Institute of Biochemistry, University of
Lausanne, BIL Biomedical Research Center,1
and Apotech Biochemicals,
Biopôle,2 CH-1066 Epalinges, Switzerland
Received 1 February 2001/Returned for modification 16 March
2001/Accepted 24 May 2001
 |
INTRODUCTION |
Members of the tumor necrosis factor
(TNF) receptor family and their corresponding ligands are critical
regulators of apoptosis and various other cellular processes. Some of
the receptors (Fas, TNF-R1, TRAIL-R1, TRAIL-R2, TRAMP/DR3, DR6, and
EDA-R) contain a cytoplasmic region, called the death domain (DD),
which is essential for cell death signaling (18, 22).
Signals emanating from Fas and TNF-R1 have been intensively studied
(14). Upon receptor activation, the DD of Fas undergoes
direct homotypic interaction with a DD in the adapter protein FADD,
while FADD recruitment is indirect (via TRADD) in the case of TNF-R1
(4). The death effector domain (DED) at the amino terminus
of FADD then recruits pro-caspase 8 via homotypic interaction with its
two DEDs. The high local concentration of caspase 8 zymogens
facilitates self-processing and assembly of the mature enzyme.
Activated caspase 8 initiates apoptosis by subsequent cleavage of
downstream caspases (caspase-3, -6, and -7).
Death induced by death receptors is tightly regulated by genes that are
activated by the transcription factor NF-
B (25). Modulation of the response in favor of NF-B protects cells from apoptosis; failure to do so results in increased cell death. At least
six NF-B-responsive genes are involved in this survival amplification loop (26), i.e., those that encode IAP-1 and
IAP-2, which block caspase activity (7); that which
encodes the Bcl-2 family member A1 (25); and those that
encode TRAF-1, TRAF-2 (1), and A20 (21),
which are themselves implicated in the NF-B signaling pathway.
However, overexpression of all of these genes affords, at best, partial
protection, in particular from death triggered by TNF
(25). The only known potent inhibitor of death receptor
signals is c-FLIP. Two c-FLIPs have been characterized (23,
24). The full-length 55-kDa-long form of FLIP
(FLIPL) exhibits overall structural homology to
caspase 8, containing two DEDs that interact with FADD and an inactive
caspase-like domain. An alternatively spliced short form of FLIP
(FLIPS) contains only the two DEDs and displays
reduced antiapoptotic capacity.
We undertook a series of experiments to investigate whether c-FLIP is
implicated in the antiapoptotic NF-B response. Here we provide
evidence that FLIP expression is upregulated upon the stimulation of
several signaling pathways that are known to trigger activation of the
transcription factor NF-B. Moreover, we found that cells that were
rendered highly sensitive to death ligand-induced apoptosis by blocking
NF-B activation could be rescued by expressing FLIP. FLIP may
therefore play a key role in the NF-B-mediated control of death signals.
| |
MATERIALS AND METHODS |
Antibodies and materials.
Rabbit anti-TRAF2 polyclonal
antibody C20, rabbit anti-cIAP1 polyclonal antibody H-85, and mouse
anti-TRAF1 monoclonal antibody H3 were purchased from Santa Cruz
Biotechnology (Santa Cruz, Calif.). Rat anti-cFLIP monoclonal antibody
Dave II was from Alexis, Lausen Switzerland, and anti-Phospho-IBa
antibody was from Biolab. Anti-tubulin and anti-Flag (M2) antibodies
were from Sigma (St Louis, Mo.). Anti-myc tag antibody (9E10) and
anti-hemagglutinin (anti-HA) antibody were purchased from Babco
(Berkeley, Calif.). Anti-mouse CD40 antibody (hybridoma FGK45) was a
gift from Ton Rolink (Basel, Switzerland). Human recombinant ligands
(TRAIL, Fas ligand [FasL], and TNF) were obtained from Alexis.
Lipopolysaccharide (LPS), phorbol myristate acetate (PMA), ionomycin,
and granulocyte-macrophage colony-stimulating factor were from Sigma.
Cell culture.
HeLa and HT1080 (human fibrosarcoma) cells,
wild type (wt) and I-B mutant (I-Bmut), were cultured in
Dulbecco's modified Eagle's medium (Gibco BRL Life Technologies,
Gaithersburg, Md.) supplemented with 10% fetal calf serum and
penicillin and streptomycin (each at 50 µg/ml) and grown in 5%
CO2 at 37°C. Mouse lymphoma EL4 and A20 cells
and Jurkat cells were cultured as described above in RPMI 1640 medium. Dendritic cells were obtained from purified human
CD34+ cells as described previously
(2). NEMO-deficient cells were a kind gift of S. C. Sun (8).
Retrovirus production and cell transduction.
The retroviral
vector pBabe-Puro and generation of viruses have been described
previously (17). Human Flag-tagged
FLIPL, HA-tagged FLIPS,
Flag-tagged TRAF1, Flag-tagged TRAF2, and myc-tagged c-IAP1 were
subcloned, respectively, from pcDNA3 plasmids (10, 11)
into pBabe-Puro. HT1080 wt or I-Bmut cells (1.5 × 106) (10) were transduced for
16 h with viral supernatants containing Polybrene (8 µg/ml).
Cells were washed once in phosphate-buffered saline, harvested, plated
in complete medium containing puromycin (2.5 µg/ml), and incubated
for 3 days before amplification and subsequent analysis of the
multiclonal population.
Cell death and viability assays.
Jurkat cells were
harvested, washed with RPMI medium, and plated at a density of 10 × 106/ml prior to stimulation with PMA at 20 ng/ml and 1 mM ionomycin. HT1080 fibrosarcoma cells or transfectants
derived therefrom (1.5 × 104 per well) were
seeded in 96-well microtiter plates in the presence of the indicated
reagents for 16 h, and viability was determined by the methylene
blue colorimetric assay (16). For HeLa cells, viability
was determined by the PMS-MTS method in accordance with the
manufacturer's (Promega, Madison, Wis.) instructions. In some experiments, the number of apoptotic cells was determined by Hoechst staining. To quantitate viability and apoptosis in experiments involving EL4 and A20 cells, phosphatidylserine exposure on apoptotic cells was measured as described previously (13). Briefly,
cells (2 × 105) were washed in ice-cold
HEPES buffer (10 mM HEPES, 150 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 1.8 mM CaCl2, pH
7.4) supplemented with glucose at 1 mg/ml and 0.5% bovine serum
albumin. Fluorescein isothiocyanate-labeled annexin V (Nexins Research,
Kattendijke, The Netherlands) was added to a final concentration of 2.5 µg/ml. Cells were incubated for 20 min at 4°C and washed twice with
HEPES buffer. Before analysis on a FACScan, propidium iodide (PI) was added (final concentration, 5 µg/ml) to the samples to discriminate necrotic cells (annexin V
PI+) from apoptotic cells (annexin
V+ PI and annexin
V+ PI+).
Western blotting.
Cell lysates were prepared in NP-40 buffer
(20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10% glycerol, 0.2%
NP-40) supplemented with a protease inhibitor cocktail stock
solution (Roche Biochemicals, Basel, Switzerland). Cell debris was
removed by centrifugation at 10,000 × g for 10 min,
and the protein concentration was determined by the Bradford assay
(Pierce, Rockford, Ill.). Proteins were resolved by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes by electroblotting, and nonspecific binding
sites were blocked by incubation in PBS containing 0.5% Tween
20 and 5% (wt/vol) dry milk. Immunoblot analyses were performed with
the indicated antibodies. Bound primary antibodies were visualized with
horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G
(IgG), goat anti-rat IgG, or goat anti-mouse IgG (Jackson
Immunoresearch Laboratories, West Grove, Pa.) and ECL (Amersham,
Freiburg, Germany).
RNase protection assay.
HT1080 wt or I-Bmut cells were
treated with TNF for the times indicated in Results. Total RNA was
isolated with the RNA INSTAPURE kit (Eurogentech, Seraing, Belgium) in
accordance with the manufacturer's recommendations. The presence of
transcripts of the indicated apoptosis-related genes, as well as
the internal controls L32 and glyceraldehyde-3-phosphate dehydrogenase
was analyzed by using the hApo3b, hApo5 Multi-Probe template sets
(PharMingen, San Diego, Calif.). Probe synthesis, hybridization,
and RNase treatment were performed with the RiboQuant Multi-Probe
RNase Protection Assay System (PharMingen) in accordance with the
manufacturer's recommendations. Finally, protected transcripts
were resolved by electrophoresis on denaturing polyacrylamide gels
(5%) and quantified on a PhosphorImager with the ImageQuant
software (Molecular Dynamics, Sunnyvale, Calif.). To correct signals of
protected transcripts of special interest for background intensity, the
latter was determined for each individual lane in close proximity to
the respective mRNA signal and subtracted from the value of the
protected transcript.
| |
RESULTS |
The short-lived FLIP protein is induced in response to stimuli
known to induce NF-B activation.
The murine B-cell line A20 is
highly susceptible to Fas-mediated apoptosis but can be rescued by
signaling through its surface B-cell receptor (6, 27).
This effect has been attributed to the upregulation of Bcl-2 family
members (6) and/or FLIP (27). The precise
signaling pathways which result in the upregulation of the inhibitory
proteins is, however, not known. As activation of the transcription
factor NF-B is frequently involved in the protection of cells from
apoptosis, we considered the possibility that the gene for FLIP is also
a target of the NF-B transcription factor. Stimulation of the CD40
receptor is known to strongly activate NF-B via recruitment of
several TRAF proteins. Murine A20 B cells were therefore stimulated
with agonistic antibodies to CD40 for 6 h before FasL addition.
Whereas more than 80% of the cells were killed at a FasL concentration
of 100 ng/ml, only 25% underwent apoptosis when cells had been
prestimulated with anti-CD40 antibodies (Fig.
1A). Western blot analysis revealed that
CD40-stimulated cells had a considerably increased concentration of
FLIP, whereas caspase 8 levels remained unchanged (data not shown).
Upon recruitment to death receptors, FLIPL is
cleaved by caspase 8 between the large and small subunits in the
caspase-like region, thereby inhibiting caspase 8 activity
(11). In FasL-sensitive cells not prestimulated with TNF
or CD40 and thus without increased FLIP levels, only processed FLIP
(p43) was detectable after FasL addition, indicating that the entire
cellular FLIP pool had been used up to form caspase 8-FLIP
heterodimers, thus leaving some caspase 8 unprotected. As a
consequence, caspase 8-caspase 8 homodimer formation was possible,
resulting in caspase 8 autoactivation and cell death. By contrast, in
CD40-prestimulated cells, a substantial amount of the increased
concentration of precursor FLIP was still discernible after FasL
treatment, indicating that the FLIP concentration was still
sufficiently high to block the full processing of all of the caspase 8 molecules that were recruited to the Fas signaling complex (DISC).
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FIG. 1.
Anti-CD40, TNF, or LPS stimulation upregulates FLIP and
partly protects from FasL-mediated apoptosis. (A) A20 lymphoma cells
were pretreated for 24 h with anti-CD40 at 10 µg/ml and treated
with recombinant Flag-tagged FasL (33 ng/ml) in the presence of
cross-linking anti-Flag antibodies at 1 µg/ml for 8 h. Apoptosis
was quantified by annexin V staining, and FLIP protein content was
determined by Western blotting with an anti-FLIP antibody. (B) Human
dendritic cells were either nontreated (left lane) or incubated with
TNF (40 ng/ml) or LPS (10 µg/ml) for 24 h and analyzed by
immunoblot assay for the expression of FLIP and TRAF1.
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We next investigated the capacity of two other stimuli which induce
NF-B activation to upregulate FLIP expression. Immature human
monocyte-derived dendritic cells are resistant to FasL (3, 19), possibly due to the high FLIP levels already present even in nonmature cells (Fig. 1B). Dendritic cell maturation can be achieved
by treating cells with agents that trigger NF-B activation, such as
LPS or TNF (5), and this is reflected by the strong increase in the NF-B-responsive protein TRAF1 (Fig. 1B). We observed that not only the expression of FLIPL but also
that of FLIPS was strongly increased in response
to a 2-day treatment with LPS and TNF (Fig. 1B).
Resistant dendritic cells can be rendered sensitive to FasL by the
protein synthesis inhibitor cycloheximide (CHX) (28). This
was correlated with the disappearance of FLIPL.
We confirmed these observation (data not shown). To explore whether
FLIP was also short-lived in other cells, the murine T-cell lymphoma
cell line EL-4 and the human adenocarcinoma line HeLa, which are both reasonably resistant to apoptosis when treated with FasL at 100 ng/ml,
were incubated with increasing doses of CHX (Fig.
2A and B). In both cell lines, incubation
with CHX at 0.5 µg/ml for 16 h sufficed to cause the
disappearance of detectable levels of FLIPL
protein, whereas levels of 
-tubulin or of the antiapoptotic gene
TRAF-2 remained unchanged. The same sensitivity to CHX was also
observed for FLIPS, which, in contrast to EL-4
cells, is expressed in HeLa cells. As a consequence of the addition of
the protein synthesis inhibitor, EL-4 and HeLa cells became sensitive to FasL (Fig. 2A and B) and underwent apoptosis at CHX concentrations that were identical to those required for FLIP downregulation. Thus,
FLIP appears to be short-lived in several cell lines, requiring continuous biosynthesis to ensure levels that are required for protection against apoptosis.
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FIG. 2.
FLIP downregulation by CHX enhances FasL-induced
apoptosis in EL4 and HeLa cells. (A) EL-4 cells were treated with
increasing concentrations of CHX in the presence or absence of
cross-linked (+M2) FasL at 100 ng/ml for 16 h. Cell viability was
subsequently determined by fluorescence-activated cell sorting using
annexin V staining. FLIP and tubulin protein contents were determined
by immunoblot assay. (B) HeLa cells were treated and analyzed as
described above, except that viability was determined by the
PMS-MTS method. In addition to FLIP and tubulin contents, TRAF2
protein content was analyzed by immunoblot assay. OD490, optical
density at 490 nm.
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The gene for FLIP is an NF-B-responsive gene.
The
above-described experiments suggested that FLIP is upregulated in
response to signals that lead to NF-B activation. To corroborate
this notion, we took advantage of the HT1080 fibrosarcoma cell line,
which expresses a modified form of the NF-B inhibitor I-B that
cannot be degraded due to mutated phosphorylation sites (10,
26). In this cell line (I-Bmut), translocation of NF-B into the nucleus is highly impaired, and thus, synthesis of
NF-B-responsive genes does not occur (10, 26). As a
result, the mutant cell line is highly susceptible to death signals
such as TNF and FasL (10, 26). Both wt and mutant HT1080
cells were treated with small doses of TNF to stimulate NF-B
activation, which, as expected, led to a high level of expression of
NF-B-responsive TRAF-1 in the wt cells but not in the in the
I-Bmut HT1080 cells (Fig. 3A). At the
TNF concentration used (50 ng/ml), expression of TRAF-1 was detectable
at 4 h and reached a maximum between 10 and 24 h, at a time
where no cell death was observed in the I-Bmut cells, and thus the
absence of TRAF-1 expression cannot be attributed to cell death (wt
HT1080 cells). The low concentration of FLIP which is already present
in HT1080 cells before stimulation was increased in wt but not mutant
HT1080 cells, indicating that activation of NF-B is required for
FLIP induction (Fig. 3A). Interestingly, the kinetics of induction of
the two forms of FLIP differed. While FLIPS was
already detectable 4 h after the addition of TNF, induction of
FLIPL was slower and peaked between 10 and
24 h, when the levels of FLIPS had already
decreased. TNF also induced a considerable induction of FLIP mRNA
levels (Fig. 3B), demonstrating that the increase in FLIP protein was,
at least in part, due to increased NF-B-mediated transcription of
the gene for FLIP. While the well-described increase in the TRAF-1 and
c-IAP-1 messages could be confirmed in our experimental setting, no
substantial increase, however, in TRAF-2 and c-IAP-2 was observed. When
the resistance to FasL of the HT1080 cells was subsequently analyzed,
wt but not I-Bmut cells showed increased resistance to FasL upon TNF
pretreatment (Fig. 4), correlating with
the increased FLIP levels. Taken together, these results indicate that
FLIP, similar to the antiapoptotic TRAF and IAP family members
(26), is positively regulated by NF-B following TNF
addition.
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FIG. 3.
NF-B activation leads to FLIP protein and mRNA
upregulation. (A) The HT1080 fibrosarcoma cell line (wt or I-Bmut),
stably transfected with a myc-tagged, mutated, nondegradable version of
I-B, were treated for the indicated periods of time with TNF at
50 ng/ml and analyzed for FLIPL, FLIPS, and
TRAF1 expression by immunoblot assay. (B) RNase protection assay
analysis of mRNA levels of various apoptosis-related genes in wt or
I-Bmut HT1080 cells after treatment with TNF as described above,
using different hApo multiprobe template sets. L32 and
glyceraldehyde-3-phosphate dehydrogenase (GADPH) were included in each
template set as internal controls.
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FIG. 4.
NF-B-mediated upregulation of FLIP protects from
Fas-induced apoptosis. HT1080 wt or I-Bmut cells were pretreated for
2 h with TNF at 50 ng/ml, washed with fresh medium, and
subsequently incubated in the presence of cross-linked FasL at 100 ng/ml for 8 h. Apoptosis was quantified by Hoechst staining. FLIP
and TRAF1 protein contents were analyzed by immunoblot assay. Compared
to Fig. 2, a longer exposure time was chosen to reveal the cleavage of
FLIPL into its p43 fragment upon treatment with FasL. The
values beside the lower left-hand images are molecular sizes in
kilodaltons.
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To demonstrate that the NF-B dependence of FLIP expression is a more
general phenomenon and not restricted to a single cell type, we
investigated FLIP expression in the Jurkat T-cell line. Assembly of the
IKK signalosome and successful signal transmission are dependent on
NEMO/IKK
, which acts as a scaffolding protein (15). We
therefore compared FLIP induction by two known stimuli of NF-B
activation in Jurkat cells, i.e., PMA-ionomycin and TNF, in wt Jurkat
cells and in Jurkat cells that are genetically deficient in NEMO
(8). Upon addition of PMA-ionomycin, levels of
FLIPL, which was barely detectable in
unstimulated cells, increased 10 min after stimulation and peaked after
2 to 4 h (Fig. 5A). In contrast to
HT1080 cells, FLIPS was induced after
FLIPL in Jurkat cells. This sequence of events
was previously observed in primary T cells (9), indicating
that the control of expression of the two FLIP splice variants is cell
type specific. When NF-B was activated with TNF,
FLIPL increased with similar kinetics (Fig. 5B).
The absolute increase in FLIPL was smaller than
in cells stimulated with PMA-ionomycin, which adequately reflects the
fact that the latter stimulus is more potent. No or little FLIP protein induction was observed in NEMO-deficient cells not exposed to the
stimulus (Fig. 5A and B). The absence of NF-B activity in NEMO-deficient cells rendered Jurkat cells sensitive to TNF, while the
parental cell line was completely resistant to TNF-mediated apoptosis
(Fig. 5C).
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FIG. 5.
NF-B activation leads to FLIP upregulation in Jurkat
T cells. (A) Both wt and NEMO (N.)-deficient
(def.) Jurkat cells were stimulated for the indicated
periods of time with PMA and ionomycin (Iono) (or phosphate-buffered
saline [control]) and analyzed for FLIPL,
FLIPS, and phosphorylated IB expression by immunoblot
assay. (B) Cells were stimulated with TNF at 100 ng/ml, and
FLIPL expression was monitored as in panel A. Expression of
NEMO is demonstrated on the right. (C) Cell death induced by TNF in wt
and NEMO-deficient cells, respectively.
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FLIP protects NF-B-incompetent cells against death
receptor-induced apoptosis.
Previous results have suggested that
the protective effect of NF-B stimulation against death receptor
(TNF)-induced apoptosis can be mimicked by simultaneously
overexpressing the NF-B-induced proteins TRAF-1, TRAF-2, c-IAP-1,
and c-IAP-2 in I-Bmut HT1080 cells, leading to a blockade of caspase
8 activation (26). All of these proteins, however, were,
at best, partially protective when overexpressed individually
(26). Given that FLIP appears to be the most potent
inhibitor of death receptor signaling pathways and that FLIP is induced
by NF-B (see above), we considered the possibility that FLIP plays
an even more important role than the TRAF and IAP proteins in the
NF-B antiapoptotic response. If this is so, overexpression of FLIP
alone in I-Bmut cells should have a strong protective effect against
death receptor signals. In order to avoid the selection of
apoptosis-resistant clones that frequently occurs when cells are
cultured in drug-containing medium for a longer period of time, we
chose to retrovirus infect cells and to analyze whole cell populations
without further cloning efforts. FLIPL was highly
expressed in several of the cell populations analyzed, and one example
is shown in Fig. 6A. In contrast,
FLIPS expression was always low, for reasons
which are not understood. However, the low expression levels of
FLIPS sufficed to protect the I-Bmut cells
from the apoptotic effect of TNF (Fig. 6B) while cell death still
occurred at high concentrations of FasL and TRAIL. Cells expressing
high levels of FLIPL were completely protected not only from TNF-induced cell death but also from FasL- and
TRAIL-induced cell death, in agreement with previous results
(11). We can exclude the possibility that this protection
was due to the reactivation of the NF-B response in I-Bmut cells,
as levels of I-Bmut remained unchanged after viral infection (data
not shown). Exogenous FLIP is also short-lived, and treatment of the
infected cell population with CHX led to a decrease in FLIP expression
after 4 h (data not shown) and to increased sensitivity to TNF in
mock-infected and FLIPS-infected cells (Fig. 6C).
By contrast, the amount of FLIPL remaining during
the CHX treatment (FLIPL is expressed at much
higher levels than FLIPS; Fig. 6A) was still
enough to afford complete protection from TNF-mediated cell death.
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FIG. 6.
FLIP overexpression protects from TNF-, FasL-, and
TRAIL-induced cell death. (A) HA-tagged FLIPS, Flag-tagged
FLIPL, and empty vectors were introduced into HT1080 wt or
I-Bmut cells by retrovirus infection. Cells were selected in
puromycin for 3 days before analysis of protein content and function.
FLIP expression was determined by immunoblot assay using an anti-FLIP
antibody, anti-HA antibody, or anti-Flag antibody. The values on the
right are molecular sizes in kilodaltons. (B) Infected pools of
cells were treated with increasing concentrations of TNF, FasL, and
TRAIL (control) in the presence of cross-linking antibody at 1 µg/ml
(with the exception of TNF). Cell viability was determined 16 h
after treatment. (C) Cell viability of the indicated cells pretreated
or not pretreated with CHX (20 µg/ml for 1 h) after incubation
(16 h) with increasing concentrations of TNF.
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To confirm the previous findings by Baldwin's group (26)
in the context of our experimental setting, we infected cells with viruses containing the genes for TRAF1, TRAF-2, and cIAP1 (Fig. 7). Despite reasonable expression levels
of all of these proteins, they were incapable of fully protecting
I-Bmut cells against TNF-mediated apoptosis, in agreement with the
published results.
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FIG. 7.
TRAF1, TRAF2, or cIAP1 overexpression fails to protect
HT1080 cells from TNF-, FasL-, and TRAIL-induced cell death. (A)
pBape-Flag-tagged TRAF1 and TRAF-2 vectors were introduced by virus
infection into HT1080 wt or I-Bmut cells. TRAF1 and TRAF2 expression
was determined by Western blot assay using anti-TRAF1, anti-Flag, or
anti-TRAF2 antibodies. The sensitivity to TNF-mediated cell death of
the cell pools obtained was determined essentially as described in the
legend to Fig 5. (B) HT1080 wt or I-Bmut cells were infected with
either pBape-myc-tagged cIAP1 or empty vector. Cellular pools were
analyzed for resistance to TNF as described above. The values beside
the blots are molecular sizes in kilodaltons.
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| |
DISCUSSION |
In this report, we provide evidence that FLIP is one of several
antiapoptotic genes that are under the control of the NF-B transcription factor. It appears, however, that FLIP is more efficient than TRAF1, TRAF-2, cIAP-1, and c-IAP-2, which have been previously proposed to be responsible for the inhibition of death receptor-induced cell death (26). When expressed at equal levels,
FLIPL was found to be by far the most potent
inhibitor, and thus, FLIP may have a dominant role in the observed
NF-B-mediated resistance to death ligand-induced apoptosis. c-IAPs
may be more important in mitochondrion-induced cell death, as they
interact with caspase-9 and caspase-3 but not with caspase 8 (20). Moreover, the overexpression of TRAF1, TRAF-2,
cIAP-1, and c-IAP-2 may result in their increased recruitment to
signaling complexes, possibly resulting in an augmented NF-B response. This, in turn, would upregulate FLIP levels and thus indirectly lead to the observed antiapoptotic response of the TRAF1,
TRAF-2, cIAP-1, and c-IAP-2 proteins.
We observed that FLIPS and
FLIPL are under the control of NF-B, but
interestingly, induction followed different kinetics. In HT1080 cells,
FLIPS responded considerably more rapidly to TNF
while FLIPS levels dropped at a time when
FLIPL protein levels increased. During T-cell
activation, the inverse order was observed (9). The
reasons for this cell type-specific regulation are unknown.
We have previously shown that FLIPL, but not
FLIPS, binds to TRAF2 and RIP, resulting in
strong NF-B activation upon stimulation of death receptor signaling
pathways (12). Thus, it is conceivable that FLIP levels
that are barely sufficient for protection against death receptors are
augmented by an autoamplification loop. In fact, only small variations
in FLIP protein levels may decide whether a cell is resistant or
sensitive to death mediated by Fas or TNF. It appears that the
recruitment of caspase 8-FLIP heterodimers is highly favored over the
recruitment of caspase 8-caspase 8 homodimers, and thus, FLIP levels
have to exceed caspase 8 levels to be protective. As caspase 8 levels
are quite stable, with no or little variation in the presence of
various stimuli, only a small increase in FLIP levels may decide
whether a cell will respond by dying or living. It is therefore not
surprising that FLIP levels are highly controlled. FLIP is an unstable
protein and is rapidly degraded when neosynthesis is inhibited
(conditions occurring during cell death). Preliminary results indicate
that FLIP is ubiquitin modified under certain conditions, which may explain its short half-life. The ring finger domain of IAPs has been
found to have ubiquitin E3 ligase activity, resulting in the transfer
of ubiquitin to IAP and thus in its own degradation (29).
Interestingly, the FLIP-binding protein TRAF2 also contains a ring
finger domain, raising the possibility that the limited stability of
FLIP is caused by its binding partner. We are currently testing this hypothesis.
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B Signals Induce the Expression of
c-FLIP
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Abstract
Introduction
