treatment effect. The revised IHC-based subtyping using KRT81 and HNF1A has prognostic significance for PDAC patients and may be of value in predicting treatment response to specific therapeutic agents.

Gallic Acid Can Promote Low-Density Lipoprotein Uptake in HepG2 Cells via Increasing Low-Density Lipoprotein Receptor Accumulation.

Gallic acid (GA) is a type of polyphenolic compound that can be found in a range of fruits, vegetables, and tea. Although it has been confirmed it improves non-alcoholic fatty liver disease (NAFLD), it is still unknown whether GA can improve the occurrence of NAFLD by increasing the low-density lipoprotein receptor (LDLR) accumulation and alleviating cholesterol metabolism disorders. Therefore, the present study explored the effect of GA on LDLR and its mechanism of action. The findings indicated that the increase in LDLR accumulation in HepG2 cells induced by GA was associated with the stimulation of the epidermal growth factor receptor-extracellular regulated protein kinase (EGFR-ERK1/2) signaling pathway. When the pathway was inhibited by EGFR mab cetuximab, it was observed that the activation of the EGFR-ERK1/2 signaling pathway induced by GA was also blocked. At the same time, the accumulation of LDLR protein and the uptake of LDL were also suppressed. Additionally, GA can also promote the accumulation of forkhead box O3 (FOXO3) and suppress the accumulation of hepatocyte nuclear factor-1α (HNF1α), leading to the inhibition of proprotein convertase subtilisin/kexin 9 (PCSK9) mRNA expression and protein accumulation. This ultimately results in increased LDLR protein accumulation and enhanced uptake of LDL in cells. In summary, the present study revealed the potential mechanism of GA's role in ameliorating NAFLD, with a view of providing a theoretical basis for the dietary supplementation of GA.

Krüppel-like factor 10 protects against metabolic dysfunction-associated steatohepatitis by regulating HNF4α-mediated metabolic pathways

BackgroundKrüppel-like factor 10 (KLF10), a zinc finger transcription factor, plays a pivotal role in modulating TGF-β-mediated cellular processes such as growth, apoptosis, and differentiation. Recent studies have implicated KLF10 in regulating lipid metabolism and glucose homeostasis. This study aimed to elucidate the precise role of hepatic KLF10 in developing metabolic dysfunction-associated steatohepatitis (MASH) in diet-induced obese mice. MethodsWe investigated hepatic KLF10 expression under metabolic stress and the effects of overexpression or ablation of hepatic KLF10 on MASH development and lipidemia. We also determined whether hepatocyte nuclear factor 4α (HNF4α) mediated the metabolic effects of KLF10. ResultsHepatic KLF10 was downregulated in MASH patients and genetically or diet-induced obese mice. AAV8-mediated overexpression of KLF10 in hepatocytes prevented Western diet-induced hypercholesterolemia and steatohepatitis, whereas inactivation of hepatocyte KLF10 aggravated Western diet-induced steatohepatitis. Mechanistically, KLF10 reduced hepatic triglyceride and free fatty acid levels by inducing lipolysis and fatty acid oxidation and inhibiting lipogenesis, and reducing hepatic cholesterol levels by promoting bile acid synthesis. KLF10 highly induced HNF4α expression by directly binding to its promoter. The beneficial effect of KLF10 on MASH development was abolished in mice lacking hepatocyte HNF4α. In addition, the inactivation of KLF10 in hepatic stellate cells exacerbated Western diet-induced liver fibrosis by activating the TGF-β/SMAD2/3 pathway. ConclusionsOur data collectively suggest that the transcription factor KLF10 plays a hepatoprotective role in MASH development by inducing HNF4α. Targeting hepatic KLF10 may offer a promising strategy for treating MASH.

Promoter Methylation Leads to Hepatocyte Nuclear Factor 4A Loss and Pancreatic Cancer Aggressiveness

Background and AimsEfforts to decode pancreatic ductal adenocarcinoma (PDAC) heterogeneity and the consequent therapeutic selection remains a challenge. We aimed to characterize epigenetically regulated pathways involved in PDAC progression. MethodsGlobal DNA methylation analysis in pancreatic cancer patient tissues and cell lines was performed to identify differentially methylated genes. Targeted bisulfite sequencing and in vitro methylation reporter assays were employed to investigate the direct link between site-specific methylation and transcriptional regulation. A series of in vitro loss- and gain-of function studies, and in vivo xenograft and the KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre) mouse models were used to assess pancreatic cancer cell properties. Gene and protein expression analyses were performed in three different cohorts of pancreatic cancer patients and correlated to clinicopathological parameters. ResultsWe identify Hepatocyte Nuclear Factor 4A (HNF4A) as a novel target of hypermethylation in pancreatic cancer and demonstrate that site-specific proximal promoter methylation drives HNF4A transcriptional repression. Expression analyses in patients, indicate the methylation-associated suppression of HNF4A expression in pancreatic cancer tissues. In vitro and in vivo studies reveal that HNF4A is a novel tumor suppressor in pancreatic cancer, regulating cancer growth and aggressiveness. As evidenced in both the KPC mouse model and human pancreatic cancer tissues, HNF4A expression declines significantly in the early stages of the disease. Most importantly, HNF4 loss correlates with poor overall patient survival. ConclusionsHNF4A silencing, mediated by promoter DNA methylation, drives pancreatic cancer development and aggressiveness leading to poor patient survival.

Effect of mutation at c.493T>C locus of transcription factor HNF1α gene on its protein level.

Hepatocyte nuclear factor 1α (HNF1α) is a transcription factor that is crucial for the regulation to maintain the function of pancreatic β-cell, hepatic lipid metabolism, and other processes. Mature-onset diabetes of the young type 3 is a monogenic form of diabetes caused by HNF1α mutations. Although several mutation sites have been reported, the specific mechanisms remain unclear, such hot-spot mutation as the P291fsinsC mutation and the P112L mutation and so on. In preliminary studies, we discovered one MODY3 patient carrying a mutation at the c.493T>C locus of the HNF1α gene. In this study, we analyzed the pathogenic of the mutation sites by using the Mutation Surveyor software and constructed the eukaryotic expression plasmids of the wild-type and mutant type of HNF1α to detect variations in the expression levels and stability of HNF1α protein by using Western blot. The analyses of the Mutation Surveyor software showed that the c.493T>C site mutation may be pathogenic gene and the results of Western blot showed that both the amount and stability of HNF1α protein expressed by the mutation type plasmid were reduced significantly compared to those by the wild type plasmid (P<0.05). This study suggests that the c.493T>C (p.Trp165Arg) mutation dramatically impacts HNF1α expression, which might be responsible for the development of the disease and offers fresh perspectives for the following in-depth exploration of MODY3's molecular pathogenic process.

Screening of Chelidonium majus isoquinoline alkaloids reveals berberine and chelidonine as selective ligands for the nuclear receptors RORβ and HNF4α, respectively.

The nuclear receptors hepatocyte nuclear factor 4α (HNF4α) and retinoic acid receptor-related orphan receptor-β (RORβ) are ligand-regulated transcription factors and potential drug targets for metabolic disorders. However, there is a lack of small molecular, selective ligands to explore the therapeutic potential in further detail. Here, we report the discovery of greater celandine (Chelidonium majus) isoquinoline alkaloids as nuclear receptor modulators: Berberine is a selective RORβ inverse agonist and modulated target genes involved in the circadian clock, photoreceptor cell development, and neuronal function. The structurally related chelidonine was identified as a ligand for the constitutively active HNF4α receptor, with nanomolar potency in a cellular reporter gene assay. In human liver cancer cells naturally expressing high levels of HNF4α, chelidonine acted as an inverse agonist and downregulated genes associated with gluconeogenesis and drug metabolism. Both berberine and chelidonine are promising tool compounds to further investigate their target nuclear receptors and for drug discovery.

The combination of Schisandrin C and Luteolin synergistically attenuates hepatitis B virus infection via repressing HBV replication and promoting cGAS-STING pathway activation in macrophages.

HBV infection can result in severe liver diseases and is one of the primary causes of liver cell carcinoma-related mortality. Liuwei Wuling tablet (LWWL) is a traditional Chinese medicine formula, with a protecting liver and decreasing enzyme activity, usually used to treat chronic hepatitis B with NAs in clinic. However, its main active ingredients and mechanism of action have not been fully investigated. Hence, we aimed to screen the active ingredient and effective ingredient combinations from Liuwei Wuling tablet to explore the anti-herpatitis B virus activity and mechanism. Analysis and screening of effective antiviral components in LWWL by network pharmacology, luteolin (Lut) may be a compound with significant antiviral activity. The mechanism of antiviral action of Lut was also found by real-time PCR detection and western blotting. Meanwhile, we established a co-culture model to investigate the antiviral mechanism of Schisandrin C (SC), one of the main active components of Schisandra chinensis fructus (the sovereign drug of LWWL). Next, HBV-infected mice were established by tail vein injection of pAAV-HBV1.2 plasmid and administered continuously for 20days. And their antiviral capacity was evaluated by checking serum levels of HBsAg, HBeAg, levels of HBV DNA, and liver levels of HBcAg. In this study, we conducted network pharmacology analysis on LWWL, and through in vitro experimental validation and data analysis, we found that luteolin (Lut) possessed obviously anti-HBV activity, inhibiting HBV replication by downregulating hepatocyte nuclear factor 4α (HNF4α) via the ERK pathway. Additionally, we established a co-culture system and proved that SC promoted activation of cGAS-STINIG pathway and IFN-β production in THP-1 cells to inhibit HBV replication in HepG2.2.15 cells. Moreover, we found the combination of SC and Lut shows a greater effect in inhibiting HBV compared to SC or Lut alone in HBV-infected mice. Taken together, our study suggests that combination of SC and Lut may be potential candidate drug for the prevention and treatment of chronic hepatitis B.

Bone and bone derived factors in kidney disease.

Purpose of review: Mineral and bone disorder (MBD) is a prevalent complication in chronic kidney disease (CKD), significantly impacting overall health with multifaceted implications including fractures, cardiovascular events, and mortality. Despite its pervasive nature, effective treatments for CKD-MBD are lacking, emphasizing the urgency to advance understanding and therapeutic interventions. Bone metabolism intricacies, influenced by factors like 1,25 dihydroxy vitamin D, parathyroid hormone (PTH), and fibroblast growth factor 23 (FGF23), along with intrinsic osseous mechanisms, play pivotal roles in CKD. Skeletal abnormalities precede hormonal changes, persisting even with normalized systemic mineral parameters, necessitating a comprehensive approach to address both aspects. Recent findings: In this review, we explore novel pathways involved in the regulation of systemic mineral bone disease factors, specifically examining anemia, inflammation, and metabolic pathways. Special emphasis is placed on internal bone mechanisms, such as hepatocyte nuclear factor 4α, transforming growth factor-β1, and sclerostin, which play crucial roles in the progression of renal osteodystrophy. Summary: Despite advancements, effective treatments addressing CKD-MBD morbidity and mortality are lacking, necessitating ongoing research for novel therapeutic targets.

HNF4A guides the MLL4 complex to establish and maintain H3K4me1 at gene regulatory elements

Hepatocyte nuclear factor 4A (HNF4A/NR2a1), a transcriptional regulator of hepatocyte identity, controls genes that are crucial for liver functions, primarily through binding to enhancers. In mammalian cells, active and primed enhancers are marked by monomethylation of histone 3 (H3) at lysine 4 (K4) (H3K4me1) in a cell type-specific manner. How this modification is established and maintained at enhancers in connection with transcription factors (TFs) remains unknown. Using analysis of genome-wide histone modifications, TF binding, chromatin accessibility and gene expression, we show that HNF4A is essential for an active chromatin state. Using HNF4A loss and gain of function experiments in vivo and in cell lines in vitro, we show that HNF4A affects H3K4me1, H3K27ac and chromatin accessibility, highlighting its contribution to the establishment and maintenance of a transcriptionally permissive epigenetic state. Mechanistically, HNF4A interacts with the mixed-lineage leukaemia 4 (MLL4) complex facilitating recruitment to HNF4A-bound regions. Our findings indicate that HNF4A enriches H3K4me1, H3K27ac and establishes chromatin opening at transcriptional regulatory regions.

HNF4A-Bridging the Gap Between Intestinal Metaplasia and Gastric Cancer.

Intestinal metaplasia (IM) of gastric epithelium has traditionally been regarded as an irreversible stage in the process of the Correa cascade. Exploring the potential molecular mechanism of IM is significant for effective gastric cancer prevention. The GSE78523 dataset, obtained from the Gene Expression Omnibus (GEO) database, was analyzed using RStudio software to identify the differently expressed genes (DEGs) between IM tissues and normal gastric epithelial tissues. Subsequently, gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, Gene Set Enrichment Analysis (GESA), and protein-protein interaction (PPI) analysis were used to find potential genes. Additionally, the screened genes were analyzed for clinical, immunological, and genetic correlation aspects using single gene clinical correlation analysis (UALCAN), Tumor-Immune System Interactions Database (TISIDB), and validated through western blot experiments. Enrichment analysis showed that the lipid metabolic pathway was significantly associated with IM tissues and the apolipoprotein B (APOB) gene was identified in the subsequent analysis. Experiment results and correlation analysis showed that the expression of APOB was higher in IM tissues than in normal tissues. This elevated expression of APOB was also found to be associated with the expression levels of hepatocyte nuclear factor 4A (HNF4A) gene. HNF4A was also found to be associated with immune cell infiltration to gastric cancer and was linked to the prognosis of gastric cancer patients. Moreover, HNF4A was also highly expressed in both IM tissues and gastric cancer cells. Our findings indicate that HNF4A regulates the microenvironment of lipid metabolism in IM tissues by targeting APOB. Higher expression of HNF4A tends to lead to a worse prognosis in gastric cancer patients implying it may serve as a predictive indicator for the progression from IM to gastric cancer.

Role of connexin 32 in the directional differentiation of induced pluripotent stem cells into hepatocytes.

This study aimed to explore the role of connexin 32 (Cx32) in the directional differentiation of induced pluripotent stem cells (iPSCs) into hepatocytes. Urine-derived epithelial cells were collected from the fresh urine of a healthy donor and transducted with reprogramming plasmid mixture to generate iPSCs. The iPSCs were then directionally differentiated into hepatocytes. During the differentiation, the upregulated and downregulated groups were treated with vitamin K2 (VK2) and 2-aminoethoxyboronate diphenylester (2-APB) to increase and inhibit Cx32 expression, respectively. The control group was not treated with the regulatory factor. Expression of Cx32 and hepatocyte-specific markers, including AFP, hepatocyte nuclear factor 4α (HNF-4α), albumin (ALB) and cytokeratin 18 (CK18) were detected. It indicated that Cx32 expression was not observed in iPSCs, but gradually increased during the process of hepatic differentiation from iPSCs. Upregulation of Cx32 expression by VK2 treatment promoted hepatocyte maturation and enhanced the expression of the aforementioned hepatic specific markers, whereas downregulation of Cx32 expression by 2-APB treatment had the opposite effects. In conclusion, urine-derived iPSCs could be directionally differentiated into hepatocytes. Up-regulation of Cx32 improves the efficiency and maturity of differentiation of iPSCs into hepatocytes, and Cx32 may be a promoting factor during the process of hepatic differentiation from iPSCs.

The Role of Nuclear Receptors in the Pathogenesis and Treatment of Non-alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD) is a global health burden closely linked to insulin resistance, obesity, and type 2 diabetes. The complex pathophysiology of NAFLD involves multiple cellular pathways and molecular factors. Nuclear receptors (NRs) have emerged as crucial regulators of lipid metabolism and inflammation in NAFLD, offering potential therapeutic targets for NAFLD. Targeting PPARs and FXRs has shown promise in ameliorating NAFLD symptoms and halting disease progression. However, further investigation is needed to address side effects and personalize therapy approaches. This review summarizes the current understanding of the involvement of NRs in the pathogenesis of NAFLD and explores their therapeutic potential. We discuss the role of several NRs in modulating lipid homeostasis in the liver, including peroxisome proliferator-activated receptors (PPARs), liver X receptors (LXRs), farnesoid X receptors (FXRs), REV-ERB, hepatocyte nuclear factor 4α (HNF4α), constitutive androstane receptor (CAR) and pregnane X receptor (PXR).The expanding knowledge of NRs in NAFLD offers new avenues for targeted therapies, necessitating exploration of novel treatment strategies and optimization of existing approaches to combat this increasingly prevalent disease.

Bile duct ligation elevates 5-HT levels in cerebral cortex of rats partly due to impairment of brain UGT1A6 expression and activity via ammonia accumulation

Hepatic encephalopathy (HE) is often associated with endogenous serotonin (5-HT) disorders. However, the reason for elevated brain 5-HT levels due to liver failure remains unclear. This study aimed to investigate the mechanism by which liver failure increases brain 5-HT levels and the role in behavioral abnormalities in HE. Using bile duct ligation (BDL) rats as a HE model, we verified the elevated 5-HT levels in the cortex but not in the hippocampus and striatum, and found that this cortical 5-HT overload may be caused by BDL-mediated inhibition of UDP-glucuronosyltransferase 1A6 (UGT1A6) expression and activity in the cortex. The intraventricular injection of the UGT1A6 inhibitor diclofenac into rats demonstrated that the inhibition of brain UGT1A6 activity significantly increased cerebral 5-HT levels and induced HE-like behaviors. Co-immunofluorescence experiments demonstrated that UGT1A6 is primarily expressed in astrocytes. In vitro studies confirmed that NH4Cl activates the ROS-ERK pathway to downregulate UGT1A6 activity and expression in U251 cells, which can be reversed by the oxidative stress antagonist N-acetyl-l-cysteine and the ERK inhibitor U0126. Silencing Hepatocyte Nuclear Factor 4α (HNF4α) suppressed UGT1A6 expression whilst overexpressing HNF4α increased Ugt1a6 promotor activity. Meanwhile, both NH4Cl and the ERK activator TBHQ downregulated HNF4α and UGT1A6 expression. In the cortex of hyperammonemic rats, we also found activation of the ROS-ERK pathway, decreases in HNF4α and UGT1A6 expression, and increases in brain 5-HT content. These results prove that the ammonia-mediated ROS-ERK pathway activation inhibits HNF4α expression to downregulate UGT1A6 expression and activity, thereby increasing cerebral 5-HT content and inducing manic-like HE symptoms. This is the first study to reveal the mechanism of elevated cortical 5-HT concentration in a state of liver failure and elucidate its association with manic-like behaviors in HE.

Association between HNF4A rs1800961 polymorphisms and gallstones in a Taiwanese population.

A large genetic effect of a novel gallstone-associated genetic variant, the hepatocyte nuclear factor 4α (HNF4A) rs1800961 polymorphism, has been identified through recent genome-wide association studies. However, this effect has not been validated in Asian populations. We investigated the association between the rs1800961 variant and gallstones among a Taiwanese population. A total of 20405 participants aged between 30 and 70years voluntarily enrolled in the Taiwan Biobank. Self-report questionnaires, physical examinations, biochemical tests, and genotyping were used for analysis. The association of the HNF4A rs1800961 variant and other metabolic risks with gallstone disease was analyzed using multiple logistic regression models. The minor T allele of HNF4A rs1800961 was associated with an increased risk of gallstone, and the association remained significant even after adjustment for other risk factors including age, body mass index (BMI), diabetes, hyperlipidemia, hypertension, and cigarette smoking (adjusted odds ratio [OR]=1.90, 95% confidence interval [CI]=1.31 to 2.75) in male participants. When further stratified by BMI and age, the lithogenic effect was the most significant in male participants with obesity (adjusted OR=3.55, 95% CI=1.92 to 6.56) and who were younger (adjusted OR=2.45, 95% CI=1.49 to 4.04). The novel gallstone-associated HNF4A rs1800961 variant was associated with the risk of gallstone in the Taiwanese men. Screening for the rs1800961 polymorphism may be particularly useful in assessing the risk of gallstone formation in younger or obese men.

HNF4α isoforms regulate the circadian balance between carbohydrate and lipid metabolism in the liver.

Hepatocyte Nuclear Factor 4α (HNF4α), a master regulator of hepatocyte differentiation, is regulated by two promoters (P1 and P2) which drive the expression of different isoforms. P1-HNF4α is the major isoform in the adult liver while P2-HNF4α is thought to be expressed only in fetal liver and liver cancer. Here, we show that P2-HNF4α is indeed expressed in the normal adult liver at Zeitgeber time (ZT)9 and ZT21. Using exon swap mice that express only P2-HNF4α we show that this isoform orchestrates a distinct transcriptome and metabolome via unique chromatin and protein-protein interactions, including with different clock proteins at different times of the day leading to subtle differences in circadian gene regulation. Furthermore, deletion of the Clock gene alters the circadian oscillation of P2- (but not P1-)HNF4α RNA, revealing a complex feedback loop between the HNF4α isoforms and the hepatic clock. Finally, we demonstrate that while P1-HNF4α drives gluconeogenesis, P2-HNF4α drives ketogenesis and is required for elevated levels of ketone bodies in female mice. Taken together, we propose that the highly conserved two-promoter structure of the Hnf4a gene is an evolutionarily conserved mechanism to maintain the balance between gluconeogenesis and ketogenesis in the liver in a circadian fashion.

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.

SPOP inhibits HBV transcription and replication by ubiquitination and degradation of HNF1α.

Hepatitis B virus (HBV) infection remains a significant public health burden worldwide. The persistence of covalently closed circular DNA (cccDNA) within the nucleus of infected hepatocytes is responsible for the failure of antiviral treatments. The ubiquitin proteasome system (UPS) has emerged as a promising antiviral target, as it can regulate HBV replication by promoting critical protein degradation in steps of viral life cycle. Speckle-type POZ protein (SPOP) is a critical adaptor for Cul3-RBX1 E3 ubiquitin ligase complex, but the effect of SPOP on HBV replication is less known. Here, we identified SPOP as a novel host antiviral factor against HBV infection. SPOP overexpression significantly inhibited the transcriptional activity of HBV cccDNA without affecting cccDNA level in HBV-infected HepG2-NTCP and primary human hepatocyte cells. Mechanism studies showed that SPOP interacted with hepatocyte nuclear factor 1α (HNF1α), and induced HNF1α degradation through host UPS pathway. Moreover, the antiviral role of SPOP was also confirmed in vivo. Together, our findings reveal that SPOP is a novel host factor which inhibits HBV transcription and replication by ubiquitination and degradation of HNF1α, providing a potential therapeutic strategy for the treatment of HBV infection.