Superoxide enhances interleukin 1beta-mediated transcription of the hepatocyte-inducible nitric oxide synthase gene.
OBJECTIVE
Exposure to oxidative stress, as in states of shock, ischemia-reperfusion injury, or sepsis, commonly initiates a complex cellular cascade of interlocking redox modulatory systems that detoxify electrophiles. In interleukin 1beta (IL-1beta)-treated rat hepatocytes, we have previously demonstrated that inducible nitric oxide synthase (iNOS) protein expression, steady-state iNOS messenger RNA (mRNA) levels, and NO synthesis are increased by oxidative stress induced by superoxide. The effect of hepatocellular redox state upon iNOS gene transcription has not been previously studied.


METHODS
Using rat hepatocytes in primary culture, iNOS gene transcription was induced by IL-1beta. Oxidative stress was mediated by 1,2,3-benzenetriol (BZT), an autocatalytic source of superoxide. Nuclear run-on assays and transient transfection assays using the rat hepatocyte iNOS full-length promoter and deletion constructs were designed to isolate a cis-acting regulatory element. Specificity was confirmed by site-directed mutagenesis. Gel shift analysis determined the presence of a corresponding trans-acting regulatory factor.


RESULTS
In IL-1beta-treated cells, BZT increased iNOS gene transcription without altering mRNA half-life. An antioxidant-responsive element (ARE) was found in the iNOS promoter at base pair -1347, which conferred redox sensitivity. Gel shift analysis identified a corresponding nuclear protein capable of binding to ARE in IL-1beta- and BZT-treated rat hepatocytes.


CONCLUSIONS
An ARE in the rat hepatocyte iNOS promoter confers redox sensitivity and augments IL-1beta-mediated iNOS gene and protein expression in the setting of superoxide treatment.
Kuo
P C
PC
Department of Surgery, Georgetown University Medical Center, Washington, D.C, USA. kuop@gunet.georgetown.edu
Abe
K
K
Schroeder
R A
RA
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001483:Base Sequence / Q000235:genetics; D002478:Cells, Cultured; D004790:Enzyme Induction; D017353:Gene Deletion; D007375:Interleukin-1 / Q000502:physiology*; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D018384:Oxidative Stress / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D011748:Pyrogallol / Q000494:pharmacology; D051381:Rats; D011917:Rats, Inbred Lew; D020218:Response Elements / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*; D014162:Transfection
D000975:Antioxidants; D007375:Interleukin-1; D011748:Pyrogallol; D013481:Superoxides; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat