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c-Jun NH2-terminal Kinase Activation Leads to a FADD-dependent but Fas Ligand-independent Cell Death in Jurkat T Cells

2001; Elsevier BV; Volume: 276; Issue: 11 Linguagem: Inglês

10.1074/jbc.m008431200

ISSN

1083-351X

Autores

Yun Chen, Ming‐Zong Lai,

Tópico(s)

NF-κB Signaling Pathways

Resumo

Persistent c-Jun NH2-terminal kinase (JNK) activation induces cell death. Different mechanisms are ascribed to JNK-induced cell death. Most of the JNK-apoptosis studies employ stress stimuli known to activate kinases other than JNK. Here we used overexpression of mitogen-activated protein kinase kinase 7 (MKK7) to activate selectively JNK in T lymphoma Jurkat cells. Similar to that reported previously, Fas ligand (FasL) expression was up-regulated by JNK activation. Dominant negative-FADD and caspase-8 inhibitor benzyloxycarbonyl-Ile-Glu-Thr-Asp effectively inhibited MKK7-induced cell death, supporting a major involvement of FADD cascade. However, MKK7-induced cell death was not prevented by antagonist antibody ZB4 and Fas-Fc, indicating that Fas-FasL interaction is minimally involved. Confocal microscopy revealed that persistent JNK activation led to clustering of Fas. Our results suggest that, in contrast to that reported previously, JNK alone-induced death in Jurkat cells is FADD-dependent but is not triggered by Fas-FasL interaction. Persistent c-Jun NH2-terminal kinase (JNK) activation induces cell death. Different mechanisms are ascribed to JNK-induced cell death. Most of the JNK-apoptosis studies employ stress stimuli known to activate kinases other than JNK. Here we used overexpression of mitogen-activated protein kinase kinase 7 (MKK7) to activate selectively JNK in T lymphoma Jurkat cells. Similar to that reported previously, Fas ligand (FasL) expression was up-regulated by JNK activation. Dominant negative-FADD and caspase-8 inhibitor benzyloxycarbonyl-Ile-Glu-Thr-Asp effectively inhibited MKK7-induced cell death, supporting a major involvement of FADD cascade. However, MKK7-induced cell death was not prevented by antagonist antibody ZB4 and Fas-Fc, indicating that Fas-FasL interaction is minimally involved. Confocal microscopy revealed that persistent JNK activation led to clustering of Fas. Our results suggest that, in contrast to that reported previously, JNK alone-induced death in Jurkat cells is FADD-dependent but is not triggered by Fas-FasL interaction. c-Jun NH2-terminal kinase dominant negative Fas ligand green fluorescence protein mitogen activated protein kinase kinase 7 mitogen activated protein kinase kinase kinase 1 propidium iodide PE, phycoerythrin glutathioneS-transferase JNK1 activation is always linked to cell death induced by stress. Apoptosis triggered by UV, γ-irradiation, and cytotoxic drugs is correlated with activation of JNK, and the cell death is prevented by inhibition of JNK activation (1Chen Y.-R. Meyer C.F. Tan T.-H. J. Biol. Chem. 1996; 271: 631-634Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 2Chen Y.-R. Wang X. Templeton D. Davis R.J. Tan T.-H. J. Biol. 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Neuron. 1999; 22: 667-676Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar). The exact molecular mechanism how JNK induces cell death remains largely elusive. Different apoptotic molecules have also been attributed to JNK-triggered cell death. Activation of c-Jun by JNK seems to mediate part of the apoptotic events (17Leppa S. Bohmann D. Oncogene. 1999; 18: 6158-6162Crossref PubMed Scopus (453) Google Scholar). MEKK1 or c-Jun induces FasL expression and the subsequent FasL-Fas interaction and cell death (6Faris M. Kokot N. Latinis K. Kasiblhatla S. Green D.R. Koretzky G.A. Nel A. J. Immunol. 1998; 160: 134-144PubMed Google Scholar, 12Le-Niculescu H. Bonfoco E. Kasuya Y. Claret F.X. Green D.R. Karin M. Mol. Cell. Biol. 1999; 19: 751-763Crossref PubMed Scopus (440) Google Scholar, 18Faris M. Latinis K. Kempiak S.J. Koretzky G.A. Nel A. Mol. Cell. Biol. 1998; 18: 5414-5424Crossref PubMed Google Scholar, 19Kolbus A. Herr I. Schreiber M. Debatin K.M. Wagner E.F. Angel P. Mol. Cell. Biol. 2000; 20: 575-582Crossref PubMed Scopus (146) Google Scholar). p53 and Bax may also mediate JNK-induced apoptosis following p75 neurotrophin receptor activation (11Aloyz R.S. Bamji S.X. Pozniak C.D. Toma J.G. Atwal J. Kaplan D.R. Miller F.D. J. Cell Biol. 1998; 143: 1691-1703Crossref PubMed Scopus (262) Google Scholar). Alternatively, apoptosis could be induced by translocation of JNK into mitochondria followed by phosphorylation and inactivation of Bcl-2 and Bcl-xL (20Yamamoto K. Ichijo H. Korsmeyer S.J. Mol. Cell. Biol. 1999; 19: 8469-8478Crossref PubMed Scopus (935) Google Scholar, 21Kharbanda S. Saxena S. Yoshida K. Pandey P. Kaneki M. Wang Q. Cheng K. Chen Y.-N. Campbell A. Sudha T. Yuan Z.-M. Narula J. Weichselbaum R. Nalin C. Kufe D. J. Biol. Chem. 2000; 275: 322-327Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). In addition, the UV-induced mitochondrial death pathway is abrogated in the absence of JNK, further supporting mitochondria as the target of JNK (22Tournier C. Hess P. Yang D.-D. Xu J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1560) Google Scholar). Most of JNK-inducing signals such as UV and cytotoxic drugs activate signals other than JNK. Even for the selective expression of MEKK1, Cdc42, or apoptosis signal-regulating kinase 1, activation of JNK is accompanied by stimulation of p38 and/or IκB kinase. In addition, the contribution of p38 to stress-activated apoptosis has been demonstrated (5Lee L.-F. Li G. Templeton D.J. Ying J.P.-Y. J. Biol. Chem. 1998; 273: 28253-28260Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 10Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5072) Google Scholar, 23Berra E. Diaz-Meco M.T Moscot J. J. Biol. Chem. 1998; 273: 10792-10797Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 24Cong F. Goff S.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13819-13824Crossref PubMed Scopus (54) Google Scholar). In this study, we used transient expression of MKK7 to activate JNK in Jurkat T cells. MKK7 selectively activates JNK but not other kinases (25Tournier C. Whitmarsh A.J. Cavanagh J. Barrett T. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7337-7342Crossref PubMed Scopus (342) Google Scholar, 26Holland P.M. Suzanne M. Campbell J.S. Noselli S. Cooper J.A. J. Biol. Chem. 1997; 272: 24994-24998Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 27Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar, 28Yao Z. Diener K. Wang X.S. Zukowski M. Matsumoto G. Zhou G. Mo R. Sasaki T. Nishina H. Hui C.-C. Tan T.-H. Woodgett J.P. Penninger J.M. J. Biol. Chem. 1997; 272: 32378-32383Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 29Wang Y. Su B. Sah V.P. Brown J.H. Han J. Chien K.-R. J. Biol. Chem. 1998; 273: 5423-5436Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). We confirmed the previous notion that JNK activation leads to increased FasL expression in Jurkat cells. Blockage of FADD-initiated apoptotic pathway effectively prevented JNK-induced cell death. However, blockage of FasL-Fas interaction by antagonizing antibody or Fas-Fc did not affect MKK7-induced apoptosis, suggesting that FasL is minimally involved in JNK-mediated cell death. Our results clearly suggest that JNK induces apoptosis by a FADD-dependent but FasL-independent mechanism in Jurkat cells. Jurkat cell (H6.2 clone) was a gift of Dr. Daniel Olive (INSERM U119, Marseille, France). Antibody against α-tubulin was obtained from Amersham Pharmacia Biotech. Anti-FasL antibody (C-20) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PE-conjugated anti-Fas antibody (DX2) and anti-FasL antibody (NOK-1) were purchased from eBioscience (San Diego, CA). Anti-Fas antibodies CH11 and ZB4 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Caspase-8 inhibitor Z-IETD-fluoromethyl ketone was obtained from Calbiochem. Human Fas-Fc was purchased from R & D Systems (Minneapolis, MN). Active MKK7α (S271D and T275D) (29Wang Y. Su B. Sah V.P. Brown J.H. Han J. Chien K.-R. J. Biol. Chem. 1998; 273: 5423-5436Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar) and active MKK3b were gifts of Dr. Jiahuai Han (Scripps Research Institute, La Jolla, CA). ΔMEKK1 and SEK-AL were gifts of Dr. Dennis Templeton (Case Western University, Cleveland, OH). pcDNA3-AU-FADD and DN-FADD were gifts of Dr. Vishva Dixit (Genentech, South San Francisco). FLIP plasmid was previously constructed (30Yeh J.-H. Hsu S.-C. Han S.-H. Lai M.-Z. J. Exp. Med. 1998; 188: 1795-1802Crossref PubMed Scopus (119) Google Scholar). Jurkat T cells (1 × 107) were washed and resuspended in 0.6 ml of RPMI medium containing 1% glucose, 10% fetal calf serum, and 10–20 μg of plasmid DNA. The electroporation was performed in Bio-Rad Gene Pulser II at 260 mV and 975 microfarads. The cuvette was left on ice for 15 min, washed twice with phosphate-buffered saline, and incubated for the indicated time for cell death and biochemical analysis. Jurkat T cells were transfected with pcDNA3, ΔMEKK1, or pcDNA3-MKK7α. Cell lysates were prepared 24 h after transfection, and 100–200 μg of lysate was precipitated with 1 μl of anti-JNK1 antibody 101 (31Meyer C.F. Wang X. Chang C. Templeton D. Tan T.H. J. Biol. Chem. 1996; 271: 8971-8976Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) or anti-p38 (32Jiang Y. Gram H. Zhao M. New L. Gu J. Feng L. Padova F.D. Ulevitch R.J. Han J. J. Biol. Chem. 1997; 272: 30122-30128Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar), followed by 20 μl of protein A-Sepharose. The kinase activity of the immune complexes was determined by using GST-c-Jun-(1–79) or myelin basic protein as substrates. The reaction mixtures were resolved on SDS-polyacrylamide gel electrophoresis, followed by autoradiography, and quantitated by PhosphorImager (Molecular Dynamics). Apoptosis in bulk population was determined by propidium iodide (PI) staining. At the indicated times after treatment, cells were harvested and washed in phosphate-buffered saline twice and resuspended in hypotonic fluorochrome solution (50 μg/ml PI, 0.1% sodium citrate, 0.1% Triton X-100) (33Nicoletti I. Migliorati G. Pagliacci M.C. Grignani F. Riccardi C. J. Immunol. Methods. 1991; 139: 271-279Crossref PubMed Scopus (4452) Google Scholar). Cells were placed at 4 °C in the dark overnight, and DNA content was analyzed by FACScan (Becton Dickinson, Mountain View, CA). The fraction of cells with sub-G1 DNA content was assessed using the CELLFIT program (Becton Dickinson). For apoptosis in cells transiently transfected with MKK7α or ΔMEKK1, green fluorescence protein expression vector pGreen Lanten-1 (Life Technologies, Inc.) was cotransfected. Cells were harvested at the indicated times, fixed with paraformaldehyde, and terminal dUTP nick-end labeling reaction was performed using FlowTACS kit (R & D Systems). The incorporated biotin-dUTP was labeled with Tri-Color-streptavidin (Caltag, Burlingame, CA). The green cells (GFP-positive) were then gated on FACScan, and the fraction of cell stained with Tri-Color was quantitated. Alternatively, PI staining was also used to determined the fraction of subdiploid cells in GFP-positive population. Jurkat cells were transfected with active MKK7α or MEKK1 by electroporation. We chose electroporation because the transfection efficiency was close to 30% as determined by cotransfection with GFP (Fig.1 A, R1). To assess the cell death induced by JNK activation, the population expressing GFP was gated in fluorescence-activated cell sorter, and the fraction of apoptotic cells labeled with biotin-dUTP was quantitated (Fig.1 A). Activation of JNK by MKK7α and MEKK1 in Jurkat cells led to 50% death 24 h after transfection (Fig. 1 B). We next examined whether the cell death observed was JNK-dependent. MKK7α and MEKK1 were equally effective in the JNK induction (Fig. 2 A). The specificity of MKK7α was further confirmed by its inability to activate p38 mitogen-activated protein kinase, in contrast to the effective induction of p38 by MEKK1 and MKK3 (Fig. 2 B). The activation of JNK by MEKK1 or MKK7α was prevented by cotransfection of SEK-AL (Fig. 2 A). The exact mechanism how SEK-AL prevents MKK7α-induced activation of JNK is not completely clear. Presumably, the binding of SEK-AL with JNK (34Xia Y. Wu Z. Su B. Murray B. Karin M. Gene Dev. 1998; 12: 3369-3381Crossref PubMed Scopus (178) Google Scholar) would compete with the interaction of JNK with MKK7α. Inhibition of JNK activation by SEK-AL prevented MKK7α- or MEKK1- induced cell death (Fig. 2 C), indicating that the observed cell death is JNK-specific. Because MKK7α is a more specific activator of JNK, in the following experiments mainly the results of MKK7α transfection are shown. In all the criteria evaluated, MEKK1-induced apoptosis displayed an identical character.Figure 2JNK-dependent cell death induced by active MKK7 α. Jurkat cells were transfected by electroporation with 3 μg of pGreen Lanten-1, with active pcDNA3-MKK7α, ΔMEKK1, or MKK3 (each 5 μg), with or without 10 μg of SEK-AL. Cell extracts were prepared 18 h after transfection. A, JNK activities were determined by immunoprecipitation with anti-JNK1 (31Meyer C.F. Wang X. Chang C. Templeton D. Tan T.H. J. Biol. Chem. 1996; 271: 8971-8976Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) and phosphorylation of GST-c-Jun-(1–79). B, p38 activities were determined by precipitation with anti-p38 (32Jiang Y. Gram H. Zhao M. New L. Gu J. Feng L. Padova F.D. Ulevitch R.J. Han J. J. Biol. Chem. 1997; 272: 30122-30128Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar) and phosphorylation of myelin basic protein (MBP). C, apoptosis was determined 24 h after transfection as described in Fig. 1. TC-SA, Tri-Color-streptavidin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Consistent with previous reports (6Faris M. Kokot N. Latinis K. Kasiblhatla S. Green D.R. Koretzky G.A. Nel A. J. Immunol. 1998; 160: 134-144PubMed Google Scholar, 18Faris M. Latinis K. Kempiak S.J. Koretzky G.A. Nel A. Mol. Cell. Biol. 1998; 18: 5414-5424Crossref PubMed Google Scholar), there was a significant increase of FasL expression after transfection of MKK7α and MEKK1 as determined by immunoblots (Fig. 3 A). Despite a similar degree of JNK activation (Fig. 2), the extent of FasL expression was higher for MEKK1 transfection than MKK7α transfection. A likely cause is because MEKK1 also activates NF-κB and p38 mitogen-activated protein kinase, and both contribute to activation of the FasL promoter (35Kasibhatla S. Brunner T. Genestier L. Echeverri F. Mahboubi A. Green D.R. Mol. Cell. 1998; 1: 543-551Abstract Full Text Full Text PDF PubMed Scopus (670) Google Scholar, 36Matsui K. Fine A. Zhu B. Marshak-Rothstein A. Ju S.-T. J. Immunol. 1998; 161: 3469-3473PubMed Google Scholar, 37Hsu S.-C. Gavrilin M. Lee H.-H. Wu C.-C. Han S.-H. Lai M.-Z. Eur. J. Immunol. 1999; 29: 2948-2956Crossref PubMed Google Scholar, 38Hsu S.-C. Gavrilin M. Tsai M.-H. Han J. Lai M.-Z. J. Biol. Chem. 1999; 274: 25769-25776Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Despite the increase of total cellular FasL, there was little increase in the surface FasL expression after MKK7α expression (Fig. 3 B, dark curve). As a positive control, TPA/A23187 treatment significantly promoted the surface FasL expression (Fig. 3 B, light curve). The expression of Fas is already high in Jurkat cells. Expression of active MEKK1 or MKK7α added little to the surface Fas expression (not shown). Fas-mediated apoptotic pathway is initiated by recruitment of FADD, followed by cleavage and activation of caspase-8 (39Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4577) Google Scholar). To examine whether MKK7α-induced cell death was indeed Fas-dependent, we used Fas-specific inhibitor DN-FADD, FLIP, and Z-IETD. DN-FADD competes with wild-type FADD (40Boldin M.P. Varfolomeev E.E. Pancer Z. Mett I.L. Camonis J.H. Wallach D. J. Biol. Chem. 1995; 270: 7795-7798Abstract Full Text Full Text PDF PubMed Scopus (943) Google Scholar, 41Chinnaiyan A.M. O'Roueke K. Tewari M. Dixit V.M. Cell. 1995; 81: 505-512Abstract Full Text PDF PubMed Scopus (2185) Google Scholar); FLIP specifically antagonizes Fas-dependent cell death at the stage of FADD and caspase-8 (42Irmler M. Thome M. Hahne M. Schnedier P. Hofmann K. Steiner V. Bodmer J.-L. Schroter M. Burns K. Mattmann C. Rimoldi D. French L.E. Tschopp J. Nature. 1997; 388: 190-195Crossref PubMed Scopus (2244) Google Scholar), and Z-IETD selectively inhibits caspase-8. Cotransfection with DN-FADD effectively inhibited MKK7α-induced apoptosis (Fig. 4). The expression of FLIP similarly inhibited MKK7α-induced apoptosis (not shown). The efficacy of caspase-8-specific inhibitor Z-IETD (50 μm) was first confirmed by blockage of CH11-induced apoptosis (not shown). The addition of Z-IETD 2 h after MKK7α transfection abrogated MKK7α-triggered apoptosis (Fig. 4). The inhibition of MKK7α-induced apoptosis by FLIP, DN-FADD, and Z-IETD supports the notion that FADD-mediated apoptotic pathway plays a major role in JNK-triggered cell death in Jurkat cells. To determine the role of Fas-FasL engagement in MKK7α-induced cell death, the antagonistic anti-Fas antibody ZB4 was used. Preincubation with ZB4 (250 ng/ml) effectively suppressed CH11-induced apoptosis of Jurkat cells (Fig.5 A). In contrast, the extent of Jurkat cell death resulting from MKK7α overexpression was indistinguishable in the presence or absence of ZB4 (Fig.5 B). We also used soluble Fas-Fc fusion protein to block the interaction of Fas and FasL. Fas-Fc (200 ng/ml) prevented FasL-induced cell death (Fig. 5 A), yet Fas-Fc failed to interfere with MKK7α-induced apoptosis in Jurkat cells (Fig. 5 B). Because ΔMEKK1 induced higher expression of FasL (Fig. 3), we further examined whether ΔMEKK1-induced cell death could be inhibited by Fas-Fc or ZB4. Neither Fas-Fc nor ZB4 prevented apoptosis induced by ΔMEKK1 (not shown). The observations that Fas-Fc and ZB4 did not protect Jurkat cells from apoptosis suggest that the apoptosis induced by JNK is not mediated through Fas-FasL interaction. Stress stimuli such as UV, cycloheximide, cisplatin, etoposide, vinblastine, and doxorubicin may induce cell death by triggering Fas clustering in a FasL-independent manner (43Bennett M. McDonald K. Chan S.W. Luzio J.P. Simari R. Weissberg P. Science. 1998; 282: 290-293Crossref PubMed Scopus (661) Google Scholar, 44Aragane Y. Kulms D. Metze D. Wilkes G. Poppelmann B. Luger T.A. Schwarz T. J. Cell Biol. 1998; 140: 171-182Crossref PubMed Scopus (433) Google Scholar, 45Micheau O. Solary E. Hammann A. Dimanche-Boitrel M.-T. J. Biol. Chem. 1999; 274: 7987-7992Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 46Tang D. Lahti J.M. Grenet J. Kidd V.J. J. Biol. Chem. 1999; 274: 7245-7252Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). We also examined the surface Fas distribution on a macroscopic level before and after JNK activation using confocal laser scanning microscope. Fas was evenly distributed on the surface of the untransfected Jurkat cells (Fig.6 A). Treatment with sFasL led to increased aggregation of Fas on the surface of Jurkat cells (Fig.6 B). For cells transfected with MKK7α, as those marked by GFP expression (Fig. 6 C), there was a similar increased clustering of Fas on the surface of Jurkat cells as compared with the nearby untransfected cells (Fig. 6 D). Therefore, constitutive MKK7α expression promotes the aggregation of the surface Fas. JNK has been implicated as the major mediator of cell death induced by stress (1Chen Y.-R. Meyer C.F. Tan T.-H. J. Biol. Chem. 1996; 271: 631-634Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 2Chen Y.-R. Wang X. Templeton D. Davis R.J. Tan T.-H. J. Biol. Chem. 1996; 271: 31929-31936Abstract Full Text Full Text PDF PubMed Scopus (859) Google Scholar, 3Zanke B.W. Boudreau K. Rubie E. Winnett E. Tibbles L.A. Zon L. Kyriakis J. Liu F.-F. Woodgett J.R. Curr. Biol. 1996; 6: 606-613Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar), yet the exact apoptotic mechanism triggered by JNK is not fully understood. In the present study, we used a JNK-specific activator MKK7α to induce JNK and the subsequent apoptosis. Because MKK7α selectively activates JNK (25Tournier C. Whitmarsh A.J. Cavanagh J. Barrett T. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7337-7342Crossref PubMed Scopus (342) Google Scholar, 26Holland P.M. Suzanne M. Campbell J.S. Noselli S. Cooper J.A. J. Biol. Chem. 1997; 272: 24994-24998Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 27Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar, 28Yao Z. Diener K. Wang X.S. Zukowski M. Matsumoto G. Zhou G. Mo R. Sasaki T. Nishina H. Hui C.-C. Tan T.-H. Woodgett J.P. Penninger J.M. J. Biol. Chem. 1997; 272: 32378-32383Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 29Wang Y. Su B. Sah V.P. Brown J.H. Han J. Chien K.-R. J. Biol. Chem. 1998; 273: 5423-5436Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar), we were able to address death specifically activated by JNK in the absence of other signaling such as p38 (Fig. 2 B). Our results illustrate that JNK activation induced FasL expression (Fig. 3). The requirement of Fas-FADD pathway is supported by the inhibition of MKK7-triggered cell death by DN-FADD, FLIP, and Z-IETD (Fig. 4). We have also demonstrated, likely for the first time, that JNK-induced cell death in Jurkat cells is FasL-independent (Fig. 5). Our observations that Fas-Fc and ZB4 were unable to prevent MKK7α-induced cell death (Fig. 5) are in direct contradiction to the report of Faris et al. (6Faris M. Kokot N. Latinis K. Kasiblhatla S. Green D.R. Koretzky G.A. Nel A. J. Immunol. 1998; 160: 134-144PubMed Google Scholar) that inducible expression of MEKK1 led to cell death which was prevented by soluble Fas and Fas antagonist antibody. We speculate the difference between their study and ours is likely due to the levels of Fas and FasL expression. In the study of Faris et al. (6Faris M. Kokot N. Latinis K. Kasiblhatla S. Green D.R. Koretzky G.A. Nel A. J. Immunol. 1998; 160: 134-144PubMed Google Scholar), the inducible activation of MEKK1 in Jurkat led to an increase of surface FasL levels by 50-fold. This was accompanied by a 10-fold increase of surface Fas (see Fig. 5 in Ref. 6Faris M. Kokot N. Latinis K. Kasiblhatla S. Green D.R. Koretzky G.A. Nel A. J. Immunol. 1998; 160: 134-144PubMed Google Scholar). With such high levels of Fas and FasL, Fas-FasL interaction would inevitably become the dominant process to trigger cell death. However, the levels of Fas and FasL induced by MEKK1 in their study are highly unphysiological, as judged from their report that there was a mere 50% increase of surface FasL with a 20% increase of surface Fas expression when Jurkat cells were activated with TPA/A23187 (6Faris M. Kokot N. Latinis K. Kasiblhatla S. Green D.R. Koretzky G.A. Nel A. J. Immunol. 1998; 160: 134-144PubMed Google Scholar). In the present study, despite an induction of total FasL content, surface FasL levels in Jurkat cells were minimally altered by JNK activation (Fig.3), and cell death proceeded in the absence of FasL binding. Fas-FasL interaction apparently is not essential for JNK-induced cell death in Jurkat cells. We have also repeated our observation in another T lymphoma EL4 (not shown) and reached an identical conclusion. FADD-dependent but FasL-independent cell death is triggered by UV, cycloheximide, cisplatin, etoposide, vinblastine, and doxorubicin through induced clustering of Fas (43Bennett M. McDonald K. Chan S.W. Luzio J.P. Simari R. Weissberg P. Science. 1998; 282: 290-293Crossref PubMed Scopus (661) Google Scholar, 44Aragane Y. Kulms D. Metze D. Wilkes G. Poppelmann B. Luger T.A. Schwarz T. J. Cell Biol. 1998; 140: 171-182Crossref PubMed Scopus (433) Google Scholar, 45Micheau O. Solary E. Hammann A. Dimanche-Boitrel M.-T. J. Biol. Chem. 1999; 274: 7987-7992Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 46Tang D. Lahti J.M. Grenet J. Kidd V.J. J. Biol. Chem. 1999; 274: 7245-7252Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), leading to the association of FADD with Fas and the subsequent activation of caspase-8. Fas aggregation-induced cell death is suppressed by DN-FADD or FADD antisense (44Aragane Y. Kulms D. Metze D. Wilkes G. Poppelmann B. Luger T.A. Schwarz T. J. Cell Biol. 1998; 140: 171-182Crossref PubMed Scopus (433) Google Scholar, 45Micheau O. Solary E. Hammann A. Dimanche-Boitrel M.-T. J. Biol. Chem. 1999; 274: 7987-7992Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 46Tang D. Lahti J.M. Grenet J. Kidd V.J. J. Biol. Chem. 1999; 274: 7245-7252Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Similarly, MKK7-induced apoptosis is sensitive to inhibition by DN-FADD, FLIP, and Z-IETD (Fig. 4) and MKK7 overexpression triggered Fas clustering (Fig. 6), suggesting that JNK-activated apoptosis is mediated by Fas aggregation. We do not know the exact cellular process between the JNK activation and Fas clustering. Changes in the microtubule cytoskeleton may activate MEKK1 and JNK activation (47Yujiri T. Fanger G.R. Garrington T.P. Schlesinger T.K. Gibson S. Johnson G.L. J. Biol. Chem. 1999; 274: 12605-12610Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), but whether persistent JNK activation could induce reorganization of the microtubule cytoskeleton that promotes Fas association remains to be determined. It may also be noted that our results on JNK-induced Fas oligomerization are compatible with the recent observation that Fas receptor is in trimer status before FasL binding (48Siegel R.M. Frederiksen J.K. Zacharias D.A. Chan K.K. Johnson M. Lynch D. Ysien R.Y. Lenardo M.J. Science. 2000; 288: 2354-2357Crossref PubMed Scopus (547) Google Scholar). This is supported by the observation that the engagement of Jurkat cells by FasL led to a visible aggregation of Fas under the microscope (Fig. 6 D) as compared with untreated cells, supporting that the Fas aggregation observed by others and us (43Bennett M. McDonald K. Chan S.W. Luzio J.P. Simari R. Weissberg P. Science. 1998; 282: 290-293Crossref PubMed Scopus (661) Google Scholar, 44Aragane Y. Kulms D. Metze D. Wilkes G. Poppelmann B. Luger T.A. Schwarz T. J. Cell Biol. 1998; 140: 171-182Crossref PubMed Scopus (433) Google Scholar, 45Micheau O. Solary E. Hammann A. Dimanche-Boitrel M.-T. J. Biol. Chem. 1999; 274: 7987-7992Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 46Tang D. Lahti J.M. Grenet J. Kidd V.J. J. Biol. Chem. 1999; 274: 7245-7252Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) is in a macroscopic level. Therefore, Fas is trimerized in resting Jurkat cells but further aggregation is induced by stress stimuli or JNK activation. Our results may also help resolve the controversy on the involvement of FasL in chemotherapy-induced apoptosis. Despite the earlier reports that DNA-damaging agent-induced cell death is inhibited by Fas antagonizing antibody or Fas-Fc (32Jiang Y. Gram H. Zhao M. New L. Gu J. Feng L. Padova F.D. Ulevitch R.J. 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Narula J. Weichselbaum R. Nalin C. Kufe D. J. Biol. Chem. 2000; 275: 322-327Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). Together with the results from the present study, persistent activation of JNK is capable of triggering apoptotic pathways initiated by both mitochondria (22Tournier C. Hess P. Yang D.-D. Xu J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1560) Google Scholar) and death receptor. We speculate that the exact contribution from mitochondria and Fas pathway in JNK-mediated apoptosis would be determined by variables such as type of stress, type of cell, expression of Fas, and cellular sensitivity to Fas. Further characterization will help understand the exact molecular process triggered by JNK apoptotic signal in different cells. We thank Dr. Jiahuai Han for MKK7α, MKK3b, and anti-p38 antibody; Dr. Daniel Olive for Jurkat cells; Dr. Vishva Dixit for DN-FADD; Dr. Dennis Templeton for ΔMEKK1 and SEK-AL; and Dr. Tse-Hua Tan for anti-JNK1 antiserum.

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