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B by Fas Ligation1
Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, IL 60637
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B and this
can be a prosurvival signal in some cells. Although activation of
NF-B by ligation of Fas (CD95/Apo-1), a member of the TNFR family,
has been observed in a few studies, Fas-mediated NF-B activation has
not previously been shown to protect cells from apoptosis. We examined
the Fas-induced NF-B activation and its antiapoptotic effects in a
leukemic eosinophil cell line, AML14.3D10, an AML14 subline resistant
to Fas-mediated apoptosis. EMSA and supershift assays showed that
agonist anti-Fas (CH11) induced nuclear translocation of NF-B
heterodimer p65(RelA)/p50 in these cells in both a time- and
dose-dependent fashion. The influence of NF-B on the induction of
apoptosis was studied using pharmacological proteasome inhibitors and
an inhibitor of IB
phosphorylation to block IB dissociation
and degradation. These inhibitors at least partially inhibited NF-B
activation and augmented CH11-induced cell death. Stable transfection
and overexpression of IB in 3D10 cells inhibited CH11-induced
NF-B activation and completely abrogated Fas resistance. Increases
in caspase-8 and caspase-3 cleavage induced by CH11 and in consequent
apoptotic killing were observed in these cells. Furthermore,
while Fas-stimulation of resistant control 3D10 cells led to increases
in the antiapoptotic proteins cellular inhibitor of apoptosis protein-1
and X-linked inhibitor of apoptosis protein, Fas-induced
apoptosis in IB-overexpressing cells led to the down-modulation
of both of these proteins, as well as that of the Bcl-2 family protein,
Bcl-xL. These data suggest that the resistance of these
leukemic eosinophils to Fas-mediated killing is due to induced NF-B
activation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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stimulation also can induce activation of the
transcription factor NF-B (reviewed in Ref. 1).
Although activation of NF-B has been thought to be part of the
apoptotic induction, recent evidence suggests that in most
circumstances, NF-B activation is a prosurvival response. Many
normal cells are not killed by TNF and this may be related to NF-B
activation; blockade of NF-B sensitizes cells to TNF and augments
induced apoptotic cell death (2, 3, 4). Fas (CD95/Apo-1) is a
member of the TNFR family of proteins, but the ability of Fas to
activate NF-B has been variably reported (5, 6) and
dissociated from any protective effect (6, 7, 8, 9, 10). Indeed,
death occurs in these cell types despite the activation of NF-B
(9, 10), and inhibition of NF-B has little effect on
apoptosis (7). In leukemic or neoplastic cells, resistance
to Fas has been attributed to deficiencies in constituents of the
Fas pathway, including decreased surface expression of Fas
(11), or to the presence of increased levels of
antiapoptotic proteins such as Fas-associated phosphatase-1
(FAP-1)3
(12) or certain members of the Bcl-2 family
(13). However, to our knowledge Fas resistance in such
cells has not been directly linked to activation of NF-B induced by
Fas itself.
The prototypical NF-B is composed of a p65 (RelA) and a p50 subunit
and is sequestered in the cytoplasm as an inactive form bound to IB
inhibitory protein, particularly IB (1). Upon
stimulation by a variety of extracellular agents, IB is
phosphorylated at serines 32 and 36, leading to its polyubiquitination;
this in turn leads to recognition and degradation of IB by the 26S
proteasome (14, 15, 16, 17, 18, 19). The disassociation of IB exposes
the nuclear localization sequence of NF-B, and it is transported
into the nucleus where it can activate expression of a wide variety of
genes (15, 16, 17, 18, 19). Notably, two protein families contain
NF-B-inducible, antiapoptotic family members—the inhibitor of
apoptosis proteins (IAPs) and the Bcl-2 family (20, 21, 22, 23).
NF-B-mediated regulation of the prosurvival Bcl-2 protein,
Bcl-xL for example, has been shown to be
important in survival signaling in both B (21) and T
lymphocytes (23). Recent studies have shown that NF-B
target gene products of the IAP family can inhibit the proteolytic
activities of caspases (reviewed in Ref. 10) and can
prevent apoptosis induced by Fas ligation (24). Recently,
overexpression of IB in endothelial cells suppressed expression
of iap genes and sensitized these cells to TNF--induced
apoptosis (25). In this study, we show that ligation of
the Fas receptors on the eosinophilic cell line AML14.3D10 (hereafter
referred to as "3D10" cells) induced a distinctive pattern of
activation of NF-B, and that these cells were resistant to
Fas-mediated killing. Pharmacologic blockade of NF-B activation or
overexpression of the physiologic NF-B inhibitor protein IB
abrogated the Fas resistance of the 3D10 cells. In
IB-overexpressing cells, both caspase-8 and caspase-3 were
activated following anti-Fas treatment and nearly all the cells
were killed, while in vector control there was little or no caspase
activation and the cells remained resistant to Fas ligation. This
suggests that NF-B activation is critical for protection of the 3D10
eosinophils from Fas-mediated apoptosis and that in these cells Fas
itself induces NF-B activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eosinophilic AML14 cell lines, the parental AML14 line and the
AML14.3D10 subline, were generated and kindly provided by Drs. C. Paul
and M. Baumann (Wright State University, Dayton, OH) (26, 27). The parental AML14 cell line (pAML) was established
from a patient with FAB M2 acute myeloid leukemia (26).
The 3D10 subline was isolated originally as a line with an advanced
eosinophil phenotype and a doubling time of 48 h without cytokine
supplementation (27). Cells were maintained in RPMI 1640
medium supplemented with 8% FBS, 2 mM L-glutamine,
1% (w/v) gentamicin, 10 mM sodium pyruvate, 1 mM HEPES, and 5 x
10-5 M 2-ME (Sigma-Aldrich, St. Louis, MO).
Cells were grown up to a maximum density of 1 x
106 cells/ml at 37°C, 5%
CO2 and were passaged twice a week. After 
40
passages, fresh cultures were started from frozen stocks to minimize
genetic drift and phenotypic changes.
Abs and reagents
The mouse anti-human Fas monoclonal (IgM) CH11 was obtained
from Panvera (Madison, WI). FITC- or PE-conjugated anti-Fas Abs (BD
Biosciences, San Jose, CA) were used in standard flow cytometric
protocols to measure surface expression of Fas. Recombinant TRAIL was
obtained from Alexis Biochemicals (San Diego, CA) and as a kind gift
from Dr. S. Lyman (Immunex, Seattle, WA). The mAb against IB, Abs
against NF-B subunits p65 (RelA), p50, RelB, and c-Rel,
and goat anti-human actin Ab were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-caspase-8, which recognizes both
predominant isoforms of the caspase (28) was generously
provided by Dr. M. Peter (University of Chicago, Chicago, IL).
Anti-caspase-3 and anti-Bcl-xL Abs were
purchased from BD PharMingen (San Diego, CA), and anti-cellular
FLIP (c-FLIP) Ab was purchased from Calbiochem (San Diego, CA). Abs to
cellular IAP (c-IAP)1 and -2 and to X-linked IAP (XIAP) were
obtained from R&D Systems (Minneapolis, MN) and from MBL International
(Watertown, MA), respectively. The IB phosphorylation inhibitor
(E)-3-[(4-t-butylphenylsulfonyl]-2-propenenitrile (BAY 11-7085),
proteasome inhibitors
carbobenzoxy-L-leucyl-L-leucyl-L-leucinal
(MG132), proteasome inhibitor I (PSI), and lactacystin (LC) were
purchased from Calbiochem. Plasmid DNA constructs of pCMV-IB and
pCMV vector only were generous gifts of Dr. D. Ballard (Vanderbilt
University, Nashville, TN) and provided through a collaboration with
Dr. J. Solway (University of Chicago). The plasmid pEGFP-C1 was
purchased from Clontech Laboratories (Palo Alto, CA).
Induction of apoptosis
Fas-induced cell death was determined by both trypan blue exclusion assay and flow cytometric analyses. Briefly, AML14.3D10 cells were seeded at a density of 3 x 105 cells/ml/well in 48-well cell culture plates. The mouse IgM monoclonal anti-Fas Ab, CH11, was used primarily at a range of 100 ng/ml to 1.0 µg/ml. TRAIL was added to the cells at a range of 50–100 ng/ml final concentrations. Each cell sample was divided for trypan blue exclusion assays and for standard propidium iodide (PI) DNA analyses after 24, 48, and 72 h. Total cell death was determined by trypan blue (0.2%) exclusion using a conventional light microscope. The remaining cells were centrifuged at 200 x g for 10 min and resuspended in hypotonic PI solution (50 µg/ml PI in 0.1% Na citrate, 0.1% Triton X-100). To ensure cell lysis, cells were stored overnight in the dark at 4°C before flow cytometric analysis. At least 5000 nuclei were examined for each sample to determine percentage of subG1 DNA content. In preliminary experiments, hypotonic PI analyses of cell samples closely correlated with other DNA fragmentation and morphologic criteria of apoptosis. Percentages (±SE) of cell death or survival (as percentage of viability) reported in the results are derived from the flow cytometric analyses.
Western blot analysis
Equal amounts of protein were separated by SDS-PAGE mini-gel electrophoresis and transferred onto nitrocellulose membrane (0.2-µm pore size; Sigma-Aldrich) using a semidry electrophoretic transfer system (Bio-Rad, Hercules, CA). Blots were stained with Ponceau S to check the quality of the protein and the transfer efficiency. Immunoblotting was performed according to the ECL Western blotting protocol (Amersham Pharmacia Biotech, Arlington Heights, IL). Briefly, blots were blocked in 5% nonfat dry milk in 1x TBS-Tween solution for 1 h followed by a 1-h incubation with the appropriate primary Ab. Blots were then washed for 30 min with four changes of 1x TBS-Tween solution followed by a 1-h incubation with the appropriate HRP-conjugated secondary Ab. Blots were washed again and incubated for 1 min with ECL detection reagents. The results were visualized by exposing blots to autoradiographic film (Kodak, Rochester, NY).
Pharmacologic inhibition of the NF-B activation
AML14.3D10 cells were cultured at a density of 1 x
106/ml and were preincubated for 1 h with
IB phosphorylation inhibitor BAY 11-7085 (29), or
proteasome inhibitors LC (30), MG132 (31), or
PSI (32) at a range of concentrations (0.1–20 µM)
before addition of TNF family ligands or Abs. Optimal doses, at which
augmentation of Fas-mediated killing was greatest with the least
background toxicity of inhibitors alone, were calculated and used in
certain experiments as described.
Extraction of nuclear protein
Cells were passaged and grown overnight at 7 x
105 cells/ml in cell culture flasks. After the
treatments, the cell nuclear extracts were prepared according to a
published method (33) with some modifications. Unless
indicated otherwise, all procedures were performed at 4°C. Briefly,
10 x 106 cells were harvested by
centrifugation and washed twice with ice-cold Dulbecco’s PBS buffer.
The pellet was resuspended in 4x packed cell volume of buffer A (10 mM
HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM
PMSF, 0.5 mM DTT) and incubated on ice for 10 min. The supernatant was
discarded after centrifugation at 1300 rpm for 7 min and 1x original
packed cell volume of buffer A was added. The cell suspension was
transferred to a 50-ml "woodrage" centrifuge tube and centrifuged
at 8500 rpm for 20 min in a Beckman JS 13.1 rotor (Beckman Instruments,
Palo Alto, CA). The supernatant was removed and set aside as the
cytoplasmic extract. The pellet was gently washed with buffer A an
additional time and resuspended in 1x original packed cell volume of
buffer C (20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM
MgCl2, 420 mM KCl, 0.2 mM EDTA, 0.5 mM PMSF, 0.5
mM DTT). The suspension was stirred on a rocking platform for 30 min
and then centrifuged in a Beckman rotor JA-17 (Beckman
Instruments) at 12,500 rpm for 30 min. The supernatant was
collected without disturbing the pellet and placed in dialysis tubing
(Life Technologies, Grand Island, NY). Dialysis was performed for
1 h against three changes of 200 ml of buffer D (20 mM HEPES (pH
7.9), 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT).
Following dialysis, the nuclear extract was clarified by centrifugation
at 14,000 rpm for 20 min in an Eppendorf microcentrifuge tube (Brinkman
Instruments, Westbury, NY). Protease inhibitors including
leupeptin, antipain, chymostatin, and pepstatin A (Sigma-Aldrich) were
added immediately (5 µg/ml each) to extracts before saving them at
-80°C.
EMSA
The details of the EMSA have been described elsewhere
(34). The procedure was performed with some modification.
Double-stranded NF-B synthetic oligonucleotides 5'-AGT TGA GGG GAC
TTT CCC AGG C-3' were purchased from Promega (Madison, WI) and
end-labeled with [
-32P]ATP (Amersham
Pharmacia Biotech) and T4 polynucleotide kinase (NEB, Beverly, MA). A
200-fold excess of unlabeled NF-B probe and unrelated
oligonucleotide probes for CArG was used to assess the specificity of
the DNA-binding reaction. Binding reactions were performed on ice in a
total volume of 15 µl. DNA probe (2000 cpm, 1–5 fmol) was
preincubated for 15 min with 1.5 µl binding buffer (50 mM HCL (pH
7.5), 20% Ficoll, 375 mM KCl, 5 mM EDTA, 5 mM DTT) and 1 µg
poly(dI-dC) (Promega). DNA-protein binding was initiated by adding 4
µg of nuclear extract. A total of 200-fold excess of "cold"
(unlabeled) NF-B probe was used as a specific competitor.
Electrophoresis was performed for 3 h at 100 V in 0.5 x
Tris-borate-EDTA running buffer in a 4°C cold room. The dried
gel was visualized via exposure to high performance autoradiography
film. The supershift analyses were performed by incubating the
DNA-binding reactions with optimal concentrations (determined
previously) of Abs to p65, p50, RelB, and c-rel for an
additional 20 min on ice before electrophoresis.
Luciferase reporter assay
Cells were transfected with the NF-B transcription reporter
plasmid DNA pNF-B-Luc (Clontech Laboratories) using GenePORTER
transfection kit (Gene Therapy Systems, San Diego, CA) essentially
according the method described in detail below. Transfection efficiency
was assessed by cotransfection with pEGFP-C1. After 24 h following
transfection, cells were serum starved for 8 h and treated with
either TNF- (5 ng/ml) or anti-Fas Ab CH11 (1 µg/ml). Cells
were harvested 16 h after treatment and analyzed for luciferase
activity using a luminometer and luciferase reporter assay kit obtained
from BD Biosciences as previously described (35).
Stable transfection of pCMV-IB
Wild-type human IB cDNA was cloned into a mammalian
expression vector, pCMV, and was used to transfect AML14.3D10 cells by
using the GenePORTER kit. Briefly, 1 x 106
cells/well were plated on a 12-well tissue culture plate. The pEGFP-C1
plasmid DNA was used as the reporter gene and cotransfected at a ratio
of 3:1. After 6 wk of selection for neomycin resistance in medium
containing 1200 µg/ml G418 (Sigma-Aldrich), the positively
transfected 3D10 cells were examined by FACS. The dead cells were
discarded by Percoll gradient centrifugation. The positive cells were
subsequently maintained in media supplemented with 400 µg/ml G418.
Cell viability and proliferation were carefully monitored for at least
2 mo before the first experiments and during the experimental period.
Over this period, the stable transfectants also exhibited almost
identical viability and proliferative capacity compared with
untransfected 3D10 cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, Fas ligand, TRAIL, and
IL-1
(36), and interactions between TNF family ligands
and their receptors are major regulatory mechanisms for hemopoietic
cells (37). Specifically, TNF-, Fas ligand, and TRAIL
interactions with their receptors are considered to be death signals to
many cells. In this study, we have examined the signaling effects of
the agonist anti-Fas Ab CH11, as well as TRAIL, on 3D10
eosinophils. We analyzed caspase (-8 and -3) cleavage, cell death or
survival, IB degradation, and NF-B nuclear translocation
following stimulation of these receptors under control conditions or in
the presence of various inhibitors of NF-B activation. We also
determined the effects of Fas stimulation on the expression of
antiapoptotic, NF-B target proteins, Bcl-xL,
c-FLIP, c-IAP1, c-IAP2, and XIAP. Finally, we examined these events in
cells overexpressing the physiologic inhibitor of NF-B,
IB. AML14.3D10 cells are resistant to apoptosis induced by anti-Fas Ab CH11
AML.3D10 eosinophils express surface levels of Fas which are
equivalent to those expressed by the pAML, and to peripheral blood
eosinophils (PBEs) and mature, differentiated cord blood-derived
eosinophils (CDEs). Minor (nonstatistical) differences in surface
expression (Fig. 1
A) did not
correlate to resistance or susceptibility. Of the death ligands tested,
only TRAIL induced death of large numbers of 3D10 cells (Fig. 1B). Percentage of cell death was 42.4 ± 6.6%,
67.7 ± 4.2%, and 80.9 ± 3.9% after 24-, 48-, and 72-h
treatments, respectively. In contrast, there was significantly less
cell death induced by anti-Fas (CH11) after 24-, 48-, and
72-h treatments (8.2 ± 0.67%, 12.9 ± 0.76%, and 17.3
± 1.2%, respectively). Only the 3D10 cells demonstrated resistance to
apoptosis induced by CH11 in comparisons with PBEs, the pAML, and
mature, differentiated CDEs (Fig. 1B). All eosinophil cell
types were resistant to TNF--induced cell death (data not
shown).
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Caspase activation has been described as a critical event(s) in
apoptotic cell death. TNF superfamily-induced cell apoptosis leads to
the proteolytic activation of both upstream caspases (e.g., caspase-8)
and downstream caspases (e.g., caspase-3) (38). Western
blot assays were performed to examine caspase-8 and caspase-3
activation/cleavage induced by CH11 or TRAIL in 3D10 cells. Cells were
treated with either 1 µg/ml CH11 or 100 ng/ml TRAIL for 1–24 h. No
substantial CH11-induced procaspase-8 or procaspase-3 degradation was
observed during this period (Fig. 2A) or during periods
extending to 72 h (data not shown). Because these cells are
sensitive to TRAIL, and TRAIL ligation has been shown to activate
caspases-8 and -3 in sensitive cells (39, 40), we used
TRAIL-induced apoptosis of 3D10 cells as a "positive control" to
examine cleavage of these caspases. Cleavage of procaspase-8 (both
isoforms; Ref. 28) was detected as early as 1 h and
cleavage of procaspase-3 was detectable by 4–6 h after treatment with
TRAIL (Fig. 2B). Anti-actin was used to monitor the
equivalent protein loading in gels as shown (Fig. 2).
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B activation is induced by Fas ligation in 3D10 eosinophils
NF-B can be an important factor in the suppression of apoptosis
in several cell types (2, 3, 4, 41, 42, 43, 44). We examined NF-B
activation induced by anti-Fas, as well as TNF- and TRAIL in the
3D10 eosinophils used in the apoptosis assays described above. EMSA
analyses (Fig. 3) showed that NF-B is
activated in 3D10 cells after a 1-h treatment with 1 µg/ml of CH11,
but not after treatment with 100 ng/ml of TRAIL (even after 2 h;
data not shown); no translocation of NF-B is observed in control
IgM-treated group (Fig. 3). TNF--induced nuclear translocation of
NF-B in these cells occurred as early as 5 min and reached a peak
around 20–30 min (data not shown). Treatment of Fas-susceptible
parental AML14 cells did not induce NF-B translocation above
baseline (untreated) levels (data not shown). Luciferase reporter
assays, performed as outlined above, routinely demonstrated 3-fold or
more augmentation of NF-B activity in Fas- (or TNF--) stimulated
3D10 cells compared with unstimulated cells or cells treated with IgM
control Ab only.
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B subunits in 3D10 eosinophils
There are five members of the NF-B/Rel family of proteins that
have been found expressed in mammalian cells. These NF-B/Rel
subunits are p65/RelA, c-Rel, RelB, p105/NF-B1 (which can
be processed to p50), and p100/NF-B2 (which can be processed to p52;
reviewed in Ref. 1). These subunits usually exist as
protein dimers such as the heterodimer, p65/p50, or the homodimer,
p50/p50. We examined the NF-B subunits in 3D10 cells in supershift
assays using Abs specific for p65, p50, RelB, and c-Rel.
Nuclear extract was prepared from 3D10 cells treated with 1 µg/ml
monoclonal anti-Fas CH11 for 1 h. This treatment induces the
optimal activation of NF-B as described above. These experiments
revealed that the p65/p50 heterodimer is the activated form of the
NF-B induced by CH11 in 3D10 eosinophils (Fig. 4). Faint bands in some lanes could be
shifted by anti-p50 Ab only and could represent endogenous p50/p50
homodimers (Fig. 4, open arrow). However, these bands were inconsistent
in their presence and responses in these experiments. The mouse IgM
protein (1 µg/ml) again showed no induction of NF-B activation
(Fig. 4).
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B in 3D10 cells stimulated by CH11 is time- and
dose-dependent
To determine details of the regulation of anti-Fas-induced
NF-B activation, we examined the CH11 dose response and the time
course of NF-B nuclear translocation. EMSAs were performed using the
nuclear extracts prepared from 3D10 cells treated either for 1 h
with different doses of CH11 ranging from 0.01–2.0 µg/ml, or with 1
µg/ml CH11 for periods from 10 min to 3 h. The results show that
the minimum dose of CH11 required for NF-B activation at 1 h is
0.1 µg/ml, and no significant increase occurs after treatment with
>1 µg/ml (Fig. 5A). The
peak of NF-B translocation was seen at 60 min after treatment with 1
µg/ml CH11 and decreases greatly after 3 h (Fig. 5B).
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B activation leads to an increase in CH11-induced
apoptosis in 3D10 eosinophils
Comparison of the apoptotic effects of CH11 and TRAIL on 3D10
cells suggests that there is a correlation between resistance to
apoptosis and NF-B activation. Treatment of 3D10 eosinophils with
TRAIL leads to apoptosis but not activation of NF-B (see Figs. 1
and 3). In contrast, 3D10 cells have shown strong resistance to both
TNF- and CH11, both of which induce activation of NF-B. To
further understand the role of NF-B activation in 3D10 cell
apoptosis, inhibition of NF-B activation was performed initially
with four pharmacologic inhibitors, BAY 11-7085, LC, MG132, and PSI
were used to treat 3D10 cells. A range of doses of inhibitors (20, 10,
2.5, 1, 0.1, or 0.1 µM) were used to pretreat cells for 1 h
before adding 1 µg/ml CH11. BAY 11-7085 inhibits IB
phosphorylation, preventing the degradation of IB and release of
the activated form of NF-B. LC, MG132, and PSI are proteasome
inhibitors which are specific for the 20S and/or 26S proteasome complex
and inhibit NF-B activation by blocking IB degradation. The
dosages of inhibitors were determined by toxicity assays and inhibitor
alone controls were also performed. Fig. 6A shows the results of
analyses of apoptosis of 3D10 cells pretreated with BAY 11-7085 (2.5
µM) before treatment with 1 µg/ml CH11 for 24, 48, or 72 h.
Dramatically increased induction of apoptosis was observed at all time
points. Similar results were seen for LC, MG132, and PSI, although
background killing of cells (with inhibitor only) were somewhat higher
with MG132 and PSI (data not shown). EMSAs were performed following the
treatment with the inhibitors which exhibited the least background
toxicity, namely BAY 11-7085 and LC. These results demonstrated that
inhibition of the NF-B activation occurs when cells were treated
with the optimal doses of either 2.5 µM BAY 11-7085 (Fig. 6B) or 1 µM LC (data not shown). Both BAY 11-7085 and LC
blocked NF-B translocation in a dose-dependent manner; the most
effective blockade of NF-B translocation induced by CH11 was
observed with either 10–20 µM of BAY 11-7085 or with 10 µM of LC.
However, background killing by inhibitors alone were greater at these
latter doses. These observations correlated well with Western blot
analyses of IB degradation (Fig. 6C), and suggested
that some maintenance of IB expression (at 2.5 µM and above)
was sufficient to inhibit NF-B-mediated protection. Furthermore,
Fas-induced NF-B activation, as measured by luciferase reporter
assay, was almost completely abrogated by BAY 11-7085 treatment (data
not shown).
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B blocks CH11-induced NF-B activation
and inhibits 3D10 cell resistance to CH11
To more rigorously test the role of NF-B activation in 3D10
eosinophils, we created the stably transfected cell line,
AML14.3D10-IB, by cotransfecting 3D10 cells with pCMV-IB
and pEGFP-C1. After transfection, cells were selected with G418 as
described above, and green fluorescence protein (GFP) expression in the
cells was analyzed via flow cytometry. These data showed that 86%
of the cells were positive for GFP as compared with the control cells
(Fig. 7). The selected cells were
maintained in media with 400 µg/ml of G418 as described above. The
stably transfected pCMV vector control cell line was generated using
the same method. Transfection of cells had no significant effect on
surface Fas expression (Fig. 8D). IB-transfected
expressed almost identical amounts of Fas as control cells, and
vector-only cells remained Fas-resistant. IB is the physiological
inhibitor of NF-B and only upon the phosphorylation and degradation
of IB can NF-B p65/p50 translocation occur. The overexpression
of IB in 3D10 eosinophils inhibits depletion of cytosolic
IB as compared with control group that showed significant
reduction of IB after treatment with 1 µg/ml of CH11 for 30 min
(Fig. 8A). The CH11-induced NF-B activation was inhibited
in IB-transfected 3D10 cells (Fig. 8B), while
transfection with vector alone had no effect on NF-B activation by
CH11 treatment. Many recent studies have shown that activation of
NF-B can lead to the antiapoptotic proteins such as XIAP, c-IAP1,
c-IAP2, survivin, and others (20). These antiapoptotic
proteins can inhibit the proteolytic activity of caspases, blocking
cascade activation (cleavage) and suppressing apoptosis
(20). To examine the apoptotic effect of NF-B
inhibition in IB-overexpressing cells, we compared CH11-induced
caspase activation and cell death in 3D10 cells transfected with the
pCMV control vector with pCMV-IB-transfected cells after
treatment with 1 µg/ml CH11. Western blot analyses revealed that
while neither caspase-8 nor caspase-3 activation were observed in the
vector control group (Fig. 9A), inhibition of NF-B
activation via IB overexpression led to a dramatic increase in
cleavage of the proforms of both upstream caspase-8 and downstream
caspase-3 (Fig. 9B). Enumeration of viable cells after
treatment of these two groups with CH11 (1 µg/ml) for 24, 48, and
72 h correlated closely with caspase activation, and <10% of
IB-overexpressing cells were viable, while >80% of control
(vector-only) eosinophils survived CH11 treatment (Fig. 9, C
and D).
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B activation leads to selective increases in
antiapoptotic proteins
To specifically identify potential targets of NF-B-mediated
protection in Fas-stimulated 3D10 cells, we examined expression of
several proteins for 12 h following CH11 treatment of cells stably
transfected with the pCMV control vector or with pCMV-IB. The
results of these analyses are shown in Fig. 10. Western blot analyses consistently
showed increases in both c-IAP1 and XIAP, but not in the other
potential NF-B targets, Bcl-xL, c-FLIP, or
c-IAP2. Both c-IAP1 and XIAP showed increased levels of expression by 2
or 3 h, and levels above baseline were maintained for most or all
of the 12-h period. Interestingly, both of these proteins, along with
Bcl-xL, appeared down-regulated or degraded
following anti-Fas treatment of IB-overexpressing cells (Fig. 10). Both Bcl-xL and XIAP were rapidly
down-regulated with decreased levels evident by 2–3 h, while c-IAP1
decreases appeared somewhat later, 6–12 h (Fig. 10).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B is now an accepted mechanism of protection
from apoptosis for some cell types. Inhibition of NF-B in such cells
may lead to increased cell death through a variety of mechanisms. In
this study, we present novel data that directly attribute Fas
resistance of 3D10 eosinophils to NF-B activation resulting from Fas
ligation itself, and show that inhibition of the nuclear translocation
of p65/p50 negates the Fas resistance of these cells.
Previous studies have demonstrated Fas-mediated NF-B activation, but
have dissociated one from the other. Using SV80 fibroblasts transfected
with the cDNA encoding human Fas, Rensing-Ehl et al. (6)
first demonstrated that anti-Fas induced NF-B translocation to
the nucleus. However, these cells were Fas-sensitive and inhibition of
NF-B had no effect on Fas-mediated cell death. Although NF-B was
activated by Fas ligation in resistant human bladder carcinoma T24
cells (7), again contrary to our observations, inhibition
of NF-B did not alter cell resistance or sensitivity. Furthermore,
using sensitive Jurkat cells transfected with CD40-Fas fusion protein
(CD40 extracellular domain and Fas intracellular and transmembrane
domains), Ponton et al. (7) showed that stimulation of
NF-B binding activity by extracellular Fas ligation was unrelated to
Fas sensitivity. Nevertheless, in agreement with our results, both of
these studies (6, 7), as well as a recent report of
CD40-induced NF-B regulation of Bcl-2 family proteins
(21), implicate the heterodimer p65/p50 as a prominent
NF-B complex in these interactions.
In another recent study, Fas ligation of dissociated cortical
neuroblasts was accompanied by nuclear translocation of the RelA/p65
subunit of NF-B as detected by immunofluorescence (10).
Nevertheless, ligation of Fas killed these cells, and condensed and
fragmented apoptotic nuclei also were immunoreactive for p65, directly
dissociating the Fas-mediated NF-B activation from protection.
Similarly, stimulation of TNFR or Fas on the surface of CEM-C7 T cells
led not only to the activation of NF-B, but to apoptotic death of
the cells (9). Most recently, EMSA, as well as microarray
analyses of the transcriptional effects of anti-Fas (and TNF-)
induction of HT29 colon carcinoma cells, confirmed activation of
NF-B (p65/p50) by Fas ligation (45). However, again
NF-B induction failed to protect these cells, and both TNF- and
anti-Fas induced cell death. In further contrast to our findings,
the latter authors did not observe IB degradation (for up to 4
h) after stimulation and suggested that anti-Fas treatment led to
NF-B activation through a different mechanism (45). In
3D10 eosinophils stimulated with anti-Fas, IB- degradation
progressed through the 2-h time point (Fig. 9). Thus, it is possible
that IB degradation in response to Fas-ligation, and perhaps the
protective capacity of Fas-mediated NF-B activation, varies
according to cell type.
Upstream mechanisms of Fas-induced activation of NF-B are unknown,
but receptor-associated proteins generally thought to be involved in
NF-B activation by TNFR family proteins include receptor-interacting
proteins and TNFR-associating proteins (46). NF-B
inducing kinase may link death receptor signaling to the IB kinases
(47). As we have shown in this study, the kinetics of
Fas-induced NF-B (Fig. 5) differ substantially from those of TNF-
where maximum nuclear translocation could be seen 20–30 min or earlier
(data not shown). Our data regarding the kinetics of Fas- and
TNF-induced NF-B activation confirm, in part, those of another
direct comparison of Fas and TNF (9), and suggest that
pathway intermediates in Fas-induced NF-B activation may differ from
those of TNF or other death receptors. Another molecule which
associates with the cytoplasmic region of Fas is FAP-1. Indeed, FAP-1
is the only known molecule which associates with the negative
regulatory domain of Fas (48, 49), and it is strongly
expressed by 3D10 eosinophils (data not shown). Although the
mechanism(s) by which FAP-1 inhibits apoptosis are still unclear, FAP-1
can interact with IB and enhance NF-B activation
(50). Current evidence suggests that
Tyr42 phosphorylation of IB protects
against its inducible degradation (51, 52). Nakai et al.
(53) showed that FAP-1 enhanced NF-B activation via the
common neurotrophic receptor in transfected 293T cells. Furthermore,
they have hypothesized that dephosphorylation of
Tyr42 of IB by FAP-1 leads to an increase
in the "receptivity" of IB for serine phosphorylation and
subsequent NF-B activation (53). We are currently
investigating the potential role of FAP-1 in Fas-induced NF-B in our
eosinophil systems.
Finally, we are also continuing investigations of the transcriptional
targets of Fas-induced NF-B. Transcriptional profiling recently has
suggested that among such genes, at least two (apoptosis inhibitor 2
(c-IAP2) and the cytoprotective manganese superoxide dismutase) are
up-regulated by both TNF- and anti-Fas signaling
(44). The former belongs to the IAP family, which has been
implicated in suppression of apoptosis induced by a variety of signals
(20). These proteins can directly inhibit caspases in
vitro, but their in vivo roles are largely undefined. In a
study of TNF-induced apoptosis of transfected HT1080 fibrosarcoma
cells, Wang et al. (54) found that activation of NF-B
blocked the activation of caspase-8. Furthermore, they demonstrated
that c-IAP1 (and c-IAP2) may play roles in blocking the cleavage and
activation of both caspase-8 and caspase-3. Both c-IAP1 and c-IAP2 have
been shown to directly bind to caspase-3 and -7, and inhibited their
proteolytic activation in a cell-free system (55).
Recently, the susceptibility of human enterocytes to Fas-induced
apoptosis was attributed to c-IAP1 and -2, and blockade of their
synthesis with cycloheximide augmented Fas-mediated killing
(56). XIAP also has been shown to be up-regulated by TNFR
stimluation and to directly inhibit caspase-3 (and -7) in some cells
(57). Our results support these observations, but the in
vivo specificity of interactions (i.e., which IAP inhibits which
caspase) within this group of caspases and inhibitors is still
unclear.
Other antiapoptotic proteins, which may be transcriptional targets of
NF-B, but of controversial relevance in Fas-mediated cell death
(58, 59, 60, 61), include members of the growing
mitochondria-associated Bcl-2 family. NF-B can directly regulate the
expression of prosurvival members such as Bcl-xL
and are required for rescuing certain cell types from apoptosis
(21, 22, 23). Although Fas-mediated apoptosis of some cells
can bypass significant mitochondrial involvement and, thus, the
antiapoptotic effects of Bcl-xL, in other cells
Bcl-xL can contribute to inactivation of
caspase-8 at the mitochondrial surface (62) or inhibit
Fas-mediated apoptosis by preventing mitochondrial release of the IAP
inhibitor, Smac/DIABLO (63). Although we have not
observed consistent Fas-mediated up-regulation of
Bcl-xL in wild-type 3D10 cells, a clear pattern
of degradation or down-regulation was observed in the
IB-transfected cells, and this could contribute to augmented
death of these cells, as has been previously suggested
(64). Finally, it is possible that the combined effect of
several NF-B-regulated proteins may be required for rescue from
Fas-mediated apoptosis.
Whether blockade of Fas-induced apoptosis occurs in 3D10 eosinophils
through antiapoptotic effects of Bcl-2 proteins, via caspase
inhibition, or by some other mechanism(s), it is clear that in these
cells, NF-B activation is critical to cell survival following Fas
ligation. This may have important implications in the therapeutic
approaches using apoptotic machinery in both inflammatory diseases and
hematological malignancies. Furthermore, these data suggest that the
AML14.3D10 cell line may provide a useful model for studying
antiapoptotic pathways involving NF-B activation via TNF family
receptor ligation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kimm J. Hamann, Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637. E-mail address: khamann{at}medicine.bsd.uchicago.edu
3 Abbreviations used in this paper: FAP-1, Fas-associated phosphatase-1; IAP, inhibitor of apoptosis protein; PSI, proteasome inhibitor I; LC, lactacystin; PI, propidium iodide; pAML, parental AML14 cell line; PBE, peripheral blood eosinophil; CDE, cord blood-derived eosinophil; GFP, green fluorescence protein; MG132, carbobenzoxy-L-leucyl-L-leucyl-L-leucinal; BAY 11-7085, (E)-3-[(4-t-butylphenylsulfonyl]-2-propenenitrile; c-IAP, cellular IAP; XIAP, X-linked IAP; c-FLIP, cellular FLIP.
Received for publication November 30, 2001. Accepted for publication July 26, 2002.
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