The mitochondrial permeability transition is required for tumor necrosis factor alpha-mediated apoptosis and cytochrome c release

This study assesses the controversial role of the mitochondrial permeability transition (MPT) in apoptosis, In primary rat hepatocytes expressing an I kappa B superrepressor, tumor necrosis factor alpha (TNF alpha) induced apoptosis as shown by nuclear morphology, DNA ladder formation, and caspase 3 activation. Confocal microscopy showed that TNF alpha induced onset of the MPT and mitochondrial depolarization beginning 9 h after TNF alpha treatment. Initially, depolarization and the MPT occurred in only a subset of mitochondria; however, by 12 h after TNF alpha treatment, virtually all mitochondria were affected, Cyclosporin A (CsA), an inhibitor of the MPT, blocked TNF alpha-mediated apoptosis and cytochrome c release. Caspase 3 activation, cytochrome c release, and apoptotic nuclear morphological changes were induced after onset of the MPT and were prevented by CsA. Depolarization and onset of the MPT were blocked in hepatocytes expressing Delta FADD, a dominant negative mutant of Fas-associated protein with death domain (FADD), or crmA, a natural serpin inhibitor of caspases, In contrast, Asp-Glu-Val-Asp-cho, an inhibitor of caspase 3, did not block depolarization or onset of the MPT induced by TNF alpha, although it inhibited Fell death completely. In conclusion, the MPT is an essential component in the signaling pathway for TNF alpha-induced apoptosis in hepatocytes which is required for both cytochrome c release and cell death and functions downstream of FADD and crmA but upstream of caspase 3.