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B by IFN-
in a STAT1-Independent Pathway1
Department of Cancer Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. It also acts as a signal transducer for transcription
factors NF-
B, IFN regulatory factor-1, and activating transcription
factor-2. IFN-
, a pleiotropic cytokine, elicits gene
expression by activating the Janus kinase-STAT signaling pathway.
IFN- can synergize with TNF- to activate NF-B in a number of
cell lines. Here we show that IFN- alone can activate NF-B, by a
Janus kinase-1-mediated, but Stat1-independent, mechanism. NF-B
activation by IFN- is associated with degradation of IB 
. The
IFN- response can be blocked by 2',5'-oligoadenylate-linked
antisense chimeras against PKR mRNA. There was no activation of NF-B
by IFN in PKR-null cells, indicating that PKR is required for IFN-
signaling to NF-B. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, is
immediately activated and migrates to the nucleus following exposure of
cells to diverse forms of environmental stress and activates the
transcription of cognate genes. The stimuli include cytokines,
mitogens, physical cellular stress, and exposure to bacterial and viral
products against which responses need to be elicited immediately for
protection of the host. Five members have been identified in the
NF-B family in mammalian cells, termed p65, c-Rel, Rel B, p50/p105,
and p52/100. Whereas p65, c-Rel, and Rel B are produced as
transcriptionally active proteins, p105 and p100 are processed to their
active counterparts. A variety of active homo/heterodimers are formed
that regulate transcription (1, 2, 3). These dimers are
sequestered in the cytoplasm of unstimulated cells via interaction with
inhibitory proteins IBs, a class of structurally and functionally
related polypeptides with ankyrin repeats. The interaction of the
ankyrin repeats of IBs and the rel homology domain of
NF-B is responsible for retaining NF-B in the cytosol. Upon
stimulation with different ligands, the IB kinase is activated,
which, in turn, phosphorylates the IB proteins. The phospho-IB
proteins become targets for ubiquitination and degradation (4, 5). This promotes the translocation of NF-B to the nucleus by
unmasking its nuclear localization signal. Although IB and
have been mostly studied in terms of responses associated with a
transient and persistent activation of NF-B, the specific response
depends on the nature and amount of ligand and the specific cell type
(6, 7, 8).
The pleiotropic cytokine IFN-, induces gene expression through
activation by tyrosine and serine phosphorylation of components of the
Janus kinase
(JAK)4-STAT pathway
(9, 10, 11). The expression of IFN-stimulated genes (ISGs)
confers antiviral and antiproliferative effects on cells. Other
pleiotropic cytokines, such as TNF-, can function together with
IFN- to synergistically induce the expression of ISGs. For genes
such as MHC-1, VCAM-1, and inducible NO synthase, IFN-activated Stats
synergize with TNF-activated NF-B to activate transcription
(12, 13). Although IFN- alone typically does not
activate NF-B in many cells, it can synergistically induce the
translocation of p50/p65 with TNF-. This correlates with gene
transcription and is mediated by the degradation of predominantly IB
protein (14).
The ISGs regulate cell growth, and this requires close
coordination of growth-promoting and growth-inhibiting signals
(9, 10, 11). The IFN-induced dsRNA-dependent protein kinase
PKR regulates cell growth by functioning as a signal transducer to both
cellular translational and transcriptional components, including
subunit of eukaryotic initiation factor-2, IFN regulatory factor-1
(IRF-1), NF-B, and activating transcription factor-2
(15, 16, 17). The phosphorylation of subunit of eukaryotic
initiation factor-2 by PKR activated in response to dsRNA blocks
protein synthesis initiation. Activation of NF-B by dsRNA is
mediated by PKR through the IB kinase complex, where IB
appears be the major target of PKR-mediated degradation (18, 19). In PKR-null cells there is also a deficiency in the
activation of IRF-1 and the activation and phosphorylation of
activating transcription factor-2 (17, 20, 21, 22). This
results in attenuation of the induction of genes dependent on these
transcription factors, including MHC-1, Gbp-1, and E-selectin.
Interestingly, we noted that in cells lacking Stat1, IFN- was still
able to induce IRF-1, albeit at a reduced level. Because the IRF-1
promoter contains NF-B sites in addition to a Stat1-binding
IFN--activated site (23, 24, 25, 47), we examined whether
IFN- could induce transcription of the IRF-1 gene by activating
NF-B in a PKR-dependent process.
In this report we provide evidence that IFN- alone can activate
NF-B in different cells. Using a mutant cell line defective in Stat1
we show that activation of NF-B by IFN- proceeds in a
Stat1-independent manner via the degradation of IB . Using both
2',5'-oligoadenylate (2–5A)-linked chimeric antisense against
PKR mRNA and PKR null cells we show that the IFN--dependent
activation of NF-B is dependent on PKR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HeLa S3, 2fTGH, and NIH-3T3-like fibroblasts derived from
PKR+/+ and PKRo/o mice were
grown in monolayer cultures in DMEM supplemented with 10%
heat-inactivated FCS, 2 mM L-glutamine, and 100 U/ml
penicillin and streptomycin (Life Technology, Grand Island, NY). U3A
and U4A cells were grown in the same medium containing 250 µg/ml of
hygromycin B. 2fTGH, U3A, and U4A cells were provided by George R.
Stark (Cleveland Clinic, Cleveland, OH), and
PKR+/+ and PKRo/o cells
were provided by C. Weissmann (Institut fur Molekularbiologie I,
Universitat Zurich, Zurich, Switzerland). 2fTGH, U3A, and U4A cells
were serum-starved for 18 h (for experiments described in Figs. 1
and 4), and U3A cells were grown totally in serum-free medium (for
experiments described in Fig. 3).
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(R&D Systems, Minneapolis, MN) and IFN- (Roche,
Indianapolis, IN) were purchased from the respective companies. The
IFN--inducible protein-10 (IP-10) luciferase used (containing
two copies of NF-B binding sequence from human IP-10 promoter) was
obtained from Y. Ohmori (Cleveland Clinic), and PKR Ab (monoclonal) was
a gift from Ara Hovanessian (Pasteur Institute, Paris, France). IB
Abs ( and , C-21 and C-20) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Isolation of stable transfectants
HeLa S3 cells were transfected in duplicate plates (10-cm plates with 2 x 106 cells/plate (approximately)) with 5 µg of human 1.3-kb IRF-1 promoter-luciferase or pGL2 basic (Promega, Madison, WI) as the control. Cotransfection was conducted with 0.2 µg of SV2-Neo marker plasmid. Transfection was performed using Lipofectamine Plus according to manufacturer’s instructions (Life Technologies). At 4 h post-transfection, the medium with DNA-Lipofectamine complexes was removed and replenished with fresh medium with 10% FCS, and cells were allowed to grow for 20 h. They were trypsinized, seeded at a lower density in 15-cm plates, and allowed to grow in a medium containing 400 µg/ml of G418, and medium was changed twice a week for 3–4 wk. G418-resistant clones were transferred individually to small plates and were propagated in G418 medium before analysis.
Preparation of whole cell and nuclear extracts
For preparing whole cell extract (WCE), cells were scraped off
from the plate using 2–3 ml of PBS. After 10 min of spinning at
10,000 x g, supernatant was discarded, followed by
addition of 50–70 µl of the extraction buffer (20 mM Tris-HCl (pH
8.0), 0.4 M NaCl, 1.5 mM MgCl2, 1% Triton X-100,
25 mM -glycerophosphate, 25 mM sodium fluoride, 0.5 mM sodium
orthovanadate, 1 mM PMSF, 10 µg/ml aprotinin, 10 mM
p-nitrophenylphosphate, and 10% glycerol). The cell pellet
was suspended in extraction buffer and left out on ice for 20 min.
After spinning for 20 min at 10,000 x g, supernatant
was collected and stored in frozen aliquots at -70°C.
Nuclear extract
Cells were washed twice in cold PBS and suspended in 400 µl of cold A buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF). They were allowed to swell on ice for 15 min, after which 25 µl of 10% Nonidet P-40 (Sigma) was added, and the cells were vigorously vortexed for 10 s. After centrifuging the homogenate at 10,000 x g for 2 min, the pellet was suspended in 50 µl of ice-cold buffer B (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF). Tubes were vigorously rocked at 4°C for 15 min in a shaking platform. The supernatant was saved as nuclear extract after centrifugation (10,000 x g for 5 min) and was stored as frozen aliquots at -70°C (26).
Electrophoretic mobility shift assays
Standard DNA binding assays were performed using 0.5 ng of
labeled probe and increasing amounts of unlabeled competitor DNA. The
full sequence of each oligonucleotide is shown in Table I. Extract containing 5 µg of total
protein from nuclear extract/25 µg from WCE was incubated in a
binding buffer (20 mM HEPES (pH 7.9), 5 mM MgCl2,
1 mM DTT, 1 mM EDTA (pH 8.0), and 10% glycerol) containing 1 µg of
poly(dI-dC) and -32P end-labeled probe for 30
min in ice. Protein-DNA complexes were resolved in low ionic strength
5% native acrylamide gels with 0.5x TBE as the running buffer.
Electrophoresis was performed at room temperature, Gels were dried and
exposed at -70°C. In the experiments indicated, extracts were
incubated with the specific Ab for supershift for 10 min in ice after
addition of the probe.
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Cells were plated (HeLa S3) in 10-cm plates at 20–30%
confluence and were allowed to grow for 16 h. Transfection was
performed with 5 µg of promoter plasmid(s) and 1 µg of Rous sarcoma
virus--galactosidase-expressing -galactosidase as an
internal transfection control using Lipofectamine Plus (Life
Technologies). After 3–4 h, media with DNA-Lipofectamine complexes
were removed, and cells were replenished with fresh medium with 10%
FCS and allowed to grow. After 16–18 h of transfection, cells were
serum-starved for 12 h and were treated with IFN- (1000 U/ml;
Roche) for 9 h, washed twice in PBS (pH 7.0), lysed in reporter
lysis buffer, and subjected to assay for luciferase or
-galactosidase activity (Promega).
Antisense treatment
Chimeric oligonucleotide (2 µM/treatment) was added to
cultures of HeLa S3 cells at 0, 12, 24, 36, 48, 60, and 72 h in
six-well plates. After 76 h, cells were treated with human
recombinant IFN- (1000 U/ml; Roche) for 9 h. Cells were washed
and scraped off the plate, and one-half of the extract was used for PKR
activity assay described in the next section. One-fourth of the lysate
from the other half was used for measuring luciferase activity.
Antisense oligonucleotide was designed on the basis of
binding site of the chimera 55–73 nucleotides down from
the start codon of PKR mRNA (27). The antisense PKR
chimera used was
5'-Sp(A2'p)3A-linker-GTACTACTCCCTGCTTCTG
3'-3' tail (dC)-5'. The antisense oligonucleotide was synthesized as
described previously (28). The linker consisted of two
1,4-butanediol monomers attached to each other and to
p5'(A2'p5')3A
and 3'-5' oligodeoxyribonucleotide moieties with phosphodiester bonds
(27). The anti-PKR and sense PKR sequences are
5'-GTACTACTCCCTGCTTCTG-3' and 5'-CAGAAGCAGGGAGTAGTAC-3' as
previously described (27).
Determination of PKR activity
Two hundred micrograms of total cell extract was used for
immunoprecipitation of PKR with 2 µl of a 1/20 dilution of
anti-human mAb of PKR. Ab dilutions were made in freshly prepared
mammalian cell lysis buffer (20 mM Tris-HCl (pH 7.0), 150 mM NaCl, 10%
glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM
EGTA, 100 mM NaF, 100 mM sodium pyrophosphate, 1 mM PMSF, 10 µg/ml
aprotinin, and 10 µg/ml of leupeptin). After incubating the mixture
of Ab and the cell extract in ice for 2 h, 2 vol of the lysis
buffer was added followed by addition of 20 µl of protein G-Sepharose
(1/1 in the lysis buffer; Amersham Pharmacia Biotech, Arlington
Heights, IL). The mixture was rotated in a shaker overnight and
centrifuged at 8000 x g for 2–3 min, followed by
washing three times with fresh lysis buffer. The beads were finally
washed in DBGA buffer (10 mM Tris (pH 7.6), 50 mM KCl, 2 mM
MgOAc, 20% glycerol, and 7 mM 2-ME) twice. Thirty microliters of DBGA
buffer was added to the beads with 20 µl of DBGB (2.5 µl of 1 M
MnCl2 in 1 ml of DBGA buffer), followed by
addition of 5 µl of ATP mix (200 µl of DBGA buffer, 2.5 µl of
[-32P]ATP (6000 Ci/mmol and 150 µCi/µl),
and 2 µl of 1 mM ATP). The mixture was incubated for 30 min at
30°C. After centrifuging at 10,000 x g for 5 min,
the supernatant was discarded, an equal volume of SDS-PAGE buffer was
added, and samples were analyzed in 10% SDS-PAGE. The gel was fixed in
50% methanol and 10% acetic acid, dried, and exposed in room
temperature for 2–3 h (29).
Immunoblotting
2fTGH, U3A cells were grown in 10-cm dishes to 50% confluence
and were serum-deprived 16 h before treatment with IFN- (1000
U/ml) and TNF- (10 ng/ml; Roche) for desired time periods. In the
case of fibroblasts derived from PKR wild-type (WT) and knockout mice,
10–25 ng/ml of TNF- was used with 100 U/ml of IFN- for 1 h.
Cells were then washed three times with cold PBS (pH 7.0) and were
lysed in 50 mM HEPES (pH 7.4), 1% Triton X-100, 0.4 M NaCl, 10 mM
EDTA, 10 mM
Na4P2O7,
100 mM NaF, 2 mM sodium orthovanadate, and protease inhibitors (1 mM
PMSF, 1 µg/ml aprotinin, and 2 µM leupeptin). After a 20-min
incubation at 4°C, the lysate was collected and centrifuged at
13,000 x g for 15 min at 4°C, and the supernatant
was stored at -70°C. Samples were separated on 10%
SDS-polyacrylamide gel and electroblotted on a polyvinylidene
difluoride membrane. The membrane was blocked for 16 h at 4°C in
20 mM Tris (pH 7.4), 500 mM NaCl, and 0.1% Tween 20 supplemented with
3% BSA and blotted for 3 h with the IB or IB Abs.
Bound Abs were detected with HRP-conjugated anti-rabbit IgG
followed by ECL detection (Amersham Pharmacia Biotech).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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induces binding of NF-B to diverse B binding sites
The effect of IFN- on NF-B activation was investigated
in cells previously used to study IFN signaling. These include mutant
derivatives of fibrosarcoma cells 2fTGH, including U3A, U4A, and 2,
which lack Stat1, JAK1, and JAK2, respectively, all of which are
required components for IFN--mediated JAK-Stat signaling
(9, 10, 11). Diverse NF-B binding sequences were used to
analyze binding with nuclear extracts of IFN--treated cells in EMSAs
(Table I
). The DNA binding sequences used include the consensus B
site as well as sequences (KB 1–4) derived from the human IRF-1
promoter that are located at 40, 130, 660, and 890 nucleotides upstream
of the transcription start site of the human IRF-1 gene
(25). Only KB1(-40) has been reported previously to bind
NF-B and is implicated in the regulation of the IRF-1 promoter in
the context of viral infection (23, 25). EMSA with
IFN--treated nuclear extracts from HeLa S3 cells showed specific
binding of NF-B to KB1, KB3, and KB4, whereas KB2, which overlaps
with the Stat binding site, failed to show direct binding (Fig. 1, A–C; KB2 data not shown).
Treatment with IFN- led to a 1.8- to 2.4-fold induction (determined
by phosphorimager quantitation; Fig. 1D) of NF-B binding,
which reached a maximum at 
45 min (Fig. 1, A–C). This
induction of binding is apparent in HeLa S3 cells as nuclear extracts
from untreated cells exhibit high constitutive binding even after
18 h of serum starvation. Serum starvation for 36 h before
IFN- treatment also failed to reduce constitutive binding (data not
shown). This induction of binding was demonstrable using KB3 and KB4 as
DNA binding elements. In all cases, binding of NF-B was confirmed by
supershifting of DNA-protein complexes with specific Abs against
NF-B subunits (p50/p65) and in some cases by using a mutant sequence
that no longer supports binding of the transcription factor, confirming
the specificity of binding. The constitutive complexes formed with
nuclear extracts from untreated HeLa S3 cells could also be
supershifted by the Abs of p50/p65 and could be efficiently competed
out by cold competitors (data not shown). The results demonstrate that
IFN- alone induces the binding of NF-B to divergent NF-B
binding sequences in the IRF-1 promoter, which have not yet been
characterized.
These observations were supported by experiments using U3A cells that
lack Stat1. Treatment of these cells with IFN- for 45 min led to the
induction of two distinct complexes, termed NF-B I and NF-B
II (Fig. 1E), both of which contained the p50 and p65
subunits of NF-B and could be efficiently competed by cold
competitor oligonucleotides. An Ab against the c-Rel subunit of NF-B
failed to supershift the NF-B complexes in U3A (Fig. 1E)
and in HeLa S3 (not shown). Of the two IFN--inducible complexes,
NF-B I was induced more by IFN- than was NF-B II, which
also was present at a constitutive level. Other complexes were also
detected (Fig. 1E, complexes CI and CII) that were
down-regulated by IFN- treatment. The complexes of untreated U3A
cells were also competed out with NF-B DNA element as a cold
competitor.
IFN--induced binding to NF-B in the IRF-1 promoter affects
transcriptional activation
It has previously been shown that a point mutation within the KB1
site abolishes factor binding and inhibits virus inducibility of the
IRF-1 promoter in L929 cells (23, 25). To correlate
binding of NF-B to this site with the IFN- response, a
site-directed mutant (at -40, KB1; Table I) that abolishes NF-B
binding was generated in a 1308-bp fragment of the human IRF-1
promoter, and the fragment was cloned upstream of the luciferase
reporter. The transcriptional response of this mutant (KB1) to IFN-
in transient transfection assays was reduced by 21% (Fig. 2). Mutation of the other NF-B binding
sites, KB1, KB3, and KB4 (mTM), reduced the response to 32% of the WT
response (Fig. 2). We conclude that IFN- activates NF-B and
induces its binding to different B sites to influence the total
IFN- response.
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B binding sequences of IRF-1 promoter by
IFN-
To determine the relative affinity of NF-B binding to the
different B sites of the human IRF-1 promoter, we used
IFN--treated nuclear extracts from U3A cells for competition in gel
shift assays. Binding of NF-B to the consensus B site was
competed using different concentrations of each of the cold DNA
elements spanning KB1, -2, -3, and -4 sites (Table I). The consensus
NF-B binding is competed out by 200-, 550-, and 700-fold molar
excesses of KB1, KB3, and KB4, whereas KB2, the known putative NF-B
binding site within the Stat binding element, failed to compete even at
a very large fold molar excess (even at 1000-fold; Fig. 3, A, C, and
E) (30). The NF-B complex from untreated U3A
cells is also competed out by using KB1 as the cold competitor (Fig. 3B). Using TNF--treated nuclear extracts from U3A cells
as the positive control assesses the identification of p65/p50 complex.
Supershifts with Abs against p50, p65, and c-Rel has been shown in the
same figure (Fig. 3D). The IFN--induced p65/p50 complex
also comigrated in the same position as the p65/p50 complex in U3A. A
similar supershift with Abs also confirms the induction of activation
of p65/p50 complex by IFN- alone.
These results show that different B sites are differentially active
in NF-B binding in response to IFN-. The quantitation of the
competition studies is presented in Fig. 3E.
Stat1 independent IFN- signaling of NF-B
To determine whether this signaling requires Jak activity, the
JAK1 mutant U4A cells were treated with IFN-, and NF-B activation
was measured. Because only constitutive binding to the consensus site
was seen (Fig. 4A), we
conclude that JAK1 activity is necessary for the IFN--activated
signal. The quantitation of the binding experiment (Fig. 4A)
is presented in Fig. 4B. A similar result was obtained in an
experiment performed in 2 cells that lack functional JAK2 (data not
shown). However, the induction in binding of NF-B was still present
in U3A cells lacking Stat1 (Fig. 4, A and B).
These results demonstrate that the alternate pathways of activation of
NF-B by IFN- require JAKs, but bypass the conventional
Stat1-mediated pathway. This is a transcriptionally competent pathway,
because a IFN--responsive reporter constructs (the IP-10 promoter
luciferase) transfected into U3A cells can be induced by IFN- (Fig. 4C).
The relative contributions of NF-B to the IFN--induced
transcriptional response of the IRF-1 promoter was assessed by
transfecting different IRF-1 promoter-reporter constructs into 2fTGH
and Stat1-null U3A cells. The results (Fig. 5) indicate that when this promoter is
activated by IFN-, 31% of the activity is contributed by
NF-B. This can be seen either when the WT construct is transfected
into U3A cells or when the B mutant construct (mTM) is transfected
into 2fTGH cells (Fig. 5). It is interesting that U3A cells transfected
with mTM failed to show any luciferase activity (Fig. 5B),
elaborating that the response of WT promoter to IFN- in U3A cells is
due to binding of NF-B (Fig. 5B). Thus, NF-B is a
significant contributing factor in the IFN--mediated induction of
the IRF-1 promoter.
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signaling to
the IRF-1 promoter
Experiments with PKR-null mice and derived cell lines have
implicated this kinase in IFN- signaling (20, 21).
Previously we have shown using 2–5A chimeric antisense
oligonucleotides (2–5A antisense chimera) directed against PKR that
activation of NF-B by dsRNA is PKR dependent (27, 31).
A similar experimental setup was used to determine the effect of
depletion of PKR on the IFN- response in human cells. A stable HeLa
S3 cell line expressing luciferase under the control of the human IRF-1
promoter was generated. This line exhibited 9.7-fold induction of the
luciferase reporter gene after IFN- treatment for 9 h.
Treatment with the antisense and sense chimerae of PKR resulted in 42%
and 10% decreases in luciferase activity after IFN- treatment for
9 h (Fig. 6; normalized in terms of
the same specific activity of PKR). PKR mRNA and protein levels were
similarly decreased (data not shown). A negative control other than the
sense PKR chimera, anti-PKR, is also ineffective in reducing PKR
activity (Fig. 6A; luciferase assay for it not shown). The
change in PKR activity coupled with the change in the IFN- response
demonstrate that PKR plays a role in the IFN--driven transcriptional
response. These results are in accord with those of transient
transfection assays with the human IRF-1 promoter as well as DNA
binding studies of IRF-1 in response to IFN-, which have clearly
established that PKR is required for activation of IRF-1 by IFN-
(21, 22). To determine whether PKR-dependent activation of
NF-B could be implicated in this response, nuclear extracts prepared
from IFN--treated WT and PKR-null fibroblasts were compared for
NF-B DNA binding. Although extracts from WT cells exhibit specific
binding of NF-B to the NF-B binding consensus sequence, this is
defective in extracts from PKR-null fibroblasts (Fig. 7A, lanes 6 and
12). Interestingly, TNF--induced activation of NF-B
was also found to be defective in the PKR-null cells, but some synergy
with IFN- was noted in fibroblasts (Fig. 7A, lanes
2–5 and 8–11). A synergism in NF-B activation
dependent on PKR has previously been noted in neuronal cells treated
with IFN- and TNF- (32). Nuclear extracts from
TNF-- and IFN--treated PKR+/+ mouse embryo
fibroblasts (MEFs) were used as a positive control to show the p50/p65
complex of NF-B and the presence of the same complex induced by
IFN- alone (Fig. 7B).
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treatment of U3A cells results in the degradation of
IB
Degradation of IB is associated with a transient activation
of NF-B, whereas degradation of IB is associated with a
persistent activation (6, 7, 8). IB also acts as an
inhibitor and chaperone-like protein. The chaperone-like properties
have been attributed to a differential phosphorylation of IB that
has been linked to protection of NF-B from inhibitory properties of
IB . Apart from role of phosphorylation of
IBs by different stimuli and their influence on activation of
NF-B, inhibition of polyubiquitination has recently been shown to
play a role in IB degradation/NF-B activation. This strategy is
used by nonvirulent enteric pathogens whose direct interaction with
human epithelia attenuate the synthesis of inflammatory effector
molecules elicited by diverse proinflammatory stimuli (33, 34).
In U3A cells, in which we clearly detected IFN--induced activation
of NF-B, degradation of IB was more prominent than
degradation of IB (Fig. 8A). These data suggest that
IFN- stimulation of NF-B via a PKR-dependent pathway
preferentially targets IB and thus contributes to persistent
activation of NF-B (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B regulates the
expression of genes involved in immune and inflammatory responses, a
number of which are associated with the IFN response. Many
IFN-regulated genes are modulated by NF-B binding; consequently,
activation of NF-B by IFNs might be an important determinant through
which these cytokines regulate immune reactivity and inflammation. Here
we report activation of NF-B by IFN- and show that it is mediated
by PKR. The direct activation of NF-B by IFN- is restricted in
both magnitude and cell type. However, even in cells in which IFN-
by itself has little effect on NF-B activation, it is able to
synergize with other stimuli, including TNF- (13, 14).
This synergistic activity is also dependent on PKR
(32).
Our study is focused on regulation of human IRF-1 promoter by
IFN- in a Stat-independent pathway(s). IRF-1 is a transcription
factor that binds to DNA sequence elements found in the promoter of
type 1 IFN and IFN-inducible genes. Transient up-regulation of IRF-1
gene by virus and IFN treatment causes the consequent induction of many
IFN-inducible genes involved in growth control and apoptosis. It has
been clearly shown that IFN- and all-trans-retinoic acid
inhibit cell proliferation of squamous cells by inducing apoptotic cell
death. This phenomenon correlates with increased expression of IRF-1.
Evidence has been provided showing that all-trans-retinoic
acid-induced expression is independent of activation of the Stat-1
activation pathway despite the presence of IFN--activated sequence
in the IRF-1 promoter. Activation of NF-B is responsible for this
increased expression of IRF-1, which also supports our finding that
activation of Stat-independent factors can regulate IRF-1 through
activation and binding to NF-B (35).
We have shown in this study that IRF-1 promoter requires both PKR and
NF-B for its full activation by IFN-. We have identified and
characterized the direct binding B sites in the human IRF-1 promoter
and measured their relative affinity for NF-B. The KB1 was
previously found to bind NF-B using an indirect approach in which
gel shift assays were performed with a potential NF-B site from
IRF-2 promoter using nuclear extracts of mouse L929 cells infected
with NDV. A virus-inducible complex was detected with the element,
which was competed by DNA fragment (-47 to -38) from the human IRF-1
promoter containing KB1. Site-directed mutagenesis confirmed the
importance of this site for NF-B binding during viral infection
(23, 25).
IFN--dependent cellular responses are mediated by JAKs and Stats.
Isolation and complementation of mutant human cell lines have revealed
that JAK1 and JAK2 become activated in IFN--treated cells and are
required for ligand-dependent activation of IFN--dependent genes.
Stat1 was isolated as a transcription factor that undergoes rapid
tyrosine phosphorylation and activation in IFN--treated cells. The
intracellular domains of the two IFN- receptor subunits have
constitutive, specific binding sites for JAK1 and JAK2. IFN- induces
the formation of a specific phosphotyrosine binding site on the
receptor for Stat1, thereby linking the activated receptor to the
signal transduction apparatus (9, 10, 11).
Physiologically IFN- is secreted by activated T lymphocytes and NK
cells under conditions of activation and has a variety of
immunomodulatory functions (12). However, how IFN-
regulates genes associated with protection against pathogens is still
not completely understood (36, 37, 38, 39). In this context, the
activation of NF-B by IFN- is important because of the
involvement of this transcription factor in the induction of a number
of protective genes (33, 40). For example, the induction
of efficient Ag processing for MHC-mediated Ag presentation has been
found to have a key role in pathogen clearance, and MHC-1 is known to
be regulated by NF-B and other IFN regulatory factors (41, 42). Therefore, there is a physiological link between the
IFN- response and activation of NF-B. PKR was first implicated in
the IFN- response when it was found that PKR-null mice had a defect
in the IFN--mediated antiviral response. Studies of MEFs from these
mice established that this defect was at the cellular level and
implicated PKR in mediating IFN--induced signal transduction via
IRF-1 (17, 20, 21). PKR is activated by dsRNA, and
experiments using 2–5A antisense or PKR-null MEFs have shown that the
activation of NF-B by dsRNA is PKR dependent (17, 20, 21, 22). Synergism between TNF- and IFN- in activation of
NF-B in neuronal cells also requires PKR. Thus, taken together, our
results indicate that IFN- binding to a receptor probably generates
a signal(s) that leads to activation of PKR (14, 32).
Proinflammtory agents such as TNF- and IFN-, generally act
through cell surface receptors to rapidly phosphorylate cytoplasmic
signaling intermediates by either serine/threonine or tyrosine kinases
to induce specific cellular responses. IFN- is well studied for
cytokine signaling through the JAK-STAT pathway and, consequently, for
controlling inflammatory cellular immune responses dependent on this
cytokine and its downstream signal transducers. Studies have
established the ability of TNF-, dsRNA, and IFN- to induce
transcription factor NF-B in astrocytes lacking Src homology domain
2-containing protein tyrosine phosphatase (SHP)-1. On exposure
to the inducers, NF-B is markedly induced in astrocytes from
motheaten mice lacking SHP-1 compared with normal littermate cells
expressing SHP-1. NF-B is induced in a protein synthesis-independent
manner and consists of p50 and p65 subunits. Enhanced NF-B
expression in motheaten mouse cells correlates with increased
expression of genes with functional NF-B sites, including IRF-1,
inducible NO synthase, and MHC class I. Therefore, the regulation of
not only Stats, but also NF-B, by SHP-1 is important in controlling
events promoted by proinflammatory agents in vivo. It further
emphasizes an additional role for SHP-1 in controlling specific and
nonspecific immune responses where induction of NF-B is involved
(43). It is interesting to note that PKR mediates
activation of NF-B by TNF- and dsRNA (18, 19), and
in this study we show that IFN- also signals through PKR to activate
the same transcription factor to regulate IRF-1.
The link between PKR activation and IFN- receptor engagement
remained to be determined. In most cell types PKR is expressed at a low
constitutive level, although the gene can be induced, mainly by type I
IFNs. While IFN- treatment of cells results in PKR activation
(21), how JAK activation by IFN- is connected to PKR
has yet to be established. The involvement of protein activators of PKR
is likely, because one of the activators (RAX) has been linked to the
IL-3 tyrosine phosphorylation signaling cascade (44, 45, 46).
The effects of IFN- on PKR may be accentuated by cotreatment with
other cytokines, including TNF-, although this can only be detected
by NF-B activation when cells are treated with low levels of TNF
(19). It has also been observed that TNF--induced
activation of NF-B is not inhibited by 2-aminopurine, a known
inhibitor of PKR (32). However, the synergism of IFN-
and TNF- to activate NF-B is efficiently blocked by
2-aminopurine, suggesting that IFN- transduces signals through PKR
(32). Again, the mechanisms involved in PKR activation by
TNF- have not been defined. However, PKR has been found to be
associated with the IB kinase complex, where its major contribution
appears to mediate prolonged activation of NF-B (19).
It is intriguing that activation of NF-B by dsRNA (18)
and IFN-, as shown in this study, both depend on PKR. PKR is likely
to be a common signal transducer of these two signaling pathways
through activation of NF-B. An analysis of
PKRo/o mice and their responses to different
environmental stresses known to be mediated by PKR and NF-B will
provide insights into these unexplored areas of IFN- signaling.
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Acknowledgments |
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Footnotes |
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2 Current address: Faculty of Medicine, University of Aarhus, DK-8000, Aarhus C, Denmark.
3 Address correspondence and reprint requests to Dr. Bryan R. G. Williams, Department of Cancer Biology, The Lerner Research Institute, NB4–40, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195.
4 Abbreviations used in this paper: JAK, Janus kinase; PKR, dsRNA-dependent protein kinase; 2–5A, 2',5'-oligoadenylate; IRF, IFN regulatory factor; ISG, IFN-stimulated gene; IP-10, IFN--inducible protein-10; WT, wild type; WCE, whole cell extract; MEF, mouse embryo fibroblast; SHP, Src homology domain 2-containing protein tyrosine phosphatase.
Received for publication March 21, 2000. Accepted for publication March 9, 2001.
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References |
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