scholarly journals Tumour suppressor p53 down-regulates the expression of the human hepatocyte nuclear factor 4α (HNF4α) gene

2006 ◽  
Vol 400 (2) ◽  
pp. 303-313 ◽  
Author(s):  
Yutaka Maeda ◽  
Wendy W. Hwang-Verslues ◽  
Gang Wei ◽  
Takuya Fukazawa ◽  
Mary L. Durbin ◽  
...  
Keyword(s):  
P53 Protein ◽  
Tumour Suppressor ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Protein Levels ◽  
Tumour Suppressor Protein ◽  
Toxic Agents ◽  
Hepatocyte Nuclear Factor 4Α

The liver is exposed to a wide variety of toxic agents, many of which damage DNA and result in increased levels of the tumour suppressor protein p53. We have previously shown that p53 inhibits the transactivation function of HNF (hepatocyte nuclear factor) 4α1, a nuclear receptor known to be critical for early development and liver differentiation. In the present study we demonstrate that p53 also down-regulates expression of the human HNF4α gene via the proximal P1 promoter. Overexpression of wild-type p53 down-regulated endogenous levels of both HNF4α protein and mRNA in Hep3B cells. This decrease was also observed when HepG2 cells were exposed to UV irradiation or doxorubicin, both of which increased endogenous p53 protein levels. Ectopically expressed p53, but not a mutant p53 defective in DNA binding (R249S), down-regulated HNF4α P1 promoter activity. Chromatin immunoprecipitation also showed that endogenous p53 bound the HNF4α P1 promoter in vivo after doxorubicin treatment. The mechanism by which p53 down-regulates the P1 promoter appears to be multifaceted. The down-regulation was partially recovered by inhibition of HDAC activity and appears to involve the positive regulator HNF6α. p53 bound HNF6α in vivo and in vitro and prevented HNF6α from binding DNA in vitro. p53 also repressed stimulation of the P1 promoter by HNF6α in vivo. However, since the R249S p53 mutant also bound HNF6α, binding HNF6α is apparently not sufficient for the repression. Implications of the p53-mediated repression of HNF4α expression in response to cellular stress are discussed.

scholarly journals Hepatocyte nuclear factor 4α regulates the expression of intestinal epithelial Na+/H+ exchanger isoform 3

2018 ◽  
Vol 314 (1) ◽  
pp. G14-G21 ◽  
Author(s):  
Saminathan Muthusamy ◽  
Jong Jin Jeong ◽  
Ming Cheng ◽  
Jessica A. Bonzo ◽  
Anoop Kumar ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Core Promoter ◽  
Epithelial Cell Line ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Intestinal Epithelial ◽  
Hepatocyte Nuclear Factor ◽  
Mobility Shift ◽  
Protein Levels ◽  
Hepatocyte Nuclear Factor 4Α

Na+/H+ exchanger isoform 3 (NHE3) plays a key role in coupled electroneutral NaCl absorption in the mammalian intestine. Reduced NHE3 expression or function has been implicated in the pathogenesis of diarrhea associated with inflammatory bowel disease (IBD) or enteric infections. Our previous studies revealed transcriptional regulation of NHE3 by various agents such as TNF-α, IFN-γ, and butyrate involving transcription factors Sp1 and Sp3. In silico analysis revealed that the NHE3 core promoter also contains a hepatocyte nuclear factor 4α (HNF-4α) binding site that is evolutionarily conserved in several species suggesting that HNF-4α has a role in NHE3 regulation. Nhe3 mRNA levels were reduced in intestine-specific Hnf4α-null mice. However, detailed mechanisms of NHE3 regulation by HNF-4α are not known. We investigated the regulation of NHE3 gene expression by HNF-4α in vitro in the human intestinal epithelial cell line C2BBe1 and in vivo in intestine-specific Hnf4α-null ( Hnf4αΔIEpC) and control ( Hnf4αfl/fl) mice. HNF-4α knockdown by short interfering RNA in C2BBe1 cells significantly decreased NHE3 mRNA and NHE3 protein levels. Gel mobility shift and chromatin immunoprecipitation assays revealed that HNF-4α directly interacts with the HNF-4α motif in the NHE3 core promoter. Site-specific mutagenesis on the HNF-4α motif decreased, whereas ectopic overexpression of HNF-4α increased, NHE3 promoter activity. Furthermore, loss of HNF-4α in Hnf4αΔIEpC mice decreased colonic Nhe3 mRNA and NHE3 protein levels. Our results demonstrate a novel role for HNF-4α in basal regulation of NHE3 expression. These studies represent an important and novel target for therapeutic intervention in IBD-associated diarrhea. NEW & NOTEWORTHY Our studies for the first time show that hepatocyte nuclear factor 4α directly regulates NHE3 promoter activity and its basal expression in the intestine.

scholarly journals Phosphorylation of Ser158 regulates inflammatory redox-dependent hepatocyte nuclear factor-4α transcriptional activity

2006 ◽  
Vol 394 (2) ◽  
pp. 379-387 ◽  
Author(s):  
Hongtao Guo ◽  
Chengjiang Gao ◽  
Zhiyong Mi ◽  
Philip Y. Wai ◽  
Paul C. Kuo
Keyword(s):  
Dna Binding ◽  
Kinase Inhibitor ◽  
Critical Role ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
P38 Kinase ◽  
Mobility Shift ◽  
Transactivation Potential ◽  
Hepatocyte Nuclear Factor 4Α

In IL-1β (interleukin 1β)-stimulated rat hepatocytes exposed to superoxide, we have previously identified an IRX (inflammatory redox)-sensitive DR1 [direct repeat of RG(G/T)TCA with one base spacing] cis-acting activator element (nt –1327 to –1315) in the iNOS (inducible nitric oxide synthase) promoter: AGGTCAGGGGACA. The corresponding transcription factor was identified to be HNF4α (hepatocyte nuclear factor-4α). HNF4α DNA binding activity and transactivation potential are tightly regulated by its state of phosphorylation. However, the functional consequences of IRX-mediated post-translational phosphorylation of HNF4α have not been well characterized. In the setting of IL-1β+H2O2, HNF4α functional activity is associated with a unique serine/threonine phosphorylation pattern. This indicates that an IRX-sensitive serine/threonine kinase pathway targets HNF4α to augment hepatocyte iNOS transcription. In the present study, following identification of phosphorylated residues in HNF4α, serial mutations were performed to render the target residues phosphorylation-resistant. Electrophoretic mobility-shift assays and transient transfection studies utilizing the iNOS promoter showed that the S158A mutation ablates IRX-mediated HNF4α DNA binding and transactivation. Gain-of-function mutation with the S158D phosphomimetic HNF4α vector supports a critical role for Ser158 phosphorylation. In vitro phosphorylation and kinase inhibitor studies implicate p38 kinase activity. Our results indicate that p38 kinase-mediated Ser158 phosphorylation is essential for augmentation of the DNA binding and transactivation potential of HNF4α in the presence of IL-1β+H2O2. This pathway results in enhanced iNOS expression in hepatocytes exposed to pro-inflammatory cytokines and oxidative stress.

scholarly journals Competitive Cofactor Recruitment by Orphan Receptor Hepatocyte Nuclear Factor 4α1: Modulation by the F Domain

2002 ◽  
Vol 22 (6) ◽  
pp. 1626-1638 ◽  
Author(s):  
Michael D. Ruse ◽  
Martin L. Privalsky ◽  
Frances M. Sladek
Keyword(s):  
Direct Interaction ◽  
Orphan Receptor ◽  
Receptor Interaction ◽  
Dependent Manner ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Interaction Domain ◽  
The Status

ABSTRACT For most ligand-dependent nuclear receptors, the status of endogenous ligand modulates the relative affinities for corepressor and coactivator complexes. It is less clear what parameters modulate the switch between corepressor and coactivator for the orphan receptors. Our previous work demonstrated that hepatocyte nuclear factor 4α1 (HNF4α1, NR2A1) interacts with the p160 coactivator GRIP1 and the cointegrators CBP and p300 in the absence of exogenously added ligand and that removal of the F domain enhances these interactions. Here, we utilized transient-transfection analysis to demonstrate repression of HNF4α1 activity by the corepressor silencing mediator of retinoid and thyroid receptors (SMRT) in several cell lines and on several HNF4α-responsive promoter elements. Glutathione S-transferase pulldown assays confirmed a direct interaction between HNF4α1 and receptor interaction domain 2 of SMRT. Loss of the F domain resulted in marked reduction of the ability of SMRT to interact with HNF4α1 in vitro and repress HNF4α1 activity in vivo, although the isolated F domain itself failed to interact with SMRT. Surprisingly, loss of both the A/B and F domains restored full repression by SMRT, suggesting involvement of both domains in the SMRT interaction. Finally, we show that when coexpressed along with HNF4α1 and GRIP1, CBP, or p300, SMRT can titer out HNF4α1-mediated transactivation in a dose-dependent manner and that this competition derives from mutually exclusive binding. Collectively, these results suggest that HNF4α can functionally interact with both a coactivator and a corepressor without altering the status of any putative ligand and that the presence of the F domain may play a role in discriminating between the different coregulators.

scholarly journals GATA binding protein 4 promotes the expression and transcription of hepatitis B virus by facilitating hepatocyte nuclear factor 4 alpha in vitro

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Xiaoqin Lv ◽  
Xia Xiang ◽  
Yue Wu ◽  
Yang Liu ◽  
Ruqing Xu ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Binding Protein ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
B Virus ◽  
Hepatocyte Nuclear Factor 4 ◽  
Positive Effect

Abstract Background GATA binding protein 4 (GATA4) has been reported as a potential target of gene therapy for hepatocellular carcinoma (HCC). It is well known that the main cause of HCC is the chronic infection of hepatitis B virus (HBV). However, whether the effect of GATA4 on HBV has not yet been reported. Methods In this study, the regulation of GATA4 on HBV was analyzed in vitro. In turn, the effect of HBV on GATA4 was also observed in vitro, in vivo, and clinical HCC patients. Subsequently, we analyzed whether the effect of GATA4 on HBV was related to hepatocyte nuclear factor 4 alpha (HNF4α) in vitro. Results The results showed that GATA4 significantly promoted the secretion of HBV surface antigen (HBsAg) and HBV e antigen in the cell culture medium, improved the replication of HBV genomic DNA, and increased the level of HBV 3.5 kb pre-genomic RNA and HBV total RNA (P < 0.05). Moreover, it was showed that HBV had no significant effect on GATA4 in vitro and in vivo (P > 0.05). At the same time, GATA4 expression was decreased in 78.9% (15/19) of HCC patients regardless of the HBV and HBsAg status. Among them, there were 76.9% (10/13) in HBV-associated patients with HCC (HBV-HCC), and 83.3% (5/6) in non-HBV-HCC patients. In addition, the expression of HNF4α was also up-regulated or down-regulated accordingly when stimulating or interfering with the expression of GATA4. Furthermore, stimulating the expression of HNF4α could only alleviate the HBsAg level and HBV transcription levels, but had no significant effect on GATA4. Conclusions In summary, this study found that GATA4 has a positive effect on HBV, and the potential pathway may be related to another transcription factor HNF4α that regulates HBV.

scholarly journals DNMT1 recruited by EZH2-mediated silencing of miR-484 contributes to the malignancy of cervical cancer cells through MMP14 and HNF1A

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Yang Hu ◽  
Fuxia Wu ◽  
Yankun Liu ◽  
Qian Zhao ◽  
Hua Tang
Keyword(s):  
Cervical Cancer ◽  
Cell Adhesion ◽  
Dna Methyltransferases ◽  
Epigenetic Mechanism ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Cervical Cancer Cells ◽  
Membrane Bound

Abstract Background Emerging evidence indicates that dysregulation of microRNAs (miRNAs) contributes to cervical cancer (CC) tumorigenesis and development. Previous work showed that miR-484 which regulated the EMT process was obviously downregulated in CC. However, little is known about the precise mechanism. Results We found that the deficiency of EZH2-recruited DNA methyltransferases DNMT1 reduced the CpG methylation of miR-484 promoter and then increased the miR-484 expression. Furthermore, the cell membrane-bound matrix metalloproteinase (MMP14) and the hepatocyte nuclear factor 1A (HNF1A) were found to be downregulated by miR-484. miR-484 repressed the expression of MMP14 and HNF1A inhibiting CC growth and metastasis in vitro and in vivo. Upregulation of MMP14 and HNF1A promotes the CC cell adhesion and EMT, all of which contribute to cell motility and metastasis. Moreover, miR-484 negatively regulates the WNT/MAPK and TNF signaling pathway by downregulating HNF1A and MMP14 respectively. Thus, miR-484, who is downregulated by DNMT1-mediated hypermethylation in its promoter, functions as a tumor suppressor by inhibiting MMP14 and HNF1A expression in CC. Conclusion Our finding characterizes miR-484 as a key suppressive regulator in CC metastasis and reveals a DNMT1-mediated epigenetic mechanism for miR-484 silencing, expanding our understanding of the molecular mechanism underlying CC progression and metastasis. Graphical abstract

scholarly journals Transcriptomically Revealed Oligo-Fucoidan Enhances the Immune System and Protects Hepatocytes via the ASGPR/STAT3/HNF4A Axis

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 898 ◽  
Author(s):  
Chun-Chia Cheng ◽  
Wan-Yu Yang ◽  
Ming-Chen Hsiao ◽  
Kuan-Hao Lin ◽  
Hao-Wei Lee ◽  
...  
Keyword(s):  
Immune System ◽  
Brown Seaweed ◽  
Underlying Mechanism ◽  
Sulfated Polysaccharide ◽  
Driver Gene ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Hepatocyte Nuclear Factor 4

Oligo-fucoidan, a sulfated polysaccharide extracted from brown seaweed, exhibits anti-inflammatory and anti-tumor effects. However, the knowledge concerning the detailed mechanism of oligo-fucoidan on liver cells is obscure. In this study, we investigate the effect of oligo-fucoidan in normal hepatocytes by transcriptomic analysis. Using an oligo-fucoidan oral gavage in wild-type adult zebrafish, we find that oligo-fucoidan pretreatment enhances the immune system and anti-viral genes in hepatocytes. Oligo-fucoidan pretreatment also decreases the expression of lipogenic enzymes and liver fibrosis genes. Using pathway analysis, we identify hepatocyte nuclear factor 4 alpha (HNF4A) to be the potential driver gene. We further investigate whether hepatocyte nuclear factor 4 alpha (HNF4A) could be induced by oligo-fucoidan and the underlying mechanism. Therefore, a normal hepatocyte clone 9 cell as an in vitro model was used. We demonstrate that oligo-fucoidan increases cell viability, Cyp3a4 activity, and Hnf4a expression in clone 9 cells. We further demonstrate that oligo-fucoidan might bind to asialoglycoprotein receptors (ASGPR) in normal hepatocytes through both in vitro and in vivo competition assays. This binding, consequently activating the signal transducer and activator of transcription 3 (STAT3), increases the expression of the P1 isoform of HNF4A. According to our data, we suggest that oligo-fucoidan not only enhances the gene expression associated with anti-viral ability and immunity, but also increases P1-HNF4A levels through ASGPR/STAT3 axis, resulting in protecting hepatocytes.

scholarly journals Modulation of Transcriptional Activation and Coactivator Interaction by a Splicing Variation in the F Domain of Nuclear Receptor Hepatocyte Nuclear Factor 4α1

1999 ◽  
Vol 19 (10) ◽  
pp. 6509-6522 ◽  
Author(s):  
Frances M. Sladek ◽  
Michael D. Ruse ◽  
Luviminda Nepomuceno ◽  
Shih-Ming Huang ◽  
Michael R. Stallcup
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Transcriptional Activation ◽  
Regulatory Region ◽  
Physical Contact ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Splicing Variants

ABSTRACT Transcription factors, such as nuclear receptors, often exist in various forms that are generated by highly conserved splicing events. Whereas the functional significance of these splicing variants is often not known, it is known that nuclear receptors activate transcription through interaction with coactivators. The parameters, other than ligands, that might modulate those interactions, however, are not well characterized, nor is the role of splicing variants. In this study, transient transfection, yeast two-hybrid, and GST pulldown assays are used to show not only that nuclear receptor hepatocyte nuclear factor 4 α1 (HNF4α1, NR2A1) interacts with GRIP1, and other coactivators, in the absence of ligand but also that the uncommonly large F domain in the C terminus of the receptor inhibits that interaction. In vitro, the F domain was found to obscure an AF-2-independent binding site for GRIP1 that did not map to nuclear receptor boxes II or III. The results also show that a natural splicing variant containing a 10-amino-acid insert in the middle of the F domain (HNF4α2) abrogates that inhibition in vivo and in vitro. A series of protease digestion assays indicates that there may be structural differences between HNF4α1 and HNF4α2 in the F domain as well as in the ligand binding domain (LBD). The data also suggest that there is a direct physical contact between the F domain and the LBD of HNF4α1 and -α2 and that that contact is different in the HNF4α1 and HNF4α2 isoforms. Finally, we propose a model in which the F domain of HNF4α1 acts as a negative regulatory region for transactivation and in which the α2 insert ameliorates the negative effect of the F domain. A conserved repressor sequence in the F domains of HNF4α1 and -α2 suggests that this model may be relevant to other nuclear receptors as well.

scholarly journals p53 regulates its own activator: transcriptional co-activator PC4, a new p53-responsive gene

2007 ◽  
Vol 406 (3) ◽  
pp. 437-444 ◽  
Author(s):  
A. Hari Kishore ◽  
Kiran Batta ◽  
Chandrima Das ◽  
Shipra Agarwal ◽  
Tapas K. Kundu
Keyword(s):  
Binding Sites ◽  
Feedback Loop ◽  
Cellular Responses ◽  
Protein Levels ◽  
Cycle Arrest ◽  
Tumour Suppressor Protein ◽  
Mediate Cell ◽  
Inducible Gene

The tumour suppressor protein p53 regulates the expression of several genes that mediate cell cycle arrest, apoptosis, DNA repair and other cellular responses. Recently, we have shown that human transcriptional co-activator PC4 is a unique activator of p53 function. In the present study, we report that PC4 is a p53-inducible gene. Bioinformatics analysis reveals multiple p53-binding sites in the PC4 promoter. We have found that indeed p53 binds to all the identified sites in vitro and in vivo with varying affinities. p53 acts as an activator of PC4 transcription. Both PC4 mRNA and protein levels increase in response to stimuli that result in p53 induction. Furthermore, PC4 enhances p53 recruitment to the PC4 promoter. Our results thus establish the first report of a positively regulated feedback loop to control p53 function.

scholarly journals Protein kinase A-dependent phosphorylation modulates DNA-binding activity of hepatocyte nuclear factor 4.

1997 ◽  
Vol 17 (8) ◽  
pp. 4208-4219 ◽  
Author(s):  
B Viollet ◽  
A Kahn ◽  
M Raymondjean
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Wild Type ◽  
Dna Binding Activity ◽  
Hepatocyte Nuclear Factor 4 ◽  
Kinase A

Hepatocyte nuclear factor 4 (HNF4), a liver-enriched transcription factor of the nuclear receptor superfamily, is critical for development and liver-specific gene expression. Here, we demonstrate that its DNA-binding activity is modulated posttranslationally by phosphorylation in vivo, ex vivo, and in vitro. In vivo, HNF4 DNA-binding activity is reduced by fasting and by inducers of intracellular cyclic AMP (cAMP) accumulation. A consensus protein kinase A (PKA) phosphorylation site located within the A box of its DNA-binding domain has been identified, and its role in phosphorylation-dependent inhibition of HNF4 DNA-binding activity has been investigated. Mutants of HNF4 in which two potentially phosphorylatable serines have been replaced by either neutral or charged amino acids were able to bind DNA in vitro with affinity similar to that of the wild-type protein. However, phosphorylation by PKA strongly repressed the binding affinity of the wild-type factor but not that of HNF4 mutants. Accordingly, in transfection assays, expression vectors for the mutated HNF4 proteins activated transcription more efficiently than that for the wild-type protein-when cotransfected with the PKA catalytic subunit expression vector. Therefore, HNF4 is a direct target of PKA which might be involved in the transcriptional inhibition of liver genes by cAMP inducers.

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