Co-operative regulation of the transcription of human dihydrodiol dehydrogenase (DD)4/aldo–keto reductase (AKR)1C4 gene by hepatocyte nuclear factor (HNF)-4α/γ and HNF-1α

2001 ◽  
Vol 355 (2) ◽  
pp. 537-544 ◽  
Author(s):  
Takeshi OZEKI ◽  
Yoshiki TAKAHASHI ◽  
Toshiyuki KUME ◽  
Kazuo NAKAYAMA ◽  
Tsuyoshi YOKOI ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Binding Motif ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Nuclear Extracts ◽  
Redox Active ◽  
Supershift Assay ◽  
Polycyclic Aromatic ◽  
Gel Shift Assay ◽  
Dihydrodiol Dehydrogenase

Human dihydrodiol dehydrogenase (DD) 4/aldo-keto reductase (AKR) 1C4 is a major isoform of hepatic DD that oxidizes trans-dihydrodiols of polycyclic aromatic hydrocarbons to reactive and redox-active o-quinones and that reduces several ketone-containing drugs. To investigate the mechanism of transcriptional regulation of the human DD4 gene, the 5′-flanking region of the gene was fused to the luciferase gene. The results of luciferase assays using HepG2 cells and of 1,10-phenanthroline-copper footprinting indicated that two positive regulatory regions were located in regions from -701 to -684 and from -682 to -666. The former region contained a putative hepatocyte nuclear factor (HNF)-4 binding motif, and the latter region contained an HNF-1 consensus binding sequence. DNA fragments of the HNF-4 or HNF-1 motif gave a shifted band in a gel-shift assay with nuclear extracts from HepG2 cells. The formation of the DNA-protein complex was inhibited by the HNF-4 or HNF-1 motif of the α1-antitrypsin gene. A supershift assay using antibodies to human HNF-4α, HNF-4γ and HNF-1α showed that HNF-4α and HNF-4γ bound to the HNF-4 motif, and that HNF-1α interacted with the HNF-1 motif. Introduction of mutations into the HNF-4 or HNF-1 motif lowered the luciferase activity to 10 or 8% respectively of that seen with the intact human DD4 gene. These results indicate that HNF-4α, HNF-4γ and HNF-1α regulate co-operatively the transcription of the human DD4 gene in HepG2 cells.

scholarly journals Upstream region of rat serum albumin gene promoter contributes to promoter activity: presence of functional binding site for hepatocyte nuclear factor-3

1999 ◽  
Vol 338 (2) ◽  
pp. 241-249 ◽  
Author(s):  
Chin-Hui HSIANG ◽  
Norman W. MARTEN ◽  
Daniel S. STRAUS
Keyword(s):  
Serum Albumin ◽  
Hepg2 Cells ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Upstream Region ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Rat Serum ◽  
Albumin Gene ◽  
Rat Serum Albumin

Transcription of the serum albumin gene occurs almost exclusively in the liver and is controlled in part by a strong liver-specific promoter. The upstream region of the serum albumin gene promoter is highly conserved among species and is footprinted in vitro by a number of nuclear proteins. However, the role of the upstream promoter region in regulating transcription and the identity of the transcription factors that bind to this region have not been established. In the present study, deletion analysis of the rat serum albumin promoter in transiently transfected HepG2 cells demonstrated that elimination of the region between -207 and -153 bp caused a two-fold decrease in promoter activity (P< 0.05). Additional analysis of the -207 to -124 bp promoter interval led to the identification of two potential binding sites for hepatocyte nuclear factor-3 (HNF-3) located at -168 to -157 bp (site X) and -145 to -134 bp (site Y). Electrophoretic mobility-shift assays performed with the HNF-3 X and Y sites demonstrated that both sites are capable of binding HNF-3α and HNF-3β. Placement of a single copy of the HNF-3 X site upstream from a minimal promoter increased promoter activity by about four-fold in HepG2 cells, and the reporter construct containing this site could be transactivated if co-transfected with an HNF-3 expression construct. Furthermore, inactivation of the HNF-3 X site by site-directed mutagenesis within the context of the -261 bp albumin promoter construct resulted in a 40% decrease in transcription (P< 0.05). These results indicate that the positive effect of the -207 to -153 bp promoter interval is attributable to the presence of the HNF-3 X site within this interval. Additional results obtained with transfected HepG2 cells suggest that the HNF-3 Y site plays a lesser role in activation of transcription than the X site.

scholarly journals Co-operative regulation of the transcription of human dihydrodiol dehydrogenase (DD)4/aldo‒keto reductase (AKR)1C4 gene by hepatocyte nuclear factor (HNF)-4α/γ and HNF-1α

2001 ◽  
Vol 355 (2) ◽  
pp. 537 ◽  
Author(s):  
Takeshi OZEKI ◽  
Yoshiki TAKAHASHI ◽  
Toshiyuki KUME ◽  
Kazuo NAKAYAMA ◽  
Tsuyoshi YOKOI ◽  
...  
Keyword(s):  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Dihydrodiol Dehydrogenase

scholarly journals Thyroid hormones act indirectly to increase sex hormone-binding globulin production by liver via hepatocyte nuclear factor-4α

2009 ◽  
Vol 43 (1) ◽  
pp. 19-27 ◽  
Author(s):  
David M Selva ◽  
Geoffrey L Hammond
Keyword(s):  
Thyroid Hormones ◽  
Hepg2 Cells ◽  
Sex Hormone ◽  
Sex Hormone Binding Globulin ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Hormone Binding ◽  
Metabolic State ◽  
Hepatocyte Nuclear Factor 4Α

Thyroid hormones increase hepatic sex hormone-binding globulin (SHBG) production, which is also regulated by hepatocyte nuclear factor-4α (HNF-4α) in response to changes in the metabolic state of the liver. Since the human SHBG promoter lacks a typical thyroid hormone response element, and because thyroid hormones influence metabolic state, we set out to determine whether thyroid hormones mediate SHBG expression indirectly via changes in HNF-4α levels in HepG2 human hepatoblastoma cells, and in the livers of transgenic mice that express a 4.3 kb human SHBG transgene under the control of its own 0.8 kb promoter sequence. Thyroid hormones (triiodothyronine (T3) and thyroxine (T4)) increase SHBG accumulation in HepG2 cell culture medium over 5 days, and increase cellular SHBG mRNA levels. In addition, T4 treatment of HepG2 cells for 5 days increased HNF-4α mRNA and HNF-4α levels in concert with decreased cellular palmitate levels. Plasma SHBG levels were also increased in mice expressing a human SHBG transgene after 5 days treatment with T3 along with increased hepatic HNF-4α levels. In HepG2 cells, the human SHBG promoter failed to respond acutely (within 24 h) to T4 treatment, but a 4-day pre-treatment with T4 resulted in a robust response that was prevented by co-treatment with HNF-4α siRNA, or by blocking the β-oxidation of palmitate through co-treatment with the carnitine palmitoyltransferase I inhibitor, etomoxir. These data lead us to conclude that thyroid hormones increase SHBG production indirectly by increasing HNF-4α gene expression, and by reducing cellular palmitate levels that further contribute to increased HNF-4α levels in hepatocytes.

scholarly journals Hepatocyte nuclear factor-4α is a central transactivator of the mouse Ntcp gene

2008 ◽  
Vol 295 (2) ◽  
pp. G226-G233 ◽  
Author(s):  
Andreas Geier ◽  
Ina V. Martin ◽  
Christoph G. Dietrich ◽  
Natarajan Balasubramaniyan ◽  
Sonja Strauch ◽  
...  
Keyword(s):  
Sodium Taurocholate ◽  
Regulatory Elements ◽  
Upstream Region ◽  
Uptake System ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Gel Mobility Shift ◽  
Fold Activation

Sodium taurocholate cotransporting polypeptide (Ntcp) is the major uptake system for conjugated bile acids. Deletions of hepatocyte nuclear factor (HNF)-1α and retinoid X receptor-α:retinoic acid receptor-α binding sites in the mouse 5′-flanking region corresponding to putatively central regulatory elements of rat Ntcp do not significantly reduce promoter activity. We hypothesized that HNF-4α, which is increasingly recognized as a central regulator of hepatocyte function, may directly transactivate mouse ( mNtcp). A 1.1-kb 5′-upstream region including the mouse Ntcp promoter was cloned and compared with the rat promoter. In contrast to a moderate 3.5-fold activation of mNtcp by HNF-1α, HNF-4α cotransfection led to a robust 20-fold activation. Deletion analysis of mouse and rat Ntcp promoters mapped a conserved HNF-4α consensus site at −345/−326 and −335/−316 bp, respectively. p-475bp mNtcpLUC is not transactivated by HNF-1α but shows a 50-fold enhanced activity upon cotransfection with HNF-4α. Gel mobility shift assays demonstrated a complex of the HNF-4α-element formed with liver nuclear extracts that was blocked by an HNF-4α specific antibody. HNF-4α binding was confirmed by chromatin immunoprecipitation. Using Hepa 1–6 cells, HNF-4α-knockdown resulted in a significant 95% reduction in NTCP mRNA. In conclusion, mouse Ntcp is regulated by HNF-4α via a conserved distal cis-element independently of HNF-1α.

scholarly journals Cell Type-specific Target Selection by Combinatorial Binding of Smad2/3 Proteins and Hepatocyte Nuclear Factor 4α in HepG2 Cells

2011 ◽  
Vol 286 (34) ◽  
pp. 29848-29860 ◽  
Author(s):  
Anna Mizutani ◽  
Daizo Koinuma ◽  
Shuichi Tsutsumi ◽  
Naoko Kamimura ◽  
Masato Morikawa ◽  
...  
Keyword(s):  
Hepg2 Cells ◽  
Target Selection ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Cell Type ◽  
Specific Target ◽  
Cell Type Specific ◽  
Hepatocyte Nuclear Factor 4Α

scholarly journals Hepatic Expression of the Mouse Angptl8 Gene Is Regulated by Insulin Through Increases in Hepatocyte Nuclear Factor-1

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A47-A48
Author(s):  
Takuya Watanabe ◽  
Takuya Watanabe ◽  
Atsushi Ozawa ◽  
Yuri Kondo ◽  
Kazuhiko Horiguchi ◽  
...  
Keyword(s):  
Promoter Region ◽  
Promoter Activity ◽  
Time Course ◽  
Binding Motif ◽  
Light Cycle ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Primary Hepatocytes ◽  
Expression Levels ◽  
Hepatic Expression

Abstract Angiopoietin-like proteins (ANGPTLs) are a family of proteins structurally similar to angiopoietins. ANGPTL8 is an important regulator of circulating triglyceride (TG) levels in mammals. Increasing evidence revealed an association between ANGPTL8 expression and serum lipid profiles, especially in subjects with metabolic syndrome. Several mice studies demonstrated that Angptl8 is suppressed by fasting and induced by long term refeeding, however the detailed mechanism is still unclear. In humans, ANGPTL8 is mainly expressed in the liver. Therefore, this study aims to investigate the mechanisms that control the refeeding induced increase in the hepatic Angptl8 gene expression. Methods and Results: Twenty-week-old male C57/BL6 mice were used in this study. Mice were fasted for 12h during the dark cycle and re-fed for 30, 60, 120, 240 and 360 minutes during the light cycle. Mice were euthanized after each refeeding time course and tissues were collected. We found even short refeeding times (~60 min) enhanced the expression levels of hepatic Angptl8 in mice. We cloned the mouse Angptl8 gene promoter region. Promoter deletion analyses showed that the basal promoter activity was significantly attenuated by a deletion of -309/-60 region in hepatocytes. A computational motif search revealed the presence of a potential binding motif for hepatocyte nuclear factor 1α/1β (HNF-1α/β) at -84/-68 bp of the promoter. Mutations of the HNF-1 binging site significantly decreased the promoter activity in mouse hepatoma cells (Hepa1-6) and mouse primary hepatocytes, and the promoter carrying the mutated HNF-1 site was not transactivated by co-transfected HNF-1 in a non-hepatic cell line. These findings indicated that HNF-1 was essential and critical factor for the basal expression of Angptl8 in murine liver. In fact, knockdown of Hnf-1 using siRNA method in mouse Hepa1-6 and mouse primary hepatocytes reduced Angptl8 protein levels. We also performed Electrophoretic mobility-shift assays and confirmed the direct binding of Hnf-1 to its Angptl8 promoter binding motif. To elucidate whether refeeding could enhance HNF-1, we checked the expression levels of Hnf-1 in mouse liver. Hnf-1 expression levels of both mRNA and protein were increased after short-term refeeding, paralleling the enhanced expression of the Angptl8. Moreover, insulin-stimulated primary hepatocytes showed increased expression of Angptl8 protein, but knockdown of Hnf-1 completely abolished this enhancement by insulin. Chromatin immunoprecipitation (ChIP) analyses confirmed the recruitment of endogenous Hnf-1 to the Angptl8 promoter region and it was strongly induced by insulin. Conclusion: HNF-1 plays essential role in hepatocyte-specific and refeeding-induced rapid increases in Angptl8 expression via insulin.

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