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 permea bility transition (MPT) in apoptosis, In primary rat hepatocytes expre ssing an I kappa B superrepressor, tumor necrosis factor alpha (TNF al pha) induced apoptosis as shown by nuclear morphology, DNA ladder form ation, and caspase 3 activation. Confocal microscopy showed that TNF a lpha induced onset of the MPT and mitochondrial depolarization beginni ng 9 h after TNF alpha treatment. Initially, depolarization and the MP T occurred in only a subset of mitochondria; however, by 12 h after TN F alpha treatment, virtually all mitochondria were affected, Cyclospor in A (CsA), an inhibitor of the MPT, blocked TNF alpha-mediated apopto sis and cytochrome c release. Caspase 3 activation, cytochrome c relea se, and apoptotic nuclear morphological changes were induced after ons et of the MPT and were prevented by CsA. Depolarization and onset of t he MPT were blocked in hepatocytes expressing Delta FADD, a dominant n egative 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 o nset of the MPT induced by TNF alpha, although it inhibited Fell death completely. In conclusion, the MPT is an essential component in the s ignaling 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.