From Chiron Technologies, Chiron Corporation, Emeryville, California 94608
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ABSTRACT |
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Fas ligand and tumor necrosis factor (TNF)
bind to members of the TNF receptor superfamily. Stimulation by Fas
ligand results in apoptosis, whereas TNF induces multiple effects
including proliferation, differentiation, and apoptosis. Activation of
the c-Jun N-terminal kinase (JNK) and p38 kinase pathways is common to
Fas and TNF signaling; however, their role in apoptosis is
controversial. Fas receptor cross-linking induces apoptosis in the
absence of actinomycin D and activates JNK in a
caspase-dependent manner. In contrast, TNF requires
actinomycin D for apoptosis and activates JNK and p38 kinase with
biphasic kinetics. The first phase is transient, precedes apoptosis,
and is caspase-independent, whereas the second phase is coincident with
apoptosis and is caspase-dependent. Inhibition of early
TNF-induced JNK and p38 kinases using MKK4/MKK6 mutants or the p38
inhibitor SB203580 increases TNF-induced apoptosis, whereas expression
of wild type MKK4/MKK6 enhances survival. In contrast, the Mek
inhibitor PD098059 has no effect on survival. These results demonstrate
that early activation of p38 kinase (but not Mek) are necessary to
protect cells from TNF-mediated cytotoxicity. Thus, early stress kinase
activation initiated by TNF plays a key role in regulating
apoptosis.
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INTRODUCTION |
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Fas ligand (FasL)1 and
tumor necrosis factor (TNF) bind to members of the TNF receptor
superfamily (1). Fas/CD95/APO-1 receptor oligomerization induced by
FasL or by agonist antibodies results in apoptosis in a variety of
cells, including T, B, and NK cells, macrophages, and fibroblasts (2).
TNF binds to two ubiquitously expressed receptors, TNF receptor I
(TNFRI/p55) and TNF receptor II (TNFRII/p75), that do not share any
homology within their cytoplasmic domains (3). Unlike FasL, TNF elicits
a wide range of cellular effects, including apoptosis, proliferation, differentiation, inflammation, and chemotaxis (4).
FasL and TNF activate apoptotic signaling pathways through a similar
mechanism. Fas and TNF interact either directly or indirectly with the
adapter protein FADD/MORT1, which recruits caspase 8 to the receptor
complex (reviewed in Ref. 3). The resulting cascade of caspase
activation causes cleavage of cytosolic, cytoskeletal, and nuclear
proteins and leads to apoptosis (5). TNFRI and TNFRII also associate
with molecules that do not interact with Fas. TRAF2 binds directly to
TNFRII and is indirectly associated with TNFRI via TRADD (3). TRAF2
mediates rapid activation of NF-B, JNK, and p38 kinases in response
to TNF (6-8). The Raf-Mek-Erk (MAPK) cascade can be activated through
FAN, a protein that binds to TNFRI and activates neutral
sphingomyelinase (9).
Although JNK and p38 kinase pathways are activated by both Fas and TNF
receptor oligomerization (10-13), it is not clear whether this occurs
through a common mechanism. JNK and p38 kinase are key mediators of the
inflammatory response and are activated by cytokines, growth factors,
and a variety of cellular stresses, including UV and, ionizing
radiation, hyperosmolarity, and heat shock (reviewed in Ref. 14). JNKs
are activated by phosphorylation on Tyr and Thr by the dual specificity
kinases, MKK4/SEK1 (15, 16) and the newly identified MKK7 (17).
Similarly, p38 kinases are activated by MKK3 and MKK6/SAPKK3; MKK6 is
the predominant activator of p38 (18-20). Downstream effectors of
the stress pathways include the transcription factors c-Jun, ATF-2, and
Elk-1, which are phosphorylated and activated in response to the JNK
cascade, whereas ATF-2, Elk-1, Max, and the kinases MAPKAP-2/3 and
MNK1/2 (21, 22) are activated by the p38 cascade (reviewed in Ref. 14).
The role of the stress-activated kinases during apoptosis induced by
various stimuli is controversial. Several published reports propose
that overexpression of constitutively active forms of stress kinase
regulators, such as MEKK1 (23-26), ASK1 (27), and MKK6b (28), result
in apoptosis. Conversely, stable cell lines expressing dominant
negative mutants of MKK4 (29) or JNK1 (30) inhibit apoptosis stimulated
by various agents, suggesting that stress kinases play a proactive role
during apoptosis. In contrast, other reports suggest that stress
kinases do not promote apoptosis. For example, transient expression of
catalytically inactivated MEKK1, MKK4, or c-Jun mutants does not impair
Fas- or TNF-induced apoptosis (6, 31) but can inhibit
-radiation-induced and UV-C radiation-induced apoptosis (24).
Furthermore, thymocytes from mice deficient in MKK4 are more
susceptible to Fas- and CD3-induced apoptosis than wild type
thymocytes, suggesting that the JNK pathway may protect cells from
apoptosis (32). These widely varying conclusions highlight the
importance of understanding differences in stress kinase signaling in
response to various apoptotic stimuli.
This report defines an important new role for JNK and p38 kinases during TNF-induced apoptosis. Our results indicate that JNK and p38 kinases are activated through distinct mechanisms during Fas- and TNF-induced apoptosis. While Fas receptor cross-linking induces apoptosis in the absence of actinomycin D (Act. D) and activates JNK in a caspase-dependent manner, TNF requires Act. D for apoptosis and activates JNK and p38 kinase with biphasic kinetics. The first phase is transient, precedes apoptosis, and is caspase-independent, whereas the second phase is coincident with apoptosis and is caspase-dependent. Inhibition of early TNF-induced JNK and p38 kinase by expression of dominant negative mutants of MKK4, MKK6, or the p38 inhibitor SB203580 increased TNF-induced apoptosis, whereas expression of wild type MKK4 and MKK6 enhanced cell survival in the presence of TNF. In contrast, the Mek inhibitor PD098059 had no effect on survival. In addition, neither JNK nor p38 kinase is required during Fas- or TNF-induced apoptosis; rather, late phase activation is a response to caspases. These results demonstrate that early activation of JNK and p38 kinase (but not Mek) mediates cell survival signals during exposure to TNF.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and Reagents--
KYM-1 cells were obtained from
the ATCC (Rockville, MD) and were maintained in RPMI 1640 medium
supplemented with 10% fetal calf serum (Summit Biotechnologies, Ft.
Collins, CO), 8 mM L-glutamine, penicillin/streptomycin (50 µg/ml each; Irvine Scientific, Santa Ana,
CA). NIH-3T3 cells (ATCC) were maintained in Dulbecco's minimal essential medium (high glucose), 10% calf serum (Summit
Biotechnologies) supplemented as above. L929-cyt16 cells were
maintained in Dulbecco's minimal essential medium supplemented with
10% fetal calf serum (Summit Biotechnologies), 8 mM
L-glutamine, penicillin/streptomycin (50 µg/ml), and
Geneticin (400 µg/ml, Life Technologies, Inc.). L929 cells were
starved in Dulbecco's minimal essential medium (0.1% bovine serum
albumin, penicillin/streptomycin, 10% Opti-MEM) and KYM-1 cells in
AIM-V medium (Life Technologies, Inc.) 20-24 h prior to induction of
apoptosis. Fas-induced apoptosis in L929-cyt16 cells was initiated by
cross-linking the chimeric receptors using 500 ng/ml rat anti-murine
CD4 (Caltag, South San Francisco, CA) for 30 min at 37 °C followed
by 5 µg/ml goat anti-rat IgG+IgM (The Jackson Laboratory, West Grove,
PA). TNF-induced apoptosis was initiated with 20 ng/ml TNF (murine,
Boehringer Mannheim; human, Chiron) ± 0.5 µg/ml Act. D (Sigma). The
caspase inhibitor Z-VAD-FMK (Kamiya, Seattle, WA) was preincubated with
cells for 1 h prior to antibody cross-linking or TNF addition.
Cell extracts were prepared after the appropriate incubation periods by
rinsing cells in cold phosphate-buffered saline followed by lysis in
Triton lysis buffer ((20 mM Tris-HCl (pH 7.5), 137 mM NaCl, 15% glycerol, 1% Triton), Complete inhibitor
mixture (Boehringer Mannheim), 1 µM pepstatin (Boehringer
Mannheim), 1 mM sodium vanadate, and 25 mM
-glycerophosphate). Extracts were then cleared at 14,000 rpm for 10 min and quick frozen. Protein concentration was determined by Coomassie
Plus Assay (Pierce) using bovine serum albumin as a standard.
Caspase Activity Assay-- Caspase 3 activity was measured in vitro by incubating 10 µg of cell extract with 20 µM Z-DEVD-AFC fluorescent substrate (Kamiya) in 100 µl of Triton lysis buffer supplemented with 1 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 2 mM dithiothreitol (33). Substrate cleavage was allowed to occur for 20 min at room temperature, and released fluorescence was measured in a 96-well plate fluorometer (excitation, 400 nm; emission, 505 nm). The assay was found to be linear at maximum caspase activation under these conditions.
Plasmid Constructs--
Flag tagged MKK4, JNK1, and
GST-jun(1-79) cDNAs were a generous gift from R. Davis.
Flag-tagged MKK4, MKK4KR, and NTRAF2 were inserted into the CMV
promoter-driven expression vector pCG (8, 34). cDNA encoding
I
B
(a gift from S. Haskill) was PCR amplified and inserted
in-frame into the pCGF vector (pCG modified to include an N-terminal
Flag tag). I
B
phosphorylation sites Ser-32 and Ser-36 were
mutated to Ala (I
B
SR) (35) using the Quickchange PCR mutagenesis
kit (Stratagene, La Jolla, CA). Full-length MKK6 was cloned from an
expressed sequence tag (GenBankTM accession no. R42128, Genome
Systems, St. Louis, MO) by PCR amplification and inserted in-frame into
pCGF. MKK6 mutants to replace Lys-82 with Met (MKK6KM) (18) were made
by two step PCR mutagenesis using express sequence tag R42128 as a
template and inserted into pCGF. JNK1 and p38
cDNAs were PCR
amplified and inserted in-frame into pCGN (pCG modified to include an
N-terminal HA tag). The nucleotide sequence of all PCR-generated
inserts was verified by sequence analysis. Expression of each construct was verified in cell lysates by Western blotting using an anti-Flag antibody (Eastman Kodak Co.).
Cell Transfections and TNF Sensitivity-- NIH-3T3 cells were co-transfected with a green fluorescent protein expression construct pEGFP-N1 (CLONTECH, Palo Alto, CA) and either pCDNA3 (vector) or the various test constructs described above, using LipofectAMINE transfection reagent (Life Technologies, Inc.). Following a 12-h expression period, murine TNF (0.5 ng/ml) was added to the culture medium for 10 h (Boehringer Mannheim). Transfected cells were viewed by fluorescent microscopy, and sensitivity to TNF was measured by counting the number of surviving transfected cells in 10 representative microscopic fields with and without TNF treatment. TNF-induced apoptosis is expressed as the decrease in cell viability in relation to an untreated control sample for each test plasmid. To assess JNK and p38 kinase activity, NIH cells were co-transfected with HA-JNK1 or HA-p38 and various test constructs as described above. Following a 16-h expression period, cells were starved in Dulbecco's minimal essential medium supplemented with 0.3% calf serum for 24 h and then treated with 20 ng/ml murine TNF for 15 min. Cells were rinsed twice in cold phosphate-buffered saline and then lysed in Triton lysis buffer, and kinase activity was measured as described below.
JNK and p38 Kinase Assays--
The activity of endogenous JNKs
was assessed by solid phase pull-down kinase assays as described
previously (36). Briefly, 100 µg of cell extract was incubated in the
presence of 8 µg of recombinant GST-c-Jun (1-79) as substrate and
GSH-Sepharose beads (Sigma) for 3 h at 4 °C. GST-Sepharose
beads were then washed in JNK reaction buffer (25 mM HEPES,
pH 7.4, 25 mM -glycerophosphate, 25 mM
MgCl2, 2 mM dithiothreitol, and 0.1 mM sodium vanadate). The in vitro kinase
reaction was carried out in 30 µl of JNK reaction buffer supplemented
with 20 µM [
-32P]ATP (Amersham Pharmacia
Biotech) for 20 min at 32 °C. Transfected JNK1 and p38
kinases
were immunoprecipitated with anti-HA antibodies (Babco, Richmond, CA)
and protein A-Sepharose beads (Amersham Pharmacia Biotech) 3 h at
4 °C. The beads were rinsed twice in Triton lysis buffer and once in
JNK reaction buffer. The in vitro kinase reaction was
carried out as above but with 40 µM ATP using GST-jun as
a substrate for JNK and Phas-I (Stratagene) as a substrate for p38
.
Phosphorylated substrates were resolved by SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose, and relative phosphorylation was quantitated using the Bio-Rad Molecular Imager system. The expression levels of JNK and p38
were verified by Western blotting using the anti-HA antibody (Babco).
MTT Assay-- Approximately 5 × 104 L929-cyt16 cells per well were plated into 96-well plates, allowed to grow for 36 h, and then pretreated for 30-60 min with Me2SO, Act. D (0.5 µg/ml), or various concentrations of the inhibitors PD098059 (New England Biolabs, Beverley, MA) or SB203580 (Calbiochem) in starvation medium prior to TNF addition. Cells were incubated in TNF at 0, 1, 10, or 100 ng/ml in triplicate for 6 h, after which, 10 µl of WST-1 reagent (Boehringer Mannheim) was added for an additional 2 h. The optical density of each sample (in triplicate) was determined at 450 nm using a Thermomax microplate reader (Molecular Devices, Menlo Park, CA).
Annexin-V Apoptosis Assay-- L929-cyt16 cells were starved for 20-24 h in starvation medium and then pretreated with the inhibitors PD098059 and SB203580 for 30 min prior to addition of TNF (10 ng/ml; Boehringer Mannheim). After 5 h of TNF treatment, cells were rinsed once in phosphate-buffered saline, stained with the ApoAlert Annexin V apoptosis kit (CLONTECH) according to the manufacturer's instructions, and then analyzed on a fluorescence-activated cell sorter (Epics, Coulter, Miami, FL).
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RESULTS |
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The kinetics of Fas-induced JNK activity correlate with caspase activation and apoptosis. The relationship between Fas-induced apoptosis, caspases, and JNK activation was investigated in a murine fibroblast cell line stably expressing a chimeric receptor consisting of the extracellular and transmembrane domains of murine CD4 fused to the cytoplasmic domain of murine Fas (L929-cyt16) (37). Within 30 min after receptor cross-linking, using anti-CD4 and secondary cross-linking antibodies (Fig. 1A), the cells began to exhibit membrane blebbing and nuclear condensation. Nearly 90% of the cells were apoptotic within 90 min in either the presence or absence of Act. D (Fig. 1A). Therefore, Fas-induced apoptosis is neither enhanced nor inhibited by signaling pathways leading to new gene expression.
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The kinetics of JNK and caspase activation were measured in cell extracts from L929-cyt16 cells made over a similar time course. Initiation of the Fas pathway activated DEVD-specific caspases with kinetics that preceded the appearance of membrane blebs (compare Fig. 1A and 1B). The kinetics of endogenous JNK activation closely resembled those of caspase activation but were slightly delayed compared with them, beginning at approximately 30 min after antibody cross-linking. Similar results were also obtained using the agonist CH-11 anti-Fas antibody in Jurkat cells expressing endogenous Fas (data not shown). Therefore, caspase activity precedes JNK activation during Fas-induced apoptosis.
The Caspase Inhibitor Z-VAD-FMK Inhibits Fas-induced JNK Activity-- To investigate whether JNK activation induced by Fas was caspase-dependent, L929-cyt16 cells were pretreated with various concentrations of the caspase inhibitor Z-VAD-FMK, followed by chimeric receptor cross-linking. Fas-induced JNK activation and apoptosis were inhibited in a dose-dependent manner up to 100 µM Z-VAD-FMK (Fig. 1C). Although caspase activity was inhibited at lower concentrations of Z-VAD-FMK, our assay measured only DEVD-directed caspases, which may be more susceptible than other caspases to Z-VAD-FMK inhibition. These results demonstrate that caspase activity, in addition to that which cleaves DEVD-containing peptides, is required for Fas-induced JNK activation.
TNF Induces JNK and p38 Kinase Activity with Biphasic Kinetics-- The kinetics of stress kinase activation and their dependence on transcription and caspase activation were also examined in response to TNF. L929-cyt16 cells were treated over a 6-h time course with TNF in the presence or absence of Act. D, and apoptotic cells were counted. In the absence of Act. D, 15% of cells died, whereas in the presence of Act. D, apoptosis increased to greater than 95% (Fig. 2). This is in contrast to Fas-induced apoptosis in the same cell line where Act. D had no effect (Fig. 1A). These results suggest that immediate early transcriptionally mediated events protect cells from TNF-induced cytotoxicity and are consistent with similar effects in other fibroblast cell lines (38-40).
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TNF-induced JNK Activation Occurs by Caspase-dependent and Caspase-independent Means-- To confirm the relationship between JNK and caspase activation during TNF-induced apoptosis, extracts were prepared from KYM-1 cells treated with TNF in the presence or absence of Z-VAD-FMK. Z-VAD-FMK did not affect the early phase of TNF-induced JNK activity at 15 min (Fig. 3), but it did inhibit late phase JNK at 4 h by almost 50%. Similar results were observed in L929-cyt16 cells. Early JNK was not affected by caspase inhibition; however, late phase JNK could not be assessed because of the obvious cytotoxicity of ZVAD-FMK after 3 h of TNF treatment (data not shown). Taken together, our data demonstrate that early phase JNK activation is caspase-independent, whereas late phase JNK is partially caspase-dependent.
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Inhibition of JNK and p38 Kinase Pathways Sensitize Cells to
TNF-induced Apoptosis--
The results described above suggest that
TNF induces an early peak of stress kinase activation that does not
promote apoptosis. To investigate whether the early phase of JNK and
p38 kinase protects cells from apoptosis, we inhibited each pathway by
transiently expressing dominant negative mutants of MKK6 (MKK6KM) or
MKK4 (MKK4KR) (15, 18). NIH-3T3 cells were used because L929-cyt16 cells were difficult to transfect to high efficiency. The expression levels from each construct were equalized as indicated by Western blotting using an anti-Flag antibody (Fig.
4B). Treatment of cells with
TNF alone (0.5 ng/ml) for 10 h induced cytotoxicity in
approximately 30% of cells transfected with vector (Fig.
4A). However, in cells expressing MKK6KM or MKK4KR,
TNF-induced apoptosis increased to 60%, suggesting that JNK and p38
pathways are required for survival in response to TNF. Consistent with
this view, expression of either MKK6-wt or MKK4-wt reduced apoptosis to
15 and 25%, respectively, indicating that JNK and p38 pathways enhance
cell survival during TNF exposure. A similar increase in TNF-induced
apoptosis (30% over vector control) was also observed using a
super-repressor IB
containing phosphorylation site mutations
(I
B
SR) that prevent inducible NF-
B DNA binding activity (35)
(Fig. 4A). These data are consistent with those reported by
others (41, 42). In vitro kinase assays indicate that
overexpression of either MKK6-wt or MKK4-wt stimulates both JNK and p38
kinases; similarly, expression of MKK6KM or MKK4KR inhibits both JNK
and p38 kinase (Fig. 4, C and D). Therefore, in
transient expression analysis, we cannot distinguish between the role
of JNK and p38 kinase. Although it appears that I
B
-SR expression
inhibits JNK and p38 kinase activity, it visibly impairs expression of
the HA-JNK and HA-p38 reporter constructs and therefore may indirectly
affect kinase activity.
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Chemical Inhibitors of p38 but Not MEK Sensitize Cells to
TNF-induced Apoptosis--
In addition to JNK and p38, the MAPK
cascade is activated in response to TNF (8, 9). We made use of specific
kinase inhibitors to investigate the role of Mek during TNF-induced
apoptosis and to address the specific protective role of p38 kinase in
L929-cyt16 cells. The inhibitor PD098059 is specific for Mek1/Mek2
(43), whereas SB203580 specifically inhibits p38 kinase (44, 45). L929-cyt16 cells were pretreated with each inhibitor for 30 min and
then with various concentrations of TNF for several hours. TNF
sensitivity was then assessed in two different assays: 1) cell
viability in a MTT assay (46) (Fig.
5, A and B), and 2) Annexin V binding, an early marker of apoptosis (47) (Fig.
5C). The p38 inhibitor SB203580 increased TNF-induced cell
death in a dose-dependent manner (Fig. 5, A and
C). Endogenous JNK activity was not affected by SB203580 at
either concentration or by PD098059 (data not shown), demonstrating
that p38 kinase is required for protective signals during TNF exposure.
The MEK inhibitor PD098059 did not affect TNF-induced apoptosis even at
30 µM, a concentration at which TNF-induced
phosphorylation of Erk1/Erk2 was completely inhibited (data not shown).
These results demonstrate that the MAPK cascade is not involved in a
protective response to TNF (Fig. 5, B and C).
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DISCUSSION |
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This report outlines differences in stress kinase activation
between Fas and TNF and defines an important protective role for JNK
and p38 kinases during TNF signaling. Our results demonstrate that
early p38 kinase but not Mek signaling pathways are necessary for cell
survival in the presence of TNF (Figs. 4 and 5). Previous work shows
that TNF-induced NF-B is also necessary for cell survival (6, 41,
42, 48). More recent results indicate that cells from TRAF2-deficient
mice activate NF-
B but not JNK activity in response to TNF and are
more susceptible to apoptosis than cells from wild type mice (49, 50).
Both reports suggest the existence of TRAF2-dependent
signaling pathways distinct from NF-
B that also protect cells from
apoptosis. JNK and p38 kinase may be such protective pathways. Although
the use of dominant negative mutants in transient expression assays
does not permit us to discriminate between the activities of JNK and
p38, it is likely that their activities overlap because of common
downstream effectors (14). However, the specificity of the SB203580
inhibitor allows us to define a specific requirement for p38 kinase as
a mediator of protective signals. Our data suggest that although early
stress kinase activation is necessary, it may not be sufficient to
confer complete protection against TNF-induced apoptosis (Fig. 4).
Most cultured cell lines require inhibitors of transcription or
translation for a cytotoxic response to TNF (Fig. 3) (39, 40, 51, 52),
suggesting that protective mechanisms may involve new gene induction.
The NF-B regulated genes A20 (53), manganese superoxide
dismutase (54) and c-IAP2 (55) are up-regulated following
TNF treatment and protect cells from apoptosis. Although studies show
that JNK, p38 kinase, and NF-
B pathways diverge downstream of TRAF2
(6, 8, 56), each pathway leads to activation of transcription factors,
such as c-Jun, ATF-2, and NF-
B, which can interact and may cooperate
during transcription (57, 58). Surprisingly, the MAPK pathway does not
contribute to these survival mechanisms (Fig. 5), suggesting that
downstream effectors of the MAPK pathway, some of which are common to
JNK and p38 (14), are not involved in protective gene induction. However, our results also suggest that protective mechanisms for TNF
may be posttranscriptionally regulated (Fig. 6). The recently identified p38 substrates Mnk1 and Mnk2 can phosphorylate eIF-4E (22),
an essential step during translation initiation (59). In addition, p38
kinase enhances translation of specific messages containing the AUUUA
repeat motif in their 3'-untranslated region (45). Therefore, early
coordinate activation of JNK, p38 kinase, and NF-
B pathways is a key
regulatory mechanism for TNF-induced apoptosis.
Our results demonstrate that JNK and p38 kinases are activated through distinct mechanisms during Fas- and TNF-induced apoptosis. Fas-induced JNK activation is entirely caspase-dependent in L929-cyt16 cells (Fig. 1). Fas-mediated JNK, MKK6b, and p38 kinase activation is also caspase-dependent in the lymphoid cell lines SKW 6.4 (60) and Jurkat (13, 24, 28, 31). In contrast, this is the first demonstration that early induction of JNK and p38 activity by TNF is independent of caspases and apoptosis, whereas late phase JNK and p38 correlate with caspase activation, are partially caspase-dependent, and occur only during apoptosis (Figs. 2 and 3 and data not shown). The lack of early stress kinase activation during Fas signaling may be explained by evidence linking TRAF2 to TNF but not Fas receptor complexes (61, 62).
There are several mechanisms by which caspases may activate stress response pathways. Caspases cleave and activate D4-GDI/Ly-GDI (63), hPAK65/PAK2 (64, 65), and MEKK-1 (26) and can generate ceramide (66, 67); these are four upstream stimulators of JNK and p38 kinases. Our results show that late phase TNF-stimulated JNK activity cannot be completely inhibited by Z-VAD-FMK (Fig. 3). This phase of JNK may be independently stimulated by oxygen radicals released from the mitochondria, which in L929 cells begins after 1 h of TNF treatment (68). Mitochondrial breakdown may also explain why late phase JNK activation slightly precedes caspase activation (Fig. 2). Our results demonstrate that neither JNK nor p38 activity is required for Fas- or TNF-induced apoptosis (Figs. 4-6). This is in agreement with recent results indicating that short term inhibition of JNK or p38 pathways cannot prevent TNF- or Fas-induced apoptosis in fibroblasts and T cells (6, 13, 31, 69). Therefore, late phase Fas- and TNF-induced stress kinase activation is a response to caspase activation and does not contribute to cell death.
Although Fas and TNF share homology within their cytoplasmic domains,
as well as some common associated molecules, our results show that
signaling to apoptosis through the two receptors is clearly distinct in
at least three ways. First, the kinetics of Fas-induced apoptosis in
L929 cells are more rapid than those induced by TNF under permissive
conditions (Figs. 1 and 2). This observation holds in L929 cells
expressing human Fas/APO-1 (70) and in several other cell lines
expressing endogenous Fas and TNFRI (71). Second, signaling to JNK and
p38 kinase is initiated within 10 min of TNF treatment, long before
caspase activation; these pathways are not initiated prior to the
induction of caspase activity after Fas cross-linking (Figs. 1 and 2)
(13, 70). Third, most cultured cell lines require inhibitors of
transcription or translation for a cytotoxic response to TNF (Fig. 3)
(39, 40, 51, 52), whereas Fas will generally cause apoptosis without a
strict requirement for metabolic inhibitors (Fig. 1A) (72,
73). TNF-stimulated stress kinase activation and NF-B may lead to
protective gene expression, suggesting a mechanism by which Act. D can
enhance TNF-mediated but not Fas-mediated apoptosis in L929-cyt16 cells
(Figs. 1 and 2). In fact, evidence suggests that early TNF signaling
may induce survival mechanisms that prevent TNF-induced but not
Fas-induced apoptosis (73). Together, these data outline clear
distinctions in the mechanism regulating Fas- and TNF-induced
apoptosis, a mechanism that, for TNF, involves early coordinate
activation of JNK, p38 kinase, and NF-
B pathways.
In summary, this study characterizes differences in signaling
mechanisms between Fas and TNF that regulate apoptosis (Fig. 7). We have identified a new role for JNK
and p38 kinases as essential components of an early response to TNF
that, in combination with NF-B, mediate survival signals that
protect cells from apoptosis. Furthermore, MAPK is not involved in this
protective response. Because the onset of TNF-induced apoptosis is
delayed, early activation of protective signals may have time to act in
the absence of caspase activation, possibly by regulating new gene
expression. Activation of JNK and p38 kinase during Fas-induced
apoptosis and during the late phase of TNF-induced apoptosis is not
required for cell death but rather is a stress response to caspase
activation. Tight regulation of survival versus apoptotic
signals may contribute to the ability of TNF to elicit a wide range of
cellular effects. The identification of genes involved in survival
mechanisms may revive the therapeutic potential for TNF in cancer
treatment.
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ACKNOWLEDGEMENTS |
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We are very grateful to Dr. Roger Davis for
cDNAs encoding MKK4, JNK1, and GST-jun(1-79); Dr. Steve Haskill
for providing the IB
cDNA; and Dr. Keting Chu for the
L929-cyt16 cell line. We thank Dr. Marty Giedlin and Tim Brown for help
with fluorescence-activated cell sorter analysis. We are also grateful
to Drs. Mike Kavanaugh, Steve Harrison, Klaus Giese, Bert Pronk, and
Wendy Fantl for helpful comments on the manuscript.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed: Chiron Corporation,
4560 Horton St., M-338, Emeryville, CA 94608. Tel.: 510-923-4039; Fax:
510-923-4115; E-mail: Anne_Roulston{at}cc.chiron.com.
1
The abbreviations used are: FasL, Fas ligand;
TNF, tumor necrosis factor ; TNFRI, TNF receptor I; MAPK,
mitogen-activated protein kinase; Act. D, actinomycin D; PCR,
polymerase chain reaction; GST, glutathione S-transferase;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium
bromide.
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REFERENCES |
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