title: Regulation of a novel pathway for cell death by lysosomal aspartic and cysteine proteinases.
abstract: PC12 cells undergo apoptosis when cultured under conditions of serum deprivation. In this situation, the activity of caspase-3-like proteinases was elevated, and the survival rate could be maintained by treatment with acetyl-DEVD-cho, a specific inhibitor of caspase-3. In a culture of PC12 cells treated with acetyl-DEVD-cho, where caspase-3-like proteinases are not activated, CA074, a specific inhibitor of cathepsin B induced active death of the cells. Cathepsin B antisense oligonucleotides showed a similar effect to CA074 on the induction of active cell death. By double staining of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling and activated caspase-3, the dying cells treated with CA074 were positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling staining but negative for activated caspase-3. Ultrastructurally, the cells were relatively large and had nuclei with chromatin condensation. The initiation of cell death by CA074 or the cathepsin B antisense were inhibited by the addition of pepstatin A, a lysosomal aspartic proteinase inhibitor, or by cathepsin D antisense. To examine whether this cell death pathway was present in cell types other than PC12 cells, we analysed dorsal root ganglion neurons obtained from rat embryos on the 15th gestational day, a time when they require nerve growth factor for survival and differentiation in culture. When cultured in the absence of nerve growth factor, the neurons survived in the presence of acetyl-DEVD-cho or acetyl-YVAD-cho. Under these conditions, CA074 reduced the survival rate of the neurons, which was subsequently restored by the further addition of pepstain A. These results suggest that a novel pathway for initiating cell death exists which is regulated by lysosomal cathepsins, and in which cathepsin D acts as a death factor. We speculate that this death-inducing activity is normally suppressed by cathepsin B.
authors.affiliation: Department of Cell Biology and Anatomy I, Osaka University Medical School, Suita, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Isahara
authors.forename: Y
authors.initials: Y
authors.lastname: Ohsawa
authors.forename: S
authors.initials: S
authors.lastname: Kanamori
authors.forename: M
authors.initials: M
authors.lastname: Shibata
authors.forename: S
authors.initials: S
authors.lastname: Waguri
authors.forename: N
authors.initials: N
authors.lastname: Sato
authors.forename: T
authors.initials: T
authors.lastname: Gotow
authors.forename: T
authors.initials: T
authors.lastname: Watanabe
authors.forename: T
authors.initials: T
authors.lastname: Momoi
authors.forename: K
authors.initials: K
authors.lastname: Urase
authors.forename: E
authors.initials: E
authors.lastname: Kominami
authors.forename: Y
authors.initials: Y
authors.lastname: Uchiyama
mesh_terms: D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000502:physiology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002401:Cathepsin B / Q000096:biosynthesis / Q000235:genetics; D002402:Cathepsin D / Q000096:biosynthesis / Q000235:genetics; D016923:Cell Death / Q000502:physiology; D002467:Cell Nucleus / Q000187:drug effects / Q000648:ultrastructure; D003546:Cysteine Endopeptidases / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D008247:Lysosomes / Q000201:enzymology*; D008854:Microscopy, Electron; D009414:Nerve Growth Factors / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D016716:PC12 Cells; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats
chemical_list: D015853:Cysteine Proteinase Inhibitors; D004792:Enzyme Precursors; D009414:Nerve Growth Factors; D016376:Oligonucleotides, Antisense; D011480:Protease Inhibitors; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D002401:Cathepsin B; D016282:Aspartic Acid Endopeptidases; D002402:Cathepsin D