Genetic inactivation of RelA/p65 sensitizes adult mouse hepatocytes to TNF-induced apoptosis in vivo and in vitro.
OBJECTIVE
The transcription factor nuclear factor (NF)-kappaB plays a critical role in mediating survival of hepatocytes in response to tumor necrosis factor (TNF)-alpha during development because mice deficient for the NF-kappaB subunit RelA/p65 die in utero because of TNF-induced liver apoptosis. For the adult liver, conflicting concepts exist as to whether soluble TNF can trigger apoptosis when NF-kappaB activation is impaired. By creating a mouse model in which the transactivating NF-kappaB subunit RelA/p65 can be genetically inactivated in hepatocytes using the Cre/lox system, we sought to clarify the role of NF-kappaB in TNF-mediated hepatocyte apoptosis.


METHODS
Deletion of RelA/p65 in the liver was achieved using an inducible conditional knockout system (rela(F/F)MxCre mice) or, hepatocyte-specifically, using a developmental conditional knockout system (rela(F/F)AlbCre mice).


RESULTS
Disruption of RelA/p65 rendered mice sensitive to lethal liver injury upon TNF administration. Primary RelA/p65-deficient hepatocytes showed no NF-kappaB activation and undergo rapid apoptosis after TNF treatment. In contrast, hepatocytes deficient for I kappa B-kinase beta (IKK beta), displayed residual NF-kappaB activity and consecutively only mild apoptosis in response to TNF. TNF-induced apoptosis in RelA/p65-deficient hepatocytes was accompanied by prolonged activation of c-jun activating kinase (JNK) and rapid, largely proteasome-independent elimination of the long splice form of the antiapoptotic cellular FLICE inhibitor protein (c-FLIP(L)). Gene silencing of caspase-8, caspase-inhibitors, inhibition of JNK, or administration of antioxidants inhibited apoptosis and elimination of c-FLIP(L).


CONCLUSIONS
RelA/p65 is essential for TNF-induced NF-kappaB activation in adult hepatocytes. Genetic deletion of a functional RelA/p65 sensitizes these cells to apoptosis in response to soluble TNF in vivo and in vitro.
Geisler
Fabian
F
Second Department of Internal Medicine, Klinikum rechts der Isar, Technical University of Munich, Munich.
Algül
Hana
H
Paxian
Stephan
S
Schmid
Roland M
RM
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002083:Butylated Hydroxyanisole / Q000494:pharmacology; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000037:antagonists & inhibitors / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D002478:Cells, Cultured; D056486:Chemical and Drug Induced Liver Injury; D004789:Enzyme Activation; D020868:Gene Silencing; D022781:Hepatocytes / Q000378:metabolism / Q000502:physiology*; D051550:I-kappa B Kinase / Q000172:deficiency; D066298:In Vitro Techniques; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism / Q000502:physiology; D008025:Ligases / Q000172:deficiency / Q000235:genetics*; D051379:Mice; D018345:Mice, Knockout; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D000975:Antioxidants; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506387:Cflar protein, mouse; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D002083:Butylated Hydroxyanisole; D051550:I-kappa B Kinase; C496562:Ikbkb protein, mouse; D048031:JNK Mitogen-Activated Protein Kinases; D053181:Caspase 8; D008025:Ligases; C028812:guanosine 3',5'-polyphosphate synthetases