From the Turku Centre for Biotechnology, POB 123, FIN-20521, the ¶ Turku Graduate School of Biomedical Science, the
§ Department of Biology, Åbo Akademi University, BioCity,
FIN-20520, and the Departments of
Biology,
Laboratory of Animal Physiology, FIN-20014 and ** Medical Biochemistry
and Dermatology, FIN-20520, University of Turku, Turku, Finland
Received for publication, November 15, 2000, and in revised form, December 27, 2000
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ABSTRACT |
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The tumor necrosis factor (TNF), Fas, and
TNF-related apoptosis-inducing ligand (TRAIL) receptors (R) are
highly specific physiological mediators of apoptotic signaling.
We observed earlier that a number of FasR-insensitive cell lines
could redirect the proapoptotic signal to an anti-apoptotic ERK1/2
signal resulting in inhibition of caspase activation. Here we determine
that similar mechanisms are operational in regulating the apoptotic
signaling of other death receptors. Activation of the FasR,
TNF-R1, and TRAIL-R, respectively, rapidly induced subsequent
ERK1/2 activation, an event independent from caspase activity. Whereas
inhibition of the death receptor-mediated ERK1/2 activation was
sufficient to sensitize the cells to apoptotic signaling from
FasR and TRAIL-R, cells were still protected from apoptotic
TNF-R1 signaling. The latter seemed to be due to the strong activation
of the anti-apoptotic factor NF- Programmed cell death or apoptosis is a self-destruction process
implanted in most cells, ready to be activated to eliminate unwanted
cells (1). Various activators and inhibitors, however, strictly
regulate this elimination mechanism in response to external and
internal signals. Proapoptotic signals may be transduced through a
subset of cytokine receptors of the tumor necrosis factor
(TNF)1 family, termed death
receptors (DRs). Members of the TNF receptor family are characterized
by similar extracellular domains containing cysteine-rich repeats (2).
The DRs also share a common intracellular domain, the death domain
(DD), which confers to them the ability to induce apoptosis. By way of
DD interaction, proteins of the death-inducing signaling complex (DISC)
will be recruited to the receptor, and the apoptotic machinery will be
activated. In parallel to this, other adaptor molecules may bind to the
complex and modulate the response, some of them inducing survival. The
number of known DRs has been growing since the first one was
discovered, and it seems that additional receptors are yet to be
discovered (for reviews, see Refs. 3-5). The Fas receptor (FasR) or
CD95/APO-1, TNF receptor 1 (TNF-R1) and TRAIL receptors 1 and 2 (TRAIL-R1 or DR4, TRAIL-R2 or DR5) are members of this family of
proteins (6-8). Although the four receptors share some common features in their structures, they also have specific characteristics. The FasR
binds the adaptor protein FADD (9), which in turn recruits and
activates procaspase-8 (7, 10). TNF-R1, however, does not bind
FADD directly, but TRADD has to be engaged before FADD (11) and
procaspase-8 (12) can be recruited to the receptor. Defining the
components of the TRAIL-R DISC is still a controversial matter. Both
caspase-8 and caspase-10 have been implicated as crucial mediators of
TRAIL-induced apoptotic signaling, and both caspases have been
identified as part of the TRAIL DISC (13-15). It has been proposed
that TRAIL would induce apoptosis through FADD-dependent
and -independent pathways (16, 17). However, recent studies suggest
that both TRAIL-R1 and TRAIL-R2 recruit FADD and caspase-8, although
TRAIL-R1 could still induce FADD-independent apoptosis in some
situations (14, 15, 18). In all cases, recruitment and activation of
caspase-8 leads to induction of effector caspases and ultimately to
apoptosis (19).
Besides the FADD/caspase-8 signaling cascade, a number of other
signaling pathways are activated by the DRs, most likely involving adaptor/regulator proteins specific to each receptor. Especially the
TNF-R has been implied to have several important signaling functions
apart from apoptotic signaling. TRAF2 and RIP, the latter which was
first identified as a component of the Fas DISC, have been shown to
bind to TRADD and are thus recruited to the TNF-R, both of them
contributing to JNK and NF- We have previously shown that ERK1/2 activation is able to suppress
Fas-induced apoptosis in activated T-cells (34, 35). Therefore, we
wanted to examine whether the same protective effect could be seen in
cancer cells. As members of the TNF receptor family are involved in
removal of tumor cells by the immune system, development of resistance
to such killing would impair the defense mechanism. In this context, we
recently demonstrated that ERK1/2 has a role in rendering cells
insensitive to FasR killing, as inhibition of ERK1/2 activation
sensitizes HeLa cells to FasR-mediated apoptosis (36). Since HeLa cells
also express TNF and TRAIL receptors (37), we wanted to investigate in
this cell model whether ERK1/2 could protect against apoptosis induced
through these other DRs in the same way as in Fas signaling, especially because the apoptotic signaling pathways induced by the three cytokines present common features in term of their DISC composition. In
this study we show that inhibition of ERK1/2 is able to enhance the
sensitivity of HeLa cells to TRAIL, as well as to Fas, whereas TNF
treated cells remain insensitive. The insensitivity in the case of the
TNF-R seems to be caused by the additional protection provided by
NF- Cell Culture and Reagents--
HeLa cells were cultured in
Dulbecco's Modified Eagle's Medium (Sigma) supplemented with 10%
inactivated fetal calf serum, 2 mM L-glutamine,
100 units/ml penicillin, and 100 µg/ml streptomycin, in a humidified
incubator with 5% CO2 in air at 37 °C. HeLa cells were
incubated with an agonistic anti-human FasR immunoglobulin M antibody
(100 ng/ml, MBL, Watertown, MA), TNF Adenovirus-mediated Gene Transfer--
Recombinant
replication-deficient adenovirus RAdlacZ (RAd35) (38), which contains
the Escherichia coli DNA Fragmentation Analysis--
For microscopy, cells grown on
coverslips were fixed with 3% paraformaldehyde in PBS for 30 min,
permeabilized with 0.1% Triton-100 in PBS for 5 min, and blocked with
3% bovine serum albumin in PBS overnight at 4 °C. Staining of
MKK1-HA positive cells and nuclei was performed by 90-min incubation
with anti-HA, three times PBS wash, and further incubation for 60 min
with fluorescein isothiocyanate-conjugated anti-mouse secondary
antibody (Zymed Laboratories Inc., San Francisco, CA)
and Hoechst 33342 (Molecular Probes, Eugene, OR). After three washes,
cells were mounted in Mowiol (Sigma) and visualized with a fluorescence
microscope (Leica, Wetzlar, Germany). Flow cytometry of propidium
iodide (PI; Molecular Probes) stained nuclei was as described (41, 42).
Briefly, cells were harvested in PBS, 5 mM EDTA, washed
with PBS, and the cell pellet (1 × 106 cells) was
gently resuspended in 1 ml of hypotonic fluorochrome solution (PI 50 µg/ml in 0.1% sodium citrate plus 0.1% Triton X-100). The cells
were incubated at 4 °C overnight before analyzing on a FACScan flow
cytometer (Becton Dickenson). The subdiploid peak was considered to be
apoptotic cells as previously described (42).
Immunoblotting for ERK1/2 and Caspase Detection--
For caspase
assay, whole cell extracts were made by freeze-thaw cycles in
resuspension buffer (150 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4) with 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml each of leupeptin, aprotinin,
and pepstatin. 50 µg of protein as determined by Bradford assay were
boiled with Laemmli sample buffer and resolved on 12.5%
SDS-polyacrylamide gel electrophoresis. For phospho-ERK1/2 and ERK2
Western blot, 106 cells were lysed in radioimmune
precipitation assay buffer (PBS, pH 7.4, 1% Nonidet P-40, 0.5% sodium
deoxycholate, 1 mM Na3VO4, 0.1%
SDS, 1 mM EDTA, 1 mM EGTA, 20 mM
NaF, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml
leupeptin, aprotinin, and pepstatin), and 3× Laemmli sample buffer was
added to the supernatant before separation on 10% SDS-polyacrylamide
gel electrophoresis. Proteins were then transferred to nitrocellulose
membrane, blocked in PBST (PBS, 0.1% Tween 20) with 5% nonfat dry
milk, incubated 16 h with C15 caspase-8 antibody (a kind gift from
Peter Krammer, German Cancer Research Center, Heidelberg, Germany; Ref.
43), phospho-p44/42-MAPK antibody (New England BioLabs, Boston, MA), or
ERK2 antibody, washed in PBST, and incubated 1 h with appropriate
horseradish peroxidase-coupled secondary antibody (Pierce). Detection
was performed by enhanced chemiluminescence reaction (ECL, Amersham Pharmacia Biotech).
Electrophoretic Mobility Shift Assay--
Cells were harvested
in cold PBS/EDTA, lysed by freeze-thaw in buffer C (25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 20 mM HEPES) containing
phenylmethylsulfonyl fluoride and dithiothreitol (0.5 mM
each), and supernatant was recovered by centrifugation at 4 °C.
Whole cell extract (15 µg) was incubated with a
32P-labeled oligonucleotide reproducing the consensus
NF- Inhibition of MAPK kinase 1 (MKK1) Sensitizes HeLa
Cells to Fas and TRAIL-mediated Apoptosis but Not TNF--
We compared
the kinetics of apoptosis induction by Fas, TNF, or TRAIL receptor,
after sensitization with either the protein synthesis inhibitor CHX or
the MKK1 inhibitor PD98059. In the absence of any specific treatment,
HeLa cells were resistant to FasR and TNF-R1 stimulation, but showed
some sensitivity to TRAIL-R-mediated apoptosis (Fig.
1), although to a lesser degree than many
other tested TRAIL-responsive cell lines (data not shown). As
previously shown, HeLa cells were markedly sensitized to Fas-induced
apoptosis by cotreatment with PD98059. Cotreatment with PD98059
rendered HeLa cells even more sensitive to TRAIL-R-mediated apoptosis, showing that ERK1/2 signaling has a role in the protection against both
TRAIL-R- and FasR-induced apoptosis. In contrast, cells remained resistant to TNF Adenovirus-mediated Expression of Constitutively Active MKK1
Rescues HeLa Cells from Fas, TRAIL, and TNF-induced
Apoptosis--
Whereas ERK1/2 inhibition was not sufficient to
sensitize the cells to stimulation by TNF The ERK1/2 Pathway Is Independent from the NF- All Death Receptors Can Activate ERK1/2--
Because the
resistance mechanism rendering a tumor cell line insensitive to
DR-induced apoptosis could be caused by a high basal level of ERK1/2 in
those cells, we studied the activation state of ERK1/2 in HeLa cells
after receptor triggering. We treated the cells with anti-Fas, TNF Activation of ERK1/2 Blocks the Apoptotic Cascade Above the Level
of Caspase-8--
Cleavage and activation of caspase-8 is an early
step in the apoptotic process triggered by the FasR, TNF-R1, and
TRAIL-R, occurring at the level of DISC recruitment (10, 12, 15). The
full-length procaspase is first cleaved once, releasing an intermediate
43-kDa fragment, which is further processed into the active 18-kDa
fragment (51). To find out at what level activation of the ERK1/2
pathway would stop the cell death cascade, we infected HeLa cells with
RAd-CA-MKK1-HA and observed whether procaspase-8 would still be cleaved
into the active form. In nontransfected cells, the amount of cleaved
caspase in the absence of sensitization is consistent with the amount
of apoptotic cells measured earlier in the same conditions (Figs.
5 and 2A), suggesting that in
resistant cells the apoptotic signal does not reach the activation of
caspase-8 but is stopped at an earlier stage. Sensitization of the
cells to DR-induced apoptosis by CHX induced a massive appearance of p18 and disappearance of the full-length procaspase-8. However, when
CA-MKK1 is expressed, the amount of active fragment diminishes, and
more procaspase-8 can be detected (Fig. 5).
The ERK1/2 pathway is of major importance in controlling cellular
differentiation and growth (52-54), and it has also been shown to act
as an important modulator of various apoptosis-inducing signals in
different systems (32, 55, 56). Our interest was to study whether this
signaling cascade is involved in the signaling from other DRs, in
addition to its established role as a regulator of FasR signaling.
Although we and others described the ERK1/2 pathway as a mechanism
preventing cell death induced by the FasR (34-36, 57, 58), the
involvement of ERK1/2 in protection against TNF or TRAIL
receptor-induced cell death has not been established so far, except for
a study suggesting that ERK1/2 is involved in FGF-2-mediated protection
against TNF Differences between Death Receptors in the Response to Receptor
Activation and Inhibition of Mitogenic Signaling--
HeLa cells have
been shown to express the FasR, the TNF-R1, the TRAIL-R1, and the
TRAIL-R2, each of them containing a death domain and able to induce
apoptosis (45). However, there are some fundamental differences between
the signaling responses elicited by activation of the respective
receptors. As we show in this article, triggering of the receptors
induce different responses in HeLa cells. Whereas HeLa cells were
completely resistant to both Fas and TNF, they showed some sensitivity
toward TRAIL-mediated apoptosis, although the degree of survival was
still higher than in other tested TRAIL-sensitive cell lines (data not
shown). In addition, ERK1/2 inhibition could sensitize HeLa cells to
Fas and TRAIL killing, whereas it did not affect the resistance of the
cells to TNF-mediated apoptosis. These results suggest differences in
the modulation of DR responses. Interestingly, the responses we
observed to correlate with the known physiological functions of each
receptor. The primary function of the FasR has long been characterized
as induction of apoptosis in different situations that include the
immune response and regulation of the immune system. It has been
suggested that tumor cells have developed several strategies to escape
the immune surveillance by turning down this apoptotic pathway (59,
60), one strategy clearly being ERK-dependent protection.
The TNF-R1, however, seems to be mainly involved in inflammatory
responses, through activation of the NF- Several Survival Pathways Can Modulate/Reinforce the Sensitivity of
the Cells at a Given Time--
TNF-R1 has been known more generally to
direct its signaling toward transcription rather than apoptosis, as
mentioned above, mainly by activating the transcription factor NF- Mechanism and Function of ERK1/2-mediated Survival--
We have
shown earlier that the ERK1/2 activation, which protects cells from
apoptotic signal generated by the FasR, originated from activation of
the receptor itself (36). We show here that the same applies to TNF-R1
and TRAIL-R, i.e. each receptor rapidly activates ERK1/2
upon stimulation. The fact that Fas-induced ERK1/2 activation was more
rapid and transient in the current study than in the previous one, is
likely to be explained by the difference in the Fas antibody used in
the experiment.
The mechanism of DR-mediated ERK1/2 activation is not yet understood. A
recent study suggests that FLIP could mediate rapid activation of
ERK1/2 by recruitment of Raf-1 to the Fas DISC (67). Additional
candidates for a role in ERK1/2 activation are the 14-3-3 proteins. It
has been reported that 14-3-3 would be necessary for serum-stimulated
ERK1/2 activation, and that dominant-negative 14-3-3 would sensitize
NIH3T3 cells to TNF-R1-mediated apoptosis (68). Moreover, 14-3-3 has
been implicated in Raf-1-mediated mitotic response (69). Concerning the
downstream targets of ERK1/2 activation in this context, our data
suggest that the apoptotic cascade is stopped either at an early stage
before activation of the initiating factor caspase-8, or at the level
of caspase-8. In a previous study (35), we showed that although the
ERK1/2-mediated protection is active, the FasR-induced DISC can be
assembled without overall activation of caspase-8. Therefore, the
apoptotic signal is inhibited either at the level of caspase-8 or at
some upstream step of the feedback amplification loop, such as that
involving cleavage of Bid and activation of cytochrome c
release. The exact mechanism remains, however, to be investigated.
Intervention by ERK1/2 in the proapoptotic signaling cascades mediated
by each of the three DRs adds to the similarities observed between the
members of this family of proteins. It is interesting to note that the
strong resemblance between the complexes assembled around the receptors
do not prevent the differences in final functions. The complexity of
the signals involved at the level and downstream of the respective
DISCs allow precise modulation of the outcome and differences in the
functions with possibility of redundancy. Although many interacting
pathways related to DR stimulation have been deciphered, much remains
to be resolved, both in normal cells as well as in cancer cells, in
which disruption of some apoptotic signaling pathways have reduced the
susceptibility to be killed by the immune system. Understanding the
mechanism of ERK1/2-mediated protection both in normal and in
pathological situations could lead to a better understanding of
pathological conditions related to defects in the apoptotic machinery.
B, which remained inactive in FasR
or TRAIL-R signaling. However, when the cells were sensitized with
cycloheximide, which is sufficient to sensitize the cells also to
apoptosis by TNF-R1 stimulation, we noticed that adenovirus-mediated
expression of constitutively active MKK1 could rescue the cells from
apoptosis induced by the respective receptors by preventing caspase-8
activation. Taken together, our results show that ERK1/2 has a dominant
protecting effect over apoptotic signaling from the death receptors.
This protection, which is independent of newly synthesized
proteins, acts in all cases by suppressing activation of the caspase
effector machinery.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B activation (11, 20). Likewise, new
signaling functions are emerging for the FasR. Among the molecules
involved in Fas-mediated signaling, RIP (21), Daxx (22, 23), and FAP-1
(24, 25) have been shown to bind to the FasR, modulating its signal.
Some inhibitor proteins act by mimicking one or another protein of the
apoptotic cascade, diverting it into inactivation. Among them, FLIP, a
caspase-8-like protein lacking proteolytic activity has been shown to
block caspase-8 activation (26, 27). Members of the mitogen-activated
protein kinase (MAPK) family have also been shown to be involved in the signaling downstream of the Fas or TNF receptors (28-31).
Particularly, the MAPK ERK1/2, which is known to induce cell growth and
differentiation, has been shown to promote survival in a number of
situations (32, 33). It has also been suggested that TRAIL-R could,
with the intermediary of FADD, recruit TRADD and activate NF-
B in
this way (17). However, TRADD and RIP were reported absent from the TRAIL-induced DISC in vivo (14), although it could exist in different cell lines than the ones investigated.
B activation. However, when protein synthesis was blocked and
cells were sensitized to Fas, TNF, and TRAIL, activation of ERK1/2 was
able to protect the cells in all the cases. Furthermore, all three
receptors were able to activate ERK1/2. The protection seems to occur
at the level or upstream of caspase-8. Thus, ERK1/2 activation has a
dominant-negative effect on the apoptotic signaling of all receptors,
although differences could be detected in the degree of ERK1/2
activation as well as anti-apoptotic effect on the different DRs.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(100 ng/ml; Upstate Biotechnology, Lake Placid, NY) or with TRAIL (100 ng/ml; Alexis Corporation, Läuflingen, Switzerland) along with cross-linking FLAG-tagged antibody M2 (Sigma), with or without 30 µM
PD98059 (CalBiochem, La Jolla, CA), 5 µM cycloheximide
(CHX; Sigma), or 20 µM Z-VAD-fmk (CalBiochem).
-galactosidase (lacZ) gene under the control of cytomegalovirus (CMV) IE promoter was kindly
provided by Gavin W. G. Wilkinson (University of Cardiff, Wales).
Construction and characterization of replication-deficient adenoviruses
RAdMEK1ca containing the coding region of constitutively active MKK1
with the HA-tagged (RAd-CA-MKK1-HA) gene driven by the CMV IE promoter
(a kind gift from Marco Foschi, University of Florence, Italy) has been
described previously (39). In experiments, adenovirus RAdLacZ was used
to determine the multiplicity of infection (MOI) required to infect
100% HeLa cells. RAdLacZ virus was serially diluted at different MOI
in culture medium, and HeLa cells were incubated for 16 h with
different concentrations as described previously (40). Cells were then
washed with PBS, fixed with glutaraldehyde and stained for LacZ
expression with X-gal. For experiments with RAd-CA-MKK1-HA, we chose
300 plaque-forming units per cell, which gives 100% transduction
efficiency in this cell line. Cells were incubated for 16 h with
RAd-CA-MKK1-HA, then washed with PBS and further incubated in medium
with the treatments described.
B binding sequence. The protein-DNA complexes were then resolved
on 4% polyacrylamide native gel electrophoresis.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-induced apoptosis after PD98059-mediated inhibition of ERK1/2 activation, revealing that the TNF-R has additional survival
mechanisms in action. To confirm the presence of functional DRs, we
treated the cells with CHX, as many cells resistant to DR stimulation
have been found to be sensitized by treatment with CHX. Cotreatment
with CHX was efficient in sensitizing the cells to FasR-, TNF-R1-, and
TRAIL-R-mediated cell killing, respectively, demonstrating that the
receptors were present and functional, and that receptor stimulation
was able to promote cell death (Fig. 1).
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Fig. 1.
Sensitivity of HeLa cells to FasR, TNF-R1,
and TRAIL-R-induced apoptosis. A, HeLa cells were
incubated 16 h with anti-FasR antibody, TNF , or TRAIL ligand
(100 ng/ml each), with or without cycloheximide (CHX, 5 µM) or PD98059 (30 µM). The percentage of
apoptotic cells was determined by FACScan flow cytometry of PI-stained
nuclei. Representative histograms from treated cells are shown.
Fragmented apoptotic nuclei appear as subdiploid counts covered by the
double arrow. B, the data obtained are
represented in the bar graph as mean value (mean ± S.E.) from a minimum of three separate experiments.
, there is still the
possibility that ERK1/2 could act as a secondary survival pathway in
the TNF-R1 signaling. Because HeLa cells can be sensitized to Fas, TNF,
and TRAIL-mediated apoptosis by treatment with CHX, we asked whether the survival mechanism activated by ERK1/2 would be able to override the CHX-induced sensitization. For this purpose, we used the
RAd-CA-MKK1-HA adenovirus for expression of hemagglutinin (HA)-tagged
constitutively active (CA) MKK1, the ERK1/2 activator, into HeLa cells.
The amount of virus particles necessary for infection of 90-100% of
the cells was determined by using an adenovirus construct containing
the LacZ gene, followed by X-gal in situ staining.
Surprisingly, we observed that expression of CA-MKK1-HA was not as
efficient under the same conditions of infection because only 25-35%
of the cells expressed CA-MKK1-HA (Table
I). However, a higher penetrance of
CA-MKK1-HA-positive cells could be obtained by longer incubation after
infection (Table I). As the aim was to study direct signaling effects
without involvement of ERK-mediated transcriptional activation, a short
time period after expression of the initiating signaling protein was
used. Despite the relatively low penetrance of CA-MKK1-HA expression at
these early time points, the percentage of positive cells in the sample
was sufficient to detect the effects of CA-MKK1-HA by microscopy and
Western blot analysis (Figs. 2, and 5).
Cells were sensitized to apoptosis by CHX, subjected to Fas, TNF, and TRAIL treatments, respectively and analyzed by fluorescence microscopy. This method enabled us to segregate the cells that actually express CA-MKK1-HA from the negative cells. Representative micrographs clearly
show that the apoptotic cells did not express MKK1 (Fig. 2A), whereas the HA-positive cells were not apoptotic.
Quantitative data obtained by manual counting of apoptotic cells, as
determined by Hoechst staining, confirmed that only a minute fraction
of apoptotic cells (<2%) was found to be HA-positive (Table I). As a
comparison to this assay, we counted surviving cells remaining on equal
areas of each coverslip (Fig. 2B). Because TNF
alone did
not induce apoptosis in HeLa cells, we used TNF-treated CA-MKK1-HA expressing cells as a positive control to calculate the
percentage of survival after the respective treatments (Fig.
2B). The results show that expression of CA-MKK1-HA rescued
the cells from CHX-induced sensitization. Taken together, because the
protection occurred shortly after infection when the expressed kinase
did not have time to induce newly synthesized proteins and also in the
presence of CHX, these data show that ERK1/2-mediated protection is
independent of protein synthesis. Furthermore, the effect of CA-MKK1-HA
expression not only counteracted the CHX sensitization, but it also
protected against the normally occurring apoptosis induced by TRAIL
alone (Fig. 2B). Interestingly, activation of ERK1/2 was
also able to rescue the cells from TNF-induced apoptosis in
CHX-sensitized cells. The survival data also provided a control showing
that the adenovirus by itself did not affect our results because the RAdLacZ-transfected cells reacted in a similar way as the
nontransfected population in Fig. 2B as well as
untransfected cells (data not shown).
The expression of HA-MKK1-CA in control cells and in DR-stimulated
apoptotic cells
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Fig. 2.
Expression of constitutively active MKK1
rescues HeLa cells from Fas, TNF, and TRAIL-induced
apoptosis. An adenovirus construct was used to transfer the
CA-MKK1-HA mutant gene in HeLa cells. Control cells and cells infected
with RAd-CA-MKK1-HA were treated with anti-Fas, TNF , or TRAIL (100 ng/ml each), with or without CHX (5 µM) or PD98059 (30 µM) for 10 h. A, micrographs of the
samples show the CA-MKK1-HA expression (HA) and nuclear
staining (Hoechst) of the same area. Examples of apoptotic
cells, detected by their fragmented nuclear morphology, are shown with
white arrows. These cells clearly do not express the
constitutively active MKK1. B, HeLa cells plated on
coverslips and infected with either RAd-CA-MKK1-HA or RAdLacZ as
control were subjected to treatments with TNF
alone or TNF
,
anti-Fas, and TRAIL with CHX. After fixation, the cells were stained
for the HA tag of the CA-MKK1-HA mutant together with nuclear staining
(Hoechst). Surviving cells from LacZ (black
boxes), CA-MKK1-HA-negative (hatched boxes) and
-positive (white boxes) cells were counted. The graph
represents mean of survival compared with survival of the
TNF
-treated samples.
B Anti-apoptotic
Pathway--
Triggering of the TNF-R1 is generally known to activate
the NF-
B signaling cascade, a major anti-apoptotic pathway of the TNF-R1. TRAIL-R1, TRAIL-R2, and FasR have also been suggested to
activate NF-
B under specific conditions and treatments (44-46). The
activation steps involve induction of a kinase cascade comprising NIK
and IKK, resulting in phosphorylation of the NF-
B inhibitor I
B
and release of the transcription factor (47). Some reports suggest that
NIK could in some situations be replaced by MEKK1 (48, 49). We wanted
to know whether the difference in sensitivity toward stimulation of the
respective receptors was because of activation of NF-
B. As expected
from earlier studies (6, 20), TNF induced a marked binding activity of
the NF-
B transcription factor with rapid kinetics (Fig.
3). However, neither Fas nor TRAIL
activated NF-
B, thereby providing an explanation of why inhibition
of ERK1/2 activation did not sensitize HeLa cells to TNF-induced
apoptosis. Furthermore, the results suggest that the ERK1/2 survival
mechanism would be independent from NF-
B activation.
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Fig. 3.
Only TNF strongly activates
NF- B. HeLa cells were treated for
15 min, 1 h, 3 h, and 6 h with anti-Fas, TNF
, or
TRAIL-L (100 ng/ml each). The NF-
B activity was measured by
electrophoretic mobility shift assay with a specific oligonucleotide
probe. A representative autoradiograph is shown. As reported
previously, TNF rapidly activates NF-
B, whereas neither Fas nor
TRAIL has any effect on the transcription factor.
,
or TRAIL for different time periods. The activation of ERK1/2 was
determined by Western blot using a phospho-ERK1/2 antibody, which
specifically recognizes the activated form of the kinase (Fig.
4A). Stimulation of
FasR, TNF-R1, as well as TRAIL-R rapidly induced ERK1/2
phosphorylation. The activation of ERK1/2 appeared 5 min after
treatment and was down-regulated after 1 h. The overall results
show that the survival mechanism is not constitutive to the cell but
rather activated by the apoptotic stimuli in itself. Because ERK1/2
activation could be mediated by caspases in the same way as indicated
for the stress-activated protein kinases (SAPK; Refs. 37, 50), we
tested the effect of the general caspase inhibitor Z-VAD-fmk on
Fas-induced ERK1/2 phosphorylation. Although Z-VAD-fmk was an efficient
inhibitor FasR-induced cleavage of caspase-8 (Fig. 4B), it
clearly did not affect FasR-mediated activation of ERK (Fig.
4A).
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Fig. 4.
Fas, TNF, and TRAIL activate ERK1/2.
A, the phosphorylation state of ERK1/2 in HeLa cells was
detected by Western blotting with a phosphospecific ERK1/2 antibody
after 5 min, 15 min, 1, and 4 h of anti-Fas, TNF or TRAIL
treatment. Fas-induced activation of ERK1/2 was also followed for 5 min, 15 min, and 1 h in the presence of 20 µg/ml of the caspase
inhibitor Z-VAD-fmk. The same samples were probed with ERK2 antibody as
a loading control. B, control of the activity of Z-VAD-fmk
by caspase-8 Western blot.
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Fig. 5.
Activation of ERK1/2 by adenovirus-based
transfer of constitutively active ERK1/2 kinase inhibits Fas, TNF, and
TRAIL-mediated caspase activation. HeLa cells expressing
CA-MKK1-HA and control cells were treated for 10 h with anti-Fas,
TNF or TRAIL (100 ng/ml each) and sensitized to apoptosis by CHX (5 µM). Caspase activation was detected by Western blotting
as appearance of the active p18 fragment of caspase-8, as well as
disappearance of the proform.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-induced apoptosis (33). We now provide evidence that
ERK1/2 controls the responses from the other DRs too. ERK1/2 is likely
to represent a mode of apoptosis regulation, which would be important
especially during dynamic situations, when cells have to rapidly switch
off the apoptotic signaling machinery. This inhibitor mechanism would then act in concert with inhibitor proteins, such as FLIP. The involvement of several regulatory pathways provides a multifaceted control system to direct the signals from these receptors.
B transcription factor (61),
which diverts its signaling from the death pathway in most cell lines
(6). Recent studies suggest that the TRAIL-Rs, the more novel of the
four DRs, are especially important in removing virus-infected cells, as
well as tumor cells (62-64), which would explain the higher
sensitivity of HeLa cells toward this receptor. The existence of two
DRs both activated by TRAIL renders the interpretation more delicate,
as the resulting effect observed is most likely a combination of different responses from the respective TRAIL-Rs, rather than one
single and redundant effect from both receptors or an effect from one
receptor alone.
B,
whereas both FasR and the TRAIL receptors are considered to be
primarily apoptosis-directed, although they have also been suggested to
have the capacity to activate NF-
B (17, 65, 66). It has been
suggested that a Fas and TRAIL-mediated FADD-dependent
activation of NF-
B in HeLa cells could be triggered by CHX, combined
to caspase inhibition to prevent apoptosis, by way of an unknown
CHX-sensitive factor. This experimental setup is, however, far from the
physiological situation (45, 46). Our system reflects the more common
responses of these receptors, as only TNF
was able to induce
activation of the transcription factor NF-
B. This would explain why
inhibition of ERK1/2 activation was not sufficient to induce
TNF-mediated apoptosis, whereas it did sensitize cells to Fas and TRAIL
killing. However, TNF-induced apoptosis could be triggered by CHX
cotreatment, and expression of CA-MKK1-HA was able to protect the cells
in the same way as it could protect against Fas and TRAIL-mediated apoptosis. This is interesting, because it shows that ERK1/2 has a
generally protective effect on DR-induced cell death, which could be
used under specific conditions, when DR responses have to be rapidly
modulated. It is interesting to speculate that, in case of failure of
the main anti-apoptotic pathway, when NF-
B is not functional, the
ERK1/2 pathway could take over the protection system. Additional
protein synthesis-dependent anti-apoptotic factor(s) seem
to be involved in DR signaling, considering the fact that CHX was able
to sensitize the cells to Fas and TRAIL. FLIP has been suggested as a
candidate for such a factor (46). The protecting effect of ERK1/2,
however, is independent of protein synthesis, as cells can be rescued
by CA-MKK1 even in the presence of CHX.
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ACKNOWLEDGEMENTS |
---|
We thank Peter Krammer (German Cancer Research Center, Heidelberg, Germany) for caspase-8 antibody, Marco Foschi (University of Florence, Italy) for RAdMKK1-CA, and Gavin W. G. Wilkinson (University of Cardiff, Wales) for RAdLacZ. We also thank Lea Sistonen and other members of our laboratories for critical comments on the manuscript and technical help during the course of this study.
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FOOTNOTES |
---|
* This work was supported by Grant 35718 from the Academy of Finland, the Sigrid Jusélius Foundation, the Erna and Victor Hasselblad Foundation, the Finnish Cancer Foundation, the Nordic Academy for Advanced Study (NorFA), the Cell Signaling Program of Åbo Akademi University, EVO Grant 13336 from the Turku University Central Hospital, and the Turku Graduate School of Biomedical Sciences (to S. E. F. T., T. H. H., and M. A.).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.
Current address: Dept. of Cell Biology, Max-Planck-Institut
for Biochemistry, D-82152, Martinsried, Germany.
§§ To whom correspondence should be addressed: Dept. of Biology, Laboratory of Animal Physiology, Science Bldg. 1, University of Turku, FIN-20014 Turku, Finland. Tel.: 358-2-333-8036; Fax: 358-2-333-8000; E-mail: john.eriksson@utu.fi.
Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M010384200
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ABBREVIATIONS |
---|
The abbreviations used are:
TNF, tumor necrosis
factor;
MAPK, mitogen-activated protein kinase;
ERK1/2, extracellular-regulated kinase 1 and 2;
MKK1, mitogen-activated protein
kinase kinase 1;
CA, constitutively active;
HA, hemagglutinin;
TRAIL, TNF-related apoptosis-inducing ligand;
DR, death receptor;
DISC, death
inducing signaling complex;
DD, death domain;
CHX, cycloheximide;
PI, propidium iodide;
PBS, phosphate-buffered saline;
X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside;
Z-VAD-fmk, benzyloxycarbonyl-VAD-fluoromethylketone;
FADD, Fas-associated death domain protein;
JNK, c-Jun N-terminal kinase;
TRADD, TNFR1-associated death domain protein.
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