Mechanism of mitochondrial glutathione-dependent hepatocellular susceptibility to TNF despite NF-kappaB activation.
OBJECTIVE
Nuclear factor kappaB (NF-kappaB) is the master regulator of tumor necrosis factor (TNF) susceptibility. Although mitochondrial glutathione (mGSH) depletion was shown to sensitize hepatocytes to TNF despite NF-kappaB activation, the mechanisms involved, particularly the role of Bax oligomerization and mitochondrial outer membrane (MOM) permeabilization, 2 critical steps in cell death, remained unexplored.


METHODS
TNF signaling at the premitochondrial and mitochondrial levels was analyzed in primary mouse hepatocytes with or without mGSH depletion.


RESULTS
Unexpectedly, we observed that TNF activates caspase-8 independently of NF-kappaB inactivation, causing Bid cleavage and mitochondrial Bax oligomerization. However, their predicted consequences on MOM permeabilization, cytochrome c release, caspase-3 activation, and hepatocellular death occurred only on mGSH depletion. These events were preceded by stimulated mitochondrial reactive oxygen species that predominantly oxidized cardiolipin, changes not observed in acidic sphingomyelinase (ASMase)(-/-) hepatocytes. Oxidized cardiolipin potentiated oligomerized Bax-induced MOM-like liposome permeabilization by restructuring the lipid bilayer, without effect on membrane Bax insertion or oligomerization. ASMase(-/-) mice with mGSH depletion by cholesterol loading were resistant to TNF-induced liver injury in vivo.


CONCLUSIONS
Thus, MOM-localized oligomeric Bax is not sufficient for TNF-induced MOM permeabilization and cell death requiring mGSH-controlled ASMase-mediated mitochondrial membrane remodeling by oxidized cardiolipin generation.
Marí
Montserrat
M
Liver Unit and Centro de Investigaciones Biomédicas Esther Koplowitz, IMDiM, Hospital Clínic i Provincial, and CIBEREHD, IDIBAPS, Barcelona, Spain.
Colell
Anna
A
Morales
Albert
A
Caballero
Francisco
F
Moles
Anna
A
Fernández
Anna
A
Terrones
Oihana
O
Basañez
Gorka
G
Antonsson
Bruno
B
García-Ruiz
Carmen
C
Fernández-Checa
José C
JC
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002308:Cardiolipins / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005234:Fatty Liver / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology; D005786:Gene Expression Regulation; D005978:Glutathione / Q000378:metabolism*; D022781:Hepatocytes / Q000378:metabolism* / Q000473:pathology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D018613:Microscopy, Confocal; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D012313:RNA / Q000235:genetics*; D017382:Reactive Oxygen Species / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology* / Q000633:toxicity; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D002308:Cardiolipins; D016328:NF-kappa B; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D051028:bcl-2-Associated X Protein; D012313:RNA; D005978:Glutathione