NF-kappaB stimulates inducible nitric oxide synthase to protect mouse hepatocytes from TNF-alpha- and Fas-mediated apoptosis.
OBJECTIVE
Hepatocyte apoptosis is induced by tumor necrosis factor alpha (TNF-alpha) and Fas ligand. Although nuclear factor-kappaB (NF-kappaB) activation protects hepatocytes from TNF-alpha-mediated apoptosis, the NF-kappaB responsive genes that protect hepatocytes are unknown. Our aim was to study the role of NF-kappaB activation and inducible nitric oxide synthases (iNOSs) in TNF-alpha- and Fas-mediated apoptosis in hepatocytes.


METHODS
Primary cultures of hepatocytes from wild-type and iNOS knockout mice were treated with TNF-alpha, the Fas agonistic antibody Jo2, a nitric oxide (NO) donor (S-nitroso-N-acetylpenicillamine), an NO inhibitor (N(G)-methyl-L-arginine acetate), and/or adenovirus-expressing NF-kappaB inhibitors.


RESULTS
The IkappaB superrepressor and a dominant-negative form of IkappaB kinase beta (IKKbeta) inhibited NF-kappaB binding activity by TNF-alpha or Jo2 and sensitized hepatocytes to TNF-alpha- and Jo2-mediated apoptosis. TNF-alpha and Jo2 induced iNOS messenger RNA and protein levels through the induction of NF-kappaB. S-nitroso-N-acetylpenicillamine inhibited Bid cleavage, the mitochondrial permeability transition, cytochrome c release, and caspase-8 and -3 activity, and reduced TNF-alpha- and Fas-mediated death in hepatocytes expressing IkappaB superrepressor. N(G)-methyl-L-arginine acetate partially sensitized hepatocytes to TNF-alpha- and Fas-mediated cell killing. TNF-alpha alone or Jo2 alone induced moderate cell death in hepatocytes from iNOS(-)/(-) mice.


CONCLUSIONS
NO protects hepatocytes from TNF-alpha- and Fas-mediated apoptosis. Endogenous iNOS, which is activated by NF-kappaB via IKKbeta, provides partial protection from apoptosis.
Hatano
E
E
Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Bennett
B L
BL
Manning
A M
AM
Qian
T
T
Lemasters
J J
JJ
Brenner
D A
DA
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D022781:Hepatocytes / Q000166:cytology / Q000201:enzymology*; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000201:enzymology; D016296:Mutagenesis / Q000502:physiology; D016328:NF-kappa B / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010396:Penicillamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000494:pharmacology*; D019014:fas Receptor / Q000276:immunology / Q000494:pharmacology*
D000906:Antibodies; D051023:BH3 Interacting Domain Death Agonist Protein; C494815:Bid protein, mouse; D002352:Carrier Proteins; D003574:Cytochrome c Group; D020672:I-kappa B Proteins; D016328:NF-kappa B; D020030:Nitric Oxide Donors; C110881:S-nitro-N-acetylpenicillamine; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C505409:Casp3 protein, mouse; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D010396:Penicillamine