Activation of caspase-3 by lysosomal cysteine proteases and its role in 2,2'-azobis-(2-amidinopropane)dihydrochloride (AAPH)-induced apoptosis in HL-60 cells.
We previously reported that in addition to mitochondrial cytochrome c dependent activation, lysosomal cysteine proteases were also involved in the activation of caspase-3. In this study, we have separately obtained the lysosomal and mitochondrial caspase-3 activating factors in a crude mitochondrial fraction and characterized their ability to activate pro-caspase-3 in the in vitro assay system. When a rat liver crude mitochondrial fraction containing lysosomes (ML) was treated with a low concentration of digitonin, lysosomal factors were selectively released without the release of a mitochondrial factor (cytochrome c, Cyt.c). Treatment of ML with Ca(2+) in the presence of inorganic phosphate (P(i)), in contrast, released mitochondrial Cyt.c without the release of lysosomal factors. The obtained lysosomal and mitochondrial factors activated caspase-3 in different manners; caspase-3 activation by lysosomal and mitochondrial factors was specifically suppressed by E-64, a cysteine protease inhibitor, and caspase-9 inhibitor, respectively. Thus, the activation of caspase-3 by lysosomal factors was found to be distinct from the activation by mitochondrial Cyt.c dependent formation of the Apaf-1/caspase-9 complex. To further determine whether or not the activation of caspase-3 by lysosomal cysteine proteases is involved in cellular apoptosis, the effect of E-64-d, a cell-permeable inhibitor of cysteine protease, on 2,2'-azobis-(2-amidinopropane)dihydrochloride (AAPH)-induced apoptosis in HL-60 cells was investigated. As a result, DNA fragmentation induced by AAPH was found to be remarkably (up to 50%) reduced by pretreatment with E-64-d, indicating the participation of lysosomal cysteine proteases in AAPH-induced apoptosis in HL-60 cells.
Ishisaka
R
R
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yoshida, Yamaguchi 753-8515, Japan. utsumi@agr.yamaguchi-u.ac.jp
Kanno
T
T
Akiyama
J
J
Yoshioka
T
T
Utsumi
K
K
Utsumi
T
T
D000578:Amidines / Q000494:pharmacology*; D017209:Apoptosis; D002118:Calcium / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism* / Q000502:physiology; D003546:Cysteine Endopeptidases / Q000378:metabolism* / Q000502:physiology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology; D004347:Drug Interactions; D004789:Enzyme Activation; D018922:HL-60 Cells; D006801:Humans; D007202:Indicators and Reagents / Q000494:pharmacology; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism
D000578:Amidines; D015853:Cysteine Proteinase Inhibitors; D003574:Cytochrome c Group; D007202:Indicators and Reagents; D010710:Phosphates; D011506:Proteins; C018589:lysosomal proteins; C046728:2,2'-azobis(2-amidinopropane); C505407:CASP3 protein, human; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D007930:Leucine; D004072:Digitonin; C108192:aloxistatin; D002118:Calcium