Two CD95 (APO-1/Fas) signaling pathways.

C Scaffidi

S Fulda

A Srinivasan

C Friesen

Abstract

We have identified two cell types, each using almost exclusively one of two different CD95 (APO-1/Fas) signaling pathways. In type I cells, caspase-8 was activated within seconds and caspase-3 within 30 min of receptor engagement, whereas in type II cells cleavage of both caspases was delayed for approximately 60 min. However, both type I and type II cells showed similar kinetics of CD95-mediated apoptosis and loss of mitochondrial transmembrane potential (DeltaPsim). Upon CD95 triggering, all mitochondrial apoptogenic activities were blocked by Bcl-2 or Bcl-xL overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl-2 or Bcl-xL blocked caspase-8 and caspase-3 activation as well as apoptosis. In type I cells, induction of apoptosis was accompanied by activation of large amounts of caspase-8 by the death-inducing signaling complex (DISC), whereas in type II cells DISC formation was strongly reduced and activation of caspase-8 and caspase-3 occurred following the loss of DeltaPsim. Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas, normally resistant to CD95-mediated apoptosis by overexpression of Bcl-xL, converted these cells into true type I cells in which apoptosis was no longer inhibited by Bcl-xL. In summary, in the presence of caspase-3 the amount of active caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway is used (type I cells) or not (type II cells).

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Adachi S, Cross AR, Babior BM, Gottlieb RA. Bcl-2 and the outer mitochondrial membrane in the inactivation of cytochrome c during Fas-mediated apoptosis. J Biol Chem. 1997 Aug 29;272(35):21878–21882. [PubMed] [Google Scholar]
Armstrong RC, Aja T, Xiang J, Gaur S, Krebs JF, Hoang K, Bai X, Korsmeyer SJ, Karanewsky DS, Fritz LC, et al. Fas-induced activation of the cell death-related protease CPP32 Is inhibited by Bcl-2 and by ICE family protease inhibitors. J Biol Chem. 1996 Jul 12;271(28):16850–16855. [PubMed] [Google Scholar]
Boldin MP, Varfolomeev EE, Pancer Z, Mett IL, Camonis JH, Wallach D. A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain. J Biol Chem. 1995 Apr 7;270(14):7795–7798. [PubMed] [Google Scholar]
Boldin MP, Goncharov TM, Goltsev YV, Wallach D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell. 1996 Jun 14;85(6):803–815. [PubMed] [Google Scholar]
Boise LH, Thompson CB. Bcl-x(L) can inhibit apoptosis in cells that have undergone Fas-induced protease activation. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3759–3764.[PMC free article] [PubMed] [Google Scholar]
Cahill MA, Peter ME, Kischkel FC, Chinnaiyan AM, Dixit VM, Krammer PH, Nordheim A. CD95 (APO-1/Fas) induces activation of SAP kinases downstream of ICE-like proteases. Oncogene. 1996 Nov 21;13(10):2087–2096. [PubMed] [Google Scholar]
Chinnaiyan AM, O'Rourke K, Tewari M, Dixit VM. FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell. 1995 May 19;81(4):505–512. [PubMed] [Google Scholar]
Chinnaiyan AM, Tepper CG, Seldin MF, O'Rourke K, Kischkel FC, Hellbardt S, Krammer PH, Peter ME, Dixit VM. FADD/MORT1 is a common mediator of CD95 (Fas/APO-1) and tumor necrosis factor receptor-induced apoptosis. J Biol Chem. 1996 Mar 1;271(9):4961–4965. [PubMed] [Google Scholar]
Chinnaiyan AM, Orth K, O'Rourke K, Duan H, Poirier GG, Dixit VM. Molecular ordering of the cell death pathway. Bcl-2 and Bcl-xL function upstream of the CED-3-like apoptotic proteases. J Biol Chem. 1996 Mar 1;271(9):4573–4576. [PubMed] [Google Scholar]
Chinnaiyan AM, O'Rourke K, Lane BR, Dixit VM. Interaction of CED-4 with CED-3 and CED-9: a molecular framework for cell death. Science. 1997 Feb 21;275(5303):1122–1126. [PubMed] [Google Scholar]
Chiu VK, Walsh CM, Liu CC, Reed JC, Clark WR. Bcl-2 blocks degranulation but not fas-based cell-mediated cytotoxicity. J Immunol. 1995 Mar 1;154(5):2023–2032. [PubMed] [Google Scholar]
Cosulich SC, Green S, Clarke PR. Bcl-2 regulates activation of apoptotic proteases in a cell-free system. Curr Biol. 1996 Aug 1;6(8):997–1005. [PubMed] [Google Scholar]
Enari M, Hug H, Nagata S. Involvement of an ICE-like protease in Fas-mediated apoptosis. Nature. 1995 May 4;375(6526):78–81. [PubMed] [Google Scholar]
Enari M, Talanian RV, Wong WW, Nagata S. Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis. Nature. 1996 Apr 25;380(6576):723–726. [PubMed] [Google Scholar]
Estoppey S, Rodriguez I, Sadoul R, Martinou JC. Bcl-2 prevents activation of CPP32 cysteine protease and cleavage of poly (ADP-ribose) polymerase and U1-70 kD proteins in staurosporine-mediated apoptosis. Cell Death Differ. 1997 Jan;4(1):34–38. [PubMed] [Google Scholar]
Fernandes-Alnemri T, Takahashi A, Armstrong R, Krebs J, Fritz L, Tomaselli KJ, Wang L, Yu Z, Croce CM, Salveson G, et al. Mch3, a novel human apoptotic cysteine protease highly related to CPP32. Cancer Res. 1995 Dec 15;55(24):6045–6052. [PubMed] [Google Scholar]
Fernandes-Alnemri T, Armstrong RC, Krebs J, Srinivasula SM, Wang L, Bullrich F, Fritz LC, Trapani JA, Tomaselli KJ, Litwack G, et al. In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7464–7469.[PMC free article] [PubMed] [Google Scholar]
Hasegawa J, Kamada S, Kamiike W, Shimizu S, Imazu T, Matsuda H, Tsujimoto Y. Involvement of CPP32/Yama(-like) proteases in Fas-mediated apoptosis. Cancer Res. 1996 Apr 15;56(8):1713–1718. [PubMed] [Google Scholar]
Huang DC, Cory S, Strasser A. Bcl-2, Bcl-XL and adenovirus protein E1B19kD are functionally equivalent in their ability to inhibit cell death. Oncogene. 1997 Jan 30;14(4):405–414. [PubMed] [Google Scholar]
Itoh N, Tsujimoto Y, Nagata S. Effect of bcl-2 on Fas antigen-mediated cell death. J Immunol. 1993 Jul 15;151(2):621–627. [PubMed] [Google Scholar]
Irmler M, Thome M, Hahne M, Schneider P, Hofmann K, Steiner V, Bodmer JL, Schröter M, Burns K, Mattmann C, et al. Inhibition of death receptor signals by cellular FLIP. Nature. 1997 Jul 10;388(6638):190–195. [PubMed] [Google Scholar]
Jättelä M, Benedict M, Tewari M, Shayman JA, Dixit VM. Bcl-x and Bcl-2 inhibit TNF and Fas-induced apoptosis and activation of phospholipase A2 in breast carcinoma cells. Oncogene. 1995 Jun 15;10(12):2297–2305. [PubMed] [Google Scholar]
Kamada S, Washida M, Hasegawa J, Kusano H, Funahashi Y, Tsujimoto Y. Involvement of caspase-4(-like) protease in Fas-mediated apoptotic pathway. Oncogene. 1997 Jul 17;15(3):285–290. [PubMed] [Google Scholar]
Kim CN, Wang X, Huang Y, Ibrado AM, Liu L, Fang G, Bhalla K. Overexpression of Bcl-X(L) inhibits Ara-C-induced mitochondrial loss of cytochrome c and other perturbations that activate the molecular cascade of apoptosis. Cancer Res. 1997 Aug 1;57(15):3115–3120. [PubMed] [Google Scholar]
Kischkel FC, Hellbardt S, Behrmann I, Germer M, Pawlita M, Krammer PH, Peter ME. Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. EMBO J. 1995 Nov 15;14(22):5579–5588.[PMC free article] [PubMed] [Google Scholar]
Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science. 1997 Feb 21;275(5303):1132–1136. [PubMed] [Google Scholar]
Kluck RM, Martin SJ, Hoffman BM, Zhou JS, Green DR, Newmeyer DD. Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system. EMBO J. 1997 Aug 1;16(15):4639–4649.[PMC free article] [PubMed] [Google Scholar]
Kuida K, Zheng TS, Na S, Kuan C, Yang D, Karasuyama H, Rakic P, Flavell RA. Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice. Nature. 1996 Nov 28;384(6607):368–372. [PubMed] [Google Scholar]
Krippner A, Matsuno-Yagi A, Gottlieb RA, Babior BM. Loss of function of cytochrome c in Jurkat cells undergoing fas-mediated apoptosis. J Biol Chem. 1996 Aug 30;271(35):21629–21636. [PubMed] [Google Scholar]
Kroemer G, Zamzami N, Susin SA. Mitochondrial control of apoptosis. Immunol Today. 1997 Jan;18(1):44–51. [PubMed] [Google Scholar]
Lacronique V, Mignon A, Fabre M, Viollet B, Rouquet N, Molina T, Porteu A, Henrion A, Bouscary D, Varlet P, et al. Bcl-2 protects from lethal hepatic apoptosis induced by an anti-Fas antibody in mice. Nat Med. 1996 Jan;2(1):80–86. [PubMed] [Google Scholar]
Lee RK, Spielman J, Podack ER. Bcl-2 protects against Fas-based but not perforin-based T cell-mediated cytolysis. Int Immunol. 1996 Jul;8(7):991–1000. [PubMed] [Google Scholar]
Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 1997 Nov 14;91(4):479–489. [PubMed] [Google Scholar]
Li F, Srinivasan A, Wang Y, Armstrong RC, Tomaselli KJ, Fritz LC. Cell-specific induction of apoptosis by microinjection of cytochrome c. Bcl-xL has activity independent of cytochrome c release. J Biol Chem. 1997 Nov 28;272(48):30299–30305. [PubMed] [Google Scholar]
Liu X, Kim CN, Yang J, Jemmerson R, Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell. 1996 Jul 12;86(1):147–157. [PubMed] [Google Scholar]
Los M, Van de Craen M, Penning LC, Schenk H, Westendorp M, Baeuerle PA, Dröge W, Krammer PH, Fiers W, Schulze-Osthoff K. Requirement of an ICE/CED-3 protease for Fas/APO-1-mediated apoptosis. Nature. 1995 May 4;375(6526):81–83. [PubMed] [Google Scholar]
Mandal M, Maggirwar SB, Sharma N, Kaufmann SH, Sun SC, Kumar R. Bcl-2 prevents CD95 (Fas/APO-1)-induced degradation of lamin B and poly(ADP-ribose) polymerase and restores the NF-kappaB signaling pathway. J Biol Chem. 1996 Nov 29;271(48):30354–30359. [PubMed] [Google Scholar]
Marchetti P, Castedo M, Susin SA, Zamzami N, Hirsch T, Macho A, Haeffner A, Hirsch F, Geuskens M, Kroemer G. Mitochondrial permeability transition is a central coordinating event of apoptosis. J Exp Med. 1996 Sep 1;184(3):1155–1160.[PMC free article] [PubMed] [Google Scholar]
Medema JP, Scaffidi C, Kischkel FC, Shevchenko A, Mann M, Krammer PH, Peter ME. FLICE is activated by association with the CD95 death-inducing signaling complex (DISC). EMBO J. 1997 May 15;16(10):2794–2804.[PMC free article] [PubMed] [Google Scholar]
Medema JP, Scaffidi C, Krammer PH, Peter ME. Bcl-xL acts downstream of caspase-8 activation by the CD95 death-inducing signaling complex. J Biol Chem. 1998 Feb 6;273(6):3388–3393. [PubMed] [Google Scholar]
Memon SA, Moreno MB, Petrak D, Zacharchuk CM. Bcl-2 blocks glucocorticoid- but not Fas- or activation-induced apoptosis in a T cell hybridoma. J Immunol. 1995 Nov 15;155(10):4644–4652. [PubMed] [Google Scholar]
Monney L, Otter I, Olivier R, Ravn U, Mirzasaleh H, Fellay I, Poirier GG, Borner C. Bcl-2 overexpression blocks activation of the death protease CPP32/Yama/apopain. Biochem Biophys Res Commun. 1996 Apr 16;221(2):340–345. [PubMed] [Google Scholar]
Moreno MB, Memon SA, Zacharchuk CM. Apoptosis signaling pathways in normal T cells: differential activity of Bcl-2 and IL-1beta-converting enzyme family protease inhibitors on glucocorticoid- and Fas-mediated cytotoxicity. J Immunol. 1996 Nov 1;157(9):3845–3849. [PubMed] [Google Scholar]
Muzio M, Chinnaiyan AM, Kischkel FC, O'Rourke K, Shevchenko A, Ni J, Scaffidi C, Bretz JD, Zhang M, Gentz R, et al. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death--inducing signaling complex. Cell. 1996 Jun 14;85(6):817–827. [PubMed] [Google Scholar]
Muzio M, Salvesen GS, Dixit VM. FLICE induced apoptosis in a cell-free system. Cleavage of caspase zymogens. J Biol Chem. 1997 Jan 31;272(5):2952–2956. [PubMed] [Google Scholar]
Newmeyer DD, Farschon DM, Reed JC. Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria. Cell. 1994 Oct 21;79(2):353–364. [PubMed] [Google Scholar]
Nicholson DW, Thornberry NA. Caspases: killer proteases. Trends Biochem Sci. 1997 Aug;22(8):299–306. [PubMed] [Google Scholar]
Perry DK, Smyth MJ, Wang HG, Reed JC, Duriez P, Poirier GG, Obeid LM, Hannun YA. Bcl-2 acts upstream of the PARP protease and prevents its activation. Cell Death Differ. 1997 Jan;4(1):29–33. [PubMed] [Google Scholar]
Peter ME, Hellbardt S, Schwartz-Albiez R, Westendorp MO, Walczak H, Moldenhauer G, Grell M, Krammer PH. Cell surface sialylation plays a role in modulating sensitivity towards APO-1-mediated apoptotic cell death. Cell Death Differ. 1995 Jul;2(3):163–171. [PubMed] [Google Scholar]
Peter ME, Heufelder AE, Hengartner MO. Advances in apoptosis research. Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12736–12737.[PMC free article] [PubMed] [Google Scholar]
Peter ME, Kischkel FC, Scheuerpflug CG, Medema JP, Debatin KM, Krammer PH. Resistance of cultured peripheral T cells towards activation-induced cell death involves a lack of recruitment of FLICE (MACH/caspase 8) to the CD95 death-inducing signaling complex. Eur J Immunol. 1997 May;27(5):1207–1212. [PubMed] [Google Scholar]
Rodriguez I, Matsuura K, Khatib K, Reed JC, Nagata S, Vassalli P. A bcl-2 transgene expressed in hepatocytes protects mice from fulminant liver destruction but not from rapid death induced by anti-Fas antibody injection. J Exp Med. 1996 Mar 1;183(3):1031–1036.[PMC free article] [PubMed] [Google Scholar]
Scaffidi C, Medema JP, Krammer PH, Peter ME. FLICE is predominantly expressed as two functionally active isoforms, caspase-8/a and caspase-8/b. J Biol Chem. 1997 Oct 24;272(43):26953–26958. [PubMed] [Google Scholar]
Schlegel J, Peters I, Orrenius S, Miller DK, Thornberry NA, Yamin TT, Nicholson DW. CPP32/apopain is a key interleukin 1 beta converting enzyme-like protease involved in Fas-mediated apoptosis. J Biol Chem. 1996 Jan 26;271(4):1841–1844. [PubMed] [Google Scholar]
Shimizu S, Eguchi Y, Kamiike W, Waguri S, Uchiyama Y, Matsuda H, Tsujimoto Y. Bcl-2 blocks loss of mitochondrial membrane potential while ICE inhibitors act at a different step during inhibition of death induced by respiratory chain inhibitors. Oncogene. 1996 Jul 4;13(1):21–29. [PubMed] [Google Scholar]
Shimizu S, Eguchi Y, Kamiike W, Matsuda H, Tsujimoto Y. Bcl-2 expression prevents activation of the ICE protease cascade. Oncogene. 1996 Jun 6;12(11):2251–2257. [PubMed] [Google Scholar]
Spector MS, Desnoyers S, Hoeppner DJ, Hengartner MO. Interaction between the C. elegans cell-death regulators CED-9 and CED-4. Nature. 1997 Feb 13;385(6617):653–656. [PubMed] [Google Scholar]
Srinivasan A, Li F, Wong A, Kodandapani L, Smidt R, Jr, Krebs JF, Fritz LC, Wu JC, Tomaselli KJ. Bcl-xL functions downstream of caspase-8 to inhibit Fas- and tumor necrosis factor receptor 1-induced apoptosis of MCF7 breast carcinoma cells. J Biol Chem. 1998 Feb 20;273(8):4523–4529. [PubMed] [Google Scholar]
Srinivasula SM, Ahmad M, Fernandes-Alnemri T, Litwack G, Alnemri ES. Molecular ordering of the Fas-apoptotic pathway: the Fas/APO-1 protease Mch5 is a CrmA-inhibitable protease that activates multiple Ced-3/ICE-like cysteine proteases. Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14486–14491.[PMC free article] [PubMed] [Google Scholar]
Strasser A, Harris AW, Huang DC, Krammer PH, Cory S. Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis. EMBO J. 1995 Dec 15;14(24):6136–6147.[PMC free article] [PubMed] [Google Scholar]
Susin SA, Zamzami N, Castedo M, Hirsch T, Marchetti P, Macho A, Daugas E, Geuskens M, Kroemer G. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J Exp Med. 1996 Oct 1;184(4):1331–1341.[PMC free article] [PubMed] [Google Scholar]
Susin SA, Zamzami N, Castedo M, Daugas E, Wang HG, Geley S, Fassy F, Reed JC, Kroemer G. The central executioner of apoptosis: multiple connections between protease activation and mitochondria in Fas/APO-1/CD95- and ceramide-induced apoptosis. J Exp Med. 1997 Jul 7;186(1):25–37.[PMC free article] [PubMed] [Google Scholar]
Takahashi A, Hirata H, Yonehara S, Imai Y, Lee KK, Moyer RW, Turner PC, Mesner PW, Okazaki T, Sawai H, et al. Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8. Oncogene. 1997 Jun 12;14(23):2741–2752. [PubMed] [Google Scholar]
Takayama S, Sato T, Krajewski S, Kochel K, Irie S, Millan JA, Reed JC. Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity. Cell. 1995 Jan 27;80(2):279–284. [PubMed] [Google Scholar]
Trauth BC, Klas C, Peters AM, Matzku S, Möller P, Falk W, Debatin KM, Krammer PH. Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science. 1989 Jul 21;245(4915):301–305. [PubMed] [Google Scholar]
Vander Heiden MG, Chandel NS, Williamson EK, Schumacker PT, Thompson CB. Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria. Cell. 1997 Nov 28;91(5):627–637. [PubMed] [Google Scholar]
Vaux DL, Weissman IL, Kim SK. Prevention of programmed cell death in Caenorhabditis elegans by human bcl-2. Science. 1992 Dec 18;258(5090):1955–1957. [PubMed] [Google Scholar]
Vincenz C, Dixit VM. Fas-associated death domain protein interleukin-1beta-converting enzyme 2 (FLICE2), an ICE/Ced-3 homologue, is proximally involved in CD95- and p55-mediated death signaling. J Biol Chem. 1997 Mar 7;272(10):6578–6583. [PubMed] [Google Scholar]
Wu D, Wallen HD, Nuñez G. Interaction and regulation of subcellular localization of CED-4 by CED-9. Science. 1997 Feb 21;275(5303):1126–1129. [PubMed] [Google Scholar]
Xue D, Shaham S, Horvitz HR. The Caenorhabditis elegans cell-death protein CED-3 is a cysteine protease with substrate specificities similar to those of the human CPP32 protease. Genes Dev. 1996 May 1;10(9):1073–1083. [PubMed] [Google Scholar]
Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, Jones DP, Wang X. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science. 1997 Feb 21;275(5303):1129–1132. [PubMed] [Google Scholar]
Yang X, Khosravi-Far R, Chang HY, Baltimore D. Daxx, a novel Fas-binding protein that activates JNK and apoptosis. Cell. 1997 Jun 27;89(7):1067–1076.[PMC free article] [PubMed] [Google Scholar]
Yuan JY, Horvitz HR. The Caenorhabditis elegans genes ced-3 and ced-4 act cell autonomously to cause programmed cell death. Dev Biol. 1990 Mar;138(1):33–41. [PubMed] [Google Scholar]
Zamzami N, Susin SA, Marchetti P, Hirsch T, Gómez-Monterrey I, Castedo M, Kroemer G. Mitochondrial control of nuclear apoptosis. J Exp Med. 1996 Apr 1;183(4):1533–1544.[PMC free article] [PubMed] [Google Scholar]
Zou H, Henzel WJ, Liu X, Lutschg A, Wang X. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell. 1997 Aug 8;90(3):405–413. [PubMed] [Google Scholar]

Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

Abstract

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