Redox-mediated upregulation of hepatocyte iNOS transcription requires coactivator PC4

Abstract

Background. Redox-mediated upregulation of transcription of hepatocyte inducible nitric oxide synthase (iNOS) requires hepatocyte nuclear factor IV-alpha (HNF-4α). In this setting, PC4 is often isolated with HNF-4α 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-4α-regulated redox-sensitive hepatocyte iNOS transcription. Methods. Murine CCL9.1 hepatocytes were stimulated with interleukin-1β (IL-1β; 1000 U/mL) in the presence and absence of peroxide (H2O2; 50 nmol/L). Antisense and sense oligonucleotides to HNF-4α and PC4 were added selectively. Coimmunoprecipitation (Co-IP) studies determined the association between HNF-4α 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-4α to the iNOS promoter region. Results. Ablation of either HNF-4α or PC4 blunted the peroxide-mediated increase in the activation of the iNOS promoter. In IL-1β+H2O2 only, co-IP studies demonstrated the presence of an HNF-4α-PC4 protein complex, and chromatin immunoprecipitation assays demonstrated that this complex binds to the genomic iNOS promoter. Conclusions. Redox-mediated upregulation of hepatocyte iNOS transcription requires an HNF-4α-PC4 transcriptional complex. © 2005 Mosby, Inc. All rights reserved.