 |
INTRODUCTION |
Virus replication within infected cells results in generation of
double-stranded RNA (dsRNA)1
molecules that can trigger host antiviral responses (1). Such dsRNA-activated responses can be mediated by
dsRNA-dependent enzymes such as the interferon-inducible
protein kinase (PKR) which phosphorylates key cellular substrates
(e.g. eukaryotic protein synthesis initiation factor-2
) (2). PKR can also activate the NF-
B transcription factor (see below) resulting in induction of type I interferon gene
expression that can prevent further virus infection (3). In addition,
dsRNA can also induce apoptosis in a PKR-dependent manner
(4-6). Thus PKR is required for both dsRNA-mediated induction of gene
expression and induction of apoptotic cell death. PKR-mediated apoptosis of virus-infected cells by dsRNA may thus limit virus infection by preventing virus replication within host cells.
Apoptosis is a genetically controlled process that plays an essential
role in regulating homeostasis and in protecting the host against
microbial infections (7, 8). Apoptotic cells manifest characteristic
morphological changes, such as nuclear condensation and fragmentation,
which are mediated by proteases belonging to the caspase family (9,
10). Although caspases are normally present in an inactive form, they
can be activated by proteolysis triggered by cell death inducers (10,
11). Activated caspases cleave key cellular substrates and thus provide a safe and efficient mechanism for eliminating surplus or infected cells. Although inhibitors of caspase proteases have been shown to
prevent apoptosis induced by many different agents (10, 12, 13), recent
studies have demonstrated that in certain cell lines, inhibition of
caspase activity induces necrotic killing of cells by TNF
(14-16).
Such necrosis can occur in cell lines that are normally resistant to
TNF
killing, is accompanied by increased production of reactive
oxygen species (ROSs), and can be prevented by antioxidants (14-16).
Importantly, similar to TNF
, dsRNA also induces necrosis in the
presence of caspase inhibitors in dsRNA-resistant wild-type fibroblasts
(16). Both TNF
and dsRNA-induced necrosis may represent host
strategies for eliminating cells infected with viruses encoding caspase
inhibitors (16).
The NF-
B family of transcription factors is a key regulator of genes
involved in immune and inflammatory responses (17, 18). Recent studies
have also demonstrated a critical role for NF-
B in regulating
apoptotic cell death. Mice deficient in the RelA (p65) subunit of
NF-
B die prenatally because of massive hepatocyte apoptosis (19),
which appears related to the cytotoxic effect of TNF
(20).
Fibroblasts or macrophages derived from RelA
/
mice or cells
overexpressing a super-repressor form of the inhibitory I
B protein
are also highly susceptible to TNF
-killing (21-24). These studies
have demonstrated an essential role for NF-
B in protecting cells
from TNF
-induced killing. In addition, NF-
B can also mediate
pro-apoptotic effects. A recent report has shown that NF-
B is
critically important for mediating p53-induced apoptosis (25). Our
recent studies have demonstrated an essential role for RelA in
induction of the death receptor Fas expression and in subsequent
apoptosis after Fas ligation (26). Taken together, these studies
suggest that NF-
B-mediated anti-apoptosis and pro-apoptosis may
be context-dependent.
The NF-
B proteins also play a key role in mediating cellular
responses to conserved microbial structures such as dsRNA and LPS (17).
Stimulation of cells with dsRNA or LPS results in rapid nuclear
translocation of NF-
B proteins and induction of NF-
B target gene
expression. Although both dsRNA-mediated NF-
B activation and
apoptosis require PKR activity, less is known about mechanisms
important for regulating dsRNA-induced apoptosis. Recent studies have
suggested, however, that PKR-dependent induction of Fas
expression may be involved in dsRNA-induced apoptosis (6, 27, 28).
Since NF-
B regulates Fas expression, these studies suggest that
NF-
B may play a pro-apoptotic role in dsRNA-induced cell death.
To determine a possible role for NF-
B in dsRNA-induced apoptosis, we
have utilized embryonic fibroblasts and macrophages from mice deficient
in the RelA subunit of NF-
B. We show here that dsRNA-induced Fas
expression was dramatically reduced in RelA
/
MEFs. Surprisingly,
dsRNA specifically induced apoptosis in NF-
B RelA
/
but not in
RelA+/+ MEFs. In addition, inhibition of macromolecule synthesis also
rendered RelA+/+ MEFs susceptible to dsRNA-induced killing. These
results suggest that dsRNA-induced apoptosis may be prevented by
NF-
B mediated induction of anti-apoptotic gene expression, rather
than induced by expression of pro-apoptotic genes, such as
fas. These results demonstrate the existence of a
novel anti-apoptotic role for NF-
B in the dsRNA-induced cell death pathway.
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EXPERIMENTAL PROCEDURES |
Cells and Materials
Mouse embryonic fibroblasts, fetal liver macrophages, and 3T3
fibroblasts were derived as described previously (21). Fibroblasts were
cultured in high glucose Dulbecco's modified Eagle's medium containing L-glutamate (2 mM), penicillin (100 units/ml), streptomycin (100 µg/ml), and calf serum (10%). Human
TNF
was obtained from R & D Systems and used at concentration of 10 ng/ml in all experiments. dsRNA poly(I-C) was purchased from Sigma and
used at a final concentration of 100 µg/ml. Caspase inhibitors,
Z-DEVD-fmk, Z-VAD-fmk, and biot-VAD-fmk, (Enzyme System Products) were
dissolved in dimethyl sulfoxide at 20 mM and used at 100 and 1 µM, respectively. Actinomycin D and antioxidant
butylated hydroxyanisole (BHA) were obtained from Sigma and used at 2 µg/ml and 100 µM, respectively. The nuclear dye
4',6'-diamidino-2-phenylindole (DAPI) and the reactive oxygen species
dye dihydrorhodamine 123 (DHR) were obtained from Molecular Probes.
pLPC expression vector was a gift from Dr. S. Lowe (Cold Spring
Harbor Laboratory, New York). pRelA was constructed by cloning the
mouse RelA cDNA into pLPC. pPKRDN, which has a six-amino acid
deletion as described previously (29), was a gift from Dr. A. García-Sastre (Mount Sinai Medical Center, New York).
Analysis of Cell Death
Nucleus Morphology--
Cells in tissue culture plates were
rinsed with PBS, fixed with 3.7% formaldehyde, and permeabilized with
0.2% Triton X-100 for 5 min. They were then washed and incubated with
a DAPI labeling solution (2 µg/ml in PBS) for 5 min and examined
under a fluorescence microscope.
Cell Viability Experiments--
Approximately 2 × 105 cells were plated on each well of a 6-well plate 1 day
before the experiments. The caspase inhibitors, Z-DEVD-fmk or
Z-VAD-fmk, or macromolecule synthesis inhibitor, actinomycin D, was
added 1 h before the addition of dsRNA. After the indicated
periods, the cells were trypsinized (fibroblasts) or scraped
(macrophages), and viable cells were counted by trypan blue exclusion.
Four independent readings within a single experiment were used to
calculate the S.D.
Transfection Experiments--
RelA
/
MEFs were cotransfected
with a GFP expression vector (0.5 µg) and pLPC, pRelA, or pPKRDN (0.5 µg) using Fugene 6 (Roche Molecular Biochemicals). 24 h later,
cells were either left untreated or treated with dsRNA for 12 h.
Viable GFP-positive cells from four randomly chosen fields were counted
and used to calculate S.D.
Determination of Reactive Oxygen Species Levels
DHR was added to a final concentration of 2 µM
before RelA
/
MEFs were treated with the indicated agents. 12 h
later, cells were trypsinized, washed and resuspended in PBS before
FACS analysis.
EMSA, Northern Blots, Affinity Blots, and Western Blots
Electrophoretic mobility shift assay (EMSA) was carried out as
described previously (19). RelA-specific antisera were purchased from
Santa Cruz Biotechnology. Northern blotting was carried out as
described (26) with probes generated from cDNA fragments by reverse
transcriptase-polymerase chain reaction using gene-specific primers.
Affinity blotting was performed essentially as described previously
(30). Briefly, ~5 × 106 cells were harvested after
the treatments indicated. The cells were then washed once with PBS and
pelleted, and the pellet was snap-frozen on dry ice. An equal volume of
1 µM biot-VAD-fmk in MDB buffer (50 mM NaCl,
2 mM MgCl2, 5 mM EGTA, 10 mM HEPES, 1 mM dithiothreitol (pH 7)) was added
to the cell pellet, and the cells were lysed by three cycles of
freezing and thawing. The lysates were incubated at 37 °C for 15 min
and centrifuged. 20 µg of protein lysates from the supernatant were
separated by SDS-PAGE and transferred to nitrocellulose membrane. The
membrane was blocked with TBST (20 mM Tris, 150 mM NaCl, 0.05% Tween 20) supplemented with 2% nonfat dry
milk for 30 min and then incubated in avidin-Neutralite (Molecular
Probes) at 1 µg/ml in TBST supplemented with 1% nonfat dry milk for
1 h. The membrane was then washed and incubated in biotinylated
horseradish peroxidase (Molecular Probes) at 25 ng/ml in TBST for
1 h. The labeled protein was visualized by ECL (Amersham Pharmacia
Biotech). For Western blotting, membranes were blocked with TBST
supplemented with 0.5% casein. Caspase 3-p17 antibody (New England
Biolabs), which specifically recognizes the large subunit of activated
caspase 3, was used at a 1 to 10,000 dilution in subsequent steps.
Identification of p18
Fifty 10-cm plates of RelA
/
3T3 fibroblasts grown to
1-2 × 107/plate were either left untreated or
treated with TNF
(10 ng/ml) for 6 h. Cells were collected by
centrifugation, lysed in the presence of 1 µM
biot-VAD-fmk to label caspases, and the lysates clarified by
centrifugation at 100,000 × g for 1 h at 4 °C.
The protein concentrations of the pooled supernatant were 4.7 mg/ml for
untreated and 2.9 mg/ml for TNF
-treated samples. To remove unbound
biot-VAD-fmk, the lysates were dialyzed with four changes of MDB buffer
(see above) supplemented with a proteinase/inhibitor mixture (Roche
Molecular Biochemicals). Dialyzed extracts were run through an
Immunopure Immobilized Streptavidin Column (Pierce). The column was
washed with 150 ml of washing buffer (50 mM sodium phosphate, 0.4 M urea, 50 µM
phenylmethylsulfonyl fluoride (pH 7)), and bound proteins were eluted
by boiling in the washing buffer with 2% SDS. The eluted proteins were
precipitated with 0.25 volume of trichloroacetic acid solution (100%
trichloroacetic acid, 0.4% sodium deoxycholate) and washed twice with
acetone. The precipitates were resuspended in 50 µl of the
SDS-loading buffer. 5 µl of either the untreated or TNF
-treated
samples were resolved by SDS-PAGE. The gel was silver-stained to test
for the purity and amount of p18 (approximate 10 ng). The rest of the samples were also subjected to SDS-PAGE and transferred to
polyvinylidene difluoride membrane (Problott, Applied Biosystem). p18
was excised and wet with 1 µl of methanol. The band was reduced and
alkylated with isopropyl acetamide followed by digestion in 20 µl of
0.05 M ammonium bicarbonate containing 0.5% Zwittergent
3-16 (Calbiochem) with 0.2 µg of trypsin (Frozen Promega Modified) at
37 °C for 17 h. Peptides generated from in situ
tryptic digests were separated on a C18 0.18 × 150-mm capillary
column (LC Packing, Inc.). The high pressure liquid chromatography
consisted of a prototype capillary gradient high pressure liquid
chromatography system (Waters Associates) and a model 783 UV detector
equipped with a Z-shaped flow cell (LC Packing, Inc.). A 30-cm length
of 0.025-mm ID glass capillary was connected to the outlet of the
Z-shaped cell inside the detector housing to minimize the delay volume.
Solvent A was 0.1% aqueous trifluoroacetic acid, and solvent B was
acetonitrile containing 0.08% trifluoroacetic acid. Peptides were
eluted using a linear gradient of 0-80% solvent B in 60 min and
detected at 195 nm. Fractions were collected automatically by a BAI
Protocol onto pre-made spots of matrix (0.5 µl of 20 mg/ml
-cyano-4-hydroxycinammic acid + 5 mg/ml nitrocellulose in 50%
acetone, 50% 2-propanol) on the target plate. Ions were formed by
matrix-assisted laser desorption/ionization with a nitrogen laser, 337 nm. Spectra were acquired with a Perspective Biosystems Voyager Elite
time-of-flight mass spectrometer, operated in reflector delayed
extraction mode. Peptides detected by MALDI-TOF MS were subjected to
collision-induced dissociation in an ion trap mass spectrometer (LCQ,
Finnigan MAT). A 1-µl aliquot (5%) of the P150 tryptic digest was
loaded onto a 100-µm inner diameter, 360-µm outer diameter, 30-cm
length of fused silica capillary packed with 15 cm of POROS 10R2
reverse phase beads (Perspective Biosystems). Peptides were eluted with an acetonitrile gradient at a flow rate of 500 nl/min for 15 min. A
data-dependent experiment was performed to obtain
structural information for selected peptides. Ions with m/z
values corresponding to peptides observed by MALDI-TOF MS were
monitored in full mass range scans and automatically subjected to
collision-induced dissociation as each eluted from the capillary
column. Peptide masses and selected b and y series fragments were used
to search an in-house protein and DNA sequence data base with an
enhanced version of the FRAGFIT (31) and the SEQUEST program. The mouse
caspase 3 was identified by a data base search of data obtained from a
liquid chromatography/MS/MS analysis of a tryptic digest of the
18-kDa band. MS/MS analysis of MH+ 1118.9 was found to
correspond to residues 65-75 (SGTDVDAANLR) of mouse caspase 3. MALDI
MS analysis identified an additional eight masses that matched with the
caspase 3 protein.
 |
RESULTS |
The RelA Subunit of NF-
B Is Required for dsRNA-induced Gene
Expression--
Our recent studies have revealed a critical role for
the RelA subunit of NF-
B in the regulation of TNF
and LPS-induced
Fas expression (26). Previous studies have also suggested that
dsRNA-induced apoptosis could be mediated by induction of Fas
expression (6, 27). We therefore tested the possible involvement of
NF-
B in dsRNA-dependent induction of Fas expression and
apoptosis. To this end, we first determined whether RelA was a
component of NF-
B complexes activated by dsRNA. RelA+/
mouse
embryonic fibroblasts (MEFs) (19) were treated with dsRNA for 2 h,
after which nuclear extracts were tested for
B-site binding activity
by EMSA. As expected, dsRNA strongly activated NF-
B (Fig.
1A). Activated NF-
B was
supershifted by antisera generated against RelA (Fig. 1A),
demonstrating the presence of RelA in dsRNA-activated NF-
B complexes. Furthermore, as previously observed following TNF
treatment (19), dsRNA-treated RelA
/
MEFs showed a significantly lower level of NF-
B activation (Fig. 1A, compare
lane 2 and lane 5). To determine whether RelA was
important for dsRNA-induced Fas expression, we treated RelA+/
or
RelA
/
MEFs with dsRNA for 6 h, after which Fas mRNA
expression was determined. Similar to TNF
and LPS, dsRNA-induced Fas
expression was dramatically reduced in RelA
/
cells (Fig.
1B). In addition, dsRNA-mediated induction of the
neutrophil-specific chemokine, MIP-2, was also found to be dependent on
RelA (Fig. 1B). These results thus demonstrate an important
role for the RelA subunit of NF-
B in mediating dsRNA-induced gene
expression.

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Fig. 1.
RelA is critical for dsRNA-induced gene
expression in MEFs. A, RelA+/ or RelA / MEFs were
either left untreated (UT) or treated with dsRNA for 2 h. B-site binding activity was determined by EMSA. RelA-specific
antisera were used to demonstrate the presence of RelA. B,
RelA+/ or RelA / MEFs were either left untreated (UT)
or treated with dsRNA for 6 h after which cellular RNA was tested
for the expression of fas, mip-2, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes by
Northern blotting.
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|
RelA Is Essential for Inhibiting dsRNA-induced
Cytotoxicity--
We wanted to determine whether Fas expression was
responsible for controlling susceptibility of MEFs to dsRNA-induced
cell death. Surprisingly, a 12-h dsRNA treatment significantly reduced viability of RelA
/
but not RelA+/+ MEFs (Fig.
2A). These results demonstrate
the existence of a previously unrecognized function of RelA in
inhibiting dsRNA-induced cytotoxicity. Recent studies have demonstrated
a critical role for RelA in protecting cells from TNF
-induced
killing (21), through induction of survival gene expression (32-36).
Consistent with a role for survival gene expression in preventing
TNF
-induced killing, inhibition of RNA or protein synthesis
sensitizes normally resistant cells to TNF
-induced killing (37).
Similar to TNF
, treatment of RelA+/+ MEFs with an RNA synthesis
inhibitor actinomycin D (thus resulting in inhibition of RelA-mediated
transcription) also rendered them susceptible to dsRNA-induced killing
(Fig. 2A). TNF
is cytotoxic to both RelA
/
fibroblasts
and macrophages (21). To determine whether dsRNA could also induce
cytotoxicity to RelA
/
macrophages, we generated fetal liver
macrophages from embryonic day 14 (E14) RelA+/+ and RelA
/
mice. As
seen with MEFs, RelA
/
macrophages readily lost viability in the
presence of dsRNA, whereas RelA+/+ cells were not affected (Fig.
2B). Taken together, these results demonstrate a new role
for RelA in preventing dsRNA-induced cytotoxicity, which similar to
TNF
may also be mediated by regulation of survival gene expression.
They also suggest that induction of pro-apoptotic genes, such as Fas,
may not play an important role in mediating dsRNA-induced killing.

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Fig. 2.
RelA is essential for protecting MEFs and
macrophages from dsRNA-induced cytotoxicity. A,
RelA / or RelA+/+ MEFs were either left untreated (UT),
treated with dsRNA alone, treated with actinomycin D (Act D)
alone, or treated with dsRNA and actinomycin D for 12 h. Viable
cells remaining after treatment are shown as a percentage of viable
untreated cells. B, RelA / or RelA+/+ macrophages
(M ) were either left untreated (UT) or treated
with dsRNA for 12 h. C, TNFR1+/+RelA / or
TNFR1 / RelA / MEFs were either left untreated (UT) or
treated with dsRNA or TNF for 12 h.
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|
These results have revealed strikingly similar mechanisms for
inhibiting cell death induced by TNF
and dsRNA. However, it is also
possible that dsRNA-induced killing of RelA
/
cells is somehow
mediated by the TNF
signaling pathway. This could be accomplished by
dsRNA-induced release of pre-synthesized TNF
or by
dsRNA-dependent mechanisms, for example, which could lead to activation of TNF receptors. Two TNF
receptors have been
identified and named TNFR1 and TNFR2 (38, 39). Unlike TNFR2, TNFR1
contains a death domain that can induce cytotoxicity in many cell types (40). To determine a possible involvement of TNFR1-mediated signaling
in dsRNA-induced cytotoxicity, we tested the sensitivity of both
TNFR1+/+RelA
/
and TNFR1
/
RelA
/
MEFs to dsRNA or
TNF
-induced cell death (41). As expected, TNF
induced significant
cytotoxicity in TNFR1+/+RelA
/
but not in TNFR1
/
RelA
/
MEFs2 (Fig. 2C).
In contrast, dsRNA treatment efficiently killed both cell types. These
results thus suggest that dsRNA induces a cell death pathway that does
not depend on TNFR1-induced signaling.
dsRNA-induced Cell Death Requires PKR Activity--
To determine
whether dsRNA-induced killing of RelA
/
cells was in fact due to the
absence of RelA, we tested whether ectopic expression of RelA was
sufficient to protect RelA
/
MEFs from dsRNA-induced cytotoxicity. A
GFP-expressing vector (to identify transfected cells) was cotransfected
with either a control pLPC vector or a vector expressing RelA (pRelA).
24 h later, cells were left untreated or treated with dsRNA for
another 12 h after which the viability of GFP-positive cells was
determined. As expected, RelA
/
MEFs transfected with the control
pLPC vector readily lost viability after dsRNA treatment (Fig.
3). In contrast, cotransfection of pRelA
significantly protected RelA
/
MEFs from dsRNA-induced cytotoxicity
(Fig. 3). These results suggest that RelA plays a direct role in
inhibiting dsRNA-induced cell death.

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Fig. 3.
PKR activity is critical for dsRNA-induced
cytotoxicity in MEFs. RelA / MEFs were cotransfected with a GFP
expression plasmid and pLPC, pRelA, or pPKRDN. 24 h later, cells
were either left untreated (UT) or treated with dsRNA for
12 h. Viable GFP-positive cells after treatment are shown as a
percentage of viable untreated GFP-positive cells.
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PKR activity is thought to be important for induction of cell death by
dsRNA (6). We were also interested in determining whether PKR was
important for dsRNA-induced killing of RelA
/
MEFs. To this end, we
cotransfected RelA
/
MEFs with a construct encoding a PKR
dominant-negative mutant, pPKR-DN. Importantly, expression of PKR-DN
significantly enhanced viability of RelA
/
MEFs following dsRNA
treatment (Fig. 3). These results thus suggest that PKR is required for
induction of cell death by dsRNA in RelA
/
MEFs.
dsRNA Induces Apoptosis and Caspase Activation in RelA
/
MEFs--
Apoptotic cell death results in characteristic morphological
changes such as membrane blebbing, nuclear fragmentation, and chromatin
condensation (42). DAPI staining of dsRNA-treated RelA
/
MEF nuclei
revealed significant nuclear fragmentation and chromatin condensation
(Fig. 4A), suggesting that
dsRNA induces apoptotic cell death in RelA
/
MEFs. Activation of
cysteine proteinases belonging to the caspase family has been shown to
be critically important for induction of apoptosis (43). To determine
whether caspase proteases are activated in RelA
/
MEFs following
dsRNA treatment, we used a biotinylated caspase inhibitor,
biot-Val-Ala-Asp(OMe)-fmk (biot-VAD-fmk). This inhibitor can covalently
associate with activated caspases and thus provides a sensitive method
for detection of caspase activation (9). dsRNA treatment of RelA
/
MEFs resulted in a time-dependent increase of a
biot-VAD-fmk binding activity of ~18 kDa (p18) (Fig. 4B).
Importantly, the molecular weight of p18 corresponded to that of the
larger subunit of most activated caspases (also see below).
Significantly, p18 could also be induced by TNF
treatment of
RelA
/
MEFs (Fig. 4B), suggesting that both TNF
and
dsRNA may induce a common caspase in these cells. In contrast, dsRNA
treatment of RelA+/+ MEFs did not lead to increased levels of p18, but
cotreatment with actinomycin D resulted in a dramatic increase of p18
(Fig. 4C). These observations thus demonstrate a critical
role for RelA in inhibiting dsRNA and TNF
-induced caspase
activation.

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Fig. 4.
dsRNA induces caspase activation and
apoptotic cell death in RelA /
MEFs. A, RelA / MEFs were either left
untreated (UT) or treated with dsRNA for 6 h before
stained with DAPI. Nuclear morphology of apoptotic cells is indicated
by arrows. B, RelA / MEFs were either left
untreated (UT), treated with dsRNA for 3, 6, or 12 h,
or treated with TNF for 12 h. Extracts were made in the
presence of biot-VAD-fmk (1 mM), and lysates (20 mg of
protein) were examined by a biot-avidin affinity blotting. The
predominant caspase activated is indicated as p18. C,
RelA+/+ MEFs were either left untreated (UT), treated with
dsRNA alone, treated with actinomycin D (Act D) alone, or
treated with dsRNA and actinomycin D for 6 h. Extracts were tested
for biot-VAD-fmk binding as in B. p18 is indicated by an
arrow.
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|
Caspase 3, a Major Caspase Activated by dsRNA and TNF
, Is
Essential for Apoptotic Nuclear Fragmentation but Dispensable for
dsRNA-induced Cytotoxicity--
To characterize further p18, we used a
streptavidin affinity column to purify p18. After extensive washing to
remove activities bound nonspecifically to streptavidin, an 18-kDa
activity could be detected in TNF
-treated extracts subjected to
SDS-PAGE (Fig. 5A). Peptide
sequencing and mass spectrometry analysis (not shown) of this 18-kDa
protein revealed that the sequence of one peptide was identical to
residues 65-75 of mouse caspase 3 (SGTDVDAANLR). Western analysis
using an antibody specific for the p17 subunit of activated caspase 3 confirmed its activation following both TNF
and dsRNA treatment
(Fig. 5B) (the apparent molecular weight difference between
p18 and p17 is likely due to association of biot-VAD-fmk, which has a
molecular weight of 672, to p18). These results thus demonstrate that
caspase 3 is a major caspase activated by both dsRNA and TNF
treatment of RelA
/
MEFs.

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Fig. 5.
Caspase 3 is the major caspase activated in
RelA / MEFs by dsRNA or
TNF . A, biot-VAD-fmk-labeled
extracts from either untreated (UT) or TNF -treated
RelA / fibroblasts were subjected to purification with a
streptavidin affinity column. The purity of the extracts before or
after purification was tested by SDS-PAGE followed by silver staining.
p18 is indicated with an arrow. B, RelA / MEFs
were either left untreated (UT) or treated with TNF or
dsRNA for 6 h. Whole cell lysates were analyzed by Western
blotting with a caspase 3-specific antibody. The caspase 3 p17 is
indicated by an arrow.
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|
Caspase 3 has been proposed to be a major downstream effector caspase
responsible for executing the apoptotic cell death program (10, 44,
45). To determine the functional significance of caspase 3 in
dsRNA-induced apoptosis, we used the caspase 3 inhibitor, Z-DEVD-fmk
(9). Z-DEVD-fmk completely inhibited binding of p18 (caspase 3) to
biot-VAD-fmk induced by dsRNA in RelA
/
MEFs (Fig.
6A). Interestingly, Z-DEVD-fmk
did not affect processing of caspase 3 (Fig. 6B) suggesting
that Z-DEVD-fmk does not inhibit upstream caspases that are responsible
for proteolytic processing of caspase 3 but rather specifically
inhibits caspase 3 activity. Although Z-DEVD-fmk completely inhibited
caspase 3 activity, Z-DEVD-fmk did not protect RelA
/
MEFs from
dsRNA-induced cytotoxicity (Fig. 6C). These results suggest
that caspase 3 activity is not essential for dsRNA-induced killing of
RelA
/
MEFs. However, Z-DEVD-fmk completely inhibited nuclear
fragmentation (Fig. 6D, compared with Fig. 4A).
These results thus demonstrate that caspase 3 activity is essential for
apoptotic nuclear changes but dispensable for dsRNA-induced
cytotoxicity in RelA
/
MEFs.

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Fig. 6.
Caspase 3 activity is essential for
dsRNA-induced apoptotic nuclear changes but dispensable for
cytotoxicity in RelA /
MEFs. A, RelA / MEFs were either left
untreated (UT), treated with dsRNA alone, or treated with
dsRNA and Z-DEVD-fmk for 6 h. Extracts were tested for
biot-VAD-fmk binding as in Fig. 4B. p18 is indicated by an
arrow. B, RelA / MEFs were treated as in
A. Whole cell lysates were analyzed for caspase 3 activation
by Western blotting. C, RelA / MEFs were either left
untreated (UT), treated with dsRNA alone, or treated with
dsRNA and Z-DEVD-fmk for 12 h before cell viability determined.
D, RelA / MEFs were treated with dsRNA and Z-DEVD-fmk for
6 h before stained with DAPI. Nuclear morphology is indicated by
arrows.
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Combined Treatment of RelA
/
MEFs with Z-DEVD-fmk and
Antioxidants Completely Inhibits dsRNA-induced
Cytotoxicity--
Z-DEVD-fmk is not a broad specificity caspase
inhibitor, raising the possibility that the inability of Z-DEVD-fmk to
inhibit killing of RelA
/
MEFs was due to incomplete blockage of
caspase activity. We therefore tested the effect of the broader
specificity Z-VAD-fmk caspase inhibitor (9) on dsRNA-induced caspase
activation and killing of RelA
/
MEFs. Unlike Z-DEVD-fmk, Z-VAD-fmk
inhibited caspase 3 processing (Fig.
7A, compared with Fig.
6B), demonstrating its ability to inhibit the activity of
upstream caspases responsible for caspase 3 processing and activation.
However, Z-VAD-fmk did not inhibit, but rather significantly enhanced,
cytotoxicity to RelA
/
MEFs following dsRNA treatment (Fig.
7B). In addition, both Z-DEVD-fmk and Z-VAD-fmk did not
affect NF-
B activation by dsRNA (data not shown), suggesting that
the enhanced cytotoxicity by Z-VAD-fmk was not due to inhibition of
NF-
B. This is consistent with our recent observations showing that
Z-VAD-fmk could sensitize RelA+/+ fibroblasts to dsRNA-induced necrotic
killing (16). These results also indicate that caspase inhibition may
not be sufficient to protect RelA
/
MEFs from dsRNA-induced
cytotoxicity.

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Fig. 7.
Cotreatment of
RelA / MEFs with
Z-DEVD-fmk and BHA prevents dsRNA-induced cytotoxicity.
A, RelA / MEFs were either left untreated
(UT), treated with dsRNA alone, or treated with dsRNA and
Z-VAD-fmk for 6 h. Whole cell lysates were analyzed for caspase 3 activation by Western blotting. B, RelA / MEFs were
either left untreated (UT), treated with dsRNA alone, or
treated with dsRNA and Z-VAD-fmk for 12 h before cell viability
determined. C, RelA / MEFs were either left untreated
(UT), treated with dsRNA alone, treated with Z-DEVD-fmk in
the presence or absence of dsRNA, treated with Z-VAD-fmk in the
presence or absence of dsRNA, treated with BHA alone, or treated with
BHA, dsRNA, and Z-DEVD-fmk for 12 h. Intracellular levels of
reactive oxygen species were determined by DHR staining followed by
FACS analysis. D, RelA / MEFs were either left untreated
(UT), treated with dsRNA alone, treated with dsRNA and
Z-DEVD-fmk, treated with dsRNA and BHA, or treated with dsRNA,
Z-DEVD-fmk, and BHA for 12 h before cell viability was
determined.
|
|
To understand further the mechanisms involved in dsRNA-induced killing
of RelA
/
MEFs, we wished to identify noncaspase cytotoxic mechanisms that may be potentially responsible for inducing cell death.
One such cytotoxic mechanism involves enhanced generation of reactive
oxygen species (ROSs), which is also important in induction of necrotic
cell death by TNF
(14-16). We therefore tested whether treatment of
RelA
/
MEFs with dsRNA resulted in enhanced ROSs production.
However, no significant increase in ROSs production was noticed
following dsRNA treatment of RelA
/
MEFs (Fig. 7C).
Similarly, dsRNA + Z-DEVD-fmk-treated RelA
/
MEFs also showed no
significant increase in ROSs production (Fig. 7C). In
contrast, treatment of RelA
/
MEFs with dsRNA + Z-VAD-fmk significantly enhanced ROSs production (Fig. 7C).
Furthermore, Z-VAD-fmk, but not Z-DEVD-fmk, also enhanced ROSs
production and necrotic cell death in RelA+/+ MEFs (data not shown).
Thus in the presence of broad specificity caspase inhibitors such as
Z-VAD-fmk, dsRNA treatment can enhance ROSs production and induce
necrotic cell death in both RelA+/+ and RelA
/
MEFs. Nevertheless,
our results suggest that such enhanced ROSs production may not be responsible for killing of RelA
/
MEFs by dsRNA or dsRNA + Z-DEVD-fmk.
It was, however, possible that dsRNA-induced killing of RelA
/
MEFs
may result not from increased generation of ROSs but from enhanced
susceptibility to constitutively produced ROSs. Importantly, in the
presence of the antioxidant BHA, constitutive generation of ROSs was
significantly reduced in both untreated and dsRNA + Z-DEVD-fmk-treated
RelA
/
MEFs (Fig. 7C). To determine a role for such
constitutively generated ROSs in dsRNA-induced killing, we tested
whether BHA could inhibit cell death induced by dsRNA. Interestingly,
BHA treatment substantially enhanced survival of RelA
/
MEFs
suggesting that dsRNA-induced killing may indeed result from enhanced
susceptibility to constitutively produced ROSs (Fig. 7D).
Importantly, combined treatments with Z-DEVD-fmk and BHA resulted in
almost complete protection from dsRNA-induced cytotoxicity (Fig.
7D). These results indicate that inhibition of dsRNA-induced
cytotoxicity requires inhibition of both caspase-dependent
and ROSs-dependent mechanisms and raise the possibility
that anti-apoptotic functions of NF-
B are mediated by simultaneous
inhibition of both cytotoxic pathways.
 |
DISCUSSION |
The results presented here provide evidence for a novel function
of the RelA subunit of NF-
B in inhibiting dsRNA-induced apoptosis.
Although the role of NF-
B in inhibiting apoptosis by endogenously
produced factors such as TNF
(i.e. host-derived) is well
established, we provide the first evidence for an
NF-
B-dependent function in inhibiting apoptosis induced
by an exogenous agent (i.e. microbe-derived factor). Our
results thus suggest that NF-
B-mediated inhibition of apoptosis may
be an important mechanism for regulating cell survival during viral
infection. Interestingly, and similar to TNF
, we have found that
dsRNA also induces caspase activation and apoptotic changes in
RelA
/
cells. In addition, and also similar to TNF
, dsRNA also
triggers apoptosis in RelA+/+ cells in the presence of an RNA synthesis
inhibitor. These results reveal a striking similarity in cellular
mechanisms responsible for inhibiting cell death induced by TNF
and
dsRNA. These results thus indicate that dsRNA-induced cell death may be
inhibited by NF-
B-mediated survival gene expression, rather than
enhanced by NF-
B activation and induction of Fas expression as
previously reported (6, 27, 28).
dsRNA can be generated during infection with virtually any kind of
virus (1). NF-
B activated by dsRNA in infected cells allows
activation of antiviral gene expression to limit further infection
(46). However, our results suggest that NF-
B-mediated induction of
survival gene expression may be important for inhibiting apoptosis of
infected cells and may thus enhance viral infection and virulence.
Consistent with such a function of NF-
B, a previous study has
demonstrated a critical role for NF-
B in maintaining virulence of
encephalomyocarditis virus by preventing virus-induced apoptosis (47).
It will thus be interesting to determine whether NF-
B-mediated
inhibition of dsRNA-induced apoptosis is a mechanism important for
enhancing viral virulence. However, it is also possible that inhibition
of NF-
B-dependent anti-apoptosis in virus-infected cells
may lead to eradication of infected cells. As shown here, inhibition of
macromolecule synthesis sensitized cells to dsRNA-induced killing.
Since shutdown of host macromolecule synthesis is one of the key events
in late stage virus replication (48), it is likely that under these
conditions the NF-
B-induced protective pathway is also inhibited.
Under these conditions, dsRNA may thus mediate cytotoxicity to
virus-infected cells. Interestingly, TNF
has also been shown to kill
vesicular stomatitis virus-infected cells (49, 50). Since NF-
B also
protects cells from TNF
-induced apoptosis, it is possible that
killing of virus-infected cells by either dsRNA or TNF
is due to
viral inhibition of the NF-
B-induced protective pathway. Additional
studies aimed at determining the impact of these
NF-
B-dependent pathways in controlling viral virulence
may thus be required to understand fully the physiological functions of
these key anti-apoptotic pathways.
We have shown here that induction of apoptosis by dsRNA in RelA
/
cells is mediated by activation of caspases. In particular, we have
identified caspase 3 as a major caspase activated by dsRNA treatment
that was found to be critically important for dsRNA-induced nuclear
fragmentation in RelA
/
fibroblasts. These results are consistent
with a previous study using caspase 3-deficient cells that demonstrated
an important role for caspase 3 in mediating apoptotic nuclear changes
by death inducers (44). Importantly, a recent study has identified a
caspase 3 substrate, acinus, as the factor responsible for induction of
chromatin condensation and fragmentation (51). It will thus be
interesting to test whether acinus is also processed and activated in
RelA
/
fibroblasts by dsRNA. Caspase activation involves a cascade
of proteolytic events. Upstream initiator caspases, such as caspase 8 or 9 (which are generally activated by oligomerization) (52, 53),
induce proteolysis and subsequent activation of downstream effector
caspases, such as caspase 3 (10, 11). Although upstream caspase(s)
involved in dsRNA-induced apoptosis are still not known, a recent study has demonstrated an essential role for the FADD protein in
dsRNA-induced, PKR-mediated apoptosis (54). FADD is an adapter molecule
involved in activation of caspase 8 by both TNFR1 and Fas (55-59).
These observations thus suggest a possible involvement of caspase 8 in
dsRNA-induced apoptosis of RelA
/
cells and suggest that dsRNA and
TNF
may induce apoptosis by similar mechanisms that can be inhibited
by NF-
B.
Interestingly, inhibition of caspase 3 by Z-DEVD-fmk did not prevent
dsRNA-induced cytotoxicity to RelA
/
MEFs. However, combined
Z-DEVD-fmk and antioxidant treatment resulted in complete protection
from dsRNA-induced cytotoxicity, even though dsRNA treatment did not
result in a significant increase in ROSs generation. In RelA+/+ MEFs,
dsRNA + actinomycin D-induced cytotoxicity was also not
inhibited by Z-DEVD-fmk alone, whereas cotreatment with Z-DEVD-fmk and
BHA resulted in significant suppression of cell death (data not shown).
We have also found that inhibition of TNF
-induced killing of
RelA
/
MEFs requires simultaneous inhibition of caspase proteases
and ROSs generation.3 Similar
to dsRNA, TNF
treatment of RelA
/
MEFs did not enhance ROSs
production. These results thus suggest that dsRNA or TNF
treatment
may enhance the susceptibility of RelA
/
MEFs to constitutively generated ROSs. These results also suggest that NF-
B-regulated anti-apoptotic genes may include those that can provide protection from
ROSs-induced cytotoxicity. Such an NF-
B-regulated gene may be
manganous superoxide dismutase, which functions as a potent scavenger of superoxide anions and has previously been shown to inhibit
TNF
-induced cell death in certain cell lines (60). It is also
possible that caspases activated during apoptosis can inhibit cellular
damage induced by constitutively generated ROSs, perhaps to prevent
necrotic lysis. In either case, our results suggest that caspase
inhibition is not sufficient to inhibit apoptosis of RelA
/
cells
and suggest that ROSs may provide an additional mechanism that plays a
key role in the regulation of apoptosis induced by dsRNA and TNF
.
Caspase inhibitors are currently being considered for possible
therapeutic use in the treatment of tissue-degenerative diseases (61).
Our results suggest, however, that combined inhibition of both
caspase-dependent and ROSs-dependent pathways
may be required for inhibiting cell death induced by certain agents.
In conclusion, we have provided evidence of a novel function for the
RelA subunit of NF-
B in preventing dsRNA-induced apoptosis by
potentially inhibiting both caspase-dependent and
ROSs-dependent mechanisms. These results further underscore
the key role played by NF-
B proteins in inhibiting apoptosis induced
by diverse agents. Our results also highlight the importance of NF-
B
proteins in regulating cellular responses to viral products that may be
important for controlling viral virulence and pathogenesis.