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

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.