Mechanism of mitochondrial glutathione-dependent hepatocellular susceptibility to TNF despite NF-κB Activation

Abstract

[Background & Aims]: Nuclear factor κB (NF-κB) is the master regulator of tumor necrosis factor (TNF) susceptibility. Although mitochondrial glutathione (mGSH) depletion was shown to sensitize hepatocytes to TNF despite NF-κB 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-κB 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.