Abstract
Effective execution of apoptosis requires the activation of caspases. However, in many cases, broad-range caspase inhibitors such as Z-VAD.fmk do not inhibit cell death because death signaling continues via basal caspase activities or caspase-independent processes. Although death mediators acting under caspase-inhibiting conditions have been identified, it remains unknown whether they trigger a physiologically relevant cell death that shows typical signs of apoptosis, including phosphatidylserine (PS) exposure and the removal of apoptotic cells by phagocytosis. Here we show that cells treated with ER stress drugs or deprived of IL-3 still show hallmarks of apoptosis such as cell shrinkage, membrane blebbing, mitochondrial release of cytochrome c, PS exposure and phagocytosis in the presence of Z-VAD.fmk. Cotreatment of the stressed cells with Z-VAD.fmk and the serine protease inhibitor Pefabloc (AEBSF) inhibited all these events, indicating that serine proteases mediated the apoptosis-like cell death and phagocytosis under these conditions. The serine proteases were found to act upstream of an increase in mitochondrial membrane permeability as opposed to the serine protease Omi/HtrA2 which is released from mitochondria at a later stage. Thus, despite caspase inhibition or basal caspase activities, cells can still be phagocytosed and killed in an apoptosis-like fashion by a serine protease-mediated mechanism that damages the mitochondrial membrane.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Abbreviations
- ER:
-
endoplasmic reticulum
- PS:
-
phosphatidylserine
- PFA:
-
paraformaldehyde
- DMEM:
-
Dulbecco's modified Eagle's medium
- FCS:
-
fetal calf serum
- Z-VAD.fmk:
-
benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone
- Z-D-dcb:
-
benzyloxycarbonyl-Asp-dichlorobenzoyloxy-methane
- BAF:
-
t-butyloxy-carbonyl-Asp-fluoromethylketone
- Pefabloc:
-
AEBSF (4-(2-aminoethyl)-benzenesulfonyl fluoride)
- PMSF:
-
phenylmethylsulfonylfluoride
- Z-VF-CHO (MDL28170):
-
benzyloxycarbonyl-Val-Phe-aldehyde
- TPCK:
-
N-tosyl-L-phenylalanine-chloromethylketone
- TLCK:
-
N-alpha-p-tosyl-L-lysinechloromethyl ketone
- Z-APF-cmk:
-
benzyloxycarbonyl-Ala-Pro-Phe-chloromethylketone
- BFA:
-
brefeldin A
- CHX:
-
cycloheximide
- PMA:
-
phorbol 12-myristate 13-acetate
- TNF:
-
tumor necrosis factor
- AIF:
-
apoptosis-inducing factor
- FADD:
-
Fas-associated death domain
- RIP:
-
receptor-interacting protein
- IAP:
-
inhibitor of apoptosis proteins
- BH:
-
Bcl-2 homology domain
- GFP:
-
green fluorescent protein
- PI:
-
propidium iodide, FITC, fluorescein isothiocyanate
- ECL:
-
enhanced chemiluminescence
- TAMRA:
-
5-(and -6)-carboxytetramethylrhodamine succinimidyl ester
- DEVD-AMC:
-
acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin
- VDVAD-AMC:
-
acetyl-Val-Asp-Val-Ala-Asp-AMC
- VEID-AMC:
-
acetyl-Val-Glu-Ile-Asp-AMC
- IETD-AMC:
-
acetyl-Ile-Glu-Thr-Asp-AMC
- LEHD-AMC:
-
ACETYL-Leu-Glu-His-Asp-AMC
- AEVD-AFC:
-
acetyl-Ala-Glu-Val-Asp-7-amino-4-trifluorometh-coumarin
References
Duvall E and Wyllie AH (1986) Death and the cell. Immunol. Today 7: 115–119
Savill J and Fadok V (2000) Corpse clearance defines the meaning of cell death. Nature 407: 784–788
Earnshaw WC, Martins LM and Kaufmann SH (1999) Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem. 68: 383–424
Krammer PH (2000) CD95's deadly mission in the immune system. Nature 407: 789–795
Donepudi M, Sweeney AM, Briand C and Grutter MG (2003) Insights into the regulatory mechanism for caspase-8 activation. Mol. Cell 11: 543–549
Boatright KM, Renatus M, Scott FL, Sperandio S, Shin H, Pedersen IM, Ricci JE, Edris WA, Sutherlin DP, Green DR and Salvesen GS (2003) A unified model for apical caspase activation. Mol. Cell 11: 529–541
Puthalakath H and Strasser A (2002) Keeping killers on a tight leash: transcriptional and post-translational control of the pro-apoptotic activity of BH3-only proteins. Cell Death Differ. 9: 505–512
Wang X (2001) The expanding role of mitochondria in apoptosis. Genes Dev. 15: 2922–2933
Zheng TS, Hunot S, Kuida K and Flavell RA (1999) Caspase knockouts: matters of life and death. Cell Death Differ. 6: 1043–1053
Ellis HM and Horvitz HR (1986) Genetic control of programmed cell death in the nematode C. elegans. Cell 44: 817–829
Oppenheim RW, Flavell RA, Vinsant S, Prevette D, Kuan CY and Rakic P (2001) Programmed cell death of developing mammalian neurons after genetic deletion of caspases. J. Neurosci. 21: 4752–4760
Wolf BB and Green DR (2002) Apoptosis: letting slip the dogs of war. Curr. Biol. 12: R177–R179
Lassus P, Opitz-Araya X and Lazebnik Y (2002) Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization. Science 297: 1290–1291
Leist M and Jaattela M (2001) Four deaths and a funeral: from caspases to alternative mechanisms. Nat. Rev. Mol. Cell. Biol 2: 589–598
Waterhouse NJ, Goldstein JC, von Ahsen O, Schuler M, Newmeyer DD and Green DR (2001) Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process. J. Cell. Biol 153: 319–328
Ekert PG, Silke J and Vaux DL (1999) Caspase inhibitors. Cell Death Differ. 6: 1081–1086
Nicholson DW (1999) Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ. 6: 1028–1042
Garcia-Calvo M, Peterson EP, Leiting B, Ruel R, Nicholson DW and Thornberry NA (1998) Inhibition of human caspases by peptide-based and macromolecular inhibitors. J. Biol. Chem. 273: 32608–32613
Borner C and Monney L (1999) Apoptosis without caspases: a inefficient molecular guillotine. Cell Death Differ. 6: 497–507
Vier J, Furmann C and Hacker G (2000) Baculovirus P35 protein does not inhibit caspase-9 in a cell-free system of apoptosis. Biochem. Biophys. Res. Commun. 276: 855–861
Shi Y (2002) Mechanisms of caspase activation and inhibition during apoptosis. Mol. Cell 9: 459–470
Hara H, Friedlander RM, Gagliardini V, Ayata C, Fink K, Huang Z, Shimizu-Sasamata M, Yuan J and Moskowitz MA (1997) Inhibition of interleukin 1beta converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage. Proc. Natl. Acad. Sci. USA 94: 2007–2012
Yaoita H, Ogawa K, Maehara K and Maruyama Y (1998) Attenuation of ischemia/reperfusion injury in rats by a caspase inhibitor. Circulation 97: 276–281
McCarthy NJ, Whyte MK, Gilbert CS and Evan GI (1997) Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak. J. Cell. Biol 136: 215–227
Hacki J, Egger L, Monney L, Conus S, Rosse T, Fellay I and Borner C (2000) Apoptotic crosstalk between the endoplasmic reticulum and mitochondria controlled by Bcl-2. Oncogene 19: 2286–2295
Lamkanfi M, Declercq W, Kalai M, Saelens X and Vandenabeele P (2002) Alice in caspase land. A phylogenetic analysis of caspases from worm to man. Cell Death Differ. 9: 358–361
Martinon F, Burns K and Tschopp J (2002) The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol. Cell 10: 417–426
Riedl SJ, Fuentes-Prior P, Renatus M, Kairies N, Krapp S, Huber R, Salvesen GS and Bode W (2001) Structural basis for the activation of human procaspase-7. Proc. Natl. Acad. Sci. USA 98: 14790–14795
Bursch W (2001) The autophagosomal–lysosomal compartment in programmed cell death. Cell Death Differ. 8: 569–581
Sperandio S, de Belle I and Bredesen DE (2000) An alternative, nonapoptotic form of programmed cell death. Proc. Natl. Acad. Sci. USA 97: 14376–14381
Reddien PW, Cameron S and Horvitz HR (2001) Phagocytosis promotes programmed cell death in C. elegans. Nature 412: 198–202
Hoeppner DJ, Hengartner MO and Schnabel R (2001) Engulfment genes cooperate with ced-3 to promote cell death in Caenorhabditis elegans. Nature 412: 202–206
Schlegel RA and Williamson P (2001) Phosphatidylserine, a death knell. Cell Death Differ. 8: 551–563
Turner C, Devitt A, Parker K, MacFarlane M, Giuliano M, Cohen GM and Gregory CD (2003) Macrophage-mediated clearance of cells undergoing caspase-3-independent death. Cell Death Differ. 10: 302–312
Renatus M, Stennicke HR, Scott FL, Liddington RC and Salvesen GS (2001) Dimer formation drives the activation of the cell death protease caspase 9. Proc. Natl. Acad. Sci. USA 98: 14250–14255
Marsden VS, O'Connor L, O'Reilly LA, Silke J, Metcalf D, Ekert PG, Huang DC, Cecconi F, Kuida K, Tomaselli KJ, Roy S, Nicholson DW, Vaux DL, Bouillet P, Adams JM and Strasser A (2002) Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome. Nature 419: 634–637
Deiss LP, Galinka H, Berissi H, Cohen O and Kimchi A (1996) Cathepsin D protease mediates programmed cell death induced by interferon-gamma, Fas/APO-1 and TNF-alpha. EMBO J. 15: 3861–3870
Guicciardi ME, Deussing J, Miyoshi H, Bronk SF, Svingen PA, Peters C, Kaufmann SH and Gores GJ (2000) Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J. Clin. Invest. 106: 1127–1137
Holler N, Zaru R, Micheau O, Thome M, Attinger A, Valitutti S, Bodmer JL, Schneider P, Seed B and Tschopp J (2000) Fas triggers an alternative, caspase-8-independent cell death pathway using the kinase RIP as effector molecule. Nat. Immunol. 1: 489–495
Johnson DE (2000) Noncaspase proteases in apoptosis. Leukemia 14: 1695–1703
Nakagawa T and Yuan J (2000) Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis. J. Cell Biol. 150: 887–894
Gao G and Dou QP (2000) N-terminal cleavage of bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes bcl-2-independent cytochrome c release and apoptotic cell death. J. Cell. Biochem. 80: 53–72
Wright SC, Wei QS, Zhong J, Zheng H, Kinder DH and Larrick JW (1994) Purification of a 24-kD protease from apoptotic tumor cells that activates DNA fragmentation. J. Exp. Med. 180: 2113–2123
Park IC, Park MJ, Choe TB, Jang JJ, Hong SI and Lee SH (2000) TNF-alpha induces apoptosis mediated by AEBSF-sensitive serine protease(s) that may involve upstream caspase-3/CPP32 protease activation in a human gastric cancer cell line. Int. J. Oncol. 16: 1243–1248
Hughes FM, Evans-Storms RB and Cidlowski JA (1998) Evidence that non-caspase proteases are required for chromatin degradation during apoptosis. Cell Death Differ. 5: 1017–1027
Dong Z, Saikumar P, Patel Y, Weinberg JM and Venkatachalam MA (2000) Serine protease inhibitors suppress cytochrome c-mediated caspase-9 activation and apoptosis during hypoxia-reoxygenation. Biochem. J. 347: 669–677
Rideout HJ, Zang E, Yeasmin M, Gordon R, Jabado O, Park DS and Stefanis L (2001) Inhibitors of trypsin-like serine proteases prevent DNA damage-induced neuronal death by acting upstream of the mitochondrial checkpoint and of p53 induction. Neuroscience 107: 339–352
Stefanis L, Troy CM, Qi H and Greene LA (1997) Inhibitors of trypsin-like serine proteases inhibit processing of the caspase Nedd-2 and protect PC12 cells and sympathetic neurons from death evoked by withdrawal of trophic support. J. Neurochem. 69: 1425–1437
Kagaya S, Kitanaka C, Noguchi K, Mochizuki T, Sugiyama A, Asai A, Yasuhara N, Eguchi Y, Tsujimoto Y and Kuchino Y (1997) A functional role for death proteases in s-Myc- and c-Myc-mediated apoptosis. Mol. Cell. Biol. 17: 6736–6745
Torriglia A, Perani P, Brossas JY, Altairac S, Zeggai S, Martin E, Treton J, Courtois Y and Counis MF (2000) A caspase-independent cell clearance program. The LEI/L-DNase II pathway. Ann. N.Y. Acad. Sci. 926: 192–203
Heibein JA, Goping IS, Barry M, Pinkoski MJ, Shore GC, Green DR and Bleackley RC (2000) Granzyme B-mediated cytochrome c release is regulated by the Bcl-2 family members bid and Bax. J. Exp. Med. 192: 1391–1402
Sutton VR, Davis JE, Cancilla M, Johnstone RW, Ruefli AA, Sedelies K, Browne KA and Trapani JA (2000) Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation. J. Exp. Med. 192: 1403–1414
Darmon AJ, Nicholson DW and Bleackley RC (1995) Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B. Nature 377: 446–448
Bidere N, Briet M, Durrbach A, Dumont C, Feldmann J, Charpentier B, de Saint-Basile G, Senik A and Venkatachalam M (2002) Selective inhibition of dipeptidyl peptidase I, not caspases, prevents the partial processing of procaspase-3 in CD3-activated human CD8(+) T lymphocytes. J. Biol. Chem. 277: 32339–32347
Sarin A, Williams MS, Alexander-Miller MA, Berzofsky JA, Zacharchuk CM and Henkart PA (1997) Target cell lysis by CTL granule exocytosis is independent of ICE/Ced-3 family proteases. Immunity 6: 209–215
Beresford PJ, Xia Z, Greenberg AH and Lieberman J (1999) Granzyme A loading induces rapid cytolysis and a novel form of DNA damage independently of caspase activation. Immunity 10: 585–594
Reeves EP, Lu H, Jacobs HL, Messina CG, Bolsover S, Gabella G, Potma EO, Warley A, Roes J and Segal AW (2002) Killing activity of neutrophils is mediated through activation of proteases by K+ flux. Nature 416: 291–297
Biggs JR, Yang J, Gullberg U, Muchardt C, Yaniv M and Kraft AS (2001) The human brm protein is cleaved during apoptosis: the role of cathepsin G. Proc. Natl. Acad. Sci. USA 98: 3814–3819
Zhou Q and Salvesen GS (1997) Activation of pro-caspase-7 by serine proteases includes a non-canonical specificity. Biochem. J. 324: 361–364
Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ, Roth KA, MacGregor GR, Thompson CB and Korsmeyer SJ (2001) Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292: 727–730
Desagher S and Martinou JC (2000) Mitochondria as the central control point of apoptosis. Trends Cell. Biol. 10: 369–377
Kaufmann T, Schlipf S, Sanz J, Neubert K and Borner C (2003) Molecular dissection of the signal that directs Bcl-x, but not Bcl-2, to the outer mitochondrial membrane. J. Cell Biol. 160: 53–64
O'Reilly LA, Ekert P, Harvey N, Mardsen V, Cullen L, Vaux DL, Hacker G, Magnusson C, Pakusch M, Cecconi F, Kuida K, Strasser A, Huang DCS and Kumar S (2002) Caspase-2 is not required for thymocyte or neuronal apoptosis even though cleavage of caspase-2 is dependent on both Apaf-1 and caspase-9. Cell Death Differ. 9: 832–841
Ernst JD, Yang L, Rosales JL and Broaddus VC (1998) Preparation and characterization of an endogenously fluorescent annexin for detection of apoptotic cells. Anal. Biochem. 260: 18–23
Acknowledgements
We thank Don Nicholson for anti-caspase-3, Andreas Strasser and Lorraine O'Reilly for anticaspase-2, Peter Krammer for anti-CD95 and anticaspase-8 antibodies, Joel Ernst for the GFP-annexin cDNA, Shujaath Mehdi for the MDL 28170 calpain inhibitor, Kurt Ballmer and Jürgen Muser for the FDC-P1 cells. We are grateful to Torsten Loop for this help with the statistical analysis and David Grubb, Anna Schinzel, Laurent Monney, Thomas Reinheckel, Andreas Hecht, Denis Grandgirard, Thomas Kaufmann and Ivonne Petermann for critical comments on the manuscript. This work was supported by a grant from the Swiss National Science Foundation (#31-57236.99) (to CB), the German–Israeli Foundation (GIF) (to CB) and the Maria Scheel Cancer Foundation (to JS).
Author information
Authors and Affiliations
Corresponding author
Additional information
Edited by Fadok/Knight
Appendix
Appendix
Supplementary Figure (online): Caspase-independent apoptotic features in HeLa cells. Phase contrast representation (200 × magnification) (a) and counting by visual inspection (b) of HeLa cells that remained attached to the culture plate after a 48 h treatment with BFA/CHX (B/C) in the absence or presence of Z-VAD.fmk (Z) or a combination of Z-VAD.fmk and Pefabloc (Z+Pef). Data are presented as the mean±SD of five determinations. (c) Co-staining of the HeLa cells (1000 × magnification) treated as in (a) with anticytochrome c (red), Hoechst 33342 (blue) and His GFP-annexin-V (green).
Rights and permissions
About this article
Cite this article
Egger, L., Schneider, J., Rhême, C. et al. Serine proteases mediate apoptosis-like cell death and phagocytosis under caspase-inhibiting conditions. Cell Death Differ 10, 1188–1203 (2003). https://doi.org/10.1038/sj.cdd.4401288
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/sj.cdd.4401288
Keywords
This article is cited by
-
Autophagy Contributes to Oxidative Stress-Induced Apoptosis in Porcine Granulosa Cells
Reproductive Sciences (2021)
-
BH3 mimetics efficiently induce apoptosis in mouse basophils and mast cells
Cell Death & Differentiation (2018)
-
IL-4 enhances survival of in vitro-differentiated mouse basophils through transcription-independent signaling downstream of PI3K
Cell Death & Disease (2018)
-
Overtraining elevates serum protease level, increases renal p16INK4α gene expression and induces apoptosis in rat kidney
Sport Sciences for Health (2018)
-
Immunoproteomic analysis of the excretory-secretory products of Trichinella pseudospiralis adult worms and newborn larvae
Parasites & Vectors (2017)