RETRACTED: Phosphorylation of Ser158 regulates inflammatory redox-dependent hepatocyte nuclear factor-4α transcriptional activity (Retracted article. See vol. 461, pg. 347, 2014)

In IL-1 beta (interleukin 1 beta)-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 HNF4 alpha (hepatocyte nuclear factor-4 alpha). HNF4 alpha 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 HNF4 alpha have not been well characterized. In the setting of IL-1 beta + H2O2, HNF4 alpha functional activity is associated with a unique serine/threonine phosphorylation pattern. This indicates that an IRX-sensitive serine/threonine kinase pathway targets HNF4 alpha to augment hepatocyte iNOS transcription. In the present study, following identification of phosphorylated residues in HNF4 alpha, 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 HNF4 alpha DNA binding and transactivation. Gain-of-function mutation with the S158D phosphomimetic HNF4a vector supports a critical role for Ser(158) phosphorylation. In vitro phosphorylation and kinase inhibitor studies implicate p38 kinase activity. Our results indicate that p38 kinase-mediated Ser(158) phosphorylation is essential for augmentation of the DNA binding and transactivation potential of HNF4 alpha in the presence of IL-1 beta + H2O2. This pathway results in enhanced iNOS expression in hepatocytes exposed to pro-inflammatory cytokines and oxidative stress.