Liver-enriched transcription factor expression relates to chronic hepatic failure in humans.
ABSTRACT: The mechanisms by which the liver fails in end-stage liver disease remain elusive. Disruption of the transcription factor network in hepatocytes has been suggested to mediate terminal liver failure in animals. However, this hypothesis remains unexplored in human subjects. To study the relevance of transcription factor expression in terminal stages of chronic liver failure in humans, we analyzed the expression of liver-enriched transcription factors (LETFs) hepatocyte nuclear factor (HNF)4?, HNF1?, forkhead box protein A2 (FOXA2), CCAAT/enhancer-binding protein (CEBP)?, and CEBP?. We then selected downstream genes responsible for some hepatic functions (ornithine transcarbamylase [OTC], cytochrome P450 3A4 [CYP3A4], coagulation factor VII [F7], cadherin 1 [CDH1], phospho-ezrin (Thr567)/radixin (Thr564)/moesin (Thr558) [p-ERM], phospho-myosin light chain [p-MLC], low-density lipoprotein receptor-related protein 1 [LRP1]) in liver tissue from patients at different stages of decompensated liver function based upon Child-Pugh classification, Model for End-Stage Liver Disease score, and degree of inflammatory activity/fibrosis. We first examined differential expression of LETF and determined whether a relationship exists between transcript and protein expression, and liver function. We found HNF4? expression was down-regulated and correlated well with the extent of liver dysfunction (P = 0.001), stage of fibrosis (P = 0.0005), and serum levels of total bilirubin (P = 0.009; r = 0.35), albumin (P < 0.001; r = 0.52), and prothrombin time activity (P = 0.002; r = 0.41). HNF4? expression also correlated with CYP3A4, OTC, and F7 as well as CDH1 RNA levels. The Rho/Rho-associated protein kinase pathways, which have been implicated in the regulation of HNF4?, were also differentially expressed, in concert with LRP1, a reported upstream regulator of RhoA function. Conclusion: HNF4? and other members of the LETFs appear to be important regulators of hepatocyte function in patients with chronic hepatic failure. (Hepatology Communications 2018;2:582-594).
Project description:Alcoholic hepatitis (AH) is a life-threatening condition characterized by profound hepatocellular dysfunction for which targeted treatments are urgently needed. Identification of molecular drivers is hampered by the lack of suitable animal models. By performing RNA sequencing in livers from patients with different phenotypes of alcohol-related liver disease (ALD), we show that development of AH is characterized by defective activity of liver-enriched transcription factors (LETFs). TGF?1 is a key upstream transcriptome regulator in AH and induces the use of HNF4? P2 promoter in hepatocytes, which results in defective metabolic and synthetic functions. Gene polymorphisms in LETFs including HNF4? are not associated with the development of AH. In contrast, epigenetic studies show that AH livers have profound changes in DNA methylation state and chromatin remodeling, affecting HNF4?-dependent gene expression. We conclude that targeting TGF?1 and epigenetic drivers that modulate HNF4?-dependent gene expression could be beneficial to improve hepatocellular function in patients with AH.
Project description:Liver-enriched transcription factors (LETF) play a crucial role in the control of liver-specific gene expression and for hepatocytes to retain their molecular and cellular functions complex interactions with extra cellular matrix (ECM) are required However, during cell isolation ECM interactions are disrupted and for hepatocytes to regain metabolic competency cells are cultured on ECM substrata. The regulation of LETFs in hepatocytes cultured on different ECM has not been studied in detail. We therefore compared two common sources of ECM and evaluated cellular morphology and hepatocyte differentiation by investigating DNA binding activity of LETFs at gene specific promoters and marker genes of hepatic metabolism. Furthermore, we studied testosterone metabolism and albumin synthesis to assess the metabolic competence of cell cultures. Despite significant difference in morphological appearance and except for HNF1? (p<0.001) most LETFs and several of their target genes did not differ in transcript expression after Bonferroni adjustment when cultured on collagen or Matrigel. Nonetheless, Western blotting revealed HNF1?, HNF3?, HNF3?, HNF4?, HNF6 and the smaller subunits of C/EBP? and C/EBP? to be more abundant on Matrigel cultured cells. Likewise, DNA binding activity of HNF3?, HNF3?, HNF4?, HNF6 and gene expression of hepatic lineage markers were increased on Matrigel cultured hepatocytes. To further investigate hepatic gene regulation, the effects of Aroclor 1254 treatment, e.g. a potent inducer of xenobiotic defense were studied in vivo and in vitro. The gene expression of C/EBP-? increased in rat liver and hepatocytes cultured on collagen and this treatment induced DNA binding activity of HNF4?, C/EBP? and C/EBP? and gene expression of CYP1A1 and CYP1A2 in vivo and in vitro. Taken collectively, two sources of ECM greatly affected hepatocyte morphology, activity of liver enriched transcription factors, hepatic gene expression and metabolic competency that should be considered when used in cell biology studies and drug toxicity testing.
Project description:microRNA (miRNA) mediated regulation of gene expression has emerged as a significant mechanism contributing to variation in gene expression. In this study, we evaluated the potential role of miRNAs in regulating expression of hepatic cytochromes P450 and their transcriptional regulatory genes. We screened the Targetscan database for high scoring miRNA binding site predictions in selected hepatic DMEs and transcription factors. Expression profiling for candidate miRNAs (n=22) and their target genes (n=20) was performed in 50 human liver samples (25 female, 25 male). Significant negative correlations were observed between expression levels of several CYPs/hepatic transcription factors and the hepatic miRNAs studied. Interestingly, hepatic miR-34a demonstrated significant negative correlation with expression levels of multiple hepatic transcription factors (including NR1I2 and HNF4?) and DMEs (CYP3A4, CYP2C19). miR-34a expression was also significantly higher in males than in females in congruence with previous observations of higher CYP3A4 expression in females versus males. A mediation analysis revealed that miR-34a was involved in significant mediation of the association observed between CYP2C19 and several hepatic transcription factors (HNF4?, NR1I2). miR-34a may thus play a key regulatory role and be a key contributory factor to the inter-individual variability observed in expression of key drug metabolizing genes in humans.
Project description:Levels of enzymes that determine testosterone catabolism such as CYP3A4 have been associated with prostate cancer (PCa) risk. Although some studies have related CYP3A4*1B allele, a gene polymorphism that modifies CYP3A4 expression level, with PCa risk, others have failed, suggesting that additional genetic variants may be involved. Expression of CYP3A4 is largely due to the activation of Pregnane X Receptor (PXR). Particularly, rs2472677 and rs7643645 PXR polymorphisms modify CYP3A4 expression levels. To evaluate whether PXR-HNF3?/T (rs2472677), PXR-HNF4/G (rs7643645), and CYP3A4*1B (rs2740574) polymorphisms are associated with PCa a case control-study was performed. The multiple testing analysis showed that the PXR-HNF4/G polymorphism was associated with higher levels of prostate-specific antigen (PSA) in patients with PCa (OR?=?3.99, p?=?0.03). This association was stronger in patients diagnosed at the age of 65 years or older (OR?=?10.8, p?=?0.006). Although the CYP3A4*1B/*1B genotype was overrepresented in PCa patients, no differences were observed in the frequency of this and PXR-HNF3?/T alleles between controls and cases. Moreover, no significant association was found between these polymorphisms and PSA, Gleason grade, or tumor lymph node metastasis.
Project description:The ornithine transcarbamylase (OTC) gene is on the X chromosome and its product catalyzes the formation of citrulline from ornithine and carbamylphosphate in the urea cycle. About 10%-15% of patients, clinically diagnosed with OTC deficiency (OTCD), lack identifiable mutations in the coding region or splice junctions of the OTC gene on routine molecular testing. We collected DNA from such patients via retrospective review and by prospective enrollment. In nine of 38 subjects (24%), we identified a sequence variant in the OTC regulatory regions. Eight subjects had unique sequence variants in the OTC promoter and one subject had a novel sequence variant in the OTC enhancer. All sequence variants affect positions that are highly conserved in mammalian OTC genes. Functional studies revealed reduced reporter gene expression with all sequence variants. Two sequence variants caused decreased binding of the HNF4 transcription factor to its mutated binding site. Bioinformatic analyses combined with functional assays can be used to identify and authenticate pathogenic sequence variants in regulatory regions of the OTC gene, in other urea cycle disorders or other inborn errors of metabolism.
Project description:The in vitro generation of terminally differentiated hepatocytes is an unmet need. We investigated the contribution of oxygen concentration to differentiation in human liver cell lines HepaRG and C3A. HepaRG cells were cultured under hypoxia (5%O2), normoxia (21%O2) or hyperoxia (40%O2). Cultures were analysed for hepatic functions, gene transcript levels, and protein expression of albumin, hepatic transcription factor CEBP?, hepatic progenitor marker SOX9, and hypoxia inducible factor (HIF)1?. C3A cells were analysed after exposure to normoxia or hyperoxia. In hyperoxic HepaRG cultures, urea cycle activity, bile acid synthesis, CytochromeP450 3A4 (CYP3A4) activity and ammonia elimination were 165-266% increased. These effects were reproduced in C3A cells. Whole transcriptome analysis of HepaRG cells revealed that 240 (of 23.223) probes were differentially expressed under hyperoxia, with an overrepresentation of genes involved in hepatic differentiation, metabolism and extracellular signalling. Under hypoxia, CYP3A4 activity and ammonia elimination were inhibited almost completely and 5/5 tested hepatic genes and 2/3 tested hepatic transcription factor genes were downregulated. Protein expression of SOX9 and HIF1? was strongly positive in hypoxic cultures, variable in normoxic cultures and predominantly negative in hyperoxic cultures. Conversely, albumin and CEBP? expression were highest in hyperoxic cultures. HepaRG cells that were serially passaged under hypoxia maintained their capacity to differentiate under normoxia, in contrast to cells passaged under normoxia. Hyperoxia increases hepatocyte differentiation in HepaRG and C3A cells. In contrast, hypoxia maintains stem cell characteristics and inhibits hepatic differentiation of HepaRG cells, possibly through the activity of HIF1?.
Project description:Metabolic diseases result from multiple genetic and environmental factors. We report here that one manner in which environmental factors can contribute to metabolic disease progression is through modification to chromatin. We demonstrate that high fat diet leads to chromatin remodeling in the livers of C57BL/6J mice, as compared with mice fed a control diet, and that these chromatin changes are associated with changes in gene expression. We further show that the regions of greatest variation in chromatin accessibility are targeted by liver transcription factors, including HNF4?, CCAAT/enhancer-binding protein ? (CEBP/?), and FOXA1. Repeating the chromatin and gene expression profiling in another mouse strain, DBA/2J, revealed that the regions of greatest chromatin change are largely strain-specific and that integration of chromatin, gene expression, and genetic data can be used to characterize regulatory regions. Our data indicate dramatic changes in the epigenome due to diet and demonstrate strain-specific dynamics in chromatin remodeling.
Project description:Hepatocyte nuclear factor 4 alpha (HNF4?) is a master regulator of liver-specific gene expression with potent tumor suppressor activity, yet many liver tumors express HNF4?. This study reveals that P1-HNF4?, the predominant isoform expressed in the adult liver, inhibits expression of tumor promoting genes in a circadian manner. In contrast, an additional isoform of HNF4?, driven by an alternative promoter (P2-HNF4?), is induced in HNF4?-positive human hepatocellular carcinoma (HCC). P2-HNF4? represses the circadian clock gene ARNTL (BMAL1), which is robustly expressed in healthy hepatocytes, and causes nuclear to cytoplasmic re-localization of P1-HNF4?. We reveal mechanisms underlying the incompatibility of BMAL1 and P2-HNF4? in HCC, and demonstrate that forced expression of BMAL1 in HNF4?-positive HCC prevents the growth of tumors in vivo. These data suggest that manipulation of the circadian clock in HNF4?-positive HCC could be a tractable strategy to inhibit tumor growth and progression in the liver.
Project description:Hepatocyte nuclear factor 4? (HNF4?) is a master regulator of liver function and a tumor suppressor in hepatocellular carcinoma (HCC). In this study, we explore the reciprocal negative regulation of HNF4? and cyclin D1, a key cell cycle protein in the liver. Transcriptomic analysis of cultured hepatocyte and HCC cells found that cyclin D1 knockdown induced the expression of a large network of HNF4?-regulated genes. Chromatin immunoprecipitation-sequencing (ChIP-seq) demonstrated that cyclin D1 inhibits the binding of HNF4? to thousands of targets in the liver, thereby diminishing the expression of associated genes that regulate diverse metabolic activities. Conversely, acute HNF4? deletion in the liver induces cyclin D1 and hepatocyte cell cycle progression; concurrent cyclin D1 ablation blocked this proliferation, suggesting that HNF4? maintains proliferative quiescence in the liver, at least, in part, via repression of cyclin D1. Acute cyclin D1 deletion in the regenerating liver markedly inhibited hepatocyte proliferation after partial hepatectomy, confirming its pivotal role in cell cycle progression in this in vivo model, and enhanced the expression of HNF4? target proteins. Hepatocyte cyclin D1 gene ablation caused markedly increased postprandial liver glycogen levels (in a HNF4?-dependent fashion), indicating that the cyclin D1-HNF4? axis regulates glucose metabolism in response to feeding. In AML12 hepatocytes, cyclin D1 depletion led to increased glucose uptake, which was negated if HNF4? was depleted simultaneously, and markedly elevated glycogen synthesis. To summarize, mutual repression by cyclin D1 and HNF4? coordinately controls the cell cycle machinery and metabolism in the liver.
Project description:Hepatocyte nuclear factor 4 alpha (HNF4?) is a transcription factor that plays a critical role in hepatocyte function, and HNF4?-based reprogramming corrects terminal liver failure in rats with chronic liver disease. In the livers of patients with advanced cirrhosis, HNF4? RNA expression levels decrease as hepatic function deteriorates, and protein expression is found in the cytoplasm. These findings could explain impaired hepatic function in patients with degenerative liver disease. In this study, we analyzed HNF4? localization and the pathways involved in post-translational modification of HNF4? in human hepatocytes from patients with decompensated liver function. RNA-sequencing analysis revealed that AKT-related pathways, specifically phospho-AKT, is down-regulated in cirrhotic hepatocytes from patients with terminal failure, in whom nuclear levels of HNF4? were significantly reduced, and cytoplasmic expression of HNF4? was increased. cMET was also significantly reduced in failing hepatocytes. Moreover, metabolic profiling showed a glycolytic phenotype in failing human hepatocytes. The contribution of cMET and phospho-AKT to nuclear localization of HNF4? was confirmed using Spearman's rank correlation test and pathway analysis, and further correlated with hepatic dysfunction by principal component analysis. HNF4? acetylation, a posttranslational modification important for nuclear retention, was also significantly reduced in failing human hepatocytes when compared with normal controls. Conclusion: These results suggest that the alterations in the cMET-AKT pathway directly correlate with HNF4? localization and level of hepatocyte dysfunction. This study suggests that manipulation of HNF4? and pathways involved in HNF4? posttranslational modification may restore hepatocyte function in patients with terminal liver failure.