Differential caspase-9-dependent signaling pathway between tumor necrosis factor receptor- and Fas-mediated hepatocyte apoptosis in mice.
OBJECTIVE
Two apoptosis signaling pathways, which are used by different cell types, are identified. The activation of caspases is critical for the apoptosis process. The aim of this study was to investigate the effects of the caspase-9 inhibitor Ac-LEHD-CHO on tumor necrosis factor receptor (TNFR)- and Fas-mediated hepatocyte apoptosis in vivo, in order to evaluate the similarities and distinctions between TNFR- and Fas-mediated signaling pathways.


METHODS
BALB/c mice were intravenously injected with d-galactosamine (GalN, 20 mg/mouse)/tumor necrosis factor-alpha (TNF-alpha, 0.5 microg/mouse), or alphaFas (10 microg/mouse) 30 min after treatment with the caspase-9 inhibitor Ac-LEHD-CHO or pan-caspase inhibitor Z-VAD-fmk. Liver injury was assessed biochemically and histologically. Cytochrome c release and processing of procaspases in the liver were analyzed by Western blotting. Activities of caspases were measured using a fluorogenic peptide substrate.


RESULTS
Pretreatment with Z-VAD-fmk prevented liver injury and hepatocyte apoptosis induced by either GalN/TNF-alpha or alphaFas. On the other hand, pretreatment with Ac-LEHD-CHO prevented GalN/TNF-alpha-induced hepatotoxicity and hepatocyte apoptosis but not alphaFas-induced liver injury and apoptosis. Both inhibitors reduced the activities of caspase-9 and -3 in the livers of mice administered by GalN/TNF-alpha. However, unlike Z-VAD-fmk, Ac-LEHD-CHO did not inhibit caspase-3 activation in alphaFas-treated mice, although this inhibitor attenuated caspase-9.


CONCLUSIONS
Fas may rely on both caspase-8 activation (extrinsic pathway) and mitochondria (intrinsic pathway) to activate caspase-3. If the mitochondria-dependent pathway is blocked, the other pathway can compensate. In contrast, TNFR may mediate hepatocellular apoptosis mainly through the mitochondria-mediated caspase-9 activation pathway alone.
Imao
Motohiro
M
First Department of Internal Medicine, Gifu University School of Medicine, Gifu, Japan.
Nagaki
Masahito
M
Imose
Motoaki
M
Moriwaki
Hisataka
H
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004195:Disease Models, Animal; D004789:Enzyme Activation; D005688:Galactosamine; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011897:Random Allocation; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism*; D015398:Signal Transduction; D019014:fas Receptor / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D015853:Cysteine Proteinase Inhibitors; D018124:Receptors, Tumor Necrosis Factor; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019014:fas Receptor; D005688:Galactosamine; C506434:Casp9 protein, mouse; D053453:Caspase 9; D020169:Caspases