From the Antibiotics Laboratory, Discovery Research
Institute, RIKEN, Wako-shi, Saitama 351-0198, Japan and the
§ Research Center for Experimental Biology, Tokyo Institute
of Technology, 4259 Nagatsuta-cho, Midori-ku,
Yokohama 226-8501, Japan
Received for publication, September 19, 2002, and in revised form, January 24, 2003
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ABSTRACT |
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Death receptors belong to the
tumor necrosis factor receptor family. They can induce apoptosis
following engagement with specific ligands and are known to play an
important role in the regulation of the immune system. Here we report
that epoxycyclohexenone (ECH) inhibits apoptosis induced by anti-Fas
antibody, Fas ligand (FasL), or tumor necrosis factor- Apoptosis can be induced by death receptors, a subgroup of the
tumor necrosis factor (TNF)1
receptor superfamily (1, 2). Fas (CD95, Apo-1) is a well characterized
member of the death receptor family that contains an extracellular
cysteine-rich motif and a cytoplasmic death domain that is essential
for transmission of the death signal (3, 4). Stimulation of Fas with
its cognate Fas ligand (FasL) leads to a clustering of Fas molecules
that induces the recruitment of the adaptor protein FADD via a
homophilic death domain interaction (5). The death effector
domain of FADD, in turn, interacts with the death effector domain of
pro-caspase-8 (6), resulting in the formation of the death-inducing
signaling complex (DISC) (7). Following DISC formation, pro-caspase-8
is proteolytically processed, resulting in the formation of an active
dimer (8). Subsequently, activated caspase-8 cleaves downstream
substrates such as effector caspases and initiates the apoptotic
cascade that ultimately leads to cell death.
In Fas-mediated apoptosis, two types of cells are proposed to transmit
distinct death signals (9, 10). In type I cells, such as SKW6.4, the
level of activation of pro-caspase-8 initiated at the DISC is
sufficient to cleave pro-caspase-3 directly. However, in type II cells,
such as Jurkat, less pro-caspase-8 is activated, and the mitochondrial
pathway is required to amplify the weak death signal. The small amount
of activated caspase-8 is able to cleave Bid efficiently, the truncated
form of which translocates to mitochondria and induces the release of
cytochrome c. Released cytochrome c then forms a
complex called the "apoptosome" that contains Apaf-1 and
pro-caspase-9, and it is this complex that generates active caspase-9
that subsequently cleaves and activates pro-caspase-3.
The Fas signaling pathway is a complex process that is regulated by
cellular and viral proteins such as FLICE inhibitory proteins (FLIPs) (11, 12), cytokine response modifier A (CrmA) (13), and
inhibitor of apoptosis proteins (IAPs) (14). Functional and structural
analyses of these regulators have contributed to our understanding of
the molecular basis of Fas-mediated apoptosis. However, several
non-peptide small molecules that modulate apoptosis also have been
reported (15-21). These molecules are useful for dissecting the
apoptosis signal transduction pathway and also may be potential
candidates for therapeutic use. To find specific inhibitors of
Fas-mediated apoptosis, we have screened a library of microbial
secondary metabolites and identified epoxycyclohexenone (ECH).
Previously, it was reported that ECH inhibits Fas-mediated apoptosis,
although the inhibitory mechanism of ECH remains obscure (22). In this
study, we show that ECH selectively inhibits Fas-mediated apoptosis by
preventing the activation of pro-caspase-8 in the DISC.
Cells--
Human T lymphoma Jurkat cells and Burkitt's
lymphoma SKW6.4 cells were cultured in RPMI 1640 medium (Sigma). Human
embryonic kidney 293T cells were cultured in Dulbecco's modified
Eagle's medium (Sigma) in 5% CO2 at 37 °C. Each medium
was supplemented with 10% (v/v) heat-inactivated fetal calf serum (JRH
Bioscience, Lenexa, KS), 50 units/ml penicillin, and 50 µg/ml
streptomycin (Sigma).
Reagents--
TNF- Antibodies--
Agonistic anti-human Fas IgM antibody CH-11 and
anti-caspase-8 antibody were obtained from Medical & Biological
Laboratories Co. Ltd. (Nagoya, Japan). Anti-FLAG M2 monoclonal antibody
and anti- Purification of ECH and Synthesis of Biotinylated ECH--
ECH
was isolated from the culture broth of a producing fungal strain using
bioassay-guided purification procedures. Biotinylated ECH was
synthesized by a coupling reaction using an activated biotin reagent
(Pierce). The structures of ECH and biotinylated ECH were determined by
their physico-chemical properties, detailed 1H- and 13C-NMR analyses
including two-dimensional techniques, and mass spectroscopies. The
details of these procedures and characterization will be reported elsewhere.
Preparation of FLAG-tagged FasL--
FLAG-tagged FasL was
prepared as reported previously (24) with the following modifications.
The extracellular domain of human FasL (amino acids 128-281) was
amplified by PCR from a Jurkat cDNA library using a 5'-forward
primer containing a ClaI site (5'-ATCGATGGAGAAGCAAATAGGCCACCCC-3') and a 3'-reverse primer containing an XbaI site (5'-TCTAGATTAGAGCTTATATAAGCCG-3') and cloned
into a modified version of pcDNA3 vector (Invitrogen) in-frame with the preprotrypsin secretion signal peptide and FLAG epitope. The pcDNA3-FLAG-FasL vector was transiently transfected into HEK293T cells using the calcium phosphate method and incubated for 72 h.
Culture supernatants were centrifuged to remove cell debris. The amount
of FLAG-tagged FasL in the culture supernatants was determined by the
cytotoxic activity as compared with purified FLAG-tagged FasL as a
standard. FLAG-tagged FasL was cross-linked with 500 ng/ml anti-FLAG M2
antibody. The cross-linked FasL was used in experiments to induce apoptosis.
Measurement of Cell Viability--
Jurkat cells or SKW6.4 cells
(5 × 104 cells, 100 µl) were cultured with
apoptosis inducers for 8 h in 96-well microtiter plates prior to treatment with 500 µg/ml
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT;
Sigma) for 2 h. MTT-formazan was solubilized in 5% SDS overnight,
and the absorbance at 595 nm was measured using a plate reader (Wallac
1420 ARVOsx; Amersham Biosciences). Cell viability (percent) was
calculated as (experimental absorbance Western Blot Analysis--
Cells were washed with
phosphate-buffered saline (PBS) and lysed in lysis buffer (100 mM Tris-HCl (pH 7.5), 1% Triton X-100, 1 mM
dithiothreitol, 1 mM EDTA, the protease inhibitor mixture (Complete; Roche Diagnostics)). The cytosolic fractions (50 µg/lane) were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were blocked with 5% non-fat dry milk in PBS containing 0.05% Tween 20 and
probed with specific antibodies. The proteins were visualized using the
ECL detection reagents (Amersham Biosciences).
Flow Cytometry--
To measure the expression level of Fas on
the cell surface, Jurkat cells were treated with anti-Fas antibody
(B-10) for 45 min on ice, washed with PBS, and then treated with
FITC-conjugated secondary antibody (Molecular Probes, Eugene, OR) for
45 min on ice. The cells were washed with PBS, and the surface
expression of Fas was detected by flow cytometry (Profile II; Coulter
Co., Hialeah, FL). The Fas-FasL interaction was measured as follows: Jurkat cells were treated with FLAG-tagged FasL and anti-FLAG M2
antibody for 60 min, washed with PBS, and chilled on ice. The cells
were treated subsequently with FITC-conjugated secondary antibody for
45 min on ice. The cells were washed with PBS, and surface binding of
FasL was assessed by flow cytometry.
DISC Analysis--
SKW6.4 cells were treated with 2 µg/ml
FLAG-tagged FasL in the presence of anti-FLAG M2 antibody (2 µg/ml)
for the indicated times. In the negative control, anti-FLAG M2 antibody
was added after lysis. Following incubation, the cells were rapidly
cooled down by the addition of 5 volumes of ice-cold PBS and then lysed in lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM sodium vanadate, 1 mM
NaF, 10% glycerol, 0.5% Nonidet P-40, the protease inhibitor
mixture). Cytosolic fractions were precleared with Sepharose 6B (Sigma)
for 60 min and then incubated with protein A-Sepharose CL-4B (Amersham
Biosciences) for 3 h. Sepharose beads were washed four times with
the lysis buffer. Proteins were separated using 10% SDS-PAGE and
blotted onto nitrocellulose membranes (Amersham Biosciences) followed
by Western blotting using anti-caspase-8 and anti-FADD antibodies.
Detection of Fas Clustering--
Fas clustering was
visualized as described previously (25). In brief, SKW6.4 cells were
treated with 1 µg/ml FLAG-tagged FasL and 1 µg/ml anti-FLAG M2
antibody for 30 min on ice and washed with PBS to remove unbound FasL.
The cells were stained with FITC-conjugated secondary antibody for 30 min on ice and washed with PBS. The cells were then warmed up and kept
at 37 °C for 30 min to trigger Fas stimulation. After stimulation,
the cells were adhered to glass slides precoated with
poly-L-lysine (Sigma) and fixed in acetone-methanol (1:1).
The glass slides were washed with PBS, mounted, and observed under
fluorescence microscopy (Olympus, Tokyo, Japan)
Construction of Fas-casp8 Expression Vector--
The full-length
human pro-caspase-8 was amplified by PCR from a Jurkat cDNA library
using a 5'-forward primer containing a NheI site
(5'-GCTAGCATGGACTTCAGCAGAAATC-3') and a 3'-reverse primer containing an
XbaI site (5'-TCTAGATTAATCAGAAGGGAAGAC-3') and cloned into
pCI-neo vector (Promega, Madison, WI), designated pCI-caspase-8. The
extracellular and transmembrane domain of human Fas (amino acids
1-184) was amplified by PCR from a Jurkat cDNA library using a
5'-forward primer containing a NheI site
(5'-GCTAGCATGCTGGGCATCTGGACCCTC-3') and a 3'-reverse primer containing
an EcoRI site (5'-GAATTCTCTGCATGTTTTCTGTACTTCC-3') and
cloned into the pCI-caspase-8 vector digested with NheI and EcoRI.
Measurement of Caspase Activity in Vitro--
Recombinant human
active caspase-3, -8, or -9 were mixed with
Ac-DEVD-methyl-coumaryl-7-amide (MCA), Ac-IETD-MCA, or Ac-LEHD-MCA, respectively (Peptide Institute Inc.) in reaction buffer (20 mM PIPES (pH 7.5), 100 mM NaCl, 0.1% CHAPS,
10% sucrose, 1 mM EDTA) for 60 min. The release of
7-amino-4-methyl-coumarin (AMC) was measured by a plate reader using an
excitation filter (360 nm) and an emission filter (460 nm).
Preparation of Recombinant Pro-caspase-8--
A DNA fragment of
full-length human caspase-8 was amplified by PCR from a Jurkat cDNA
library using a 5'-forward primer containing a BamHI site
(5'-GGATCCGATGGACTTCAGCAGAAATC-3') and a 3'-reverse primer
containing a HindIII site
(5'-AAGCTTTCAATCAGAAGGGAAGAC-3') and cloned into the pRSET-C vector
(Invitrogen) containing an N-terminal His6 tag,
designated pRSET-caspase-8. Cell lysates were prepared from an
Escherichia coli BL21(DE3) strain harboring pRSET-caspase-8
following induction with
isopropyl-thio- Caspase-8 Binding Assay--
For the in vitro binding
assay, recombinant pro-caspase-8 (800 ng) and active caspase-8 (800 ng)
were pretreated in the presence or the absence of 500 µM
ECH for 1 h as the competitor in the reaction buffer and then
treated with various concentrations of biotinylated ECH for 2 h on
ice. Protein samples were separated by SDS-PAGE and detected by Western
blotting using anti-caspase-8 antibody and avidin-conjugated
horseradish peroxidase (HRP) (Pierce).
For the in situ binding assay, HEK293T cells transiently
transfected with FLAG-tagged pro-caspase-8 or SKW6.4 cells were lysed in lysis buffer (0.5% Nonidet P-40, 20 mM Tris-HCl (pH
7.4), 150 mM NaCl, 2 mM sodium vanadate, 1 mM NaF, 10% glycerol, the protease inhibitor mixture). The
cell lysates (500 µg) were treated with 20 µM
biotinylated ECH for 1 h on ice and then immunoprecipitated with
anti-FLAG M2 antibody or anti-caspase-8 antibody together with protein
A-agarose for 4 h. The agarose beads were then washed four times
with the lysis buffer. Proteins were separated by SDS-PAGE and detected
by Western blotting using anti-caspase-8 antibody or anti-FLAG M2
antibody. The amount of biotinylated ECH bound to pro-caspase-8 was
detected by avidin-conjugated HRP.
ECH Inhibits Fas-mediated Apoptosis in Both Type I and Type II
Cells--
Human Burkitt's lymphoma SKW6.4 cells are classified as
type I, in which Fas ligation is able to trigger sufficient activation of pro-caspase-8 to induce the direct activation of pro-caspase-3. In
contrast, human T lymphoma Jurkat cells are classified as type II
since, due to weak activation of pro-caspase-8, activation of
pro-caspase-3 requires amplification via the mitochondrial pathway. To
obtain specific inhibitors of Fas-mediated apoptosis, we screened a
library of microbial metabolites and identified ECH as a compound that
will inhibit Fas-mediated apoptosis in Jurkat cells. Agonistic anti-Fas
antibody CH-11 and cross-linked FasL are both able to induce apoptosis
in Jurkat cells (Fig. 1A). We
found that ECH inhibited the apoptosis induced by CH-11 or FasL at
equivalent concentrations and did not decrease cell viability under
these conditions (Fig. 1B). Similar results were obtained with SKW6.4 cells that underwent apoptosis upon treatment with CH-11 or
FasL (Fig. 1, C and D). Both Jurkat and SKW6.4
cells treated with CH-11 or FasL showed cell body shrinkage and
chromatin condensation, and this was also reduced by ECH (Fig.
1E). Clonogenic assays revealed that ECH increases the
survival rates of FasL-treated SKW6.4 cells, although ECH alone had
little effect on cell proliferation in long term cultures (Fig.
1F).
ECH Selectively Inhibits Death Receptor-mediated
Apoptosis--
Jurkat cells are highly sensitive to a variety of
apoptotic stimuli. In addition to Fas-mediated apoptosis, we
examined the inhibitory effects of ECH on various apoptotic pathways
induced by TNF-
Upon ligand binding, death receptors such as TNF receptor 1 initiate
two distinct signals: the caspase-8-dependent death signal and the survival signal that activates transcription factor NF- ECH Inhibits Death Receptor-mediated Apoptosis Upstream of
Pro-caspase-8 Activation--
To investigate the molecular target of
ECH during the inhibition of Fas-mediated apoptosis, we first examined
whether ECH treatment decreases the expression level of cell surface
Fas. Jurkat cells were pretreated with ECH and then treated with
anti-Fas antibody (B-10). Following assessment by flow cytometry, we
concluded that ECH did not produce observable changes in the level of
cell surface Fas (Fig. 3A). We
then examined whether ECH disrupts the Fas-FasL interaction. For this,
Jurkat cells were pretreated with ECH and incubated with cross-linked
FasL followed by staining with FITC-conjugated secondary antibody.
Equal amounts of cell surface FasL binding were detected in both the
control and ECH-treated cells, suggesting that ECH did not affect the
interaction between Fas and FasL (Fig. 3B).
Therefore, to determine which step of the Fas-mediated apoptosis is
blocked by ECH, caspase-dependent cleavage of proapoptotic molecules was assessed by Western blot analysis. CH-11, FasL, and
TNF- ECH Blocks the Activation of Pro-caspase-8 in the DISC--
The
engagement of FasL with Fas initiates the recruitment of FADD and
pro-caspase-8 to Fas, enabling formation of the DISC. Immediately
following DISC formation, pro-caspase-8 is placed in a configuration
that facilitates self-processing, resulting in the generation of its
active form. In SKW6.4 cells, both FADD and pro-caspase-8 were rapidly
recruited to Fas upon treatment with FasL, and pro-caspase-8 was
processed to yield the intermediate cleaved form, p43 (Fig.
4A). Although the recruitment
of FADD and pro-caspase-8 to Fas was observed to be unaffected in
ECH-treated cells, the self-processing of pro-caspase-8 was markedly
blocked in a dose-dependent manner (Fig. 4A).
DISC formation reached a maximum at 15 min and was reduced after 60 min
(Fig. 4B). However, in ECH-treated cells, the DISC remained
stable for 60 min without any processing of pro-caspase-8 (Fig.
4B). These results suggest that ECH blocks self-processing
of pro-caspase-8 after its recruitment to FADD.
Next we investigated whether or not ECH inhibited Fas clustering. Under
non-stimulatory conditions, Fas was distributed equally on the cell
surface (Fig. 4C, panel b). FasL
engagement triggered the formation of Fas clustering at the cell
surface (Fig. 4C, panel d). However, ECH markedly
reduced this clustering (Fig. 4C, panel f), as
did the pan-caspase inhibitor, zVAD-fmk (Fig. 4C,
panel h). Therefore, these observations support the idea
that ECH blocks the self-processing of pro-caspase-8 during
Fas-mediated apoptosis.
ECH Inhibits Self-activation of Pro-caspase-8--
The above
observations suggest that the primary target of ECH in Fas-mediated
apoptosis may be the self-processing of pro-caspase-8 following its
binding to FADD. To support this hypothesis, we constructed a
Fas-caspase-8 fusion protein (Fas-casp8) consisting of the caspase-8
catalytic domain (amino acids 180-479) and the Fas extracellular and
transmembrane domains (amino acids 1-184) (Fig.
5A). The assay system can
detect directly that cross-linked FasL induces FADD-independent
activation of pro-caspase-8. Although transfection of Fas-casp8 in
HEK293T cells slightly induced caspase-8 activity, exogenous addition
of FasL strongly enhanced the activity. ECH significantly suppressed
the proteolytic activity of caspase-8 in Fas-casp8-transfected cells
(Fig. 5B). Under the same conditions, self-processing of
Fas-casp8 was monitored by Western blot analysis. ECH markedly
suppressed the appearance of the cleaved form of Fas-casp8 (Fig.
5C). These results support the notion that ECH targets
self-activation of pro-caspase-8 in the DISC.
ECH Inhibits the Activation of Pro-caspase-8 but Does Not Inhibit
Active Caspase-8 in Intact Cells--
We examined the direct effect of
ECH on recombinant active caspase-8. ECH inhibited the enzymatic
activity of active caspase-8 in a dose-dependent manner
(Fig. 6A). Unexpectedly, ECH
also inhibited recombinant active caspase-3 and recombinant active
caspase-9 (Fig. 6, B and C). The IC50
value of active caspase-8 (24 µM) was slightly lower than
that of active caspase-3 (50 µM) and active caspase-9 (62 µM); however, the IC50 difference might be
insufficient to explain the selectivity of ECH in the cell-based assay.
Thus, we examined the effect of ECH on pro-caspase-8 and active
caspase-8 in intact cells. Jurkat cells were preincubated with FasL for 60 min (during which time caspase-8 activity steadily increases) and
then treated with ECH or zVAD-fmk. ECH inhibited further activation of
pro-caspase-8 but did not affect the already activated caspase-8 (Fig.
6D). In contrast, zVAD-fmk reduced caspase-8 activity to background levels, thus demonstrating that zVAD-fmk is able to inhibit
both pro-caspase-8 and active caspase-8. Therefore, these results
suggest that ECH inhibits the activation of pro-caspase-8 but does not
affect active caspase-8 in intact cells.
ECH Preferentially Binds to Pro-caspase-8 in Intact Cells--
To
compare the binding capacity of ECH with pro-caspase-8 and active
caspase-8, we synthesized a biotin-labeled form of ECH (Fig.
7A). The biotinylated ECH
bound to both the recombinant pro-caspase-8 and the recombinant
caspase-8 large subunit (p18) that contains the active site cysteine.
However, pro-caspase-8 had a relatively higher affinity to ECH than did
caspase-8 p18 (Fig. 7B). A competition assay using
non-labeled ECH showed that the binding of ECH to pro-caspase-8 and
caspase-8 p18 is specific. We then examined whether ECH is able to bind
to pro-caspase-8 in crude cell lysates. For this, HEK293T cells were
transiently transfected with FLAG-tagged pro-caspase-8. The cell
lysates were treated with biotinylated ECH and subjected to
immunoprecipitation with anti-FLAG antibody to pull down FLAG-tagged
pro-caspase-8. FLAG-tagged pro-caspase-8 was clearly detected by
avidin-HRP (Fig. 7C). In addition, endogenous pro-caspase-8
was also detectable by avidin-HRP after immunoprecipitation with
anti-caspase-8 antibody from cell lysates of SKW6.4 cells (Fig.
7D). To examine the selectivity of ECH on each pro-caspase,
we performed a depletion analysis by using biotinylated ECH and avidin
beads in the cell lysates. The depletion efficiency of pro-caspase-8
was more than 10-fold higher as compared with that of pro-caspase-3 and
pro-caspase-9 (Fig. 7E), suggesting that ECH preferentially
affects pro-caspase-8 in intact cells. Pro-caspase-8 is a member of the
cysteine protease family, which possesses a cysteine residue at its
active center. Therefore, it is possible that ECH attacks the cysteine
residue of pro-caspase-8. In agreement with this hypothesis, addition of glutathione or cysteine, but not serine, attenuated the binding activity of ECH on pro-caspase-8 (Fig. 7F).
Bioprobes are low molecular weight chemical inhibitors that can be
used for the functional analysis of complex cellular processes (28). To
date, various bioprobes that are able to modulate apoptosis have been
reported (21, 29). Caspase substrate-mimicking peptide inhibitors have
been most frequently used to block apoptosis. Radical scavengers such
as N-acetylcysteine and metalloporphyrin can protect against
apoptosis induced by reactive oxygen species (15-17). Isatin
sulfonamide derivatives directly inhibit effector caspases such as
active caspase-3 and active caspase-7 and are able to block apoptosis
induced by cycloheximide or camptothecin (18). It has been reported
that protein kinase C activators, such as phorbol esters, suppress
Fas-mediated apoptosis via blocking the oligomerization of Fas and
recruitment of FADD and pro-caspase-8 into the DISC (19, 20). In this
report, we show that ECH inhibits Fas-mediated apoptosis by blocking
the activation of pro-caspase-8. Since ECH is structurally different
from the apoptosis modulators thus far reported, ECH is a novel type of
non-peptide apoptosis inhibitor.
ECH inhibited Fas-mediated apoptosis in all of the cell lines tested,
such as Jurkat, SKW6.4, Raji, U937, and HepG2 cells (Fig. 1 and data
not shown). The inhibitory effect of ECH was highly selective for death
receptor-mediated apoptosis (Fig. 2). Consistent with these
observations, ECH blocked the activation of pro-caspase-8, which is the
specific initiator caspase for death receptor-mediated apoptosis (6).
Activation of pro-caspase-8 is known to be the first step in the
cascade of apoptosis events induced by Fas stimulation. Upon FasL
binding, pro-caspase-8 is recruited to the DISC multiprotein complex.
The recruited pro-caspase-8 associates with the cytoplasmic portion of
the Fas receptor via the adaptor protein FADD. In the DISC,
pro-caspase-8 is proteolytically auto-processed to the active form (7,
8). In ECH-treated cells, although recruitment of FADD and
pro-caspase-8 was unaffected, the self-processing of pro-caspase-8 was
blocked (Fig. 4, A and B), suggesting that the
molecular target of ECH is part of the DISC component. As shown in Fig.
3, A and B, ECH affected neither Fas expression
nor Fas-FasL interaction. Therefore, FADD and pro-caspase-8 were both
possible candidates as target molecules for the action of ECH. To
clarify this issue, we constructed a Fas-casp8 fusion protein, which
allows caspase-8 to be activated without the involvement of FADD, by
the addition of cross-linked FasL as described previously (30). ECH
blocked the FADD-independent caspase-8 activation (Fig. 5), indicating
that the inhibitory target of ECH is pro-caspase-8 rather than FADD.
This is consistent with the observation that ECH suppressed Fas
clustering, which was reported previously to be a
caspase-8-dependent event (25).
ECH inhibited the enzymatic activity of recombinant active caspase-8 at
a slightly lower concentration than its inhibition of recombinant
active caspase-3 and caspase-9 (Fig. 6, A-C). However, ECH
was not able to block apoptosis mediated by active caspase-3 and active
caspase-9 in cells treated with chemical drugs and UV irradiation
(Figs. 2A and 3C). Similar results have been
reported in the case of zVAD-fmk (31, 32). To resolve this discrepancy, we examined whether ECH inhibits active caspase-8 in FasL-treated cells. In contrast to the caspase inhibitor zVAD-fmk, ECH inhibited activation of pro-caspase-8 but did not inhibit the activity of already
active caspase-8 (Fig. 6D). Binding analysis revealed that
ECH preferentially bound recombinant pro-caspase-8 rather than the
large subunit of recombinant active caspase-8 (Fig. 7B). Moreover, ECH bound pro-caspase-8 in the cell lysates (Fig. 7, C and D), and pro-caspase-8 was more efficiently
depleted from these lysates as compared with pro-caspase-3 and
pro-caspase-9 (Fig. 7E). These results indicate that ECH
possesses a higher affinity to pro-caspase-8 than to active caspase-8,
pro-caspase-3, or pro-caspase-9 and that it inactivates the
intrinsic proteolytic activity of pro-caspase-8. It may also be
possible that the cellular amount of pro-caspase-8 is less than that of
pro-caspase-3 and pro-caspase-9 and that the total pool of
pro-caspase-8 in the cell is predominantly inactivated by ECH. ECH has
a molecular structure of Death receptor-mediated apoptosis has important functions for the
homeostasis of the immune system and immune surveillance. Such
functions include the elimination of harmful cells by cytotoxic T-lymphocytes and the maturational selection of T- and B-lymphocytes. Consequently, derailment of death receptor-mediated apoptosis leads to
severe diseases. For therapeutic use, the peptide-based caspase
inhibitors including zVAD-fmk are first generation drugs, and a number
of studies describe their potency in reducing cell death in acute
situations of fulminant hepatitis, ischemia, and bacterial meningitis
(33-35). These caspase inhibitors may also be useful in reducing cell
death in organs awaiting transplant (36) and in reducing apoptosis of
normal cells caused by chemotherapeutic drugs for cancer treatment. As
a potential pharmacological drug, ECH has important characteristics. It
is highly selective for death receptor-mediated apoptosis, it is a
membrane-permeable non-peptide compound, and it is a small molecule
that promises easy modification. Recently, death receptor-independent
activation of pro-caspase-8 in neuronal diseases such as Huntington
diseases has been reported (37, 38). In such diseases, expansion of polyglutamine repeats induces unfavorable protein aggregation that
eventually recruits pro-caspase-8 and activates apoptosis independently
of death receptors, resulting in neuronal cell death. Consistent
with this model, the caspase-8 inhibitory proteins, CrmA and c-FLIP, as
well as the dominant-negative mutant of FADD, prevent neuronal cell
death (37, 38). Therefore, ECH, a novel non-peptide inhibitor
preventing the activation of pro-caspase-8, could be a good candidate
for therapeutic use to treat such neuronal diseases.
but not by
staurosporine, MG-132, C2-ceramide, or UV irradiation. These results
suggest that ECH specifically blocks death receptor-mediated apoptosis.
Neither the surface expression of Fas nor the Fas-FasL interaction was
influenced by ECH. However, ECH did block the activation of
pro-caspase-8 in the death-inducing signaling complex, although
recruitment of Fas-associating death domain (FADD) and pro-caspase-8
was not affected. ECH inhibited the enzymatic activity of recombinant active caspase-8 at slightly lower concentrations than it did for
active caspase-3 and active caspase-9 in vitro. However, in FasL-treated cells, ECH was only able to inhibit the activation of
pro-caspase-8, and it had no effect on the already activated caspase-8
at a concentration that is effective at inhibiting Fas-induced apoptosis. ECH directly bound the large subunit of active caspase-8 that contains the active center cysteine and had a relatively higher
affinity to pro-caspase-8. Moreover, compared with pro-caspase-3 and
pro-caspase-9, pro-caspase-8 was predominantly depleted by biotinylated
ECH with avidin beads in the cell lysates, suggesting that ECH
preferentially affects pro-caspase-8. Thus, our results suggest that
ECH blocks the self-activation of pro-caspase-8 in the death-inducing
signaling complex and thus selectively inhibits death
receptor-mediated apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was obtained from R&D Systems
(Minneapolis, MN). C2-ceramide was purchased from Sigma. Staurosporine
was purified as reported in our previous study (23). MG-132 and
zVAD-fmk were obtained from Peptide Institute Inc. (Osaka, Japan).
-tubulin antibody were purchased from Sigma.
Anti-Fas (B-10), anti-caspase-3, anti-Bid, and anti-poly(ADP-ribose)
polymerase (PARP) antibodies were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-cytochrome c antibody
was purchased from BD Biosciences. Anti-FADD antibody was from
Pharmingen. Anti-I
B antibody was obtained from Cell Signaling
Technology (Beverly, MA).
background
absorbance)/(control absorbance
background absorbance) × 100.
-D-galactopyranoside. The
His6 tag-tagged pro-caspase-8 was purified with
nickel-nitrilotriacetic acid agarose (Qiagen, Hilden, Germany) and
desalted with the PD-10 column (Amersham Biosciences) according to
the manufacturer's instructions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
ECH inhibits Fas-mediated
apoptosis. Jurkat (A) and SKW6.4 (C) cells
were treated with various concentrations of agonistic anti-Fas antibody
CH-11 (closed circles) or cross-linked FasL (closed
squares) for 8 h. Cell viability was measured by using MTT
reagent. Data points represent the mean ± S.D. of triplicate
determinations. Jurkat (B) and SKW6.4 (D) cells
were pretreated with various concentrations of ECH for 30 min and then
incubated in the absence (open circles) or in the presence
of 300 ng/ml CH-11 (closed circles) or 500 ng/ml
cross-linked FasL (closed squares) for 8 h. Cell
viability was measured by using MTT reagent. Data points
represent the mean ± S.D. of triplicate determinations. As shown
in E, Jurkat (panels a-c) and SKW6.4
(panels d-f) cells were treated with 20 µM
ECH (panels c and f) for 30 min and then
incubated in the absence (panels a and d) or
in the presence of 500 ng/ml cross-linked FasL (panels b,
c, e, and f) for 6 h. Cells were
fixed with methanol-acetone and stained with Hoechst33258 followed
by observation under fluorescent microscopy. Arrows indicate
apoptotic cells. As shown in F, SKW6.4 cells were pretreated
with 10 µM ECH for 30 min and then incubated with 500 ng/ml cross-linked FasL for 2 h. Cells were washed with PBS and
cultured in 96-well microtiter plates (1 cell/well) for 10 days. The
number of colonies formed was counted. Data columns
represent the mean ± S.D. of triplicate determinations.
, staurosporine (a protein kinase inhibitor), MG-132
(a proteasome inhibitor), C2-ceramide (a cell-permeable analogue of
ceramide), and UV irradiation. All apoptosis inducers tested caused
cell death in Jurkat cells within 8 h and reduced the cell viability. As observed with Fas-mediated apoptosis (Fig.
1B), ECH markedly inhibited apoptosis induced by TNF-
in a dose-dependent manner (Fig.
2A). In contrast, ECH did not
significantly block apoptosis induced by staurosporine, MG-132,
C2-ceramide, or UV irradiation (Fig. 2A). A pan-caspase
inhibitor, zVAD-fmk, was able to prevent apoptosis irrespective of the
inducer used (data not shown). These results suggest that in contrast
to zVAD-fmk, ECH blocks death receptor-mediated apoptosis
selectively.
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Fig. 2.
ECH selectively inhibits Fas- and TNF
receptor-mediated apoptosis. As shown in A, Jurkat
cells were pretreated with various concentrations of ECH for 30 min and then incubated without apoptosis inducers (open
circles) or with the following apoptosis inducers for 8 h:
100 ng/ml TNF- plus 10 nM cycloheximide (closed
circles), 300 nM staurosporine (open
triangles), 3 µM MG-132 (closed
triangles), 100 µM C2-ceramide (open
squares), 10 mJ/cm2 of UV irradiation (closed
squares). Cell viability was measured by using MTT reagent.
Data points represent the mean ± S.D. of triplicate
determinations. As shown in B, Jurkat cells were pretreated
with 20 µM ECH or 10 µM MG-132 for 30 min.
The cells were then incubated with 10 ng/ml TNF-
for 15 min.
Cytosolic fractions were separated by SDS-PAGE and analyzed by Western
blotting. I
B (upper panel) and
-tubulin (lower
panel) were detected by specific antibodies.
B (26). The degradation of I
B is a prerequisite event for NF-
B activation (27). Therefore, to examine whether ECH has an effect on the
survival signal activated by TNF-
, we monitored the cellular levels
of I
B. We observed degradation of I
B within 15 min of TNF-
stimulation in Jurkat cells (Fig. 2B). A proteasome
inhibitor, MG-132, inhibited such TNF-
-induced I
B degradation;
however, in ECH cells, degradation was still observed (Fig.
2B). These data indicate that ECH blocks the TNF-
-induced
death signal but had no effect on the survival signal.
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Fig. 3.
ECH inhibits signaling pathway upstream of
pro-caspase-8 activation in Fas- and TNF receptor-mediated
apoptosis. As shown in A, Jurkat cells were
pretreated with (lower panel) or without (upper
panel) 20 µM ECH for 30 min. The cells were treated
with (black areas) or without (gray areas)
anti-Fas antibody (B-10) for 45 min on ice and washed with PBS to
remove unbound antibody. The cells were then treated with
FITC-conjugated secondary antibody for 45 min on ice. Expression of
cell surface Fas was assessed by flow cytometry. As shown in
B, Jurkat cells were pretreated with (lower
panel) or without (upper panel) 20 µM ECH
for 30 min and then incubated with (black areas) or without
(gray areas) FLAG-tagged FasL plus anti-FLAG M2 antibody for
60 min followed by washing with PBS to remove unbound FasL. The cells
were treated with FITC-conjugated secondary antibody for 45 min on ice.
Fas-FasL binding was measured by flow cytometry. As shown in
C, Jurkat cells were pretreated with 20 µM ECH
(E) or 20 µM zVAD-fmk (Z) for 30 min or left untreated (N). The cells were then incubated
with apoptosis inducers for 4 h: 100 ng/ml CH-11, 500 ng/ml
cross-linked FasL, 100 ng/ml TNF- , plus 10 nM
cycloheximide, 300 nM staurosporine (SSP), 3 µM MG-132, 100 µM C2-ceramide, 10 mJ/cm2 of UV irradiation. Cytosolic fractions were
separated by SDS-PAGE and analyzed by Western blotting.
induced the cleavage of pro-caspase-8 into intermediate cleaved forms, which leads to triggering of the downstream cascade such
as the cleavage of Bid, the release of cytochrome c into the
cytosol, the cleavage of pro-caspase-3, and the cleavage of poly
(ADP-ribose) polymerase, a substrate of active caspase-3 (Fig.
3C). ECH blocked the self-cleavage of pro-caspase-8, and thus, the downstream events were all suppressed. Treatment with staurosporine, MG-132, C2-ceramide, and UV irradiation also triggered the apoptotic cascade; however, ECH did not affect all events under
these conditions. These results were consistent with the observations made above (Fig. 2A), which demonstrated that
ECH was not able to inhibit death receptor-independent apoptosis. In
contrast, the pan-caspase inhibitor, zVAD-fmk, was able to block all of
the steps initiated by any of the apoptotic stimuli tested. These
results suggest that ECH targets an early signaling event(s) upstream
of pro-caspase-8 activation in the apoptosis pathways via the Fas and
TNF receptor.
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Fig. 4.
ECH inhibits self-processing of pro-caspase-8
in the DISC. As shown in A, SKW6.4 cells were
pretreated with the indicated concentrations of ECH for 30 min and
incubated with 2 µg/ml FLAG-tagged FasL in the presence or the
absence of 2 µg/ml anti-FLAG M2 antibody for 15 min. As shown in
B, SKW6.4 cells were pretreated with or without 40 µM ECH for 30 min and then incubated with 2 µg/ml
FLAG-tagged FasL in the presence or the absence of 2 µg/ml anti-FLAG
M2 antibody for the indicated time periods. Postnuclear lysates were
precleared with Sepharose 6B for 1 h, and the DISC was
immunoprecipitated (IP) using protein A-Sepharose CL-4B for
3 h. Proteins were separated by SDS-PAGE. The DISC and whole
lysates were analyzed by Western blotting using anti-caspase-8 and
anti-FADD antibodies. The nonspecific band is indicated with an
asterisk. As shown in C, Jurkat cells were
pretreated with 20 µM ECH (panels e and
f) or 20 µM zVAD-fmk (panels g and
h) for 30 min or left untreated (panels a-d).
The cells were then incubated with 1 µg/ml FLAG-tagged FasL and 1 µg/ml anti-FLAG M2 antibody (panels c-h) followed by
addition of FITC-conjugated secondary antibody for 45 min on ice. The
cells were then warmed up to 37 °C for 30 min and mounted on
poly-L-lysine-coated glass slides. Cell morphology and Fas
distribution were observed under phase contrast microscopy
(panels a, c, e, and g) and
fluorescent microscopy (panels b, d,
f, and h), respectively.
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Fig. 5.
ECH inhibits self-activation of
pro-caspase-8. A, schematic diagram of
Fas-caspase-8 fusion protein (Fas-casp8). TM, transmembrane
domain; DD, death domain; DED, death effector
domain. As shown in B, control vector and Fas-casp8 vector
were transiently transfected into HEK293T cells. After 12 h, the
cells were treated with (closed bars) or without (open
bars) 40 µM ECH for 2 h and then incubated with
or without 3 µg/ml cross-linked FasL for 10 h. Postnuclear
lysates were incubated with Ac-IETD-MCA for 60 min. The enzymatic
activity of caspase-8 was measured by hydrolysis of Ac-IETD-MCA.
Data columns represent the mean ± S.D. of triplicate
determinations. As shown in C, the same lysates were
subjected to Western blotting for caspase-8.
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Fig. 6.
ECH does not inhibit active caspase-8 in
intact cells. Recombinant active caspase-8 (A),
caspase-3 (B), and caspase-9 (C) were pretreated
with various concentrations of ECH for 30 min and then incubated with
Ac-DEVD-MCA, Ac-IETD-MCA, or Ac-LEHD-MCA, respectively, for 60 min.
Each caspase activity was measured by the detection of released AMC.
Data points represent the mean ± S.D. of triplicate
determinations. As shown in D, Jurkat cells were treated
with 300 ng/ml cross-linked FasL for 60 min, and then 20 µM ECH (closed circles) or 20 µM
zVAD-fmk (closed squares) was added. The cells were
harvested at the indicated time periods, and caspase-8 activity was
measured with Ac-IETD-MCA. Data points represent the
mean ± S.D. of triplicate determinations.
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Fig. 7.
Biotinylated ECH preferentially binds to
pro-caspase-8. A, structure of biotinylated ECH. As
shown in B, recombinant pro-caspase-8 and active caspase-8
were pretreated with or without 500 µM ECH for 1 h
as the competitor and then incubated with the indicated concentrations
of biotinylated ECH for 2 h on ice. Protein samples were separated
by SDS-PAGE and analyzed by Western blotting (WB) using
anti-caspase-8 antibody (upper panel) and
avidin-conjugated HRP (lower panel). MS,
molecular size. As shown in C, HEK293T cells were
transiently transfected with FLAG-tagged caspase-8. The cell lysates
(500 µg of protein) were treated with 20 µM
biotinylated ECH and immunoprecipitated (IP) with anti-FLAG
M2 antibody. The immunoprecipitated complexes were separated by
SDS-PAGE and analyzed by Western blotting using anti-FLAG M2 antibody
and avidin-conjugated HRP. As shown in D, the cell lysates
(500 µg of protein) of SKW6.4 cells were treated with 20 µM biotinylated ECH and immunoprecipitated with
anti-caspase-8 antibody. The immunoprecipitated complexes were
separated by SDS-PAGE and analyzed using anti-caspase-8 antibody and
avidin-conjugated HRP. As shown in E, the cell lysates (500 µg of protein) of SKW6.4 cells were treated with various
concentrations of biotinylated ECH for 1 h, and biotinylated ECH
was depleted with avidin-agarose for 4 h at 4 °C. Then, the
supernatants were separated by SDS-PAGE and analyzed by anti-caspase-3,
caspase-8, caspase-9, and -tubulin antibodies. As shown in
F, recombinant pro-caspase-8 was pretreated with various
concentrations of glutathione (left panel) or cysteine
(middle panel) or serine (right panel) for 1 h on ice and then incubated with 30 µM ECH for 2 h
on ice. Protein samples were separated by SDS-PAGE and analyzed by
Western blotting using anti-caspase-8 antibody (upper panel)
and avidin-conjugated HRP (lower panel).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
-unsaturated ketone and epoxide that
are highly reactive to addition of nucleophiles. Therefore, ECH
may act by binding to the thiol group of cysteine residues within the
active site of pro-caspase-8. Consistent with this hypothesis, the
addition of either glutathione or cysteine was able to attenuate the
binding of ECH on pro-caspase-8 (Fig. 7F). Thus, we conclude
that ECH is able to bind to cysteine residues possibly within the
active center of pro-caspase-8 and thereby inhibits the
self-activation of pro-caspase-8 in the DISC.
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ACKNOWLEDGEMENTS |
---|
We thank Y. Ichikawa and R. Nakazawa for DNA sequencing (Bioarchitect Research Group, RIKEN, Saitama, Japan).
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FOOTNOTES |
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* This work was supported in part by a grant for Multibioprobes and a Special Grant for Promotion of Research from RIKEN and by a grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan.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.
¶ Partly supported by a grant of the 21st Century COE Program, from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
To whom correspondence should be addressed. Tel.:
81-48-467-9541; Fax: 81-48-462-4669; E-mail:
antibiot@postman.riken.go.jp.
Published, JBC Papers in Press, January 24, 2003, DOI 10.1074/jbc.M209610200
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ABBREVIATIONS |
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The abbreviations used are: TNF, tumor necrosis factor; FADD, Fas-associating death domain; FasL, Fas ligand; AMC, 7-amino-4-methyl-coumarin; DISC, death-inducing signaling complex; ECH, epoxycyclohexenone; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; MCA, 4-methyl-coumaryl-7-amide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phosphate buffered saline; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethyl ketone; PIPES, 1,4-piperazinediethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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