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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Article

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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Cat B contributes to TNF-α/AcD–induced hepatocyte apoptosis. Isolated he...
Cat B contributes to TNF-α/AcD–induced hepatocyte apoptosis. Isolated hepatocytes from catB+/+ and catB–/– mice were incubated in the absence (control) or presence of TNF-α (28 ng/ml) and AcD (0.2 μg/ml) for up to 12 hours. (a) Intracellular cat B activity was measured fluorometrically in the cells after 4 hours of treatment using the fluorogenic substrate VLK-CMAC and digitized video microscopy, as described in Methods. (b) At the indicated time points, cytosolic extracts were prepared by selective permeabilization with digitonin as described in Methods and subjected to immunoblot analysis using an anti–cat B antiserum. Locations of 30-kDa (p30) and 27-kDa (p27) active fragments of cat B are indicated. Immunoblot analysis of β-actin was performed as a control for protein loading. Cont., control. (c) Cultured McNtcp.24 cells grown on collagen-coated glass coverslips were transfected with the plasmid construct encoding the cat B-GFP fusion protein (control, TNF-α) or double-transfected with the cat B-GFP plasmid and the plasmid encoding the viral protein CrmA (CrmA + TNF-α). Forty-eight hours later, cells were incubated in the absence (control) or presence of TNF-α/AcD at 37°C for 2 hours and transferred to the stage of an inverted confocal microscope. Cat B-GFP fluorescence was imaged as described in Methods.