Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c

TNF-alpha -induced apoptosis is thought to involve mediators from acidic ve sicles. Cathepsin B (cat B), a lysosomal cysteine protease, has recently be en implicated in apoptosis. To determine whether cat B contributes to TNF-a lpha -induced apoptosis, we exposed mouse hepatocytes to the cytokine in vi tro and in vivo. Isolated hepatocytes treated with TNF-alpha in the presenc e of the transcription inhibitor actinomycin D (AcD) accumulated cat B in t heir cytosol. Further experiments using cell-free systems indicated that ca spase-8 caused release of active cat B from purified lysosomes and that cat B, in turn, increased cytosol-induced release of cytochrome c from mitocho ndria. Consistent with these observations, the ability of TNF-alpha /AcD to induce mitochondrial release of cytochrome c, caspase activation, and apop tosis of isolated hepatocytes was markedly diminished in cells from CatB(-/ -) mice. Deletion of the CatB gene resulted in diminished Liver injury and enhanced survival after treatment in vivo with TNF-alpha and an adenovirus construct expressing the I kappaB superrepressor. Collectively, these obser vations suggest that caspase-mediated release of cat B from lysosomes enhan ces mitochondrial release of cytochrome c and subsequent caspase activation in TNF-alpha -treated hepatocytes.