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TNFR80-Dependent Enhancement of
TNFR60-Induced Cell Death Is Mediated by
TNFR-Associated Factor 2 and Is Specific for
TNFR60
Tilo Weiss, Matthias Grell, Katrin Siemienski, Frank
Mühlenbeck, Horst Dürkop, Klaus Pfizenmaier, Peter
Scheurich and Harald Wajant
J Immunol 1998; 161:3136-3142; ;
http://www.jimmunol.org/content/161/6/3136
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Copyright © 1998 by The American Association of
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References
TNFR80-Dependent Enhancement of TNFR60-Induced Cell
Death Is Mediated by TNFR-Associated Factor 2 and Is
Specific for TNFR601
Tilo Weiss,2* Matthias Grell,2* Katrin Siemienski,* Frank Mühlenbeck,* Horst Dürkop,†
Klaus Pfizenmaier,* Peter Scheurich,* and Harald Wajant3*
T
umor necrosis factor is a pleiotropic cytokine that is
mainly produced by activated macrophages and lymphocytes. TNF initiates inflammatory, immune regulatory,
and pathophysiologic responses by binding to two distinct cell surface receptors of 60 kDa (TNFR60) and 80 kDa (TNFR80) (reviewed in Ref. 1). The TNFR are the name-giving members of a
receptor superfamily whose members have in common three to six
extracellular copies of a canonical motif of cysteine-rich pseudorepeats, each comprising six conserved cysteines in a stretch of
about 40 amino acids. Further members of the TNFR superfamily
are CD40, CD30, CD27, Apo1/Fas, OX40, 4-1BB, nerve growth
factor receptor, LT-bR, and several viral gene products (reviewed
in Refs. 2 and 3). The ligands of these receptors also form a complementary family of molecules, most of which are expressed primarily as biologically active type II membrane proteins, from
which soluble forms are produced by proteolytic cleavage or alternative splicing (reviewed in Refs. 2 and 3).
Until recently, the molecular mechanisms of intracellular signal
initiation by binding of TNF and TNF-related cytokines to their
cognate receptors remained undefined. In fact, none of the TNFR
*Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany; and †Institute of Pathology, Universitätsklinikum Benjamin Franklin, Berlin,
Germany
Received for publication December 31, 1997. Accepted for publication May 19, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by Deutsche Forschungsgemeinschaft Grant Wa 1025/3-1
and by Bundesministerium für Bildung, Forschung und Technologie, Germany, Grant
0310816.
2
T.W. and M.G. contributed equally to this manuscript.
3
Address correspondence and reprint requests to Dr. H. Wajant, Institute of Cell
Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart,
Germany. E-mail address: [email protected]
Copyright © 1998 by The American Association of Immunologists
superfamily members possesses sequences implying any known
catalytic activity. However, during the last 4 yr, a rapidly growing
number of proteins has been identified that directly or indirectly
associate with the cytoplasmic domains of the TNFR superfamily
members. Most of these associated factors belong to two groups of
signal-transduction molecules, the so-called death domain proteins
and the TNFR-associated factor (TRAF)4 family. Both groups are
defined by distinct sequence motifs that are involved in further
protein/protein interactions (reviewed in Ref. 4). The death domain
has been defined originally as a part of the intracellular regions of
TNFR60 and Apo1/Fas, and is responsible for the generation of the
apoptotic signal by these receptors (5, 6). TRADD (TNFR1-associated death domain protein; 7), FADD/MORT1 (Fas-associated
protein with death domain/mediator of receptor-induced toxicity;
8 –10), and RIP (receptor-interacting protein; 11) are death domain-containing proteins that have been identified by yeast twohybrid screens using the intracellular domain of TNFR60 or Apo1/
Fas as baits. Overexpression of these molecules leads to activation
of NF-kB and/or induction of apoptosis (7–11). FADD/MORT1 is
part of the inducible Apo1/Fas death-inducing signaling complex
(12), and is believed to represent the physical link to proapoptotic
proteases of the caspase family (13, 14).
There are several lines of evidence that two TNFR60-signaling
cascades bifurcate at TRADD, one leading to the activation of
NF-kB and the other coupling via FADD/MORT1 to the apoptotic
caspase cascade (15, 16). The serine threonine kinase RIP is recruited to the TNFR60-signaling complex in a ligand-dependent
manner, and might also be involved in the activation of NF-kB as
4
Abbreviations used in this paper: TRAF, TNF receptor-associated factor; Apo2L,
Apo2 ligand; FADD, Fas-associated protein with death domain; IAP, inhibitors of
apoptosis; JNK, c-Jun amino-terminal kinase; MORT, mediator of receptor-induced
toxicity; NDPF, nuclear factor-kB-dependent cell death protective factor; RIP, receptor-interacting protein; TRADD, TNF receptor 1-associated death domain protein;
TRAIL, TNF-related apoptosis-inducing ligand.
0022-1767/98/$02.00
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Costimulation of TNFR80 can strongly enhance TNFR60-induced cell death. In this study, we show that this enhancement is
TNFR60 selective, as neither TNF-related apoptosis-inducing ligand/Apo2 ligand-, Apo1/Fas-, ceramide-, nor daunorubicin-mediated cell death was affected by costimulation of TNFR80. We further demonstrate that TNFR-associated factor 2 (TRAF2) is
critically involved in both negative and positive regulation of TNF-induced cell death. Overexpression of TRAF2 and of a TRAF2
mutant, deficient in nuclear factor-kB activation, selectively desensitized and enhanced, respectively, TNFR60-induced cell death
in HeLa cells. However, upon costimulation of TNFR80, which mediates activation of nuclear factor-kB and the c-Jun aminoterminal kinase via TRAF2, TNF-induced cell death is drastically enhanced in parental and TRAF2-transfected, but not in TRAF2
(87–501)-transfected cells. These data point to a critical role of TRAF2 in the apoptotic TNFR cross talk, whereby the TNFR80dependent enhancement of TNFR60-induced cell death is due to TNFR80-mediated negative regulation of TRAF2 function(s). An
interference with TRAF2 function was confirmed independently by analysis of c-Jun amino-terminal kinase activation via
TNFR60 upon prestimulation of TNFR80. We propose that the apoptotic TNFR cross talk is based on TNFR80-mediated abrogation of antiapoptotic TRAF2-dependent signaling pathways initiated by TNFR60, but not Apo1/Fas or the apoptotic TNFrelated apoptosis-inducing ligand receptors. The Journal of Immunology, 1998, 161: 3136 –3142.
The Journal of Immunology
Materials and Methods
Abs and reagents
The TNFR80-specific agonistic mAb MR2-1 was kindly provided by W.
Buurman (University of Limburg, Maasstricht, The Netherlands). FITClabeled goat anti-mouse IgG plus IgM Ab was obtained from Dianova
(Hamburg, Germany). All other reagents were obtained from Sigma (Deisenhofen, Germany), if not otherwise stated. A 1:1 mixture of purified
recombinant Flag-tagged human TRAIL (10 mg/ml) and anti-Flag M2 Ab
(20 mg/ml; Kodak International Biotechnologies, New Haven, CT) was
incubated at room temperature for 10 min and diluted to the final concentrations indicated in the figure legends.
Cells
HeLa cells and transfectants derived thereof were grown at 37°C in a
humidified 5% CO2 incubator in Click RPMI 1640 medium supplemented
with 5% heat-inactivated FCS, 2 mM L-glutamine, 50 U/ml penicillin, and
50 mg/ml streptomycin. Additionally, transfectants were permanently cultured in the presence of the appropriate selection drug.
DNA transfection
Cells were transfected by a standard electroporation procedure. In brief,
5 3 106 cells were incubated in HEBS buffer (135 mM NaCl, 5 mM KCl,
0.75 mM Na2HPO4, 5 mM dextrose, and 20 mM HEPES, pH 7.05) with 10
to 20 mg of vector DNA and, when necessary, 1 mg of a selection plasmid
carrying the Escherichia coli neomycin resistance gene (pMAMneo; Clontech, Palo Alto, CA) or the Zeocin resistance gene (pZeoSV; Invitrogen,
San Diego, CA). Electroporation was performed by a prepulse of 320 V, 3
mF capacitance, and 800 V; incubation for 5 min on ice; and a final pulse
of 220 V, 960 mF, and 400 V (Gene Pulsar; Bio-Rad, München, Germany).
Transfected cells were selected in culture medium containing 600 mg/ml
G418 (Life Technologies, Eggenheim, Germany), 500 mg/ml Zeocin (Invitrogen, San Diego, CA), 200 mg/ml hygromycin B (Boehringer Mannheim, Mannheim, Germany), or a combination thereof. After 2 to 3 wk of
selection, individual drug-resistant colonies were isolated. Transfectants of
receptor constructs were pooled and enriched for high expressors by cell
sorting using a FACStarPlus (Becton Dickinson, San Jose, CA). Transfectants overexpressing intracellular proteins were individually expanded and
characterized.
Cytotoxicity assay
HeLa cells and transfectants derived thereof were plated at a density of
1.5 3 104 cells/well in triplicates in 96-well microtiter plates in 100 ml of
Click RPMI 1640 overnight at 37°C. On the next day, the reagents of
interest were added in the presence of 2.5 mg/ml cycloheximide. The plates
were incubated for additional 12- to 24-h culture, and cell viability was
determined by crystal violet staining. Briefly, supernatants were discarded
and the cells were washed once with PBS, followed by crystal violet staining (20% methanol, 0.5% crystal violet) for 15 min. The wells were washed
with H2O and air dried. Residual dye was diluted with methanol for 15 min,
and OD at 550 nm was measured with a R5000 ELISA plate reader (Dynatech, Guernsey, U.K.).
IL-6 assay
Cells were plated at a density of 1.5 3 104 cells/well in triplicates in
96-well microtiter plates in 100 ml of Click RPMI 1640 overnight at 37°C.
On the next day, the cells were treated with the reagents of interest for
additional 12 to 24 h. Finally, supernatants were removed and cleared by
centrifugation at 15,000 rpm for 10 min, and IL-6 concentration was determined using a commercially available ELISA kit, according to the manufacturer’s recommendations (PharMingen, Hamburg, Germany).
Transfections, luciferase assays, and protein kinase assay
HeLa cells (0.8 3 105) were seeded in 24-well tissue culture plates. On the
following day, the cells were transfected using the SuperFect reagent (Qiagen, Hilden, Germany) with a 33 NF-kB-luciferase reporter plasmid and
a SV40 promoter-driven b-galactosidase expression plasmid to normalize
the transfection efficiency. After additional 24 h, the cells were harvested
in PBS, lysed in luciferase lysis buffer (Promega, Mannheim, Germany),
and assayed for luciferase and b-galactosidase activities using a LUMAT
9501 Luminometer (Berthold, Bad Wildbad, Germany). JNK activity was
measured by immunocomplex kinase assay with glutathione-S-transferase
c-jun (1–79) as substrate, as described elsewhere (38).
Results
Stimulation of TNFR80 enhances TNFR60-induced cell death,
but not TRAIL/Apo2L-, Apo1/Fas-, and ceramide/daunorubicinmediated cell death
We have demonstrated recently a synergistic enhancement of
TNF/TNFR60 signaling by costimulation of TNFR80 by the membrane form of TNF- or TNFR80-specific agonistic Abs (35, 37).
To obtain insight into the molecular mechanisms of this TNFR
cooperation, we asked whether similar cooperative mechanisms
exist between TNFR80 and other death-inducing members of the
TNFR family. Whereas costimulation of TNFR80 in HeLa cells
transfected with TNFR80 (HeLa-TNFR80) resulted in a strong enhancement of TNF-induced cell death (Fig. 1A; Ref. 37), no enhancement of the cytotoxic effects induced by TRAIL/Apo2 ligand
(TRAIL/Apo2L), a member of the TNF ligand family (39, 40), or
stimulation of the Apo1/Fas Ag could be observed (Fig. 1, B and
C). This is remarkable because it has been shown that TNF,
TRAIL/Apo2L, and stimulation of Apo1/Fas can induce cell death
using similar pathways, including the molecule FADD and different members of the caspase family (41, 42). Accordingly, in our
cell systems, cytotoxicity by the aforementioned stimuli could be
inhibited completely by the caspase inhibitor peptide z-VAD-fmk
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well as induction of cell death (17), although the position of RIP
within the signaling cascades is yet rather unclear (18).
The TRAF protein family currently comprises six members that
interact with molecules of the TNFR superfamily or with the IL1R. Rothe et al. (19) have isolated the first two members of the
TRAF protein family based on their interaction with the cytoplasmic domain of TNFR80. TRAF3, also designated as CD40bp,
CAP-1, LAP1, or CRAF1, was identified by its association with
CD40 (20 –23) and TRAF5 by binding to the LT-bR (24). TRAF4,
also termed CART1, was isolated by differential screening of libraries of malignant and nonmalignant breast tissues (25) and
TRAF6 by homology screening of an expressed sequence tag library (26). Overexpression studies and analysis of various deletion
mutants of members of the TNFR superfamily suggested that
TRAF2, 5, and 6 are critically involved in the activation of NF-kB
by CD40 (27, 28), CD30 (29), LT-bR (24), TNFR80 (27), and
TNFR60 (15), as well as the membrane receptor for IL-1 (26). The
C-terminal TRAF domain of TRAF proteins comprises about 230
amino acid residues, and can be divided in the N- and C-TRAF
domain. For most of the TRAF molecules, the capability to interact
with several members of the TNFR superfamily and other TRAFs
has been established (reviewed in Ref. 4). Moreover, some of the
TRAF molecules bind to cytoplasmic proteins that are capable or
suspected to modulate TNF-initiated apoptosis or TNF-dependent
activation of NF-kB. These include the inhibitors of apoptosis
(IAP; 30), the antiapoptotic molecule A20 (31), and the molecule
TANK (TNF activator of NF-kB)/I-TRAF (TRAF-interacting protein) (32, 33), as well as the TRAF-interacting protein TRIP (34),
respectively. In particular, Hsu et al. (15) have shown that interaction of the death domain protein TRADD with TRAF2 is critically involved in TNFR60-dependent activation of NF-kB.
The common usage of the above-described signal-transduction
molecules by various members of the TNFR superfamily opens up
the possibility of a complex pattern of receptor cross talk. In fact,
we and others have recently described synergistic action of
TNFR80, CD40, and TNFR60 in the induction of cell death (35–
37). In this study, we demonstrate that TRAF2 is involved in the
enhancement of TNFR60-mediated cell death by TNFR80. This
apoptotic cross talk occurs upstream of FADD/MORT1 in the
TNFR60-signaling pathway or at a pathway selectively used by
TNFR60 because no interference with the apoptotic signaling induced by the cytokines Apo1/Fas and TRAIL/Apo2 ligand or with
ceramide- and UV-induced cell death was observed.
3137
3138
TNFR60/TNFR80 CROSS TALK
(Fig. 1D). Together, these data indicate that the TNFR80-dependent enhancement of TNFR60-induced cytotoxicity modulates a
signaling pathway selectively used by TNFR60 or that the synergistic action of TNFR80 occurs upstream of FADD/MORT1 and
the apoptotic caspases engaged by TNFR60, Apo1/Fas, and the
receptors for TRAIL/Apo2L.
Metabolites of the sphingomyelin pathways have been implicated in the induction of apoptosis, and several studies have presented evidence that the second messenger ceramide is also involved in signaling the cytotoxic effects of TNFR60 and Apo1/Fas
(43– 46). Therefore, we analyzed whether TNFR80 stimulation
would modulate apoptosis induced by exogenously added ceramide. As shown in Figure 1E, ceramide-induced cell death was
not enhanced by costimulation of TNFR80. Likewise, the cytotoxic effects of daunorubicin, a drug that has been suggested to
induce apoptosis by elevating the intracellular ceramide level (47,
48), were not modulated by TNFR80 stimulation (Fig. 1F). Moreover, neither ceramide- nor daunorubicin-induced cell death was
affected by the addition of z-VAD-fmk (Fig. 1, E and F), suggesting that ceramide acts downstream of the caspase cascade or on a
parallel pathway.
TRAF2-mediated protection from TNFR60-induced cell death is
antagonized in the context of TNFR80 coactivation
In a recent study, we have shown that the TRAF2 binding domain of
TNFR80 is essential for the TNFR80-mediated enhancement of
TNFR60-induced cell death (37). In fact, overexpression of wild-type
TRAF2 in HeLa-TNFR80 cells resulted in a reduced sensitivity to the
cytotoxic effects of TNF (Fig. 2A). Moreover, introduction of a deletion mutant of TRAF2 (TRAF2 (87–501)) that is still capable of associating with TNFR, but is deficient in the activation of NF-kB and
JNK due to the lack of the RING finger domain (27), resulted in a
dramatic enhancement of TNFR60-mediated cytotoxicity (Fig. 2A),
with no change in the sensitivity toward TRAIL/Apo2L-, UV-, ceramide-, and daunorubicin-induced cell death (data not shown). Hence,
upon stimulation of TNFR60, both apoptotic and antiapoptotic pathways are activated simultaneously, with TRAF2 being critically involved in the transduction of protective signals. To evaluate the potential role of TRAF2 in the TNFR80-mediated enhancement of
TNFR60-induced apoptosis, the double transfectants HeLa-TNFR80TRAF2 and HeLa-TNFR80-TRAF2 (87–501) were investigated under conditions of specific coactivation of both TNFRs. Interestingly,
HeLa-TNFR80-TRAF2 double transfectants, although less sensitive
for TNF-induced cytotoxicity (Fig. 2A), were still highly susceptible
toward TNFR80-mediated enhancement of TNFR60-induced apoptosis (Fig. 2B). Overexpression of TRAF2 (87–501) resulted in a reduction of both the proliferation rate (Fig. 2C) and TNF-induced gene
induction (Fig. 2D), which is in good accordance with a critical role
of TRAF2 in TNF-induced activation of NF-kB and JNK. In the same
cells, which show an increased sensitivity against TNF, no further
enhancement was noted upon costimulation of TNFR80 (Fig. 2B).
These data indicate that the TRAF2 (87–501) molecule interferes with
the TRAF2-dependent induction of antiapoptotic molecules (Fig. 2A).
The TNF sensitivity of TRAF2 (87–501) transfectants resembles the
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FIGURE 1. A and C, Effect of costimulation of
TNFR80 on TNF-, anti-Apo1-, and TRAIL/Apo2L-induced cell death. HeLa-TNFR80 cells (A, B) and
TNFR80-Apo1/Fas cells (C) were plated in 96-well microtiter plates at a density of 1.5 3 104 cells/well and
cultured overnight at 37°C. On the next day, the cells
were treated in triplicates for additional 24 h with various concentrations of TNF (A, open circles), TRAIL/
Apo2L-M2 complex (B, open squares), anti-Apo1 (C,
open triangles), or a combination of these reagents with
the agonistic anti-TNFR80 Ab MR2-1 (1 mg/ml; filled
symbols) in the presence of 2.5 mg/ml cycloheximide.
The viable cells were quantified by staining with crystal
violet. D, Effect of z-VAD-fmk on cytokine-induced
cell death. HeLa-TNFR80 or HeLA-TNFR80-Apo1/
Fas cells were plated in 96-well microtiter plates at a
density of 1.5 3 104 cells/well in triplicate overnight at
37°C. On the next day, the cells were treated for additional 24 h with concentrations of TNF (10 ng/ml), antiApo1 (50 ng/ml), TRAIL/Apo2L-M2 complex, or TNF
(0.5 ng/ml) plus mAb MR2-1 (1 mg/ml) sufficient to
induce maximal cell death, in the presence of z-VADfmk (20 mM; hatched bars) or without further treatment
(filled bars). Cell death was investigated in the presence
of 2.5 mg/ml cycloheximide. The viable cells were
quantified by staining with crystal violet. E and F, Effects of z-VAD-fmk and costimulation of TNFR80 on
ceramide (E)- and daunorubicin (F)-induced cell death.
HeLa-TNFR80 cells were plated in 96-well microtiter
plates at a density of 1.5 3 104 cells/well overnight at
37°C. On the next day, the cells were treated in triplicate for additional 24 h with various concentrations of
ceramide or daunorubicin (filled circles) in the presence
of z-VAD-fmk (20 mM; open squares) or in the presence of the agonistic anti-TNFR80 Ab MR2-1 (1 mg/
ml; open triangles). The viable cells were quantified by
staining with crystal violet. In E, the open circles show
the effect of dihydroceramide as a control.
The Journal of Immunology
3139
sensitivity of TNF in wild-type cells after enhancement by costimulation of TNFR80. Therefore, it appears that the TRAF2-dependent
protective response induced concomitantly with the apoptotic pathway is overruled in the context of TNFR80 stimulation. It is important
to note that TNF-induced apoptosis only occurs in the presence of the
protein synthesis inhibitor cycloheximide in all of our HeLa transfectants, a fact that, at the first view, is in conflict with NF-kB-dependent
induction of antiapoptotic proteins. However, in our experiments, we
used only 2.5 mg/ml cycloheximide, a concentration at which protein
synthesis is only reduced, but not prevented. We have confirmed this
by determination of IL-6 production and synthesis of a NF-kB-dependent reporter protein, which were reduced under these conditions
only for 5–40% and 10%, respectively (Fig. 3).
Stimulation of TNFR80 abrogates TNF- but not IL-1-induced
activation of the amino-terminal c-jun kinase
A recent study has shown that transient cotransfection of TNFR80
and TRAF2 results in a rapid depletion of TRAF2 (49). It is tempting to speculate that TNFR80-induced degradation/inactivation of
TRAF2 and possibly other associated proteins contributes to the
apoptotic TNFR cross talk investigated in our study. To test this
hypothesis, we analyzed the effect of TNFR80 costimulation on
TNF-mediated JNK activation since the TNF-induced JNK activation is indicative for TRAF2 action (50, 51). As shown in Figure
4, prestimulation of TNFR80 strongly interferes with JNK activation by TNF. TNF-induced JNK activation was reduced by ap-
proximately 50% after prestimulation of TNFR80 for 0.5 h, and
was almost completely abrogated after 6-h prestimulation of
TNFR80. In contrast, IL-1-induced activation of JNK, which is
independent of TRAF2, remained largely unaffected upon
TNFR80 prestimulation (Fig. 4).
Discussion
TNFR80-dependent enhancement of TNFR60-mediated cell
death occurs upstream of FADD/MORT1 or on a separate
TNFR60-induced pathway
As shown in Figure 1, TNFR80 triggering enhances TNFR60-mediated, but neither Apo1/Fas-mediated nor TRAIL/Apo2L-induced
cell death. FADD/MORT1 is a common component of the apoptotic pathways utilized by TNFR60 and Apo1/Fas (15, 16, 52,
53). Hence, it is most likely that the TNFR60-TNFR80 cross talk
occurs upstream of FADD/MORT1 or on a separate TNFR60-induced pathway, being distinct from, but coinitiated with the
FADD/MORT1-dependent apoptotic pathway. The existence of
mechanisms able to selectively modulate TNFR60- or Apo1/Fasinduced cell death at a point at which both pathways have not
merged, is also indicated by the fact that a number of cellular
systems have been described in which TNF-sensitive cells are
Apo1/Fas resistant and vice versa (54 –57). One possible branching point is the cytoplasmic domain of TNFR60. It has been shown
recently that a nine-amino-acid binding motif outside the death
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FIGURE 2. Expression of TRAF2 (87–501) renders HeLa-TNFR80 cells more sensitive toward TNF and abolishes TNFR80-dependent enhancement
of TNFR60-mediated cell death, whereas TRAF2 desensitizes against TNFR60-induced cell death. A, Data are from three independent clones stably
transfected with TRAF2 (87–501) (open symbols; circles, clone 3; triangles, clone 19; square rhombs, clone 21) and two clones stably transfected with
TRAF2 (filled symbols; squares, clone 48; triangles, clone 49). Cells were plated in 96-well microtiter plates at a density of 1.5 3 104 cells/well in triplicates
overnight at 37°C. On the next day, the cells were treated for additional 24 h with various concentrations of TNF in the presence of 2.5 mg/ml cycloheximide. The dotted line shows the TNF sensitivity of the parental HeLa-TNFR80 cells. B, To compare the synergistic effect of TNFR80 costimulation
on TNFR60-mediated cytotoxicity in HeLa-TNFR80, HeLa-TNFR80-TRAF2 (87–501), and HeLa-TNFR80-TRAF2 transfectants, cells were stimulated
with a TNF concentration sufficient to induce 20% cell death in the respective clone (clone 3, 1 pg/ml; clone 19, 0.5 pg/ml; clone 21, 0.05 pg/ml; clones
48 and 49, 1 ng/ml; control, 100 pg/ml) in the absence (open bars) or presence (hatched bars) of the anti-TNFR80 Ab MR2-1 (1 mg/ml). Viable cells were
quantified by staining with crystal violet. C and D, Proliferation and IL-6 production of HeLa-TNFR80-TRAF2 (87–501) cells are reduced significantly
compared with the parental HeLa-TNFR80 cells (C). The proliferation rates of three independent HeLa-TNFR80 clones transfected with TRAF2 (87–501)
(open symbols) and of the parental cell line (filled symbol) were monitored for several days by staining with crystal violet (D). For determination of
constitutive and TNF-induced IL-6 production, cells were cultured in 96-well microtiter plates overnight at 37°C in triplicate. On the next day, medium
(open bars), TNF (30 ng/ml; horizontally hatched), or the agonistic anti-TNFR80 Ab MR2-1 (1 mg/ml; diagonally hatched) was added and final IL-6
concentrations in the supernatants were determined by an ELISA after 12 h.
3140
TNFR60/TNFR80 CROSS TALK
FIGURE 3. Effect of cycloheximide on synthesis of a reporter gene product (luciferase) (A) and IL-6 production (B). A, HeLa cells were transfected with
33 NF-kB-luciferase reporter. One day later, some of the cultures were stimulated for 6 h with the indicated concentrations of TNF in the presence (filled
bars) or absence (open bars) of 2.5 mg/ml cycloheximide. Then luciferase activity was determined and normalized against a cotransfected b-galactosidase
standard. B, For determination of constitutive and cytokine-induced IL-6 production, cells were cultured in 96-well microtiter plates overnight at 37°C in
triplicate. On the next day, cells were treated with 10 ng/ml TNF for 16 h in the presence (filled bars) or absence (open bars) of 2.5 mg/ml cycloheximide.
IL-6 concentrations in supernatants were determined by an ELISA.
TRAF2 is involved both in positive and negative regulation of
TNF-induced apoptosis
In the HeLa-TNFR80 transfectants used in this study, no further
TNFR80-dependent enhancement of TNFR60-mediated cytotoxicity can be detected in cells, in which a negative regulator of
NF-kB activation (TRAF2 (87–501)) is expressed. These transfectants already display a high susceptibility toward TNF in the absence of TNFR80 stimulation (Fig. 2). Furthermore, we have
shown recently that TNFR cross talk is dependent on the TRAF2
binding site of TNFR80 (37). These data suggest that TNFR80
exerts its proapoptotic capability by a TRAF2-dependent interaction with a NF-kB-dependent gene product. The interference of a
FIGURE 4. Effect of TNFR80 prestimulation on TNF- and IL-1-mediated JNK activation. HeLa-TNFR80 cells prestimulated for the indicated
times with MR2-1 (1 mg/ml) in the presence of 2.5 mg/ml cycloheximide.
Subsequently, cells were treated for 20 min with TNF (10 ng/ml) or IL-1b
(3 ng/ml). Finally, JNK activity was measured by immunocomplex kinase
assay with glutathione-S-transferase c-jun (1–79) as a substrate.
NF-kB-dependent gene product with TNF-induced cytotoxicity is
in good accordance with recent results from several groups, demonstrating that inhibition of NF-kB activation increases the sensitivity toward TNF-induced apoptosis (61– 65). The fact that overexpression of TRAF2 (87–501) resulted in a dramatic
enhancement of TNFR60-mediated cytotoxicity (Fig. 2A), thus resembling the TNFR80-mediated enhancement of TNF-induced cytotoxicity in cells overexpressing TRAF2 or only endogenous
TRAF2 (Fig. 2B), can be explained by the following hypothesis
(Fig. 5).
The central regulator of TNF-mediated cell death is the TRAF2
molecule, which can interfere with TNFR60-induced cytotoxicity
directly at two levels: 1) induction of protective proteins via
NF-kB and JNK activation, and 2) recruitment of these proteins to
the TNFR60 death-signaling complex. In cells coexpressing
TNFR80, however, appropriate stimulation of this receptor results
in binding and degradation of TRAF2 and TRAF2-associated proteins, thereby reducing the availability of protective factors at the
TNFR60 death-signaling complex. This promotes the apoptotic
TNF response (Fig. 5). According to this model, in the cellular
system studied in this investigation, exclusive stimulation of
TNFR60 in HeLa-TNFR80 cells concomitantly induces pro- and
antiapoptotic pathways. As overexpression of TRAF2 (87–501)
drastically enhances TNF sensitivity, but not responsiveness to
anti-Apo1 or TRAIL/Apo2L, an NF-kB-dependent cell death protective factor (NDPF) that is selective for TNF must be postulated
(Fig. 5A). In addition, the efficient activation of the death proteases
is counteracted by constitutively expressed, cycloheximide-sensitive negative regulator(s), for example a cellular homologue of the
CrmA gene product. This is evident from the fact that induction of
cell death by TNF, anti-Apo1, and TRAIL/Apo2L in HeLa cells
can occur only when protein translation is reduced. Putative candidates for this/these protective protein(s) are the IAP molecules
(30), TRAF-interacting protein (TRIP) (34), TRAF1 (19), cellular
FLICE-inhibitory protein/casper (66, 67), and A20 (31), because
these molecules are able to form complexes with TRAF2. In fact,
Speiser et al. (68) have shown recently in transgenic mice that
overexpression of TRAF1 inhibits Ag-induced apoptosis by CD81
T lymphocytes, a process in which TNF is critically involved (69, 70).
Moreover, Chu et al. (71) have shown that c-IAP2 counteracts TNFinduced cell death. According to the model proposed in this work, a
rather balanced cellular response with intermediate sensitivity to cell
death induction will be shifted toward a vigorous apoptotic response
upon costimulation of TNFR80 due to TNFR80-dependent inactivation of TRAF2: 1) TRAF2 will be recruited and/or degraded by this
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
domain is required to couple the TNFR60 to neutral sphingomyelinase (58, 59), whereas acidic sphingomyelinase, NF-kB activation, and induction of apoptosis have been assigned to the death
domain of the receptor (6, 60). Hsu et al. (15) have described an
additional bifurcation point downstream of the death domain of
TNFR60, namely the TNFR60-associated death domain protein
TRADD. This molecule directly associates in a ligand-dependent
manner with TNFR60 (15) and is critically involved in both
NF-kB and JNK activation as well as induction of apoptosis (7,
61). In particular, amino acid residues 1–169 of TRADD are sufficient to associate with TRAF2, which is involved in NF-kB activation by TNFR60, TNFR80, and CD40 (27, 15). The N-terminal
death domain of TRADD (amino acid residues 195–312) links to
FADD/MORT1, which transduces the apoptotic signal to the
caspase cascade (13, 14).
The Journal of Immunology
3141
receptor, thus reducing the production of NDPFs (e.g., TRAF1, A20,
and c-IAP2); 2) NPDFs bound to TRAF2 may also be degraded; 3)
still existing NDPFs cannot be recruited sufficiently to their relevant
site of action, the apoptotic TNFR60-signaling complex, because of
lack of the essential adapter, the TRAF2 molecule.
Acknowledgments
We thank I.-M. von Broen (Knoll AG, Ludwigshafen, Germany) for recombinant TNF; W. Buurman (University of Limburg, Maastricht, The
Netherlands) for mAb MR2-1; and P. H. Krammer (German Cancer Research Center, Germany) for mAb anti-Apo1. The N-terminal Flag-tagged
soluble TRAIL and M2 anti-Flag Ab were generous gifts from Craig A.
Smith (Immunex, Seattle, WA). The expression vectors encoding human
TRAF2, human TRAF (87–501), and Fas/Apo1, respectively, were a kind
gift of Ulrich Grunwald (Institute of Immunology, University of Munich,
Munich, Germany).
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TNFR60/TNFR80 CROSS TALK