 |
INTRODUCTION |
Nitric oxide (NO)1 has
been identified as an important signaling molecule involved in
regulating a wide range of biological activities in the immune,
vascular, and neural system (1-3). NO and its metabolites are central
to the antimicrobial and tumoricidal activity of activated macrophages
and are implicated in the pathogenesis of tissue damage associated with
acute and chronic inflammation (4-9). Macrophages generate NO from
L-arginine through the inducible isoform of nitric oxide
synthase (iNOS), which is regulated transcriptionally. iNOS gene
expression is induced by a variety of stimuli, including lipopolysaccharide (LPS), bacterial wall products, and cytokines. In
particular, interferon-
(IFN-
) is a potent inducer of iNOS gene
expression (10, 11), although the molecular mechanism for the effect is
still not understood fully.
The murine iNOS promoter region contains several binding sites for
transcription factors implicated in iNOS regulation, including two
NF-
B binding sites. The NF-
B site situated closer to the transcriptional start site is required for iNOS gene induction by LPS,
because deleting the downstream site essentially abolishes iNOS
transcription (12). Deletion of the upstream NF-
B site reduces but
does not abolish iNOS transcription (12-14). The upstream portion of
the iNOS promoter contains an enhancer region, with several
transcription factor binding sites that mediate transcriptional responses to IFN-
. These sites include a
-interferon-activated site element and two IFN-stimulated response elements (ISRE) (15, 16).
Deletion and mutational analysis have shown the importance of the
promoter sequence from
1029 to
913 in the induction of iNOS by
IFN-
(13). Site-directed mutagenesis and competition experiments
have shown that the ISRE site between
923 and
913 is required for
the full synergistic effects of IFN-
and LPS in the transcription of
the iNOS gene (16).
The cytokine IFN-
plays an essential role in innate and adaptive
immunity (17). IFN signaling, which involves a variety of
trans- and cis-acting factors, is mediated
through DNA motifs, designated ISRE and IFN-
-activated
sequence, which are found in promoters of IFN-inducible genes. The
interferon consensus sequence-binding protein (ICSBP or IRF-8) is a
transcription factor belonging to the IFN regulatory factor (IRF)
family, which also includes the IRF-1, IRF-2, IRF-3,
interferon-stimulated gene factor 3
, and pip/IRF-4 proteins (18,
19). Unlike the other members of the IRF family, ICSBP expression is
limited to activated macrophages, B cells, and T cells (20-23).
Proteins of the IRF family, including ICSBP, bind to the ISRE (18). The
tyrosine-phosphorylated form of ICSBP does not bind to DNA
independently but requires complex formation with other members of the
IRF family (IRF-1 and IRF-2) or with other transcriptional elements
(24). Previous studies found that ICSBP represses transcriptional
activity of ISRE promoters in various cell types. However, recent
studies suggest that ICSBP can activate genes required for the
development and function of macrophages (25, 26). ICSBP
/
mice were
shown to be highly susceptible to infection with several pathogens
including Listeria monocytogenes (27, 28) and are impaired
in the production by macrophages of IFN-
-induced NO (29). However,
the molecular mechanism for the regulation of NO by ICSBP is not clear.
In the present study we have analyzed iNOS gene expression in
macrophages induced by IFN-
and have identified an ICSBP binding site in the iNOS promoter region. Transduction of ICSBP with a retroviral vector in ICSBP
/
macrophages rescues IFN-
-induced iNOS expression. Surprisingly, iNOS expression is not induced by
transduction of ICSBP alone but requires co-transduction of ICSBP with
IRF-1. This study provides new insights into the molecular mechanism of
iNOS gene induction by IFN-
, which indicates the importance of an
ICSBP·IRF-1 complex in the regulation of iNOS gene expression.
 |
MATERIALS AND METHODS |
Plasmid Constructs--
iNOS promoter fragments were inserted
into the luciferase reporter vector pGL2B (Promega) as described
previously (13). The IRF-1 expression plasmid was a gift from T. Taniguchi (University of Tokyo, Tokyo, Japan). All site-directed mutant
plasmids were generated by two-step polymerase chain reaction using
overlapping internal primers containing the mutant site (30).
Cell Lines and Reagents--
The RAW264.7 murine macrophage cell
line (American Type Culture Collection) was maintained in RPMI 1640 supplemented with 10% fetal bovine serum and penicillin-streptomycin.
ICSBP
/
macrophages (CL-2 cells) were established from bone marrow
cells obtained from ICSBP
/
mice following coincubation with the
murine J2 virus, harboring v-raf and v-myc genes
(25). The CL-2 cells were maintained in the same media as above,
supplemented with recombinant mouse M-CSF (6 ng/ml; R & D Systems,
Minneapolis, MN) and recombinant mouse GM-CSF (6 ng/ml; Peprotech,
Rocky Hill, NJ) (25). IFN-
and IL-4 were obtained from R & D
Systems. LPS was purchased from Sigma. Antibodies against iNOS, ICSBP,
IRF-1, and IRF-2 were from Santa Cruz Biotechnology.
Nitrite Determination--
Nitrite concentration, a measure of
NO synthesis, was assayed by a standard Griess reaction adapted to
microplates, as described previously (31). Nitrite accumulation was
determined by using NaNO2 as standard, and the data were
expressed as concentration of nitrite.
Transfection--
RAW267.4 cells were transiently transfected
using Superfect (Qiagen). For each transfection, 2.5 µg of plasmid
was mixed with 100 µl of RPMI 1640 (without serum and antibiotics)
and 10 µl of Superfect reagent. The mixture was incubated at room
temperature for 10 min, 600 µl of RPMI 1640 complete medium was added
and immediately placed onto the cells in 6-well plates, and luciferase activity was measured 16-24 h later. For CL-2 cell transfection, reporter (10 µg) and expression vectors (30 µg) were transfected into 107 CL-2 cells by electroporation, and luciferase
activity was measured 16 h later. When indicated, murine IFN-
(10 ng/ml) was added to the culture for 6-12 h before harvest. The
cells were harvested with reporter lysis buffer (Promega), and 20 µl
of extract was assayed for luciferase as described (30). Cells were
co-transfected with a constitutively active cytomegalovirus
promoter-
-galactosidase reporter plasmid to normalize experiments
for transfection efficiency.
Retroviral Transduction--
For retroviral transduction we used
a derivative of the Moloney murine leukemia virus vector pMMP412 (32),
courtesy of Dr. Adrain Ting (Mount Sinai School of Medicine). To
prepare pseudotyped virus human 293 EbnaT cells were seeded at a
density of 4 × 106 cells in a 10-cm dish. The next
day, cells were transfected using calcium phosphate with a mixture of
2.5 µg of plasmid pMD.G encoding vesicular stomatitis virus G
protein, 7.5 µg of plasmid pMD.OGP encoding gag-pol, and 10 µg of
the retroviral expression construct. 48 h post-transfection, the
viral supernatant was collected, centrifuged at 800 × g, and used to infect cells. RAW264.7 and CL-2 cells (5 × 106 cells) were resuspended in 12 ml of viral
supernatant in the presence of polybrene (4 µg/ml), aliquoted into
six-well plates, and spun at 800 × g in a microtiter
rotor for 1 h at room temperature.
Electrophoretic Mobility Shift Assays (EMSA)--
RAW264.7 cell
nuclear extracts were prepared as described previously (33). EMSA
probes were prepared by annealing complementary single-stranded
oligonucleotides with 5'GATC overhangs (Genosys Biotechnologies, Inc.)
and were labeled by filling in with [
-32P]dGTP and
[
-32P]dCTP using Klenow enzyme. Labeled probes were
purified with Nuctrap purification columns (Roche Molecular
Biochemicals). The sequence of the EMSA probe (
935 to
905) is 5'gatcACACTGTCTCA-ATATTTCACTTTCATAATGGAAAAT. Electrophoretic mobility shift assays were performed as described previously (34), using 105-cpm probe and 5 µg of nuclear
extracts per reaction. DNA binding complexes were separated by 5%
polyacrylamide-Tris/glycine-EDTA gel run at 4 °C for 4 h at 150 volts. Gels were dried and exposed to Eastman Kodak Co. X-AR 5 film at
80 °C in the presence of an intensifying screen.
Western Blotting--
Nuclear protein extracts and pre-stained
molecular weight markers were denatured in Laemmli buffer (10%
glycerol, 2% SDS, 0.1 M dithiothreitol, 65 mM
Tris, 0.01 mg/ml bromphenol blue) at 90 °C, separated by SDS-PAGE,
and transferred to a nitrocellulose membrane. After protein transfer,
the membranes were blocked with 5% nonfat milk in TBST
(Triton-containing Tris-buffered saline), incubated with anti-iNOS,
anti-ICSBP, anti-IRF-1, or anti-IRF-2 antibodies (1:6000) for 1-2 h,
washed with TBST, and stained with anti-rabbit or anti-mouse IgG
conjugated to peroxidase (1:6000). Immunoreactivity was visualized by
enhanced chemiluminescence reaction (enhanced chemiluminescence kit;
Santa Cruz Biotechnology).
RT-PCR--
For RT-PCR assays, total RNA was extracted using
TRIzol (Invitrogen). cDNA was prepared from 1-3 µg of RNA
with Superscript II RT (Invitrogen) and random hexamer primers
(Promega). The following primers were used for PCR amplification: IL-12
p40, 5'-TCGCAGCAAGATGTG-3' and 5'-GAGCAGCAGATGT-GAGTGGC; iNOS,
5'-CCCTTCCGAAGTTTCTGGCAGCAGC-3' and 5'-GGCTGTC-AGAGCCTCGTGGCTTTGG-3'.
PCR was carried out by a standard protocol for appropriate
cycles. An equal aliquot of cDNA was amplified with
-actin
primers. Aliquots of PCR reactions were resolved on a 1% agarose gel
and visualized with UV light after staining with ethidium bromide.
Co-immunoprecipitation--
RAW264.7 cells were activated with
IFN-
for 4 h. Then cells were washed with cold
phosphate-buffered saline and lysed for 15 min on ice in 0.5 ml of
lysis buffer (50 mM Tris, pH 8.0, 280 mM NaCl,
0.5% Nonidet P-40, 0.2 mM EDTA, 2 mM EGTA,
10% glycerol, and 1 mM dithiothreitol) containing protease
inhibitors. Cell lysates were clarified by centrifugation at 4 °C
for 15 min at 14,000 rpm. Cells lysates were incubated with 2 µg of
IRF-1 antibody (Santa Cruz Biotechnology) in the presence of 20 µl of
50% (v/v) protein G-agarose overnight at 4 °C with gentle rocking.
After three washings with lysis buffer, precipitated complexes were solubilized by boiling in SDS buffer, fractionated by 10% SDS-PAGE, and transferred to nitrocellulose. Western blotting was performed using
ICSBP antibody.
Chromatin Immunoprecipitation Assay--
The experiments were
performed using a chromatin immunoprecipitation (Chip) assay kit from
Upstate Biotechnology according to the manufacturer's protocol.
RAW264.7 cells were cultured in complete Dulbecco's modified Eagle's
medium, activated with IFN-
for 4 h, fixed with 1%
formaldehyde for 30 min at room temperature, and immunoprecipitated
with anti-ICSBP antibody. The immunoprecipitated DNA was amplified by
PCR with primers spanning from
1000 to
788 in the murine iNOS p40
promoter region.
 |
RESULTS |
Characterization of ICSBP Binding Site in the iNOS Promoter
Region--
Macrophages from ICSBP
/
mice are impaired in
IFN-
-induced NO production (29), which suggests that ICSBP is
involved directly in the regulation of iNOS gene expression. Because
ICSBP is a transcription factor, we hypothesize that the murine iNOS
promoter region may have an ICSBP site to which ICSBP can bind,
resulting in the IFN-
-induced activation of the iNOS promoter. The
mouse iNOS promoter has an enhancer and basal promoter, within which a
number of response elements have been localized. Those known to be
active include NF-
B sites located both in the enhancer and basal
promoter and two ISRE. The distal ISRE element (
923 to
913) is a
strong activator, whereas the proximal one has no significant effect on
iNOS promoter activation (16, 35). In the present study, the ISRE site
between
923 and
913 was selected for the analysis, because this
site is important for full induction of iNOS gene expression by
IFN-
. This site was also reported previously (16) as a binding site
for IRF-1. RAW264.7 cells were activated with IFN-
, LPS, or IFN-
plus LPS for 4 h, and nuclear proteins were extracted for EMSA,
which was performed by using probe spanning
935 to
905 of the iNOS
promoter region containing
923 to
913 of the ISRE site. A
protein·oligonucleotide complex was formed that is inducible by
IFN-
and unaffected by LPS (Fig.
1A). When we used probe (
935
to
905) with mutations of ISRE site (TTTCACTTTC to GCCGTAGGCA), there
was no DNA·protein complex formation (data not shown). Furthermore,
ICSBP antibody blocked the formation of DNA·protein complex (Fig.
1B), which suggests that ICSBP can bind to this ISRE site.
To confirm this result, we performed chromatin co-immunoprecipitation
experiments. PCR analysis showed that ICSBP antibody precipitated the
iNOS promoter region (
2000 to
788) from RAW264.7 cells activated with IFN-
for 4 h. (Fig. 1C). Western blotting
experiments revealed that IFN-
induced ICSBP protein expression in
RAW264.7 cells (Fig. 1D). These results strongly suggest
that the ISRE site (
923 to
913) of the iNOS promoter region is a
binding site for ICSBP.

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Fig. 1.
ICSBP binding to the ISRE site ( 923 to
913) of the iNOS promoter region. A, IFN- induces
DNA·protein complex formation. RAW264.7 cells were activated with
IFN- (10 ng/ml), LPS (5 µg/ml), or IFN- /LPS for 4 h, and
nuclear protein was extracted for EMSA. 5 µg of nuclear protein were
added to the 32P-labeled probe ( 935 to 905 of the iNOS
promoter). Extract and probe were incubated with 0.5 µg of
poly(dI-dC) at room temperature for 30 min. B, ICSBP
antibody (Ab) prevents DNA·protein complex formation.
RAW264.7 cells were activated with IFN- for 4 h, and nuclear
protein was extracted for EMSA. 5 µg of nuclear protein were
incubated with 2 µg of ICSBP antibody, and 32P-labeled
probe ( 935 to 905 of iNOS promoter) was added. C,
chromatin co-immunoprecipitation (IP) assays. RAW264.7 cells
were activated with IFN- for 4 h. Formaldehyde was added to 1%
final concentration for 30 min at room temperature. Cells were
harvested, and the chromatin was sheared by sonication and purified for
immunoprecipitation with an antibody against ICSBP. After reversal of
cross-linking, DNA was amplified by PCR with primers spanning the iNOS
promoter ( 1000 to 788). D, IFN- -induced ICSBP protein
expression. RAW cells were activated with IFN- , LPS, or IFN- /LPS
for 4 h, and nuclear protein was extracted for Western blotting.
20 µg of nuclear protein was subject to 10% SDS-PAGE, transferred to
nitrocellulose membrane, and blotted with an antibody against
ICSBP.
|
|
Overexpression of ICSBP Increases IFN-
-induced iNOS Promoter
Activity--
The above data indicate that ICSBP can bind to the ISRE
site (
923 to
913) in the iNOS promoter region. The effect of
overexpressing ICSBP on iNOS promoter activity was analyzed next.
RAW264.7 cells were co-transfected with an iNOS promoter luciferase
reporter construct and an ICSBP expression plasmid for 12 h and
activated with IFN-
for 8 h prior to determination of
luciferase activity. Co-transfection of ICSBP strongly enhanced
IFN-induced iNOS promoter activity (Fig.
2A). Showing the importance of
the ISRE site, IFN-
-induced activity from an iNOS promoter with a
mutated ISRE site (CTTTC into TGCCT) was inhibited 90%, and
co-transfection with the ICSBP expression plasmid had no effect on the
IFN-
-induced activity (Fig. 2, A and B). The
synergistic induction of the mutated iNOS promoter activation by
IFN-
and LPS was reduced by 40%, but the mutation has no effect on
LPS-induced iNOS promoter activity (Fig. 2B). The results
indicate that ICSBP is important for IFN-
-induced iNOS promoter
activation.

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Fig. 2.
ICSBP is important for
IFN- -induced iNOS promoter activation.
An ICSBP expression plasmid was co-transfected with an iNOS
promoter luciferase construct or an iNOS promoter with a mutated ISRE
site ( 923 to 913) into RAW264.7 cells and incubated for 18 to
24 h. The transfected cells were activated with IFN- (10 ng/ml), LPS (5 µg/ml), or IFN- /LPS for 12 h. Extracts from
activated cells were analyzed by luciferase assay. Results (normalized
for -galactosidase activity) are expressed as the luciferase
activity (A) or percentage of wild-type iNOS promoter
activity (B). Each result represents the mean of data from
four to five experiments.
|
|
iNOS Promoter Activity Is Impaired in ICSBP
/
Macrophages--
To analyze further the importance of ICSBP in the
activation of the iNOS promoter, we tested ICSBP
/
macrophages for
IFN-
induced NO production and iNOS mRNA and protein expression.
Both RAW264.7 cells and CL-2 cells were activated with IFN-
(10 ng/ml) for 12 h (protein) or 72 h (nitrite). CL-2 cells had a
reduced iNOS protein expression and nitrite accumulation induced by
IFN-
compared with RAW264.7 cells (Fig.
3, A and B). To
determine iNOS promoter activation in CL-2 cells, RAW264.7 cells and
CL-2 cells were transfected with iNOS promoter luciferase reporter
construct. iNOS promoter activation induced by IFN-
was strongly
impaired in CL-2 cells (Fig. 3C). However, co-transfection
of ICSBP moderately rescued the iNOS promoter activation induced by
IFN-
in CL-2 cells. All these results suggest that ICSBP is
important for iNOS expression induced by IFN-
.

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Fig. 3.
iNOS promoter activation is impaired in
ICSBP / macrophages. A, nitrite accumulation was
strongly reduced in ICSBP / macrophages. Both RAW264.7 cells and
ICSBP / macrophages were activated with IFN- (10 ng/ml) for
48 h. Supernatants were then collected, and nitrite accumulation
was determined using Griess reagent. B, iNOS protein
expression was impaired in ICSBP / macrophages. Both RAW264.7 cells
and ICSBP / macrophages were activated with IFN- for 12 h,
and cellular protein was extracted for Western blotting. 20 µg of
cellular protein was subjected to 10% SDS-PAGE, transferred to
nitrocellulose membrane, and blotted with iNOS antibody. C,
IFN- -induced iNOS promoter activity was strongly impaired in
ICSBP / macrophages. Both RAW264.7 cells and ICSBP / macrophages
were co-transfected with an iNOS promoter luciferase plasmid and an
ICSBP expression plasmid. The cells were incubated for 18 to 24 h
and activated with IFN- for 12 h. Extracts from the activated
cells were analyzed by luciferase activity. Results (normalized for
-galactosidase activity) were expressed as luciferase activity. Each
result represents the mean of data from four to five experiments.
|
|
Transduction of ICSBP in ICSBP
/
Macrophages Rescues
IFN-
-induced iNOS Gene Expression--
The data above showed that
CL-2 cells were deficient in NO production induced by IFN-
. To
demonstrate the importance of ICSBP for iNOS gene expression, we
transduced ICSBP into ICSBP
/
macrophages to rescue iNOS gene
expression. The transduction efficiency judged by GFP fluorescence was
around 70-80% by fluorescence-activated cell sorter (data not shown).
Cells were activated with IFN-
and analyzed for iNOS mRNA and
protein and nitrite accumulation. After transduction of ICSBP,
IFN-
-induced iNOS expression and nitrite accumulation were rescued
(Fig. 4, A-C). In addition, the synthesis of IL-12 expression, another IFN-
-regulated gene, was
rescued, as well (Fig. 4D). Unexpectedly, transduction of ICSBP alone did not rescue iNOS expression, although ICSBP protein was
clearly expressed (Fig. 4, A-C). These results suggest that ICSBP alone is not sufficient for iNOS gene expression.

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Fig. 4.
Transduction of ICSBP into ICSBP /
macrophages rescues IFN- -induced iNOS
expression. CL-2 cells were transduced with retrovirus-ICSBP or
retrovirus-GFP as control. A, the transduced cells were
activated with IFN- for 48 h. Supernatants were then collected,
and nitrite accumulation was determined using Griess reagent.
B, the transduced cells were activated with IFN- for
8 h. Total cellular RNA was then extracted, and RT-PCR was
performed for the detection of iNOS mRNA expression. C,
the transduced cells were activated with IFN- for 12 h, and
Western blotting was performed for the detection of iNOS protein
expression. D, the transduced cells were activated with
IFN- /LPS for 8 h, total cellular RNA was extracted, and RT-PCR
was performed for the detection of IL-12 mRNA expression.
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|
ICSBP·IRF-1 Complex Formation Is Required for iNOS Gene
Expression--
To explain the requirement for both IFN-
and ICSBP
to rescue iNOS transcription and NO production in CL-2 cells, we
suggest that either another transcription factor may be needed or that IFN-
induces post-translational modification of ICSBP. We first examined whether ICSBP can bind to the ISRE site of the iNOS promoter. After transduction with ICSBP or GFP as a control, CL-2 cells were
incubated with or without IFN-
for 4 h, nuclear protein was
extracted, and EMSA was performed with a probe (
935 to
905) containing the iNOS promoter ISRE site (
923 to
913). Interestingly, EMSA experiments indicated that transduction with ICSBP alone did not
result in DNA·protein complex formation, which was, however, seen
after activation with IFN-
(Fig.
5A). Furthermore,
cycloheximide prevented the formation of this complex (Fig.
5B), suggesting that synthesis of another IFN-induced
transcription factor is needed for ICSBP to bind to the ISRE site of
iNOS promoter.

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Fig. 5.
IFN- induces
DNA·protein complex formation in ICSBP / macrophages transduced
with ICSBP. CL-2 cells were transduced with ICSBP or GFP as a
control, and the cells were activated with IFN- (10 ng/ml) for
4 h. The nuclear proteins were extracted, and electrophoretic
mobility shift assay was performed using a probe between 935 and
905 of the iNOS promoter. 32P-Labeled probes were
incubated with 0.5 µg of poly(dI-dC) and 5 µg of nuclear extracts
(A). The transduced cells were pretreated with cycloheximide
(10 µg/ml) for 1 h and activated with IFN- (10 ng/ml) for
4 h. The nuclear proteins were then extracted, and electrophoretic
mobility shift assay was performed using probe between 935 and 905
of the iNOS promoter. 32P-Labeled probes were incubated
with 0.5 µg of poly(dI-dC) and 5 µg of nuclear extracts
(B).
|
|
ICSBP is known to form complexes with IRF-1 or IRF-2. When RAW264.7
cells were activated with IFN-
for 4 h, Western blotting results indicated that IFN-
strongly induces IRF-1 protein
expression (Fig. 6A). However,
because IFN-
has no effect on IRF-2 protein expression (Fig.
6A), IRF-1 is therefore the more likely candidate as the
ICSBP partner. The ISRE site in the iNOS promoter region (
923 to
913) was identified as an IRF-1 binding site in previous studies
(16), and IRF-1
/
mice are defective in NO production. In addition,
IRF-1 binds to the same ISRE site that we identified as an ICSBP site.
To confirm the binding of IRF-1 to the ISRE site, we transduced
ICSBP
/
macrophages with ICSBP and activated the cells with IFN-
for 4 h. Nuclear protein was extracted, and EMSA was performed
using a probe (
935 to
905) bracketing the ISRE site (
923 to
913). Both ICSBP and IRF-1 antibodies prevented the DNA·protein
complex formation (Fig. 6B), showing the presence of both
proteins in the DNA·protein complex. In addition,
co-immunoprecipitation experiments demonstrated that a complex formed
between ICSBP and IRF-1 in nuclear proteins from IFN-
-activated
RAW264.7 cells (Fig. 6C). When RAW264.7 cells were
co-transfected with an iNOS luciferase reporter and both ICSBP and
IRF-1, strong luciferase activity was observed, whereas transfection
with ICSBP or IRF-1 alone only weakly induced iNOS promoter activation
(Fig. 6D). Co-transduction with ICSBP and IRF-1 induced iNOS
expression and nitrite accumulation, whereas transduction of with
either ICSBP or IRF-1 alone was not sufficient to induce iNOS
expression (Fig. 6F). These results suggest that the complex
formation of ICSBP with IRF-1 is essential for iNOS gene
expression.

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Fig. 6.
The complex formation of ICSBP with IRF-1 is
important for iNOS expression. A, RAW264.7 cells were
activated with IFN- for 4 h, and nuclear protein was extracted
for Western blotting. 20 µg of nuclear protein was subjected to 10%
SDS-PAGE, transferred to nitrocellulose, and blotted with IRF-1 or
IRF-2 antibodies. B, CL-2 cells were transduced with ICSBP
or GFP as a control, and the cells were activated with IFN- (10 ng/ml) for 4 h. The nuclear proteins were then extracted, and
electrophoretic mobility shift assay was performed using a probe
between 935 and 905 of the iNOS promoter. 5 µg of nuclear
extracts was incubated with either 2 µg of ICSBP or 2 µg of IRF-1
antibody for 40 min before adding 32P-labeled probe ( 935
to 905). C, RAW264.7 cells were activated with IFN- for
4 h, and nuclear protein was extracted. 500 µg of nuclear
protein was immunoprecipitated with 2 µg of IRF-1 antibody. The
immunoprecipitated protein was subjected to 10% SDS-PAGE, transferred
to nitrocellulose membrane, and blotted with IRF-1 antibody.
D, the iNOS promoter luciferase reporter was co-transfected
with ICSBP and IRF-1 into RAW264.7 cells. The cells were incubated for
24 h, and the cellular extracts were analyzed for luciferase
activity. E, RAW264.7 cells were co-transduced with ICSBP
and IRF-1, and the cells were incubated for 48 h. Supernatants
were collected for the determination of nitrite accumulation using the
Griess reagent, and cellular protein was extracted for Western
blotting. 20 µg of protein were subjected to 10% SDS-PAGE,
transferred to nitrocellulose membrane, and blotted with an antibody
against iNOS.
|
|
The ICSBP-IRF-1 Interaction Is Important for the Inhibition of iNOS
Expression by IL-4--
IL-4 is a Th2 cytokine that can counteract the
effects of IFN-
and thus serves as an anti-inflammatory molecule.
IL-4 can suppress iNOS expression in both primary macrophages and
RAW264.7 cells treated with IFN-
. In the present study, we
demonstrated that complex formation of ICSBP with IRF-1 is essential
for iNOS induction by IFN-
. To test whether the ICSBP·IRF-1
complex is a target of IL-4, we first analyzed the effect of IL-4 on
iNOS gene expression. RAW264.7 cells pretreated with IL-4 (10 ng/ml) for 1 h were activated with IFN-
, after which iNOS mRNA and
protein expression and nitrite accumulation were determined. IL-4
clearly inhibited iNOS expression and nitrite accumulation induced by IFN-
(Fig. 7, A-C). We
next analyzed the effect of IL-4 on IFN-
-induced DNA·protein
complex formation by EMSA with probe covering iNOS promoter ISRE site
(
923 to
913). The results showed that IL-4 prevented the
DNA·protein complex formation between ICSBP and IRF-1 (Fig.
7D). Co-immunoprecipitation experiments indicated that the
physical binding of ICSBP with IRF-1 was attenuated by the pretreatment
with IL-4 (Fig. 7E). These results suggest that the
interaction of ICSBP with IRF-1 may be the target for the IL-4-mediated inhibition of IFN-
-induced iNOS expression.

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Fig. 7.
IL-4 attenuates the physical interaction of
ICSBP with IRF-1. A, RAW264.7 cells were pretreated
with IL-4 (10 ng/ml) for 1 h and activated with IFN- for
48 h. Supernatants were then collected, and nitrite accumulation
was determined using the Griess reagent. B, RAW264.7 cells
were pretreated with IL-4 (10 ng/ml) for 1 h and activated with
IFN- (10 ng/ml) for 6 h. Total cellular RNA was then extracted,
and RT-PCR was performed for the detection of iNOS mRNA expression.
C, RAW264.7 cells were pretreated with IL-4 (10 ng/ml) for
1 h and activated with IFN- for 12 h. Total cellular
protein was then extracted for Western blotting. 20 µg of protein
were subjected to 10% SDS-PAGE, transferred to a nitrocellulose
membrane, and blotted with an antibody against iNOS. D,
RAW264.7 cells were pretreated with IL-4 (10 ng/ml) and activated with
IFN- for 4 h. The nuclear proteins were then extracted, and
electrophoretic mobility shift assay was performed using a probe
between 935 and 905 of the iNOS promoter. 32P-Labeled
probes were incubated with 0.5 µg of poly(dI-dC) and 5 µg of
nuclear extracts. E, RAW264.7 cells were pretreated with
IL-4 (10 ng/ml) for 1 h and activated with IFN- . Nuclear
extracts were prepared and immunoprecipitated with 2 µg of antibody
to IRF-1 and 20 µl of protein G-agarose beads. Beads were washed
extensively and boiled with 1× SDS-PAGE sample buffer, subjected to
10% SDS-PAGE, and immunoblotted with an antibody against ICSBP. For
controls, 20 µg of nuclear protein was subjected to 10% SDS-PAGE and
immunoblotted either with antibodies against ICSBP or IRF-1.
|
|
 |
DISCUSSION |
This study describes the role of ICSBP in the regulation of murine
macrophage iNOS gene expression. An ICSBP binding site in the iNOS
promoter region (
923 to
913) was identified, and we show that the
complex formed by ICSBP and IRF-1 is essential for IFN-
-induced iNOS
gene expression. IL-4, a Th2 cytokine that inhibits IFN-
-induced
iNOS gene expression, attenuates the physical interaction of ICSBP with
IRF-1. Thus, complex formation of ICSBP with IRF-1 is a central element
in iNOS gene expression and offers a novel example of cooperation among
transcription factors resulting in the regulation of target gene expression.
The transcriptional regulation of iNOS has been studied extensively and
characterized by a promoter with numerous protein-DNA and
protein-protein interactions. Although many potential
cis-regulatory elements have been identified, six enhancer
elements have been shown to be important in IFN-
and LPS induction
of iNOS. In the upstream enhancer element known as region II, located
between positions
1029 and
913, one IFN-stimulated response element and an IFN-
-activated sequence have been identified (15, 16, 35).
For LPS transcriptional regulation of iNOS, two NF-
B sites, one
located in the basal enhancer region I between positions
48 and
209
and another located in region II, are required (36). More recently,
another cis-regulatory element, an Oct site, which binds the
basal transcriptional element octomer, was found to be required for
maximal iNOS transcription by LPS. NF-
B has also been shown to be
required in the regulation of the human iNOS gene (37). Although it has
become clear that many transcription factors are important for the
activation of iNOS promoter, how these factors work together to
regulate iNOS gene expression still remains to be elucidated.
IFN-
, a potent inducer of NO production, also has synergistic
effects with other proinflammatory cytokines, such as IL-1 and tumor
necrosis factor-
(38). IFN-
strongly activates IRF family
members, including IRF-1 and ICSBP. Both IRF-1- and ICSBP-deficient mice are vulnerable to bacterial infections (16, 27, 28). These mice do
not generate a Th1 immune response against intracellular pathogens, and
macrophage production of IL-12 in response to LPS and IFN-
activation is seriously compromised (39, 40). Interestingly both
IRF-1
/
and ICSBP
/
macrophages display selective impairment of
IFN-
-induced NO production (16, 29). Kamijo et al. (16) reported that NO production induced by IFN-
and LPS was strongly impaired in IRF-1
/
mice, and they also identified the IRF-1 binding
site in the ISRE site (
923 to
913) of the iNOS promoter region.
Other reports suggest that IRF-1 is required, along with NF-
B, for
the synergistic effect of IFN-
with tumor necrosis factor-
in the
induction of iNOS expression (41). However, the mechanism responsible
for the IRF-1 effect on iNOS gene expression is still not understood fully.
IRF-1
/
mice and ICSBP
/
mice both display clear impairment of
IFN-
-induced NO production (29), which suggests that ICSBP is
involved directly in iNOS gene regulation. Because ICSBP is an
IFN-
-induced transcription factor, we hypothesized that there should
be an ICSBP binding site in the iNOS promoter region, and we have now
identified ICSBP binding site between
923 and
913, which is
required for maximal effects of IFN-
induction of the iNOS gene
(35). EMSA and chromatin co-immunoprecipitation experiments show that
ICSBP binds to this typical ISRE site. Overexpression of ICSBP greatly
enhanced IFN-
-induced iNOS promoter activation, whereas
overexpression of ICSBP had no effect on the mutant iNOS promoter with
a mutated ISRE site (
923 to
913) in the iNOS promoter. Furthermore,
macrophages from ICSBP
/
mice are severely compromised in
IFN-
-induced iNOS promoter activation and nitric oxide production. These results suggest that ICSBP is important for iNOS gene expression and that ISRE site at
923 to
913 is a binding site for ICSBP.
As we expected, when we transduce ICSBP
/
macrophages with ICSBP,
IFN-
-induced iNOS expression was rescued. But, unexpectedly, transduction with ICSBP alone did not induce iNOS gene expression, although ICSBP was expressed. EMSA experiments indicate that without IFN-
activation, ICSBP did not bind to the ISRE site (
923 to
913) in the iNOS promoter. Furthermore, IFN-
-induced DNA·protein complex formation is sensitive to cycloheximide, suggesting that another transcription factor is needed to form the complex. EMSA experiments demonstrated that this complex also contains IRF-1. In
addition, co-immunoprecipitation experiments indicated the presence of
an ICSBP·IRF-1 complex in the nucleus of IFN-
-activated RAW264.7
cells. All these results suggest that a complex between ICSBP and IRF-1
is essential for the iNOS expression induced by IFN-
.
Transcription of critical cytokines and other important genes is often
regulated by synergistic combination of transcription factors, which
form a nuclear protein complex called an "enhanceosome" (42). For example, NF-
B alone or IRF-1 alone induces transcription of the IFN-
gene at much lower levels than both transcription factors together. In the present study we found that ICSBP and IRF-1
synergize to activate the iNOS promoter. ICSBP was cloned from a
cDNA expression library screened with a labeled ISRE DNA element
from a major histocompatibility complex class I promoter. Specific
interaction with different IFN type I-induced genes was demonstrated by
Southern-Western analysis. The stimulatory effects of ICSBP on the
transcriptional activity of both IRF-1 and IRF-2 suggest that different
DNA-binding heterocomplexes were involved, which led to the
identification of specific protein·protein complexes between these
factors. In vitro translated IRF-1, IRF-2, and ICSBP were
able to form strong interacting complexes that were also identified in
nuclear extracts from various cell lines. In the present study, direct
physical interaction of ICSBP with IRF-1 was found in nuclear extracts
from RAW264.7 cells activated with IFN-
. This suggests that the
effects of ICSBP and IRF-1 on the iNOS gene expression are based on the
direct physical interaction of ICSBP with IRF-1.
ICSBP was regarded initially as a transcription repressor (21).
Contursi et al. (29) also reported that ICSBP represses IFN-
-induced ISRE activity. However, it has been demonstrated recently that ICSBP can interact with PU.1, a hematopoietic
cell-specific member of the Ets family (43), resulting in the
activation of transcription. Accumulated evidence indicates that ICSBP
and PU.1 activate transcription through Ets-IRF DNA elements and
related elements in IFN-
-stimulated macrophages, as reported for
IL-1
and the respiratory oxidase gene gp91 phox (44-46). ICSBP and
PU.1 may also have a role in enhancing expression of the toll-like receptor 4 and IL-18 through a similar element (47, 48). This activation seems to involve other factors, notably the
co-activator/histone acetylase cAMP-response element-binding
protein-binding protein/p300, with which PU.1 has been shown to
interact (49). The present study demonstrated that ICSBP and IRF-1 have
a synergistic effect on the activation of iNOS promoter, which agrees
with the results reported for activation of the IL-12 promoter (25).
These findings suggest ICSBP has a dual role in the regulation of
transcription. On one hand, ICSBP represses ISRE promoter activity
resulting in the inhibition of transcription, but ICSBP can also
activate iNOS, IL-12, and IL-1
promoter activities. Therefore, the
exact molecular mechanism for the regulation of transcription by ICSBP still needs to be elucidated. Our future study will focus on analysis of the ISRE sites in different promoters to find differences in ISRE
sequence or flanking sequence and correlate these differences with the
regulation of transcription by ICSBP. We will also search for new
partner that ICSBP recruits following macrophage IFN-
activation. We
hope to address the mechanism responsible for the dual role of ICSBP in
the regulation of transcription.
Regulation of inflammatory responses involves intercellular
communication through a network of secreted cytokines. To avoid detrimental inflammatory and cytotoxic reactions of activated macrophages, the production of proinflammatory cytokines and
chemokines, as well as of reactive oxygen and nitrogen intermediates,
are tightly regulated (50, 51). This regulation is at least partially dependent on the balance between proinflammatory and anti-inflammatory cytokines. Although IL-4-mediated anti-inflammatory function has been
found to include both transcriptional and translational events, the
transcriptional regulation represents a major target (52, 53). In the
present study, we found IL-4 inhibits iNOS gene expression in part by
attenuating the interaction of ICSBP with IRF-1. However, we cannot
exclude that other mechanisms may play a role in the inhibition of iNOS
gene expression by IL-4.
Taken together, our results provide a novel insight on the molecular
mechanism for the induction of iNOS gene expression by IFN-
. ICSBP,
a member of IRF family, can form a complex with IRF-1 that is essential
for iNOS gene expression. IL-4, an anti-inflammatory cytokine, inhibits
iNOS gene expression by disrupting the interaction of ICSBP with IRF-1.
These results may provide a framework for future anti-inflammatory therapy.