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1.
Drug Metab Dispos ; 50(5): 704-715, 2022 05.
Article in English | MEDLINE | ID: mdl-34949673

ABSTRACT

Ritonavir (RTV), a pharmacoenhancer used in anti-HIV regimens, can induce liver damage. RTV is primarily metabolized by cytochrome P450 3A4 (CYP3A4) in the liver. HNF4A antisense RNA 1 (HNF4A-AS1) and HNF1A antisense RNA 1 (HNF1A-AS1) are long noncoding RNAs that regulate the expression of pregnane X receptor (PXR) and CYP3A4. This study investigated the role and underlying mechanisms of HNF4A-AS1 and HNF1A-AS1 in RTV-induced hepatotoxicity. HNF4A-AS1 and HNF1A-AS1 were knocked down by small hairpin RNAs in Huh7 and HepG2 cells. Lactate dehydrogenase and reactive oxygen species assays were performed to assess RTV-induced hepatotoxicity. Chromatin immunoprecipitation quantitative real-time polymerase chain reaction was used to detect PXR enrichment and histone modifications in the CYP3A4 promoter. HNF4A-AS1 knockdown increased PXR and CYP3A4 expression and exacerbated RTV-induced cytotoxicity, whereas HNF1A-AS1 knockdown generated the opposite phenotype. Mechanistically, enrichment of PXR and trimethylation of histone 3 lysine 4 (H3K4me3) in the CYP3A4 promoter was increased, and trimethylation of histone 3 lysine 27 (H3K27me3) was decreased after HNF4A-AS1 knockdown. However, PXR and H3K4me3 enrichment decreased after HNF1A-AS1 knockdown. Alterations in RTV-induced hepatotoxicity caused by decreasing HNF4A-AS1 or HNF1A-AS1 were reversed by knockdown or overexpression of PXR. Increased susceptibility to RTV-induced liver injury caused by the PXR activator rifampicin was attenuated by HNF4A-AS1 overexpression or HNF1A-AS1 knockdown. Taken together, these results revealed that HNF4A-AS1 and HNF1A-AS1 modulated RTV-induced hepatotoxicity by regulating CYP3A4 expression, primarily by affecting the binding of PXR and histone modification status in the CYP3A4 promoter. SIGNIFICANCE STATEMENT: HNF4A-AS1 and HNF1A-AS1, transcribed separately from neighboring antisense genes of the human transcription factor genes HNF4A and HNF1A, were identified as long noncoding RNAs that can affect RTV-induced hepatotoxicity and susceptibility to RTV-induced hepatotoxicity caused by rifampicin exposure, mainly by affecting the expression of CY3A4 via alterations in PXR enrichment and histone modification status in the CYP3A4 promoter. This discovery provides directions for further research on the mechanisms of RTV-induced liver injury.


Subject(s)
Carcinoma, Hepatocellular , Chemical and Drug Induced Liver Injury, Chronic , Liver Neoplasms , RNA, Long Noncoding , Receptors, Steroid , Carcinoma, Hepatocellular/genetics , Cytochrome P-450 CYP3A/genetics , Cytochrome P-450 CYP3A/metabolism , Hepatocyte Nuclear Factor 1/metabolism , Hepatocyte Nuclear Factor 4/genetics , Hepatocyte Nuclear Factor 4/metabolism , Histones/metabolism , Humans , Liver Neoplasms/genetics , Lysine , RNA, Antisense/genetics , RNA, Long Noncoding/genetics , Receptors, Steroid/metabolism , Rifampin/toxicity , Ritonavir/toxicity
2.
Sci Rep ; 10(1): 9999, 2020 06 19.
Article in English | MEDLINE | ID: mdl-32561878

ABSTRACT

Brief refeeding times (~60 min) enhanced hepatic Angptl8 expression in fasted mice. We cloned the mouse Angptl8 promoter region to characterise this rapid refeeding-induced increase in hepatic Angptl8 expression. Deletion of the -309/-60 promoter region significantly attenuated basal promoter activity in hepatocytes. A computational motif search revealed a potential binding motif for hepatocyte nuclear factor 1α/1ß (HNF-1α/ß) at -84/-68 bp of the promoter. Mutation of the HNF-1 binding site significantly decreased the promoter activity in hepatocytes, and the promoter carrying the mutated HNF-1 site was not transactivated by co-transfection of HNF-1 in a non-hepatic cell line. Silencing Hnf-1 in hepatoma cells and mouse primary hepatocytes reduced Angptl8 protein levels. Electrophoretic mobility-shift assays confirmed direct binding of Hnf-1 to its Angptl8 promoter binding motif. Hnf-1α expression levels increased after short-term refeeding, paralleling the enhanced in vivo expression of the Angptl8 protein. Chromatin immunoprecipitation (ChIP) confirmed the recruitment of endogenous Hnf-1 to the Angptl8 promoter region. Insulin-treated primary hepatocytes showed increased expression of Angptl8 protein, but knockdown of Hnf-1 completely abolished this enhancement. HNF-1 appears to play essential roles in the rapid refeeding-induced increases in Angptl8 expression. HNF-1α may therefore represent a primary medical target for ANGPTL8-related metabolic abnormalities. The study revealed the transcriptional regulation of the mouse hepatic Angptl8 gene by HNF-1.


Subject(s)
Angiopoietin-like Proteins/genetics , Gene Expression Regulation , Hepatocyte Nuclear Factor 1/genetics , Liver/metabolism , Transcription, Genetic/genetics , Angiopoietin-Like Protein 8 , Angiopoietin-like Proteins/metabolism , Animals , Cell Line , Chromatin Immunoprecipitation , Hepatocyte Nuclear Factor 1/metabolism , Hepatocytes/metabolism , Mice , Promoter Regions, Genetic
3.
J Appl Toxicol ; 39(12): 1640-1650, 2019 12.
Article in English | MEDLINE | ID: mdl-31385618

ABSTRACT

Acetaminophen (APAP) is an antipyretic and analgesic, which is commonly associated with drug-induced hepatic injury. C2-ceramide plays a key role in mediating cell life activities, and oltipraz was extensively studied as a cancer chemopreventive agent. Glutathione S-transferase A1 (GSTA1) acts as a vital liver detoxification enzyme. Hepatocyte nuclear factor 1 (HNF-1) regulates various cellular signaling pathways. In this study, we investigated the effects of C2-ceramide and oltipraz on APAP-induced hepatocyte injury and the changes of HNF-1 and GSTA1. Results showed that C2-ceramide (6 µmol/L) exacerbated APAP-induced hepatocyte injury and caused a significant decrease (P < .01) in HNF-1 and GSTA1 expressions. Meanwhile, GSTA1 content in supernatant was significantly increased (P < .01). In contrast, oltipraz (8 µmol/L) reduced the injury and significantly elevated (P < .01) HNF-1 and GSTA1 expressions while GSTA1 content in supernatant was significantly decreased (P < .01). In conclusion, these findings revealed that C2-ceramide inhibited HNF-1 and GSTA1 expression and exacerbated hepatocyte injury, while oltipraz treatment results in the reduction of hepatocyte injury, and promoted HNF-1 and GSTA1 expression. Additionally, the changes in HNF-1 and GSTA1 were related to APAP-induced hepatocyte injury. These results were useful to investigate the mechanism of an antipyretic and analgesic drug combination.


Subject(s)
Acetaminophen/toxicity , Analgesics, Non-Narcotic/toxicity , Glutathione Transferase/metabolism , Hepatocyte Nuclear Factor 1/metabolism , Hepatocytes/drug effects , Pyrazines/pharmacology , Sphingosine/analogs & derivatives , Antioxidants/metabolism , Cell Culture Techniques , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Combinations , Drug Interactions , Gene Expression/drug effects , Glutathione Transferase/genetics , Hep G2 Cells , Hepatocyte Nuclear Factor 1/genetics , Hepatocytes/metabolism , Hepatocytes/pathology , Humans , Sphingosine/pharmacology , Thiones , Thiophenes
4.
Andrology ; 7(5): 741-747, 2019 09.
Article in English | MEDLINE | ID: mdl-31050198

ABSTRACT

BACKGROUND: The epithelial lining of the human epididymis is critical for sperm maturation. This process requires distinct specialized functions in the head, body, and tail of the duct. These region-specific properties are maintained by distinct gene expression profiles which are governed by transcription factor networks, non-coding RNAs, and other factors. MATERIALS AND METHODS: We used genome-wide protocols including DNase-seq, RNA-seq and ChIP-seq to characterize open (active) chromatin, the transcriptome and occupancy of specific transcription factors (TFs) respectively, in caput, corpus, and cauda segments of adult human epididymis tissue and primary human epididymis epithelial (HEE) cell cultures derived from them. RNA-seq following TF depletion or activation, combined with gene ontology analysis also determined TF targets. RESULTS: Among regional differentially expressed transcripts were epithelial-selective transcription factors (TFs), microRNAs, and antiviral response genes. Caput-enriched TFs included hepatocyte nuclear factor 1 (HNF1) and the androgen receptor (AR), both of which were also predicted to occupy cis-regulatory elements identified as open chromatin in HEE cells. HNF1 targets were identified genome-wide using ChIP-seq, in HEE cells. Next, siRNA-mediated depletion of HNF1 revealed a pivotal role for this TF in coordinating epithelial water and solute transport in caput epithelium. The importance of AR in HEE cells was shown by AR ChIP-seq, and by RNA-seq after synthetic androgen (R1881) treatment. AR has a distinct transcriptional program in the HEE cells and likely recruits different co-factors (RUNX1 and CEBPß) in comparison to those used in prostate epithelium. DISCUSSION AND CONCLUSION: Our data identify many transcription factors that regulate the development and differentiation of HEE cells. Moreover, a comparison between immature and adult HEE cells showed key TFs in the transition to fully differentiated function of this epithelium. These data may help identify new targets to treat male infertility and have the potential to open new avenues for male contraception.


Subject(s)
Computational Biology/methods , Epididymis/metabolism , Gene Expression Regulation/genetics , Gene Regulatory Networks/genetics , Gene Expression Profiling , Hepatocyte Nuclear Factor 1/metabolism , Humans , Male , Sperm Maturation/genetics , Transcriptome/genetics
5.
Cardiovasc Res ; 115(11): 1646-1658, 2019 09 01.
Article in English | MEDLINE | ID: mdl-30715251

ABSTRACT

AIMS: We previously reported that sodium-dependent glucose cotransporter 1 (SGLT1) is highly expressed in cardiomyocytes and is further up-regulated in ischaemia. This study aimed to determine the mechanisms by which SGLT1 contributes to ischaemia/reperfusion (I/R) injury. METHODS AND RESULTS: Mice with cardiomyocyte-specific knockdown of SGLT1 (TGSGLT1-DOWN) and wild-type controls were studied. In vivo, the left anterior descending coronary artery was ligated for 30 min and reperfused for 48 h. Ex vivo, isolated perfused hearts were exposed to 20 min no-flow and up to 2 h reperfusion. In vitro, HL-1 cells and isolated adult murine ventricular cardiomyocytes were exposed to 1 h hypoxia and 24 h reoxygenation (H/R). We found that TGSGLT1-DOWN hearts were protected from I/R injury in vivo and ex vivo, with decreased infarct size, necrosis, dysfunction, and oxidative stress. 5'-AMP-activated protein kinase (AMPK) activation increased SGLT1 expression, which was abolished by extracellular signal-related kinase (ERK) inhibition. Co-immunoprecipitation studies showed that ERK, but not AMPK, interacts directly with SGLT1. AMPK activation increased binding of the hepatocyte nuclear factor 1 and specificity protein 1 transcription factors to the SGLT1 gene, and HuR to SGLT1 mRNA. In cells, up-regulation of SGLT1 during H/R was abrogated by AMPK inhibition. Co-immunoprecipitation studies showed that SGLT1 interacts with epidermal growth factor receptor (EGFR), and EGFR interacts with protein kinase C (PKC). SGLT1 overexpression activated PKC and NADPH oxidase 2 (Nox2), which was attenuated by PKC inhibition, EGFR inhibition, and/or disruption of the interaction between EGFR and SGLT1. CONCLUSION: During ischaemia, AMPK up-regulates SGLT1 through ERK, and SGLT1 interacts with EGFR, which in turn increases PKC and Nox2 activity and oxidative stress. SGLT1 may represent a novel therapeutic target for mitigating I/R injury.


Subject(s)
Myocardial Infarction/metabolism , Myocardial Reperfusion Injury/metabolism , Myocytes, Cardiac/metabolism , Sodium-Glucose Transporter 1/metabolism , AMP-Activated Protein Kinases/metabolism , Animals , Cell Line , Disease Models, Animal , ELAV-Like Protein 1/metabolism , ErbB Receptors/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Hepatocyte Nuclear Factor 1/metabolism , Male , Mice, Knockout , Myocardial Infarction/genetics , Myocardial Infarction/pathology , Myocardial Infarction/physiopathology , Myocardial Reperfusion Injury/genetics , Myocardial Reperfusion Injury/pathology , Myocardial Reperfusion Injury/physiopathology , Myocytes, Cardiac/pathology , NADPH Oxidase 2/metabolism , NADPH Oxidases/metabolism , Necrosis , Oxidative Stress , Protein Kinase C/metabolism , Signal Transduction , Sodium-Glucose Transporter 1/deficiency , Sodium-Glucose Transporter 1/genetics
6.
Biochem Biophys Res Commun ; 495(2): 1758-1765, 2018 01 08.
Article in English | MEDLINE | ID: mdl-29233692

ABSTRACT

The chipmunk hibernation-related proteins (HPs) HP-20 and HP-27 are components of a 140-kDa complex that dramatically decreases in the blood during hibernation. The HP-20 and HP-27 genes are expressed specifically in the liver and are downregulated in hibernating chipmunks. Hibernation-associated physiological changes are assumed to be under genetic control. Therefore, to elucidate the molecular mechanisms of hibernation, here we examined the mechanisms behind the altered HP-20 and HP-27 gene expression in nonhibernating versus hibernating chipmunks. Chromatin immunoprecipitation (ChIP) analyses revealed that histone H3 on the HP-20 and HP-27 gene promoters was highly acetylated at lysine (K) 9 and K14 and highly trimethylated at K4 in the liver of nonhibernating chipmunks, while these active histone modifications were nearly absent in hibernating chipmunks. Furthermore, histone acetyltransferases and a histone methyltransferase were associated with the HP-20 and HP-27 gene promoters primarily in nonhibernating chipmunks. Consistent with a previous finding that HNF-1 and USF can activate HP-20 and HP-27 gene transcription by binding to the proximal promoter region, ChIP-quantitative PCR (qPCR) analyses revealed that significantly less HNF-1 and USF were bound to these gene promoters in hibernating than in nonhibernating chipmunks. These findings collectively indicated that the hibernation-associated HP-20 and HP-27 gene expression is epigenetically regulated at the transcriptional level by the binding of HNF-1 and USF to their proximal promoters, and that histone modification has a key role in hibernation-associated transcriptional regulation.


Subject(s)
Blood Proteins/genetics , Blood Proteins/physiology , Hibernation/genetics , Hibernation/physiology , Sciuridae/genetics , Sciuridae/physiology , Animals , Base Sequence , Epigenesis, Genetic , Gene Expression , Hepatocyte Nuclear Factor 1/metabolism , Histones/metabolism , Male , Promoter Regions, Genetic , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription, Genetic , Upstream Stimulatory Factors/metabolism
7.
Int J Mol Med ; 39(3): 749-756, 2017 Mar.
Article in English | MEDLINE | ID: mdl-28204827

ABSTRACT

Proprotein convertase subtilisin/kexin type 9 (PCSK9) impedes low­density lipoprotein (LDL) receptor (LDLR)-mediated LDL-cholesterol uptake and has hence emerged as a critical regulator of serum cholesterol levels and a new therapeutic target for the treatment of hypercholesterolemia. Statins have been shown to elevate circulating PCSK9 levels by stimulating PCSK9 gene transcription, which reduces the clinical efficacy of statin in LDL­cholesterol reduction. The transcription of PCSK9 is partially controlled by the hepatocyte nuclear factor 1 (HNF1) binding site embedded in the proximal region of its promoter. In this study, we utilized adenoviral shRNA delivery vectors to generate liver-specific knockdown of HNF1α (Ad­shHNF1α) or HNF1ß (Ad­shHNF1ß) in hamsters to examine the impact of reduced hepatic expression of HNF1 transcription factors on statin­induced elevation of PCSK9 expression and serum cholesterol levels. We showed that the administration of rosuvastatin (RSV) to normolipidemic hamsters significantly augmented hepatic PCSK9 expression and serum PCSK9 levels. In addition, RSV treatment increased hepatic HNF1α protein levels without a clear effect on HNF1α mRNA expression. Injection of Ad-shHNF1α or Ad­shHNF1ß into hamsters both blunted RSV­induced elevation of PCSK9 serum concentration and hepatic mRNA and protein levels, which led to significant increases in liver LDLR protein abundance. Furthermore, hepatic depletion of HNF1 factors lowered circulating total cholesterol and non­high density lipoprotein cholesterol levels in RSV­treated hamsters. Our study demonstrates that both HNF1α and HNF1ß are positive regulators of hepatic PCSK9 transcription in hamster species and that transient, liver-specific knockdown of either HNF1α or HNF1ß could antagonize the RSV­induced elevation of serum PCSK9 and reduce circulating cholesterol levels.


Subject(s)
Gene Expression Regulation/drug effects , Hepatocyte Nuclear Factor 1/metabolism , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Proprotein Convertase 9/genetics , Rosuvastatin Calcium/pharmacology , Adenoviridae/genetics , Animals , Base Sequence , Cholesterol/blood , Cloning, Molecular , Cricetinae , Gene Knockdown Techniques , Gene Silencing , Genetic Vectors/genetics , Hepatocyte Nuclear Factor 1/genetics , Hepatocyte Nuclear Factor 1-alpha/genetics , Hepatocyte Nuclear Factor 1-alpha/metabolism , Hepatocyte Nuclear Factor 1-beta/genetics , Hepatocyte Nuclear Factor 1-beta/metabolism , Liver/metabolism , Male , Proprotein Convertase 9/blood , Proprotein Convertase 9/metabolism , RNA, Small Interfering/genetics , Receptors, LDL/metabolism , Signal Transduction , Sterol Regulatory Element Binding Proteins/metabolism , Transduction, Genetic
8.
Br Poult Sci ; 58(1): 19-25, 2017 Feb.
Article in English | MEDLINE | ID: mdl-27844479

ABSTRACT

1. Albumin (ALB) is a serum protein most highly expressed in liver and regarded as an effective indicator for liver pathologies. The objectives of this study were to determine the expression of duck ALB gene (duALB) in various non-hepatic tissues and identify the potential cis-regulatory elements in the promoter. 2. A model was established to assess duALB promoter activity in different cell lines by construction of a duALB promoter-driven GFP (Green Fluorescent Protein)-expressing vector, which exhibited high expression activity in liver-derived cells and lower expression in other cells. Through the firefly luciferase reporter gene driven by a series of constructs carrying progressive deletions, the core transcriptional regulatory region within the duALB promoter was identified. Mutations in candidate-binding sites were made by site-directed mutagenesis. 3. The core transcriptional regulatory region was located in the -190/-51 bp region. This region contains three potential transcription factor-binding sites, one each for hepatocyte nuclear factor (HNF-3ß) (-158/-149), CCAAT/Enhancer-binding protein element (C/EBPα) (-119/-107) and nuclear factor-1 (HNF-1) (-67/-57). Site-directed mutagenesis of HNF-1 and C/EBPα-binding sites resulted in a significant reduction in duALB promoter activity. Two potential cis-regulatory elements (C/EBPα and HNF-1) were responsible for its transcriptional activity in liver-derived cells. 4. These findings contribute to the further understanding of the fundamental mechanisms of ALB gene regulation and the use of tissue-specific gene promoters to regulate tissue-specific expression of exogenous genes in vivo.


Subject(s)
Ducks/genetics , Gene Expression/genetics , Promoter Regions, Genetic/genetics , Serum Albumin/genetics , Animals , Binding Sites/genetics , CCAAT-Enhancer-Binding Protein-alpha/metabolism , DNA/metabolism , Gene Expression Regulation/genetics , Genetic Vectors , Green Fluorescent Proteins/genetics , Hepatocyte Nuclear Factor 1/metabolism , Hepatocyte Nuclear Factor 3-beta/metabolism , Liver/metabolism , Mutagenesis, Site-Directed
9.
Mol Cell Endocrinol ; 425: 94-102, 2016 Apr 15.
Article in English | MEDLINE | ID: mdl-26808453

ABSTRACT

The luminal environment of the epididymis participates in sperm maturation and impacts male fertility. It is dependent on the coordinated expression of many genes encoding proteins with a role in epithelial transport. We identified cis-regulatory elements for critical genes in epididymis function, by mapping open chromatin genome-wide in human epididymis epithelial (HEE) cells. Bioinformatic predictions of transcription factors binding to the regulatory elements suggested an important role for hepatocyte nuclear factor 1 (HNF1) in the transcriptional program of these cells. Chromatin immunoprecipitation and deep sequencing (ChIP-seq) revealed HNF1 target genes in HEE cells. In parallel, the contribution of HNF1 to the transcriptome of HEE cells was determined by RNA-seq, following siRNA-mediated depletion of both HNF1α and HNF1ß transcription factors. Repression of these factors caused differential expression of 1892 transcripts (902 were downregulated and 990 upregulated) in comparison to non-targeting siRNAs. Differentially expressed genes with HNF1 ChIP-seq peaks within 20 kb were subject to gene ontology process enrichment analysis. Among the most significant processes associated with down-regulated genes were epithelial transport of water, phosphate and bicarbonate, all critical processes in epididymis epithelial function. Measurements of intracellular pH (pHi) confirmed a role for HNF1 in regulating the epididymis luminal environment.


Subject(s)
Epididymis/metabolism , Gene Regulatory Networks , Hepatocyte Nuclear Factor 1/genetics , Hepatocyte Nuclear Factor 1/metabolism , Regulatory Elements, Transcriptional , Cells, Cultured , Chromatin Immunoprecipitation , Computational Biology/methods , Epididymis/cytology , Gene Expression Regulation , High-Throughput Nucleotide Sequencing , Humans , Male , Transcription, Genetic
10.
J Pharmacol Exp Ther ; 355(3): 429-41, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26427720

ABSTRACT

Cytosolic sulfotransferase 1C2 (SULT1C2) is expressed in the kidney, stomach, and liver of rats; however, the mechanisms regulating expression of this enzyme are not known. We evaluated transcriptional regulation of SULT1C2 by mevalonate (MVA)-derived intermediates in primary cultured rat hepatocytes using several cholesterol synthesis inhibitors. Blocking production of mevalonate with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin (30 µM), reduced SULT1C2 mRNA content by ∼40% whereas the squalene synthase inhibitor squalestatin (SQ1, 0.1 µM), which causes accumulation of nonsterol isoprenoids, increased mRNA content by 4-fold. Treatment with MVA (10 mM) strongly induced SULT1C2 mRNA by 12-fold, and this effect was blocked by inhibiting squalene epoxidase but not by more distal cholesterol inhibitors, indicating the effects of MVA are mediated by postsqualene metabolites. Using rapid amplification of cDNA ends (RACE), we characterized the 5' end of SULT1C2 mRNA and used this information to generate constructs for promoter analysis. SQ1 and MVA increased reporter activity by ∼1.6- and 3-fold, respectively, from a construct beginning 49 base pairs (bp) upstream from the longest 5'-RACE product (-3140:-49). Sequence deletions from this construct revealed a hepatocyte nuclear factor 1 (HNF1) element (-2558), and mutation of this element reduced basal (75%) and MVA-induced (30%) reporter activity and attenuated promoter activation following overexpression of HNF1α or 1ß. However, the effects of SQ1 were localized to a more proximal promoter region (-281:-49). Collectively, our findings demonstrate that cholesterol biosynthetic intermediates influence SULT1C2 expression in rat primary hepatocytes. Further, HNF1 appears to play an important role in mediating basal and MVA-induced SULT1C2 transcription.


Subject(s)
Cholesterol/biosynthesis , Gene Expression Regulation, Enzymologic/physiology , Hepatocytes/enzymology , Sulfotransferases/biosynthesis , Sulfotransferases/genetics , Animals , Anticholesteremic Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Genes, Reporter , Hepatocyte Nuclear Factor 1/genetics , Hepatocyte Nuclear Factor 1/metabolism , Hepatocytes/drug effects , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Male , Mevalonic Acid/pharmacology , Primary Cell Culture , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , Squalene Monooxygenase/antagonists & inhibitors , Sulfotransferases/drug effects , Transfection , Tricarboxylic Acids/pharmacology
11.
Drug Metab Pharmacokinet ; 30(2): 188-97, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25989892

ABSTRACT

Constitutive androstane receptor (CAR) is one of the principal regulators of hepatic cytochrome P450s (CYPs) 3A (CYP3A). cDNA-mediated expression of a mature rat CAR (rCAR) into rat hepatoma cells induced CYP3A1 and CYP2B mRNAs. Aberrant rCAR failed in these inductions. Three important human CYP3A4 regulatory elements (REs), proximal ER6 (proER6), xenobiotic responsive enhancer module (XREM) and constitutive liver enhancer module (CLEM), support constitutive and inducible expression of CYP3As mediated by CAR and pregnane X receptor (PXR). NHR-scan software predicted proER6, XREM and CLEM at -255 b, -8 kb and -11.5 kb, respectively of CYP3A4, but neither XREM nor CLEM was predicted in rat CYP3A. A luciferase reporter construct carrying a 5'-flanking sequence of CYP3A1 (-31,739 to -31,585 from its transcription initiation site) revealed important for the rCAR-dependent transactivation of CYP3A1. This region includes two putative binding motifs of nuclear receptors (DR4 and DR2), a putative hepatocyte nuclear factor-1 binding motif (HNF1), nuclear factor-kappa B binding motif (NFκB), activator protein 1 binding motif (AP-1), and ecotropic viral integration site 1 binding motif (Evi1). We hereby conclude DR4 and/or DR2 motifs being primarily responsible and HNF1 being synergistically functioning elements for the rCAR-mediated transcription of CYP3A1.


Subject(s)
5' Flanking Region , Cytochrome P-450 CYP3A/genetics , Hepatocytes/enzymology , Receptors, Cytoplasmic and Nuclear/genetics , Response Elements , Animals , Binding Sites , Carcinoma, Hepatocellular/enzymology , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Constitutive Androstane Receptor , Cytochrome P-450 CYP3A/metabolism , Dexamethasone/pharmacology , Gene Expression Regulation, Enzymologic , Genes, Reporter , Hepatocyte Nuclear Factor 1/metabolism , Hepatocytes/drug effects , Liver Neoplasms/enzymology , Liver Neoplasms/genetics , Male , Protein Binding , RNA, Messenger/metabolism , Rats, Inbred F344 , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription, Genetic , Transcriptional Activation , Transfection , Tricarboxylic Acids/pharmacology
12.
Int J Cancer ; 135(3): 585-97, 2014 Aug 01.
Article in English | MEDLINE | ID: mdl-24382740

ABSTRACT

Targeted approaches have revealed frequent epigenetic alterations in ovarian cancer, but the scope and relation of these changes to histologic subtype of disease is unclear. Genome-wide methylation and expression data for 14 clear cell carcinoma (CCC), 32 non-CCC and four corresponding normal cell lines were generated to determine how methylation profiles differ between cells of different histological derivations of ovarian cancer. Consensus clustering showed that CCC is epigenetically distinct. Inverse relationships between expression and methylation in CCC were identified, suggesting functional regulation by methylation, and included 22 hypomethylated (UM) genes and 276 hypermethylated (HM) genes. Categorical and pathway analyses indicated that the CCC-specific UM genes were involved in response to stress and many contain hepatocyte nuclear factor (HNF) 1-binding sites, while the CCC-specific HM genes included members of the estrogen receptor alpha (ERalpha) network and genes involved in tumor development. We independently validated the methylation status of 17 of these pathway-specific genes, and confirmed increased expression of HNF1 network genes and repression of ERalpha pathway genes in CCC cell lines and primary cancer tissues relative to non-CCC specimens. Treatment of three CCC cell lines with the demethylating agent Decitabine significantly induced expression for all five genes analyzed. Coordinate changes in pathway expression were confirmed using two primary ovarian cancer datasets (p < 0.0001 for both). Our results suggest that methylation regulates specific pathways and biological functions in CCC, with hypomethylation influencing the characteristic biology of the disease while hypermethylation contributes to the carcinogenic process.


Subject(s)
Adenocarcinoma, Clear Cell/genetics , DNA Methylation , Epigenomics , Ovarian Neoplasms/genetics , Adenocarcinoma, Clear Cell/pathology , Female , Hepatocyte Nuclear Factor 1/genetics , Hepatocyte Nuclear Factor 1/metabolism , Humans , Neoplasm Staging , Ovarian Neoplasms/pathology , Real-Time Polymerase Chain Reaction , Tumor Cells, Cultured
13.
Meat Sci ; 94(4): 474-9, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23628452

ABSTRACT

The aim of this study was to investigate the association and expression of HNF1A gene as a candidate gene for meat and carcass quality traits in pigs. Statistical analysis revealed that the g.8260 A>G polymorphism significantly associated with pH 24(H), meat percentage and muscle area in the F2 Duroc × Pietrain (DuPi, n=313) and with pH 24(L), fat area and backfat thickness in the Pietrain (Pi, n=110) population. HNF1A mRNA and protein expressions were higher (p<0.05) in animals with the low post-mortem muscle pH 24(L). The promoter methylation profiling suggested that methylation was not involved on HNF1A expression regulation (p>0.05) in animal with divergent muscle pH. In conclusion, polymorphism in porcine HNF1A gene could be used as a candidate marker to improve the meat and carcass quality traits, with the consideration of breed-specific effect.


Subject(s)
Adipose Tissue/metabolism , Breeding , Gene Expression , Hepatocyte Nuclear Factor 1/genetics , Meat/analysis , Muscle, Skeletal/metabolism , Polymorphism, Single Nucleotide , Animals , Diet , Dietary Fats/analysis , Gene Expression Regulation , Genetic Association Studies , Hepatocyte Nuclear Factor 1/metabolism , Hydrogen-Ion Concentration , Meat/standards , Methylation , Promoter Regions, Genetic , RNA, Messenger/metabolism , Sus scrofa
14.
Hum Mutat ; 32(10): 1153-60, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21681857

ABSTRACT

N-acetylglutamate synthase (NAGS) catalyzes the conversion of glutamate and acetyl-CoA to NAG, the essential allosteric activator of carbamyl phosphate synthetase I, the first urea cycle enzyme in mammals. A 17-year-old female with recurrent hyperammonemia attacks, the cause of which remained undiagnosed for 8 years in spite of multiple molecular and biochemical investigations, showed markedly enhanced ureagenesis (measured by isotope incorporation) in response to N-carbamylglutamate (NCG). This led to sequencing of the regulatory regions of the NAGS gene and identification of a deleterious single-base substitution in the upstream enhancer. The homozygous mutation (c.-3064C>A), affecting a highly conserved nucleotide within the hepatic nuclear factor 1 (HNF-1) binding site, was not found in single nucleotide polymorphism databases and in a screen of 1,086 alleles from a diverse population. Functional assays demonstrated that this mutation decreases transcription and binding of HNF-1 to the NAGS gene, while a consensus HNF-1 binding sequence enhances binding to HNF-1 and increases transcription. Oral daily NCG therapy restored ureagenesis in this patient, normalizing her biochemical markers, and allowing discontinuation of alternate pathway therapy and normalization of her diet with no recurrence of hyperammonemia. Inc.


Subject(s)
Amino-Acid N-Acetyltransferase/genetics , Enhancer Elements, Genetic , Glutamates/therapeutic use , Sequence Deletion , Urea Cycle Disorders, Inborn/drug therapy , Urea Cycle Disorders, Inborn/genetics , Adolescent , Alleles , Base Sequence , Binding Sites , Cell Line, Tumor , Child , Female , Gene Frequency , Glutamates/metabolism , Hep G2 Cells , Hepatocyte Nuclear Factor 1/metabolism , Humans , Nucleotide Motifs , Polymorphism, Single Nucleotide , Sequence Alignment , Treatment Outcome , Urea Cycle Disorders, Inborn/metabolism
15.
J Biol Chem ; 286(19): 17259-69, 2011 May 13.
Article in English | MEDLINE | ID: mdl-21454713

ABSTRACT

HDL is a negative risk factor for atherosclerosis because of its multiple atheroprotective functions. Inflammation converts HDL particles from anti-atherogenic to pro-atherogenic, and this transformation is associated with changes in HDL structure and composition. Apolipoprotein M (apoM) has been recently shown to play a role in the maturation of HDL in plasma and to protect from atherosclerosis. ApoM gene is expressed primarily in the liver and kidney and is down-regulated by pro-inflammatory signals. We now show that the human apoM promoter harbors a dual specificity regulatory element in the proximal region that binds hepatocyte nuclear factor 1 (HNF-1) and members of the AP-1 family of pro-inflammatory transcription factors (c-Jun and JunB). Overexpression of c-Jun or JunB repressed both the basal and the HNF-1-mediated transactivation of the human apoM promoter. Treatment of HepG2 cells with potent inflammation-inducing phorbol esters or overexpression of PKCα was associated with a marked inhibition of apoM gene expression in a c-Jun/JunB-dependent manner. We provide evidence for a novel mechanism of inflammation-induced transcriptional repression that is based on the competition between HNF-1 and Jun proteins for binding to the same regulatory region. A similar mechanism accounts for the down-regulation of the liver-specific apolipoprotein A-II gene by Jun factors. Our studies provide novel insights on the mechanisms that control the expression of liver-specific apolipoprotein genes during inflammation and could affect the maturation and the functionality of HDL particles.


Subject(s)
Apolipoproteins/metabolism , Gene Expression Regulation , Hepatocyte Nuclear Factor 1/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Apolipoproteins M , Cell Line , Cholesterol, HDL/metabolism , Humans , Inflammation , Lipocalins , Models, Biological , Mutation , Phorbol Esters/metabolism , Promoter Regions, Genetic , Transcription Factor AP-1/metabolism , Transcriptional Activation
16.
J Biol Chem ; 286(8): 6049-60, 2011 Feb 25.
Article in English | MEDLINE | ID: mdl-21163946

ABSTRACT

Resveratrol, a polyphenol compound, is known for its effects on energy homeostasis. With properties of energy sensors mediating effects of calorie restriction, sirtuins are targets of resveratrol. The mammalian sirtuin homolog SIRT1 is a protein deacetylase playing a role in glucose metabolism and islet function. Here, we investigated the effects of resveratrol and possible link with SIRT1 in ß-cells. Insulinoma INS-1E cells and human islets were cultured with resveratrol before analyzing their physiological responses. Treatment of INS-1E cells for 24 h with 25 µM resveratrol resulted in marked potentiation of glucose-stimulated insulin secretion. This effect was associated with elevated glycolytic flux, resulting in increased glucose oxidation, ATP generation, and mitochondrial oxygen consumption. Such changes correlated with up-regulation of key genes for ß-cell function, i.e. Glut2, glucokinase, Pdx-1, Hnf-1α, and Tfam. In human islets, chronic resveratrol treatment similarly increased both the glucose secretory response and expression of the same set of genes, eventually restoring the glucose response in islets obtained from one type 2 diabetic donor. Overexpression of Sirt1 in INS-1E cells potentiated resveratrol effects on insulin secretion. Conversely, inhibition of SIRT1 achieved either by expression of an inactive mutant or by using the EX-527 inhibitor, both abolished resveratrol effects on glucose responses. Treatment of INS-1E cells with EX-527 also prevented resveratrol-induced up-regulation of Glut2, glucokinase, Pdx-1, and Tfam. Resveratrol markedly enhanced the glucose response of INS-1E cells and human islets, even after removal of the compound from the medium. These effects were mediated by and fully dependent on active SIRT1, defining a new role for SIRT1 in the regulation of insulin secretion.


Subject(s)
Glucose/metabolism , Insulin-Secreting Cells/metabolism , Insulin/metabolism , Sirtuin 1/metabolism , Animals , Carbazoles/pharmacology , Cell Line, Tumor , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Enzyme Inhibitors/pharmacology , Glucokinase/genetics , Glucokinase/metabolism , Glucose/pharmacology , Glucose Transporter Type 2/genetics , Glucose Transporter Type 2/metabolism , Hepatocyte Nuclear Factor 1/genetics , Hepatocyte Nuclear Factor 1/metabolism , High Mobility Group Proteins/genetics , High Mobility Group Proteins/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Insulin/genetics , Insulin Secretion , Insulin-Secreting Cells/cytology , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Oxygen Consumption/drug effects , Oxygen Consumption/physiology , Rats , Resveratrol , Sirtuin 1/antagonists & inhibitors , Sirtuin 1/genetics , Stilbenes/pharmacology , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Up-Regulation/drug effects , Up-Regulation/physiology
17.
Exp Biol Med (Maywood) ; 235(1): 32-9, 2010 Jan.
Article in English | MEDLINE | ID: mdl-20404016

ABSTRACT

The incidence of hepatocellular carcinoma is rising due to alcohol drinking, hepatitis C viral infection and metabolic syndrome. Differential expression of CYP2E1 may play a pleiotropic role in the multistep process of liver carcinogenesis. Considerable attention has focused on the antitumor effect of trichostatin A (TSA) as well as CYP2E1 expression-induced apoptosis of cancer cells. However, very few studies have examined the mechanisms by which TSA has an antitumor effect and its association to CYP2E1 expression. The current study examined the action of TSA on CYP2E1 expression and the role of CYP2E1 in inducing apoptosis of HepG2 cells. Our data showed that TSA selectively induced CYP2E1 in four studied human hepatocellular carcinoma (HCC) cell lines (Huh7, PLC/PRF/5, Hep3B and HepG2), but not in normal primary human hepatocytes. TSA-mediated up-regulation of CYP2E1 expression was associated with histone H3 acetylation and the recruitment of HNF-1 and HNF-3beta to the CYP2E1 promoter in HepG2 cells. siRNA-mediated knockdown experiments showed that TSA-induced caspase-3 cleavage was decreased due to reduced expression of CYP2E1 in HepG2 cells. Moreover, down-regulation of CYP2E1 was accompanied by decreased production of mitochondrial reactive oxygen species. These results suggest that histone modification is involved in CYP2E1 gene expression and that CYP2E1-dependent mitochondrial oxidative stress plays a role in TSA-induced apoptosis.


Subject(s)
Apoptosis/physiology , Carcinoma, Hepatocellular/metabolism , Cytochrome P-450 CYP2E1/genetics , Histones/metabolism , Liver Neoplasms/metabolism , Acetylation , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Base Sequence , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Caspase 3/metabolism , Cell Line , Cell Line, Tumor , Cytochrome P-450 CYP2E1/biosynthesis , Cytochrome P-450 CYP2E1 Inhibitors , DNA Primers/genetics , Enzyme Induction/drug effects , Gene Expression , Hepatocyte Nuclear Factor 1/metabolism , Hepatocyte Nuclear Factor 3-beta/metabolism , Hepatocytes/cytology , Hepatocytes/drug effects , Hepatocytes/metabolism , Histones/chemistry , Humans , Hydroxamic Acids/pharmacology , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Oxidative Stress , RNA, Small Interfering/genetics
18.
Mol Nutr Food Res ; 54(10): 1445-51, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20425755

ABSTRACT

Maltase and glucoamylase are derived from the same mRNA and are responsible for digestion of starch in the small intestine. Their jejunal activities in rodents are induced by a high-starch/low-fat (HS)-diet. However, it is unknown whether jejunal expression of the maltase-glucoamylase (Mgam) gene is enhanced by the HS-diet. In this study, we found that jejunal Mgam mRNA was increased by a HS-diet in mice. We showed that the HS-diet increased acetylation of histones, bindings of a coactivator, Creb binding protein (CREBBP), and the transcriptional factors caudal type homeobox 2 (CDX2) and HNF1 homeobox (HNF1) in the promoter/enhancer and transcriptional regions of Mgam gene. This suggests that the increase in the jejunal activity of maltase and glucoamylase caused by a HS-diet in mice is regulated at the mRNA level through histone acetylation and binding of CREBBP, CDX2 and HNF1 in the promoter/enhancer and transcriptional regions of Mgam gene.


Subject(s)
Diet , Enzyme Induction , Jejunum/enzymology , Starch/administration & dosage , alpha-Glucosidases/biosynthesis , Acetylation , Animals , CDX2 Transcription Factor , CREB-Binding Protein/metabolism , DNA-Binding Proteins/metabolism , Diet/adverse effects , Diet, Fat-Restricted , Hepatocyte Nuclear Factor 1/metabolism , Histones/metabolism , Homeodomain Proteins/metabolism , Jejunum/metabolism , Male , Mice , Mice, Inbred C57BL , Open Reading Frames , Promoter Regions, Genetic , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Starch/adverse effects , Transcription Factors/metabolism , alpha-Glucosidases/genetics
19.
BMC Mol Biol ; 11: 9, 2010 Jan 22.
Article in English | MEDLINE | ID: mdl-20096102

ABSTRACT

BACKGROUND: UDP-glucuronosyltransferase 1A1 (UGT1A1) is a pivotal enzyme involved in metabolism of SN-38, the active metabolite of irinotecan commonly used to treat metastatic colorectal cancer. We previously demonstrated aberrant methylation of specific CpG dinucleotides in UGT1A1-negative cells, and revealed that methylation state of the UGT1A1 5'-flanking sequence is negatively correlated with gene transcription. Interestingly, one of these CpG dinucleotides (CpG -4) is found close to a HNF1 response element (HRE), known to be involved in activation of UGT1A1 gene expression, and within an upstream stimulating factor (USF) binding site. RESULTS: Gel retardation assays revealed that methylation of CpG-4 directly affect the interaction of USF1/2 with its cognate sequence without altering the binding for HNF1-alpha. Luciferase assays sustained a role for USF1/2 and HNF1-alpha in UGT1A1 regulation in colon cancer cells. Based on the differential expression profiles of HNF1A gene in colon cell lines, we also assessed whether methylation affects its expression. In agreement with the presence of CpG islands in the HNF1A promoter, treatments of UGT1A1-negative HCT116 colon cancer cells with a DNA methyltransferase inhibitor restore HNF1A gene expression, as observed for UGT1A1. CONCLUSIONS: This study reveals that basal UGT1A1 expression in colon cells is positively regulated by HNF1-alpha and USF, and negatively regulated by DNA methylation. Besides, DNA methylation of HNF1A could also play an important role in regulating additional cellular drug metabolism and transporter pathways. This process may contribute to determine local inactivation of drugs such as the anticancer agent SN-38 by glucuronidation and define tumoral response.


Subject(s)
Colonic Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Glucuronosyltransferase/genetics , Hepatocyte Nuclear Factor 1/metabolism , Azacitidine/analogs & derivatives , Azacitidine/pharmacology , Base Sequence , Colonic Neoplasms/metabolism , CpG Islands , Cytidine Triphosphate/analogs & derivatives , Cytidine Triphosphate/pharmacology , DNA Methylation , Electrophoretic Mobility Shift Assay , Glucuronosyltransferase/metabolism , HCT116 Cells , HT29 Cells , Hepatocyte Nuclear Factor 1/genetics , Humans , Molecular Sequence Data , Promoter Regions, Genetic , Up-Regulation
20.
Arterioscler Thromb Vasc Biol ; 30(3): 568-74, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20007910

ABSTRACT

OBJECTIVE: Our aim was to elucidate mechanisms involved in the acquisition of lipid transport properties during enterocyte differentiation. METHODS AND RESULTS: We show that lipid mobilization via apolipoprotein B lipoproteins is dependent on the expression of microsomal triglyceride transfer protein (MTP) during differentiation of Caco-2 cells into enterocyte-like cells. Mechanistic studies showed that binding of the nuclear receptor family 2 group F member 1 (NR2F1) to the DR1 element in the MTTP promoter suppresses MTTP expression in undifferentiated cells. During cellular differentiation, NR2F1 expression and its binding to MTTP promoter decline and MTP induction ensues. Moreover, undifferentiated cells express inositol-requiring enzyme 1beta (IRE1beta), a protein that posttranscriptionally degrades MTP mRNA, and its expression substantially decreases during differentiation, contributing to MTP induction. Immunohistochemical studies revealed a significant negative relationship between the expressions of MTP and NR2F1/IRE1beta in undifferentiated and differentiated Caco-2 cells, as well as in crypt-villus and jejunum-colon axes of mouse intestine. CONCLUSIONS: We propose that transcriptional and posttranscriptional mechanisms involving NR2F1 and IRE1beta ensure low MTP expression in undifferentiated intestinal cells and avoid apolipoprotein B lipoprotein biosynthesis.


Subject(s)
COUP Transcription Factor I/metabolism , Carrier Proteins/metabolism , Cell Differentiation , Endoribonucleases/metabolism , Intestinal Mucosa/metabolism , Lipoproteins/metabolism , Membrane Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Apolipoproteins B/metabolism , Base Sequence , Caco-2 Cells , Colon/cytology , Colon/metabolism , Enterocytes/cytology , Enterocytes/metabolism , Hepatocyte Nuclear Factor 1/genetics , Hepatocyte Nuclear Factor 1/metabolism , Hepatocyte Nuclear Factor 4/genetics , Hepatocyte Nuclear Factor 4/metabolism , Humans , Jejunum/cytology , Jejunum/metabolism , Mice , Mice, Inbred C57BL , Models, Animal , Molecular Sequence Data
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