THE JOURNAL OF BIOLOGICAL CHEMISTRY

2003 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 278, No. 8, Issue of February 21, pp. 56225629, 2003

Printed in U.S.A.

Disruption of HSP90 Function Reverts Tumor Necrosis

Factor-induced Necrosis to Apoptosis*
Received for publication, August 30, 2002, and in revised form, November 12, 2002
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M208925200

Tom Vanden Berghe, Michael Kalai, Geert van Loo, Wim Declercq, and Peter Vandenabeele
From the Molecular Signaling and Cell Death Unit, Department of Molecular Biomedical Research, VIB, Gent University,
B-9000 Gent, Belgium

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

* 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 QLG1CT-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. This
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: 32-9-26453-48; E-mail: peter.vandenabeele@dmb.rug.ac.be.

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-B activation. In the present study, we analyzed whether GA-induced
inhibition of HSP90 function could block TNFR1-induced
1
The abbreviations used are: zVAD-fmk, benzyloxycarbonyl-Val-AlaAsp-(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, 90kDa heat shock protein; IKK, inhibitory B kinase; JNK, c-Jun NH2terminal 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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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 proteasomedependent decrease in the levels of several TNFR1interacting proteins including the kinases receptorinteracting protein, inhibitor of B kinase-, inhibitor of
B kinase-, and to a lesser extent the adaptors NF-B
essential modulator and tumor necrosis factor receptorassociated 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-8dependent 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.

HSP90 Inhibition Reverts TNF-induced Necrosis to Apoptosis

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.
EXPERIMENTAL PROCEDURES

was removed by centrifugation, and caspase activity was determined by

incubating 15 l of the soluble fraction with 50 M Ac-DEVD-amc in 150
l 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 (F/min).
Light Microscopic AnalysesThe cells were seeded in 6-well adherent plates at a density of 2 105 cells per well. 1 M GA was added 16 h
before stimulation. After preincubation for 1 h with or without 25 M
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).
RESULTS

Disruption of HSP90 Function in L929sA Cells Reverts

TNFR1-induced Necrosis to ApoptosisA 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 caspaseindependent necrosis even without the addition of caspase inhibitors (6). Therefore, we analyzed the effect of GA on TNFR1induced 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 apoptosisrelated 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 LevelIn 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

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CellsL929sA 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 (57). L929sA and derivatives were cultured in Dulbeccos modified Eagles 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 ReagentsRecombinant 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 (Mu nster, Germany). GA and MG132
were obtained from Sigma and used at 1 and 20 M, respectively.
Propidium iodide (PI; BD Biosciences) was dissolved at 3 mM in phosphate-buffered saline and was used at 30 M. The caspase peptide
inhibitor zVAD-fmk was supplied by Bachem (Bubendorf, Switzerland)
and used at 25 M. 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 M.
Anti-murine caspase-9 antibodies and polyclonal antibodies against
JNK, p38MAPK, ERK, IKK-, 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 dEconomie Rurale (Laboratoire dHormonologie 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).
Induction of Apoptosis or Necrosis for Fluorescence-activated Cell
Sorter Analysis and Western BlottingThe cells were kept in suspension by seeding 105 cells in uncoated 24-well tissue culture plates
(Sarstedt Inc., Newton, NC). 1 M 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 FluorocytometryCell death was analyzed for loss of membrane integrity by PI
uptake. PI (30 M) 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 AnalysisCells were washed in cold phosphate-buffered
saline A and lysed in 150 l 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 ActivityThe 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 l of caspase lysis buffer. Cell debris

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HSP90 Inhibition Reverts TNF-induced Necrosis to Apoptosis

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-, IKK-,
NEMO, and TRAF2 Results in the Abolishment of TNF-mediated Activation of NF-B and the MAPKs in the Presence of
GAThe above results show that an efficient necrotic response
requires a functional HSP90 and suggest that one or more of
the chaperones 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

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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 M
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.

HSP90 Inhibition Reverts TNF-induced Necrosis to Apoptosis

5625

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-, N, and TRAF2 are

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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 M 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 M GA, cells were pretreated for 1 h with 25 M zVAD-fmk,
followed by treatment with 104 units/ml
hTNF (left). L929sACrmA cells were
treated for 16 h with or without 1 M GA,
followed by treatment with 104 units/ml
hTNF (right).

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.
DISCUSSION

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

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HSP90 Inhibition Reverts TNF-induced Necrosis to Apoptosis

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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 M 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 M GA for 15 h and incubated for 1 h in the
presence or absence of 20 M of the proteasome inhibitor MG132.

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

HSP90 Inhibition Reverts TNF-induced Necrosis to Apoptosis

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

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 antiapoptotic 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.

whether necrosis or apoptosis occurs. FADD or caspase-8 deficiency has been reported to protect Jurkat cells from Fasmediated apoptosis (3538), 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-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.
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-,
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/

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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
M 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 M 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.

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HSP90 Inhibition Reverts TNF-induced Necrosis to Apoptosis

TNFR1 complex, whereas in the absence of GA, a pronecrotic
TNFR1 complex is formed in which RIP and/or other HSP90interacting proteins are preferably recruited.
AcknowledgmentsWe 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).
REFERENCES
1. Schweichel, J. U., and Merker, H. J. (1973) Teratology 7, 253266
2. Fiers, W., Beyaert, R., Declercq, W., and Vandenabeele, P. (1999) Oncogene 18,
7719 7730
3. Kerr, J. F., Wyllie, A. H., and Currie, A. R. (1972) Br. J. Cancer 26, 239 257
4. Wyllie, A. H., Kerr, J. F., and Currie, A. R. (1980) Int. Rev. Cytol. 68, 251306
5. Denecker, G., Vercammen, D., Steemans, M., Vanden Berghe, T., Brouckaert,
G., Van Loo, G., Zhivotovsky, B., Fiers, W., Grooten, J., Declercq, W., and
Vandenabeele, P. (2001) Cell Death Differ. 8, 829 840
6. Vercammen, D., Beyaert, R., Denecker, G., Goossens, V., Van Loo, G.,
Declercq, W., Grooten, J., Fiers, W., and Vandenabeele, P. (1998) J. Exp.
Med. 187, 14771485
7. Vercammen, D., Brouckaert, G., Denecker, G., Van de Craen, M., Declercq, W.,
Fiers, W., and Vandenabeele, P. (1998) J. Exp. Med. 188, 919 930
8. Holler, N., Zaru, R., Micheau, O., Thome, M., Attinger, A., Valitutti, S.,
Bodmer, J. L., Schneider, P., Seed, B., and Tschopp, J. (2000) Nat. Immunol.
1, 489 495
9. Whitesell, L., Mimnaugh, E. G., De Costa, B., Myers, C. E., and Neckers, L. M.
(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8324 8328
10. Grenert, J. P., Sullivan, W. P., Fadden, P., Haystead, T. A., Clark, J.,
Mimnaugh, E., Krutzsch, H., Ochel, H. J., Schulte, T. W., Sausville, E.,
Neckers, L. M., and Toft, D. O. (1997) J. Biol. Chem. 272, 2384323850
11. Prodromou, C., Roe, S. M., OBrien, R., Ladbury, J. E., Piper, P. W., and Pearl,
L. H. (1997) Cell 90, 6575
12. Stebbins, C. E., Russo, A. A., Schneider, C., Rosen, N., Hartl, F. U., and
Pavletich, N. P. (1997) Cell 89, 239 250
13. Hartl, F. U. (1996) Nature 381, 571579
14. Schneider, C., Sepp-Lorenzino, L., Nimmesgern, E., Ouerfelli, O., Danishefsky,
S., Rosen, N., and Hartl, F. U. (1996) Proc. Natl. Acad. Sci. U. S. A. 93,
14536 14541
15. Schulte, T. W., An, W. G., and Neckers, L. M. (1997) Biochem. Biophys. Res.
Commun. 239, 655 659
16. Mimnaugh, E. G., Chavany, C., and Neckers, L. (1996) J. Biol. Chem. 271,
22796 22801
17. Richter, K., and Buchner, J. (2001) J. Cell. Physiol. 188, 281290
18. Garrido, C., Gurbuxani, S., Ravagnan, L., and Kroemer, G. (2001) Biochem.
Biophys. Res. Commun. 286, 433 442
19. Lewis, J., Devin, A., Miller, A., Lin, Y., Rodriguez, Y., Neckers, L., and Liu,
Z. G. (2000) J. Biol. Chem. 275, 10519 10526
20. Poupart, P., Vandenabeele, P., Cayphas, S., Van Snick, J., Haegeman, G.,
Kruys, V., Fiers, W., and Content, J. (1987) EMBO J. 6, 1219 1224
21. OReilly, L. A., Ekert, P., Harvey, N., Marsden, V., Cullen, L., Vaux, D. L.,
Hacker, G., Magnusson, C., Pakusch, M., Cecconi, F., Kuida, K., Strasser,
A., Huang, D. C., and Kumar, S. (2002) Cell Death Differ. 9, 832 841
22. Kalai, M., Van Loo, G., Vanden Berghe, T., Meeus, A., Burm, W., Saelens, X.,
and Vandenabeele, P. (2002) Cell Death Differ. 9, 981994
23. Gross, A., Yin, X. M., Wang, K., Wei, M. C., Jockel, J., Milliman, C.,
Erdjument-Bromage, H., Tempst, P., and Korsmeyer, S. J. (1999) J. Biol.
Chem. 274, 1156 1163
24. Bossy-Wetzel, E., and Green, D. R. (1999) J. Biol. Chem. 274, 17484 17490
25. Rothe, M., Wong, S. C., Henzel, W. J., and Goeddel, D. V. (1994) Cell 78,
681 692
26. Rothwarf, D. M., Zandi, E., Natoli, G., and Karin, M. (1998) Nature 395,
297300
27. Yamaoka, S., Courtois, G., Bessia, C., Whiteside, S. T., Weil, R., Agou, F., Kirk,
H. E., Kay, R. J., and Israel, A. (1998) Cell 93, 12311240
28. Mercurio, F., Murray, B. W., Shevchenko, A., Bennett, B. L., Young, D. B., Li,
J. W., Pascual, G., Motiwala, A., Zhu, H., Mann, M., and Manning, A. M.
(1999) Mol. Cell. Biol. 19, 1526 1538
29. Li, Y., Kang, J., Friedman, J., Tarassishin, L., Ye, J., Kovalenko, A., Wallach,
D., and Horwitz, M. S. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 10421047
30. Kelliher, M. A., Grimm, S., Ishida, Y., Kuo, F., Stanger, B. Z., and Leder, P.
(1998) Immunity 8, 297303
31. Devin, A., Cook, A., Lin, Y., Rodriguez, Y., Kelliher, M., and Liu, Z. (2000)
Immunity 12, 419 429
32. Liu, Z. G., Hsu, H., Goeddel, D. V., and Karin, M. (1996) Cell 87, 565576
33. Yuasa, T., Ohno, S., Kehrl, J. H., and Kyriakis, J. M. (1998) J. Biol. Chem. 273,
2268122692
34. Roulston, A., Reinhard, C., Amiri, P., and Williams, L. T. (1998) J. Biol. Chem.
273, 1023210239
35. Juo, P., Kuo, C. J., Yuan, J., and Blenis, J. (1998) Curr. Biol. 8, 10011008
36. Juo, P., Woo, M. S., Kuo, C. J., Signorelli, P., Biemann, H. P., Hannun, Y. A.,
and Blenis, J. (1999) Cell Growth Differ. 10, 797 804
37. Matsumura, H., Shimizu, Y., Ohsawa, Y., Kawahara, A., Uchiyama, Y., and
Nagata, S. (2000) J. Cell Biol. 151, 12471256
38. Kawahara, A., Ohsawa, Y., Matsumura, H., Uchiyama, Y., and Nagata, S.
(1998) J. Cell Biol. 143, 13531360
39. Lin, Y., Devin, A., Rodriguez, Y., and Liu, Z. G. (1999) Genes Dev. 13,
2514 2526

Downloaded from http://www.jbc.org/ by guest on February 8, 2015

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.
RIP, IKK-, 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.
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-FADDcaspase-8 pathway and thus apoptosis (Fig. 5, right). This
fact implies that in the absence of GA one or more HSP90interacting 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 (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.
One of the currently emerging questions is why two distinct
cell death programs did evolve that seem to negatively influence each others 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

HSP90 Inhibition Reverts TNF-induced Necrosis to Apoptosis

40. Martinon, F., Holler, N., Richard, C., and Tschopp, J. (2000) FEBS Lett. 468,
134 136
41. Kim, J. W., Choi, E. J., and Joe, C. O. (2000) Oncogene 19, 4491 4499
42. Chen, G., Cao, P., and Goeddel, D. V. (2002) Mol. Cell 9, 401 410
43. Duan, H., and Dixit, V. M. (1997) Nature 385, 86 89
44. Chang, L., and Karin, M. (2001) Nature 410, 37 40
45. English, J., Pearson, G., Wilsbacher, J., Swantek, J., Karandikar, M., Xu, S.,
and Cobb, M. H. (1999) Exp. Cell Res. 253, 255270
46. Beg, A. A., Sha, W. C., Bronson, R. T., Ghosh, S., and Baltimore, D. (1995)
Nature 376, 167170
47. Wang, C. Y., Guttridge, D. C., Mayo, M. W., and Baldwin, A. S., Jr. (1999) Mol.
Cell. Biol. 19, 59235929

5629

48. Micheau, O., Lens, S., Gaide, O., Alevizopoulos, K., and Tschopp, J. (2001) Mol.
Cell. Biol. 21, 5299 5305
49. Lin, H., Chen, C., Li, X., and Chen, B. D. (2002) Exp. Cell Res. 272, 192198
50. Ekert, P. G., Silke, J., and Vaux, D. L. (1999) Cell Death Differ. 6,
10811086
51. Green, D. R., and Evan, G. I. (2002) Cancer Cell 1, 19 30
52. Franc, N. C. (2002) Front. Biosci. 7, d1298 1313
53. Sauter, B., Albert, M. L., Francisco, L., Larsson, M., Somersan, S., and
Bhardwaj, N. (2000) J. Exp. Med. 191, 423 434
54. Todryk, S., Melcher, A. A., Hardwick, N., Linardakis, E., Bateman, A.,
Colombo, M. P., Stoppacciaro, A., and Vile, R. G. (1999) J. Immunol. 163,
1398 1408

Downloaded from http://www.jbc.org/ by guest on February 8, 2015

MECHANISMS OF SIGNAL
TRANSDUCTION:
Disruption of HSP90 Function Reverts
Tumor Necrosis Factor-induced Necrosis to
Apoptosis
Tom Vanden Berghe, Michael Kalai, Geert
van Loo, Wim Declercq and Peter
Vandenabeele
J. Biol. Chem. 2003, 278:5622-5629.
doi: 10.1074/jbc.M208925200 originally published online November 18, 2002

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