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Redox regulation of the rat hepatocyte iNOS promoter
C Punzalan1, C Cai, R A Schroeder
1Department of Surgery, Georgetown University Medical Center, Washington, DC, USA.
Background:
The purpose of this study was to define the redox sensitive cis-acting transcriptional mechanisms that regulate inducible nitric oxide synthase (iNOS) promoter function in the hepatocyte.
Methods:
Clonal deletion constructs of the rat hepatocyte iNOS promoter (Genbank X95629; 1845 base pair)-reporter plasmid were transiently transfected into HepG2 cells treated with IL-1 beta and IL-1 beta + hydrogen peroxide.
Results:
A segment of the promoter upstream from nucleotide -1126 was associated with redox-sensitive augmentation of promoter activity. Site-directed mutagenesis of 2 antioxidant response elements (AREs) was combined with transfection analysis to demonstrate that mutation of the ARE at nt-1347 ablated oxidant stress-mediated activation of the iNOS promoter.
Conclusions:
This ARE conveys the redox-sensitive response of the rat iNOS promoter. Hepatocyte iNOS expression is a novel and, as yet, poorly described antioxidant mechanism that is cytokine and redox sensitive and that plays a pleuripotent regulatory role in hepatocellular function in the face of sepsis and shock.