Early mitochondrial activation and cytochrome c up-regulation during apoptosis

Apoptosis induced by many stimuli requires the mitochondrial respiratory chain (MRC) function. While studying the molecular mechanisms underlying this AIRC-dependent apoptotic pathway, we find that apoptosis in multiple cell types induced by a variety of stimuli is preceded by an early induction of MRC proteins such as cytochrome c (which is encoded by a nuclear gene) and cytochrome c oxidase subunit II (COX II) (which is encoded by the mitochondrial genome). Several non-MRC proteins localized in the mitochondria, e.g. Smac, Bim, Bak, and Bcl-2, are also rapidly up-regulated. The up-regulation of many of these proteins (e.g. cytochrome c, COX II, and Bim) results from transcriptional activation of the respective genes. The up-regulated cytosolic cytochrome c rapidly translocates to the mitochondria, resulting in an accumulation of holocytochrome c in the mitochondria accompanied by increasing holocytochrome c release into the cytosol. The increased cytochrome c transport from cytosol to the mitochondria does not depend on the mitochondrial protein synthesis or MRC per se. In contrast, cytochrome c release from the mitochondria involves dynamic changes in Bcl-2 family proteins (e.g. up-regulation of Bak, Bcl-2, and Bcl-x(L)), opening of permeability transition pore, and loss of mitochondrial membrane potential. Overexpression of cytochrome c enhances caspase activation and promotes cell death in response to apoptotic stimulation, but simple up-regulation of cytochrome c using an ecdysone-inducible system is, by itself, insufficient to induce apoptosis. Taken together, these results suggest that apoptosis induced by many stimuli involves an early mitochondrial activation, which may be responsible for the subsequent disruption of MRC functions, loss of Deltapsi(m), cytochrome c release, and ultimately cell death.