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INTRODUCTION |
Apoptosis is an evolutionarily conserved and highly controlled
form of cell death that plays a crucial role in development, maintenance of homeostasis, and immunological responses in
multicellular organisms. Dysregulation of apoptosis is implicated in a
range of diseases, including cancer, neurodegenerative disorders, and autoimmune diseases (1). Apoptosis-inducing signals can be divided into
two main groups: (i) those that lead to the formation of a
death-inducing signaling complex at the cytoplasmic side of
death receptors, resulting in the direct activation of a cascade of
caspases responsible for cell death, and (ii) those that
initially lead to the release of apoptogenic molecules from the
mitochondria (2). In certain cell types, however, even death
receptor-initiated apoptotic signals may be dependent on the
mitochondria to induce cell death (3). Such cell types are termed
"type II"-cells, in contrast to "type I"-cells where death
receptor-induced cell death is independent of the mitochondria (3). One
of the best characterized type II-cells is the Jurkat T cell
(4), where apoptosis induced by ligation of the Fas/CD95 death receptor
(5) is inhibited by interference with pro-apoptotic mitochondrial changes (3, 6, 7).
Fas-mediated apoptosis plays a pivotal role in a number of
physiological processes and seems to be particularly important for
immune function (8). Inhibition of Fas-mediated cell death is critical
for T lymphocytes to survive activation, because activation of T cells
leads to a markedly increased expression of both the Fas-receptor and
its ligand (9-12). Accordingly, stimulation through the T cell
receptor (TCR)1 has been
shown to confer resistance of mature T lymphocytes toward Fas-induced
apoptosis (9, 11, 13). The mechanism of this anti-apoptotic effect has
been studied in Jurkat T cells, where Fas-induced apoptosis can be
inhibited by stimulation with antibodies against the TCR (14), the
TCR-binding and mitogenic lectin concanavalin A (ConA) (15), or by the
PKC-activating phorbol ester TPA (16-23).
In two early reports (15, 21), the inhibiting effect of TPA or ConA on
Fas-mediated apoptosis in Jurkat cells was ascribed solely to the
MEK-ERK pathway. However, a number of studies have challenged the
significance of this pathway in protection against Fas-mediated cell
death (13, 16, 20, 22, 24-27).
The aim of the current study was therefore to (i) assess the importance
of the MEK-ERK pathway in protection against Fas-mediated apoptosis and
(ii) determine possible contributions from other anti-apoptotic
signaling pathways that are activated independently of MEK. We report
that the ERK pathway plays a significant, but not exclusive, role in
both TPA- and ConA-mediated suppression of Fas-induced apoptosis
in Jurkat cells. Furthermore, we identify NF
B as an equally
important anti-apoptotic player and show that ERK and NF
B are
activated independently of each other. Finally, we demonstrate that
simultaneous inhibition of ERK and NF
B fully abrogates the ability
of TPA to antagonize Fas-induced cell death. These results suggest that
T cells make use of (at least) two independently activated signaling
pathways to inhibit Fas-induced apoptosis.
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MATERIALS AND METHODS |
Reagents, Antibodies, and Plasmids--
CH11 was purchased from
Upstate Biotechnology, Inc. (Lake Placid, NY). TPA, ConA, LY294002,
wortmannin, and calphostin C were obtained from Sigma. PD98059,
U0126, BAY11-7082, SB202190, and bisindolylmaleimide I were purchased
from Calbiochem. TPA, calphostin C, and bisindolylmaleimide I were
dissolved in ethanol, and ConA was dissolved in phosphate-buffered
saline. LY294002, wortmannin, PD98059, U0126, BAY11-7082, and SB202190
were dissolved in Me2SO. All stock solutions were
aliquoted, stored at
20 °C, and diluted in cell culture medium
immediately prior to use. PD98059 was always diluted directly to its
final concentration (25 µM) without sub-dilutions, because of its poor solubility in aqueous solution. JC-1 (Molecular Probes Inc., Eugene, OR) was dissolved in Me2SO to 2 mg/ml,
and aliquots were stored at
20 °C. Polyclonal rabbit antibodies
recognizing human phospho-p44/42 MAP kinase (Thr-202/Tyr-204), total
ERK, or cleaved poly(ADP-ribose) polymerase (PARP) (89 kDa; Asp-214) were obtained from Cell Signaling Technology (Beverly, MA). pEGFP-N1 was from BD Clontech (Palo Alto, CA);
pCMV4-HA-I
B
-SS32/36AA was a generous gift from
Dr. W. C. Greene, University of California; pRL-CMV and pCMV-
Gal
were from Promega (Madison, WI); 3×
B-TATA was a kind gift
from Dr. Thomas Wirth, Ulm University, Germany; and pAP-1-luc and
pFC-MEKK were obtained from Stratagene (La Jolla, CA).
Cell Culture and Treatment--
The Jurkat E6-1 cell line (28)
was purchased from American Type Culture Collection (Manassas, VA) and
was cultured in RPMI 1640 medium supplemented with 10%
heat-inactivated fetal bovine serum (Invitrogen), 2 mM
glutamine, 125 units/ml penicillin, and 125 µg/ml streptomycin at
37 °C in a humidified incubator with 5% CO2. Except for
transfection studies, experiments were performed on cells that had been
diluted to 2 × 105 cells/ml 1 day earlier.
Cell-permeable protein kinase inhibitors were added 1-2 h prior to TPA
or ConA, which were added 5 min before CH11.
Determination of Apoptotic Cells by Morphological
Criteria--
Jurkat cells were pipetted to single cell suspension,
and putatively 10,000 cells were analyzed for size (forward scatter) and granularity (side scatter) using a FACSCalibur flow cytometer (BD
Biosciences). Based on the scatter profiles two populations, viable
cells and apoptotic cells, were identified, where the apoptotic cells
exhibit decreased forward scatter (size) and increased side scatter
(granularity) compared with the viable cells. In our experiments, the
percentage of necrotic cells (exhibiting increased forward scatter
compared with viable cells) was always less than 0.5%.
TUNEL Assay--
The in situ cell death detection kit
Fluorescein (Roche Molecular Biochemicals) was used to detect DNA
strand breaks that are associated with apoptosis (36). The assay was
performed as recommended by the manufacturer, with the exception that
cells were permeabilized with 0.1% saponin (in phosphate-buffered
saline) instead of Triton X-100.
Analysis of Mitochondrial Membrane Potential--
Jurkat cells
were stained for 10 min at 37 °C with 10 µg/ml of the cationic,
mitochondrial membrane potential sensor dye, JC-1, and 10,000 cells
were analyzed by fluorescence-activated cell sorter (FACS). In advance,
compensation for the overlap between FL1 and FL2 had been set by FACS
analyses of Jurkat cells stained with anti-CD3-FITC-Ab (BD Biosciences)
for FL1 and anti-CXCR4-PE-Ab (BD PharMingen) for FL2.
Western Blot Analysis--
Jurkat cells (2 ml) were washed once
with phosphate-buffered saline and lysed by gentle rotation at 4 °C
for 15 min in 50 µl of lysis buffer (50 mM Tris-Cl, pH
7.4, 0.1% Triton X-100, 250 mM NaCl, 10 µg/ml leupeptin,
9.5 µg/ml aprotinin, 0.2 mM phenylmethylsulfonyl fluoride, 5 mM NaF, 0.1 mM
Na3VO4, 10 mM
-glycerophosphate). Cell lysates were cleared by centrifugation at
4 °C for 10 min at 14 000 × g and boiled for 5 min
in 1× Laemmli sample buffer (29). Equal amounts of protein (~25
µg) were separated by 12% (for ERK) or 9% (for PARP) SDS-PAGE.
Proteins were transferred to a nitrocellulose membrane (Amersham
Biosciences) using a semi-dry transfer cell (Bio-Rad), and the
membranes were probed with antibodies from Cell Signaling Technologies
as recommended by the manufacturer. Immunoreactive proteins were
visualized with the ECL-plus detection system (Amersham
Biosciences).
Transfections--
Exponentially growing Jurkat cells (1 × 107) were washed in RPMI 1640 (saving the conditioned
medium), resuspended in 400 µl of RPMI, mixed with a total of 10-20
µg of DNA, and electroporated at 250 V and 950 µF using a Bio-Rad
GenePulser. The cells were then transferred to 2 ml of conditioned
medium on ice. Subsequently, 8 ml of fresh medium (with 10% fetal
bovine serum) was added, and the cells were allowed to rest for 24 h before experimental treatments were initiated.
Green Fluorescent Protein (GFP) Cotransfection
Studies--
Jurkat cells were cotransfected with pEGFP-N1 and
pCMV4-I
B
-SS32/36AA or pRL-CMV as a control. After
stimulation with TPA and/or CH11, cells were analyzed by flow
cytometry. Cells that expressed high levels of pEGFP-N1
(i.e. showing high FL-1 intensity) were also expected to
express high levels of the mutant I
B-construct, thus resulting in a
high degree of NF
B inhibition. These cells were gated and analyzed
for forward and side scatter properties.
Reporter Assays--
A dual luciferase reporter assay system
(Promega) was used as described in the manufacturer's protocol.
Luciferase activities were measured with a Turner Designs
(Sunnyvale, CA) luminometer.
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RESULTS |
MEK-inhibitors PD98059 and U0126 Decrease the Ability of TPA and
ConA to Suppress Fas-mediated Apoptosis--
Fas-induced apoptosis of
Jurkat cells can be inhibited by the phorbol ester TPA or by the
TCR-binding lectin ConA. Investigations of the role of the MEK-ERK
pathway as a potential anti-apoptotic mediator in this process have
yielded conflicting results (15, 16, 21, 22, 27). Therefore, to
understand how Fas-induced apoptosis is regulated in Jurkat cells,
we first assessed the importance of the MEK-ERK pathway in both TPA-
and ConA-mediated protection against apoptosis. ERK is a
serine/threonine protein kinase that is activated through
phosphorylation by the dual-specificity protein kinase MEK (30). To
date, the only MEK substrate recognized is ERK (31), and therefore
specific inhibition of MEK will also specifically inhibit ERK
phosphorylation and thus ERK activity. We tested the effect of two
structurally unrelated cell-permeable MEK-inhibitors, PD98059
(32, 33) and U0126 (34), on various features of apoptosis. First
we examined the overall morphology of the cells by flow cytometric
analysis of cell size (forward scatter) and granularity (side scatter),
apoptotic cells being smaller and more granular than viable cells. Dot
blots from one such experiment are shown in Fig.
1A, and the result from three independent experiments is graphically presented in Fig. 1B.
Stimulation of Jurkat cells with agonistic anti-Fas antibodies (CH11)
alone increased the percentage of apoptotic cells from 4 to 65% after 12 h. In the presence of TPA or ConA, however, CH11-induced
apoptosis was reduced to 24%. Both PD98059 and U0126 partly reversed
the anti-apoptotic effect of TPA and, to a lesser extent, that of ConA.
The largest effect was seen when cells were pretreated with U0126
before TPA. Under these circumstances, CH-11-induced apoptosis was
increased to 52%, i.e. the TPA effect was reversed by 68%. When examining the levels of the apoptosis-specific 89-kDa C-terminal caspase cleavage product of PARP (35) as a second means of analyzing apoptotic features, qualitatively the same results were obtained. As
shown in Fig. 1B, CH11-induced PARP cleavage was strongly
reduced by both TPA and ConA (compare lanes 3 and
6 with lane 2), an effect which was partly
abrogated by both PD98059 and U0126 (compare lanes 4 and
5 with lane 3 and lanes 7 and
8 with lane 6).

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Fig. 1.
MEK-inhibitors PD98059 and U0126 decrease the
ability of TPA and ConA to inhibit Fas-mediated apoptosis. Jurkat
cells were stimulated with Fas-Ab (25 ng/ml CH11), TPA (1 nM), ConA (4 µg/ml), PD98059 (25 µM), or
U0126 (10 µM) as indicated. A, after treatment
with CH11 for 12 h, cells were harvested and their forward- and
side-scatter profiles were analyzed by flow cytometry as described
under "Materials and Methods." The percentages of cells gated as
apoptotic are indicated. One representative experiment is shown.
B, average percentages of apoptotic cells from three
independent experiments like the one described above are presented
graphically with S.E. of the mean as indicated. Protein levels of
(phosphorylated and total) ERK and cleaved PARP were determined by
Western blot analyses after 0.5 and 2 h of CH11 treatment,
respectively. TPA or ConA treatment alone led to a slight induction of
apoptosis (5-10%), which was not significantly altered by MEK
inhibition (data not shown). C, after treatment with CH11
for 6 h, cells were subjected to the TUNEL assay as described
under "Material and Methods." The percentages of cells gated as
TUNEL-positive are indicated. One representative experiment is shown.
D, average percentages of TUNEL-positive cells from three
independent experiments like the one described in panel C
are presented graphically with S.E. of the mean as indicated.
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As a third means of analyzing apoptotic features, we examined the
cleavage of genomic DNA, a hallmark of apoptosis (36), by the TUNEL
assay. In this assay, DNA strand breaks generated by DNA fragmentation
are labeled with fluorescein dUTP, and the amount of DNA fragmentation
(reflected by fluorescence intensity) can then be determined at the
single-cell level by flow cytometry. Under the same experimental
conditions as described in Fig. 1, A and B, cells
were subjected to the TUNEL assay after 6 h of CH11 treatment. An
example of dot plots from one such experiment is shown in Fig.
1C, and the results from three independent experiments are
graphically presented in Fig. 1D. Whereas only 5% of the
cells were gated as positive for DNA fragmentation in untreated Jurkat cells, 51% of the cells were TUNEL-positive after treatment with CH11
alone. Pretreatment with TPA reduced the amount of TUNEL-positive cells
to 18%. Both PD98059 and U0126 were able to substantially counteract
the TPA effect. Again, U0126 was the most efficient inhibitor. In the
presence of U0126 the percentage of TUNEL-positive cells rose to 39%,
i.e. the TPA-effect was reversed by 64%.
To ensure that the MEK-inhibitors actually inhibited ERK activity
in the cells, we examined the levels of phosphorylated ERK protein by
Western blot analyses. Indeed, as shown in Fig. 1B, both
PD98059 and U0126 substantially inhibited both TPA- and ConA-induced phosphorylation of ERK after 30 min of stimulation. The inhibiting effect was also observed at earlier time points (10 and 20 min) and was
sustained for at least 2 h (data not shown). U0126 was a more
efficient inhibitor of ERK phosphorylation than PD98059 (Fig.
1B, compare lanes 4 and 7 with
lanes 5 and 8, respectively). This could be the
reason why U0126 also was observed to be superior to
PD98059 in abrogating the anti-apoptotic effects of TPA and ConA. To
test whether there was a connection between the extent of ERK
inhibition and augmentation of apoptosis, we examined the dose-dependence of U0126-mediated reversion of TPA's protective effect
against Fas-induced apoptosis with that of ERK inhibition. Indeed, as
shown in Fig. 2, there was an excellent
correlation between U0126-mediated ERK inhibition and abolishment of
the anti-apoptotic effect of TPA.

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Fig. 2.
U0126-mediated inhibition of ERK
phosphorylation correlates with reversion of the anti-apoptotic effect
of TPA against Fas-induced apoptosis. Jurkat cells were treated
with CH11 (25 ng/ml) and TPA (1 nM) in the absence or
presence of increasing concentrations of U0126 (0.1, 0.5, 2, and 10 µM, respectively). After treatment
with CH11 for 0.5 or 12 h, respectively, protein levels of
phosphorylated and total ERK and percentages of apoptotic cells were
determined as described in the legend to Fig. 1, A and
B. Average percentages of apoptotic cells from three
independent experiments, with S.E. of the mean as indicated, are
shown.
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We could therefore conclude that the MEK-ERK pathway was involved in
both TPA- and ConA-mediated inhibition of Fas-induced apoptosis in
Jurkat cells. However, even at concentrations of U0126 that almost
completely inhibited ERK phosphorylation, the MEK-inhibitor was never
able to fully abolish the protective effect of TPA or ConA against
Fas-induced apoptosis, as determined by examination of cell morphology,
PARP-cleavage, and DNA fragmentation. This suggested that at least one
signaling pathway, other than the MEK-ERK pathway, is involved in the
anti-apoptotic effect of TPA and ConA in Jurkat cells.
Inhibition of NF
B Reverses TPA- and ConA-mediated Suppression of
Fas-induced Apoptosis--
In addition to the MEK-ERK pathway, three
other signaling pathways have been reported to be involved in
regulation of Fas-induced apoptosis in human T cells, i.e.
those that lead to the activation of PI3K/Akt, p38/MAPK, and NF
B,
respectively (13, 24-26). We therefore tested whether cell-permeable
inhibitors of PI3K, p38/MAPK, or NF
B would prevent TPA- or
ConA-mediated suppression of Fas-induced apoptosis in Jurkat cells. The
inhibitors were used at concentrations that are known to specifically
inhibit PI3K (10 µM LY294002 or 100 nM
wortmannin), p38/MAPK (5 µM SB202190), or NF
B (2.5 µM BAY11-7082), respectively, and apoptosis was analyzed
by examination of cellular morphology. Whereas inhibitors against PI3K
and p38/MAPK had no effect, the NF
B inhibitor BAY11-7082 markedly
abrogated both TPA- and ConA-mediated
protection against Fas-induced apoptosis (Fig. 3). To
examine whether this effect could be attributed to specific NF
B
inhibition, we assessed whether there was a correlation between the
ability of BAY11-7082 to inhibit NF
B and its ability to reverse
TPA-mediated suppression of Fas-induced apoptosis. To determine NF
B
activity, Jurkat cells were transiently transfected with a luciferase
reporter plasmid controlled by three repeated NF
B response elements
(3×
B-TATA), together with a constitutively active renilla
luciferase reporter (pRL-CMV) to normalize for variability in
transfection efficiencies. Treatment with TPA and CH11 stimulated the
NF
B reporter about 6-fold above basal levels, and BAY11-7082
inhibited this activation in a dose-dependent manner (Fig.
4A). Thus at 3 µM, BAY11-7082 inhibited NF
B activity by 86%. At
concentrations above 3 µM, BAY11-7082 was toxic to
Jurkat cells (data not shown). Treatment with CH11 alone did not
increase NF
B reporter activity (rather a slight inhibition was
observed), and TPA-induced activation of the reporter was not
significantly influenced by CH11 (Fig. 4A). When we tested
the same concentrations of BAY11-7082 for its ability to inhibit the
anti-apoptotic effect of TPA (Fig. 4B), we observed that
there was excellent correlation between BAY11-7082-induced inhibition
of NF
B activity and reversion of TPA-mediated suppression of
Fas-induced apoptosis (compare Fig. 4A and
B).

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Fig. 3.
Suppression of Fas-induced apoptosis is
reversed by an NF B-inhibitor but not by
inhibitors of PI3K or p38/MAPK. Jurkat cells were treated with
CH11 (25 ng/ml), TPA (1 nM), ConA (4 µg/ml), LY294002 (10 µM), wortmannin (100 nM), SB202190 (5 µM), and BAY11-7082 (2.5 µM) as indicated.
After 24 h of treatment with CH11, apoptosis was determined by
analyzing the forward- and side-scatter profiles of the cells as
described under "Materials and Methods." Average percentages of
apoptotic cells from six independent experiments, with S.E. of the mean
as indicated, are shown.
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Fig. 4.
Inhibition of NF B
activity correlates with reversion of the anti-apoptotic effect of TPA
against Fas-induced apoptosis. A, Jurkat cells (1 × 107) were cotransfected with 1 µg of pRL-CMV and 9 µg of 3× B-TATA as described under "Material and Methods" and
treated as indicated. Luciferase activities were measured after 5 h of stimulation. Renilla luciferase activities were used to normalize
for variations in transfection efficiencies. Normalized LUC activity in
unstimulated cells was arbitrarily set to 100. Average relative LUC
activities from three independent experiments, with S.E. of the mean as
indicated, are shown. B, Jurkat cells were stimulated as
indicated. After 12 h of treatment with CH11, apoptosis was
determined by analyzing the forward- and side-scatter profiles of the
cells. Average percentages of apoptotic cells from three independent
experiments are shown. C, Jurkat cells (1 × 107) were cotransfected with 5 µg of pEGFP-N1 and 15 µg
of pCMV4-I B -SS32/36AA or 15 µg of pRL-CMV as a
control, and cells were stimulated as indicated. Apoptosis of
transfected cells was determined by analyzing the forward- and
side-scatter profiles of the cells. Results from four independent
experiments, with S.E. of the mean as indicated, are shown.
D, Jurkat cells (1 × 107) were
cotransfected with 5 µg of 3× B-TATA and 15 µg of
pCMV4-I B -SS32/36AA or 15µg of pCMV- Gal as a
control and treated as indicated. Luciferase activities were measured
after 5 h of stimulation. LUC activity in unstimulated cells was
arbitrarily set to 100. The result from five independent experiments is
shown.
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To further establish the role of NF
B in the anti-apoptotic effect of
TPA, Jurkat cells were transiently transfected with an expression
vector encoding a non-degradable I
B
mutant (
I
B
). To be
able to detect transfected cells, the cells were cotransfected with
pEGFP-N1, an expression vector encoding GFP. As a control, cells were
transfected with pEGFP-N1 and a plasmid expressing renilla luciferase.
As shown in Fig. 4C, the ability of TPA to suppress
Fas-induced apoptosis was reduced in cells transfected with the
I
B expression vector. Thus, the percentage of apoptotic cells in
the presence of TPA and CH11 increased from 18% in control-transfected cells to 31% in cells transfected with the
I
B-construct (Fig. 4C). To ensure that the I
B
mutant inhibited NF
B
activity,
I
B
was cotransfected with 3×
B-TATA, and a
luciferase reporter assay was performed similar to that described
above. Indeed, cotransfection of
I
B
strongly inhibited the
ability of TPA to activate the NF
B reporter (Fig. 4D).
Together these experiments strongly suggested that NF
B plays a
significant role in TPA-mediated repression of Fas-induced apoptosis.
TPA Activates NF
B through Protein Kinase C--
It has recently
been suggested (37, 38) that phorbol esters elicit some of their
biological effects through PKC-independent pathways. We therefore
assessed whether TPA activated NF
B through a
PKC-dependent or -independent pathway. For this purpose, we employed two functionally unrelated and highly potent inhibitors of
PKC, calphostin C and bisindolylmaleimide I, which specifically bind to
the regulatory and catalytic regions of PKC, respectively (39, 40). As
shown in Fig. 5A, both
PKC-inhibitors strongly inhibited TPA-induced activation of the NF
B
reporter, indicating that TPA activates NF
B through a
PKC-dependent pathway in Jurkat cells.

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Fig. 5.
TPA activates NF B
through a PKC-dependent pathway, and BAY11-7082 does not
inhibit AP-1 activity. A, Jurkat cells (1 × 107) were cotransfected with 1 µg of pRL-CMV and 9 µg
of 3× B-TATA and treated as indicated. Luciferase activities were
measured after 5 h of stimulation. Normalized LUC activity in
unstimulated cells was arbitrarily set to 100. Average relative LUC
activities from four independent experiments, with S.E. of the mean as
indicated, are shown. B, Jurkat cells (1 × 107) were transfected with 0.5 µg of pRL-CMV, 4.5 µg of
pAP-1-luc, and 5 µg of pFC-MEKK or 5 µg of pEGFP-N1 as a control.
Subsequently, cells were treated as indicated, and luciferase
activities were determined after 5 h. Normalized LUC activity in
unstimulated pFC-MEKK-transfected cells was arbitrarily set to 100. Results shown represent three independent experiments.
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NF
B and MEK/ERK Are Independent Signaling
Pathways--
Although we had shown that both the NF
B and the
MEK/ERK pathways were involved in repression of Fas-induced apoptosis,
we could not exclude the possibility that this could be due to ERK and
NF
B being part of the same anti-apoptotic signaling pathway instead
of being parallel pathways that both contribute to the inhibition of
apoptosis. To resolve this issue, we assessed whether inhibition of the
MEK/ERK pathway would affect NF
B activity and, conversely, whether
inhibition of NF
B would affect the activity of AP-1, a transcription
factor that is known to be dependent on ERK for its maximal activity
(41). As shown in Fig. 4A, whereas BAY11-7082 strongly
inhibited NF
B activity in a dose-dependent manner, the
MEK-inhibitor U0126 instead slightly augmented TPA-induced NF
B
activation. To determine AP-1 transactivating activity, a transient
transfection reporter assay with an AP-1-driven luciferase reporter
construct was performed. For optimal activation of AP-1, the cells were
cotransfected with an expression vector encoding a constitutively
active form of MEKK (pFC-MEKK), and luciferase activities were measured
after treatment with BAY11-7082 or U0126 for 5 h. U0126 inhibited
MEKK-induced AP-1 activity in a dose-dependent manner (Fig.
5B). However, as anticipated, U0126 did not completely inhibit AP-1 activity, because U0126 does not inhibit MEKK-induced activation of c-Jun N-terminal kinase (JNK), a major contributor to
AP-1 activity (41). Importantly, BAY11-7082 did not inhibit AP-1
activity (Fig. 5B). Furthermore, BAY11-7082 had no effect on TPA-stimulated phosphorylation of ERK (data not shown). These data
indicate that ERK and NF
B are components of parallel, and not
sequentially activated, signaling pathways and that they therefore contribute to the anti-apoptotic effect of TPA independently of each other.
Simultaneous Inhibition of MEK and NF
B Completely
Abolishes TPA-mediated Suppression of Fas-induced Apoptosis--
We
had shown that inhibition of neither ERK nor NF
B alone was able to
completely reverse TPA-mediated suppression of Fas-induced apoptosis.
We therefore investigated whether the anti-apoptotic effect of TPA
could be a result of cooperative actions between ERK and NF
B.
Simultaneous inhibition of MEK and NF
B was enforced by combined
treatment with U0126 and BAY11-7082, and TPA-mediated repression of
Fas-induced apoptosis was analyzed. Whereas U0126 or BAY11-7082
alone only partly reversed TPA-mediated suppression of CH11-induced
apoptosis, the combination of the two inhibitors completely abolished
the anti-apoptotic effect of TPA (Fig.
6A). U0126 was also tested in
combination with inhibitors of PI3K (LY294002 and wortmannin) or
p38/MAPK (SB202190), but none of these inhibitors potentiated the
effect of U0126 on TPA-mediated suppression of Fas-induced cell death
(data not shown).

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Fig. 6.
Simultaneous inhibition of MEK and
NF B completely abolishes TPA-mediated
suppression of Fas-induced apoptosis. Jurkat cells were treated
with Fas-Ab (25 ng/ml CH11), TPA (1 nM), U0126 (3 µM), or BAY11-7082 (2.5 µM) as indicated.
A, after 12 h of treatment with CH11, apoptosis was
determined by analyzing the forward- and side-scatter profiles of the
cells as described under "Materials and Methods." B,
after 8 h of treatment with CH11 the percentage of cells
containing mitochondria with depolarized membranes was determined by
JC-1 staining as described under "Materials and Methods." Results
from three independent experiments are shown. C, dot plots
from flow cytometrical analysis of JC-1-stained cells. One
representative experiment is shown.
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ERK and NF
B Inhibit Apoptosis Upstream of Mitochondrial Membrane
Depolarization in a Cooperative Manner--
Fas-induced apoptosis in
Jurkat and other type II cells is dependent on the mitochondria (3, 6,
7). One of the major pro-apoptotic mitochondrial events is the
disruption of the mitochondrial membrane potential, 
m
(42, 43). 
m can be monitored by the use of cationic
dyes, among which JC-1 is the most specific for monitoring
mitochondrial versus plasma membrane potential (44). In
cells that contain mitochondria with a normal 
m, JC-1
forms red fluorescent aggregates. A decrease in 
m,
however, leads to the disintegration of these aggregates concomitantly with an accumulation of green fluorescent JC-1 monomers.

m was analyzed by flow cytometry as shown in Fig.
6C. Cells stained with JC-1 that displayed decreased red
fluorescence concomitant with increased green fluorescence were
classified as cells containing mitochondria with depolarized membranes.
By analyzing the effects of U0126 and BAY11-7082 on TPA-mediated
inhibition of Fas-induced depolarization of the mitochondrial membrane
(Fig. 6, B and C), we observed that the effects
were strikingly similar to those detected when apoptosis was analyzed
by morphological criteria (Fig. 6A). Thus, whereas treatment
with either U0126 or BAY11-7082 alone only partially reversed
TPA-mediated suppression of Fas-induced loss of 
m,
the presence of both inhibitors simultaneously almost completely
abrogated the TPA effect (Fig. 6B). This suggests that the
anti-apoptotic effects of ERK and NF
B are upstream of the mitochondrial membrane depolarization event. Based on our results, a
model is depicted (Fig. 7) of how we
believe TPA protects Jurkat cells from Fas-induced apoptosis.

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Fig. 7.
Molecular mechanism of TPA-mediated
suppression of Fas-induced apoptosis in Jurkat T cells. TPA
inhibits Fas-receptor-stimulated depolarization of the mitochondrial
membrane potential and apoptosis through a parallel activation of two
anti-apoptotic pathways, i.e. those that involve NF B and
ERK, respectively. Therefore, the simultaneous inhibition of both
NF B and ERK is necessary to completely abolish TPA-mediated
suppression of Fas-induced apoptosis.
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DISCUSSION |
Although much studied, the mechanisms that regulate Fas-induced
apoptosis in T cells are incompletely understood. Progress during
recent years has implicated several different signal transduction pathways in protection against Fas-induced apoptosis in T cells (13-15, 21, 24-26). However, in all these studies the anti-apoptotic signaling pathways have only been explored individually. In the present
study we showed that in Jurkat T cells the disruption of one signaling
pathway alone was never sufficient to fully abolish the strong
repression of Fas-induced apoptosis mediated by TPA. We therefore
hypothesized that the anti-apoptotic effect of TPA could only be
explained by the involvement of at least two distinct and cooperative
signaling pathways. Indeed, we report here that simultaneous inhibition
of ERK and NF
B was both necessary and sufficient to fully prevent
TPA-mediated suppression of Fas-induced apoptosis in Jurkat cells.
Other signaling transduction pathways, including the PI3/Akt and
p38/MAPK pathways (13, 25, 26), have been implicated in repression of
Fas-induced cell death in T cells. However, from our results, neither
of these pathways seems to be involved in either TPA- or ConA-mediated
protection from Fas-induced apoptosis in Jurkat cells. First of all,
two widely used inhibitors of PI3K, LY294002 and wortmannin, had no
effect on either TPA- or ConA-mediated suppression of Fas-induced
apoptosis. Second, in agreement with a recent report (20), TPA
failed to activate Akt as determined by phospho-Akt Western blot
analysis and an in vitro Akt kinase assay (data not shown).
Third, we showed that TPA- and ConA-mediated suppression of
Fas-induced apoptosis were unaffected by the p38/MAPK-inhibitor, SB202190. This is also in line with a previous report (15) in which
another widely used p38/MAPK-inhibitor, SB203580, did not influence
ConA-mediated protection from Fas-induced apoptosis in Jurkat cells. In
addition to the PI3K/Akt and p38/MAPK pathway, the JNK/SAPK pathway has
been implicated in protection from Fas-induced apoptosis in some cell
types (45, 46). However, in agreement with a recent study (20), we
found that JNK activity was unaffected by TPA in Jurkat cells (data not
shown). Thus, neither the PI3K/Akt pathway nor the stress-activated MAP
kinases, p38/MAPK and JNK, seem to play any significant role in
TPA-mediated suppression of Fas-induced apoptosis in Jurkat T cells.
It was not clear to us why there have been conflicting reports
regarding the involvement of the MEK/ERK pathway in TPA-mediated protection from Fas-induced apoptosis in Jurkat cells (15, 16, 21, 22,
27). One possible reason could be that previous studies have been based
on the use of only PD98059 as a MEK inhibitor. We, and others (31),
have experienced that PD98059 can be difficult to work with because of
its very limited solubility in aqueous solution. When PD98059 was not
properly solubilized (i.e. crystals were formed in the
medium) it had no effect on TPA-mediated suppression of Fas-induced
apoptosis (data not shown). We therefore used two structurally
unrelated MEK-inhibitors (PD98059 and U0126) to analyze the effect on
apoptosis. Moreover, we assessed apoptosis by three different methods,
based on morphological (cell size and granularity), biochemical (PARP
cleavage), and molecular (DNA fragmentation) criteria, respectively.
Because we obtained essentially the same results with both inhibitors
on all three apoptotic criteria, we concluded that the MEK/ERK pathway
is indeed required for TPA-mediated suppression of Fas-induced
apoptosis in Jurkat cells.
When utilizing small, cell-permeable inhibitors, there is a
general problem of potential unspecific effects. However, we strongly believe that the inhibitor-induced effects we observed in the present
study were due to specific interference with the activities of the
targeted proteins. First, in a recent study, U0126, PD98059, wortmannin, and SB202190 were all found to be among the most specific of commonly used protein kinase inhibitors (47). Second, we demonstrated the specificity of the effects of U0126 and BAY11-7082 by
the excellent correlations between inhibitor-induced repression of ERK
and NF
B activities, respectively, and their abilities to antagonize
the anti-apoptotic effect of TPA. Finally, we directly assessed the
ability of U0126 to inhibit NF
B activity and, conversely, that of
BAY11-7082 to inhibit AP-1 activity. Indeed, we did not find any
unspecific inhibitory effects; if any, U0126 and BAY11-7082 rather
slightly increased the activities of NF
B and AP-1, respectively, which in fact may be due to an unleashing of the competition of AP-1
and NF
B for a common co-factor, such as p300 (48).
In conclusion, we propose a model where TPA-mediated
suppression of Fas-induced apoptosis in Jurkat T cells is dependent on the activation and cooperative action of both ERK and NF
B (Fig. 7).
In this model, the anti-apoptotic effects of ERK and NF
B are
suggested to lie upstream of the mitochondrial membrane disruption event. It will be important to identify the downstream targets that are
responsible for the anti-apoptotic effects of ERK and NF
B to further
understand how Fas-induced apoptosis is regulated in human T cells.