Nitric oxide from the inducible nitric oxide synthase (iNOS) increases the expression of cytochrome P450 2E1 in iNOS-null hepatocytes in the absence of inflammatory stimuli.
Nitric oxide (NO) can modulate numerous genes through several pathways, yet some genes may be modulated only in the presence of the inflammatory stimuli that upregulate the inducible nitric oxide synthase (iNOS) rather than by NO alone. Furthermore, the role of prior expression of iNOS in the modulation of genes by NO is unknown. We addressed these issues in hepatocytes harvested from iNOS-null (iNOS(-/-)) mice exposed to NO by treatment with NO donors or by infection with an adenovirus-expressing human iNOS (Ad-iNOS), rather than by stimulation with inflammatory cytokines. Differential display and gene array analyses performed on mRNA derived from iNOS(-/-) hepatocytes demonstrated that infection with Ad-iNOS, but not infection with a control adenovirus expressing the beta-galactosidase gene (Ad-LacZ), induced a gene fragment identical to cytochrome P450 2E1 (CYP2E1). Northern analysis performed with this fragment demonstrated that treatment of iNOS(-/-) hepatocytes with Ad-iNOS or with the NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP), but not control treatment or infection with Ad-LacZ, resulted in increased expression of CYP2E1. Inhibition of soluble guanylyl cyclase partially blocked the induction of CYP2E1 mRNA by Ad-iNOS. Rat hepatocytes treated with SNAP also exhibited increased expression of CYP2E1 mRNA. Preliminary studies, however, suggest that the induction of CYP2E1 in the rat hepatocytes treated with cytokines was not reduced in the presence of a NOS inhibitor. Our results suggest that CYP2E1 can be induced solely by NO derived from iNOS, at least partly in a cyclic GMP-dependent manner and independently of inflammatory stimuli or of prior exposure to NO.
Zamora
R
R
Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA. zamorar@pitt.edu
Vodovotz
Y
Y
Alarcon
L
L
Betten
B
B
Loughran
P A
PA
Aulak
K S
KS
Stuehr
D J
DJ
Gibson
K F
KF
Billiar
T R
TR
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000096:biosynthesis* / Q000235:genetics; D004790:Enzyme Induction; D022781:Hepatocytes / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D020411:Oligonucleotide Array Sequence Analysis; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D015854:Up-Regulation
D020030:Nitric Oxide Donors; D012333:RNA, Messenger; C110881:S-nitro-N-acetylpenicillamine; D009569:Nitric Oxide; D019392:Cytochrome P-450 CYP2E1; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C496318:Nos2 protein, rat; D010396:Penicillamine; D006152:Cyclic GMP