Necrotic Death Pathway in Fas Receptor Signaling

View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central Necrotic Death Pathway in Fas Receptor Signaling Hirotaka Matsumura,* Yusuke Shimizu,* Yoshiyuki Ohsawa,‡ Atsuo Kawahara,* Yasuo Uchiyama,‡ and Shigekazu Nagata*§ *Department of Genetics and ‡Department of Cell Biology and Anatomy, Osaka University Medical School, Osaka 565-0871, Japan; and §Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Osaka 565-0871, Japan Abstract. A caspase 8–deﬁcient subline (JB6) of hu- with Fas ligand or anti–mouse Fas antibodies, the cells man Jurkat cells can be killed by the oligomerization of died, showing characteristics of apoptosis. A broad Fas-associated protein with death domain (FADD). caspase inhibitor (z-VAD–fmk) blocked the apoptotic This cell death process is not accompanied by caspase morphological changes and the release of cytochrome activation, but by necrotic morphological changes. c. However, the cells still died, and this cell death pro- Here, we show that the death effector domain of cess was accompanied by a strong reduction in ⌬⌿m, as FADD is responsible for the FADD-mediated necrotic well as necrotic morphological changes. The presence pathway. This process was accompanied by a loss of mi- of z-VAD–fmk and pyrrolidine dithiocarbamate to- tochondrial transmembrane potential (⌬⌿m), but not gether blocked cell death, suggesting that both apop- by the release of cytochrome c from mitochondria. Pyr- totic and necrotic pathways can be activated through rolidine dithiocarbamate, a metal chelator and antioxi- the Fas death receptor. dant, efﬁciently inhibited the FADD-induced reduction of ⌬⌿m and necrotic cell death. When human Jurkat, Key words: apoptosis • caspase • Fas • mitochondrial or its transformants, expressing mouse Fas were treated membrane potential • necrosis Introduction Based on morphological changes, cell death in mammals is is now known that a family of cysteine proteases with as- classified into two types: apoptosis and necrosis (Wyllie et partate specificity are the major effectors of the process al., 1980; Duval and Wyllie, 1986). Apoptosis is character- (Thornberry and Lazebnik, 1998). Caspases exist as inac- ized by plasma membrane blebbing, condensation, and tive precursors in proliferating cells and are activated in a fragmentation of cells and nuclei, as well as extensive deg- cascade. Upon exposure to apoptotic stimuli, initiator radation of chromosomal DNA into nucleosomal units caspases are activated by the formation of a complex (Wyllie, 1980). On the other hand, necrosis is accompa- with other molecules. Once the initiator caspases are nied by the swelling of cells and organelles, in addition to activated, they cause the processing of downstream cas- the ultimate disintegration of the plasma membrane. pases, which cleave a set of cellular proteins, leading to Apoptosis can be triggered by a variety of stimuli, in- morphological changes and the degradation of chromo- cluding genotoxic agents, factor deprivation, and death somal DNA. Necrosis is known to occur as a result of factors (Nagata, 1997; Ashkenazi and Dixit, 1998; Green complement attack, severe hypoxia, hyperthermia, lytic and Reed, 1998; Raff, 1998). It is involved in the removal viral infection, or exposure to a variety of toxins and res- of surplus cells generated during mammalian develop- piratory poisons. Tumor necrosis factor (TNF)1 can acti- ment, and in the removal of virally infected or cancer vate the necrotic death program in some cell lines, such cells as part of the immune response (Raff et al., 1993; as mouse L929 cells (Vercammen et al., 1998a). An in- Nagata, 1997). Apoptotic signal transduction has been volvement of necrosis in the removal of interdigital cells extensively studied biochemically and genetically, and it in the mouse embryo has also been suggested (Chautan Address correspondence to Shigekazu Nagata, Osaka University Medical 1Abbreviations used in this paper: BHA, butylated hydroxyanisole; School, B-3, Department of Genetics, 2-2 Yamada-oka, Suita, Osaka 565- DD, death domain; DED, death effector domain; FADD, Fas-associ- 0871, Japan. Tel.: 81-6-68-79-33-10. Fax: 81-6-68-79-33-19. E-mail: nagata ated protein with death domain; FasL, Fas ligand; FKBP, FK506-bind- @genetic.med.osaka-u.ac.jp ing protein; LZ-FasL, FasL tagged with a leucine-zipper motif; PDTC, A. Kawahara’s present address is Laboratory of Molecular Genetics, pyrrolidine dithiocarbamate; TNF, tumor necrosis factor; z-VAD–fmk, National Institute of Child Health and Human Development, Building carbobenzoyl-Val-Ala-Asp-fluoromethylketone; ⌬⌿m, mitochondrial 6B, Room 420, NIH, Bethesda, MD 20892. transmembrane potential. The Rockefeller University Press, 0021-9525/2000/12/1247/9 $5.00 The Journal of Cell Biology, Volume 151, Number 6, December 11, 2000 1247–1255 http://www.jcb.org/cgi/content/full/151/6/1247 1247 et al., 1999). In contrast to apoptotic cell death, the mo- Materials and Methods lecular mechanism of necrosis is not well understood, though several mechanisms, including the release of lyso- Cell Lines and Materials somal enzymes, the generation of toxic oxygen radicals, Jurkat, a human T cell lymphoma cell line, and its derivative, JB6, which is and the activation of calcium-dependent phospholipases, deficient in caspase 8 (Kawahara et al., 1998), were maintained in RPMI have been proposed (Fiers et al., 1999). 1640 medium containing 10% FCS (EQUITECH-BIO, Inc.). AP1510, Fas ligand (FasL) is a member of the TNF family (Na- a dimerizer of FK506-binding protein (FKBP) (Amara et al., 1997), gata and Golstein, 1995; Nagata, 1997) and was originally was provided by Dr. M. Gilman (ARIAD Pharmaceuticals, Inc., Cam- bridge, MA). PDTC and carbobenzoyl-Val-Ala-Asp-fluoromethylketone identified as a cytokine that triggers apoptosis by binding (z-VAD–fmk) were purchased from Sigma-Aldrich and Takara Shuzo, to Fas (Suda et al., 1993). Studies on FasL-induced apop- Co., respectively. Monoclonal antibodies against human caspase 3 and tosis have revealed the following mechanism. The bind- FADD were purchased from Transduction Laboratories. The monoclonal ing of FasL or agonistic anti-Fas antibody to Fas rapidly antibodies against human cytochrome c, human cytochrome oxidase sub- recruits procaspase 8 through an adaptor called FADD unit II, and the FLAG epitope were from BD PharMingen, Molecular Probes, and Sigma-Aldrich, respectively. Horseradish peroxidase–conju- (Fas-associated protein with death domain) (Boldin et gated anti–mouse IgG used as a secondary antibody was obtained from Dako. al., 1995; Chinnaiyan et al., 1995). FADD carries a do- The recombinant human FasL tagged with a leucine-zipper motif (LZ- main called a death domain (DD) at its COOH terminus, FasL) (Walczak et al., 1999) was produced by transfecting COS cells with the FasL-expression plasmid. The concentration of FasL in the superna- which is involved in the binding to Fas. The NH2-termi- tant of the transfected COS cells was determined by ELISA using anti– nal part of FADD is called the death effector domain human FasL antibodies, as described previously (Tanaka et al., 1996). (DED) and is responsible for recruiting procaspase 8 (Boldin et al., 1996; Muzio et al., 1996). Thus, procaspase Establishment of Stable Transformants 8 oligomerized at the plasma membrane is autoactivated to a mature enzyme (Muzio et al., 1998; Yang et al., The plasmid pEF-FK-FADD carries the coding sequence (FKBP-FADD) 1998). Two pathways have been shown for the signal for a myristilation-targeting peptide, two tandem repeats of FKBP12, and human FADD in the pEF-BOS mammalian expression vector (Kawahara transduction downstream of caspase 8, which are used in et al., 1998). The DNA fragments coding for the DED of FADD (FADD- different cell types (types I and II) (Scaffidi et al., 1998). DED, amino acids 1–117), its truncated form (FADD-⌬DED, amino acids In type I cells, caspase 8 directly activates procaspase 3. 79–117), and the DD of FADD (FADD-DD, amino acids 105–208) were However, in type II cells, caspase 8 cleaves Bid, a proap- prepared from human FADD cDNA (Boldin et al., 1995; Chinnaiyan et al., 1995). They were fused to a DNA fragment carrying a myristilation- optotic member of the Bcl-2 family (Li et al., 1998; Luo targeting peptide and two tandem repeats of FKBP12, and inserted into et al., 1998; Gross et al., 1999). The cleaved Bid translo- pEF-BOS to generate pEF-FK-DED, pEF-F-FK-DED, pEF-F-FK- cates to the mitochondria and stimulates the release of ⌬DED, and pEF-FK-DD (Fig. 1 A). In pEF-F-FK-DED and pEF-F-FK- cytochrome c. Cytochrome c, together with Apaf-1, acti- ⌬DED, a DNA fragment coding for the FLAG epitope (DYKDDDDK) vates procaspase 9 (Li et al., 1997), which leads to the was inserted between the myristilation-targeting peptide and FKBP. The stable cell transformants expressing FADD-DD, FADD-DED, downstream processing of procaspase 3. and FADD-⌬DED were established, as previously described (Kawahara We and others have recently established sublines et al., 1998). In brief, cells were cotransfected with 50 ␮g of the respective from human Jurkat cells that do not express caspase 8 expression vectors with 1 ␮g of the hygromycin-resistant gene (pMiwhyg) (Juo et al., 1998; Kawahara et al., 1998). Engagement of by electroporation. Cells were cultured in the presence of 0.8–1.0 mg/ml of hygromycin, and the transformant clones expressing FADD-DD, FADD- Fas by agonistic anti–human Fas antibodies in these cell DED, or FADD-⌬DED were identified by Western blotting using anti- lines does not activate caspases, and, thus, cannot in- FADD or anti-FLAG antibodies. Jurkat cell transformants expressing duce apoptotic cell death. On the other hand, when high levels of mouse Fas were established by introducing the mouse Fas FADD was artificially oligomerized, these cells died. expression plasmid (Watanabe-Fukunaga et al., 1992).

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