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 3

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catB–/– mouse hepatocytes are more resistant to TNF-α–induced apoptosis....
catB–/– mouse hepatocytes are more resistant to TNF-α–induced apoptosis. Isolated hepatocytes from catB+/+ and catB–/– mice were incubated in the absence (control) or presence of TNF-α and AcD. (a) Apoptosis was quantitated in catB+/+ and catB–/– hepatocytes at different times of incubation after staining with both FITC-annexin V (dotted lines) and DAPI (solid lines). Cells were considered apoptotic if either externalization of phosphatidylserine residues on the plasma membrane or chromatin condensation and nuclear fragmentation occurred. At least 300 cells in six high-power fields were counted by an individual blinded to the experimental conditions. (b) Apoptosis was quantitated in catB+/+ and catB–/– hepatocytes by DAPI staining after 24 hours of incubation in medium lacking (control) or containing either TNF-α and AcD (TNF-α) or AcD alone (AcD). catB+/+ hepatocytes were also treated with TNF-α and AcD after a 30-minute preincubation with CA-074, a pharmacological inhibitor of cat B. Results are representative of at least three independent experiments using cells from three separate isolations and are expressed as mean ± SEM. Data were compared using a one-tail t test. AP < 0.05, catB–/– vs. catB+/+; BP < 0.05, catB+/+ + CA-074 vs. catB+/+. (c) Isolated mouse hepatocytes from catB+/+ (filled bars) and catB–/– (open bars) mice were infected with an adenovirus expressing the IκB-superrepressor (Ad5IκB) or with an empty adenovirus (Ad5ΔE1) as a negative control, and treated with TNF-α (28 ng/ml) for 12 hours. Apoptosis was quantitated after staining with DAPI. Results are representative of three independent experiments performed in triplicate from separate isolations and are expressed as mean ± SEM.