From the Molecular Signaling and Cell Death Unit, Department of Molecular Biomedical Research, VIB, Gent University, B-9000 Gent, Belgium
Received for publication, August 30, 2002, and in revised form, November 12, 2002
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ABSTRACT |
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Triggering tumor necrosis factor
receptor-1 (TNFR1) induces apoptosis in various cell lines. In
contrast, stimulation of TNFR1 in L929sA leads to necrosis. Inhibition
of HSP90, a chaperone for many kinases, by geldanamycin or radicicol
shifted the response of L929sA cells to TNF from necrosis to apoptosis.
This shift was blocked by CrmA but not by BCL-2 overexpression,
suggesting that it occurred through activation of procaspase-8.
Geldanamycin pretreatment led to a
proteasome-dependent decrease in the levels of several
TNFR1-interacting proteins including the kinases
receptor-interacting protein, inhibitor of The primary paradigm of natural programmed cell death (PCD) is
observed during normal embryogenesis. Schweichel and Merker (1, 2)
identified three pathways of programmed cell death. The first pathway
was characterized by the condensation of nucleus and cytoplasm and
corresponds to apoptosis. The second pathway was characterized by
abundant autophagic vacuoles and no or minimal nuclear changes (as
often seen in cells dying by necrosis), whereas the third pathway was a
more rare variant of necrotic cell death. Apoptosis is morphologically
characterized by membrane blebbing, shrinking of the cell and its
organelles, internucleosomal degradation of DNA, and disintegration of
the cell after which the fragments are phagocytosed by neighboring
cells (3, 4). Necrosis is characterized by swelling of the cell and the
organelles and results in disruption of the cell membrane and in lysis
(5). One of the cell lines intensively studied in our laboratory is the
L929 fibrosarcoma cell line. In these cells apoptotic as well as
necrotic cell death can be induced. Stimulation of TNFR1 in L929sA
cells leads to necrotic cell death. This process is strongly sensitized by pretreatment with the pancaspase inhibitor
benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk)1 or overexpression
of CrmA (6). In L929sAhFas cells transfected with human Fas, the
apoptotic cell death pathway can be induced by clustering of FAS with
agonistic anti-FAS antibodies. This apoptotic cell death process can be
reverted to necrosis by inhibiting caspase-activity (7).
Holler and colleagues (8) demonstrated that pretreatment of human
Jurkat T-cell lymphoma cells with geldanamycin (GA) protects these
cells from FASL-mediated caspase-independent cell death in the presence
of zVAD-fmk. The specific target of GA has been identified as the
90-kDa heat shock protein (HSP90) (9-12). Heat shock proteins are a
group of chaperone proteins that help to maintain protein stability, to
renature unfolded proteins, or to target them for degradation when
cells are subjected to heat shock or other types of stress (13).
Geldanamycin prevents the ATP-dependent release of the
client protein undergoing refolding from HSP90 (14). The stabilized
complex is then ubiquitinated and degraded (15, 16). Unlike the better
characterized HSP70 and HSP60 chaperones, HSP90 displays considerable
specificity for its client proteins, including steroid hormone
receptors and kinases (reviewed in Refs. 13, 17, and 18). Lewis and
colleagues reported (19) that one of the HSP90 client proteins is the
death domain kinase receptor-interacting protein (RIP) (19). They showed that disruption of HSP90 function by the addition of GA results
in the degradation of RIP and blockage of TNF-induced NF- Cells--
L929sA is a murine fibrosarcoma cell line, derived
from L929, which was selected for its sensitivity to the cytotoxic
activity of TNF (5). L929sA was transfected with the human
FAS receptor cDNA with or without human BCL-2
cDNA and with cDNA encoding CrmA from cowpox
virus, resulting in L929sAhFas, L929AhFasBCL2, and L929sACrmA clones,
as described previously (5-7). L929sA and derivatives were cultured in
Dulbecco's modified Eagle's medium supplemented with 5% newborn
bovine serum, 5% fetal calf serum, penicillin (100 units/ml),
streptomycin (0.1 mg/ml), and L-glutamine (0.03%).
Antibodies, Cytokines, and Reagents--
Recombinant hTNF was
produced in Escherichia coli and purified to at least 99%
homogeneity. The specific biological activity was 2.3 × 107 units/mg as determined in a standardized cytotoxicity
assay on L929sA cells. Secreted IL-6 in the supernatant was quantified using a bioassay, viz. IL-6-dependent growth of
7TD1 cells (20). Anti-human Fas antibody (clone 2R2) was purchased from
Cell Diagnostica (Münster, Germany). GA and MG132 were obtained
from Sigma and used at 1 and 20 µM, respectively.
Propidium iodide (PI; BD Biosciences) was dissolved at 3 mM
in phosphate-buffered saline and was used at 30 µM. The
caspase peptide inhibitor zVAD-fmk was supplied by Bachem (Bubendorf,
Switzerland) and used at 25 µM. The caspase fluorogenic substrate
acetyl-Asp(OMe)- Glu-(OMe)-Val-Asp(OMe)-aminomethylcoumarin (Ac-DEVD-amc) was obtained from Peptide Institute (Osaka, Japan) and
used at 50 µM. Anti-murine caspase-9 antibodies and
polyclonal antibodies against JNK, p38MAPK, ERK, IKK- Induction of Apoptosis or Necrosis for Fluorescence-activated
Cell Sorter Analysis and Western Blotting--
The cells were kept in
suspension by seeding 105 cells in uncoated 24-well tissue
culture plates (Sarstedt Inc., Newton, NC). 1 µM GA was
added 16 h before stimulation. After preincubation for
1 h with or without zVAD-fmk, human TNF (10,000 units/ml) was added to the cells for different time intervals.
Measurement of Cell Death and Hypoploidy by Flow
Fluorocytometry--
Cell death was analyzed for loss of membrane
integrity by PI uptake. PI (30 µM) was added 10 min
before measuring and was detected at 610 nm on a FACScalibur® flow
fluorocytometer (BD Biosciences) equipped with a 488-nm argon ion
laser. DNA histograms were obtained by subjecting the samples to one
freeze-thaw cycle in the presence of PI and analyzing PI fluorescence
of the cells as described above.
Western Analysis--
Cells were washed in cold
phosphate-buffered saline A and lysed in 150 µl of caspase lysis
buffer (1% Nonidet P-40, 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 0.3 mM
aprotinin, and 1 mM leupeptin). Equal amounts were loaded
per lane on 12.5% SDS-PAGE, and after separation and blotting the
different proteins were detected with the indicated antibodies and
developed by ECL (Amersham Biosciences). For detection of the
phosphorylated forms of p38MAPK and JNK, cells were lysed in SDS sample
buffer (62.5 mM Tris-HCl, pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% w/v bromphenol blue).
Fluorogenic Substrate Assay for Caspase Activity--
The
fluorogenic substrate assay for caspase activity was carried out as
described previously (7). Cells were transferred to Eppendorf tubes,
washed in cold phosphate buffer, and lysed in 150 µl of caspase lysis
buffer. Cell debris was removed by centrifugation, and caspase activity
was determined by incubating 15 µl of the soluble fraction with 50 µM Ac-DEVD-amc in 150 µl of cell-free system buffer
containing 10 mM Hepes, pH 7.4, 220 mM
mannitol, 68 mM sucrose, 2 mM NaCl, 2.5 mM KH2PO4, 0.5 mM EGTA, 2 mM MgCl2, 0.5 mM sodium pyruvate,
0.5 mM L-glutamine, and 10 mM
dithiothreitol. The release of fluorescent aminomethylcoumarin was
measured for 1 h at 2-min intervals by fluorometry
(excitation at 360 nm and emission at 480 nm) (Cytofluor; PerSeptive
Biosystems, Cambridge, MA); the maximal rate of increase in
fluorescence was calculated ( Light Microscopic Analyses--
The cells were seeded in 6-well
adherent plates at a density of 2 × 105 cells per
well. 1 µM GA was added 16 h before stimulation.
After preincubation for 1 h with or without 25 µM
zVAD-fmk, hTNF (10,000 units/ml) was added to the cells for different
time intervals. Light microscopic pictures were taken on different time
points (Integrated Modulation Contrast, ×400).
Disruption of HSP90 Function in L929sA Cells Reverts TNFR1-induced
Necrosis to Apoptosis--
A previous report demonstrated that
pretreatment of human Jurkat T-cell lymphoma cells with the pancaspase
inhibitor zVAD-fmk shifts their response to FASL from apoptosis to
necrosis. However, pretreating these cells with the combination of
zVAD-fmk and the HSP90 inhibitor geldanamycin protects them from
FAS-mediated necrosis (8). L929sA cells respond to TNF by
caspase-independent necrosis even without the addition of caspase
inhibitors (6). Therefore, we analyzed the effect of GA on
TNFR1-induced necrosis in these cells. Preincubation of the cells with
GA during 16 h strongly sensitized the cells to the cytotoxic
effect of TNF. However, analysis of the cell morphology by light
microscopy revealed that the cells did not die by necrosis but instead
responded by apoptosis characterized by membrane blebbing and nuclear
condensation (Fig. 1A). The
morphological assessment of apoptotic cell death after pretreatment
with GA was confirmed using biochemical parameters such as (in order of
appearance) caspase activation, tBid generation, cytochrome
c release, DNA hypoploidy, and cell membrane
permeabilization (Fig. 1, B and C). None of these
typically apoptosis-related events was detected in TNF-induced necrosis
in the absence of GA (Fig. 1, B and C). To
exclude the possibility that the effect described may not arise from a
side effect of GA, we analyzed the effect of a structurally unrelated
inhibitor of HSP90, viz. radicicol. Pretreatment of the
cells with radicicol also caused a shift from necrosis to apoptosis in
response to TNF (data not shown). These results confirm that inhibition
of HSP90 in L929sA cells reverts TNFR1-induced necrotic cell death to
apoptosis.
The GA-induced Shift from Necrosis to Apoptosis after Stimulation
with TNF Occurs at a Premitochondrial Level--
In a previous report
we demonstrated that the antioxidant butylated hydroxyanisole can shift
the response of L929sAhFas cells to treatment with the combination of
interferon and dsRNA from necrosis to apoptosis. This shift occurred at
the mitochondrial level and was completely blocked by overexpression of
BCL-2 (5, 22). Because tBid generation and cytochrome c
release were observed also in cells treated with the combination of GA
and TNF (Fig. 1C), we investigated whether or not
TNFR1-induced apoptosis in the presence of GA is dependent on the
mitochondrial apoptotic pathway. We analyzed the effect of GA on
TNF-induced necrosis in L929sAhFasBCL2 cells stably transfected with
Bcl-2. Signaling through the intrinsic pathway, originating
at the mitochondria, is attenuated in these cells (5, 22). In
comparison with parental L929sAhFas, a substantial delay in apoptotic
cell death induced by TNF in the presence of GA was observed in
L929sAhFasBCL2 cells, as revealed by uptake of propidium iodide and
hypoploidy (Fig. 2A,
left). Although overexpression of BCL-2 had a
partial inhibitory effect on apoptosis induced by TNF and
GA, as demonstrated by the decrease in caspase-3 activity (Fig.
2, A and B, left), the cells still
responded by apoptosis. A similar effect on FAS-mediated apoptosis was
observed before (5, 22), suggesting that the mitochondrial apoptotic
pathway amplifies the apoptotic death process, and in its absence the
process is delayed. Moreover, tBid generation during TNF/GA treatment
clearly preceded cytochrome c release (Fig. 1C),
suggesting that the shift to apoptosis occurred upstream to the
mitochondria. Caspase-8 is required for death receptor-induced
apoptosis and is a known Bid-cleaving protease (23, 24). Therefore, we
tested whether the apoptotic process was initiated by caspase-8 by
analyzing the effect of GA on TNFR1-induced cell death response in
L929sACrmA cells. CrmA, the viral serpin-like inhibitor of caspase-8,
should block death receptor-induced apoptosis in these cells.
Contrary to their parental L929sA cells, the L929sACrmA cells still
died by necrosis when treated with TNF after pretreatment with GA.
Indeed, no hypoploidy, caspase-3 activity, or caspase-3 cleavage was
detected (Fig. 2, A and B, right).
Inhibition of caspase activation by pretreatment with zVAD-fmk yielded
similar results in L929sA cells (Fig. 2C). In both cases,
cells responded to TNF by necrosis even after pretreatment with GA.
However, cell death was delayed in comparison with cells treated with
TNF in the absence of GA (Fig. 2C). Thus, inhibition of
caspase-8 blocks TNFR1-induced apoptosis in the presence of GA. This
finding suggests that the apoptotic process induced by TNF in the
presence of GA is mediated through activation of caspase-8, a
receptor-mediated event.
Decrease of the Expression Levels of RIP, IKK- Our results demonstrate that pretreatment with the HSP90
inhibitors GA or radicicol shifts the response to TNF from necrosis to
apoptosis in L929sA cells. Several lines of evidence suggest that the
shift occurs at the receptor level. First, cleavage of Bid preceded
cytochrome c release. Second, overexpression of the caspase-8 inhibitor CrmA abolished the shift from necrosis to apoptosis. Third, BCL-2 overexpression decreased the apoptotic response
but did not block it, although BCL-2 overexpression in these cells was
able to block the conversion of necrotic to apoptotic signaling induced
by dsRNA and interferon in the presence of the antioxidant butylated
hydroxyanisole (22). Taken together, these observations indicate that
in L929sA cells the shift from necrotic to apoptotic cell death in the
case of TNF/GA occurs at the receptor level, whereas the shift induced
on dsRNA/interferon/butylated hydroxyanisole treatment occurs at the
mitochondrial level.
Inhibition of necrotic cell death by GA has been reported in Jurkat
cells treated with FasL in the presence of zVAD-fmk (8). In addition,
GA can protect caspase-8-deficient Jurkat cells from dsRNA-induced
necrosis (22). Several reports suggest that the availability of certain
proteins such as FADD, caspase-8, RIP, and/or still unknown proteins
can determine whether necrosis or apoptosis occurs. FADD or
caspase-8 deficiency has been reported to protect Jurkat cells from
Fas-mediated apoptosis (35-38), whereas RIP deficiency protects these
cells from necrosis (8). This finding implies that death receptors can
initiate two alternative cell death pathways, one relying on FADD and
caspase-8 and the other dependent on the kinase RIP. Indeed,
FADD-deficient Jurkat cells respond to TNF by necrosis (8, 22).
Similarly, dsRNA-induced apoptosis shifts to necrosis in FADD- or
caspase-8-deficient Jurkats, whereas dsRNA-induced necrosis is blocked
in RIP-deficient Jurkats (22). Moreover, accumulating evidence suggests
that signaling compounds in necrosis and apoptosis compete and
counteract each other. In necrotic cell death, RIP leads to
anti-apoptotic signals by the activation of NF- GA prevents the ATP-dependent release from HSP90 of a
client protein undergoing refolding leading to its degradation
(14-16). Here, we analyzed the effect of the inhibition of HSP90 by GA on the expression levels of TNFR1-related adaptor proteins, kinases, and caspases. Our results show that GA induces a decrease in the expression levels of RIP, IKK- RIP, IKK- Our results show that TRADD, FADD, and caspase-3, -8, and
-9, which are involved in the apoptotic-signaling pathway, are not affected by the pretreatment with GA. This finding suggests that inhibition of HSP90 alters the composition of the TNFR1 complex, favoring the TRADD-FADD-caspase-8 pathway and thus apoptosis (Fig. 5, right). This fact implies
that in the absence of GA one or more HSP90-interacting proteins needed
for the induction of necrosis induced by TNF are probably recruited
more efficiently to the TNFR1-complex (Fig. 5, left). These
proteins may prevent apoptosis by competing with FADD or caspase-8 for
recruitment to the TNFR1-complex and by initiating anti-apoptotic
mechanisms through NF-B kinase-
,
inhibitor of
B kinase-
, and to a lesser extent the adaptors
NF-
B essential modulator and tumor necrosis factor
receptor-associated factor 2. As a consequence, NF-
B, p38MAPK, and
JNK activation were abolished. No significant decrease in the levels of
mitogen-activated protein kinases, adaptor proteins TNFR-associated
death domain and Fas-associated death domain, or caspase-3, -8, and -9 could be detected. These results suggest that HSP90 client proteins
play a crucial role in necrotic signaling. We conclude that inhibition
of HSP90 may alter the composition of the TNFR1 complex, favoring the
caspase-8-dependent apoptotic pathway. In the absence of geldanamycin,
certain HSP90 client proteins may be preferentially recruited to the
TNFR1 complex, promoting necrosis. Thus, the availability of proteins
such as receptor-interacting protein, Fas-associated death domain, and caspase-8 can determine whether TNFR1 activation will lead to apoptosis
or to necrosis.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
B
activation. In the present study, we analyzed whether GA-induced inhibition of HSP90 function could block TNFR1-induced necrosis in
L929 fibrosarcoma cells. We show that inhibition of the function of
HSP90 leads to a switch from TNFR1 necrotic cell death to apoptotic cell death. This correlates with the preferential decrease of the
expression levels of several TNFR1 signaling-associated proteins, resulting in the abolishment of TNF-mediated activation of NF-
B, JNK, p38MAPK, and necrosis. Moreover, GA pretreatment does not affect
the expression levels of proteins that constitute the apoptotic signaling axis, such as TRADD, FADD, and procaspase-8, and thus promotes a TNFR1-induced apoptotic response.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
, and IKK-
were obtained from New England Biolabs (Beverly, MA). Antibodies
against TRADD, TRAF2, RAF-1, and NEMO were from Santa Cruz
Biotechnology (Santa Cruz, CA), antibodies against mouse Bid
were from R&D Systems (Minneapolis, MN), and antibodies against
cytochrome c were from Amersham Biosciences. Rabbit
polyclonal antibodies against recombinant murine caspase-3 and -8 were
prepared at the Centre d'Economie Rurale (Laboratoire d'Hormonologie
Animale, Marloie, Belgium). Anti-caspase-2 antibody was kindly provided
by Professor Dr. Kumar Sharad (The Hanson Centre for Cancer Research,
Institute of Medical and Veterinary Science, Adelaide, Australia) (21).
Anti-murine RIP antibodies were obtained from Transduction Laboratories
(Lexington, KY).
F/min).
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ABSTRACT
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Fig. 1.
GA shifts TNF-induced necrosis to
apoptosis. Mouse L929sA fibrosarcoma cells were pretreated
during 16 h with or without 1 µM GA followed by
treatment with 104 units/ml hTNF for 1, 2, 4, 6, and 8 h. A, light microscopy of L929sA cells (integrated
modulation contrast). Morphology of cells treated with TNF alone (8 h)
is necrotic, whereas that of a combined treatment with TNF (2 h) and GA
(16 h) is apoptotic. B, analysis of caspase activity
(relative DEVDase activity), DNA degradation (% hypoploidy), and loss
of membrane impermeability (% PI-positive cells). C,
Western blot (WB) analysis of cytosolic lysates detecting
Bid cleavage and cytochrome c release. Results are
representative for three independent experiments.
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Fig. 2.
TNFR1-induced shift from necrosis to
apoptosis is blocked in L929sACrmA cells but not in
L929sAhFasBcl2 cells. A, cell lines were treated
for 16 h with 1 µM GA followed by treatment
with 104 units/ml hTNF for 1, 2, 4, 6, and 8 h. Cells
were analyzed by flow fluorocytometry for loss of membrane
impermeability (% PI-positive cells), DNA degradation (% hypoploidy),
and caspase activity (relative DEVDase activity). B,
caspase-3 cleavage was analyzed by Western blotting on cytosolic
lysates (procaspase-3, black triangle; activated form of
caspase-3, white triangle). The disappearance of
procaspase-3 at 4, 6, and 8 h in L929sACrmA cells is caused by
leakage of the cellular contents by the strong necrotic response.
C, fluorocytometric analysis for loss of membrane integrity
by PI. After treatment for 16 h of L929sA cells with or without 1 µM GA, cells were pretreated for 1 h with 25 µM zVAD-fmk, followed by treatment with 104
units/ml hTNF (left). L929sACrmA cells were treated for
16 h with or without 1 µM GA, followed by treatment
with 104 units/ml hTNF (right).
, IKK-
, NEMO,
and TRAF2 Results in the Abolishment of TNF-mediated Activation of
NF-
B and the MAPKs in the Presence of GA--
The above results
show that an efficient necrotic response requires a functional HSP90
and suggest that one or more of the chaperone's client proteins are
involved in the TNFR1 complex signaling to necrosis or in the
prevention of apoptosis. Therefore, we investigated the effect of HSP90
inhibition by GA on the expression levels of different TNFR1
complex-associated adaptor proteins, kinases, and caspases. Western
blot analysis of different kinases revealed that the detected levels of
RIP, RAF-1, IKK-
, and IKK-
are decreased in the presence of GA,
whereas those of ERK, p38MAPK, JNK, and dsRNA-activated protein kinase
are not affected (Fig. 3A).
No, or a minor effect on the expression levels of adaptors recruited to
the TNFR1 complex, such as TRADD and FADD, was detected (Fig.
3A). However, a decrease in the expression levels of two RIP-interacting proteins, TRAF2 and NEMO, was clearly apparent in cells
treated with GA (Fig. 3A). TRAF2 is an adaptor protein known
also to interact with TRADD and TNFR2 (25), whereas NEMO is a scaffold
protein playing a central role in the assembly of the IKK complex
(26-29). We could not detect any change in the levels of caspase-3,
-8, and -9. Surprisingly, caspase-2 levels clearly dropped in the
presence of GA. Several reports demonstrated that the decrease in
expression levels of HSP90 client proteins is caused by degradation by
the proteasome (15, 16). Indeed, 1 h of treatment with the
proteasome inhibitor MG132 was sufficient to restore approximately half
of the expression level of RIP that was lost because of 15 h of
preincubation with GA (Fig. 3B). Several reports using
knockout mice demonstrated that RIP and TRAF2 are involved in the
activation of NF-
B (30, 31). RIP was also shown to be involved in
the activation of JNK and p38MAPK (32, 33). Moreover, disruption of
HSP90 function was reported to result in blockage of NF-
B activation
(19). Therefore, we investigated whether degradation of RIP, IKK-
,
IKK-
and, to a lesser extent, NEMO and TRAF2 in the presence of GA
abrogated the activation of NF-
B, JNK, and p38MAPK in L929sA cells.
First, we analyzed the effect of GA on the TNF-induced secretion of
IL-6, one of the major NF-
B-regulated cytokines. In the presence of
GA, TNF-induced IL-6 production was blocked completely; also, the basal
level of IL-6 was decreased (Fig.
4A). Next we analyzed the
activation of JNK and p38MAPK by Western blotting using phosphospecific
antibodies. In the absence of GA, TNF treatment led to an early
transient activation of JNK and p38MAPK (Fig. 4B). The
activation of JNK was more prolonged than that of p38MAPK. The primary
transient activation of p38MAPK was followed by a secondary activation
(Fig. 4B). Such a biphasic activation pattern for MAPKs in
response to a combined treatment with TNF and cycloheximide was
reported previously (34). The activation of both MAPKs by TNF was
prevented by pretreatment with GA. In conclusion, these results show
that the expression levels of TNFR1 adaptor protein FADD and the
initiator caspase-8 required to initiate apoptosis are not affected by
the pretreatment of GA. However, the expression levels of RIP, IKK-
, IKK-
, NEMO, and TRAF2 are reduced by pretreatment with GA. As a consequence, NF-
B, p38MAPK, and JNK activation are abolished. The
restoration of the expression level of RIP by inhibiting the proteasome
suggests that treatment with GA leads to degradation of HSP90 client
proteins.
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Fig. 3.
Expression levels of RIP,
IKK- , IKK-
, NEMO, and
TRAF2 are lower in L929sA cells treated with GA. A, cells
were treated with and without 1 µM GA for 16 h.
Equal amounts of cytosolic lysates were analyzed by Western blotting
with antibodies specific for different DISC-related proteins, kinases,
and caspases, viz. RIP, IKK-
, IKK-
, ERK, JNK, p38MAPK,
RAF-1, dsRNA-activated protein kinase, TRAF-2, FADD, TRADD, NEMO,
procaspase-2, -3, -8, and -9. As a control for equal loading, lysates
were analyzed with antibodies against actin. Immunoreactive signals
were quantified by densitometry. B, cells were treated with
and without 1 µM GA for 15 h and incubated for
1 h in the presence or absence of 20 µM of the
proteasome inhibitor MG132.
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Fig. 4.
Preincubation of L929sA cells with GA blocks
activation of NF- B, p38MAPK, and JNK.
A, cells were pretreated with 1 µM GA for
16 h followed by addition of a serial dilution of hTNF ranging
between 3.2 and 104 units/ml during 24 h. Secreted
IL-6 in the supernatant was quantified using a bioassay. B,
cells were treated with and without 1 µM GA for 16 h
followed by treatment with hTNF for the indicated time points. Equal
amount of total lysates were loaded and analyzed by Western blotting
(WB) using antibodies that specifically recognize the
phosphorylated form of p38MAPK and JNK. As a control for equal loading,
lysates were analyzed with antibodies against the nonphosphorylated
form of p38MAPK.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and the MAPKs (30,
33). In apoptosis, caspase-8 cleaves RIP and thus may block these
anti-apoptotic signals (39-41). These results suggest that
TNFR1-induced necrosis and apoptosis use distinct proteins/components
in their signaling pathways.
, IKK-
and, to a lesser extent, of
NEMO, TRAF2, and RAF-1, whereas no significant drop in the expression
levels of the MAPKs, dsRNA-activated protein kinase, and adaptors of
the TNFR1 complex such as TRADD and FADD was observed. The degradation
of RIP and RAF-1 in the presence of GA confirms previous reports (15,
19). The strong drop of the expression levels of IKK-
/
in the
presence of GA suggests that both kinases are also client proteins of
HSP90. Indeed, a recent report demonstrated that the IKK complex, which
contains the two catalytic subunits IKK-
and IKK-
and a
regulatory subunit NEMO, forms a ~900-kDa heterocomplex with Cdc37
and HSP90 (42). Contrary to our results, the authors could not detect
any degradation of the IKK-
/
in HeLa cells after 15 h of GA
treatment. Nevertheless, inhibition of HSP90 in the latter cells
prevented the recruitment of the IKK complex to TNFR1. The observation
that NEMO and TRAF2 are significantly less degraded in the presence of
GA than RIP, IKK-
, and IKK-
, suggests that no direct interaction
occurs between HSP90 and TRAF2 or NEMO. The latter might be degraded
because of their recruitment to complexes containing either RIP or
IKK-
and IKK-
. Similarly, the decrease in the level of caspase-2
in cells treated with GA may be caused by an indirect interaction of
caspase-2 with RIP via RIP-associated Ich-1/CED-3 homologous protein
with a death domain (43). Holler and colleagues (8) suggested that GA
inhibits necrotic cell death because of the degradation of RIP.
However, our data show that necrotic cell death can still occur in the
presence of zVAD-fmk or CrmA, although RIP is degraded by GA treatment.
This finding suggests that other proteins (kinases) may have an
important role in signaling to necrosis or that the small amount of RIP
still present in the GA-treated cells is sufficient to allow the
necrotic process to occur.
, IKK-
, NEMO, and TRAF2 are involved in the signaling to
NF-
B, p38MAPK, and JNK (44, 45). NF-
B activation has been
implicated in the suppression of apoptosis. For example, RelA
/
mice die at embryonic day 15 as
a result of extensive liver apoptosis (44, 46). Other studies indicate
that an early but brief activation of JNK and/or p38MAPK may protect
cells from TNF-induced apoptosis (34). Indeed, pretreatment with GA
blocks TNF-induced activation of NF-
B, p38MAPK, and JNK in our
cells, again favoring the apoptotic cell death process.
B (30, 31) and activation of JNK and/or
p38MAPK (32, 33). Additionally, they may actively contribute to the
signaling to and the execution of the necrotic process. In L929sA cells
we have shown before that butylated hydroxyanisole blocks necrosis
induced by TNF (6) or anti-Fas plus zVAD-fmk (7) and shifts the
response to dsRNA/interferon from necrosis to apoptosis (22). These
observations argue for a role of oxygen radical production in the
execution of the necrotic process.
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Fig. 5.
Putative model of TNF-induced cell death in
the presence or absence of GA in L929sA cells. Stimulation of
TNFR1 leads to necrosis and activation of NF- B and MAPKs. In these
conditions TRADD, RIP, TRAF-2, and the IKK complex are recruited to the
TNFR1 complex and would initiate a necrotic response and
activation of NF-
B and MAPKs. The latter two also govern
anti-apoptotic transcriptional activation. In the presence of GA, TNF
leads to a fast apoptotic response initiated by caspase-8. Activation
of NF-
B and the MAPKs is blocked because of degradation of RIP,
IKK
/
, and, to a lesser extent, of TRAF-2 and NEMO.
One of the currently emerging questions is why two distinct cell death
programs did evolve that seem to negatively influence each other's
pathway. Apoptosis might be blocked in several pathophysiological conditions as these often coincide with inflammation. During
inflammation NF-B- and MAPK-dependent transcription
occurs, leading to the expression of several anti-apoptotic proteins
such as X-linked inhibitor of apoptosis, Bcl-2, and cellular
Flice-like inhibitory protein (47-49). Certain viruses up-regulate
endogenous inhibitors of apoptosis and/or encode for caspase inhibitors
(e.g. p35, CrmA, viral Flice-like inhibitory protein, viral
inhibitor of apoptosis) (50). Moreover, tumor cells often carry
mutations leading to the inactivation of apoptosis-initiating proteins
or caspases (51). Thus, necrosis may function as a backup program
resistant to or even enhanced by the factors blocking the apoptotic
signaling (6). Apoptosis coupled to phagocytosis is an efficient way of
clearing dying cells (52). In necrosis, cytosolic constituents leak
into the intercellular space through damaged plasma membrane and may
provoke a strong immune response that may be physiologically important
during certain dangerous situations such as viral or bacterial
infection, traumas, and cancer (53, 54).
The present results show that TNF can lead to either necrosis or
apoptosis. The choice between these cell death pathways depends on the
availability of certain adaptor proteins. We suggest that inhibition of
HSP90 by GA is favoring an apoptotic TNFR1 complex, whereas in the
absence of GA, a pronecrotic TNFR1 complex is formed in which RIP
and/or other HSP90-interacting proteins are preferably recruited.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. G. M. Cohen (University of Leicester) for fruitful discussion. We are grateful to Ann Meeus and Wilma Burm for expert technical assistance. Anti-caspase-2 antibody was kindly provided by Professor Dr. Kumar Sharad (The Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, Australia).
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FOOTNOTES |
---|
* This work was supported in part by the Interuniversitaire Attractiepolen V, the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (Grant 3G.0006.01 and Grant 3G.021199), an EC-RTD Grant QLG1-CT-1999-00739, a university BOF cofinancing EU Project (011C0300), and a university GOA Project (12050502).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Fellow with the Vlaams Instituut voor de Bevordering van het
Wetenschappelijk-technologisch Onderzoek in de Industrie.
§ Both authors share senior authorship.
¶ To whom correspondence should be addressed: K. L. Ledeganckstraat 35, B-9000 Gent, Belgium. Tel.: 32-9-264-51-31; Fax: 32-9-264-53-48; E-mail: peter.vandenabeele@dmb.rug.ac.be.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M208925200
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ABBREVIATIONS |
---|
The abbreviations used are:
zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone;
Bcl-2, B-cell
lymphoma;
CrmA, cytokine response modifier A;
ERK, extracellular
signal-regulated kinase;
FADD, Fas-associated death domain;
GA, geldanamycin;
HSP90, 90-kDa heat shock protein;
IKK, inhibitory B
kinase;
JNK, c-Jun NH2-terminal kinase;
MAPK, mitogen-activated protein kinase;
NEMO, nuclear factor-
B essential
modulator;
NF-
B, nuclear factor-
B;
PI, propidium iodide;
RAF-1, Ras-associated factor 1;
RIP, receptor interacting protein;
TNF, tumor necrosis factor;
TNFR1, 55-kDa tumor necrosis factor receptor;
TRADD, TNFR-associated death domain;
TRAF2, TNF receptor-associated
factor 2;
dsRNA, double-stranded RNA;
hTNF, human tumor necrosis
factor;
IL, interleukin;
Ac-DEVD-amc, acetyl-Asp
(OMe)-Glu(OMe)-Val-Asp(OMe)-aminomethylcoumarin..
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