Phosphorylation of Ser158 regulates inflammatory redox-dependent hepatocyte nuclear factor-4alpha transcriptional activity

Abstract

In IL-1beta (interleukin 1beta)-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 HNF4alpha (hepatocyte nuclear factor-4alpha). HNF4alpha 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 HNF4alpha have not been well characterized. In the setting of IL-1beta+H2O2, HNF4alpha functional activity is associated with a unique serine/threonine phosphorylation pattern. This indicates that an IRX-sensitive serine/threonine kinase pathway targets HNF4alpha to augment hepatocyte iNOS transcription. In the present study, following identification of phosphorylated residues in HNF4alpha, 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 HNF4alpha DNA binding and transactivation. Gain-of-function mutation with the S158D phosphomimetic HNF4alpha 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 HNF4alpha in the presence of IL-1beta+H2O2. This pathway results in enhanced iNOS expression in hepatocytes exposed to pro-inflammatory cytokines and oxidative stress.

Figure 1. HNF4α DNA binding

(A) ChIP assay of iNOS promoter HNF4α binding. Chromatin from hepatocytes was fixed and immunoprecipitated using the ChIP assay kit as recommended by the manufacturer. Goat anti-human polyclonal HNF4α antibody (5 μg, Santa Cruz Biotechnology) was used for each immunoprecipitation; 5 μl of rabbit IgG served as a control. The DNA was recovered and subjected to analysis by PCR. The primers had the following sequence: 5′-tgaccaattgactggtatgtgtg-3′ (sense strand) and 5′-gctgggctggggagatggctga-3′ (antisense strand) to yield a PCR product of 280 bp. The input fraction corresponded to 0.1% of the chromatin solution before immunoprecipitation. After DNA purification, the presence of the selected DNA sequence was assessed by PCR. The blot is representative of four experiments. H₂O₂ (50 μM); STA, staurosporine (0.5 μM). (B) EMSA analysis of in vitro WT and mutant HNF4α binding. EMSAs were performed using 10 μg of FLAG-tagged WT or mutant HNF4 peptide. Unlabelled target oligonucleotide was added at 200 M excess in specific or non-specific competition assays (Spec Comp or Nonspec Comp respectively). For supershift experiments, FLAG antibody was included in the binding reaction. Probe was prepared by end-labelling the WT 28 bp double-stranded ARE (antioxidant response element) with [γ-³²P]ATP (2500 Ci/mmol) using T4 polynucleotide kinase, followed by gel purification on 15% polyacrylamide. The gel is representative of five experiments. Ab, antibody.

Figure 2. Transient transfection analysis of iNOS promoter activity in COS-1 cells

Cells were transfected using the Lipofectamine™ technique. After cells were washed twice with MEM, 10 μg of plasmid DNA containing the iNOS promoter construct (1845 bp; GenBank® accession no. X95629) coupled with a CAT reporter gene were added. In selected instances, an empty vector or an HNF4α expression vector (10 μg) encoding FLAG-tagged WT or mutant variants was co-transfected with the iNOS promoter plasmid construct. After 24 h, cells were stimulated with IL-1β (1000 units/ml) in the presence and absence of H₂O₂ (50 μM). Unstimulated cells served as controls. After 6 h, the supernatant was assayed for CAT activity using a CAT ELISA technique. Transfection efficiency was normalized by co-transfection of a β-galactosidase reporter gene with a constitutively active early SV40 promoter. All values are expressed as pg of CAT/mg of protein. Results are expressed as means±S.E.M. for three experiments (*P<0.01 versus control, IL-1β- or H₂O₂-treated cells).

Figure 3. Kinase inhibitors and in vitro DNA binding of WT and mutant HNF4α peptides

COS-1 cells were transfected with empty vector or FLAG-tagged WT or mutant (S158A or S304A) HNF4α expression vectors. Cells were treated with IL-1β+H₂O₂ in the presence or absence of increasing concentrations of p38, PKA or PKC inhibitors. Unstimulated cells served as a control. EMSAs were performed using 10 μg of isolated HNF4 peptide. For the supershift experiments, anti-FLAG antibody (Kodak) was included in the binding reaction. Probe was prepared by end-labelling WT 28 bp double-stranded ARE with [γ-³²P]ATP (2500 Ci/mmol) using T4 polynucleotide kinase, followed by gel purification on 15% polyacrylamide. The gel is representative of five experiments. NP, not performed; Ab, antibody.

Figure 4. In vitro DNA binding of an S158D phosphomimetic mutant of HNF4α

COS-1 cells were transfected with empty vector or FLAG-tagged WT or mutant (S158A or S158D) HNF4α expression vectors. Cells were treated as indicated. Unstimulated cells served as a control. EMSAs were performed using 10 μg of isolated HNF4 peptide. Probe was prepared by end-labelling the WT 28 bp double-stranded ARE with [γ-³²P]ATP (2500 Ci/mmol) using T4 polynucleotide kinase, followed by gel purification on 15% polyacrylamide. The gel is representative of three experiments.

Figure 5. HNF4α phosphorylation and transactivation

(A) In vitro phosphorylation of HNF4α. Purified FLAG-tagged WT or mutant HNF4α protein was incubated with 1 μl of protein kinase (p38, JNK, or ERK1/2). The reaction products were immunoprecipitated with anti-FLAG antibody at 0 and 20 min, followed by separation on SDS/4–20% polyacrylamide gels; the gel was then dried and analysed by autoradiography. The blot is representative of three experiments. (B) Transient transfection analysis of iNOS promoter activity in COS-1 cells. Cells were transfected using the Lipofectamine™ technique. After cells were washed twice with medium, 10 μg of plasmid DNA containing the iNOS promoter construct (1845 bp; GenBank® accession no. X95629) coupled with a CAT reporter gene were added. In selected instances, an empty vector or an HNF4α expression vector (10 μg) encoding FLAG-tagged WT or mutant variants was co-transfected with the iNOS promoter plasmid construct. In selected instances, the p38 kinase inhibitor was also added. Cells were stimulated with IL-1β (1000 units/ml) in the presence and absence of H₂O₂ (50 μM). Unstimulated cells served as controls. After 6 h, the supernatant was assayed for CAT activity using a CAT ELISA technique. Transfection efficiency was normalized by co-transfection of a β-galactosidase reporter gene with a constitutively active early SV40 promoter. All values are expressed as pg of CAT/mg of protein. Results are expressed as means±S.E.M. for three experiments (*P<0.01 versus control or H₂O₂-treated cells; **P<0.01 versus control, IL-1β- or H₂O₂-treated cells).

Figure 6. Co-immunoprecipitation of HNF4α and PC4

HepG2 cells were transfected with FLAG-tagged WT-HNF4α or S158A-HNF4α by Lipofectin® (Invitrogen) and exposed to IL-1β+H₂O₂. Immunoprecipitation (IP) was performed with anti-FLAG M2–agarose affinity gel (Sigma). Polyclonal anti-PC4 antibody (Santa Cruz Biotechnology) was used for immunoblot (IB) analysis. Membranes were then exposed to secondary antibody coupled with horseradish peroxidase and visualized. The blot is representative of three experiments.