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 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.

Orphan nuclear receptor SHP interacts with and represses hepatocyte nuclear factor-6 (HNF-6) transactivation

2008 ◽  
Vol 413 (3) ◽  
pp. 559-569 ◽  
Author(s):  
Yong-Soo Lee ◽  
Don-Kyu Kim ◽  
Yong Deuk Kim ◽  
Ki Cheol Park ◽  
Minho Shong ◽  
...  
Keyword(s):  
Nuclear Receptor ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Target Genes ◽  
Consensus Sequence ◽  
Nuclear Factor ◽  
Orphan Nuclear Receptor ◽  
Hepatocyte Nuclear Factor ◽  
Mobility Shift ◽  
Endocrine Differentiation

SHP (small heterodimer partner; NR0B2) is an atypical orphan NR (nuclear receptor) that functions as a transcriptional co-repressor by interacting with a diverse set of NRs and transcriptional factors. HNF-6 (hepatocyte nuclear factor-6) is a key regulatory factor in pancreatic development, endocrine differentiation and the formation of the biliary tract, as well as glucose metabolism. In this study, we have investigated the function of SHP as a putative repressor of HNF-6. Using transient transfection assays, we have shown that SHP represses the transcriptional activity of HNF-6. Confocal microscopy revealed that both SHP and HNF-6 co-localize in the nuclei of cells. SHP physically interacted with HNF-6 in protein–protein association assays in vitro. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays demonstrated that SHP inhibits the DNA-binding activity of HNF-6 to an HNF-6-response element consensus sequence, and the HNF-6 target region of the endogenous G6Pase (glucose 6-phosphatase) promoter respectively. Northern blot analysis of HNF-6 target genes in cells infected with adenoviral vectors for SHP and SHP siRNAs (small inhibitory RNAs) indicated that SHP represses the expression of endogenous G6Pase and PEPCK (phosphoenolpyruvate carboxykinase). Our results suggest that HNF-6 is a novel target of SHP in the regulation of gluconeogenesis.

Abstract 248: Hepatocyte Nuclear Factor 4α Regulates Lipoprotein Metabolism and Atherosclerosis

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Yang Xu ◽  
Munaf Zalzala ◽  
Yuanyuan Li ◽  
Yanqiao Zhang
Keyword(s):  
Critical Role ◽  
Lipoprotein Metabolism ◽  
Clinical Importance ◽  
Nuclear Hormone Receptor ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Lipid Disorder ◽  
Atherosclerotic Lesions ◽  
Hepatocyte Nuclear Factor 4Α ◽  
Hepatocyte Nuclear Factor 4Alpha

Hepatocyte nuclear factor 4alpha (HNF4a) is a nuclear hormone receptor and highly expressed in the liver. Mutations in human HNF4a causes maturity-onset diabetes of the young type 1 (MODY1) and lipid disorder. We investigated the role of loss of hepatic HNF4a in lipid and lipoprotein metabolism using both adenovirus-mediated gene knockdown (Ad-shHNF4a) and liver-specific HNF4a knockout (L-HNF4a-/-) mice. Our data show that loss of hepatic HNF4a caused hypolipidemia and fatty liver disease. Plasma total cholesterol and triglyceride levels were significantly reduced whereas hepatic triglyceride levels were markedly increased. There effects were caused by impaired VLDL secretion. We further determined the effect of loss of hepatic HNF4a in atherosclerosis using ApoE-/- or Ldlr-/- mice. Our data show that loss of hepatic HNF4a caused a marked reduction in the atherosclerotic lesions when mice were challenged with a Western diet. Our data indicate that hepatic HNF4a plays a critical role in modulating lipid and lipoprotein metabolism and the development of atherosclerosis. Since human HNF4a mutations are found in MODY1 patients, this study may be of clinical importance.

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 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.

scholarly journals Hepatocyte Nuclear Factor 4α, a Key Factor for Homeostasis, Cell Architecture, and Barrier Function of the Adult Intestinal Epithelium

2009 ◽  
Vol 29 (23) ◽  
pp. 6294-6308 ◽  
Author(s):  
Anne-Laure Cattin ◽  
Johanne Le Beyec ◽  
Frederick Barreau ◽  
Susan Saint-Just ◽  
Anne Houllier ◽  
...  
Keyword(s):  
Intestinal Epithelium ◽  
Cell Function ◽  
Integrated Control ◽  
Cell Lineage ◽  
Cell Junctions ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Cell Architecture ◽  
Hepatocyte Nuclear Factor 4Α

ABSTRACT Hepatocyte nuclear factor 4α (HNF-4α) is a transcription factor which is highly expressed in the intestinal epithelium from duodenum to colon and from crypt to villus. The homeostasis of this constantly renewing epithelium relies on an integrated control of proliferation, differentiation, and apoptosis, as well as on the functional architecture of the epithelial cells. In order to determine the consequences of HNF-4α loss in the adult intestinal epithelium, we used a tamoxifen-inducible Cre-loxP system to inactivate the Hnf-4a gene. In the intestines of adult mice, loss of HNF-4α led to an increased proliferation in crypts and to an increased expression of several genes controlled by the Wnt/β-catenin system. This control of the Wnt/β-catenin signaling pathway by HNF-4α was confirmed in vitro. Cell lineage was affected, as indicated by an increased number of goblet cells and an impairment of enterocyte and enteroendocrine cell maturation. In the absence of HNF-4α, cell-cell junctions were destabilized and paracellular intestinal permeability increased. Our results showed that HNF-4α modulates Wnt/β-catenin signaling and controls intestinal epithelium homeostasis, cell function, and cell architecture. This study indicates that HNF-4α regulates the intestinal balance between proliferation and differentiation, and we hypothesize that it might act as a tumor suppressor.

Hepatocyte nuclear factor-4α regulates the human organic anion transporter 1 gene in the kidney

2007 ◽  
Vol 292 (6) ◽  
pp. F1819-F1826 ◽  
Author(s):  
Ken Ogasawara ◽  
Tomohiro Terada ◽  
Jun-ichi Asaka ◽  
Toshiya Katsura ◽  
Ken-ichi Inui
Keyword(s):  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Organic Anion ◽  
Critical Role ◽  
Direct Repeat ◽  
Organic Anion Transporter ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Mobility Shift ◽  
Anion Transporter

Human organic anion transporter 1 (OAT1, SLC22A6), which is localized to the basolateral membranes of renal tubular epithelial cells, plays a critical role in the excretion of anionic compounds. OAT1 is regulated by various pathophysiological conditions, but little is known about the molecular mechanisms regulating the expression of OAT1. In the present study, we investigated the transcriptional regulation of OAT1 and found that hepatocyte nuclear factor (HNF)-4α markedly transactivated the OAT1 promoter. A deletion analysis of the OAT1 promoter suggested that the regions spanning −1191 to −700 base pairs (bp) and −140 to −79 bp were essential for the transactivation by HNF-4α. These regions contained a direct repeat separated by two nucleotides (DR-2), which is one of the consensus sequences binding to HNF-4α, and an inverted repeat separated by eight nucleotides (IR-8), which was recently identified as a novel element for HNF-4α, respectively. An electrophoretic mobility shift assay showed that HNF-4α bound to DR-2 and IR-8 under the conditions of HNF-4α overexpression. Furthermore, under normal conditions, HNF-4α bound to IR-8, and a mutation in IR-8 markedly reduced the OAT1 promoter activity, indicating that HNF-4α regulates the basal transcription of OAT1 via IR-8. This paper reports the first characterization of the human OAT1 promoter and the first gene in the kidney whose promoter activity is regulated by HNF-4α.

scholarly journals The G115S mutation associated with maturity-onset diabetes of the young impairs hepatocyte nuclear factor 4α activities and introduces a PKA phosphorylation site in its DNA-binding domain

2004 ◽  
Vol 383 (3) ◽  
pp. 573-580 ◽  
Author(s):  
Bénédicte OXOMBRE ◽  
Mostafa KOUACH ◽  
Ericka MOERMAN ◽  
Pierre FORMSTECHER ◽  
Bernard LAINE
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Transcriptional Activity ◽  
Phosphorylation Site ◽  
Binding Domain ◽  
Nuclear Factor ◽  
Dna Binding Domain ◽  
Maturity Onset Diabetes ◽  
Hepatocyte Nuclear Factor ◽  
Hepatocyte Nuclear Factor 4Α

HNF4α (hepatocyte nuclear factor 4α) belongs to a complex transcription factor network that is crucial for the function of hepatocytes and pancreatic β-cells. In these cells, it activates the expression of a very large number of genes, including genes involved in the transport and metabolism of glucose and lipids. Mutations in the HNF4α gene correlate with MODY1 (maturity-onset diabetes of the young 1), a form of type II diabetes characterized by an impaired glucose-induced insulin secretion. The MODY1 G115S (Gly115→Ser) HNF4α mutation is located in the DNA-binding domain of this nuclear receptor. We show here that the G115S mutation failed to affect HNF4α-mediated transcription on apolipoprotein promoters in HepG2 cells. Conversely, in pancreatic β-cell lines, this mutation resulted in strong impairments of HNF4α transcriptional activity on the promoters of LPK (liver pyruvate kinase) and HNF1α, with this transcription factor playing a key role in endocrine pancreas. We show as well that the G115S mutation creates a PKA (protein kinase A) phosphorylation site, and that PKA-mediated phosphorylation results in a decreased transcriptional activity of the mutant. Moreover, the G115E (Gly115→Glu) mutation mimicking phosphorylation reduced HNF4α DNA-binding and transcriptional activities. Our results may account for the 100% penetrance of diabetes in human carriers of this mutation. In addition, they suggest that introduction of a phosphorylation site in the DNA-binding domain may represent a new mechanism by which a MODY1 mutation leads to loss of HNF4α function.

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