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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Hepatocyte nuclear factor 4 alpha (HNF4α), the master regulator of hepatocyte differentiation, has been recently shown to inhibit hepatocyte proliferation by way of unknown mechanisms. We investigated the mechanisms of HNF4α-induced inhibition of hepatocyte proliferation using a novel tamoxifen (TAM)-inducible, hepatocyte-specific HNF4α knockdown mouse model. Hepatocyte-specific deletion of HNF4α in adult mice resulted in increased hepatocyte proliferation, with a significant increase in liver-to-body-weight ratio. We determined global gene expression changes using Illumina HiSeq-based RNA sequencing, which revealed that a significant number of up-regulated genes following deletion of HNF4α were associated with cancer pathogenesis, cell cycle control, and cell proliferation. The pathway analysis further revealed that c-Myc-regulated gene expression network was highly activated following HNF4α deletion. To determine whether deletion of HNF4α affects cancer pathogenesis, HNF4α knockdown was induced in mice treated with the known hepatic carcinogen diethylnitrosamine (DEN). Deletion of HNF4α significantly increased the number and size of DEN-induced hepatic tumors. Pathological analysis revealed that tumors in HNF4α-deleted mice were well-differentiated hepatocellular carcinoma (HCC) and mixed HCC-cholangiocarcinoma. Analysis of tumors and surrounding normal liver tissue in DEN-treated HNF4α knockout mice showed significant induction in c-Myc expression. Taken together, deletion of HNF4α in adult hepatocytes results in increased hepatocyte proliferation and promotion of DEN-induced hepatic tumors secondary to aberrant c-Myc activation. (HEPATOLOGY 2013;57:2480–2490)

Hepatocyte nuclear factor 4 alpha (HNF4α, NR2A1) is considered the master regulator of hepatocyte differentiation.1, 2 It plays an important role in the regulation of many hepatocyte-specific genes including those involved in glycolysis, gluconeogenesis, ureagenesis, fatty acid metabolism, bile acid synthesis, drug metabolism, apolipoprotein synthesis, and blood coagulation.3-7 Because of its important role in liver development and homeostasis, disruption of HNF4α has been linked to various disorders of the liver including metabolic syndrome, type 2 diabetes, mature onset diabetes in the young (MODY), and hepatocellular carcinoma (HCC).8-12

Recent studies suggest a novel role of HNF4α in the regulation of cell proliferation within multiple tissues including liver, pancreas, and kidney.3, 4, 10-17 It is known that HCC progression is associated with down-regulation of HNF4α in rodents and humans.10, 12, 14, 16 More recently, Ning et al.11 reported that overexpression of HNF4α suppresses diethylnitrosamine (DEN)-induced HCC in rats. These data suggest that HNF4α may have the ability to inhibit hepatocyte proliferation within the liver; however, the mechanisms are yet to be determined.

Because of its fundamental role in liver development and homeostasis, whole-body deletion of HNF4α results in an embryonic lethal phenotype.18 Liver-specific deletion of HNF4α under an albumin promoter-driven cre recombinase results in severe hepatic metabolic disruption and lethality between 6 and 8 weeks of age.4, 18 In these mice produced using constitutively active albumin-cre, HNF4α is deleted during early postnatal development, making it difficult to decipher the effect of improper hepatic differentiation and aberrant hepatic proliferation on the observed phenotype. To overcome these issues, we developed an inducible knockout (KO) of HNF4α where HNF4α is deleted in the mature mouse liver using a tamoxifen (TAM)-inducible cre recombinase (ERT2-Cre), first described by Bonzo et al.17 Using this novel mouse model of hepatocyte specific HNF4α deletion in the adult liver combined with RNA sequencing mediated transcriptomics, we investigated the mechanism of HNF4α-mediated inhibition of hepatocyte proliferation. We also studied the significance of the role of HNF4α-mediated regulation of hepatocyte proliferation using a chemical carcinogenesis model. Our studies indicate that apart from its role in hepatic differentiation, HNF4α actively inhibits hepatocyte proliferation and plays a critical role in maintenance of hepatic homeostasis.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Animals, Treatments, and Tissue Collection.

The HNF4αFl/Fl mice (provided by Dr. Frank Gonzalez of NCI-NIH) and the TAM-inducible albumin cre mice (AlbCreERT2+, provided by Dr. Pierre Chambon, IGBMC-France) used in these studies have been described.4 The HNF4αFl/Fl, AlbCreERT2+ mice were produced by standard animal breeding and identified using polymerase chain reaction (PCR)-based genotyping of tail biopsies. All animals were housed in Association for Assessment and Accreditation of Laboratory Animal Care-accredited facilities at the University of Kansas Medical Center under a standard 12-hour light/dark cycle with access to chow and water ad libitum. The Institutional Animal Care and Use Committee approved all of the studies.

Three-month-old male, HNF4αFl/Fl, AlbERT2-Cre+ mice were treated with TAM (6 μg/mouse, intraperitoneal, referred to as HNF4α-KO), or with vehicle alone (corn oil, intraperitoneal, referred to as Control) subcutaneously. To account for changes induced by TAM, 3-month-old male, HNF4αFl/Fl, AlbERT2-Cre mice were treated with TAM (6 μg/mouse, intraperitoneal, referred to as TAM Control). Mice were killed by cervical dislocation under isoflurane anesthesia and livers were collected 7 days postinjection. For the DEN-induced HCC protocol, male HNF4αFl/Fl, AlbERT2-Cre+ mice were injected subcutaneously with 15 μg/g DEN (in 0.9% saline) at postnatal days 12-15 and allowed to grow until 8 months of age. At 8 months these mice were divided into two groups and treated with either TAM (6 μg/mouse) or corn oil and sacrificed 2 months later at 10 months of age. Liver and serum samples were obtained and processed as described.19 Liver and body weights of mice were noted at the time of sacrifice and used to determine liver/body weight ratios. Liver injury and function were determined by serum alanine aminotransferase (ALT), serum bilirubin, and serum glucose levels measured using the Infinity ALT (GPT) and the Infinity Glucose kit (Thermo Scientific; Middletown, VA) according to the manufacturer's protocol.

Western Blotting.

RIPA extracts obtained from whole liver tissues were used for western blot analysis and western blots were performed using the described protocol.20 The antibodies used in this study were: HNF4α (1:1,000; R&D Systems, Minneapolis, MN; Cat. no. PP-H1415-00), Cyclin D1 (Cat. no. 2978), c-Myc (Cat. no. 5605), and β-Actin (Cat. no. 4970) (1:1,000; Cell Signaling, Danvers, MA).

Staining Procedures.

Paraffin-embedded liver sections (4-μm thick) were used for hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), and immunohistochemical staining of proliferating cell nuclear antigen (PCNA) as described.20 After staining for PCNA, positive cells were quantified by counting four 40× fields per slide for each liver sample (n = 3 per group). Fresh-frozen sections (5-μm thick) were used to detect lipid accumulation by staining with Oil Red O and Ki-67 immunofluorescence as described.19, 20 Apoptosis was measured using the In Situ Cell Death Detection Kit, TMR red (Roche Applied Science, Indianapolis, IN; Cat. no. 12156792910) according to the manufacturer's protocol.

RNA-Sequencing (RNA-Seq) and Functional Analysis.

Total RNA was isolated from liver tissue using the phenol/chloroform extraction protocol. Integrity of RNA was analyzed by the Microarray Core Facility at KUMC (Kansas City, KS) using an Agilent Bioanalyzer 2100 (Agilent Technologies; Santa Clara, CA).

We performed two separate and independent RNA-Seq experiments for the same treatment conditions, Cre+/Tamoxifen, Cre−/Tamoxifen, and Cre+/Corn Oil. In the first instance (Run1), the total processed RNA extracted from pooled mouse liver samples (3 mice per group) treated with Cre+/Tamoxifen, Cre−/Tamoxifen, and Cre+/Corn Oil was sequenced in an Illumina HiSeq 2000 sequencing machine (Illumina, San Diego, CA). The initial library of 10 nM concentration for each of the three samples was split into two diluted concentrations of 5 pM and 3 pM and sequenced separately at a 2 × 100 basepair (bp) paired-end resolution and the output of the sequencing runs combined for downstream analysis. In order to complement the initial RNA-Seq analysis, we ran a second RNA-seq experiment (Run2) on biological replicate samples (n = 2) of mouse liver treated with Cre+/Tamoxifen, Cre−/Tamoxifen, and Cre+/Corn Oil. These samples were sequenced at a 50 bp single-end resolution. The RNA-Seq data obtained from both experiments were for the bioinformatics analysis. Reads from both experiments were mapped to the mouse reference genome (NCBI37/mm9) using TopHat v. 1.4.1.21 TopHat was run with default parameters and for Run1 with paired reads, a mate inner distance of −40 was set to accommodate the 260 bp average fragment length. The binary output files generated by TopHat were passed to CuffDiff21 for calculating differential gene expression. For samples from both runs, differential gene expression was first calculated for Cre+/Tamoxifen against Cre+/Corn Oil and Cre+/Tamoxifen against Cre−/Tamoxifen. For each run, genes that were significantly differentially expressed in both these measures based on an absolute fold change of at least 1.5 and a q-value (the P-value adjusted for multiple hypothesis correction using the Benjamini-Hochberg procedure) less than or equal to 0.05 were selected. This data filtering resulted in 1,096 genes from Run1 and 3436 genes from Run2. Of these, 877 genes were common to both runs (right tailed Fisher's exact test significance P < 1E-100). These genes were uploaded to Ingenuity Pathways Analysis (IPA, Ingenuity Systems, v. 7.6; www.ingenuity.com) for gene set enrichment analysis. Out of the 877 genes uploaded, 864 genes qualified for analysis in IPA. The analysis was performed in IPA with default parameters. The RNA-Seq data have been submitted to the Sequence Read Archive (SRA) of the NCBI.

RNA-Seq/ChIP-Seq Comparative Analysis.

We analyzed publicly available ChIP-Seq data (SRA008281) from Hoffman et al.22 to obtain an unbiased whole genome mapping of Hnf4α binding sites in mouse. Sequences were aligned using Bowtie2 (v. 2.0.2) to the latest mouse reference genome (GRCm38/mm10) using default parameters.23 Peak detection was performed using the Model-based Analysis of ChIP-Seq (MACS) algorithm with the peak detection P-value cutoff set at 1e-5 (default).24 This resulted in a set of 9,281 significant (false discovery rate [FDR] less than 1 in a 100) Hnf4α binding sites. We searched for the Hnf4α consensus sequence within a 250 bp region from either side of the called peaks using a weight-matrix match with at least 80% similarity. The Hnf4α weight matrix obtained from the JASPAR database25 was used as a surrogate to model Hnf4α binding sites. A substantial proportion (92%) of the highly enriched Hnf4α binding sites consisted of at least one Hnf4α consensus site. All identified Hnf4α binding sites were annotated with their closest upstream, downstream, and overlapping genes using Ensembl gene annotations.

We looked at how many of the 877 putative Hnf4α perturbed genes identified in our experiment were among the putative Hnf4α target genes identified by the ChIP-Seq experiment. The significance of the overlap of genes between the two studies was calculated using the right tailed Fisher's exact test. The statistic was calculated separately for genes with upstream, downstream, and overlapping Hnf4α targets and in combination and is shown in Supporting Table 2. The IPA analysis was also used to determine the most activated and most inhibited transcription factor gene networks using activation of Z-score criteria (described in the Supporting Material).

Statistical Analysis.

For all experiments not associated with RNA sequencing, such as ALT measurements, the results are expressed as mean ± standard deviation. Student's t test was applied to all analyses with P < 0.001 being considered significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

HNF4α Deletion Results in Increased Cell Proliferation.

Treatment of HNF4αFl/Fl, AlbERT2-Cre+ mice with TAM resulted in deletion of HNF4α as demonstrated by western blot analysis (Fig. 1B). Data shows ∼80%-90% decrease in HNF4α protein level in the KO, as compared to controls. HNF4αFl/Fl AlbERT2-Cre+ treated with corn oil and HNF4αFl/Fl AlbERT2-Cre treated with TAM was observed 7 days after TAM or corn oil injection. HNF4α deletion was also confirmed by immunohistochemical staining of paraffin-embedded sections (data not shown).

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Figure 1. Deletion of HNF4α using TAM-driven albumin cre mice. (A) Scheme of HNF4α deletion using the HNF4αFl/Fl, AlbERT2-Cre+ mice. (B) Western blot analysis of HNF4α using nuclear proteins isolated from HNF4αFl/Fl, AlbERT2-Cre+ mice treated with either TAM or corn oil and HNF4αFl/Fl, AlbERT2-Cre mice treated TAM. See Materials and Methods for details. (C) Liver weight to body weight ratios (D) serum ALT levels, and (E) serum glucose levels of control, HNF4α-KO and TAM control mice 7 days after either TAM or corn oil injection.

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Deletion of HNF4α resulted in a significant increase in liver-to-body-weight ratio (Fig. 1C) but did not result in significant liver injury as indicated by serum ALT and glucose concentrations (Fig. 1D,E). Staining of liver sections indicated that there was no cell death or inflammation following deletion of HNF4α. There was no apparent apoptosis, necrosis, or infiltration of immune cells, all which are hallmark signs of injury (Fig. 2; H&E). Also, we did not observe an increase in terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL)-positive cells following deletion of HNF4α (Fig. 3D). However, the hepatocytes exhibited extensive vacuolization giving them an “empty” appearance. Further analysis indicated a significant decrease in hepatic glycogen accumulation and a significant increase in lipid accumulation demonstrated by PAS and Oil Red O staining, respectively, after HNF4α deletion (Fig. 2; PAS and Oil Red O). Finally, deletion of HNF4α resulted in a dramatic increase in cell proliferation as demonstrated by an ∼20% increase in the amount of PCNA-positive cells (Fig. 3A,B). These data were corroborated by Ki-67 staining (Fig. 3C).

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Figure 2. Histopathological analysis of livers after HNF4α deletion. Representative photographs of paraffin-embedded liver sections from the livers of control, HNF4α-KO, and TAM control livers 7 days after either TAM or corn oil treatment were used to stain for H&E (upper panel), and PAS for glycogen staining (middle panel). Frozen sections from the same samples were used for Oil Red O staining (lower panel). All images are 600×.

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Figure 3. Increased cell proliferation after HNF4α deletion. (A) Representative photomicrographs of PCNA immunohistochemistry and (B) bar graph showing percentage of PCNA-positive cells in control, HNF4α-KO, and TAM control livers 7 days after either TAM or corn oil treatment. Frozen sections from same liver were used to stain for Ki-67 for detection of cell proliferation (C) and TUNEL assay for detection of apoptosis (D).

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HNF4α Deletion Results in Increased Promitogenic Gene Expression.

High-throughput sequencing generated 117, 179, and 136 million reads for the Cre+/TAM, Cre−/TAM, and Cre+/Corn Oil samples, respectively. Of these, TopHat was able to map 103, 163, and 121 million reads to the mouse reference genome, respectively. Further statistics on the quality of the RNA-Seq data is provided in Supporting Table 1.

Deletion of HNF4α resulted in the down-regulation of many genes known to be involved in hepatocyte function, such as xenobiotic metabolism, cholesterol metabolism, coagulation, bile acid synthesis, etc. (Table 1). Interestingly, many of the up-regulated genes are known to be involved in the cell cycle and cancer (Table 2). A complete list of gene expression changes can be found in Supporting Table 6.

Table 1. Down-regulated Genes Following Deletion of HNF4α in Adult Mouse Liver
Gene NameGene SymbolFold ChangeP-value
UDP Glucuronosyltransferase 2 Family, Polypeptide B4Ugt2b1−159.92<1.00E-20
Apolipoprotein A-IIApoa2−54.52<1.00E-20
Coagulation Factor XIIF12−22.06<1.00E-20
Cytochrome P450, Family 8, Subfamily B, Polypeptide 1Cyp8b1−25.47<1.00E-20
Apolipoprotein A-IVApoa4−3.93<1.00E-20
Sulfotransferase Family, Cytosolic, 1A, Phenol-Preferring, Member 1Sult1a1−11.72<1.00E-20
Claudin 2Cldn2−4.186.08E-12
Apolipoprotein BApob−7.131.15E-14
Claudin 1Cldn1−4.79<1.00E-20
Cytochrome P450, Family 7, Subfamily B, Polypeptide 1Cyp7b1−13.68<1.00E-20
Table 2. Up-regulated Genes Following Deletion of HNF4α in Adult Mouse Liver
Gene NameGene SymbolFold Changeq-value
Cyclin B1Ccnb158.75<1.00E-20
Cell Division Cycle Homolog 25 CCdc25c21.077.96E-05
Cyclin-Dependent Kinase Inhibitor 3Cdkn3144.662.28E-03
Cell Division Cycle 20 HomologCdc2074.59<1.00E-20
Antigen Identified by Antibody Ki-67Ki-6723.71<1.00E-20
Polo-Like Kinase 1Plk112.901.71E-14
Cyclin B2Ccnb221.79<1.00E-20
Cyclin A2Ccna220.89<1.00E-20
Epithelial Cell Transforming Sequence 2Ect220.53<1.00E-20
Cell Division Cycle Associated 3Cdca313.16<1.00E-20
Cell Division Cycle Associated 8Cdca88.021.82E-10
Early Growth Response 1Egr15.44<1.00E-20
Myelocytomatosis Viral Oncogene HomologMyc3.84<1.00E-20

RNA-Seq/ChIP-Seq comparative analysis revealed that of the total gene changes observed following deletion of HNF4α (877), ∼53% of these (462) contained a putative HNF4α binding site within 50 kb of the transcriptional start site (TSS) (Supporting Table 2). Further, ∼45% (395) contained a putative HNF4α binding site within 10 kb of the TSS.

To identify patterns in gene expression changes, we used IPA (Ingenuity Systems, www.ingenuity.com). Functional analysis of gene expression changes revealed genes to be involved with cancer pathogenesis to be one of the most significant groups of genes to be changed (Fig. 4A). Other groups of genes found to be significantly changed included genes involved in cell cycle, cellular growth and proliferation, and lipid metabolism (Fig. 4A,B). IPA further revealed changes in major transcription factor activity following HNF4α deletion (Supporting Tables 3, 4). The c-Myc-regulated gene expression network showed the most significant changes in gene expression that correlate with activation of c-Myc following HNF4α deletion (Fig. 3B,C). This included several genes involved in cell proliferation including ccnb1, ccnb2, fus, and set oncogene. Also, many other transcription factor networks known to be involved in cell proliferation and cancer were significantly activated (Supporting Table 3). As expected, gene network associated with HNF4α was inhibited (regulation z-score −5.0, Supporting Table 4). Other factors inhibited include CDKN1A (p21), Smarcb1, Tob1, and CDKN2A (p16), all of which have been shown to be associated with cancer pathogenesis.

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Figure 4. Gene expression change in livers following HNF4α deletion. Global gene expression analysis was conducted using Illumina-based RNA sequencing as described in Materials and Methods. The data were used for IPA. (A) Bar graph showing top categories of genes changed following HNF4α deletion. (B) c-MycGene network showing various genes either up-regulated (red) or down-regulated (green) in HNF4α-KO mice as compared to control. (C) Table showing up-regulated promitogenic genes within the c-Myc-regulated gene network.

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HNF4α Deletion Results in Increased Progression of DEN-Induced HCC.

To determine the effect of HNF4α deletion on hepatic tumor progression we used a DEN-induced HCC model. HNF4αFl/Fl, AlbERT2-Cre+ mice were treated with a known hepatic carcinogen, DEN, at postnatal day 15 and then treated with TAM (HNF4α-KO) or corn oil (control) at 8 months of age followed by tissue collection 2 months later at 10 months of age (Fig. 4A). Deletion of HNF4α only for a 2-month period resulted in increased HCC progression demonstrated by an increase in tumor number and size (Supporting Table 6; Fig. 4B, arrows), along with an ∼2-fold increase in liver/body weight ratio (Supporting Table 6; Fig. 4C). HNF4α-KO livers display advanced tumor morphology and significantly increased proliferation when compared to control livers by way of H&E (Fig. 5D) and PCNA staining, respectively (Fig. 5E). The control mice treated with DEN exhibited mainly regenerative nodules and a few high-grade dysplastic nodules with few early-stage HCCs. In contrast, the HNF4α-KO mice treated with DEN exhibited extensive dyspastic nodules, HCCs (Fig. 5D-ii, iv), and tumors with mixed HCC-cholangiocarcinoma morphology (Fig. 5D-iii, v). The tumors in HNF4α-KO mice exhibited distinct histological features including expansion of oval cell population (Fig. 5D-ii, iv, E-iii) and the presence of inflammatory cell foci (Fig. 5D-vi).

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Figure 5. HNF4α deletion promotes DEN-induced hepatic tumors in mice. (A) Scheme showing protocol of DEN-induced hepatic tumor induction. (B) Representative photographs of livers of control mice (left) and HNF4α-KO mice (right) treated with DEN. (C) Liver to body weight ratios of control and HNF4α-KO mice treated with DEN. (D) Representative photomicrographs of paraffin-embedded liver sections stained for H&E from control (i) and HNF4α-KO mice (ii to vi). Large arrowheads show oval cell-like cells in (ii) and (iv). Arrows point to biliary duct proliferation in the mixed type tumors in (iii) and (v). Small arrowheads indicate inflammatory cell focus in (vi). (E) Representative photomicrographs of PCNA immunohistochemistry on paraffin section from control (i) and HNF4α-KO mice (ii and iii). Arrowheads point to proliferating biliary duct cells.

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HNF4α Deletion Results in Increased Promitogenic Signaling in DEN-Induced HCC.

We hypothesized that increased progression of HCC in HNF4α-KO mice treated with DEN may be due to increased promitogenic signaling. Western blot analysis of control, control+DEN (normal tissue), tumor tissues, and surrounding normal tissues from HNF4α-KO mice treated with DEN indicated increased Cyclin D1 and c-Myc expression only in normal liver tissue surrounding the tumors and in the tumors observed in HNF4α-KO mice treated with DEN (Fig. 6A). Real-time PCR analysis showed that expression of several genes up-regulated following HNF4α deletion were further increased in the tumors observed in HNF4α-KO mice (Fig. 6B-G).

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Figure 6. Increased promitogenic gene expression in HNF4α-KO tumors. (A) Western blot analysis of HNF4α, Cyclin D1, and c-Myc using either nuclear proteins (HNF4α) or total liver extracts (all other) of control mice, control mice treated with DEN, normal liver tissue of HNF4α-KO treated with DEN, and tumor tissue of HNF4α-KO treated with DEN. (B-G) Real-time PCR analysis of putative negative targets of HNF4α identified by combined RNA-seq-ChIP-seq bioinformatics analysis.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

HNF4α is known as the master regulator of hepatocyte differentiation because it regulates many hepatocyte-specific genes involved in bile acid metabolism, drug metabolism, blood coagulation, and lipid metabolism. Recent studies suggest a novel function of HNF4α in the regulation of hepatocyte proliferation and indicate that HNF4α may actively inhibit hepatocyte proliferation; however, the mechanisms of HNF4α-mediated inhibition of hepatocyte proliferation are not known. In this study we explored the mechanisms by which HNF4α inhibits hepatocyte proliferation using a novel mouse model combined with RNA sequencing.

Previous models of HNF4α deletion, produced using constitutively active albumin-cre, result in deletion of HNF4α soon after birth, when the liver is still growing and differentiating, leading to early postnatal lethality.4 This makes it difficult to distinguish the role of HNF4α in the regulation of hepatocyte differentiation from its role in the regulation of cell proliferation. We have independently developed a novel model of HNF4α deletion using a TAM inducible albumin-cre, first described by Bonzo et al.17 In this model, HNF4α is deleted in 3-month-old male mice by activating albumin-cre using TAM treatment, which allows us to study the role of HNF4α in adult, fully mature livers. Our study clearly indicates that deletion of HNF4α in adult mouse liver results in initiation of hepatocyte proliferation and leads to an increase in liver/body weight ratio as early as 1 week following deletion of HNF4α. Because we could not detect any liver injury either biochemically or histologically, the increase in cell proliferation is not a compensatory regeneration in response to injury; however, we did observe biochemical changes in liver following HNF4α deletion. Hepatocytes in normal liver store significant amounts of glycogen, but hepatocytes in HNF4α-KO mice exhibited a decrease in glycogen and a substantial increase in hepatic fat content. These data are reflective of the metabolic changes induced in the liver due to a lack of HNF4α, which regulates many of the genes involved in glycogen synthesis (Gys2)26 and lipid transport (Apoa2, Apoa4, Apob, Apoc2, Apoc3, and MTP) and are consistent with the observations made by Bonzo et al.17

To determine the mechanisms by which HNF4α inhibits hepatocyte proliferation, we performed a global gene expression study using Illumina Hiseq2000-based RNA sequencing combined with functional and pathway analysis. A majority of the genes down-regulated after HNF4αdeletion are previously known HNF4α targets, mainly involved in hepatic differentiation. Interestingly, many of the genes up-regulated are involved in cell cycle control and cancer (Table 2). IPA-mediated functional analysis reveals that the major classes of genes changed following HNF4α deletion are in the cancer and cell proliferation category. The up-regulation of promitogenic genes explains the significant increase in proliferation within the liver of HNF4α-KO mice. This observation also raises questions regarding the mechanism by which HNF4α is regulating promitogenic genes. Whereas beyond the scope of this study, a closer look at the up-regulated genes in HNF4α-KO mice raises the possibility that HNF4α inhibits hepatocyte proliferation by way of both direct and indirect inhibition for select subpopulations of genes.

Bonzo et al.17 first reported the observation that deletion of HNF4α results in an increase in hepatocyte proliferation due to an increase in promitogenic gene expression. The data obtained in this study further confirmed that HNF4α inhibits proliferation through the inhibition of genes involved in cell cycle control. Analysis within the Bonzo et al. study was performed 19 days following initial TAM exposure. Our study strengthens their findings by showing that hepatocyte proliferation and changes in promitogenic gene expression occur as early as 7 days after HNF4α deletion. This suggests that the increased promitogenic gene expression and hepatocyte proliferation may be due directly to the loss of HNF4α as opposed to another factor that HNF4α may regulate. We have recently made similar observation using an adeno-associated virus 8-mediated Cre system.19

Our analysis revealed that a large number of the genes up-regulated after HNF4α deletion are regulated by c-Myc. The RNA-Seq data showed a 3.8-fold increase in c-Myc gene expression, corroborating these results. Previous studies have indicated that HNF4α competes with c-Myc for binding on the promoter of cell cycle inhibitor p21/WAF1.27 Further analysis revealed that several genes up-regulated in the c-Myc gene network are involved in stimulation of cell proliferation and cancer pathogenesis including the set oncoprotein, fus, ccnb1, and ccnb2. These data indicate that HNF4α may indirectly down-regulate these genes by way of suppressing c-Myc activation in normal adult hepatocytes.

It has been speculated that deletion of HNF4α will result in rapid liver failure, making it difficult to directly study its role in the pathogenesis of HCC.17 Whether HNF4α deletion itself can result in hepatocarcinogenesis is not known and may be difficult to study due to limitations of the model system; therefore, we decided to investigate whether HNF4α deletion can promote existing tumors in the liver and can be tested using the two-stage DEN-induced chemical carcinogenesis model. Our studies indicate that HNF4α deletion during the late stage of HCC progression can substantially promote DEN-induced hepatic tumor formation. Our results show that deletion of HNF4α in mice treated with DEN results in a vast expansion in tumor size and tumor number. Furthermore, the tumors, as well as surrounding tissues in HNF4α-KO mice showed extensive up-regulation of c-Myc and Cyclin D1. These data further support the hypothesis that HNF4α inhibits hepatocyte proliferation by inhibiting the c-Myc gene network.

RNA-seq analysis revealed several up-regulated genes, which are potentially negatively regulated by HNF4α. A few of these genes have a putative HNF4α binding site on their promoter and may be targets of direct inhibition by HNF4α (Ect2 and Cdc20), one of which we have confirmed in previous studies using ChIP (Ect2)19; however, a vast number of the up-regulated genes do not have an HNF4α binding site, including Cyclin D1 and c-Myc, and direct regulation of these genes at the level of transcription is unlikely. It is possible that HNF4α may regulate these genes indirectly by way of an intermediary pathway, or by way of microRNAs (miRNAs), as shown by Hatziapostolou et al.28 They provide evidence of an “HNF4α circuit” involving miR-124, IL6R, STAT3, and miR-24/miR-629 in the regulation of hepatocarcinogenesis. They show a correlation between the down-regulation of HNF4α and miR-24 and an up-regulation of IL6R and STAT3 associated with the progression of HCC. We cannot comment on the expression of miRs in our model at this time, but we do not observe an increase in IL6R or STAT3. This may be due to a lack of inflammatory responses within our model, which may be a mediating event in the activation of the “HNF4α circuit.” With this said, it is still very much a possibility that HNF4α is regulating many of the gene expression changes that we observe by an indirect mechanism involving miRNAs.

Taken together, our data indicate that HNF4α is not only an important factor in the regulation of hepatocyte differentiation, but also critical for inhibition of hepatic proliferation. Our study sheds light on the mechanism of HNF4α-mediated inhibition of cell proliferation and indicates that HNF4α inhibits hepatocyte proliferation by down-regulation of promitogenic genes such as c-Myc. These data suggest a novel role as a tumor suppressor and highlight HNF4α as a potential therapeutic target, as well as a prognostic marker, for liver cancers.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

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