1 Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring
31, 70569 Stuttgart, Germany
2 National Institute for Medical Research, Division of Membrane Biology, The
Ridgeway, Mill Hill, London NW7 1AA, UK
3 Institut für Gefäßbiologie und Thromboseforschung, University
of Vienna, 1235 Vienna, Austria
* These authors contributed equally to this work
Author for correspondence (e-mail:
harald.wajant{at}po.uni-stuttgart.de
)
Accepted 27 March 2002
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Summary |
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Key words: Caspase-8, Cell death, TNF, TNF-R2, TRAF2
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Introduction |
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After ambiguous results concerning the apoptosis-inducing capabilities of
the non-death-domain-containing receptor TNF-R2
(Heller et al., 1992;
Tartaglia et al., 1993
), the
use of agonistic TNF-R2-specific antibodies clearly showed that exclusive
triggering of this receptor is sufficient in some cells to induce cell death
(Bigda et al., 1994
;
Grell et al., 1993
;
Medvedev et al., 1994
).
However, stimulation of TNF-R2 does not directly engage the apoptotic program,
but relies on the induction of endogenous TNF, which subsequently activates
TNF-R1 (Grell et al., 1999
;
Vercammen et al., 1995
). In
addition, activation of TNF-R2 can lead to a tremendous enhancement of
TNF-R1-induced cell death independent of endogenous TNF by a TRAF2-dependent
intracellular mechanism (Chan and Lenardo,
2000
; Declercq et al.,
1998
; Duckett and Thompson,
1997
; Vandenabeele et al.,
1995
; Weiss et al.,
1997
).
Here, we show that stimulation of TNF-R2 results in a strong recruitment of TRAF2 that is accompanied by a strong depletion of cytosolic TRAF2. Moreover, we give evidence that TNF-R1 and TNF-R2 can compete for TRAF2-dependent recruitment of the anti-apoptotic proteins cIAP1 and cIAP2, which suggests that TNF-R2-induced TRAF2-mediated depletion of cIAP1/2 underlies the apoptotic TNF receptor crosstalk. In accordance with this model we show that TNF-R1-induced activation of procaspase-8 is slower than TNF-R2-induced TRAF2 depletion.
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Materials and Methods |
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For generation of HeLa-CD30 cells, HeLa cells were stably transfected with a CD30 expression plasmid. After G418 selection more than 100 primary clones were pooled, expanded and enriched for cells with high expression of cell surface CD30 by three rounds of cell sorting using a FACStarPlus (Becton Dickinson, San Jose, CA) and the CD30-specific mAb Ki-1. Expression plasmids encoding TRAF2-GFP, TRAF2-YFP, TNF-R1-YFP, cIAP1(NT)-GFP and cIAP2(NT)-GFP were generated by proofreading PCR-based amplification of the corresponding cDNA regions from HeLa cDNA and insertion of the obtained amplicons into the pEGFP-N1 and pEYFP-N1 vectors (Clontech, Heidelberg, Germany). Forward primers contained a BamHI (BglII for cIAP1 plasmids; NheI for TNF-R1 plasmid) site and reverse primers contained a SacII (SacI for TNF-R1 plasmids) site in their 5' overhangs to allow oriented insertion into the respective sites of the pEGFP-N1 and pEYFP-N1 vectors.
The deletion mutant of TRAF2, where the C-terminal TRAF domain is
substituted by GFP (TRAF2-NT-GFP), was generated by proofreading PCR based
amplification of a cDNA fragment encoding amino acids 1-272 of TRAF2 and
insertion of the obtained amplicon into pEGFP-N1. Again primers were used with
BamHI and SacII overhangs to allow in frame insertion into
the BglII and SacII sites of pEGFP-N1. To generate the
N-terminal deletion mutant of TRAF2 in which the TRAF domain of the molecule
was C-terminally fused to GFP, a cDNA fragment comprising TRAF2 amino acids
186-501 was amplified by proofreading PCR with HeLa cDNA as template. The
forward primers used contained a BamHI site and the reverse primers
used contained a NotI site. The BamHI and NotI
sites were used for subsequent cloning of the amplicon in a modified version
of the recently described pcDNA3.1-GFPFADD expression construct
(Wajant et al., 1998
). In this
modified version a BamHI/BglII hybrid site between GFP and
FADD was restored to a complete BamHI site, and a
BamHI site 5' to the start codon of the fusion protein was
destroyed. Thus, in this modified version the
FADD part could be
substituted by the respective TRAF2 amplicon.
The expression construct CFP-TRADD-DD was obtained by introducing a
respective cDNA amplicon into the BglII and SacII sites of
pECFP-N1. The expression vector pECFP-Mem encoding ECFP targeted to cellular
membranes by 20 amino acids of neuromodulin was from Clontech (Heidelberg,
Germany). Rabbit polyclonal anti-TNF-R2 IgG was already described elsewhere
(Grell et al., 1995).
Flag-tagged recombinant soluble TRAIL, FasL and CD40L were generously supplied
by P. Schneider and J. Tschopp (University of Lausanne, Switzerland). If not
otherwise stated, all other reagents were from Sigma (Deisenhofen,
Germany).
Cytotoxicity assay
Cells were seeded in 96-well microtiter plates (20x103
HeLa cells per well; 15x103 Kym1 cells per well) and
cultivated over night. The next day HeLa cells were treated with 2.5 µg/ml
cycloheximide and Kym1 cells remained untreated. After 1 hour, TNF or FasL
were added and costimulation of TNF-R2, CD30 or CD40 was performed as follows:
TNF-R2 was stimulated with an agonistic TNF-R2-specific rabbit IgG fraction (2
µg/ml); CD40 was stimulated with Flag-tagged recombinant soluble CD40L (100
ng/ml) crosslinked with the Flag-specific mAb M2 (1 µg/ml; Sigma); and CD30
was stimulated with the agonistic CD30-specific mAb Ki-1 (3 µg/ml). FasL
was used as Flag-tagged recombinant soluble molecule crosslinked with 1
µg/ml M2 (Schneider et al.,
1998). After an additional 18 hours, cells were washed with PBS
followed by crystal violet staining (20% methanol, 0.5% crystal violet) for 15
minutes. The wells were washed with H2O and air-dried. The dye was
eluted with methanol for 15 minutes and optical density at 550 nm was measured
with a R5000 ELISA plate reader (Dynatech, Guernsey, GB).
Transient transfection and reporter gene assays
HeLa-TNFR2 cells were seeded in 96-well plates with a density of
1.5x104 cells per well. The next day cells were transfected
with 200 ng/well of the indicated expression plasmid, 35 ng/well of a
luciferase reporter plasmid driven by three consensus NF-B sites and 15
ng/ml of ß-galactosidase expression vector driven by the SV40 promoter
using the Superfect reagent (Qiagen, Hilden, Germany) according to the
manufacturer's instructions. After 1 day, cells were stimulated as indicated
and cell extracts were prepared by the addition of 50 µl of luciferase
lysis solution (Galactolight-Kit, Tropix, Bedford, MA) and one freeze-thaw
cycle. A portion of the extracts (25 µl) was mixed with 50 µl of
luciferase substrate (Luciferase Assay System, Promega) and the luminescence
was determined in the single photon mode using an Anthos microplate
luminometer (Lucy 2). In parallel, 25 µl of each cell extract was incubated
for 1 hour with a 1:100 dilution of Galacton substrate in reaction buffer and
mixed with 100 µl of accelerator II solution to determine relative
ß-galactosidase activity (Galactolight-Kit) again with the Anthos
microplate luminometer (Lucy 2). Luciferase activities were normalized based
on the ß-galactosidase activities.
Western blot analysis
For detection of IB, caspase-8 and caspase-3 pellets of
2x106 cells were lysed in lysis buffer containing 150 mM
NaCl, 50 mM Tris-HCl, pH 8.0, 1% NP-40 and 0.1% SDS, and lysates were
clarified by centrifugation (4°C, 10 minutes, 13,000 rpm). Protein
concentrations were determined using a Bradford based protein assay (Bio-Rad)
and equal amounts of protein were resolved by SDS-polyacrylamide gel
electrophoresis and transferred on nitrocellulose membranes. After blocking
with 5% nonfat dry milk in PBS-Tween-20 (0.05%) for 1 hour, membranes were
probed with the indicated antibodies. The antibody-antigen complexes were
finally detected using alkaline phosphatase labeled secondary antibodies (0.1
µg/ml, Sigma-Aldrich, Deisenhofen, Germany) and BCIP and NBT as substrates.
For analyzing total cellular TRAF2 content by western blot, cell pellets were
directly solubilized in Laemmli sample buffer (106 cells per 50
µl buffer) by help of a sonicator. The equivalent of
0.8x106 cells per group was applied on the gel.
For preparation of TRAF2 cytoplasmic extracts and the corresponding
detergent-insoluble fraction cells were lysed in 150 mM NaCl, 50 mM Tris-HCl
pH 7.7, 1 mM EDTA and 1% Triton X-100 at 4°C for 1 hour. Then, the
cytoplasmic fraction was cleared by centrifugation (4°C, 30 minutes,
10,000 g). The obtained detergent-insoluble pellet was washed
twice and solubilized in Laemmli sample buffer. Protein concentrations were
determined using a Bradford based protein assay (Bio-Rad) and equal amounts of
protein (100 µg cytoplasmic extract or the corresponding amount of
insoluble pellet) were resolved by SDS-PAGE and analyzed by western blotting.
Anti-caspase-3 mAb clone 19 was from Transduction Laboratories (Heidelberg,
Germany) and anti-IB was from Santa Cruz (Heidelberg, Germany). The
anti-caspase-8 mAb was a kind gift from Klaus Schulze-Osthoff (University of
Tübingen, Germany).
Gelfiltration analysis of TRAF2 containing complexes
Cells (100-300x106) were treated with the reagents of
interest for the indicated times and subsequently scraped with a rubber
policeman into the medium. Cells were washed in an ice-cold solution
containing 50 mM Tris, pH 7.4, 400 mM NaCl and 10% glycerol, and the cell
pellet was resupended in 0.5-1-fold its volume of the same ice-cold solution.
All the following procedures were performed on ice or at 4°C. Cells were
lysed with Nonident-P40 to a final concentration of 0.1% and a protease
inhibitor cocktail (Boehringer Mannheim, Germany) was added according to the
recommendations of the supplier. The lysates were cleared by centrifugation
(8000 g, 10 minutes, 4°C) and the S-100 supernatants were
obtained by centrifugation at 100,000 g for 1 hour in a TL-100
rotor at 4°C (Beckman, Munich, Germany). 200 µl of the S-100
supernatants were then separated by size exclusion chromatography on a
Superdex 200 HR 10/30 column (Pharmacia, Freiburg, Germany) in 50 mM Tris, 400
mM NaCl and 10% glycerol, pH 7.4 at 0.5 ml/minute. Samples were collected in
fractions of 0.5 ml and analyzed by immunoblotting with a polyclonal rabbit
TRAF2-specific IgG fraction (Santa Cruz). The column was previously calibrated
with thyroglobulin (663 kDa), apoferritin (443 kDa), alcohol dehydrogenase
(150 kDa), bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa) and
cytochrome c (12.4 kDa), all purchased from Sigma.
Immunofluorescence and confocal microscopy
Transfected cells were fixed onto coverslips in 3% paraformaldehyde,
permeablized with 0.1% NP-40 for 10 minutes and blocked for a further 10
minutes with PBS/1% BSA. Primary rabbit antisera specific for Lck and TNF-R2
were diluted 1:150 in TBST (150 mM NaCl, 50 mM Tris-HCl pH 8.0, 0.1% Tween-20)
and applied for 1 hour. Cells were then washed in PBS/0.05% Tween-20 for 15
minutes, incubated for 45 minutes with rabbit-specific antisera (diluted
1:200) conjugated with AlexaFluor 546 (A-11060), washed and mounted onto
microscope slides. Alternatively, cells expressing GFP fusion proteins were
maintained in a conditioned chamber (37°C, 5% CO2) for up to 2
hours on the microscope stage. Fluorescent specimens were analyzed with a
Leica SP2 confocal microscope and photographed using the Leica TCS
software.
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Results |
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TNF-R2 stimulation depletes high molecular mass TRAF2-containing
complexes
It has been shown that the transient overexpression of TNF-R2 or CD30
degraded co-transfected TRAF2 (Duckett and
Thompson, 1997). Owing to the importance of TRAF2 for
TNF-R1-mediated NF-
B activation
(Devin et al., 2000
;
Devin et al., 2001
;
Tada et al., 2001
),
receptor-induced degradation of TRAF2 may account for the enhancement of
TNF-R1-induced cell death by triggering of CD30, CD40 and TNF-R2. We therefore
analyzed endogenous TRAF2 levels upon selective stimulation of TNF-R2 with
agonistic mAbs. For this purpose we prepared S-100 supernatants from untreated
HeLa-TNF-R2 cells and cells that were previously stimulated with
TNF-R2-specific antibodies for 6 hours. Upon separation of the SN-100
supernatants over a superdex 200 HR10/30 column the fractions obtained were
analyzed by immunoblotting. In accordance with data from the literature
(Shu et al., 1996
), we found
that, in untreated cells, most of the cytosolic TRAF2 eluated in fractions
corresponding to 300-500 kDa (Fig.
3A), suggesting that the majority of TRAF2 is part of a
multiprotein complex in HeLa-TNF-R2 cells. But more importantly, when
identical amounts of protein lysates were compared, obtained from
receptor-stimulated and untreated cells, we found a significant depletion of
TRAF2-containing complexes in the TNF-R2-stimulated group
(Fig. 3B). TRAF2 depletion was
most pronounced in the high molecular mass fractions
(Fig. 3B), whereas a minority
of TRAF2, which was eluted around 60 kDa, was not affected (data not shown).
It is tempting to speculate that the `monomeric' TRAF2 fraction represents a
pool of TRAF2 that is unable to interact with TNF-R2. However, further studies
will be necessary to clarify the importance and function of this monomeric
TRAF2 fraction. No TNF-R2-dependent TRAF2 depletion was found in HeLa cells
stably expressing a deletion mutant of TNF-R2 lacking the cytoplasmic domain
of the molecule (Fig. 3C).
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In the stable transfectants expressing CD30 (HeLa-CD30) and CD40 (HeLa-CD40), respectively, we also found a depletion of high molecular mass TRAF2-containing complexes upon stimulation with agonistic antibodies (Fig. 3D,E). In these cases we were unable to detect `monomeric' TRAF2 even in nonstimulated cells (data not shown). Compared with HeLa-TNF-R2 cells, we observed reduced levels of TRAF2 in unstimulated cells of both HeLa-CD30 and HeLa-CD40. This correlates with the observation that, in particular, HeLa-CD30 and, to a lesser extent, HeLa-CD40, but not TNF-R2-expressing cells exerted some ligand-independent constitutive receptor signaling (data not shown), possibly caused by the overexpression of these molecules. As `monomeric' TRAF2 represents a minor fraction of total TRAF2, it is possible that it was below the detection threshold of our western blot analysis. The `ligand'-induced depletion of TRAF2-containing complexes started to become apparent 1 hour after receptor triggering, and was almost complete after 6 hours (Fig. 3F). Depletion of TRAF2 occurred in the absence as well as in the presence of cycloheximide [i.e. under conditions where TNF-treatment could induce apoptosis in this cell line (data not shown)].
Receptor-induced depletion of cytoplasmic TRAF2-containing complexes could
be caused by translocation of TRAF2 to a detergent-insoluble cellular
compartment or/and by proteolytic degradation. We therefore analyzed total
TRAF2 levels in cells by direct solubilization of cell pellets in SDS-PAGE
sample buffer and determined TRAF2 levels in cytoplasmic extracts and
detergent-resistant membrane fractions. Activation of TNF-R2 resulted in
transient accumulation of TRAF2 in the detergent-resistant membrane fraction
after 1-3 hours (Fig. 3G,H).
However, 6-18 hours after receptor stimulation, a decrease in the TRAF content
of total cell lysates was observed (Fig.
3G,H) suggesting that, after the first few hours of TNF-R2
stimulation, depletion of `free' cytoplasmic TRAF2 pools was mainly caused by
recruitment of the protein by TNF-R2 into a detergent-insoluble compartment,
followed by TRAF2 degradation leading to sustained downregulation of this
signaling molecule. Nevertheless, TNF-R2-dependent depletion of TRAF2 also
seems to continue at late time points, because the total content of TRAF2
dropped down by more than 60% (Fig.
3H) after 18 hours, whereas TRAF2 levels in the DIC fraction were
comparable with those before stimulation
(Fig. 3G). This implies that
the distribution of TRAF2 between the soluble and the DIC fraction is still
significantly shifted towards the DIC fraction. As an alternative to TRAF2
degradation, reduced de novo synthesis of TRAF2 could also explain the
reduction of total TRAF2 protein, found at later time points, but we observed
no changes in the TRAF2 mRNA content at any time after TNF-R2 stimulation
(data not shown). In agreement with these findings Arch et al. have found that
TRAF2 is redistributed from the cytoplasm into detergent-insoluble aggregates
after co-transfection with constitutively active forms of CD30, 4-1BB and OX40
(Arch et al., 2000). In this
report redistribution of TRAF2 correlated with an increased sensitivity
towards TNF. Reduction in cytoplasmic TRAF2 levels after CD30 stimulation was
also associated with increased TNF sensitivity in anaplastic large cell
lymphoma cells (Mir et al.,
2000
). In accordance with the idea that the availability of
cytoplasmic TRAF2 determines the TNF sensitivity, it has been shown that HeLa
transfectants stably expressing increased amounts of TRAF2 have a reduced
sensitivity against soluble TNF but can still be sensitized to this death
ligand by TNF-R2 stimulation (Weiss et
al., 1998
).
It has recently been shown that TRAF proteins can be the target of caspases
[TRAF1 (Irmler et al., 2000),
TRAF3 (Lee et al., 2001
)] or
the proteasome [TRAF6 (Takayanagi et al.,
2000
), TRAF2 (Brown et al.,
2001
)]. However, the ligand-induced depletion of TRAF2 observed in
our system was not affected by the pan caspase-inhibitor z-VAD-fmk nor the
proteasome inhibitor MG-132, even at concentrations tenfold higher than
necessary to completely inhibit induction of apoptosis or activation of
NF-
B, respectively (data not shown).
Temporal order of stimulation of TNF-R1 and TNF-R2 determines the
outcome of TNF-R1 signaling
We next analyzed the effects of TNF-R2 stimulation and TRAF2 depletion on
TNF-R1-induced signaling in detail. To analyze TNF-R1-induced NF-B
activation in a situation where TRAF2 was already depleted, we selectively
prestimulated TNF-R2 with agonistic antibodies overnight, followed by TNF-R1
activation using either the TNF-R1-specific agonistic mAb HTR1 or soluble TNF.
Under these conditions TNF-R1-mediated activation of a NF-
B-driven
reporter gene was found to be reduced by up to 90% over a wide range of TNF
(HTR1) concentrations (Fig.
4A,C). Comparable results were obtained in the presence of low
doses of CHX (2.5 µg/ml) [i.e. under conditions where apoptosis is induced
by TNF-R1 but protein synthesis is only modestly inhibited (data not
shown)].
|
The situation was different when both TNF receptors were stimulated at the
same time. Under these conditions there was no sign of TNF-R2-dependent
inhibition of TNF-R1-induced NF-B activation
(Fig. 4B,D). Again, the
presence of CHX did not change the outcome of the experiment (data not shown).
As stimulation of TNF-R1 induces transient activation of the IKK complex
within 5-15 minutes, which in turn leads to sustained NF-
B binding
activity in the nucleus, it is feasible that the slowly proceeding
TNF-R2-mediated TRAF2 depletion (Fig.
3) does not interfere with TNF-R1-induced NF-
B activation
when both receptors are triggered at the same time. Indeed, the kinetics of
TNF-R2-induced TRAF2 depletion (Fig.
3F) are in good agreement with the time dependency of
NF-
B-inhibitory effect of TNF-R2 prestimulation. While TRAF2 depletion
requires 1-3 hours, half maximal inhibition of TNF-R1-induced NF-
B
activation by prestimulation of TNF-R2 became evident after about 2 hours
(Fig. 4E). In accordance with
the anti-apoptotic function of the NF-
B pathway, prestimulation of
TNF-R2 for 8 hours resulted in a significantly greater enhancement of
TNF-R1-induced cell death than a simultaneous stimulation of both TNF
receptors (Fig. 4F). Nevertheless, as already shown in Figs
1 and
2, there is a clear enhancement
of TNF-R1-induced cell death under costimulatory conditions where
TNF-R1-induced NF-
B activation was not inhibited. Obviously, the
apoptotic TNF-R1/TNF-R2 crosstalk is not only related to inhibition of the
NF-
B pathway, but also based on an additional TNF-R2-dependent,
NF-
B-independent mechanism, capable of enhancing TNF-R1-induced cell
death at the post-transcriptional level.
TNF-R2 stimulation accelerates caspase-8 activation by TNF-R1
In transient co-transfection studies it has been found that TRAF2 can be
part of a complex containing cIAP1, cIAP2 and TRAF1, which inhibits
TNF-R1-mediated apoptosis (Wang et al.,
1998). TNF-R2-induced TRAF2 depletion/degradation could lead to
inhibition of the formation of this anti-apoptotic complex and consequently to
an enhancement of TNF-R1-induced activation of caspase-8. Under prestimulation
conditions, where cytosolic TRAF2 is significantly depleted, the formation of
the anti-apoptotic complex might be affected in two ways: (1) by reduced
NF-
B-dependent induction of TRAF1, cIAP1 and cIAP2; and (2) by the lack
of TRAF2 itself. In contrast, under costimulatory conditions only the lack of
available TRAF2 may limit TRAF1/TRAF2/cIAP1/cIAP2 complex formation. However,
when this is the case, the kinetics of TNF-R1-induced caspase-8 activation
have to be slow or delayed to give the TNF-R2-dependent TRAF2 depletion time
to take place and to interfere with the formation of, or to disrupt, the
TRAF1/2/cIAP1/2 complex.
Slow or delayed caspase-8 activation by `activated' TNF-R1 is not
self-evident. In the case of Fas-mediated apoptosis, a quantitative activation
of procaspase-8 within a few minutes has been described
(Medema et al., 1997). In HeLa
cells treated with CHX, caspase-8 processing is detectable by cleavage of its
full-length 55- and 53 kDa isoforms leading to disappearance of the respective
proteins and concomitant appearance of the active p18 subunit of
caspase-8.
As shown in Fig. 5A, almost all full-length caspase-8 was processed 1 hour after apoptosis-induction in HeLa-Fas cells by treatment with Fas-specific agonistic mAbs in the presence of CHX. In contrast, caspase-8 activation in TNF/CHX-treated cells was detectable only after 3 hours and complete activation was not achieved at all (Fig. 5A). Similar results were obtained by directly measuring caspase activity (Fig. 5B). The differential kinetics and extent of caspase-8 activation by stimulation of TNF-R1 and Fas were also observed in Jurkat, Kym1 (TNF-R1) and SKW (Fas) cells, where induction of apoptosis does not require the presence of CHX (data not shown). Similar results were obtained when the activity of the excutioner caspase-3 was monitored (Fig. 5A,B) or when crosslinked soluble FasL was used instead of Fas-specific agonistic antibodies (data not shown). The differences in kinetics and extent of caspase activation by TNF and anti-Apo1/FasL correlated with the variations in time, when the broad range caspase inhibitor z-VAD-fmk had to be added to prevent apoptosis. After 1 hour of Fas/Apo1 stimulation, z-VAD-fmk treatment was able to rescue only 60-70% of the cells and after 3 hours there was no protective effect at all (Fig. 5C). In contrast, upon TNF-R1 triggering a significant proportion of the challenged cells were protected by z-VAD-fmk even after 8 hours (Fig. 5C). Hence, under costimulatory conditions, the slow and ineffective activation of caspase-8 by TNF via TNF-R1 would allow TNF-R2-induced TRAF2 degradation to interfere with the action of the anti-apoptotic cIAP/TRAF complex. In accordance with this concept, we found that costimulation and, to an even greater extent, prestimulation of TNF-R2 enhanced TNF-R1-induced caspase-8 activation (Fig. 5A,B).
|
TRAF2 is necessary for the recruitment of cIAP1 and cIAP2 to
TRADD
To investigate the role of TRAF2 for the recruitment of antiapoptotic
factors into the TNF-R1 signaling complex we used confocal fluorescence
microscopy and GFP-tagged forms of components of this complex. As shown in
Fig. 6A, cotransfection of
TNF-R1-YFP and CFP-TRADD-DD led to a colocalization of both molecules.
Interestingly, overexpression of CFP-TRADD-DD
(Fig. 6A) or full-length
myc-tagged TRADD (data not shown) resulted in the formation of TRADD
filaments. As transiently overexpressed CFP-TRADD-DD and full-length
myc-tagged TRADD showed effects similar to those of overexpressed TNF-R1 or
ligand-stimulated TNF-R1, such as activation of the NF-B
(Fig. 6B), JNK and apoptosis
pathways (data not shown) (Hsu et al.,
1996
), we postulate that TRADD filaments have some properties
related to receptor-recruited endogenous TRADD. Importantly, we found that
GFP-tagged IKK1, a part of the NF-
B-inducing IKK complex, was also
partially recruited into the TRADD filaments in the presence of cotransfected
TRAF2 (Fig. 6D). This is in
accordance with a recent study using TRAF2-/- fibroblasts, which
showed that TRAF2 is necessary to recruit the IKK complex into the TNF-R1
signaling complex (Devin et al.,
2001
). Again this emphasizes our assumption that TRADD filaments
are functionally equivalent to TNF/TNF-R1/TRADD signaling complexes.
|
We therefore used the artificial filament formation of TRADD to investigate
the interaction of TRADD with TRAF2, cIAP1 and cIAP2. We first studied the
interaction of untagged TRADD and GFP fusion protein of TRAF2. N-terminally as
well as C-terminally GFP-linked TRAF2 behaved similarly to untagged TRAF2 in
respect to homo- and heterooligomerization, NF-B activation and binding
of a TNF-R2 GST fusion protein (data not shown). In unstimulated cells, TRAF2
N-terminally linked to GFP (TRAF2-GFP) was found in the cytoplasm
(Fig. 6C). In addition,
TRAF2-GFP accumulates in a few, large round patches
(Fig. 6C). Most likely these
TRAF2-GFP patches are artefacts caused by overexpression and may represent the
proportion of transiently expressed TRAF2 molecules that is responsible for
ligand-independent activation of NF-
B and JNK. We found that TRAF2-GFP
was recruited into TRADD filaments under critical involvement of its TRAF
domain (Fig. 6C). Important for
the idea that TRAF2 depletion by non-death receptors interferes with the
formation/action of TNF-R1/TRADD-recruitable anti-apoptotic
cIAP/TRAF-complexes, we found that TRAF2 dramatically enhanced the recruitment
of cIAP1-GFP and cIAP2-GFP into TRADD filaments. While in cells coexpressing
TRADD and cIAP1-GFP or cIAP2-GFP, only a small proportion of the GFP-tagged
IAP proteins were associated with the TRADD filaments, coexpression of TRAF2
leads to a quantitative recruitment of cIAP1-GFP and cIAP2-GFP into TRADD
complexes (Fig. 6E,F).
Obviously, TRAF2 has an essential role in the recruitment of cIAP1 and cIAP2
into the TNF-R1/TRADD complex, data that are in good accordance with the
postulated importance of the TRAF1/TRAF2/cIAP1/cIAP2 complex discussed above
and with previous data showing ligand-induced recruitment of cIAP1 into the
TNF-R1 signaling complex (Shu et al.,
1996
).
TRAF2 is necessary for the recruitment of cIAP1 and cIAP2 to the
receptor complex of TNF-R2 in living cells
Next we studied directly alterations of the intracellular localization of
GFP-linked TRAF2 induced by stimulation of TNF-R2. Real-time monitoring of
TRAF2-GFP by confocal laser microscopy showed that, in HeLa-TNF-R2 cells,
TRAF2-GFP becomes compartmentalized in small aggregates in response to
selective activation of TNF-R2 (Fig.
7A), whereas Ki-1 or a control IgG preparation had no effect on
HeLa-TNF-R2 cells (Fig. 7B,C).
Similar results were obtained upon stimulation of CD30 or CD40. The
TNF-R2-induced TRAF2-GFP containing aggregates represent complexes of TNF-R2
and TRAF2-GFP, as is evident from colocalization with the plasma membrane
marker CFP-Mem (Fig. 7D) and
TNF-R2-specific antibodies (Fig.
7E). Of particular interest, soluble TNF, which is unable to
properly activate TNF-R2 in HeLa-TNFR2 cells, failed to induce
receptor/TRAF2-GFP complexes (Fig.
9B). Importantly, cIAP1-GFP and cIAP2-GFP (data not shown) as well
as GFP fusion proteins of the IAP1/2 binding domain for TRAF2
(Fig. 8) were recruited into
the TNF-R2 signaling complex only when TRAF2 was coexpressed. This is in
accordance with data from the literature showing ligand-independent
interaction of TNF-R2 and cIAP proteins in the presence of TRAF2 in transient
overexpression experiments (Rothe et al.,
1995). Analysis of cytoplasmic cIAP1-GFP and cIAP2-GFP revealed a
significant depletion of these molecules 30 minutes after TNF-R2 stimulation
(Table 1). In six independent
experiments, reduction of cytoplasmic cIAP-GFP proteins ranged between 45 and
81% (average 52% for cIAP1-GFP and 56% for cIAP2-GFP. Depletion of IAP1/2-GFP
in the presence of TRAF2 was typically more efficient than depletion of
TRAF2-GFP (Table 1). Thus, the
possibility arises that TRAF2/cIAP complexes interact better with TNF-R2 than
with IAP-free TRAF2 complexes. However, it cannot be ruled out that this is
caused by higher expression of TRAF2-YFP compared with the non-tagged TRAF2
mediating TNF-R2-recruitment of TRAF2/cIAP-GFP complexes in the IAP depletion
experiments. Further experiments will be necessary to clarify this.
|
|
|
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Discussion |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Exclusive stimulation of TNF-R1 (Fig.
9A) with soluble TNF immediately activates the NF-B pathway
leading to the synthesis of NF-
B-dependent genes. However, the
formation of TNF-R1 complexes that can signal apoptosis occurs with a delay of
several hours. This is evident from the time course of TNF-induced caspase-8
activation (Fig. 5). TNF-induced NF-
B activation results in the upregulation of
anti-apoptotic factors such as TRAF1
(Schwenzer et al., 1999
;
Wang et al., 1998
), cIAP1
(Wang et al., 1998
), cIAP2
(Chu et al., 1997
;
Wang et al., 1998
), cFLIP
(Kreuz et al., 2001
), and
Bfl-1/A1 (Lee et al., 1999
).
An anti-apoptotic function has been published for all these proteins and, with
the exception of Bfl1/A1, all these molecules can interact with TRAF2. With
respect to the apoptotic TNF-R1/TNF-R2 crosstalk, Bfl1/A1 can most likely be
neglected as it acts independently of TRAF2, is barely detectable in HeLa
cells and is not induced by TNF in this cell line (data not shown). Although
cFLIP can bind to TRAF2 it is certainly not directly involved in the apoptotic
TNF-R1/TNF-R2 crosstalk for several reasons: (1) In Jurkat-TNF-R2 cells, in
which the apoptotic crosstalk occurs in the absence of CHX, cFLIP is not
detectable and is not induced by TNF. (2) In many cell lines, including HeLa
cells, which have been used in this study, at least low concentrations of CHX
are necessary for the induction of death receptor-induced apoptosis. It is
commonly assumed that this reflects the necessity to downregulate an
anti-apoptotic factor with rapid turnover below a critical threshold
concentration. A prime candidate for this anti-apoptotic factor is cFLIP, as
we and others have recently found that cFLIP has a rapid turnover and is
therefore its expression is highly sensitive to metabolic inhibitors such
actinomycin D or CHX (Griffith et al.,
1998
; Kreuz et al.,
2001
; Leverkus et al.,
2000
; Wajant et al.,
2000
). It is noteworthy that the requirement for this compound for
induction of apoptosis remains unchanged upon prestimulation of TNF-R2,
indicating that this CHX-sensitive factor is distinct from the
NF-
B-dependent anti-apoptotic factors mediating the apoptotic
TNF-R1/TNF-R2 crosstalk. Moreover, the apoptotic TNF-R1/TNF-R2 crosstalk is
FLIP-independent, which is in agreement with the facts that TNF- but not Fas-
and TRAIL-induced apoptosis are enhanced by prestimulation of TNF-R2, and that
apoptosis induction in Hela cells by all these reagents requires CHX.
Therefore, according to our model, we suggest that the anti-apoptotic factors
cIAP1, cIAP2, and TRAF1 regulate the apoptotic TNF-R1/TNF-R2 crosstalk. All
these factors bind to TRAF2 and become recruited into the TNF-R1 signaling
complex, thereby preventing efficient activation of caspase-8. The outcome is
a modest apoptotic response. Hence, the balance between protective mechanisms
and activation of caspase-8, the trigger of the apoptotic program, is shifted
towards apoptosis only in a small fraction of the cells. cIAP1 and cIAP2 bind
and inhibit caspase-3 and caspase-7 but not caspase-8
(Roy et al., 1997
). Thus, it
is tempting to speculate that in the TNF-R1 signaling complex
TRAF1/2-recruited IAPs and TRADD/FADD-recruited caspase-8 come into close
proximity, which allows IAP proteins to inhibit caspase-8 activation, possibly
without a direct interaction between the two. In accordance with the scenario
described above, Wang et al. have found in NF-
B-activation-deficient
p65-/- fibroblasts that the individual transient overexpression of
TRAF1, TRAF2, cIAP1 and cIAP2 has no significant inhibitory effect on
TNF-induced apoptosis, whereas the coexpression of these molecules protects
the cells from TNF-induced cell death
(Wang et al., 1998
).
The consequences of TNF-R1 stimulation, as described above, are quite
different when TNF-R1 becomes triggered several hours after activation of
TNF-R2 (Fig. 9B): prestimulated
TNF-R2 has then depleted TRAF2. This has two consequences for TNF-R1
signaling. First, owing to the important role of TRAF2 in TNF-R1-induced
NF-B activation (Devin et al.,
2000
; Devin et al.,
2001
; Tada et al.,
2001
), this pathway is inhibited
(Fig. 4) and therefore the
production of anti-apoptotic factors is reduced. Second, low levels of
pre-existing or still reduced levels of induced anti-apoptotic proteins cannot
work properly as they need the support of TRAF2 [e.g. for targeting to the
TNF-R1 signaling complex (Fig.
6)]. Reduced NF-
B activation and impaired formation of
TRAF1/TRAF2/cIAP1/cIAP2 complexes fails to inhibit caspase-8 activation and
the apoptotic pathway becomes activated in all cells.
The situation changes again when TNF-R1 is stimulated together with TNF-R2
(Fig. 9C). TNF-R1-induced
NF-B activation per se is not inhibited under these conditions
(Fig. 4), as this process is
clearly faster than depletion/degradation of TRAF2. Hence, TNF-R1 triggering
leads to normal production of anti-apoptotic factors. However, the
biosynthesis of these factors needs time, and during this time TRAF2 is
depleted/degraded by TNF-R2. Thus, although anti-apoptotic factors are
produced they cannot act properly as they need TRAF2 for their action at the
post-transcriptional level (see above). The outcome is an enhanced but,
compared with the prestimulation scenario, reduced sensitization of the cells
for the apoptotic effects of TNF-R1.
All the experiments described in this study and all experiments related to
TNF receptor cooperation and TRAF2 depletion described in the literature were
performed in cancer cell lines. Thus, the question of the physiological
relevance of TRAF2 depletion and TNF receptor crosstalk was raised. In this
regard, the available literature points to at least two physiological
processes that could be related to this TNF receptor cooperation. First,
induction of activation-induced cell death in CD8+ T cells. In
accordance with an involvement of TRAF2 depletion and TNF receptor
cooperation, it has been shown in TNF-R1
(Speiser et al., 1996) and
TNF-R2 (Zheng et al., 1995
)
knockout mice that both receptors can have a major impact on this TNF-mediated
apoptotic process. Moreover, Sarin et al. have shown that TNF-R1 and TNF-R2
synergistically induce cell death in T cell blasts in vitro
(Sarin et al., 1995
). Second,
TNF-R2-induced TRAF2 depletion and inhibition of TNF-R1-induced NF-
B
activation under non-apoptotic conditions
(Fig. 4) could explain the
finding that in a mylelin oligodendrocyte glycoprotein (MOG) model of
experimental autoimmune encephalomyelitis (EAE) TNF- or TNF-R1-deficiency led
to delay in disease onset, whereas TNF-R2-deficiency results in a more severe
phenotype (Eugster et al.,
1999
).
![]() |
Acknowledgments |
---|
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