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


METHODS
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.


RESULTS
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.


CONCLUSIONS
Redox-mediated upregulation of hepatocyte iNOS transcription requires an HNF-4alpha-PC4 transcriptional complex.
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
Carlos E
CE
Marroquin
Philip Y
PY
Wai
Paul C
PC
Kuo
Hongtao
H
Guo
D000818:Animals; D004268:DNA-Binding Proteins / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D051557:Hepatocyte Nuclear Factor 4; D022781:Hepatocytes / Q000201:enzymology / Q000502:physiology*; D017874:Immediate-Early Proteins; D047468:Immunoprecipitation; D008565:Membrane Proteins; D051379:Mice; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D010084:Oxidation-Reduction; D010750:Phosphoproteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology; D014162:Transfection; D015854:Up-Regulation
D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C490042:Ifrd2 protein, mouse; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse