From the Lineberger Comprehensive Cancer Center,
University of North Carolina School of Medicine, Chapel Hill,
North Carolina 27599, and the ¶ Department of Cancer Biology,
Lerner Research Institute, Cleveland, Ohio 44195
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ABSTRACT |
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Tumor necrosis factor- The transcription factor nuclear factor- The pleiotropic cytokines tumor necrosis factor- An important signal transduction molecule that is targeted by IFNs is
the double-stranded RNA (dsRNA)-activated protein kinase (PKR). This
serine/threonine kinase was first discovered as a translation inhibitor
because of its ability to phosphorylate and deactivate the translation
initiation factor, eIF-2 (for review, see Ref. 28). It plays a role in
cellular antiviral responses and growth control and is a candidate
tumor supressor gene (for review, see Refs. 29 and 30). PKR is
IFN-inducible, is present at low levels in most cells, and is found in
the nucleus as well as in the cytoplasm (31). Recent evidence indicates
that activation of NF- In this report, we provide evidence supporting a role for PKR
involvement in the synergistic activation of NF- Cell Culture and Treatments--
The rat preneuronal adrenal
pheochromocytoma cell line, PC12 (CRL 1721, American Type Culture
Collection, Rockville, MD) (38), was maintained in Dulbecco's modified
Eagle's medium F-12 supplemented with 10% fetal bovine serum and
antibiotics. The central nervous system-derived rat preneuronal cell
line B12 (gift of Dave Schubert, The Salk Institute, La Jolla, CA) (39)
was maintained in Dulbecco's modified Eagle's medium H supplemented
with 10% fetal bovine serum and antibiotics. The human vascular
endothelial cell line EA.hy926 (gift of Cora-Jean S. Edgell, University
of North Carolina, Chapel Hill) (40) was maintained in Dulbecco's
modified Eagle's medium H supplemented with 10% fetal bovine serum,
1× hypoxanthine-aminopterin-thymidine medium supplement (Boehringer
Mannheim), and antibiotics.
Cells were incubated for the times indicated under "Results" with
0.025-10 ng/ml human recombinant TNF- Nuclear and Cytoplasmic Extracts--
The day before treatment,
cells were plated in 10 ml of complete media in 100-mm tissue culture
plates at 1 × 107 cells/plate (PC12), 1 × 106 cells/plate (B12), or 2 × 106
cells/plate (EA.hy926). After treatment, nuclear and cytoplasmic extracts were made using a procedure described previously (27). Briefly, cells were washed with phosphate-buffered saline, scraped from
plates, transferred to microcentrifuge tubes, and lysed on ice in 3 pellet volumes of cytoplasmic extraction buffer (10 mM Hepes, pH 7.6, 60 mM KCl, 1 mM EDTA, 0.1%
Nonidet P-40, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, and 2.5 µg/ml each of aprotinin, leupeptin, and pepstatin). Nuclei were pelleted, and cytoplasmic supernatants were transferred to fresh tubes. Nuclei were washed with
100 µl of extraction buffer without Nonidet P-40 and then repelleted.
Supernatants were discarded, and nuclear pellets were resuspended by
vortexing in 2 pellet volumes of nuclear extraction buffer (20 mM Tris, pH 8.0, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 25% glycerol, and 2.5 µg/ml each of aprotinin, leupeptin, and pepstatin) in which the final
salt concentration was adjusted to ~400 mM NaCl. All cytoplasmic and nuclear extracts were cleared and transferred to fresh
tubes. Next, glycerol was added to the cytoplasmic extracts to a final
concentration of 20%, protein concentrations were determined by the
Bradford assay using the Bio-Rad protein assay dye reagent (500-0006),
and all extracts were stored at EMSAs--
Electrophoretic mobility shift assays (EMSAs) were
performed as described previously (27). Briefly, equal amounts of
nuclear extracts were incubated for 15 min at room temperature with a 32P-labeled probe containing a Western Blot Analysis--
Equal amounts of cytoplasmic extracts
were electrophoresed in 10% polyacrylamide-SDS gels and transferred to
nitrocellulose membranes (Schleicher & Schuell) (27). The upper half of
each membrane was probed with an antibody specific for I Transient Transfections and Luciferase Assays--
Transient
transfection of PC12 cells was accomplished using 20 µl/ml
LipofectAMINE reagent (Life Technologies, Inc.) and a total of 6 µg
of DNA for each sample. The MHC-NF- Stable Transfectants--
PC12 cells stably expressing
MHC-NF- TNF- TNF- PKR May Play a Role in the Synergistic Activation of
To explore further the requirement for PKR during
TNF- The cooperation between TNF- What is the signal generated by IFN- The requirement for PKR may be cell type-specific because 2-AP
completely blocked the TNF- There is evidence that PKR can affect the activation of NF- What is PKR's target during NF- There are several possible mechanisms by which TNF- The results presented here are directed at elucidating the
mechanisms(s) whereby IFNs impinge on TNF- (TNF-
) and
-interferon (IFN-
) cooperate during a variety of biological
responses and ultimately synergistically enhance the expression of
genes involved in immune and inflammatory responses. Recently, we
demonstrated that IFN-
can significantly potentiate TNF-
-induced
nuclear factor (NF)-
B nuclear translocation in neuronal derived and
endothelial cell lines. The mechanism by which these two cytokines
exert their synergistic effect on NF-
B involves the de
novo degradation of the NF-
B inhibitor, I
B
. The
double-stranded RNA-dependent kinase PKR is IFN-inducible
and has been implicated in the activation of NF-
B; therefore, we
examined the possibility that PKR may play a role in the synergistic
activation of NF-
B during TNF-
/IFN-
cotreatment. The PKR
inhibitor 2-aminopurine (2-AP) inhibited TNF-
/IFN-
-induced
NF-
B nuclear translocation in neuronal derived cells but not in
endothelial cells. The induced degradation of I
B
, which is
normally observed upon TNF-
/IFN-
cotreatment, was blocked
completely by 2-AP in neuronal derived cells. Also, 2-AP treatment or
overexpression of a catalytically inactive PKR inhibited the
TNF-
/IFN-
-induced synergistic activation of
B-dependent gene expression. Our results suggest that
the signal generated by IFN-
during TNF-
/IFN-
cotreatment may
require PKR to elicit enhanced NF-
B activity, and this signal may
affect the stability of the I
B
protein.
INTRODUCTION
Top
Abstract
Introduction
References
B
(NF-
B)1 is activated by a
variety of stimuli including cytokines, mitogens, cellular stress, and
bacterial or viral products (for review, see Refs. 1-5). The family of
mammalian NF-
B transcription factors consists of at least five
distinct members: c-Rel, p50 (NF-
B1), p52 (NF-
B2), p65 (RelA),
and RelB, which form a variety of active homo- and heterodimers (for
review, see Refs. 1-5). Classic NF-
B exists as a p50-p65
heterodimer that is sequestered in the cytoplasm by inhibitor proteins
collectively referred to as inhibitors of kappa B (I
Bs) (for review,
see Ref. 4). The two major forms of I
B are I
B
and I
B
.
Upon stimulation, an activated I
B kinase (IKK) complex (6-9)
phosphorylates the I
B proteins, which targets these inhibitor
proteins for ubiquitination and degradation (10-13). This process
allows NF-
B to translocate to the nucleus and regulate gene-specific
transcription. Structurally, I
B
and I
B
are similar, and
both interact with p65- and c-Rel-containing dimers through similar
binding domains (14). Additionally, both forms of I
B are
phosphorylated on analogous serine residues by the activated IKK
complex (6). However, I
B
is characteristically involved in the
transient activation of NF-
B, whereas I
B
has been implicated in the persistent activation of NF-
B (14-17). There is also
evidence that the stimuli that ultimately target the I
Bs for
degradation may differ, although this may be cell type-specific or may
depend on the concentration of the inducer (14, 15, 18).
(TNF-
) and
interferon (IFN) can function together to coregulate gene expression synergistically in a variety of cell lines. Typically, the coregulatory effects involve the independent activation of NF-
B by TNF-
(for review, see Ref. 4) and of IFN-responsive factors by IFNs (for review,
see Refs. 19 and 20), permitting these transcription factors to bind
their unique sites within the promoters of target genes such as MHC
class I, ICAM-1, VCAM-1, inducible iNOS, interleukin-6, and
interleukin-8 (21-26). Recently, we reported that IFN-
, which typically does not activate NF-
B, synergistically enhances
TNF-
-induced nuclear translocation of p50-p65 NF-
B heterodimers
and synergistically activates
B-dependent gene
expression (27). We also demonstrated that the mechanism for this
synergistic activation involved the de novo degradation of
the I
B
protein and that the TNF-
/IFN-
coactivation of
NF-
B in PC12 cells is sensitive to the protein tyrosine kinase
inhibitor genistein (27).
B by dsRNA, but not by TNF-
or
interleukin-1
, may involve PKR and that PKR may phosphorylate
I
B
in vitro (32-35). Therefore, we examined the
possibility that PKR may be involved in TNF-
/IFN-
-induced synergistic activation of NF-
B.
B by TNF-
/IFN-
cotreatment in the preneuronal derived cell line, PC12. The PKR inhibitor 2-aminopurine (2-AP) (36, 37) blocks synergistic TNF-
/IFN-
-induced NF-
B nuclear translocation. The requirement for PKR may be specific for cells of neuronal origin because 2-AP was
able to block the synergy in another neuronal derived cell line (B12),
but not in an endothelial cell line (EA.hy926). The synergistic
activation of
B-dependent gene expression can be inhibited by 2-AP or by the overexpression of a catalytically inactive,
dominant negative form of PKR. Also, 2-AP inhibits the de
novo degradation of I
B
observed during TNF-
/IFN-
cotreatment in PC12 cells and B12 cells but does not affect the normal
pattern of I
B
degradation. Therefore, the mechanism by which the
IFN-inducible kinase PKR functions in this system may involve targeted
phosphorylation and degradation of I
B
. These data indicate a
novel role for PKR in the activation of NF-
B.
EXPERIMENTAL PROCEDURES
(Boehringer Mannheim), 50-100 units/ml rat recombinant IFN-
(Life Technologies, Inc.), or
10 mM 2-AP (Sigma).
70 °C until analyzed.
B site from the class I
MHC promoter (41, 42) in binding buffer (10 mM Tris, pH
7.7, 50 mM NaCl, 0.5 mM EDTA, 1 mM
dithiothreitol, and 10% glycerol) (43) plus 2 µg of
poly(dI-dC)·poly(dI-dC) (Amersham Pharmacia Biotech). Complexes were
separated in 5% polyacrylamide gels in Tris-glycine-EDTA buffer (25 mM Tris, 190 mM glycine, and 1 mM
EDTA), dried, and autoradiographed.
B
(sc-945, Santa Cruz), and the lower half was probed with an antibody
specific for I
B
(100-4167C, Rockland). Specific proteins were
visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech).
BLuc plasmid contains three
tandem repeats of the
B site from the class I MHC enhancer cloned
into a luciferase expression vector (44). Luciferase expression vector
was a gift of Bill Sugden, University of Wisconsin, Madison. The
wild-type PKR expression plasmid (wtPKR) and the catalytically inactive
Lys296
Arg mutant PKR expression plasmid (mutPKR) were
described previously (32). PC12 cells were plated in 60-mm tissue
culture plates (7 × 106 cells/plate) the day before
transfection. LipofectAMINE-DNA complexes were allowed to form for 30 min in serum-free medium before being added to plates containing cells
plus 2 ml of serum-free medium (27). Cells were incubated with the
complexes for 7-8 h, the medium was replaced with medium containing
0.5% serum, and 8 h of cytokine treatment began 36 h after
the medium change. Cells were washed, collected, resuspended in 2 pellet volumes of 0.25 M Tris pH 7.5, and subjected to
three cycles of freeze/thaw. Samples were cleared, and protein
concentrations were determined using the Bio-Rad protein assay dye
reagent. Luciferase assays were performed in duplicate on equal amounts
of protein using 200 µM D-Luciferin as a
substrate (Sigma), and relative light units were determined using an
AutoLumat LB953 luminometer (Berthold Analytical Instruments, Inc.,
Nashua, NH).
BLuc or its mutated counterpart were obtained by
LipofectAMINE cotransfections with the reporter plasmid and pcDNA3
(Invitrogen, Carlsbad, CA), which contains the neomycin resistance
gene. 2 days post-transfection, the medium was replaced with complete
medium containing 600 µg/ml Geneticin/G418 (Life Technologies) for
selection purposes. Fresh G418-containing medium was added every 4-5
days for 2 months, allowing pools of cells stably expressing
MHC-NF-
BLuc to grow out. Stably transfected cells were plated (80%
confluent in 60-mm plates) the day before treatment. Where indicated,
cells were pretreated for 1 h with 10 mM 2-aminopurine
before treatment for 8 h with TNF-
and/or IFN-
. Cells were
collected, lysed, and assayed as described for transient transfections
(see above).
RESULTS
/IFN-
-induced NF-
B Nuclear Translocation Is Inhibited
by 2-AP--
Previously, we demonstrated that IFN-
treatment
synergistically enhances TNF-
-induced nuclear translocation of
NF-
B in PC12 cells even though IFN-
, by itself, does not induce
NF-
B in these cells (27). In response to dsRNA treatment but not to
TNF-
or IL-1
treatment, the Ser/Thr protein kinase PKR can induce
NF-
B DNA binding activity, and this may occur following phosphorylation of I
B
(32-35). To determine if IFN-
-enhanced NF-
B activity involves PKR, PC12 cells were pretreated for 30 min
with 2-AP, a selective inhibitor of PKR, which can inhibit PKR
autophosphorylation and activation (36, 37), and then were treated with
TNF-
and/or IFN-
. Nuclear extracts were prepared and analyzed by
EMSA. As described previously (27), there was very little binding to a
consensus
B site with nuclear extracts from untreated PC12 cells
(Fig. 1A, lane 1).
Although it has been documented that a 2-h incubation with 10 mM 2-AP can slightly increase NF-
B DNA binding in the
human promonocytic cell line U937 (45), we do not detect a change in
binding in PC12 cells after 1 or 3.5 h of treatment (compare
lanes 1-3). Treatment with TNF-
alone for 30 min
(lane 4) induced binding of one major NF-
B-specific complex that was identified previously as p50-p65 (27). By 3 h of
TNF-
treatment the binding activity was reduced significantly (lane 6) and returned to basal levels by 16 h (data not
shown). Pretreatment with 2-AP did not affect the TNF-
-induced
NF-
B DNA binding profile (compare lane 4 with
5 and lane 6 with 7). As expected,
treatment with IFN-
alone or after pretreatment with 2-AP did not
induce binding to the NF-
B-specific probe (lanes 8-11).
Cotreatment with TNF-
and IFN-
elicited a striking synergistic effect on
B-specific binding activity after 3 h of cotreatment as reported earlier (compare lane 6 with lane 14)
(27). Pretreatment with 2-AP completely blocked the
TNF-
/IFN-
-induced synergy (compare lanes 14 and
15). Similar experiments were performed with the central
nervous system-derived B12 cell line, and the effects of cytokine
treatment with and without 2-AP treatment were nearly indistinguishable
from the PC12 cell NF-
B activation profiles (data not shown). In the
endothelial cell line, EA.hy926, the TNF-
/IFN-
-induced activation
of NF-
B was not inhibited by 2-AP but was enhanced slightly (Fig.
1B, compare lanes 3 and 4).
Pretreatment with the broad specificity serine/threonine kinase
inhibitor staurosporine had no effect on the TNF-
/IFN-
-induced
synergy in either cell type (data not shown). Collectively, the data
from these three cell lines indicate that PKR may be involved in the
regulatory mechanism for this synergistic response in cells of neural
origin.
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Fig. 1.
2-AP inhibits
TNF- /IFN-
-induced
NF-
B activation in PC12 cells but not in
EA.hy926 cells. EMSAs of equal amounts of protein from nuclear
extracts using a probe containing a consensus NF-
B binding site are
shown. The time and treatment are indicated above each lane
(UT is untreated). Where indicated, cells treated with
cytokine were preincubated with 10 mM 2-AP for 30 min.
Arrows indicate the major NF-
B-specific band
(p50/p65), a nonspecific band (n.s.), and free
probe. Panel A, for PC12 cells, TNF-
and IFN-
concentrations were 10 ng/ml and 100 units/ml, respectively. As a
control, cells not treated with cytokine were incubated with 2-AP for
1 h (lane 2) or 3.5 h (lane 3).
Panel B, for EA.hy926 cells, TNF-
and IFN-
concentrations were 25 pg/ml and 100 units/ml, respectively. As a
control, cells not treated with cytokine were incubated with 2-AP for
1.5 h (lane 2).
/IFN-
-induced Degradation of I
B
Is Inhibitied by
2-AP--
Typically, NF-
B is retained in the cytoplasm by
inhibitory proteins that are collectively referred to as I
B proteins
(for review, see Refs. 3 and 4). In general, NF-
B-inducing stimuli promote the degradation of specific I
B proteins, which allows the
release and nuclear translocation of NF-
B subunits. Previously, we
demonstrated that costimulation of NF-
B by TNF-
and IFN-
in
PC12 cells requires the de novo degradation of I
B
(27). Therefore, we investigated whether 2-AP affects I
B
or
I
B
protein degradation. Western blot analyses were performed on
cytoplasmic extracts collected at the same time as the nuclear extracts
that were analyzed for Fig. 1A. Incubation for up to 3 h with 2-AP alone had no effect on either I
B
or I
B
protein
levels (Fig. 2, compare lanes
1-3). TNF-
treatment for 30 min resulted in degradation of
I
B
but not I
B
protein levels (compare lanes 1 and 4), and 2-AP did not inhibit this degradation (compare
lanes 1, 4, and 5). In fact, 2-AP
appears to enhance I
B
degradation in the presence of TNF-
,
consistent with the slight increase in DNA binding activity observed in
Fig. 1A, lanes 4 and 5. TNF-
was
able to lead to a modest reduction in I
B
levels after 3 h of
stimulation, and this was not affected by 2-AP (Fig. 1A, lanes 6 and 7). As expected, I
B
was
resynthesized within 3 h because the expression of I
B
is
transcriptionally regulated by NF-
B (lane 6) (for review,
see Ref. 4). Treatment with IFN-
either alone or after pretreatment
with 2-AP also did not change the level of either I
B protein
(lanes 8-11). Furthermore, TNF-
/IFN-
cotreatment
caused extensive degradation of I
B
which corresponds to the
synergistic activation of NF-
B shown in Fig. 1 (Fig. 2, lane
14). Interestingly, the degradation of I
B
was inhibited by
2-AP (compare lanes 14 and 15), which corresponds to the inhibition of NF-
B activity shown in Fig. 1. Similar
experiments were performed with the central nervous system-derived B12
cell line, and the effect of 2-AP on TNF-
/IFN-
-induced I
B
degradation was nearly identical (data not shown). We were unable to
identify a higher mobility, hypophosphorylated form of I
B
which
has been detected following its initial degradation (17). Also, we have not analyzed the potential of TNF-
and IFN-
to lead to enhanced degradation of other forms of I
B, such as I
B
. Our data
indicate that the degradation of I
B
in response to
TNF-
/IFN-
cotreatment and therefore the synergistic activation of
NF-
B may be PKR-dependent in cells of neuronal
origin.
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Fig. 2.
2-AP inhibits
TNF- /IFN-
-induced
I
B
degradation.
Western analyses of equal amounts of protein from PC12 cell cytoplasmic
extracts using polyclonal antibodies specific for I
B
or I
B
are shown. The time and treatment are indicated above each
lane (UT is untreated). TNF-
, IFN-
, and
2-AP concentrations were 10 ng/ml, 100 units/ml, and 10 mM,
respectively. As a control, cells not treated with cytokine were
incubated with 2-AP for 1 h (lane 2) or 3.5 h
(lane 3). Where indicated, cells treated with cytokine were
preincubated with 2-AP for 30 min (lanes 5, 7,
9, 11, 13, and 15).
Arrows indicate each specific I
B protein.
B-dependent Gene Expression by TNF-
/IFN-
Cotreatment--
To test whether the synergistic activation of
NF-
B-dependent transcriptional responses requires PKR
activity, we examined the ability of 2-AP to inhibit
B-dependent reporter gene expression. We used cells that
were stably transfected rather than transiently transfected with a
B-dependent reporter because transient expression can be
affected by 2-AP (45-47). PC12 cells stably maintaining a luciferase
reporter construct containing three
B sites cloned in tandem in
front of the minimal luciferase promoter (MHC-NF-
BLuc) were treated
with TNF-
and/or IFN-
in the presence or absence of 2-AP. The
B sites conferred a ~35-fold induction of luciferase activity upon
treatment with TNF-
, a ~5-fold induction upon treatment with
IFN-
, and a synergistic ~95-fold induction upon cotreatment (Fig.
3). Preincubation with 2-AP significantly
reduced the TNF-
/IFN-
induction of MHC-NF-
BLuc by ~40%,
eliminating the IFN-
-supplied synergism. 2-AP did not
nonspecifically affect gene expression (data not shown).
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Fig. 3.
2-AP inhibits
TNF- /IFN-
-induced
B-specific gene expression. PC12 cells stably
expressing a
B-dependent luciferase reporter construct
(MHC-NF-
BLuc) were pretreated for 30 min with 10 mM 2-AP
or were left untreated. Subsequently, the cells were treated with 10 ng/ml TNF-
, 100 units/ml IFN-
, or a combination of both for
7 h. Lysates were assayed in duplicate for luciferase activity,
and fold activity was determined by dividing the number of relative
light units from treated samples by the number of relative light units
from untreated (UT) samples. The data shown are averages of
three independent experiments, and the S.E. of the mean are indicated
by error bars.
/IFN-
-induced
B-dependent gene expression, we
transiently cotransfected PC12 cells with MHC-NF-
BLuc plus a plasmid
that expresses wtPKR or one that expresses mutPKR. MutPKR contains a
Lys296
Arg mutation which makes it a catalytically
inactive kinase (32). The inactive PKR acts as a dominant negative
either by competing for an endogenous PKR activator (48, 49) or by
forming inactive dimers with endogenous PKR (50, 51). After
transfection, we treated the cells with TNF-
and/or IFN-
and
compared the luciferase activity relative to cells that were
transfected with the reporter construct alone. WtPKR had little effect
on the increased luciferase activity observed after cytokine treatment;
however, mutPKR inhibited TNF-
/IFN-
-induced MHC-NF-
BLuc
activity by ~80% (Fig. 4). The
expression of mutPKR also decreased TNF-
-induced luciferase
activity, suggesting that a minor component of TNF-
signaling may
involve PKR and that its inhibitory effect may be on the ability of
NF-
B to transactivate gene expression rather than on its ability to
translocate to the nucleus. This effect has been documented previously
by Kumar et al. (32). In summary, these data indicate that
IFN-
enhances TNF-
-induced NF-
B-dependent transcription through PKR.
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Fig. 4.
MutPKR inhibits
TNF- /IFN-
-induced
B-specific gene expression. PC12 cells
transiently transfected with a
B-dependent luciferase
reporter construct (MHC-NF-
BLuc) alone or in combination with a
plasmid expressing wtPKR or a catalytically inactive mutPKR were
treated with 10 ng/ml TNF-
, 100 units/ml IFN-
, or a combination
of both for 7 h. Lysates were assayed in duplicate for luciferase
activity, and fold activity was determined by dividing the number of
relative light units from treated samples by the number of relative
light units from untreated (UT) samples. The data shown are
averages of three independent experiments, and the S.E. of the mean are
indicated by error bars.
DISCUSSION
and IFN-
during many biological
responses including the regulation of gene expression is well documented (for review, see Refs. 52 and 53), and there are several
mechanisms by which these two cytokines can collaborate. For example,
cooperation can be achieved by mutual up-regulation of each other's
receptors (54-58). In the context of gene expression, the synergy
between TNF-
and IFN-
is thought to be the result of the
independent activation of NF-
B by TNF-
and of IFN regulatory factors or signal transducers and activators of transcription by
IFN-
which bind to unique promoter sites and synergistically regulate gene expression. Previously, we reported a mechanism by which
these two cytokines can synergistically activate gene expression in an
endothelial and a preneuronal cell line (27). In our model, IFNs
significantly potentiate the TNF-
-induced nuclear translocation of
NF-
B and
B-dependent gene expression. The mechanism
for this synergy involves the de novo degradation of
I
B
. This is a novel mechanism for NF-
B activation because IFN-
alone does not activate NF-
B. The net result is the targeted degradation of both I
B
and I
B
which increases the amount of NF-
B that is free to translocate into the nucleus and therefore synergistically increases
B-dependent gene expression.
binding to its receptor which
is responsible for enhanced nuclear translocation of NF-
B and the
synergistic activation of
B-dependent gene expression during TNF-
/IFN-
cotreatment? Our data indicate that in cells of
neural origin, IFN-
potentiates the ability of TNF-
to induce NF-
B activity by targeting the serine/threonine kinase, PKR. This
dsRNA-activated, IFN-inducible kinase is best known for its role during
antiviral responses where, in response to dsRNA, it homodimerizes and
autophosphorylates and then phosphorylates and deactivates its primary
target, the translation initiation factor, eIF-2. However, PKR has also
been implicated in controlling cell growth, cell differentiation, and
tumor supression (for review, see Refs. 29 and 30), and there is
evidence that it can become phosphorylated in the absence of viral
infection or dsRNA treatment (59). In PC12 and B12 cells, pretreatment
with the PKR-specific inhibitor 2-AP completely blocked
TNF-
/IFN-
-induced NF-
B nuclear translocation and reduced
B-dependent gene expression by at least 40% (Figs. 1
and 3). Because it is possible that 2-AP could affect molecules other
than PKR (36), we specifically targeted PKR by transfecting cells with
a catalytically inactive form of PKR. This dominant negative PKR
effectively reduced the level of TNF-
/IFN-
-induced
B-dependent gene expression to the level observed with
TNF-
alone (~80% reduction) (Fig. 4).
/IFN-
-induced activation of NF-
B in
cells of neural origin but not in endothelial cells (Fig. 1). Petryshyn
et al. have shown that IFN-induced PKR activity does not
occur until at least 3 h after treatment (60, 61). This might
explain the cell type-specific effect of 2-AP because the synergistic
activation of NF-
B occurs in endothelial cells within 15 min to
1 h post-stimulation, whereas the synergy in neuronal derived
cells does not occur until later than 2 h post-stimulation (27).
Also, 2-AP did not block NF-
B activity induced by TNF-
alone,
therefore 2-AP is most likely targeting a signal generated by IFN-
binding to its receptor.
B in
mouse embryo fibroblasts isolated from the PKR knockout mouse (Pkr0/0 MEFs). In Pkr0/0
MEFs, dsRNA-activated NF-
B is reduced compared with levels in wild-type MEFs (Pkr+/+ MEFs), but
TNF-
-activated NF-
B levels are normal (32). Upon pretreatment
with IFN-
or IFN-
, dsRNA-induced NF-
B activity is restored to
normal. Maran et al. (34) have used an antisense procedure
to decrease selectively the level of PKR activity in cells. In these
cells, dsRNA could not activate NF-
B, but the activation of NF-
B
by TNF-
was unaffected. Recently, several groups have shown that
I
B
may be phosphorylated by PKR in vitro in response
to dsRNA, but it is not clear whether this occurs in vivo
(32-35). There is no direct evidence that I
B
can be
phosphorylated by PKR; however, I
B
has two serine residues that
are analogous to the two serines present in I
B
which can be
inducibly phosphorylated prior to ubiquitination and degradation
(10-12, 14, 18). In an attempt to implicate PKR further during
synergistic NF-
B activation, we began to examine the effect of
TNF-
/IFN-
cotreatment in Pkr+/+ MEFs and
Pkr0/0 MEFs. The Pkr+/+
MEFs did not exhibit synergistic activation of NF-
B (at least with
the concentrations of TNF-
and IFN-
used to generate synergism in
PC12 cells), therefore we were unable to use this cell model for
further studies. These results further strengthen our hypothesis that
the mechanisms for the synergistic activation of NF-
B will be
diverse and cell type-specific.
B activation? A major component of
the TNF-
/IFN-
-induced activation of NF-
B is the mechanistic switch from I
B
degradation to I
B
degradation, leading to
persistent activation of NF-
B. This is similar to previous
documentation where I
B
is thought to be involved in the transient
activation of NF-
B, whereas I
B
is targeted during the
persistent activation of NF-
B (14-17) Also, it has been proposed
that I
B
can act either as an inhibitor or as a chaperone-like
protein. As a chaperone, I
B
could protect NF-
B from the
inhibitory properties of I
B
, and this mechanism may be explained
by the differential phosphorylation of I
B
(17). In our system,
using 2-AP to block PKR activity inhibits the de novo
degradation of I
B
during costimulation but does not affect the
pattern of I
B
degradation. Blocking I
B
degradation is
concomitant with inhibiting the synergistic and prolonged activation of
NF-
B. This leads to a model whereby signals generated from
TNF-
/IFN-
cotreatment activate PKR, which in turn either directly
or indirectly causes the induced phosphorylation and degradation of
I
B
and consequently the synergistic activation of NF-
B.
/IFN-
cotreatment could target PKR. First, PKR protein levels could be up-regulated by IFN-
or by TNF-
(62; and for review, see Refs. 29
and 30). However, our previous work using cycloheximide demonstrates
that protein synthesis is not required for this TNF-
/IFN-
synergy, therefore an increase in PKR protein levels cannot account for
this response. Second, IFN-
may lead to an increased activity of
PKR, but signals generated by the presence of both cytokines may be
required to target the NF-
B·I
B
complex. Third, signals generated by cotreatment could induce the synthesis of or change the
structure of a cellular dsRNA or other PKR activator, which could then
activate PKR (59). Also, there is evidence for endogenous proteins that
act as cellular PKR inhibitors (63, 64); therefore cotreatment could
generate signals that could counteract the inhibitory roles of these
proteins. Another possibility is that IFN-
and/or TNF-
may
generate signals that induce phosphorylation and activation of PKR. PKR
could then directly phosphorylate I
B proteins and target them for
degradation or activate a kinase that is responsible for I
B
phosphorylation. Recently, two subunits of the multiprotein complex
that forms a functional IKK have been isolated and characterized. Both
IKK
and IKK
are TNF-
-inducible and specifically phosphorylate both I
B
and I
B
on critical serine residues (6-9). It has been reported that IKK itself may require phosphorylation for activation, although the required kinase(s) has not been identified (7). Potentially, there could be distinct IKKs that are specific for
individual I
B proteins in vivo, and an IKK which
specifically targets I
B
could be a substrate for PKR. It is also
likely that the strength of the signal determines the extent to which
NF-
B will be activated. For example, TNF-
can activate IKK but
maybe only to limited levels. However, in the presence of signals
generated by IFN-
(such as increased PKR activity) the activity of
IKK could be elevated and subsequently increase and/or prolong the activity of NF-
B.
-induced activation of
NF-
B. Our data indicate a role for the IFN-inducible kinase PKR in
this response in cells of neuronal origin. Clearly there are
implications that the synergistic activation of NF-
B in neural derived cells may be important in suppressing an apoptotic mechanism. TNF-
and IFN-
are both potentially apoptotic agents; however, together they potentiate the activation of the anti-apoptotic activity
of NF-
B. Although PKR has been implicated in a mechanism for
stress-induced apoptosis (65, 66), the net cellular response to TNF-
and IFN-
may depend on the source(s) and/or strength of the signals
that are generated. Therefore, the synergistic activation of NF-
B by
TNF-
and IFN-
during an inflammation response could protect cells
of neural origin from death.
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ACKNOWLEDGEMENTS |
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We thank William E. Miller for insightful discussions and for critical reading of the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by National Institutes of Health/National Research Service Award Fellowship 1 F32 AG05745. Present address: Bayer Corp., R&D Pathogen Safety Research, Research Triangle Park, NC 27709.
Supported by National Institutes of Health Grant AI34039.
** Supported by National Institutes of Health Grant AI35098. To whom correspondence should be addressed: Lineberger Comprehensive Cancer Center, CB 7295, University of North Carolina School of Medicine, Chapel Hill, NC 27599. Fax: 919-966-0444.
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ABBREVIATIONS |
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The abbreviations used are:
NF-B, nuclear
factor kappa B;
I
B, inhibitor of kappa B;
IKK, I
B kinase;
TNF-
, tumor necrosis factor-
;
IFN, interferon;
MHC, major
histocompatibility complex;
PKR, double-stranded RNA-activated protein
kinase;
dsRNA, double-stranded RNA;
eIF-2, eukaryotic initiation
factor-2;
2-AP, 2-aminopurine;
EMSA, electrophoretic mobility shift
analysis;
wtPKR, wild-type PKR, mutPKR, mutant PKR;
Pkr0/0 MEFs, mouse embryo fibroblasts devoid of
functional PKR;
Pkr +/+ MEFs, wild-type mouse
embryo fibroblasts.
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REFERENCES |
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