HNF4α Expression Research Articles

Conjugated Linoleic Acid Treatment Attenuates Cancerous features in Hepatocellular Carcinoma Cells.

Background A growing number of hepatocellular carcinoma (HCC), and recurrence frequency recently have drawn researchers' attention to alternative approaches. The concept of differentiation therapies (DT) relies on inducing differentiation in HCC cells in order to inhibit recurrence and metastasis. Hepatocyte nuclear factor 4 alpha (HNF4α) is the key hepatogenesis transcription factor and its upregulation may decrease the invasiveness of cancerous cells by suppressing epithelial-mesenchymal transition (EMT). This study aimed to evaluate the effect of conjugated linoleic acid (CLA) treatment, natural ligand of HNF4α, on the proliferation, migration, and invasion capacities of HCC cells in vitro. Materials and Method. Sk-Hep-1 and Hep-3B cells were treated with different doses of CLA or BIM5078 [1-(2′-chloro-5′-nitrobenzenesulfonyl)−2-methylbenzimidazole], an HNF4α antagonist. The expression levels of HNF4a and EMT related genes were evaluated and associated to hepatocytic functionalities, migration, and colony formation capacities, as well as to viability and proliferation rate of HCC cells. Results In both HCC lines, CLA treatment induced HNF4α expression in parallel to significantly decreased EMT marker levels, migration, colony formation capacity, and proliferation rate, whereas BIM5078 treatment resulted in the opposite effects. Moreover, CLA supplementation also upregulated ALB, ZO1, and HNF4α proteins as well as glycogen storage capacity in the treated HCC cells. Conclusion CLA treatment can induce a remarkable hepatocytic differentiation in HCC cells and attenuates cancerous features. This could be as a result of HNF4a induction and EMT inhibition.

Network pharmacology and in vitro experiments-based strategy to investigate the mechanisms of KangXianYiAi formula for hepatitis B virus-related hepatocellular carcinoma.

The Chinese traditional medicine KangXianYiAi formula (KXYA) is used to treat hepatic disease in the clinic. Here we aim to confirm the therapeutic effects and explore the pharmacological mechanisms of KXYA on hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). We first collected and analyzed clinical data of 40 chronic hepatitis B (CHB) patients with precancerous liver lesions under KXYA treatment. Then, the cell viability, migration, cell cycle, and apoptosis of HepAD38 cells with KXYA treatment were examined. Next, we performed network pharmacological analysis based on database mining to obtain the key target pathways and genes of KXYA treatment on HBV-related HCC. We finally analyzed the expression of the key genes between normal and HBV-related HCC tissues in databases and measured the mRNA expression of the key genes in HepAD38 cells after KXYA treatment. The KXYA treatment could reduce the liver nodule size of CHB patients, suppress the proliferation and migration capabilities, and promote apoptosis of HepAD38 cells. The key pathways of KXYA on HBV-related HCC were Cancer, Hepatitis B, Viral carcinogenesis, Focal adhesion, and PI3K-Akt signaling, and KXYA treatment could regulate the expression of the key genes including HNF4A, MAPK8, NR3C1, PTEN, EGFR, and HDAC1. The KXYA exhibited a curative effect via inhibiting proliferation, migration, and promoting apoptosis of HBV-related HCC and the pharmacological mechanism was related to the regulation of the expression of HNF4A, MAPK8, NR3C1, PTEN, EGFR, and HDAC1.

Hepatic circadian and differentiation factors control liver susceptibility for fatty liver disease and tumorigenesis.

Hepatocellular carcinoma (HCC) is a leading cause of cancer deaths, and the most common primary liver malignancy to present in the clinic. With the exception of liver transplant, treatment options for advanced HCC are limited, but improved tumor stratification could open the door to new treatment options. Previously, we demonstrated that the circadian regulator Aryl Hydrocarbon-Like Receptor Like 1 (ARNTL, or Bmal1) and the liver-enriched nuclear factor 4 alpha (HNF4α) are robustly co-expressed in healthy liver but incompatible in the context of HCC. Faulty circadian expression of HNF4α- either by isoform switching, or loss of expression- results in an increased risk for HCC, while BMAL1 gain-of-function in HNF4α-positive HCC results in apoptosis and tumor regression. We hypothesize that the transcriptional programs of HNF4α and BMAL1 are antagonistic in liver disease and HCC. Here, we study this antagonism by generating a mouse model with inducible loss of hepatic HNF4α and BMAL1 expression. The results reveal that simultaneous loss of HNF4α and BMAL1 is protective against fatty liver and HCC in carcinogen-induced liver injury and in the "STAM" model of liver disease. Furthermore, our results suggest that targeting Bmal1 expression in the absence of HNF4α inhibits HCC growth and progression. Specifically, pharmacological suppression of Bmal1 in HNF4α-deficient, BMAL1-positive HCC with REV-ERB agonist SR9009 impairs tumor cell proliferation and migration in a REV-ERB-dependent manner, while having no effect on healthy hepatocytes. Collectively, our results suggest that stratification of HCC based on HNF4α and BMAL1 expression may provide a new perspective on HCC properties and potential targeted therapeutics.

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.

Expression of hepatocyte nuclear factor 4 alpha, wingless-related integration site, and β-catenin in clinical gastric cancer

BACKGROUNDGastric cancer (GC) is the second most common cause of cancer-related deaths worldwide. Hepatocyte nuclear factor 4 alpha (HNF4α) that belongs to the nuclear hormone receptor superfamily, is overexpressed in GC tissues, and might be involved in the development of GC by regulating its downstream wingless-related integration site (WNT)/β-catenin signaling.AIMTo clarify the expression of HNF4α/WNT5a/β-catenin signaling proteins in clinical GC tissues. METHODSWe immunohistochemically stained pathological blocks of GC and matched para-cancerous tissues. The intensity of HNF4α, WNT5a and β-catenin staining in the tumor cells was determined according to cell rates and staining intensity. The correlations between GC and HNF4α, WNT5a, and β-catenin expression using chi-square and paired chi-square tests. Relationships between double-positive HNF4α and WNT5a expression and types of gastric tumor tissues were assessed using regression analysis. Correlations between HNF4α and WNT5a expression at the RNA level in GC tissues found in the TCGA database were analyzed using Pearson correlation coefficients. RESULTSWe found more abundant HNF4α and WNT5a proteins in GC, especially in mucinous adenocarcinoma and mixed GC than in adjacent tissues (P < 0.001). Low and high levels of cytoplasmic β-catenin respectively expressed in GC and adjacent tissues (P < 0.001) were not significantly associated with pathological parameters. CONCLUSIONThe expressions of HNF4α and WNT5a could serve as early diagnostic biomarkers for GC.

Feedback loop between hepatocyte nuclear factor 1α and endoplasmic reticulum stress mitigates liver injury by downregulating hepatocyte apoptosis

Hepatocyte nuclear factor alpha (HNF1α), endoplasmic reticulum (ER) stress, and hepatocyte apoptosis contribute to severe acute exacerbation (SAE) of liver injury. Here, we explore HNF1α–ER stress-hepatocyte apoptosis interaction in liver injury. LO2, HepG2 and SK-Hep1 cells were treated with thapsigargin (TG) or tunicamycin (TM) to induce ER stress. Carbon tetrachloride (CCl4) was used to induce acute liver injury in mice. Low-dose lipopolysaccharide (LPS) exacerbated liver injury in CCl4-induced mice. Significant apoptosis, HNF1α upregulation, and nuclear factor kappa B (NF-κB) activation were observed in human-derived hepatocytes during ER stress. Knockdown of Rela, NF-κB p65, inhibited the HNF1α upregulation. Following CCl4 treatment ER stress, apoptosis, HNF1α expression and RelA phosphorylation were significantly increased in mice. HNF1α knockdown reduced activating transcription factor 4 (ATF4) expression, and aggravated ER stress as well as hepatocyte apoptosis in vivo and in vitro. The double fluorescent reporter gene assay confirmed that HNF1α regulated the transcription of ATF4 promoter. LPS aggravated CCl4-induced liver injury and reduced HNF1α, and ATF4 expression. Therefore, in combination, HNF1α and ER stress could be mutually regulated forming a feedback loop, which helps in protecting the injured liver by down-regulating hepatocyte apoptosis. Low-dose LPS aggravates hepatocyte apoptosis and promotes the SAE of liver injury by interfering with the feedback regulation of HNF1α and ER stress in acute liver injury.

Epithelial HNF4A shapes the intraepithelial lymphocyte compartment via direct regulation of immune signaling molecules.

Hepatocyte nuclear factor 4 α (HNF4A) is a highly conserved nuclear receptor that has been associated with ulcerative colitis. In mice, HNF4A is indispensable for the maintenance of intestinal homeostasis, yet the underlying mechanisms are poorly characterized. Here, we demonstrate that the expression of HNF4A in intestinal epithelial cells (IECs) is required for the proper development and composition of the intraepithelial lymphocyte (IEL) compartment. HNF4A directly regulates expression of immune signaling molecules including butyrophilin-like (Btnl) 1, Btnl6, H2-T3, and Clec2e that control IEC-IEL crosstalk. HNF4A selectively enhances the expansion of natural IELs that are TCRγδ+ or TCRαβ+CD8αα+ to shape the composition of IEL compartment. In the small intestine, HNF4A cooperates with its paralog HNF4G, to drive expression of immune signaling molecules. Moreover, the HNF4A-BTNL regulatory axis is conserved in human IECs. Collectively, these findings underscore the importance of HNF4A as a conserved transcription factor controlling IEC-IEL crosstalk and suggest that HNF4A maintains intestinal homeostasis through regulation of the IEL compartment.

Galectin-3 inhibition retained expression of hepatocyte nuclear factors 4α and 1α in acetaminophen induced acute liver injury

Introduction: Hepatocyte nuclear factors HNF4α and HNF1α, key transcription factors that regulate drug metabolism enzymes expression, were linked to inflammation. Galectin-3 (Gal-3) is a multi-role animal lectin which is involved in inflammation. This study aims to evaluate the impact of galectin-3 inhibition, by using modified citrus pectin (MCP), on HNF4α and HNF1α gene expression levels after acetaminophen (APAP) induced acute liver injury in Wistar rats. Materials and Methods: Sixty-four male Wistar rats were divided into four groups as follows; control, MCP, APAP and MCP plus APAP administered groups. The groups received APAP were divided into three subgroups each; in which rats were sacrificed after 24, 48 and 72 hours (h) from APAP administration. Expression levels of HNF4α and HNF1α, beside levels of Gal-3, tumor necrosis factor- α (TNF-α), Cytochrome P450 2E1 (CYP2E1), reduced glutathione (GSH), glutathione reductase (GR) and peroxidase (GPx) activities, liver function parameters were evaluated, along with histopathological study of the liver. Results: APAP high dose induced inhibition of liver HNF4α and HNF1α gene expression, CYP2E1 and GSH levels, GR and GPx activities, and increased hepatic Gal-3, TNF-α and serum liver function parameters levels, besides inducing hepatic necrosis. The toxic effects were stronger after 24 h then declined gradually after 48 h and 72 h. Inhibiting Gal-3 functionality after APAP high dose administration reduced TNF-α level and retrieved liver levels of HNF4α and HNF1α expression, CYP2E1, GSH, GR and GPx closer to normal control levels. Conclusion: Inhibiting Gal-3 functionality affects HNF4α and HNF1α gene expression levels and reduced inflammation after APAP high dose administration.

Forkhead O Transcription Factor 4 Restricts HBV Covalently Closed Circular DNA Transcription and HBV Replication through Genetic Downregulation of Hepatocyte Nuclear Factor 4 Alpha and Epigenetic Suppression of Covalently Closed Circular DNA via Interacting with Promyelocytic Leukemia Protein.

ABSTRACTNuclear located hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) remains the key obstacle to cure chronic hepatitis B (CHB). In our previous investigation, it was found that FoxO4 could inhibit HBV core promoter activity through downregulating the expression of HNF4α. However, the exact mechanisms whereby FoxO4 inhibits HBV replication, especially its effect on cccDNA, remain unclear. Here, our data further revealed that FoxO4 could effectively inhibit cccDNA mediated transcription and HBV replication without affecting cccDNA level. Mechanistic study showed that FoxO4 could cause epigenetic suppression of cccDNA. Although FoxO4-mediated downregulation of HNF4α contributed to inhibiting HBV core promoter activity, it had little effect on cccDNA epigenetic regulation. Further, it was found that FoxO4 could colocalize within promyelocytic leukemia protein (PML) nuclear bodies and interact with PML. Of note, PML was revealed to be critical for FoxO4-mediated inhibition of cccDNA epigenetic modification and of the following cccDNA transcription and HBV replication. Furthermore, FoxO4 was found to be downregulated in HBV-infected hepatocytes and human liver tissues, and it was negatively correlated with cccDNA transcriptional activity in CHB patients. Together, these findings highlight the role of FoxO4 in suppressing cccDNA transcription and HBV replication via genetic downregulation of HNF4α and epigenetic suppression of cccDNA through interacting with PML. Targeting FoxO4 may present as a new therapeutic strategy against chronic HBV infection.IMPORTANCE HBV cccDNA is a determining factor for viral persistence and the main obstacle for a cure of chronic hepatitis B. Strategies that target cccDNA directly are therefore of great importance in controlling persistent HBV infection. In present investigation, we found that FoxO4 could efficiently suppress cccDNA transcription and HBV replication without affecting the level of cccDNA itself. Further, our data revealed that FoxO4 might inhibit cccDNA function via a two-part mechanism: one is to epigenetically suppress cccDNA transcription via interacting with PML, and the other is to inhibit HBV core promoter activity via the genetic downregulation of HNF4α. Of note, HBV might dampen the expression of FoxO4 for its own persistent infection. We propose that manipulation of FoxO4 may present as a potential therapeutic strategy against chronic HBV infection.

Alisol B Alleviates Hepatocyte Lipid Accumulation and Lipotoxicity via Regulating RARα-PPARγ-CD36 Cascade and Attenuates Non-Alcoholic Steatohepatitis in Mice.

Non-alcoholic steatohepatitis (NASH) is a common chronic liver disease worldwide, with no effective therapies available. Discovering lead compounds from herb medicine might be a valuable strategy for the treatment of NASH. Here, we discovered Alisol B, a natural compound isolated from Alisma orientalis (Sam.), that attenuated hepatic steatosis, inflammation, and fibrosis in high-fat diet plus carbon tetrachloride (DIO+CCl4)-induced and choline-deficient and amino acid-defined (CDA)-diet-induced NASH mice. RNA-seq showed Alisol B significantly suppressed CD36 expression and regulated retinol metabolism in NASH mice. In mouse primary hepatocytes, Alisol B decreased palmitate-induced lipid accumulation and lipotoxicity, which were dependent on CD36 suppression. Further study revealed that Alisol B enhanced the gene expression of RARα with no direct RARα agonistic activity. The upregulation of RARα by Alisol B reduced HNF4α and PPARγ expression and further decreased CD36 expression. This effect was fully abrogated after RARα knockdown, suggesting Alisol B suppressed CD36 via regulating RARα-HNF4α-PPARγ cascade. Moreover, the hepatic gene expression of RARα was obviously decreased in murine NASH models, whereas Alisol B significantly increased RARα expression and decreased CD36 expression, along with the downregulation of HNF4α and PPARγ. Therefore, this study showed the unrecognized therapeutic effects of Alisol B against NASH with a novel mechanism by regulating RARα-PPARγ-CD36 cascade and highlighted Alisol B as a promising lead compound for the treatment of NASH.

Gandi Capsule Improved Podocyte Lipid Metabolism of Diabetic Nephropathy Mice through SIRT1/AMPK/HNF4A Pathway.

Podocyte lipid accumulation is a potential therapeutic target for diabetic nephropathy (DN). This study was aimed at clarifying the mechanism of Gandi capsule (GDC) ameliorating DN by regulating the lipid metabolism of podocytes. Network pharmacology methods were performed to screen the key molecules and potential targets of GDC for constructing the molecular-protein interaction network of GDC and conducting signal pathway enrichment analysis. GDC was predicted to ameliorate DN through SIRT1/AMPK/HNF4A pathway. Our results showed that GDC improved renal function in db/db mice. Besides, GDC exhibited effectiveness in relieving kidney tissue damage and renal lipid accumulation in db/db mice, and same effects were present in GDC-active ingredient baicalin. We further proved the new role of HNF4A in the lipid metabolism of DN mediated by SIRT1 and AMPK signaling pathways. The results suggested decreased expression of SIRT1 and p-AMPKα in the kidney tissue and increased expression of HNF4A of db/db mice compared with the control group. GDC and baicalin could reverse these expression changes. Furthermore, similar expression changes were observed in the murine podocyte cell line (MPC-5) treated with different concentrations of GDC and baicalin. Our research suggested that GDC and its active ingredient baicalin could alleviate the abnormal lipid metabolism in the kidney of db/db mice and might exert renal protection through the SIRT1/AMPK/HNF4A pathway.

Berberine retarded the growth of gastric cancer xenograft tumors by targeting hepatocyte nuclear factor 4α.

BACKGROUNDGastric cancer is the third deadliest cancer in the world and ranks second in incidence and mortality of cancers in China. Despite advances in prevention, diagnosis, and therapy, the absolute number of cases is increasing every year due to aging and the growth of high-risk populations, and gastric cancer is still a leading cause of cancer-related death. Gastric cancer is a consequence of the complex interaction of microbial agents, with environmental and host factors, resulting in the dysregulation of multiple oncogenic and tumor-suppressing signaling pathways. Global efforts have been made to investigate in detail the genomic and epigenomic heterogeneity of this disease, resulting in the identification of new specific and sensitive predictive and prognostic biomarkers. Trastuzumab, a monoclonal antibody against the HER2 receptor, is approved in the first-line treatment of patients with HER2+ tumors, which accounts for 13%-23% of the gastric cancer population. Ramucirumab, a monoclonal antibody against VEGFR2, is currently recommended in patients progressing after first-line treatment. Several clinical trials have also tested novel agents for advanced gastric cancer but mostly with disappointing results, such as anti-EGFR and anti-MET monoclonal antibodies. Therefore, it is still of great significance to screen specific molecular targets for gastric cancer and drugs directed against the molecular targets.AIMTo investigate the effect and mechanism of berberine against tumor growth in gastric cancer xenograft models and to explore the role of hepatocyte nuclear factor 4α (HNF4α)-WNT5a/β-catenin pathways played in the antitumor effects of berberine.METHODSMGC803 and SGC7901 subcutaneous xenograft models were established. The control group was intragastrically administrated with normal saline, and the berberine group was administrated intragastrically with 100 mg/kg/d berberine. The body weight of nude mice during the experiment was measured to assess whether berberine has any adverse reaction. The volume of subcutaneous tumors during this experiment was recorded to evaluate the inhibitory effect of berberine on the growth of MGC803 and SGC7901 subcutaneous transplantation tumors. Polymerase chain reaction assays were conducted to evaluate the alteration of transcriptional expression of HNF4α, WNT5a and β-catenin in tumor tissues and liver tissues from the MGC803 and SGC7901 xenograft models. Western blotting and IHC were performed to assess the protein expression of HNF4α, WNT5a and β-catenin in tumor tissues and liver tissues from the MGC803 and SGC7901 xenograft models.RESULTSIn the both MGC803 and SGC7901 xenograft tumor models, berberine significantly reduced tumor volume and weight and thus retarded the growth rate of tumors. In the SGC7901 and MGC803 subcutaneously transplanted tumor models, berberine down-regulated the expression of HNF4α, WNT5a and β-catenin in tumor tissues from both transcription and protein levels. Besides, berberine also suppressed the protein expression of HNF4α, WNT5a and β-catenin in liver tissues.CONCLUSIONBerberine retarded the growth of MGC803 and SGC7901 xenograft model tumors, and the mechanism behind these anti-growth effects might be the downregulation of the expression of HNF4α-WNT5a/β-catenin signaling pathways both in tumor tissues and liver tissues of the xenograft models.

Small Molecule-Induced Differentiation As a Potential Therapy for Liver Cancer.

Despite the efficacy demonstrated by immunotherapy recently, liver cancer still remains one of the deadliest cancers, mainly due to heterogeneity of this disease. Continuous exploration of new therapeutics is therefore necessary. Chemical‐induced cell differentiation can serve as a promising approach, with its ability to consistently remodel gene expression profile and alter cell fate. Inspired by advances in stem cell and reprogramming field, here it is reported that a small molecule cocktail (SMC) consisted of: SB431542 (TGFβ inhibitor), CHIR99021 (GSK3β inhibitor), BIX01294 (H3K9 methyltransferase/G9a inhibitor), and all‐trans retinoic acid (ATRA), can induce differentiation of liver cancer cells including cell lines, primary cancer cells, cancer stem cells, and drug resistant cells. Treated cells lose malignant characteristics and regain hepatocyte phenotype instead. When applied in vivo, SMC induces wide range of tissue necrosis or fibrosis within the tumors, while remaining tissues begin to express hepatic nuclear factor 4α (HNF4α), the hepatic nuclear marker. SMC also leads to tumor abrogation in orthotopic xenograft models and life span extension of animals. The powerful differentiation induction of SMC is exerted through modulation of Akt/mTOR/HIF1α signaling and metabolic reprogramming, as well as suppressing Snail and enhancing HNF4α expression. Together, these results highlight that chemical‐induced differentiation has the potential to effectively treat liver cancer disregard of heterogeneity.

Prenatal glucocorticoid administration accelerates the maturation of fetal rat hepatocytes.

Prenatal glucocorticoid (GC) is clinically administered to pregnant women who are at risk of preterm birth for the maturation of cardiopulmonary function. Preterm and low-birth-weight infants often experience liver dysfunction after birth because their livers are immature. However, the effects of prenatal GC administration on the liver remain unclear. We aimed to investigate the effects of prenatal GC administration on the maturation of liver hepatocytes in preterm rats. Dexamethasone (DEX) was administered to pregnant Wistar rats on gestational days 17 and 19 before cesarean section. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the mRNA levels of albumin, hepatocyte nuclear factor-4 alpha (HNF4α), hepatocyte growth factor (HGF), thymus cell antigen 1 (Thy-1), cyclin B, and Cyclin-dependent kinase 1 (CDK1) in the liver samples. Immunohistochemical staining and enzyme-linked immunosorbent assay were performed to examine protein production. The hepatocytes enlarged because of growth and prenatal DEX administration. Albumin, HNF4α, and HGF levels increased secondary to growth and prenatal DEX administration. The levels of the cell cycle markers cyclin B and CDK1 gradually decreased during growth and with DEX administration. The results suggest that prenatal GC administration leads to hepatocyte maturation via expression of HNF4α and HGF in preterm fetuses.

Hepatocyte nuclear factor 4α in the pathogenesis of non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is emerging as the most common chronic liver disease worldwide. It refers to a range of liver conditions affecting people who drink little or no alcohol. NAFLD comprises non-alcoholic fatty liver and non-alcoholic steatohepatitis (NASH), the more aggressive form of NAFLD. NASH is featured by steatosis, lobular inflammation, hepatocyte injury, and various degrees of fibrosis. Although much progress has been made over the past decades, the pathogenic mechanism of NAFLD remains to be fully elucidated. Hepatocyte nuclear factor 4α (HNF4α) is a nuclear hormone receptor that is highly expressed in hepatocytes. Hepatic HNF4α expression is markedly reduced in NAFLD patients and mouse models of NASH. HNF4α has been shown to regulate bile acid, lipid, glucose, and drug metabolism. In this review, we summarize the recent advances in the understanding of the pathogenesis of NAFLD with a focus on the regulation of HNF4α and the role of hepatic HNF4α in NAFLD. Several lines of evidence have shown that hepatic HNF4α plays a key role in the initiation and progression of NAFLD. Recent data suggest that hepatic HNF4α may be a promising target for treatment of NAFLD.

Scalable Dual-Fluorescence Assay for Functional Interpretation of HNF-4α Missense Variants.

AimThe study aimed to develop a scalable dual-fluorescence assay in cells to enable the functional interpretation of HNF-4α missense variants identified in exome sequencing, which can be used to guide clinical diagnosis.MethodsUsing mOrange2 and GFP fluorescence proteins to track the expression of HNF-4α (HNF-4α-mOrange2) and reporter activity under the control of the HNF-1α promoter (pHNF1A-GFP), respectively, we designed a dual-fluorescence assay to evaluate the expression level, cellular localization, and transcriptional function of HNF-4α simultaneously in live cells. To assess the scalable characteristic of the assay, a small library containing five previously reported mutations and wild-type HNF-4α was constructed. Cells infected with this library were sorted into different populations through fluorescence-activated cell sorting (FACS) according to the transcription activity and expression abundance. Cloning and Sanger sequencing were used to detect the mutations of the different groups. High content screening (HCS) assay was used for the validation of individual mutants in the function and expression point of view.ResultsHNF-4α-mOrange2 exhibited nuclear localization and transactivation capability on the HNF-1α promoter as physical HNF-4α does. The expression of HNF-4α-mOrange2 shows a 6-fold induction of GFP expression compared to the control without HNF-4α-mOrange2, which was significantly abolished by the known loss-of-function mutant M373R. The different performances of wild-type and mutant M373R made them distinguishable in the FACS system, empowering the scalable capability of this assay for classifying large numbers of variants combining functional stratification and sequencing. Further application of the assay in the small library showed that three cell populations were seen grouped as Normal (same transactivation as wild type), Reducedexp_nor (reduced transactivation with normal or higher expression), and Reducedexp_low (reduced transactivation with lower expression). Subsequently, Sanger sequencing showed that wild-type HNF-4α was in the Normal group, two mutations (M373R and G79C) were enriched in the Reducedexp_nor group, and three mutations (C115S, L272P, and F83C) belonged to the Reducedexp_low group. These results were validated by further imaging data using HCS assay for individual mutation.ConclusionsOur study proposes a scalable and informative approach for the characterization of the variants in HNF-4α genes in a quantitative and high-throughput manner.

Induction of peripheral blood mononuclear cells to hepatocyte-like cells and preliminary study of cell response to injury under the effect of acetaminophen

Objective: To establish a method for the induction of peripheral blood mononuclear cells to hepatocyte-like cells, and preliminarily investigate cell response to injury under the effect of acetaminophen (APAP). Methods: The surface marker CD45 of peripheral blood mononuclear cells wase detected cells by using flow cytometry and immunofluorescence methods. The cellular morphology of induced hepatocyte-like cells was observed under an inverted microscope. Real-time fluorescent quantitative PCR (RT-PCR) was used to detect the expression level of hepatocyte-specific genes, such as cytochrome (CY) P1A2, CYP3A4, CYP2C9, albumin (ALB), alpha-fetoprotein (AFP), and hepatocyte nuclear factor (HNF)4α mRNA. Immunofluorescence method was used to detect intracellular hepatocyte markers AFP, HNF4α, and ALB expression at the protein level. Biochemical analyzer was used to detect hepatocyte-specific secretory functions of AFP, ALB, and urea. Luciferase chemiluminescence method was used to detect the activity of key drug metabolizing enzyme CYP3A4. Colorimetric assay was used to detect the effect of the drug acetaminophen on hepatocyte-like cells, and alanine aminotransferase (ALT) was used as an indicator of liver cell injury. The statistical differences between the data were compared with t-test and rank-sum test. Results: The positive expression rate of CD45 cell surface markers isolated from peripheral blood mononuclear cells was about 98%, and hepatocyte-like cell morphology changes appeared on 15th day of induction. Compared with isolated mononuclear cells, CYP1A2, CYP3A4, CYP2C9, ALB, AFP and HNF4α mRNA was markedly elevated. The expression level of AFP, ALB and HNF4α protein were equally increased, and the secretory function of AFP, ALB and urea were enhanced. Compared with primary hepatocytes, CYP1A2, CYP2C9, AFP, HNF4α mRNA, and CYP3A4 mRNA did not decrease. The expression levels of AFP, ALB, and HNF4α proteins in the cells did not decrease, and the secretory function of AFP, ALB, and urea did not decrease. In addition, the CYP3A4 enzyme activity produced by hepatocyte-like cells was similar to that of primary hepatocytes. Compared with hepatocyte-like cells incubated without APAP, hepatocyte-like cells incubated with APAP had higher ALT level. Under the effect of APAP, the ALT level of hepatocyte-like cells was higher than isolated mononuclear cells. Conclusion: Peripheral blood mononuclear cells can be induced into hepatocyte-like cells with partial characteristics of hepatocytes, including the activity of CYP3A4, a key enzyme of hepatocyte drug metabolism. Additionally, preliminarily ALT secretory features reflect the hepatocytes injury under the effect of acetaminophen.

Impact of Soy β-Conglycinin Peptides on PCSK9 Protein Expression in HepG2 Cells.

Background: Dyslipidaemias, particularly elevated plasma low-density lipoprotein cholesterol (LDL-C) levels, are major risk factors for cardiovascular disease (CVD). Besides pharmacological approaches, a nutritional strategy for CVD prevention has gained increasing attention. Among functional foods, the hypocholesterolemic properties of soy are driven by a stimulation of LDL-receptor (LDL-R) activity. Aim: To characterize the effect of two soy peptides, namely, β-conglycinin-derived YVVNPDNDEN and YVVNPDNNEN on the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9), one of the key-regulators of the LDL-R. Methods: PCSK9 promoter activity (luciferase assay), PCSK9 protein expression (WB) and secretion (ELISA), PCSK9 interaction with LDL-R (binding assay) and human HepG2 cells were the objects of this investigation. Results: Treatment with YVVNPDNNEN peptide has led to a rise in PCSK9 gene expression (90.8%) and transcriptional activity (86.4%), and to a decrement in PCSK9 intracellular and secreted protein (−42.9%) levels. YVVNPDNNEN peptide reduced the protein expression of transcriptional factor HNF1α. Most changes driven by YVVNPDNDEN peptide were not statistically significant. Neither peptide inhibited the PCSK9–LDLR interaction. Conclusions: Although sharing a common effect on LDL-R levels through the inhibition of 3-hydroxy-3-methylglutaryl CoA reductase activity, only the YVVNPDNNEN peptide has an additional mechanism via the downregulation of PCSK9 protein levels.

CES2 sustains HNF4α expression to promote pancreatic adenocarcinoma progression through an epoxide hydrolase-dependent regulatory loop

ObjectiveIntra-tumoral expression of the serine hydrolase carboxylesterase 2 (CES2) contributes to the activation of the pro-drug irinotecan in pancreatic ductal adenocarcinoma (PDAC). Given other potential roles of CES2, we assessed its regulation, downstream effects, and contribution to tumor development in PDAC. MethodsAssociation between the mRNA expression of CES2 in pancreatic tumors and overall survival was assessed using The Cancer Genome Atlas. Cell viability, clonogenic, and anchorage-independent growth assays as well as an orthotopic mouse model of PDAC were used to evaluate the biological relevance of CES2 in pancreatic cancer. CES2-driven metabolic changes were determined by untargeted and targeted metabolomic analyses. ResultsElevated tumoral CES2 mRNA expression was a statistically significant predictor of poor overall survival in PDAC patients. Knockdown of CES2 in PDAC cells reduced cell viability, clonogenic capacity, and anchorage-independent growth in vitro and attenuated tumor growth in an orthotopic mouse model of PDAC. Mechanistically, CES2 was found to promote the catabolism of phospholipids resulting in HNF4α activation through a soluble epoxide hydrolase (sEH)-dependent pathway. Targeting of CES2 via siRNA or small molecule inhibitors attenuated HNF4α protein expression and reduced gene expression of classical/progenitor markers and increased basal-like markers. Targeting of the CES2-sEH-HNF4α axis using small molecule inhibitors of CES2 or sEH reduced cell viability. ConclusionsWe establish a novel regulatory loop between CES2 and HNF4α to sustain the progenitor subtype and promote PDAC progression and highlight the potential utility of CES2 or sEH inhibitors for the treatment of PDAC as part of non-irinotecan-containing regimens.

Follistatin-controlled activin-HNF4α-coagulation factor axis in liver progenitor cells determines outcome of acute liver failure.

In patients with acute liver failure (ALF) who suffer from massive hepatocyte loss, liver progenitor cells (LPCs) take over key hepatocyte functions, which ultimately determines survival. This study investigated how the expression of hepatocyte nuclear factor 4α (HNF4α), its regulators, and targets in LPCs determines clinical outcome of patients with ALF. Clinicopathological associations were scrutinized in 19 patients with ALF (9 recovered and 10 receiving liver transplantation). Regulatory mechanisms between follistatin, activin, HNF4α, and coagulation factor expression in LPC were investigated in vitro and in metronidazole-treated zebrafish. A prospective clinical study followed up 186 patients with cirrhosis for 80months to observe the relevance of follistatin levels in prevalence and mortality of acute-on-chronic liver failure. Recovered patients with ALF robustly express HNF4α in either LPCs or remaining hepatocytes. As in hepatocytes, HNF4α controls the expression of coagulation factors by binding to their promoters in LPC. HNF4α expression in LPCs requires the forkhead box protein H1-Sma and Mad homolog 2/3/4 transcription factor complex, which is promoted by the TGF-β superfamily member activin. Activin signaling in LPCs is negatively regulated by follistatin, a hepatocyte-derived hormone controlled by insulin and glucagon. In contrast to patients requiring liver transplantation, recovered patients demonstrate a normal activin/follistatin ratio, robust abundance of the activin effectors phosphorylated Sma and Mad homolog 2 and HNF4α in LPCs, leading to significantly improved coagulation function. A follow-up study indicated that serum follistatin levels could predict the incidence and mortality of acute-on-chronic liver failure. These results highlight a crucial role of the follistatin-controlled activin-HNF4α-coagulation axis in determining the clinical outcome of massive hepatocyte loss-induced ALF. The effects of insulin and glucagon on follistatin suggest a key role of the systemic metabolic state in ALF.