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The Tumor Necrosis Factor-related Apoptosis-inducing Ligand Receptors TRAIL-R1 and TRAIL-R2 Have Distinct Cross-linking Requirements for Initiation of Apoptosis and Are Non-redundant in JNK Activation

The Tumor Necrosis Factor-related Apoptosis-inducing Ligand Receptors TRAIL-R1 and TRAIL-R2 Have... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 41, Issue of October 13, pp. 32208 –32213, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Tumor Necrosis Factor-related Apoptosis-inducing Ligand Receptors TRAIL-R1 and TRAIL-R2 Have Distinct Cross-linking Requirements for Initiation of Apoptosis and Are Non-redundant in JNK Activation* Received for publication, January 18, 2000, and in revised form, May 5, 2000 Published, JBC Papers in Press, May 11, 2000, DOI 10.1074/jbc.M000482200 Frank Mu ¨ hlenbeck‡, Pascal Schneider§, Jean-Luc Bodmer§, Ralph Schwenzer‡, Angelika Hauser‡, Gisela Schubert‡, Peter Scheurich‡, Dieter Moosmayer‡, Ju ¨ rg Tschopp§, and Harald Wajant‡¶ From the ‡Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany and the §Institute of Biochemistry, University of Lausanne, Ch. des Boveresses 155, 1066 Epalinges, Switzerland Overexpression of the tumor necrosis factor (TNF)- an involvement of TRAIL in natural killer cell-, dendritic cell-, and CD41 T-cell-mediated cytotoxicity (3– 6), TRAIL may also related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2, induces apoptosis and activa- be involved in monocyte-mediated tumoricidal activity (7) and kB in cultured cells. In this study, we have tion of NF- activation-induced T cell death during HIV infection (8, 9). demonstrated differential signaling capacities by both Currently five human TRAIL receptors belonging to the TNF receptors using either epitope-tagged soluble TRAIL receptor superfamily have been identified. Two of them, (sTRAIL) or sTRAIL that was cross-linked with a mono- TRAIL-R1 (DR4, Ref. 10) and TRAIL-R2 (DR5, TRICK2, KILL- clonal antibody. Interestingly, sTRAIL was sufficient for ER; see Refs. 11–18) contain a cytoplasmic death domain and induction of apoptosis only in cell lines that were killed transmit an apoptotic signal in response to TRAIL. Two other by agonistic TRAIL-R1- and TRAIL-R2-specific IgG prep- cellular TRAIL receptors, TRAIL-R3 (TRID, DcR1; see Refs. 11, arations. Moreover, in these cell lines interleukin-6 se- 14, 17, 19), which is glycosylphosphatidylinositol (GPI)-linked kB activation were induced by cross- cretion and NF- and TRAIL-R4 (DcR2; see Refs. 20, 21), which contains a trun- linked or non-cross-linked anti-TRAIL, as well as by both cated death domain, bind TRAIL without activation of the receptor-specific IgGs. However, cross-linking of sTRAIL apoptotic machinery and seem to antagonize the death domain- was required for induction of apoptosis in cell lines that containing TRAIL receptors. In addition, osteoprotegerin, a only responded to the agonistic anti-TRAIL-R2-IgG. Inter- regulator of osteoclastogenesis, is a soluble receptor for TRAIL estingly, activation of c-Jun N-terminal kinase (JNK) was (22). TRAIL-R1- and TRAIL-R2-mediated apoptosis occurs via only observed in response to either cross-linked sTRAIL or activation of caspase-8 and subsequent activation of effector anti-TRAIL-R2-IgG even in cell lines where both receptors caspases. However, the link between the death domain-con- kB activation. were capable of signaling apoptosis and NF- taining TRAIL receptors and activation of caspase-8 is rather Taken together, our data suggest that TRAIL-R1 responds undefined. Transient transfection of TRAIL-R1 leads to activa- to either cross-linked or non-cross-linked sTRAIL which kB activation and apoptosis, whereas TRAIL-R2 tion of the apoptotic machinery in Fas-associated death domain signals NF- kB activation, apoptosis, and JNK activation signals NF- protein-deficient fibroblasts, suggesting that FADD, a death only in response to cross-linked TRAIL. domain adapter molecule, is not required for TRAIL-R1-in- duced apoptosis (23, 24). On the other hand, overexpression of a dominant-negative FADD mutant was shown to block Tumor necrosis factor-related apoptosis-inducing ligand TRAIL-mediated apoptosis (12, 13, 15, 25). It has not yet been (TRAIL), also designated as APO-2 ligand, is a member of the clarified whether FADD plays a specific role in TRAIL-R2- tumor necrosis factor (TNF) family that is capable of inducing induced apoptosis or whether a closely related adapter protein apoptosis in several cell lines (1, 2). TRAIL is widely expressed is involved in TRAIL-R1- and TRAIL-R2-mediated apoptosis. in normal cells and is highly homologous to FasL, another Because TRAIL is highly effective in killing cancer cell lines cytotoxic member of the TNF ligand family (1, 2). In addition to but has apparently no lethal effects on normal cells, TRAIL and its apoptotic receptors have attracted much attention as tar- gets for anti-cancer therapy (26, 27). In this study, we show * This work was supported by Deutsche Forschungsgemeinschaft that TRAIL-R1 and TRAIL-R2 have different capabilities for Grant Wa 1025/3-1. The costs of publication of this article were de- stimulating the JNK pathway and differ also in their cross- frayed in part by the payment of page charges. This article must linking requirements for activation by recombinant ligands. therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This is the first reported evidence of a difference between ¶ To whom correspondence should be addressed. Tel.: 49-711-685- TRAIL-R1 and TRAIL-R2 signaling activities. 7446; Fax: 49-711-685-7484; E-mail: [email protected] stuttgart.de. EXPERIMENTAL PROCEDURES The abbreviations used are: TRAIL, TNF-related apoptosis-induc- Materials—The anti-FLAG monoclonal antibody M2 was purchased ing ligand; JNK, c-Jun N-terminal kinase; TNF, tumor necrosis factor; from Sigma-Aldrich (Deisenhofen, Germany). Polyclonal sera specific MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; for JNK, p65, p50, and cRel were purchased from Santa Cruz Biotech- TRAIL-R, TRAIL receptor; FACS, fluorescence-activated cell sorter; nology (Heidelberg, Germany) and protein A-Sepharose was from Am- CHX, cycloheximide; EMSA, electrophoretic mobility shift assay; Z- ersham Pharmacia Biotech (Freiburg, Germany). The SuperFect trans- VAD-fmk, benzyloxycarbonyl-VAD-fluoromethylketone; mAb, mono- fection reagent was obtained from Qiagen (Hilden, Germany). TRAIL- clonal antibody; IL-6, interleukin-6; GST, glutathione S-transferase; ELISA, enyzme-linked immunosorbent assay. R1-Fc and TRAIL-R2-Fc were from Alexis (La ¨ ufelfingen, Switzerland). 32208 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Non-redundant TRAIL Receptor Signaling 32209 FIG.1. Cell type- and response-spe- cific effects of cross-linked TRAIL- FLAG. A, the indicated cell lines were analyzed for their sensitivity to FLAG- tagged sTRAIL in the presence (filled squares) or absence (open squares)ofag- gregating anti-FLAG antibody M2. Cell viability was determined using the MTT assay (Jurkat, Kym-1) or by staining ad- herent cells with crystal violet (HeLa, KB, HepG2, HT1080). The difference in ab- sorbance between dead and living cells was in the range of 600 –1000 mOD units for all cells. B, HeLa and KB cells were cultured overnight in 96-well plates. Cells were then incubated for 18 h with the indicated concentrations of cross-linked (filled squares) and non-cross-linked (open squares) sTRAIL-FLAG in the pres- ence of 2.5 mg/ml CHX and 10 mM Z-VAD- fmk. Finally, IL-6 concentrations in the supernatants were determined using a commercially available ELISA kit. C, HeLa cells were cultured overnight in 96- well plates. The next day, cells were transfected with a 33 NF-kB-luciferase reporter plasmid and a SV40 promoter- driven b-galactosidase expression plas- mid to normalize the transfection effi- ciency. After an additional day, cells were stimulated for 9 h with cross-linked (filled bars) and non-cross-linked TRAIL-FLAG (open bars) in the presence of CHX (2.5 mg/ml) and Z-VAD-fmk (10 mM). Finally cells were assayed for NF-kB activation. D, cell lysates were prepared from HeLa and Kym-1 cells that had been stimulated for 4 h with the indicated concentrations of cross-linked and non-cross-linked TRAIL- FLAG (TRAIL-F). JNK activity was meas- ured by immunocomplex kinase assay with GSTzc-Jun-(1–79) as substrate. Cell Lines—HeLa, HepG2, and Jurkat cells were maintained in concentration of FLAG-tagged TRAIL with the anti-FLAG monoclonal RPMI 1640 medium containing 5% (HeLa, HepG2) or 10% (Jurkat) antibody M2 to a final concentration of 1 mg/ml of antibody. After a heat-inactivated fetal calf serum. KB cells were grown in Dulbecco’s 15-min incubation at room temperature, the TRAIL-M2-complex was modified Eagle’s medium supplemented with 10% fetal calf serum and transferred to the cells. TRAIL-R1 and TRAIL-R2 IgG were added in HT1080 cells in Dulbecco’s modified Eagle’s medium-nutrient mix F12 the presence of 1 mg/ml protein A (Sigma, Deisenhofen, Germany). Cell containing 10% fetal calf serum. The Kym-1 cell line was maintained in viability was determined using the MTT method (Jurkat, Kym-1) or Click RPMI 1640 medium supplemented with 10% fetal calf serum. crystal violet staining (HepG2, HeLa, KB, HT1080) as described previ- Generation of TRAIL-R1 and TRAIL-R2-specific IgG Preparations— ously (28, 29). Using a commercial antibody production service (Eurogentec, Seraing, Immunocomplex JNK Assay—N-terminal c-Jun kinase assays were Belgium), rabbits were immunized with TRAIL-R1-Fc and TRAIL-R2- performed upon immunoprecipitation of JNK1 using a rabbit antiserum Fc. For antibody purification, TRAIL-R1-Fc and TRAIL-R2-Fc were (Santa Cruz Biotechnology, Heidelberg, Germany). GSTzc-Jun was used coupled to HiTrap N-hydroxysuccinimide (NHS)-Sepharose (Amersham as substrate in an in vitro kinase assay as described previously (30). Pharmacia Biotech, Freiburg, Germany) according to the manufactur- Transient Reporter Gene Assays—For transient reporter gene assays, er’s protocol. Fc-specific antibodies were first depleted by repeated 20,000 HeLa cells were seeded in 96-well tissue culture plates, and the passages over human IgG1-agarose (Sigma, Deisenhofen, Germany). following day the cells were transfected with a 33 NF-kB-luciferase TRAIL-R1/R2-specific antibodies were further purified on TRAIL-R1/ reporter plasmid (15% transfected DNA), a SV40 promoter-driven b-ga- R2-Fc-Sepharose, eluted in 50 mM citrate/NaOH, pH 2.7, neutralized lactosidase expression plasmid (5% transfected DNA) to normalize the with Tris-HCl, pH 9, and dialyzed against phosphate-buffered saline. transfection efficiency, and empty vector (80% transfected DNA). At concentrations below 500 ng/ml, we found no evidence for cross- Transfections were performed with SuperFect reagent according to the reactivity of the anti-TRAIL receptor IgGs even upon secondary cross- manufacturer’s recommendations (Qiagen, Hilden, Germany). After a linking with protein A. At higher concentrations (.1000 ng/ml) we 1-day recovery, cells were treated with TRAIL, TRAIL-M2-complex, observed a significant cross-reactivity of the anti-TRAIL-receptor IgGs CHX, and Z-VAD-fmk as indicated, harvested in phosphate-buffered that could be blocked by addition of TRAIL-R1-Fc to anti-TRAIL-R2 IgG saline, and then luciferase and b-galactosidase activities were deter- and vice versa (data not shown). mined using the Galacto-Light Plus reporter gene assay kit (Perkin Cytotoxic Assays—50,000 (Jurkat), 30,000 (HepG2, KB), 20,000 Elmer, Nieuwerkerk, The Netherlands) and a Lucy2 96-well luminom- (HeLa, HT1080) or 15,000 Kym-1 cells were grown overnight in 100 ml eter (Anthos, Krefeld, Germany). of culture medium in 96-well plates. The cells were then treated for 16 h Determination of Interleukin-6 Production—Cells (1.5 3 10 per well) with FLAG-tagged TRAIL, TRAIL-M2-complex, anti-TRAIL-R1- and were seeded in triplicates in 96-well tissue culture plates in 100 mlof anti-TRAIL-R2-IgG. Cell death assays with HepG2, HeLa, KB, and Click RPMI 1640 and cultured overnight. The following day the cells HT1080 cells were performed in the presence of 2.5 mg/ml cycloheximide were treated with the reagents of interest as indicated for an additional (CHX). TRAIL-M2-complex was generated by mixing the respective 12–24 h. Then the supernatants were removed, cleared by centrifuga- 32210 Non-redundant TRAIL Receptor Signaling FIG.3. TRAIL-R2 but not TRAIL-R1 signals JNK activation. A, cell lysates were prepared from Kym-1, Jurkat, HeLa, and HepG2 cells that had been stimulated for 4 h with protein A cross-linked (1 mg/ml) anti-TRAIL-R1 IgG and anti-TRAIL-R2 IgG, each at 200 ng/ml in the absence of CHX. For control purposes lysates from cells treated with protein A and non-relevant IgG were also analyzed. JNK activity was measured by immunocomplex kinase assay with GSTzc-Jun-(1–79) as a substrate. B, dose dependence of anti-TRAIL-R2 IgG-induced JNK ac- tivation. Kym-1, Jurkat, HeLa, and HepG2 cells were stimulated with the indicated concentrations of protein A cross-linked anti-TRAIL-R2 IgG for 4 h, and lysates were analyzed for JNK activity by immuno- complex kinase assays. electrophoresis in low ionic strength buffer. For supershift analyses, 10 ml of the nuclear extracts (1 mg/ml protein) were incubated on ice for 1 h with 1 mg of polyclonal antibodies specific for p65, p50, and cRel (Santa Cruz Biotechnology, Heidelberg, Germany). Then the formed complexes FIG.2. A, protein A cross-linking enhanced the agonistic capacity of were incubated with 2 mlof53 binding buffer (500 mM KCl, 50 mM anti-TRAIL-R1 and -R2 IgG. HepG2 cells were incubated overnight Tris-HCl, pH 7.4, 25 mM MgCl , 50% glycerol, 5 mM dithiothreitol) and with the indicated concentration of anti-TRAIL-R1 IgG and anti- 2 ml of poly(dI-dC) (2 mg/ml) in a final volume of 20 ml. NF-kB DNA- TRAIL-R2 IgG, respectively, with (filled bars) or without (open bars) binding activity was again analyzed by native polyacrylamide gel elec- previous aggregation with protein A in the presence of 2.5 mg/ml CHX. trophoresis and phosphorimaging (Storm 860; Amersham Pharmacia Cell viability was measured by crystal violet staining. In addition, HeLa Biotech, Freiburg, Germany). cells were treated in the same way with the anti-TRAIL-receptor IgGs in the presence of 2.5 mg/ml CHX and 10 mM Z-VAD-fmk, and superna- RESULTS AND DISCUSSION tants were analyzed for production of IL-6. B, cytotoxic effects of anti- TRAIL-R1 IgG and anti-TRAIL-R2 IgG. Various cell lines were incu- Most ligands of the TNF family are either membrane-bound bated overnight with protein A cross-linked anti-TRAIL-R1 IgG (filled or proteolytically processed into soluble proteins. Evidence sug- bars) or anti-TRAIL-R2 IgG (hatched bars) or were left untreated (open gests that artificial cross-linking of soluble ligands mimics the bars). The next day, cell viability was determined by the MTT assay distinct biological activities of the corresponding membrane- (Jurkat, Kym-1) or by staining with crystal violet (HeLa, KB, HepG2, HT1080). To allow induction of apoptosis, HepG2, HeLa, KB, or HT1080 bound ligands. For example, we have recently shown that the cells were treated in the presence of 2.5 mg/ml CHX. C, Kym-1 cells were cytotoxic activity of FLAG-tagged human Fas ligand (sFasL), treated with anti-TRAIL-R1 or anti-TRAIL-R2 IgG (aTR1-IgG or aTR2- was increased by .1000-fold in response to cross-linking with IgG, respectively; 200 ng/ml) and protein A (1 mg/ml) with Z-VAD-fmk the anti-FLAG monoclonal antibody M2. Notably, this in- (ZVAD,20 mM). After 3 h, cells were harvested and analyzed for NF-kB activation by EMSA. D, HeLa cells were treated as indicated with, creased activity was comparable with the cytotoxic potency of anti-TRAIL-R1 and anti-TRAIL-R2 IgG (200 ng/ml), protein A (1 mg/ membrane-bound FasL (32). Further, activation of TNF-R2-de- ml), CHX (2.5 mg/ml), and Z-VAD-fmk (20 mM) for 3 h. As a control, cells pendent signaling pathways by soluble FLAG-tagged TNF was were also treated with 10 ng/ml TNF for 30 min. Supershift analyses strongly increased by multimerization of this ligand by the were performed as described under “Experimental Procedures.” Super- shifted complexes of the NF-kB oligonucleotide and p65, p50, or cRel, anti-FLAG monoclonal antibody M2. In accordance with that, respectively, are indicated by arrows. we have previously shown that membrane-bound, but not sol- uble TNF, is the prime activating ligand for TNF-R2 (33, 34), tion (15,000 rpm, 10 min, 4 °C) and interleukin-6 concentrations were suggesting that cross-linked and membrane-bound ligands determined using a commercially available ELISA kit (PharMingen, have analogous effects on this receptor. Using various cell lines Hamburg, Germany). we have therefore tested whether a recombinant soluble FLAG- FACS Staining—Cells were stained for TRAIL-R1, TRAIL-R2, tagged form of TRAIL (sTRAIL) required cross-linking for its TRAIL-R3, and TRAIL-R4 expression in 100 ml of FACS buffer (phos- activity. phate-buffered saline, 5% fetal calf serum, 0.1% NaN ) with 5 mg/ml We found that several cell lines, e.g. Jurkat and Kym-1, anti-TRAIL-R1 mAb M271 (IgG2a), anti-TRAIL-R2 mAb M413 (IgG1), anti-TRAIL-R3 mAb M430 (IgG1) and anti-TRAIL-R4 mAb M445 designated in the following as group I cells, were killed by (IgG1), respectively, or the respective control IgG, followed by fluores- physiological amounts (,200 ng/ml) of sTRAIL only in the cein isothiocyanate-labeled anti-mouse antibody (5 mg/ml). FACS anal- presence of secondary cross-linking by the anti-FLAG mono- yses were performed with a FACStar plus instrument (Becton Dickin- clonal antibody M2 (Fig. 1A). However, we also identified a son, San Jose, CA). 6 second set of cell lines designated in the following as group II EMSA Analysis of NF-kB Activation—HeLa and Kym-1 cells (10 ) cells that were efficiently killed by non-cross-linked sTRAIL were seeded in 60-mm cell culture dishes and cultivated overnight to allow adherence. The next day the cells were stimulated for 3 h with the (HeLa, HepG2, HT1080, and KB; Fig. 1A). The group II cell indicated combinations of anti-TRAIL-R1 and anti-TRAIL-R2 IgG, pro- lines Hela and KB were also tested with respect to the cross- tein A (1 mg/ml), Z-VAD-fmk (20 mM) and CHX (2.5 mg/ml). Nuclear linking requirements of sTRAIL for non-apoptotic signaling. As extracts were prepared as described previously (31), and EMSA analy- shown in Fig. 1, cross-linked and non-cross-linked sTRAIL both ses were performed using a standard procedure with a high pressure have a comparable capacity to induce IL-6 production (Fig. 1B) liquid chromatography-purified NF-kB-specific oligonucleotide (59-ATC and elicited comparable NF-kB activation in a reporter gene AGG GAC TTT CCG CTG GGG ACT TTC CG-39), end-labeled with [ P]ATP. Finally, samples were separated by native polyacrylamide gel assay (Fig. 1C). The magnitude of NF-kB activation and IL-6 Non-redundant TRAIL Receptor Signaling 32211 FIG.4. A, analysis of TRAIL receptor expression on various cell lines by FACS. B, effect of CHX on the killing of Kym-1 cells by cross-linked and non-cross-linked sTRAIL. Kym-1 cells were cultured over- night in 96-well plates. Cells were then incubated for additional 18 h with the indicated concentrations of cross-linked (circles) and non-cross-linked (squares) sTRAIL in the presence (filled symbol)or absence (open symbol)of2.5 mg/ml CHX. Cell viability was measured by the MTT assay. C, Kym-1 cells were treated with anti-TRAIL-R1 IgG (200 ng/ml), protein A (1 mg/ml), and Z-VAD-fmk (ZVAD,20 mM) in the presence or absence of 2.5 mg/ml CHX. After 3 h, cells were harvested and analyzed for NF-kB activation with EMSA. production induced by cross-linked and non-cross-linked (HeLa), we found in both cell lines a requirement for cross- sTRAIL was similar to that obtained by TNF stimulation (data linked sTRAIL (Fig. 1D). As already outlined above, in group II not shown). In all group II cells investigated, TRAIL-induced cell lines, sTRAIL activated NF-kB only in the presence of apoptosis and activation of NF-kB were dependent on the pres- CHX/Z-VAD-fmk and induced cell death only if CHX was pres- ence of CHX. Activation of the NF-kB pathway is inhibited by ent. However, activation of the JNK pathway by cross-linked caspase-dependent mechanisms during apoptosis (35–38). TRAIL occurred in the absence of CHX and was therefore not Thus, TRAIL-induced activation of NF-kB was therefore only linked to cell death. observed in group II cells when in addition to CHX a caspase To analyze whether the requirement for cross-linked sTRAIL inhibitor (Z-VAD-fmk) was present (data not shown). However, correlated with a differential utilization of TRAIL-R1 and in group I cells, NF-kB activation was found in the absence of TRAIL-R2, we reexamined the cells described above using pu- CHX, provided that apoptosis was again inhibited by Z-VAD- rified IgG fractions of agonistic TRAIL-R1- and TRAIL-R2- fmk (data not shown). Notably, when we analyzed TRAIL- specific antisera. The agonistic activity of anti-TRAIL-R1 IgG mediated JNK activation in group I (Kym-1) and II cells and anti-TRAIL-R2 IgG, respectively, was significantly in- 32212 Non-redundant TRAIL Receptor Signaling creased upon aggregation with protein A, with respect to death clearly demonstrating the ability of TRAIL-R2 to transmit spe- induction and up-regulation of IL-6 production (Fig. 2A). At the cific signals in the absence of cell death. It is possible that concentrations used in this study (,200 ng/ml) the IgG frac- TRAIL-R2 is also important for non-apoptotic signal transduc- tions were not cross-reactive. Using the agonist anti-TRAIL- tion. This may involve the activation of c-Jun and other JNK- or NF-kB related downstream responses, which regulate pro- receptor IgGs, we found that group I cells were exclusively killed by anti-TRAIL-R2 IgG, whereas group II cells were sen- liferation and differentiation in normal cells. The apoptotic function of TRAIL-R2, which is cryptic in normal cells, may sitive for stimulation with both anti-TRAIL-R1 and anti- only be dominantly revealed in transformed cells. Although TRAIL-R2 IgG (Fig. 2B). Moreover, in the group II cell line HeLa, both IgG preparations induced NF-kB activation JNK was only activated via endogenous TRAIL-R2 but not endogenous TRAIL-R1, we also noted that transient overex- whereas in the group I cell line Kym-1 only anti-TRAIL-R2 IgG but not anti-TRAIL-R1 IgG was able to activate NF-kB (Fig. 2, pression of both TRAIL-R1 and TRAIL-R2 activated JNK in a ligand-independent fashion (data not shown). We can therefore C and D). Again in HeLa cells treatment with CHX and Z-VAD- not completely exclude the possibility that endogenous fmk was necessary to elicit the NF-kB response, whereas in Kym-1 cells NF-kB activation only required inhibition of the TRAIL-R1 might activate JNK in some circumstances. In conclusion, our data suggest that TRAIL-R1 responds to apoptotic pathway. Supershift analyses in HeLa cells revealed cross-linked and non-cross-linked TRAIL to signal NF-kB acti- that TRAIL-R1 and TRAIL-R2 engaged the NF-kB family vation and apoptosis, whereas TRAIL-R2 signals NF-kB acti- members p65, p50, and cRel in a comparable manner to vation, apoptosis, and JNK activation in response to cross- TNF-R1 (Fig. 2D). In group I as well as in group II cell lines the linked TRAIL only. We hypothesize that the requirement of first signs of NF-kB DNA-binding activity were detectable 1 to cross-linked sTRAIL reflects the requirement of TRAIL-R2 for 2 h upon TRAIL receptor stimulation whereas TNF induced membrane-bound TRAIL. NF-kB DNA-binding activity within 15–30 min. NF-kB DNA- binding activity induced by both cytokines sustained for sev- Acknowledgment—The authors thank David Lynch, Immunex Cor- eral hours in both type of cells (data not shown). Importantly, poration, Seattle, WA for supplying us with TRAIL receptor-specific the JNK pathway was triggered in group I and II cell lines by mAbs M271, M413, M430, and M445. stimulation of TRAIL-R2 but not by stimulation of TRAIL-R1 REFERENCES (Fig. 3). To our knowledge, this is the first reported difference 1. Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C.-P., Nicholl, in the signaling capacities of the two death domain-containing J. K., Sutherland, G. R., Smith, T. D., Rauch, C., Smith, C. A., and Goodwin, TRAIL receptors. R. G. (1995) Immunity 3, 673– 682 Based on these results, it is evident that group II cells must 2. Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J., Moore, A., and Ashkenazi, A. (1996) J. Biol. Chem. 271, 12687–12690 co-express both death domain-containing TRAIL receptors, 3. Thomas, W. D., and Hersey, P. (1998) J. Immunol. 161, 2195–2200 whereas group I cells either express no TRAIL-R1 or this mol- 4. 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Biol. 7, 693– 696 in this group I cell line also in the presence of CHX. In fact, 17. Sheridan, J. P., Marsters, S. A., Pitti, R. M., Gurney, A., Skubatch, M., Baldwin, D., Ramakrishnan, L., Gray, C. L., Baker, K., Wood, W. I., Kym-1 cells became sensitive to anti-TRAIL-R1 IgG (data not Goddard, A. D., Godowski, P., and Ashkenazi, A. (1997) Science 277, shown) and non-cross-linked sTRAIL (Fig. 4B) in the presence 818 – 821 18. Wu, G. S., Burns, T. F., McDonald, E. R. III, Jiang, W., Meng, R., Krantz, I. D., of CHX. Moreover, whereas in the absence of CHX only stim- Kao, G., Gan, D. D., Zhou, J. Y., Muschel, R., Hamilton, S. R., Spinner, ulation of TRAIL-R2 activated the NF-kB pathway (see Fig. N. B., Markowitz, S., Wu, G., and El-Deiry, W. S. (1997) Nat. Genet. 17, 2C), stimulation of TRAIL-R1 also induced NF-kB activation 141–143 19. Degli-Esposti, M. A., Smolak, P. J., Walczak, H., Waugh, J., Huang, C.-P., provided that CHX and Z-VAD-fmk were added (see Fig. 4C). DuBose, R. 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The Tumor Necrosis Factor-related Apoptosis-inducing Ligand Receptors TRAIL-R1 and TRAIL-R2 Have Distinct Cross-linking Requirements for Initiation of Apoptosis and Are Non-redundant in JNK Activation

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 41, Issue of October 13, pp. 32208 –32213, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Tumor Necrosis Factor-related Apoptosis-inducing Ligand Receptors TRAIL-R1 and TRAIL-R2 Have Distinct Cross-linking Requirements for Initiation of Apoptosis and Are Non-redundant in JNK Activation* Received for publication, January 18, 2000, and in revised form, May 5, 2000 Published, JBC Papers in Press, May 11, 2000, DOI 10.1074/jbc.M000482200 Frank Mu ¨ hlenbeck‡, Pascal Schneider§, Jean-Luc Bodmer§, Ralph Schwenzer‡, Angelika Hauser‡, Gisela Schubert‡, Peter Scheurich‡, Dieter Moosmayer‡, Ju ¨ rg Tschopp§, and Harald Wajant‡¶ From the ‡Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany and the §Institute of Biochemistry, University of Lausanne, Ch. des Boveresses 155, 1066 Epalinges, Switzerland Overexpression of the tumor necrosis factor (TNF)- an involvement of TRAIL in natural killer cell-, dendritic cell-, and CD41 T-cell-mediated cytotoxicity (3– 6), TRAIL may also related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2, induces apoptosis and activa- be involved in monocyte-mediated tumoricidal activity (7) and kB in cultured cells. In this study, we have tion of NF- activation-induced T cell death during HIV infection (8, 9). demonstrated differential signaling capacities by both Currently five human TRAIL receptors belonging to the TNF receptors using either epitope-tagged soluble TRAIL receptor superfamily have been identified. Two of them, (sTRAIL) or sTRAIL that was cross-linked with a mono- TRAIL-R1 (DR4, Ref. 10) and TRAIL-R2 (DR5, TRICK2, KILL- clonal antibody. Interestingly, sTRAIL was sufficient for ER; see Refs. 11–18) contain a cytoplasmic death domain and induction of apoptosis only in cell lines that were killed transmit an apoptotic signal in response to TRAIL. Two other by agonistic TRAIL-R1- and TRAIL-R2-specific IgG prep- cellular TRAIL receptors, TRAIL-R3 (TRID, DcR1; see Refs. 11, arations. Moreover, in these cell lines interleukin-6 se- 14, 17, 19), which is glycosylphosphatidylinositol (GPI)-linked kB activation were induced by cross- cretion and NF- and TRAIL-R4 (DcR2; see Refs. 20, 21), which contains a trun- linked or non-cross-linked anti-TRAIL, as well as by both cated death domain, bind TRAIL without activation of the receptor-specific IgGs. However, cross-linking of sTRAIL apoptotic machinery and seem to antagonize the death domain- was required for induction of apoptosis in cell lines that containing TRAIL receptors. In addition, osteoprotegerin, a only responded to the agonistic anti-TRAIL-R2-IgG. Inter- regulator of osteoclastogenesis, is a soluble receptor for TRAIL estingly, activation of c-Jun N-terminal kinase (JNK) was (22). TRAIL-R1- and TRAIL-R2-mediated apoptosis occurs via only observed in response to either cross-linked sTRAIL or activation of caspase-8 and subsequent activation of effector anti-TRAIL-R2-IgG even in cell lines where both receptors caspases. However, the link between the death domain-con- kB activation. were capable of signaling apoptosis and NF- taining TRAIL receptors and activation of caspase-8 is rather Taken together, our data suggest that TRAIL-R1 responds undefined. Transient transfection of TRAIL-R1 leads to activa- to either cross-linked or non-cross-linked sTRAIL which kB activation and apoptosis, whereas TRAIL-R2 tion of the apoptotic machinery in Fas-associated death domain signals NF- kB activation, apoptosis, and JNK activation signals NF- protein-deficient fibroblasts, suggesting that FADD, a death only in response to cross-linked TRAIL. domain adapter molecule, is not required for TRAIL-R1-in- duced apoptosis (23, 24). On the other hand, overexpression of a dominant-negative FADD mutant was shown to block Tumor necrosis factor-related apoptosis-inducing ligand TRAIL-mediated apoptosis (12, 13, 15, 25). It has not yet been (TRAIL), also designated as APO-2 ligand, is a member of the clarified whether FADD plays a specific role in TRAIL-R2- tumor necrosis factor (TNF) family that is capable of inducing induced apoptosis or whether a closely related adapter protein apoptosis in several cell lines (1, 2). TRAIL is widely expressed is involved in TRAIL-R1- and TRAIL-R2-mediated apoptosis. in normal cells and is highly homologous to FasL, another Because TRAIL is highly effective in killing cancer cell lines cytotoxic member of the TNF ligand family (1, 2). In addition to but has apparently no lethal effects on normal cells, TRAIL and its apoptotic receptors have attracted much attention as tar- gets for anti-cancer therapy (26, 27). In this study, we show * This work was supported by Deutsche Forschungsgemeinschaft that TRAIL-R1 and TRAIL-R2 have different capabilities for Grant Wa 1025/3-1. The costs of publication of this article were de- stimulating the JNK pathway and differ also in their cross- frayed in part by the payment of page charges. This article must linking requirements for activation by recombinant ligands. therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This is the first reported evidence of a difference between ¶ To whom correspondence should be addressed. Tel.: 49-711-685- TRAIL-R1 and TRAIL-R2 signaling activities. 7446; Fax: 49-711-685-7484; E-mail: [email protected] stuttgart.de. EXPERIMENTAL PROCEDURES The abbreviations used are: TRAIL, TNF-related apoptosis-induc- Materials—The anti-FLAG monoclonal antibody M2 was purchased ing ligand; JNK, c-Jun N-terminal kinase; TNF, tumor necrosis factor; from Sigma-Aldrich (Deisenhofen, Germany). Polyclonal sera specific MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; for JNK, p65, p50, and cRel were purchased from Santa Cruz Biotech- TRAIL-R, TRAIL receptor; FACS, fluorescence-activated cell sorter; nology (Heidelberg, Germany) and protein A-Sepharose was from Am- CHX, cycloheximide; EMSA, electrophoretic mobility shift assay; Z- ersham Pharmacia Biotech (Freiburg, Germany). The SuperFect trans- VAD-fmk, benzyloxycarbonyl-VAD-fluoromethylketone; mAb, mono- fection reagent was obtained from Qiagen (Hilden, Germany). TRAIL- clonal antibody; IL-6, interleukin-6; GST, glutathione S-transferase; ELISA, enyzme-linked immunosorbent assay. R1-Fc and TRAIL-R2-Fc were from Alexis (La ¨ ufelfingen, Switzerland). 32208 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Non-redundant TRAIL Receptor Signaling 32209 FIG.1. Cell type- and response-spe- cific effects of cross-linked TRAIL- FLAG. A, the indicated cell lines were analyzed for their sensitivity to FLAG- tagged sTRAIL in the presence (filled squares) or absence (open squares)ofag- gregating anti-FLAG antibody M2. Cell viability was determined using the MTT assay (Jurkat, Kym-1) or by staining ad- herent cells with crystal violet (HeLa, KB, HepG2, HT1080). The difference in ab- sorbance between dead and living cells was in the range of 600 –1000 mOD units for all cells. B, HeLa and KB cells were cultured overnight in 96-well plates. Cells were then incubated for 18 h with the indicated concentrations of cross-linked (filled squares) and non-cross-linked (open squares) sTRAIL-FLAG in the pres- ence of 2.5 mg/ml CHX and 10 mM Z-VAD- fmk. Finally, IL-6 concentrations in the supernatants were determined using a commercially available ELISA kit. C, HeLa cells were cultured overnight in 96- well plates. The next day, cells were transfected with a 33 NF-kB-luciferase reporter plasmid and a SV40 promoter- driven b-galactosidase expression plas- mid to normalize the transfection effi- ciency. After an additional day, cells were stimulated for 9 h with cross-linked (filled bars) and non-cross-linked TRAIL-FLAG (open bars) in the presence of CHX (2.5 mg/ml) and Z-VAD-fmk (10 mM). Finally cells were assayed for NF-kB activation. D, cell lysates were prepared from HeLa and Kym-1 cells that had been stimulated for 4 h with the indicated concentrations of cross-linked and non-cross-linked TRAIL- FLAG (TRAIL-F). JNK activity was meas- ured by immunocomplex kinase assay with GSTzc-Jun-(1–79) as substrate. Cell Lines—HeLa, HepG2, and Jurkat cells were maintained in concentration of FLAG-tagged TRAIL with the anti-FLAG monoclonal RPMI 1640 medium containing 5% (HeLa, HepG2) or 10% (Jurkat) antibody M2 to a final concentration of 1 mg/ml of antibody. After a heat-inactivated fetal calf serum. KB cells were grown in Dulbecco’s 15-min incubation at room temperature, the TRAIL-M2-complex was modified Eagle’s medium supplemented with 10% fetal calf serum and transferred to the cells. TRAIL-R1 and TRAIL-R2 IgG were added in HT1080 cells in Dulbecco’s modified Eagle’s medium-nutrient mix F12 the presence of 1 mg/ml protein A (Sigma, Deisenhofen, Germany). Cell containing 10% fetal calf serum. The Kym-1 cell line was maintained in viability was determined using the MTT method (Jurkat, Kym-1) or Click RPMI 1640 medium supplemented with 10% fetal calf serum. crystal violet staining (HepG2, HeLa, KB, HT1080) as described previ- Generation of TRAIL-R1 and TRAIL-R2-specific IgG Preparations— ously (28, 29). Using a commercial antibody production service (Eurogentec, Seraing, Immunocomplex JNK Assay—N-terminal c-Jun kinase assays were Belgium), rabbits were immunized with TRAIL-R1-Fc and TRAIL-R2- performed upon immunoprecipitation of JNK1 using a rabbit antiserum Fc. For antibody purification, TRAIL-R1-Fc and TRAIL-R2-Fc were (Santa Cruz Biotechnology, Heidelberg, Germany). GSTzc-Jun was used coupled to HiTrap N-hydroxysuccinimide (NHS)-Sepharose (Amersham as substrate in an in vitro kinase assay as described previously (30). Pharmacia Biotech, Freiburg, Germany) according to the manufactur- Transient Reporter Gene Assays—For transient reporter gene assays, er’s protocol. Fc-specific antibodies were first depleted by repeated 20,000 HeLa cells were seeded in 96-well tissue culture plates, and the passages over human IgG1-agarose (Sigma, Deisenhofen, Germany). following day the cells were transfected with a 33 NF-kB-luciferase TRAIL-R1/R2-specific antibodies were further purified on TRAIL-R1/ reporter plasmid (15% transfected DNA), a SV40 promoter-driven b-ga- R2-Fc-Sepharose, eluted in 50 mM citrate/NaOH, pH 2.7, neutralized lactosidase expression plasmid (5% transfected DNA) to normalize the with Tris-HCl, pH 9, and dialyzed against phosphate-buffered saline. transfection efficiency, and empty vector (80% transfected DNA). At concentrations below 500 ng/ml, we found no evidence for cross- Transfections were performed with SuperFect reagent according to the reactivity of the anti-TRAIL receptor IgGs even upon secondary cross- manufacturer’s recommendations (Qiagen, Hilden, Germany). After a linking with protein A. At higher concentrations (.1000 ng/ml) we 1-day recovery, cells were treated with TRAIL, TRAIL-M2-complex, observed a significant cross-reactivity of the anti-TRAIL-receptor IgGs CHX, and Z-VAD-fmk as indicated, harvested in phosphate-buffered that could be blocked by addition of TRAIL-R1-Fc to anti-TRAIL-R2 IgG saline, and then luciferase and b-galactosidase activities were deter- and vice versa (data not shown). mined using the Galacto-Light Plus reporter gene assay kit (Perkin Cytotoxic Assays—50,000 (Jurkat), 30,000 (HepG2, KB), 20,000 Elmer, Nieuwerkerk, The Netherlands) and a Lucy2 96-well luminom- (HeLa, HT1080) or 15,000 Kym-1 cells were grown overnight in 100 ml eter (Anthos, Krefeld, Germany). of culture medium in 96-well plates. The cells were then treated for 16 h Determination of Interleukin-6 Production—Cells (1.5 3 10 per well) with FLAG-tagged TRAIL, TRAIL-M2-complex, anti-TRAIL-R1- and were seeded in triplicates in 96-well tissue culture plates in 100 mlof anti-TRAIL-R2-IgG. Cell death assays with HepG2, HeLa, KB, and Click RPMI 1640 and cultured overnight. The following day the cells HT1080 cells were performed in the presence of 2.5 mg/ml cycloheximide were treated with the reagents of interest as indicated for an additional (CHX). TRAIL-M2-complex was generated by mixing the respective 12–24 h. Then the supernatants were removed, cleared by centrifuga- 32210 Non-redundant TRAIL Receptor Signaling FIG.3. TRAIL-R2 but not TRAIL-R1 signals JNK activation. A, cell lysates were prepared from Kym-1, Jurkat, HeLa, and HepG2 cells that had been stimulated for 4 h with protein A cross-linked (1 mg/ml) anti-TRAIL-R1 IgG and anti-TRAIL-R2 IgG, each at 200 ng/ml in the absence of CHX. For control purposes lysates from cells treated with protein A and non-relevant IgG were also analyzed. JNK activity was measured by immunocomplex kinase assay with GSTzc-Jun-(1–79) as a substrate. B, dose dependence of anti-TRAIL-R2 IgG-induced JNK ac- tivation. Kym-1, Jurkat, HeLa, and HepG2 cells were stimulated with the indicated concentrations of protein A cross-linked anti-TRAIL-R2 IgG for 4 h, and lysates were analyzed for JNK activity by immuno- complex kinase assays. electrophoresis in low ionic strength buffer. For supershift analyses, 10 ml of the nuclear extracts (1 mg/ml protein) were incubated on ice for 1 h with 1 mg of polyclonal antibodies specific for p65, p50, and cRel (Santa Cruz Biotechnology, Heidelberg, Germany). Then the formed complexes FIG.2. A, protein A cross-linking enhanced the agonistic capacity of were incubated with 2 mlof53 binding buffer (500 mM KCl, 50 mM anti-TRAIL-R1 and -R2 IgG. HepG2 cells were incubated overnight Tris-HCl, pH 7.4, 25 mM MgCl , 50% glycerol, 5 mM dithiothreitol) and with the indicated concentration of anti-TRAIL-R1 IgG and anti- 2 ml of poly(dI-dC) (2 mg/ml) in a final volume of 20 ml. NF-kB DNA- TRAIL-R2 IgG, respectively, with (filled bars) or without (open bars) binding activity was again analyzed by native polyacrylamide gel elec- previous aggregation with protein A in the presence of 2.5 mg/ml CHX. trophoresis and phosphorimaging (Storm 860; Amersham Pharmacia Cell viability was measured by crystal violet staining. In addition, HeLa Biotech, Freiburg, Germany). cells were treated in the same way with the anti-TRAIL-receptor IgGs in the presence of 2.5 mg/ml CHX and 10 mM Z-VAD-fmk, and superna- RESULTS AND DISCUSSION tants were analyzed for production of IL-6. B, cytotoxic effects of anti- TRAIL-R1 IgG and anti-TRAIL-R2 IgG. Various cell lines were incu- Most ligands of the TNF family are either membrane-bound bated overnight with protein A cross-linked anti-TRAIL-R1 IgG (filled or proteolytically processed into soluble proteins. Evidence sug- bars) or anti-TRAIL-R2 IgG (hatched bars) or were left untreated (open gests that artificial cross-linking of soluble ligands mimics the bars). The next day, cell viability was determined by the MTT assay distinct biological activities of the corresponding membrane- (Jurkat, Kym-1) or by staining with crystal violet (HeLa, KB, HepG2, HT1080). To allow induction of apoptosis, HepG2, HeLa, KB, or HT1080 bound ligands. For example, we have recently shown that the cells were treated in the presence of 2.5 mg/ml CHX. C, Kym-1 cells were cytotoxic activity of FLAG-tagged human Fas ligand (sFasL), treated with anti-TRAIL-R1 or anti-TRAIL-R2 IgG (aTR1-IgG or aTR2- was increased by .1000-fold in response to cross-linking with IgG, respectively; 200 ng/ml) and protein A (1 mg/ml) with Z-VAD-fmk the anti-FLAG monoclonal antibody M2. Notably, this in- (ZVAD,20 mM). After 3 h, cells were harvested and analyzed for NF-kB activation by EMSA. D, HeLa cells were treated as indicated with, creased activity was comparable with the cytotoxic potency of anti-TRAIL-R1 and anti-TRAIL-R2 IgG (200 ng/ml), protein A (1 mg/ membrane-bound FasL (32). Further, activation of TNF-R2-de- ml), CHX (2.5 mg/ml), and Z-VAD-fmk (20 mM) for 3 h. As a control, cells pendent signaling pathways by soluble FLAG-tagged TNF was were also treated with 10 ng/ml TNF for 30 min. Supershift analyses strongly increased by multimerization of this ligand by the were performed as described under “Experimental Procedures.” Super- shifted complexes of the NF-kB oligonucleotide and p65, p50, or cRel, anti-FLAG monoclonal antibody M2. In accordance with that, respectively, are indicated by arrows. we have previously shown that membrane-bound, but not sol- uble TNF, is the prime activating ligand for TNF-R2 (33, 34), tion (15,000 rpm, 10 min, 4 °C) and interleukin-6 concentrations were suggesting that cross-linked and membrane-bound ligands determined using a commercially available ELISA kit (PharMingen, have analogous effects on this receptor. Using various cell lines Hamburg, Germany). we have therefore tested whether a recombinant soluble FLAG- FACS Staining—Cells were stained for TRAIL-R1, TRAIL-R2, tagged form of TRAIL (sTRAIL) required cross-linking for its TRAIL-R3, and TRAIL-R4 expression in 100 ml of FACS buffer (phos- activity. phate-buffered saline, 5% fetal calf serum, 0.1% NaN ) with 5 mg/ml We found that several cell lines, e.g. Jurkat and Kym-1, anti-TRAIL-R1 mAb M271 (IgG2a), anti-TRAIL-R2 mAb M413 (IgG1), anti-TRAIL-R3 mAb M430 (IgG1) and anti-TRAIL-R4 mAb M445 designated in the following as group I cells, were killed by (IgG1), respectively, or the respective control IgG, followed by fluores- physiological amounts (,200 ng/ml) of sTRAIL only in the cein isothiocyanate-labeled anti-mouse antibody (5 mg/ml). FACS anal- presence of secondary cross-linking by the anti-FLAG mono- yses were performed with a FACStar plus instrument (Becton Dickin- clonal antibody M2 (Fig. 1A). However, we also identified a son, San Jose, CA). 6 second set of cell lines designated in the following as group II EMSA Analysis of NF-kB Activation—HeLa and Kym-1 cells (10 ) cells that were efficiently killed by non-cross-linked sTRAIL were seeded in 60-mm cell culture dishes and cultivated overnight to allow adherence. The next day the cells were stimulated for 3 h with the (HeLa, HepG2, HT1080, and KB; Fig. 1A). The group II cell indicated combinations of anti-TRAIL-R1 and anti-TRAIL-R2 IgG, pro- lines Hela and KB were also tested with respect to the cross- tein A (1 mg/ml), Z-VAD-fmk (20 mM) and CHX (2.5 mg/ml). Nuclear linking requirements of sTRAIL for non-apoptotic signaling. As extracts were prepared as described previously (31), and EMSA analy- shown in Fig. 1, cross-linked and non-cross-linked sTRAIL both ses were performed using a standard procedure with a high pressure have a comparable capacity to induce IL-6 production (Fig. 1B) liquid chromatography-purified NF-kB-specific oligonucleotide (59-ATC and elicited comparable NF-kB activation in a reporter gene AGG GAC TTT CCG CTG GGG ACT TTC CG-39), end-labeled with [ P]ATP. Finally, samples were separated by native polyacrylamide gel assay (Fig. 1C). The magnitude of NF-kB activation and IL-6 Non-redundant TRAIL Receptor Signaling 32211 FIG.4. A, analysis of TRAIL receptor expression on various cell lines by FACS. B, effect of CHX on the killing of Kym-1 cells by cross-linked and non-cross-linked sTRAIL. Kym-1 cells were cultured over- night in 96-well plates. Cells were then incubated for additional 18 h with the indicated concentrations of cross-linked (circles) and non-cross-linked (squares) sTRAIL in the presence (filled symbol)or absence (open symbol)of2.5 mg/ml CHX. Cell viability was measured by the MTT assay. C, Kym-1 cells were treated with anti-TRAIL-R1 IgG (200 ng/ml), protein A (1 mg/ml), and Z-VAD-fmk (ZVAD,20 mM) in the presence or absence of 2.5 mg/ml CHX. After 3 h, cells were harvested and analyzed for NF-kB activation with EMSA. production induced by cross-linked and non-cross-linked (HeLa), we found in both cell lines a requirement for cross- sTRAIL was similar to that obtained by TNF stimulation (data linked sTRAIL (Fig. 1D). As already outlined above, in group II not shown). In all group II cells investigated, TRAIL-induced cell lines, sTRAIL activated NF-kB only in the presence of apoptosis and activation of NF-kB were dependent on the pres- CHX/Z-VAD-fmk and induced cell death only if CHX was pres- ence of CHX. Activation of the NF-kB pathway is inhibited by ent. However, activation of the JNK pathway by cross-linked caspase-dependent mechanisms during apoptosis (35–38). TRAIL occurred in the absence of CHX and was therefore not Thus, TRAIL-induced activation of NF-kB was therefore only linked to cell death. observed in group II cells when in addition to CHX a caspase To analyze whether the requirement for cross-linked sTRAIL inhibitor (Z-VAD-fmk) was present (data not shown). However, correlated with a differential utilization of TRAIL-R1 and in group I cells, NF-kB activation was found in the absence of TRAIL-R2, we reexamined the cells described above using pu- CHX, provided that apoptosis was again inhibited by Z-VAD- rified IgG fractions of agonistic TRAIL-R1- and TRAIL-R2- fmk (data not shown). Notably, when we analyzed TRAIL- specific antisera. The agonistic activity of anti-TRAIL-R1 IgG mediated JNK activation in group I (Kym-1) and II cells and anti-TRAIL-R2 IgG, respectively, was significantly in- 32212 Non-redundant TRAIL Receptor Signaling creased upon aggregation with protein A, with respect to death clearly demonstrating the ability of TRAIL-R2 to transmit spe- induction and up-regulation of IL-6 production (Fig. 2A). At the cific signals in the absence of cell death. It is possible that concentrations used in this study (,200 ng/ml) the IgG frac- TRAIL-R2 is also important for non-apoptotic signal transduc- tions were not cross-reactive. Using the agonist anti-TRAIL- tion. This may involve the activation of c-Jun and other JNK- or NF-kB related downstream responses, which regulate pro- receptor IgGs, we found that group I cells were exclusively killed by anti-TRAIL-R2 IgG, whereas group II cells were sen- liferation and differentiation in normal cells. The apoptotic function of TRAIL-R2, which is cryptic in normal cells, may sitive for stimulation with both anti-TRAIL-R1 and anti- only be dominantly revealed in transformed cells. Although TRAIL-R2 IgG (Fig. 2B). Moreover, in the group II cell line HeLa, both IgG preparations induced NF-kB activation JNK was only activated via endogenous TRAIL-R2 but not endogenous TRAIL-R1, we also noted that transient overex- whereas in the group I cell line Kym-1 only anti-TRAIL-R2 IgG but not anti-TRAIL-R1 IgG was able to activate NF-kB (Fig. 2, pression of both TRAIL-R1 and TRAIL-R2 activated JNK in a ligand-independent fashion (data not shown). We can therefore C and D). Again in HeLa cells treatment with CHX and Z-VAD- not completely exclude the possibility that endogenous fmk was necessary to elicit the NF-kB response, whereas in Kym-1 cells NF-kB activation only required inhibition of the TRAIL-R1 might activate JNK in some circumstances. In conclusion, our data suggest that TRAIL-R1 responds to apoptotic pathway. Supershift analyses in HeLa cells revealed cross-linked and non-cross-linked TRAIL to signal NF-kB acti- that TRAIL-R1 and TRAIL-R2 engaged the NF-kB family vation and apoptosis, whereas TRAIL-R2 signals NF-kB acti- members p65, p50, and cRel in a comparable manner to vation, apoptosis, and JNK activation in response to cross- TNF-R1 (Fig. 2D). In group I as well as in group II cell lines the linked TRAIL only. We hypothesize that the requirement of first signs of NF-kB DNA-binding activity were detectable 1 to cross-linked sTRAIL reflects the requirement of TRAIL-R2 for 2 h upon TRAIL receptor stimulation whereas TNF induced membrane-bound TRAIL. NF-kB DNA-binding activity within 15–30 min. NF-kB DNA- binding activity induced by both cytokines sustained for sev- Acknowledgment—The authors thank David Lynch, Immunex Cor- eral hours in both type of cells (data not shown). 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