Inhibition of cytokine-induced nitric oxide synthase expression by gene transfer of adenoviral I kappa B alpha.
BACKGROUND
Nitric oxide is overexpressed in nearly every organ during sepsis and it has profound biologic effects. Previously, we showed that maximal inducible nitric oxide synthase (iNOS) expression is up-regulated by a combination of cytokines and that this effect is mediated by the transcription factor NF-kappa B. Therefore the purpose of this study was to establish whether gene transfer of the inhibitory molecule I kappa B would result in the abrogation of cytokine-induced iNOS expression.


METHODS
Cultured hepatocytes were infected with an adenoviral vector containing the I kappa B alpha gene (Ad5I kappa B) and after an 18-hour recovery period were stimulated with the cytokine mixture of tumor necrosis factor-alpha (500 U/mL) plus interleukin 1 beta (200 U/mL) plus interferon gamma (100 U/mL).


RESULTS
As expected, cytokine mixture induced significant hepatocyte nitrite (NO2-) and iNOS messenger RNA production. Cells infected with the I kappa B alpha gene showed a dose-dependent decrease in NO2- and iNOS messenger RNA levels. Western blot analysis showed a marked decrease in iNOS protein levels in the presence of Ad5I kappa B alpha. Gel shift assays of nuclear extracts demonstrated that Ad5I kappa B alpha decreased the cytokine-induced DNA binding activity for NF kappa B.


CONCLUSIONS
NF kappa B is an important regulator of cytokine-induced NO expression. These results identify a novel therapeutic approach where gene transfer of the inhibitory molecule I kappa B alpha can be used to down-regulate cytokine-induced iNOS expression as well as other NF kappa B-dependent genes that are up-regulated during the inflammatory response.
Taylor
B S
BS
Department of Surgery, University of Pittsburgh, PA 15261, USA.
Shao
L
L
Gambotto
A
A
Ganster
R W
RW
Geller
D A
DA
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D020672:I-kappa B Proteins; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008297:Male; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D052247:Nitric Oxide Synthase Type II; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606206:Nfkbia protein, rat; D012333:RNA, Messenger; D000072000:NF-KappaB Inhibitor alpha; D009569:Nitric Oxide; D004247:DNA; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat