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Gene Expression: IL-12-Induced STAT4 Contributes to IFN- Promoter Activation by Up-Regulating the Binding Activity of IL-18-Induced Activator Protein 11
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Department of Oncology, Osaka University Graduate School of Medicine, Osaka, Japan; and
Fujisaki Institute, Hayashibara Biochemical Laboratories, Okayama, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA transcription
by activating STAT4 and AP-1, respectively. However, it is still
unknown how STAT4/AP-1 elicit IFN- promoter activation. Using an
IL-12/IL-18-responsive T cell clone, we investigated the mechanisms
underlying synergistic enhancement of IFN- mRNA expression induced
by these two cytokines. Synergy was observed in a reporter gene assay
using an IFN- promoter fragment that binds AP-1, but not STAT4. An
increase in c-Jun, a component of AP-1, in the nuclear compartment was
elicited by stimulation with either IL-12 or IL-18, but accumulation of
serine-phosphorylated c-Jun was induced only by IL-18 capable of
activating c-Jun N-terminal kinase. The binding of AP-1 to the relevant
promoter sequence depended on the presence of STAT4. STAT4 bound with
c-Jun, and a phosphorylated c-Jun-STAT4 complex most efficiently
interacted with the AP-1-relevant promoter sequence. Enhanced cobinding
of STAT4 and c-Jun to the AP-1 sequence was also observed when
activated lymph node T cells were exposed to IL-12 plus IL-18. These
results show that STAT4 up-regulates AP-1-mediated IFN- promoter
activation without directly binding to the promoter sequence, providing
a mechanistic explanation for IL-12/IL-18-induced synergistic
enhancement of IFN- gene expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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plays an important role in host defense and inflammatory responses
through many immunoregulatory effects (1, 2). Like IL-2
production, IFN- production by T cells is induced during their
interaction with APCs. Unlike IL-2, IFN- is also produced when T
cells are stimulated with cytokines elaborated by APC. Two APC-derived
cytokines, IL-12 and IL-18, have been shown to stimulate IFN-
production (3, 4, 5).
IL-12 is a critical cytokine that is required for the promotion of Th1
development as well as IFN- expression (3, 6). This
cytokine exerts its effects depending on signaling through STAT4
(7, 8). In addition to STAT4, other factors also enhance
IL-12-mediated Th1 responses. In particular, there is growing evidence
to support a role for IL-18 in the augmentation of Th1 responses.
Although IL-12 drives Th1 differentiation directly (3, 6),
IL-18 functions as a cofactor rather than as an initiator for Th1
development (9, 10). Moreover, recent studies have shown
that these two cytokines exert a synergistic effect on IFN-
production by T cells (9, 11) and NK cells
(12). Two different aspects of the mechanisms for this
synergy have been reported thus far. First, IL-12 up-regulates IL-18R
mRNA transcription/IL-18R expression on mouse T cells (11, 13, 14). Additional support for the synergy comes from the
observation that IL-12 and IL-18 use different signaling pathways in
inducing IFN- production (15). In fact, IL-12 and IL-18
activate different transcriptional factors: the former activates STAT4
and STAT3 (16, 17), and the latter activates NF-
B
(9) and AP-1 (15). A recent study
(15) showed that STAT4 and AP-1 interact with different
ends of the -266 to -186 fragment of the human IFN- promoter,
supporting the notion of IL-12/IL-18-induced enhancement of IFN-
mRNA transcription. However, there is no apparent STAT4-binding site in
the corresponding fragment of the mouse IFN- promoter. This has
raised an important question addressing how STAT4 functions to
up-regulate IFN- promoter activation.
In the present study, we investigated the mechanisms underlying
the synergy of IL-12 and IL-18 for enhanced IFN- transcription.
Using a mouse Th1 clone capable of responding to both IL-12 and IL-18
(18), we focused on the role of STAT4 in the regulation of
IFN- promoter activation. Our results show that IFN- promoter
activation by IL-18-induced AP-1 was marginal in the absence of
IL-12-induced STAT4. Costimulation with IL-12 and IL-18 resulted in the
accumulation of tyrosine-phosphorylated STAT4 and AP-1 containing
serine-phosphorylated c-Jun in the nuclear compartment. These two
transcription factors formed a complex that exhibited much stronger
binding to the AP-1-binding promoter sequence when compared with
STAT4-free AP-1. Thus, the present study illustrates a mechanism by
which a transcription factor up-regulates promoter activation without
directly binding to the promoter sequence.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The 2D6 mouse T cell clone, initially established as an IL-12-responsive clone (18) and later found to also respond to IL-18 (11), was used. 2D6 cells maintained with IL-12 (250 pg/ml) were used after intensive washing followed by starvation of IL-12 for 30 h. In some experiments, 2D6 cells were used by harvesting from IL-12 cultures without starvation.
Reagents
Mouse rIL-12 and rIL-18 were provided by Genetics Institute (Cambridge, MA) and Hayashibara Biochemical Laboratories (Okayama, Japan), respectively. Anti-c-Jun (H-79/sc-1694 and N/sc-45x) and anti-STAT4 (C-20/sc-486 and L-18/sc-485x) Abs as well as normal rabbit Ig were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phosphotyrosine mAb (4G10) and anti-phosphoserine-c-Jun (Ser63) Ab were obtained from Upstate Biotechnology (Lake Placid, NY) and New England Biolabs (Beverly, MA), respectively.
IFN- production by 2D6 cells and measurement of IFN-
concentration
2D6 cells (2 x 105/well) were
cultured with rIL-12 (250 pg/ml), rIL-18 (100 ng/ml), or a combination
of these in 24-well culture plates (Corning 25820; Corning Glass,
Corning, NY). After 24 h, culture supernatants were harvested and
IFN- concentrations were measured by ELISA using mouse IFN- ELISA
kits (Genzyme, Cambridge, MA).
Measurement of mRNA expression
Total cellular RNA was isolated by the acid guanidium-thiocyanate-phenol-chloroform method, and mRNA levels were determined using the RNase protection assay according to the procedure described in our previous report (11).
Immunofluorescence staining and flow cytometry
The detection of IL-18R was performed as previously described (11). Briefly, 2D6 cells were incubated with 0.4 µg of rIL-18, washed, and incubated with 0.1 µg of rabbit anti-mouse IL-18 polyclonal Ab. Cells were allowed to react with 0.1 µg of biotinylated goat anti-rabbit IgG, followed by incubation with PE-conjugated streptavidin. Stained cells were analyzed with a FACSCaliber (BD Biosciences, Mountain View, CA).
Preparation of cell lysates and nuclear extracts
Nuclear extracts were prepared as follows: after washing with PBS, cells were resuspended in cell lysis buffer (20 mM of HEPES-NaOH (pH 7.9), 20 mM of NaF, 1 mM of Na3VO4, 1 mM of EDTA, and 0.1 mM of EGTA) supplemented with 0.2% NP40, 1 mM of DTT, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 0.1 mM of Pefabloc (Roche, Mannheim, Germany). The nuclei were pelleted and then extracted with vigorous agitation at 4°C in the above buffer without NP40 but containing 0.42 M NaCl, 20% glycerol, and protease inhibitors as above described.
Immunoprecipitation and immunoblotting
Nuclear extracts were immunoprecipitated with anti-c-Jun antiserum conjugated to protein A-coupled Sepharose beads. The immunoprecipitates were resolved on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF)3 membrane (Millipore, Bedford, MA). For immunoblotting with anti-c-Jun or anti-STAT4 Ab, membranes were blocked in TBS containing 5% BSA and 0.05% Tween 20, and were sequentially incubated with the Abs and HRP-conjugated donkey anti-rabbit IgG F(ab')2 (Amersham, Aylesbury, U.K.). Detection was performed using ECL (Amersham).
In vitro kinase assays
2D6 cells were lysed with lysis buffer (20 mM of Tris (pH
7.5), 1% Triton X-100, 0.15 M of NaCl, 1 mM of 
-glycerophosphate, 1
mM of EDTA, 1 mM of Na3VO4, 2.5 mM of Na
pyrophosphate, 10 µg/ml leupeptin, 1 mM of PMSF). c-Jun N-terminal
kinase (JNK) was immunoprecipitated using anti-JNK Ab (BD
Transduction Laboratories, Lexington, KY). The lysis of
immunoprecipitates and in vitro kinase assay were performed as
described (19).
EMSA
Binding reaction was performed in a total volume of 20 µl in
the following buffer: 10 mM of HEPES-NaOH (pH 7.9), 1 mM of EDTA, 30 mM
of NaCl, 0.1% NP40, 1 mM of DTT, 1 mg/ml BSA, and 5% glycerol. Each
reaction, also containing 3 µg of poly(dI-dC) and
32P end-labeled probe, was initiated by the
addition of 
9 µg of nuclear extract and was allowed to incubate at
room temperature for 30 min before electrophoretic analysis on a 4.5%
polyacrylamide gel in 0.25x Tris-borate-EDTA (TBE) buffer. The
STAT4, AP-1, and NF-B consensus oligonucleotide probes (STAT4,
5'-GAGCCTGATTTCCCCGAAATGATGAGC-3' (20); AP-1,
5'-CGCTTGATGACTCAGCCGGAA-3' (21); and NF-B,
5'-AGTTGAGGGGACTTTCCCAGGG-3' (22)) were purchased from
Santa Cruz Biotechnology. The oligonucleotide probes corresponding to
the STAT-common binding motif
(5'-CCACCCCAAATGGTGTGAAGTAAAAGTGCTTTCAGAGAATCCCA-3') and the
AP-1-binding sequence present in the mouse IFN- promoter
(5'-GCGGGGCTGTCTCATCGTCAGA-3') were prepared in our laboratory.
Plasmids and vectors
The luciferase reporter plasmid used was pGL3 (Promega, Madison,
WI). Three fragments of the IFN- promoter sequence, positions -436
to +113, positions -206 to +113, and positions -189 to +113 were
amplified by PCR from C57BL/6 mouse splenocyte genomic DNA using three
MluI site-containing upstream primers
5'-CGACGCGTCCCAAGAGTTTCCTCATGGTTTGAGAAGCC-3',
5'-CGACGCGTAGCGGGGCTGTCTCATCGTCAGAGAGCCCAA-3', and
5'-CGACGCGTGTCAGAGAGCCCAAGGAGTCGAAAGGAAACT-3', respectively,
together with a single downstream primer
5'-GAAGATCTGTCTCAGAGCTAGGCCGCAGGAGGAGAAG-3' containing the
BglII site. Each IFN- promoter fragment was cloned into
the pGEM-Teasy vector (Promega) by TA cloning. The IFN-
promoter DNA was then excised with MluI and BglII
from the pGEM-Teasy vector and it was cloned into the MluI
and BglII sites of the promoterless pGL3 luciferase reporter
gene vector.
Reporter gene assay
Twenty five micrograms of luciferase reporter plasmid and 2 µg of pRL-TK reporter plasmid were cotransfected into 2D6 cells by electroporation using a Gene Pulser (Bio-Rad, Richmond, VA) with 950 µF at 250 V. After transfection, the cells were stimulated with IL-12 (1000 pg/ml) and/or IL-18 (100 ng/ml) for 24–30 h, harvested, washed in PBS, and lysed in passive lysis buffer (Promega). A reporter gene (luciferase) assay was performed according to the procedure recommended by Promega. Briefly, luciferase activity was measured as light emission over a period of 10 s after addition of luciferase assay buffer II (Promega). Data were normalized for transfection efficiency by Renilla luciferase activity of the pRL-TK reporter plasmid (Promega). A relative luciferase activity in each cytokine-stimulation group was expressed as a ratio to a control (cytokine unstimulation) group.
Oligo DNA precipitation
The procedure was essentially the same as that previously described (23). Nuclear extracts were incubated with agarose beads coupled to an AP-1 consensus oligonucleotide (TGACTCA; Santa Cruz Biotechnology). The binding reaction was performed for 45 min at 4°C in a binding buffer containing 100 mM of NaCl, 10 mM of Tris-HCl (pH 7.5), 0.1 mM of EDTA, 1 mM of DTT, 5% glycerol, 0.1% NP40, 0.2 mM of PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM of Na3VO4, 50 mM of NaF, 1 mg/ml BSA, and 30 µg/ml poly(dI-dC). The agarose beads were washed five times with binding buffer. The bound proteins were released with SDS loading buffer, separated by 10% SDS-PAGE, transferred to PVDF membrane, and visualized with the relevant Abs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-12-unstarved or -starved 2D6 cells were stimulated with IL-12,
IL-18, or IL-12 plus IL-18 for 24 h. IL-12-unstarved 2D6 cells
produced small and large amounts of IFN- following stimulation with
IL-12 or IL-18, respectively. The level of IFN- production induced
by IL-18 alone was as high as that induced by combined stimulation with
IL-12 and IL-18. In contrast, IL-12-starved 2D6 cells exhibited very
low levels of IFN- production upon stimulation with IL-18 alone.
When stimulated simultaneously with IL-12 and IL-18, these cells
produced comparable amounts of IFN- with those induced by
IL-12-unstarved cells stimulated with IL-18 or IL-12 plus IL-18 (Fig. 1
A). Differential patterns of
IFN- production by these 2D6 cells were reflected in IFN- mRNA
expression. IL-12-unstarved and -starved 2D6 cells expressed high and
low levels of IFN- mRNA, respectively, upon stimulation with IL-18
alone (Fig. 1B). Importantly, the failure of IL-12-starved
2D6 cells to respond to IL-18 alone did not result from their reduced
IL-18R expression, because IL-12-starved and -unstarved 2D6 cells
exhibited comparable levels of IL-18R (Fig. 1A,
insets).
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IL-12 induces STAT4 phosphorylation and its binding to DNA in 2D6
cells (24). A recent study revealed that IL-18 induces
AP-1 binding to DNA in human CD4+ T cells
(15). We examined whether AP-1-binding activity can also
be induced in 2D6 cells. Nuclear extracts from 2D6 cells stimulated
with IL-12 and/or IL-18 were examined for binding to an oligonucleotide
probe corresponding to a consensus binding site for AP-1. Nuclear
extracts from IL-12-unstarved 2D6 cells that were stimulated with IL-18
alone or IL-18 combined with IL-12 contained increased amounts of
protein capable of binding to the AP-1 consensus sequence, whereas
extracts from IL-12-starved cells exhibited only marginal levels of
AP-1-binding activity when stimulated with IL-18 alone (Fig. 2A, upper panel).
Comparable levels of AP-1-binding activity with those observed in
IL-12-unstarved cells were induced in IL-12-starved cells
stimulated with IL-12 plus IL-18 (Fig. 2A, lower
panel). This was the case for the entire time course (0.5–8 h) of
cytokine stimulation (data not shown).
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is different from the
sequence for the AP-1 binding site present in the mouse IFN-
promoter. We found that a nuclear extract from IL-12/IL-18-stimulated
2D6 cells also exhibits binding to the latter sequence (Fig. 2B). Moreover, the binding to each oligonucleotide probe was
completely inhibited in the presence of an excess of unlabeled
oligonucleotide. Together, these results indicate that although IL-18
signaling leads to AP-1 induction, the binding of this transcription
factor does not occur in the absence of IL-12 signals.
Synergy between IL-12 and IL-18 for IFN- promoter activation
To investigate the regulatory mechanism for IFN- gene
expression, we constructed a plasmid containing an IFN- promoter
fragment (positions -436 to +113) upstream of a luciferase reporter
gene (pGL3-IFN-). IL-12-unstarved and -starved 2D6 cells were
transiently transfected with the pGL3-IFN- plasmid and were
immediately stimulated with IL-12, IL-18, or IL-12 plus IL-18.
Stimulation of IL-12-unstarved 2D6 cells with IL-12 or IL-18 alone
resulted in low and high IFN- promoter activation, respectively
(Fig. 3). The level of IFN- promoter
activity in IL-18-stimulated 2D6 was comparable with that observed in
IL-12/IL-18-stimulated 2D6. In contrast, IFN- promoter activity
induced by IL-18 alone in IL-12-starved 2D6 cells was low, and in these
cells, high levels of the activity were obtained only when the
IL-12/IL-18 combined stimulation was provided. The patterns of IFN-
promoter activation in the two types of 2D6 cells were similar to those
observed for IFN- expression and AP-1 induction. These results
indicate that IL-12 and IL-18 are weak and strong inducers,
respectively, of IFN- promoter activity, but the strength of
IL-18-mediated activation depends on the costimulatory activity of
IL-12.
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promoter activation
A human IFN- promoter was shown to interact with STAT4 upstream
of its AP-1 binding site (25). However, an apparent
STAT4-binding sequence does not exist upstream of the AP-1 binding site
in the mouse IFN- promoter, although a STAT-common binding motif is
present in the relevant region. We examined whether IL-12-activated
STAT4 can bind to this sequence. The gel shift pattern corresponding to
IL-12-induced STAT4 is observed using the STAT4 consensus probe, but
STAT4 only marginally reacts with the STAT-common binding motif on the
IFN- promoter (Fig. 4).
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). However, even combined stimulation
failed to activate the S-2 promoter. These results indicate that
activation of the IFN- promoter requires the presence of the binding
site for AP-1 induced by IL-18 and they suggest that IL-12 functions to
enhance the activation of this promoter without direct binding to
it.
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The above results raise the possibility that STAT4 may interact
with other transcription factors rather than bind directly to the
IFN- promoter to enhance its activation. This hypothesis is based on
the recent observation that STAT3 interacts with c-Jun, a component of
AP-1, to activate transcription (25). Therefore, we
examined whether STAT4 interacts with c-Jun to form a complex. A
nuclear extract from IL-12/IL-18-stimulated 2D6 cells was
immunoprecipitated with anti-c-Jun, blotted with anti-STAT4 and
then reblotted with anti-c-Jun. The anti-c-Jun
immunoprecipitate contained c-Jun as well as STAT4 (Fig. 6A). Next, we compared the
accumulation of c-Jun, its interaction with STAT4, and serine
phosphorylation of c-Jun in nuclear extracts from 2D6 cells stimulated
with IL-12 and/or IL-18. Anti-c-Jun immunoprecipitates were either
blotted with anti-phosphoserine c-Jun Ab followed by reblotting
with anti-c-Jun or they were blotted with anti-STAT4. Fig. 6B (middle and lower panels)
demonstrates that stimulation with either IL-12 or IL-18 induces the
accumulation of c-Jun in the nuclear compartment, and combined
stimulation results in enhanced accumulation. Furthermore, blotting
with anti-STAT4 demonstrates that c-Jun accumulating after
stimulation with IL-12 or IL-12 plus IL-18 is associated with STAT4
that is recruited after activation with IL-12. These results indicate
that IL-12-activated STAT4 interacts with c-Jun to form a
protein-protein complex.
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B shows serine
phosphorylation of the c-Jun accumulating in the nuclear fraction of
2D6 cells stimulated with IL-18 or IL-12 plus IL-18), although the
phosphorylation levels were quite low. We examined whether IL-12 or
IL-18 induces JNK activation in 2D6 cells. As shown in Fig. 6C, JNK was activated by IL-18 but not by IL-12. Together,
these results indicate that IL-12 can induce an accumulation of c-Jun
in the nuclear compartment, but it does not induce c-Jun
phosphorylation because of the failure to activate JNK. In contrast, a
detectable albeit small amount of c-Jun is serine-phosphorylated by
IL-18 that has the capacity to activate JNK. c-Jun induced by IL-12 plus IL-18 exhibits a markedly enhanced AP-1-binding activity
We compared the binding activity of c-Jun induced by IL-12 and/or
IL-18 with the AP-1 sequence by oligo DNA precipitation (Fig. 7). Nuclear extracts from 2D6 cells
stimulated with IL-12 and/or IL-18 were allowed to interact with
agarose beads coupled to the AP-1-binding oligonucleotide sequence. The
bound proteins were analyzed by immunoblotting using anti-c-Jun or
anti-STAT4. A much larger amount of c-Jun bound to the AP-1
sequence in the nuclear extract from IL-12/IL-18-stimulated 2D6 cells
than that from 2D6 cells stimulated with either cytokine alone (Fig. 7A).
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A). The binding of
c-Jun was completely inhibited by an excess of the competitive
oligonucleotide (Fig. 7B). The binding of STAT4 was also
lost along with inhibition of c-Jun binding. These observations suggest
that STAT4 interacts with the AP-1 sequence not by direct binding but
via an indirect interaction involving its association with c-Jun. Enhanced c-Jun binding to the AP-1 sequence is associated with serine phosphorylation of c-Jun
The complex of c-Jun and STAT4 was observed in nuclear extracts
from 2D6 cells stimulated with IL-12 alone and those stimulated with
IL-12 plus IL-18 (Fig. 6B), although the amount appeared
greater in the latter than the former. However, the binding of the
c-Jun/STAT4 complex to the AP-1 sequence greatly differed between these
two groups of nuclear extracts (Fig. 7, A and B).
These observations suggest that the capacity of c-Jun to bind to the
AP-1 sequence is not enhanced solely by interaction with STAT4. In view
of the additional requirement for enhanced c-Jun binding, we examined
the state of phosphorylation of the c-Jun interacting with the AP-1
binding site. In the IL-18-stimulated group, only a small amount of
c-Jun interacted with the AP-1 binding site (Fig. 7C).
Serine phosphorylation was hardly detected in this c-Jun fraction. In
contrast, in IL-12/IL-18-stimulated 2D6 cells, a large amount of c-Jun
was again found to bind to the AP-1 site and this fraction exhibited
high levels of serine phosphorylation. Simultaneously, STAT4
interacting with the AP-1 sequence together with c-Jun exhibited
tyrosine phosphorylation. Serine phosphorylation levels of c-Jun in the
total nuclear fraction from IL-18- and IL-12/IL-18-stimulated cells
were very low without any great differences (Fig. 6B,
upper panels). These observations suggest that
serine-phosphorylated c-Jun interacting with STAT4 accumulates by
binding to the AP-1 sequence, whereas c-Jun that is serine
phosphorylated but does not interact with STAT4 fails to exhibit
efficient binding to the AP-1 sequence.
c-Jun induced in primary activated T cells exhibits enhanced AP-1 binding together with STAT4
We finally investigated whether enhanced AP-1 binding seen in 2D6
cells is observed for primary activated T cells. Purified lymph node T
cells were stimulated with anti-CD3 plus anti-CD28 and they
were then exposed to IL-12 for the induction of IL-18R
(14). These activated T cells expressing both IL-12R and
IL-18R were stimulated with IL-12 and/or IL-18 after a short-term
starvation culture. Nuclear extracts obtained were examined for the
binding to the AP-1- and the NF-B (control)-binding sequences in
EMSA and were also subjected to oligo DNA precipitation (Fig. 8). Similar patterns of AP-1 binding to
those seen for nuclear extracts of 2D6 cells in Fig. 2B were
observed for nuclear extracts from primary activated T cells (Fig. 2B, top panel). Importantly, IL-18-mediated
NF-B induction (14) was comparable in stimulation with
IL-18 alone and IL-12 plus IL-18 (Fig. 2B, bottom
panel). This indicates that the IL-12 signal functions to enhance
selectively AP-1 binding.
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C, untreated nuclear
extracts exhibited enhanced binding to the AP-1-related sequence in the
IFN- promoter. Anti-c-Jun or anti-STAT4 treatment failed to
produce a supershift, although comparable levels of the decrease in
AP-1 shift were induced by either of these Abs. To confirm the
interaction of c-Jun and STAT4 with the AP-1-binding sequence, we again
took advantage of oligo DNA precipitation (Fig. 8D). In
nuclear extracts from IL-12/IL-18-stimulated T cell blasts, increased
amounts of c-Jun and STAT4 were found to bind to the AP-1-binding
sequence, whereas STAT4 phosphorylation was not detected in the
recovered sample. These results indicate that enhanced c-Jun binding to
the AP-1 site, along with the cobinding of STAT4, is also induced in
primary activated T cells when the cells are stimulated with IL-12 and
IL-18. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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promoter activation and mRNA expression when
compared with those stimulated with IL-12 plus IL-18. Decreased IFN-
promoter activation was associated with the generation of only a weak
AP-1-binding activity following IL-18 stimulation. IL-12 markedly
enhanced the activation of the IFN- promoter (standard promoter:
positions -436 to +113). The effect of IL-12 was also manifested for
the promoter lacking the fragment upstream of the AP-1-related
sequence, but not for the promoter lacking the AP-1-related sequence as
well. More importantly, IL-12-activated STAT4 interacted with
IL-18-induced c-Jun/AP-1 to form a complex. The complex of STAT4 and
c-Jun/AP-1 produced after IL-12/IL-18 stimulation exhibited strikingly
enhanced binding to the AP-1-relevant sequence compared with c-Jun/AP-1
free of STAT4. Thus, these results indicate that STAT4 functions to
enhance the binding activity of an IL-18-induced transcriptional
factor, c-Jun/AP-1 to the AP-1-related sequence, through interaction
with the c-Jun component of AP-1.
IL-12R is induced on T cells following TCR triggering (14, 26, 27). These T cells are allowed to express IL-18R upon
stimulation with IL-12 (14). The ability of IL-12 to
induce IL-18R was originally demonstrated in a cloned Th1 cell line,
2D6, that was established as an IL-12-responsive clone
(18). Although 2D6 cells maintained with IL-2 failed to
express IL-18R, 2D6 cells maintained with IL-12 consistently expressed
both IL-12R and IL-18R (11). Thus, this IL-12 function has
been regarded as providing a mechanistic explanation underlying the
synergy between IL-12 and IL-18 for IFN- gene expression. However,
it remained unclear whether other mechanisms exist for synergy that are
at a different level from that of IL-18R induction. In this study, 2D6
cells maintained with IL-12 exhibited high levels of IFN- expression
following IL-18 stimulation irrespective of whether IL-12 is
simultaneously present. In contrast, 2D6 cells that were depleted of
IL-12 signals due to IL-12 starvation (IL-12-starved 2D6 cells)
required fresh IL-12 stimulation for efficient IL-18 signaling despite
comparable levels of IL-18R expression with those in IL-12-unstarved
cells. Thus, the present model using IL-12-starved 2D6 cells permitted
us to investigate the mechanism of synergistic IL-12/IL-18 signaling
downstream of cytokine receptor expression.
A number of transcription factor binding sites have been identified in
the 5'-untranslated region of the IFN- gene. Within the immediate 5'
region (positions -108 to -40), there are some transcriptional
elements that are responsive to TCR signaling (28).
Another set of transcription factor binding sites resides between
positions -280 and -180, upstream of the above-mentioned
TCR-responsive region (29). This second region contains
STAT (STAT-common binding motif), AP-2/YY-1, AP-1, and NF-AT binding
sites. A recent study of Barbulescu et al. (15) reported
that IL-12/IL-18-mediated activation of the human IFN- promoter is
assigned to the second region of the promoter because IL-12 and IL-18
induce STAT4 and AP-1, respectively. However, their study did not
examine molecular mechanisms underlying the synergy between IL-12 and
IL-18 in the activation of the second promoter region. Moreover, it
remained unclear whether the STAT-common binding motif within the mouse
second region actually binds STAT4, because the sequence at the
relevant promoter site is not typical for the binding of STAT4.
The present study focused on the mechanism by which IL-12 and IL-18
synergize to activate the above-mentioned second region of the IFN-
promoter. Our results demonstrate that combined stimulation with IL-12
and IL-18 enhanced the synergistic activation of the promoter fragment
(positions -436 to +113) incorporating the second region (positions
-280 to -180). Synergy was not observed when the promoter fragment
was deleted of the AP-1-responsive site. In contrast, comparable levels
of synergistic activation with those observed for the standard promoter
fragment were induced in a fragment containing the AP-1-responsive site
but with its upstream element deleted. This indicated that the
so-called STAT binding site present in the element upstream of the AP-1
site is not necessarily required for the synergistic action of IL-12.
These results also suggest that IL-12-activated STAT4 can exert its
cooperative effect on transactivation of IL-18-induced AP-1 through a
mechanism other than the direct binding to a given promoter region.
The interaction of a transcription factor with others has been reported for a wide variety of transcription factors, including interaction between STAT3 and c-Jun (25). Similarly, this study demonstrates that STAT4 interacts with c-Jun/AP-1 to form a protein-protein complex. The complex of STAT4 with c-Jun/AP-1 was found in the nuclear compartment of 2D6 cells stimulated with IL-12 alone or IL-12 plus IL-18. This was due to accumulation of IL-12-activated STAT4 and IL-12- or IL-18-induced c-Jun/AP-1 in the nuclear compartment.
Furthermore, our results showed that c-Jun/AP-1 interacting with STAT4
exhibits a much stronger binding ability to the AP-1-related sequence
than c-Jun/AP-1 alone. It should be noted that considerable amounts of
the complex were detected in the nuclear compartment of 2D6 cells
stimulated with IL-12 alone. However, the binding of this complex to
the AP-1-related sequence was weak. Because IL-12 failed to induce JNK
activation, serine phosphorylation was not detected in c-Jun/AP-1
induced by IL-12. This suggested that c-Jun/AP-1 in such a complex was
not serine phosphorylated. In contrast, IL-18 stimulation induced high
levels of JNK activation. Accordingly, serine phosphorylation of
recruited c-Jun/AP-1 was observed in nuclear extracts from 2D6 cells
stimulated with IL-18 or IL-12 plus IL-18, although the detected
phosphorylation level was low. These observations suggest that unlike
IL-12 stimulation, IL-12/IL-18 stimulation produces a complex of STAT4
and serine-phosphorylated c-Jun/AP-1. In fact, such a protein-protein
complex was found to bind to the AP-1-related sequence in large
amounts, and moreover, exhibited considerable levels of c-Jun/AP-1
serine phosphorylation along with tyrosine phosphorylation of STAT4.
The fact that after oligonucleotide precipitation, larger amounts of
serine-phosphorylated c-Jun were recovered than expected from the
amount of serine-phosphorylated c-Jun present in nuclear extracts
suggests that phosphorylated c-Jun accumulated selectively due to its
efficient binding to DNA. Thus, it is conceivable that enhanced binding
of c-Jun/AP-1 to the AP-1-related sequence is achieved through two
requirements, i.e., serine phosphorylation and its association with
tyrosine-phosphorylated STAT4, both of which are induced only when 2D6
cells are stimulated with IL-12 plus IL-18 (Fig. 9).
|
Nevertheless, it is still possible that STAT4 binds directly to the
STAT binding site upstream of the AP-1-related sequence or to sequences
other than the IFN- promoter fragment used in this study, as has
been described particularly for the intron of the human IFN- gene
(15, 34). Therefore, it is possible that the
above-mentioned cooperative transcriptional activation also occurs
between STAT4 and AP-1. Thus, STAT4 may be speculated to function for
enhanced IFN- promoter activation not only by strengthening the
binding of c-Jun/AP-1, but also by inducing enhanceosome-associated
transcriptional activation. Moreover, a recent study demonstrated a
critical role for STAT4 in IL-12-induced IL-18R expression
(35). Taken together, STAT4 is central in IFN-
expression induced by the synergy between IL-12 and IL-18. This study
reinforces the notion of an unequivocal role for STAT4 in IFN-
expression by providing a new aspect of mechanisms underlying
transcriptional synergy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hiromi Fujiwara, Department of Oncology (C6), Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail address: hf{at}ongene.med.osaka-u.ac.jp
3 Abbreviations used in this paper: PVDF, polyvinylidene difluoride; JNK, c-Jun N-terminal kinase.
Received for publication July 9, 2001. Accepted for publication November 21, 2001.
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