Redox-mediated upregulation of hepatocyte iNOS transcription requires coactivator PC4.

Abstract

Redox-mediated upregulation of transcription of hepatocyte inducible nitric oxide synthase (iNOS) requires hepatocyte nuclear factor IV-alpha (HNF-4alpha). In this setting, PC4 is often isolated with HNF-4alpha in DNA-protein pull-down studies. Transcriptional coactivator PC4 facilitates activator-dependent transcription via interactions with basal transcriptional machinery that are independent of PC4-DNA binding. We hypothesized that PC4 is a necessary component of HNF-4alpha-regulated redox-sensitive hepatocyte iNOS transcription. Murine CCL9.1 hepatocytes were stimulated with interleukin-1beta (IL-1beta; 1000 U/mL) in the presence and absence of peroxide (H(2)O(2); 50 nmol/L). Antisense and sense oligonucleotides to HNF-4alpha and PC4 were added selectively. Coimmunoprecipitation (Co-IP) studies determined the association between HNF-4alpha and PC4. Transient transfection was performed with the use of a luciferase reporter construct containing the murine iNOS promoter (1.8 kb). Chromatin immunoprecipitation assays determined in vivo binding of PC4 and HNF-4alpha to the iNOS promoter region. Ablation of either HNF-4alpha or PC4 blunted the peroxide-mediated increase in the activation of the iNOS promoter. In IL-1beta+H(2)O(2) only, co-IP studies demonstrated the presence of an HNF-4alpha-PC4 protein complex, and chromatin immunoprecipitation assays demonstrated that this complex binds to the genomic iNOS promoter. Redox-mediated upregulation of hepatocyte iNOS transcription requires an HNF-4alpha-PC4 transcriptional complex.