Cathepsin B contributes to TNF-α–mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c
M. Eugenia Guicciardi, Jan Deussing, Hideyuki Miyoshi, Steven F. Bronk, Phyllis A. Svingen, Christoph Peters, Scott H. Kaufmann, Gregory J. Gores
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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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.