Received for publication, September 14, 2000, and in revised form, November 30, 2000
Persistent c-Jun NH2-terminal
kinase (JNK) activation induces cell death. Different mechanisms are
ascribed to JNK-induced cell death. Most of the JNK-apoptosis
studies employ stress stimuli known to activate kinases other than JNK.
Here we used overexpression of mitogen-activated protein kinase kinase
7 (MKK7) to activate selectively JNK in T lymphoma Jurkat cells.
Similar to that reported previously, Fas ligand (FasL) expression was
up-regulated by JNK activation. Dominant negative-FADD and caspase-8
inhibitor benzyloxycarbonyl-Ile-Glu-Thr-Asp effectively
inhibited MKK7-induced cell death, supporting a major involvement of
FADD cascade. However, MKK7-induced cell death was not prevented by
antagonist antibody ZB4 and Fas-Fc, indicating that Fas-FasL
interaction is minimally involved. Confocal microscopy revealed that
persistent JNK activation led to clustering of Fas. Our results suggest
that, in contrast to that reported previously, JNK alone-induced death
in Jurkat cells is FADD-dependent but is not triggered by
Fas-FasL interaction.
 |
INTRODUCTION |
JNK1 activation is
always linked to cell death induced by stress. Apoptosis triggered by
UV,
-irradiation, and cytotoxic drugs is correlated with activation
of JNK, and the cell death is prevented by inhibition of JNK activation
(1-5). The pivotal role of JNK is further illustrated by JNK
activation induced by active mitogen-activated protein kinase kinase
kinase 1 (MEKK1) (2, 6), active Cdc42 (7), and apoptosis
signal-regulating kinase 1 (8) that either initiates the apoptotic
process or potentiates cell death triggered by low dose stress stimuli
(9). JNK activation is implicated in growth factor deprivation-induced
cell death (10-12), in class I major histocompatibility complex
ligation-triggered apoptosis (13), or possibly in anoikis (14). The
critical role of JNK is also supported by the lack of apoptosis on
hippocampal neurons in JNK3-deficient and in JNK1/JNK2 double knockout
mice (15, 16).
The exact molecular mechanism how JNK induces cell death remains
largely elusive. Different apoptotic molecules have also been
attributed to JNK-triggered cell death. Activation of c-Jun by JNK
seems to mediate part of the apoptotic events (17). MEKK1 or c-Jun
induces FasL expression and the subsequent FasL-Fas interaction and
cell death (6, 12, 18, 19). p53 and Bax may also mediate JNK-induced
apoptosis following p75 neurotrophin receptor activation (11).
Alternatively, apoptosis could be induced by translocation of JNK into
mitochondria followed by phosphorylation and inactivation of Bcl-2 and
Bcl-xL (20, 21). In addition, the UV-induced mitochondrial death
pathway is abrogated in the absence of JNK, further supporting
mitochondria as the target of JNK (22).
Most of JNK-inducing signals such as UV and cytotoxic drugs activate
signals other than JNK. Even for the selective expression of MEKK1,
Cdc42, or apoptosis signal-regulating kinase 1, activation of JNK is
accompanied by stimulation of p38 and/or I
B kinase. In addition, the
contribution of p38 to stress-activated apoptosis has been demonstrated
(5, 10, 23, 24). In this study, we used transient expression of MKK7 to
activate JNK in Jurkat T cells. MKK7 selectively activates JNK but not
other kinases (25-29). We confirmed the previous notion that JNK
activation leads to increased FasL expression in Jurkat cells. Blockage
of FADD-initiated apoptotic pathway effectively prevented JNK-induced
cell death. However, blockage of FasL-Fas interaction by antagonizing
antibody or Fas-Fc did not affect MKK7-induced apoptosis, suggesting
that FasL is minimally involved in JNK-mediated cell death. Our results clearly suggest that JNK induces apoptosis by a
FADD-dependent but FasL-independent mechanism in Jurkat cells.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
Jurkat cell (H6.2 clone) was a gift of Dr. Daniel
Olive (INSERM U119, Marseille, France). Antibody against
-tubulin
was obtained from Amersham Pharmacia Biotech. Anti-FasL antibody (C-20)
was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
PE-conjugated anti-Fas antibody (DX2) and anti-FasL antibody (NOK-1)
were purchased from eBioscience (San Diego, CA). Anti-Fas
antibodies CH11 and ZB4 were purchased from Upstate Biotechnology, Inc.
(Lake Placid, NY). Caspase-8 inhibitor Z-IETD-fluoromethyl ketone was
obtained from Calbiochem. Human Fas-Fc was purchased from R & D Systems (Minneapolis, MN).
Plasmids--
Active MKK7
(S271D and T275D) (29) and
active MKK3b were gifts of Dr. Jiahuai Han (Scripps Research Institute,
La Jolla, CA).
MEKK1 and SEK-AL were gifts of Dr. Dennis Templeton
(Case Western University, Cleveland, OH). pcDNA3-AU-FADD and
DN-FADD were gifts of Dr. Vishva Dixit (Genentech, South San
Francisco). FLIP plasmid was previously constructed (30).
Transfection--
Jurkat T cells (1 × 107)
were washed and resuspended in 0.6 ml of RPMI medium containing 1%
glucose, 10% fetal calf serum, and 10-20 µg of plasmid DNA. The
electroporation was performed in Bio-Rad Gene Pulser II at 260 mV and
975 microfarads. The cuvette was left on ice for 15 min, washed twice
with phosphate-buffered saline, and incubated for the indicated time
for cell death and biochemical analysis.
Protein Kinase Assay--
Jurkat T cells were transfected with
pcDNA3,
MEKK1, or pcDNA3-MKK7
. Cell lysates were prepared
24 h after transfection, and 100-200 µg of lysate was
precipitated with 1 µl of anti-JNK1 antibody 101 (31) or anti-p38
(32), followed by 20 µl of protein A-Sepharose. The kinase activity
of the immune complexes was determined by using
GST-c-Jun-(1-79) or myelin basic protein as substrates. The
reaction mixtures were resolved on SDS-polyacrylamide gel electrophoresis, followed by autoradiography, and quantitated by
PhosphorImager (Molecular Dynamics).
Cell Death Measurement--
Apoptosis in bulk population was
determined by propidium iodide (PI) staining. At the indicated times
after treatment, cells were harvested and washed in phosphate-buffered
saline twice and resuspended in hypotonic fluorochrome solution (50 µg/ml PI, 0.1% sodium citrate, 0.1% Triton X-100) (33). Cells were
placed at 4 °C in the dark overnight, and DNA content was analyzed
by FACScan (Becton Dickinson, Mountain View, CA). The fraction of cells
with sub-G1 DNA content was assessed using the CELLFIT
program (Becton Dickinson). For apoptosis in cells transiently
transfected with MKK7
or
MEKK1, green fluorescence protein
expression vector pGreen Lanten-1 (Life Technologies, Inc.) was
cotransfected. Cells were harvested at the indicated times, fixed with
paraformaldehyde, and terminal dUTP nick-end labeling reaction
was performed using FlowTACS kit (R & D Systems). The incorporated
biotin-dUTP was labeled with Tri-Color-streptavidin (Caltag,
Burlingame, CA). The green cells (GFP-positive) were then gated on
FACScan, and the fraction of cell stained with Tri-Color was
quantitated. Alternatively, PI staining was also used to determined the
fraction of subdiploid cells in GFP-positive population.
 |
RESULTS |
Expression of Active MKK7
and MEKK1 Led to
JNK-dependent Cell Death--
Jurkat cells were
transfected with active MKK7
or MEKK1 by electroporation. We chose
electroporation because the transfection efficiency was close to 30%
as determined by cotransfection with GFP (Fig.
1A, R1). To assess
the cell death induced by JNK activation, the population expressing GFP
was gated in fluorescence-activated cell sorter, and the fraction of
apoptotic cells labeled with biotin-dUTP was quantitated (Fig.
1A). Activation of JNK by MKK7
and MEKK1 in Jurkat cells
led to 50% death 24 h after transfection (Fig. 1B). We
next examined whether the cell death observed was JNK-dependent. MKK7
and MEKK1 were equally effective in
the JNK induction (Fig. 2A).
The specificity of MKK7
was further confirmed by its inability to
activate p38 mitogen-activated protein kinase, in contrast to the
effective induction of p38 by MEKK1 and MKK3 (Fig. 2B). The
activation of JNK by MEKK1 or MKK7
was prevented by cotransfection
of SEK-AL (Fig. 2A). The exact mechanism how SEK-AL prevents
MKK7
-induced activation of JNK is not completely clear. Presumably,
the binding of SEK-AL with JNK (34) would compete with the interaction
of JNK with MKK7
. Inhibition of JNK activation by SEK-AL prevented
MKK7
- or MEKK1- induced cell death (Fig. 2C), indicating
that the observed cell death is JNK-specific. Because MKK7
is a more
specific activator of JNK, in the following experiments mainly the
results of MKK7
transfection are shown. In all the criteria
evaluated, MEKK1-induced apoptosis displayed an identical
character.

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Fig. 1.
Expression of active MKK7
and MEKK1 led to Jurkat cell death. Jurkat were transfected
with pcDNA3, pcDNA3-MKK7 , or MEKK1 (each 5 µg),
together with 3 µg of pGreen Lanten-1. After 24 h, cells were
fixed with paraformaldehyde and proceeded with terminal dUTP
nick-end labeling reaction. GFP-positive cells were then gated
(R1) on FACScan, and the incorporated biotin-dUTP in
apoptotic cells was analyzed by Tri-Color-streptavidin
(TC-SA) staining.
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Fig. 2.
JNK-dependent cell death induced
by active MKK7 . Jurkat cells were
transfected by electroporation with 3 µg of pGreen Lanten-1, with
active pcDNA3-MKK7 , MEKK1, or MKK3 (each 5 µg), with or
without 10 µg of SEK-AL. Cell extracts were prepared 18 h after
transfection. A, JNK activities were determined by
immunoprecipitation with anti-JNK1 (31) and phosphorylation of
GST-c-Jun-(1-79). B, p38 activities were determined by
precipitation with anti-p38 (32) and phosphorylation of myelin basic
protein (MBP). C, apoptosis was determined
24 h after transfection as described in Fig. 1. TC-SA,
Tri-Color-streptavidin.
|
|
MKK7
Expression-induced FasL Expression--
Consistent with
previous reports (6, 18), there was a significant increase of FasL
expression after transfection of MKK7
and MEKK1 as determined by
immunoblots (Fig. 3A). Despite
a similar degree of JNK activation (Fig. 2), the extent of FasL
expression was higher for MEKK1 transfection than MKK7
transfection.
A likely cause is because MEKK1 also activates NF-
B and p38
mitogen-activated protein kinase, and both contribute to activation of
the FasL promoter (35-38). Despite the increase of total cellular
FasL, there was little increase in the surface FasL expression after MKK7
expression (Fig. 3B, dark curve). As a positive
control, TPA/A23187 treatment significantly promoted the surface FasL
expression (Fig. 3B, light curve). The expression of Fas is
already high in Jurkat cells. Expression of active MEKK1 or MKK7
added little to the surface Fas expression (not shown).

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Fig. 3.
MKK7 expression
induced FasL expression in Jurkat cells. Jurkat cells were
transfected with pcDNA3, pcDNA3-MKK7 , or MEKK1 (each 5 µg), together with 3 µg of pGreen Lanten-1. A, cell
extracts were prepared 24 h after transfection, resolved by
SDS-polyacrylamide gel electrophoresis, and were transferred to a
polyvinylidene difluoride membrane. Total FasL contents were determined
using anti-FasL antibody (C-20, Santa Cruz Biotechnology). The contents
of -tubulin were used as internal control. B, cell
surface FasL was determined by staining with PE-conjugated anti-human
FasL antibody (NOK-1, eBioscience) 24 h after transfection.
Jurkat cells activated with TPA/A23187 (T/A) were used as
positive control. pcDNA3, shaded curve; MKK7, dark
curve; T/A, light curve.
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MKK7
-induced Cell Death Was Blocked by DN-FADD and Caspase-8
Inhibitor IETD--
Fas-mediated apoptotic pathway is initiated by
recruitment of FADD, followed by cleavage and activation of caspase-8
(39). To examine whether MKK7
-induced cell death was indeed
Fas-dependent, we used Fas-specific inhibitor DN-FADD,
FLIP, and Z-IETD. DN-FADD competes with wild-type FADD (40, 41); FLIP
specifically antagonizes Fas-dependent cell death at the
stage of FADD and caspase-8 (42), and Z-IETD selectively inhibits
caspase-8. Cotransfection with DN-FADD effectively inhibited
MKK7
-induced apoptosis (Fig. 4). The
expression of FLIP similarly inhibited MKK7
-induced apoptosis (not
shown). The efficacy of caspase-8-specific inhibitor Z-IETD (50 µM) was first confirmed by blockage of CH11-induced
apoptosis (not shown). The addition of Z-IETD 2 h after MKK7
transfection abrogated MKK7
-triggered apoptosis (Fig. 4). The
inhibition of MKK7
-induced apoptosis by FLIP, DN-FADD, and Z-IETD
supports the notion that FADD-mediated apoptotic pathway plays a major role in JNK-triggered cell death in Jurkat cells.

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Fig. 4.
MKK7 -induced cell
death was inhibited by DN-FADD and caspase-8 inhibitor IETD in Jurkat
cells. Jurkat cells were transfected with pcDNA3-MKK7 (5 µg) and pGreen Lanten-1 (3 µg) in the presence or absence of
DN-FADD (10 µg) by electroporation. For those treated with caspase-8
inhibitor, Z-IETD (50 µM) was added 2 h after
transfection. Cell death was quantitated 24 h after transfection
as described in Fig. 1. TC-SA, Tri-Color-streptavidin.
|
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MKK7
-induced Cell Death Was Not Prevented by Fas-antagonizing
Antibody and Fas-Fc--
To determine the role of Fas-FasL engagement
in MKK7
-induced cell death, the antagonistic anti-Fas antibody ZB4
was used. Preincubation with ZB4 (250 ng/ml) effectively suppressed
CH11-induced apoptosis of Jurkat cells (Fig.
5A). In contrast, the extent
of Jurkat cell death resulting from MKK7
overexpression was
indistinguishable in the presence or absence of ZB4 (Fig.
5B). We also used soluble Fas-Fc fusion protein to block the
interaction of Fas and FasL. Fas-Fc (200 ng/ml) prevented FasL-induced
cell death (Fig. 5A), yet Fas-Fc failed to interfere with
MKK7
-induced apoptosis in Jurkat cells (Fig. 5B). Because
MEKK1 induced higher expression of FasL (Fig. 3), we further
examined whether
MEKK1-induced cell death could be inhibited by
Fas-Fc or ZB4. Neither Fas-Fc nor ZB4 prevented apoptosis induced by
MEKK1 (not shown). The observations that Fas-Fc and ZB4 did not
protect Jurkat cells from apoptosis suggest that the apoptosis induced
by JNK is not mediated through Fas-FasL interaction.

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Fig. 5.
MKK7 -induced cell
death was not prevented by Fas-antagonizing antibody ZB4 and
Fas-Fc. A, Jurkat cells were stimulated with CH11
antibody (100 ng/ml) or sFasL (100 ng/ml) in the absence or presence of
ZB4 (250 ng/ml) or Fas-Fc (200 ng/ml) for 24 h. Cells were then
stained with PI, and DNA content was analyzed by FACScan (Becton
Dickinson). Percentage of cells with sub-G1 DNA
content was assessed using CELLFIT program (Becton Dickinson). Each
experiment was repeated at least twice. B, Jurkat cells were
transfected with MKK7 and pGreen Lanten-1, and cell death was
determined 24 h after transfection. When indicated, ZB4 (250 ng/ml) or Fas-Fc (500 ng/ml) was added right after transfection.
Results were average of the three independent experiments.
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MKK7-induced Fas Aggregation on Jurkat Cells--
Stress stimuli
such as UV, cycloheximide, cisplatin, etoposide, vinblastine, and
doxorubicin may induce cell death by triggering Fas clustering in a
FasL-independent manner (43-46). We also examined the surface Fas
distribution on a macroscopic level before and after JNK activation
using confocal laser scanning microscope. Fas was evenly distributed on
the surface of the untransfected Jurkat cells (Fig.
6A). Treatment with sFasL led
to increased aggregation of Fas on the surface of Jurkat cells (Fig.
6B). For cells transfected with MKK7
, as those marked by
GFP expression (Fig. 6C), there was a similar increased
clustering of Fas on the surface of Jurkat cells as compared with the
nearby untransfected cells (Fig. 6D). Therefore,
constitutive MKK7
expression promotes the aggregation of the surface
Fas.

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Fig. 6.
MKK7 expression
induced aggregation of Fas. Jurkat cells were transfected with
pcDNA3-MKK7 (5 µg) and pGreen Lanten-1 (3 µg). Cells were
fixed with paraformaldehyde 16 h after transfection and stained by
PE-conjugated anti-Fas antibody (DX-2, eBioscience). The cells
were then transferred to slide using Cytospin, and the expression of
Fas in GFP-positive population was analyzed by Zeiss confocal laser
scanning microscope LSM 510 with a × 100 objective. A,
untreated Jurkat cells; B, Jurkat cells treated with sFasL;
C, Jurkat transfected with GFP; D, Fas expression
on the same field as in C, and the arrow
indicates GFP-positive cells. Bar indicates 10 µ m.
|
|
 |
DISCUSSION |
JNK has been implicated as the major mediator of cell death
induced by stress (1-3), yet the exact apoptotic mechanism triggered by JNK is not fully understood. In the present study, we used a
JNK-specific activator MKK7
to induce JNK and the subsequent apoptosis. Because MKK7
selectively activates JNK (25-29), we were
able to address death specifically activated by JNK in the absence of
other signaling such as p38 (Fig. 2B). Our results illustrate that JNK activation induced FasL expression (Fig. 3). The
requirement of Fas-FADD pathway is supported by the inhibition of
MKK7-triggered cell death by DN-FADD, FLIP, and Z-IETD (Fig. 4). We
have also demonstrated, likely for the first time, that JNK-induced
cell death in Jurkat cells is FasL-independent (Fig. 5).
Our observations that Fas-Fc and ZB4 were unable to prevent
MKK7
-induced cell death (Fig. 5) are in direct contradiction to the
report of Faris et al. (6) that inducible expression of
MEKK1 led to cell death which was prevented by soluble Fas and Fas
antagonist antibody. We speculate the difference between their study
and ours is likely due to the levels of Fas and FasL expression. In the
study of Faris et al. (6), the inducible activation of MEKK1
in Jurkat led to an increase of surface FasL levels by 50-fold. This
was accompanied by a 10-fold increase of surface Fas (see Fig. 5 in
Ref. 6). With such high levels of Fas and FasL, Fas-FasL interaction
would inevitably become the dominant process to trigger cell death.
However, the levels of Fas and FasL induced by MEKK1 in their study are
highly unphysiological, as judged from their report that there was a
mere 50% increase of surface FasL with a 20% increase of surface Fas
expression when Jurkat cells were activated with TPA/A23187 (6). In the present study, despite an induction of total FasL content, surface FasL
levels in Jurkat cells were minimally altered by JNK activation (Fig.
3), and cell death proceeded in the absence of FasL binding. Fas-FasL
interaction apparently is not essential for JNK-induced cell death in
Jurkat cells. We have also repeated our observation in another T
lymphoma EL4 (not shown) and reached an identical conclusion.
FADD-dependent but FasL-independent cell death is triggered
by UV, cycloheximide, cisplatin, etoposide, vinblastine, and
doxorubicin through induced clustering of Fas (43-46), leading to the
association of FADD with Fas and the subsequent activation of
caspase-8. Fas aggregation-induced cell death is suppressed by DN-FADD
or FADD antisense (44-46). Similarly, MKK7-induced apoptosis is
sensitive to inhibition by DN-FADD, FLIP, and Z-IETD (Fig. 4) and MKK7
overexpression triggered Fas clustering (Fig. 6), suggesting that
JNK-activated apoptosis is mediated by Fas aggregation. We do not know
the exact cellular process between the JNK activation and Fas
clustering. Changes in the microtubule cytoskeleton may activate MEKK1
and JNK activation (47), but whether persistent JNK activation could induce reorganization of the microtubule cytoskeleton that promotes Fas
association remains to be determined. It may also be noted that our
results on JNK-induced Fas oligomerization are compatible with the
recent observation that Fas receptor is in trimer status before FasL
binding (48). This is supported by the observation that the engagement
of Jurkat cells by FasL led to a visible aggregation of Fas
under the microscope (Fig. 6D) as compared with
untreated cells, supporting that the Fas aggregation observed by others and us (43-46) is in a macroscopic level. Therefore, Fas is
trimerized in resting Jurkat cells but further aggregation is induced
by stress stimuli or JNK activation.
Our results may also help resolve the controversy on the involvement of
FasL in chemotherapy-induced apoptosis. Despite the earlier reports
that DNA-damaging agent-induced cell death is inhibited by Fas
antagonizing antibody or Fas-Fc (32, 49), many studies have shown that
apoptosis induced by UV and genotoxic drugs is not prevented by
neutralizing antibodies for Fas and FasL (45, 50-52). Our
demonstration that JNK triggers FADD-dependent apoptosis
through induction of Fas aggregation, which does not require FasL in
Jurkat cells, suggests one of the FasL-independent mechanisms that may
be involved in the killing of Fas-sensitive cancer cells by
chemotherapeutic agents is known to activate JNK.
Our observation on JNK-induced apoptosis in Jurkat cells, however, is
not necessarily applicable to all types of cells. In neuronal cells,
with the well established role of JNK and c-Jun in apoptosis induction
(15, 16), Fas-FasL interaction mediates part of JNK-activated apoptosis
induced by growth factor withdrawal (12). The difference in the degree
of the FasL participation in JNK-induced cell death could be due to the
different nature between T lymphocytes and cerebella granule neurons.
Notably, a pivotal role of FADD in JNK-triggered apoptosis does not
exclude other apoptotic mechanisms. MKK7-induced cell death was largely
inhibited by FADD and Z-IETD (Fig. 4), yet a small fraction of death
could still be detected even in the excess of DN-FADD and Z-IETD (not
shown), suggesting the presence of apoptotic pathway not mediated by
caspase-8. JNK is known to induce apoptosis in Fas-independent manner
by phosphorylation and inactivation of Bcl-2 and Bcl-xL (20, 21).
Together with the results from the present study, persistent activation
of JNK is capable of triggering apoptotic pathways initiated by both
mitochondria (22) and death receptor. We speculate that the exact
contribution from mitochondria and Fas pathway in JNK-mediated
apoptosis would be determined by variables such as type of stress, type
of cell, expression of Fas, and cellular sensitivity to Fas. Further
characterization will help understand the exact molecular process
triggered by JNK apoptotic signal in different cells.
We thank Dr. Jiahuai Han for MKK7
, MKK3b,
and anti-p38 antibody; Dr. Daniel Olive for Jurkat cells; Dr. Vishva
Dixit for DN-FADD; Dr. Dennis Templeton for
MEKK1 and SEK-AL; and
Dr. Tse-Hua Tan for anti-JNK1 antiserum.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M008431200
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