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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catB–/– mice are more resistant to TNF-α–induced liver damage. catB–/– a...
catB–/– mice are more resistant to TNF-α–induced liver damage. catB–/– and catB+/+ were injected via tail vein with the adenovirus Ad5IκB (0.35 × 109 pfu/mouse) encoding for an IκB superrepressor. In control experiments, mice were injected with the adenovirus Ad5ΔE1 (0.35 × 109 pfu/mouse in 0.22 ml sterile saline) or with sterile saline (0.22 ml).Twenty-four hours later, each mouse received a dose of 0.5 μg of recombinant mouse TNF-α intravenously. Mice were sacrificed after 2- and 4-hour treatment with TNF-α. (a) Serum alanine aminotransferase (ALT) levels were measured and expressed as mean ± SEM (n = 3). AP < 0.01. ALT values in control samples were < 20 IU/L, except in the Ad5ΔE1-injected mice, in which they were < 750 IU/L at 2 hours and < 1850 IU/L at 4 hours (data not shown). (b) H&E staining of Ad5IκB-injected catB+/+ (left) and catB–/– (right) mouse liver harvested 4 hours after treatment with TNF-α.