Cathepsin B contributes to TNF-α–mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c
M. Eugenia Guicciardi, … , Scott H. Kaufmann, Gregory J. Gores
M. Eugenia Guicciardi, … , Scott H. Kaufmann, Gregory J. Gores
Published November 1, 2000
Citation Information: J Clin Invest. 2000;106(9):1127-1137. https://doi.org/10.1172/JCI9914.
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Cathepsin B contributes to TNF-α–mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c

Abstract

TNF-α–induced apoptosis is thought to involve mediators from acidic vesicles. Cathepsin B (cat B), a lysosomal cysteine protease, has recently been implicated in apoptosis. To determine whether cat B contributes to TNF-α–induced apoptosis, we exposed mouse hepatocytes to the cytokine in vitro and in vivo. Isolated hepatocytes treated with TNF-α in the presence of the transcription inhibitor actinomycin D (AcD) accumulated cat B in their cytosol. Further experiments using cell-free systems indicated that caspase-8 caused release of active cat B from purified lysosomes and that cat B, in turn, increased cytosol-induced release of cytochrome c from mitochondria. Consistent with these observations, the ability of TNF-α/AcD to induce mitochondrial release of cytochrome c, caspase activation, and apoptosis 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-α and an adenovirus construct expressing the IκB superrepressor. Collectively, these observations suggest that caspase-mediated release of cat B from lysosomes enhances mitochondrial release of cytochrome c and subsequent caspase activation in TNF-α–treated hepatocytes.

Authors

M. Eugenia Guicciardi, Jan Deussing, Hideyuki Miyoshi, Steven F. Bronk, Phyllis A. Svingen, Christoph Peters, Scott H. Kaufmann, Gregory J. Gores

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Figure 7

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Cat B–induced release of cytochrome c from mitochondria is enhanced by c...
Cat B–induced release of cytochrome c from mitochondria is enhanced by cytosol and is not due to a nonspecific proteolytic effect. Isolated mitochondria from catB+/+ mouse liver (25 μg protein) were incubated at 37°C with increasing concentrations of purified recombinant cat B (5–50 ng) (a) or m-calpain (10 ng) (b), in the presence or in the absence of S-100 cytosol fraction (50 μg) as described in Methods. Mitochondria were also treated with 0.1% Triton X-100 to induce maximum release of cytochrome c (positive control). After 1 hour, mitochondria were pelleted by centrifugation at 12,000 g for 5 min. Supernatants were subjected to SDS-PAGE on 15% acrylamide gels, transferred to nitrocellulose, and immunoblotted for cytochrome c. Blots were also probed for cytochrome c oxidase (subunit IV) to exclude mitochondria contamination in the supernatant. (c) Active cat B induces release of cytochrome c from isolated catB–/– mouse liver mitochondria in the presence of cytosol. Isolated mitochondria from catB–/– mouse liver (25 μg protein) were incubated at 37°C with purified recombinant cat B (25 ng), in the presence or in the absence of S-100 cytosol fraction (50 μg) obtained from the same animal. Mitochondria were also treated with 0.1% Triton X-100 to induce maximum release of cytochrome c (positive control). After 1 hour, mitochondria were pelleted by centrifugation at 12,000 g for 5 minutes, and the resulting supernatants were subjected to SDS-PAGE and subsequent immunoblot analysis for cytochrome c as already above. Immunoblot for cytochrome c oxidase (subunit IV) was also performed to exclude mitochondria contamination in the supernatant.