The mitochondrial permeability transition augments Fas-induced apoptosis in mouse hepatocytes

Tumor necrosis factor-alpha receptor 1 and Fas recruit overlapping signaling pathways. To clarify the differences between tumor necrosis factor alpha (TNF alpha) and Fas pathways in hepatocyte apoptosis, primary mouse hepatocytes were treated with TNF alpha or an agonist anti-Fas antibody after infection with an adenovirus expressing an I kappa B superrepressor (Ad5I kappa B). Treatment with TNF alpha induced apoptosis in Ad5I kappa B-infected mouse hepatocytes, as we previously reported for rat hepatocytes. Ad5I kappa B plus anti-Fas antibody or actinomycin D plus anti-Fas antibody rapidly induced apoptosis, whereas anti-Fas antibody alone produced little cytotoxicity. The proteasome inhibitor (MG-132) and a dominant-negative mutant of nuclear factor-kappa B-inducing kinase also promoted TNF alpha- and Fas-mediated apoptosis. Expression of either crmA or a dominant-negative mutant of the Fas-associated death domain protein prevented TNF alpha- and Fas-mediated apoptosis. In addition, the caspase inhibitors, DEVD-cho and IETD-fmk, inhibited TNF alpha- and Fas-mediated apoptosis. In Ad5I kappa B-infected hepatocytes, caspases-3 and -8 were activated within 2 h after treatment with anti-Fas antibody or within 6 h after TNF alpha treatment. Confocal microscopy demonstrated onset of the mitochondrial permeability transition (MPT) and mitochondrial depolarization by 2-3 h after anti-Fas antibody treatment and 8-10 h after TNF alpha treatment, followed by cytochrome c release. The combination of the MPT inhibitors, cyclosporin A, and trifluoperazine, protected Ad5I kappa B-infected hepatocytes from TNF alpha-mediated apoptosis. After anti-Fas antibody, cyclosporin A and trifluoperazine decreased cytochrome c release but did not prevent caspase-3 activation and cell-death. In conclusion, nuclear factor-kappa B activation protects mouse hepatocytes against both TNF alpha- and Fas-mediated apoptosis. TNF alpha and Fas recruit similar but nonidentical, pathways signaling apoptosis. The MPT is obligatory for TNF alpha-induced apoptosis. In Fas-mediated apoptosis, the MPT accelerates the apoptogenic events but is not obligatory for them.