Vascular Biology Center and Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, Georgia 30912-2500
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ABSTRACT |
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In rat aortic smooth muscle cells
(RASMC), interferon (IFN)- enhanced nitrite accumulation and type II
nitric oxide synthase (iNOS) protein expression induced by interleukin
(IL)-1
. IFN-
alone had no effect on nitrite accumulation or iNOS
protein. IL-1
, but not IFN-
, induced nuclear factor (NF)-
B and
CCAAT box/enhancer binding protein (C/EBP) nuclear binding. Conversely,
IFN-
, but not IL-1
, induced signal transducer and activator of
transcription (STAT) 1 and interferon regulatory factor (IRF)-1
binding. In a
1.4-kb rat iNOS promoter segment, deletion of an
IFN-
-activated site (GAS) increased IL-1
-induced activity but
inhibited IFN-
-enhanced activity, suggesting a two-way effect of the
GAS site on iNOS induction: enhancing induction through STAT1
activation and inhibiting induction through a non-IFN-
-mediated
mechanism. Deletion of both an IRF and a C/EBP site reduced the
IL-1
-induced and the IFN-
-enhanced activities. However, IRF site
mutations decreased the IFN-
-enhanced activity without affecting the
IL-1
-induced activity. Insertion of two IRF sites increased the
IFN-
-enhanced, but not the IL-1
-induced, activity. Mutations of a
reverse NF-
B site did not significantly change IFN-
-enhanced
activity. We conclude that in RASMC, NF-
B and C/EBP mediate the
IL-1
-induced iNOS expression, whereas IRF-1 and STAT1 mediate the
IFN-
-enhanced iNOS induction.
nuclear factor-B; CCAAT box/enhancer binding protein; interferon
regulatory factor-1; signal transducer and activator of transcription
1; interferon-
activation site
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INTRODUCTION |
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NITRIC
OXIDE (NO), a multifunctional effector molecule synthesized
by nitric oxide synthase (NOS) from L-arginine
(28), transmits signals for vasorelaxation,
neurotransmission, and cytotoxicity. Three different NOS isoforms have
been identified, which fall into two distinct types, constitutive and
inducible. Unlike the constitutively expressed NOS isoforms (eNOS, or
type III NOS; nNOS, or type I NOS) (4, 29), the activity
of inducible NOS (iNOS, or type II NOS) isoform is independent of
intracellular calcium concentration and yields much higher NO output
(26, 35). The excess NO produced by iNOS is implicated in
the pathophysiological processes of cardiovascular diseases such as
sepsis (17), stroke (2), and atherosclerosis
(7). However, the mechanisms underlying iNOS expression in
vascular diseases remain unclear. Unraveling the molecular mechanisms
responsible for iNOS induction may shed light on ways to manage these
diseases. Interferon (IFN)- alone is reported to induce iNOS
expression in cultured human and rat vascular smooth muscle cells (SMC)
(6, 19) but not in cultured rat pulmonary artery SMC
(27), aortic SMC (1), and the SMC cell line
A7r5 (33). Similarly, interleukin (IL)-1
alone has been
shown to induce iNOS expression in cultured rat aortic SMC (1,
11, 16, 19) but not in cultured human vascular SMC (6), rat pulmonary artery SMC (27), and the
SMC cell line A7r5 (33). Variations in species and cell
types and in long-term cultured cell line vs. short-term cultures from
primaries may account for the observed different effects. Thus, in each
experimental model, the effect of IFN-
or IL-1
on iNOS induction
must be clarified before the molecular mechanisms of iNOS induction can be investigated.
Previous studies have shown that nuclear factor (NF)-B sites are
required for iNOS induction (12, 33, 36, 37, 39). The
importance of NF-
B sites also was confirmed by NF-
B inhibition (14). Besides NF-
B, other transcription factors, such
as interferon regulatory factor (IRF)-1, CCAAT box/enhancer binding
protein (C/EBP), and signal transducer and activator of transcription (STAT) 1, also are involved in iNOS induction. IRF-1 is required for
iNOS induction in mice, as demonstrated by the IRF-1 gene knockout
(15). Mutation of the IRF-1 binding site in murine iNOS
promoter showed that the site mediates the IFN-
-enhanced iNOS
promoter activity (24). STAT1
induction and its binding to the interferon-
activation site (GAS) in murine iNOS promoter mediates iNOS promoter induction by IFN-
and lipopolysaccharide (LPS) in RAW 246.7 cells (13). C/EBP induction and binding
to a C/EBP site in rat iNOS promoter mediates the iNOS promoter
induction by cAMP in rat mesangial cells (12).
The mechanisms of iNOS induction are cell and species specific. For
full promoter activity, different lengths of the 5' flanking regions
among murine, rat, and human iNOS promoters are required. The 1 kb of
murine iNOS promoter confers full promoter activity (22).
To confer full promoter activity in the rat, 2 kb of additional 5'
flanking region are required (40). The first 3 kb of human iNOS promoter exhibits no activity, and over 10 kb of the 5' flanking region are required for full human iNOS promoter activity (10, 21). Similarly, iNOS induction is cell specific. The Janus
kinase (JAK)/STAT pathway mediates the LPS plus IFN--induced iNOS
expression in RAW 264.7 cells (13). However, inhibition of
JAK/STAT pathway enhances iNOS induction by LPS plus IFN-
in rat
aortic smooth muscle cells (RASMC) (23).
We recently cloned the rat iNOS promoter (40). Because of
the differences in iNOS induction among species, we have used a
homologous system, rat iNOS promoter transfected into rat SMC, to
eliminate possible species differences in the regulation of iNOS. We
now report that in RASMC, IL-1 alone, but not IFN-
alone, induces
iNOS. However, IFN-
enhances iNOS induction by IL-1
. We further
present data showing that the IL-1
-induced iNOS expression is
mediated by NF-
B and C/EBP activation and that IFN-
-enhanced
promoter activity is mediated by IRF-1 and STAT1 activation.
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MATERIALS AND METHODS |
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Chemicals.
Rat recombinant IFN- was obtained from R&D Systems (Minneapolis,
MN), and (human) IL-1
was purchased from Boehringer Mannheim (Indianapolis, IN). The concentrations of cytokines used in the study
were similar to those used by other investigators in vascular SMC
(6, 33). Lipofectamine was purchased from Life
Technologies (Gaithersburg, MD). Gel shift antibodies were obtained
from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell cultures.
RASMC were harvested from Wistar rats weighing 200-225 g (Harlan,
Indianapolis, IN) by enzymatic dissociation. The cells were positively
identified as smooth muscle by indirect immunofluorescent staining for
-actin with the use of mouse anti-
-actin antibody and anti-mouse
IgG-FITC conjugate. Cells were grown in 50% Dulbecco's modified
Eagle's medium and 50% F-12 nutrient medium (DMEM/F-12) supplemented
with 10% fetal bovine serum, glutamine (0.2 g/l), penicillin (100 units/ml), and streptomycin (100 units/ml). All cultures were grown in
a humidified incubator at 37°C under 5% CO2 in air.
Cells at passages 2-4 were used in the studies.
Nitrite assay. Nitrite levels were measured by a fluorometric method (25). First, 150 µl of cell culture supernatant were diluted in 2 ml of H2O and mixed with 200 µl of 2,3-diaminonaphthalene (0.05 mg/ml 0.62 M HCl). After incubation at room temperature for 15 min, 100 µl of NaOH (2.8 M) were added to stop the reaction. The fluorescence intensity was measured at 380 nm (excitation) and 407 nm (emission) with a Spectroluminometer (Perkin-Elmer 5B).
Western blot. After the experiment protocol, RASMC were rinsed twice with cold PBS and 300 µl of the lysis buffer [20 mM Tris · HCl, pH 7.4, 2.5 mM EDTA, 1% Triton, 10% glycerol, 1% deoxycholate, 0.1% SDS, 50 mM NaF, 10 mM Na4P2O7, and 1 mM phenylmethylsulfonyl fluoride (PMSF)] were added. The lysates containing an equal amount of protein (10 µg) were subsequently loaded on 7.5% SDS polyacrylamide gels, and the resolved proteins were electrophoretically transferred to nitrocellulose membrane. iNOS protein was specifically detected by rabbit polyclonal anti-mouse iNOS antibody with 1: 5,000 dilution (Transduction Laboratories, Lexington, KY). The second antibody was a peroxidase-conjugated anti-rabbit IgG from donkey. Membrane was developed with the enhanced chemiluminescence detection system (ECL; Amersham, Piscataway, NJ) and exposed on film.
Nuclear extracts.
The nuclear protein isolation protocol was modified from the method of
Sadowski and Gilman (31). After treatment, RASMC were
rinsed with ice-cold PBS and hypotonic buffer [20 mM HEPES-KOH, pH
7.9, 20 mM NaF, 1 mM Na3VO4, 1 mM
Na4P2O7, 0.4 µM microcystin, 1 mM
EDTA, 1 mM EGTA, 1 mM dithiothreitol (DTT), 1 mM PMSF, and 1 µg/ml
each of leupeptin, aprotinin, and pepstatin]. Ice-cold hypotonic
buffer (0.5 ml) with 0.5% Nonidet P-40 was added to the dish (100 mm)
and incubated on ice for 10 min. The lysates were scraped into a glass
homogenizer and stroked 12 times. The homogenate was transferred to a
1.5-ml tube and vortexed vigorously for 10 s. The nuclei were
pelleted by centrifugation at 12,000 rpm for 2 min at 4°C, washed
with 500 µl of hypotonic buffer once, resuspended in 120 µl of
high-salt buffer (hypotonic buffer plus 420 mM NaCl and 20% glycerol),
and then rocked gently for 30 min at 4°C. The extracted nuclear
proteins were separated from residual nuclei by centrifugation at
12,000 rpm at 4°C for 30 min. The supernatant (nuclear proteins) was
collected and frozen at 80°C. Protein concentration in nuclear
extracts was measured with the Bradford assay (3).
Electrophoretic mobility shift assays.
The DNA probes were hybridized and end-labeled with T4
polynucleotide kinase in a reaction containing 2 µl of probe (1.75 pmol/µl), 1 µl of 10× T4 kinase buffer, 1 µl of
[-32P]ATP (10 mCi/ml), 5 µl of water, and 1 µl of
T4 kinase (10 units/µl). After incubation at 37°C for 30 min, the
reaction was stopped by adding 1 µl of 0.5 M EDTA and then 89 µl of
Tris base-EDTA buffer. The labeled probe was separated from
unincorporated [
-32P]ATP chromatographically on G6
spin columns (Bio-Rad, Hercules, CA).
Plasmids.
The construction of a 3.2-kb rat iNOS promoter-luciferase construct
has been described previously (40). The
1.4-kb rat iNOS
promoter-luciferase construct was generated by using the restriction
enzymes SphI and XhoI to cut the DNA from the
3.2-kb rat iNOS promoter-pGL3basic construct and was ligated with the pGL3basic vector. Sequencing confirmed the 5' end of promoter located
at
1368 bp. To generate the mutated rat iNOS promoter constructs, we
performed site-directed mutagenesis on the context of the
1.4-kb
reporter construct, using the QuickChange site-directed mutagenesis as
follows. The
1.4-kb rat iNOS promoter reporter construct was used as
the template. Two synthetic oligonucleotide primers (Table
1), which were complementary to each
other, were extended during temperature cycling by Pfu DNA
polymerase (Promega, Madison, WI). After 20 cycles at 95°C for
30 s, 55°C for 1 min, and 72°C for 10 min, the product was
treated with DpnI (Stratagene, La Jolla, CA) at 37°C for
2 h and transformed into TOP 10 Escherichia coli. All
mutations were verified by nucleotide sequencing in an ABI automated
sequencing system (model 377) in the Molecular Biology Core Facility of
the Medical College of Georgia.
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Transient transfection and luciferase assay.
The transfection experiments and luciferase assays were performed as
described previously (40), with the following
modifications. RASMC were seeded in 12-well plates at 1.5 × 105 cells/well. Each well was loaded with 500 µl of
transfection mixture that contained 0.3 µg of 1.4-kb rat iNOS
promoter-pGL3basic construct, 0.036 µg of pCMV-
-gatactosidase
construct, 2 µl of Plus reagent, and 3.5 µl of Lipofectamine (2 mg/ml). To control for the efficiency of transfections, a plasmid DNA
containing a cytomegalovirus promoter-driven
-galactosidase gene was
cotransfected. After treatment, the transfected cells were washed three
times with PBS and were lysed with 0.35 ml of 1× Cell Culture Lysis Reagent. The luciferase activity in cell lysate was measured with the
Luciferase Assay Substrate (Promega) in a TD 20/20 luminometer (Turner Designs).
Data analysis.
Transfection efficiency was estimated by normalization to the
cotransfected -galactosidase activity. Promoter (luciferase) activity was then expressed as multiples of control after normalization to the corresponding vehicle control group. The "multiples of control" were then converted to a percentage of the IL-1
-induced wild-type promoter activity by being normalized to the corresponding IL-1
-induced wild-type promoter activity. IFN-
-enhanced
activity was derived by subtracting IL-1
-induced activity from
IFN-
plus IL-1
-induced activity. It was expressed as a percentage
of corresponding IL-1
-induced promoter activity. Data were reported
as means ± SE. Statistical analyses utilized Student's
t-test or one-way ANOVA, followed by the least significant
different procedure, as appropriate, and differences at
P < 0.05 were considered statistically significant.
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RESULTS |
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IFN- enhances IL-1
-induced iNOS expression in RASMC.
RASMC were treated with or without IFN-
for 24 h. After
treatment, the medium was collected and the concentration of nitrite, the stable metabolite of NO, was measured. As shown in Fig.
1, IFN-
(rat recombinant, 250 units/ml) alone could not induce nitrite accumulation in RASMC; IL-1
alone strongly induced nitrite accumulation (121.4 ± 12.0-fold
higher than control). IFN-
significantly enhanced the
nitrite accumulation induced by IL-1
(from 121.4 ± 12.0- to
177.6 ± 11.4 fold- higher than control, P < 0.01) or IL-1
plus tumor necrosis factor (TNF)-
(from 157.3 ± 9.8- to 223.8 ± 14.0-fold higher than control,
P < 0.01) (Fig. 1). Similarly, IFN-
alone did not
induce iNOS protein but enhanced IL-1
-induced iNOS protein
expression (Fig. 2).
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Role of a GAS site in rat iNOS promoter induction.
With the use of a rat iNOS promoter fragment (941 to
922 bp)
containing a GAS site (
936 to
928 bp) as probe (Fig.
3A), electrophoretic mobility
shift assays (EMSA) showed that IFN-
, but not IL-1
, induced a
factor binding to the probe; this factor was identified as STAT1 from
the EMSA band supershifted by anti-STAT1
antibody (Fig.
3B). Deletion of the GAS site increased the IL-1
-induced promoter activity but decreased the IFN-
-enhanced promoter activity (Fig. 3, C and D). These results suggest a
two-way effect of the GAS site on iNOS induction: an enhancement
mediated by an IFN-
-dependent mechanism, presumably JAK/STAT
pathway, and an inhibition mediated by an unknown IFN-
-independent
mechanism.
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Role of IRF and C/EBP sites in IFN- rat iNOS promoter
activation.
With the use of a rat iNOS promoter fragment (
921 to
898 bp)
containing an IRF (
918 to
907 bp) and a C/EBP (
910 to
902 bp)
site as probe (Fig.
4A), EMSA
showed that IFN-
, but not IL-1
, induced IRF-1 binding to the
probe and that the binding disappeared in the presence of anti-IRF-1
antibody (Fig. 4B). Conversely, IL-1
, but not IFN-
,
induced a C/EBP binding to the probe, and IFN-
did not enhance the
IL-1
-induced C/EBP binding. The C/EBP binding band disappeared in
the presence of anti-C/EBP antibody (Fig. 4C). However, the
IRF-1 and C/EBP bands were not significantly changed in the presence of
either anti-p50 or anti-p65 antibodies (Fig. 4D). Deletion
mutation (Del-IRF-C/EBP) of
914 to
905 bp, which disrupted both the
IRF and the C/EBP sites, abolished both the IL-1
-induced and
IFN-
-enhanced promoter activities (Fig. 5A). To distinguish between
the roles of the IRF and the C/EBP sites, three loss-of-function
mutations of the IRF site were generated outside of the C/EBP motif to
disrupt only the IRF site. One mutation consisted of an insertion of an
XbaI site (IRF-Ins6) between
911 and
910 bp. The other
two were substitution mutations; one substituted AGT for TCA at
914
to
912 bp (IRF-Mu3), and the other substituted GG for TT at
916 to
915 bp (IRF-Mu2). All loss-of-function IRF site mutations abolished
the IFN-
-enhanced activity but not the IL-1
-induced activity
(Fig. 5, A and B). A gain-of-function mutation of
the IRF site also was generated. The IRF site was changed to 100%
homology with the IRF consensus sequence, and two additional such IRF
sites were inserted upstream of the original IRF site (Ins2IRF).
Ins2IRF increased the IFN-
-enhanced activity but not the
IL-1
-induced activity (Fig. 5, A and B). These
results suggest that the IRF site mediates IFN-
-enhanced iNOS
expression and that the C/EBP site contributes to IL-1
-induced iNOS
expression.
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Role of NF-B activation in iNOS induction.
With the use of a commercial NF-
B probe (Fig.
6A) and nuclear extracts from
RASMC, EMSA showed that IL-1
, but not IFN-
, induced NF-
B
binding to the probe. IFN-
did not enhance the IL-1
-induced
binding (Fig. 6B). Previously, we had demonstrated that a
reverse NF-
B site mediates IL-1
-induced rat iNOS promoter activity in RASMC (37). As shown in Table
2, reverse NF-
B site mutations did not
significantly affect the IFN-
-enhanced activity. The results suggest
that NF-
B activation mediates IL-1
-induced iNOS expression but
not the IFN-
-enhanced iNOS expression.
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DISCUSSION |
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We have demonstrated that in RASMC, IL-1 induces iNOS
expression, but IFN-
only enhances it. We also have demonstrated
that in RASMC, IL-1
, but not IFN-
, induces NF-
B and C/EBP
nuclear binding, whereas IFN-
, but not IL-1
, induces STAT1 and
IRF-1 nuclear binding. Although IFN-
enhances iNOS expression
induced by IL-1
, it does not enhance the IL-1
-induced nuclear
bindings of NF-
B and C/EBP.
The JAK/STAT pathway is a well-characterized pathway of IFN-
signaling (9). It involves IFN-
dimer binding to
IFN-
R1, IFN-
R1 and IFN-
R2 association, JAK1 and JAK2
activation, phosphorylation of Tyr457 in IFN-
R1,
recruitment and phosphorylation of Tyr701 in STAT1
,
STAT1
dimerization, and nuclear translocation. The final step of the
JAK/STAT pathway is the interaction between the STAT1
dimer and a
GAS element in the promoter regions of IFN-
-inducible genes to
initiate the induction of this family of genes. STAT1
activation and
its binding to a GAS site in murine iNOS promoter have been shown to
mediate iNOS induction by IFN-
or LPS in RAW 264.7 cells
(13); inhibition of JAK2 decreases iNOS mRNA induction in
human DLD-1 cells (18). However, inhibition of the
JAK/STAT pathway also enhances iNOS induction by IFN-
plus LPS in
RASMC (23), suggesting that the JAK/STAT pathway might
play a different role in iNOS induction in RASMC. Rat and murine iNOS
promoter have identical GAS sites. We have shown that IFN-
, but not
IL-1
, induces STAT1
binding to the GAS site in rat iNOS promoter.
As in RAW 264.7 cells, the JAK/STAT pathway appears to mediate
IFN-
-enhanced iNOS promoter activation in RASMC, because deletion of
the GAS site decreases the IFN-
-enhanced promoter activity. The GAS
element exhibits two functions in iNOS induction in RASMC. Besides
mediating IFN-
enhancement, it inhibits iNOS induction by IL-1
,
because deletion of the GAS site increases IL-1
-induced promoter
activity. The exact mechanism is not clear. It may be independent of
the JAK/STAT pathway, because IL-1
itself does not induce STAT1
binding to the GAS site.
There are seven STATs (STAT1, 2, 3, 4, 5A, 5B, and 6) and four JAKs
(JAK1, 2, 3 and Tyk2). STAT1 has two splice variants; STAT1, unlike
STAT1
, lacks the terminal 38 amino acids and cannot restore IFN-
responsiveness in U3 cells (8). The JAK/STAT pathway
mediates not only IFN transduction but also that of a large variety of
polypeptide ligands. STATs are phosphorylated not only by JAK kinases
that are associated with cytokine receptors but also by receptors with
intrinsic tyrosine activity (for example, epidermal growth factor,
platelet-derived growth factor, and colony-stimulating factor-1
receptors). JAK kinases do not seem to have specificity for a
particular STAT substrate and the specificity of receptor tyrosine
kinase activation of STATs is unclear (8). The complexity of the JAK/STAT pathway may explain why inhibition of the JAK/STAT pathway might superinduce iNOS induction by IFN-
plus LPS in RASMC
(23). The JAK/STAT pathway may not be an important
mechanism for iNOS induction in RASMC because deletion of the GAS site
does not significantly change the IFN-
plus IL-1
-induced promoter activity (Fig. 3). The reasons behind the differences between the
present study and that of Marrero et al. (23) are unclear. It is possible that the combination of LPS and IFN-
used by Marrero et al. (23) may have a very different effect from the use
of IL-1
and IFN-
in the present study. There may be other signal transduction pathways mediating IFN-
-enhanced iNOS expression in
RASMC. For example, IFN-
activates NF-
B in ChWK cells
(38). Also, the GAS element appears to be dispensable for
the expression of many IFN-
-induced genes, whereas the IRF element
is not (5).
IRF-1 has been shown to be important for iNOS induction
(15). The IRF site mediates the IFN--enhanced murine
iNOS promoter induction in RAW 264.7 cells but has no effect on the
LPS-induced murine iNOS promoter activity in RAW 264.7 cells
(24). IRF-1 is induced by IFN-
in RASMC
(32). However, the relationship between IFN-
-induced
IRF-1 activation and iNOS induction in RASMC has not been established.
In this study, IRF-1 is induced by IFN-
but not by IL-1
. Similar
to the murine iNOS promoter study (24), disruption of the
IRF site abolishes only the IFN-
-enhanced, but not the
IL-1
-induced, rat iNOS promoter activity in RASMC. Similarly,
insertion of two additional IRF sites increases the IFN-
-enhanced,
but not the IL-1
-induced, rat iNOS promoter activity in RASMC,
further confirming that in RASMC, IRF-1 activation and its binding to
the IRF site mediate the IFN-
-enhanced rat iNOS promoter activity.
The mechanism by which IFN-
induces IRF-1 still remains to be determined.
C/EBP family members are basic-leucine-zipper transcription factors
that recognize specific sequences as either homodimers or heterodimers.
The C/EBP family includes at least six members (C/EBP,
,
,
,
, and
) that dimerize in a tissue-specific manner and have
highly homologous dimerization and DNA contact domains and similar DNA
binding activities (20). C/EBP
, previously called
NF-IL6 because it mediates IL-6 gene regulation, is induced by
inflammatory agents, such as LPS (30) and IL-1
(12). There is a perfectly matched C/EBP site located at
172 to
164 bp of rat iNOS promoter, which also contains a perfectly
matched cAMP-responsive element. C/EBP
, C/EBP
, and
cAMP-responsive element binding protein bind to the C/EBP site.
Although both IL-1
and cAMP strongly induce C/EBP
and C/EBP
,
in rat mesangial cells, mutation of the C/EBP site abolished
cAMP-induced rat iNOS promoter activity only (12), which
casts doubt on whether the C/EBP site is a real C/EBP site. Another
C/EBP site exists at
910 to
902 bp of the rat iNOS promoter and is
identical to the
916- to
908-bp site of the murine iNOS promoter.
Substitution of CC for AA at
910 to
909 bp of the murine iNOS
promoter decreased IL-1 plus IFN-
plus TNF-
-induced murine iNOS
promoter activity in A7r5 cells, a rat vascular smooth muscle cell line
(34). It is quite possible that the C/EBP site in rat iNOS
promoter is functional. We have demonstrated that IL-1
, but not
IFN-
, induces C/EBP binding to the C/EBP site. The C/EBP site
overlaps with an IRF site (
918 to
907 bp of the rat iNOS promoter).
Disruption of both the IRF and C/EBP sites abolishes both
IFN-
-enhanced and IL-1
-induced rat iNOS promoter activity in
RASMC; however, the IRF site mediates only the IFN-
-enhanced
activity. We suggest that the C/EBP site contributes to IL-1
-induced
iNOS expression in RASMC.
There are three NF-B sites in the rat iNOS promoter, one upstream
(
965 to
956 bp), one reverse (
901 to
892 bp), and one downstream (
107 to
98 bp). The up- and downstream NF-
B sites are
required for iNOS induction in RASMC (41). IL-1
induces NF-
B. The reverse NF-
B site has been demonstrated to mediate the
IL-1
-induced rat iNOS promoter activity in RASMC (37); however, disruption of the reverse NF-
B site does not significantly change IFN-
-enhanced activity (Table 2). IFN-
does not induce NF-
B nuclear binding and does not enhance IL-1
-induced NF-
B nuclear binding. It is unlikely that IFN-
enhances iNOS expression in RASMC through the NF-
B pathway, although IFN-
has been shown to activate NF-
B in ChWK cells (38).
There may be two ways to enhance IL-1-induced iNOS transcription:
enhancing the IL-1
-induced transcription factors that are
responsible for iNOS gene transcription or inducing other transcription
factors that cooperate with IL-1
-induced transcription factors to
enhance the iNOS gene transcription. As shown in Fig. 7, our study demonstrates that in RASMC,
IL-1
induces iNOS gene transcription through activation of NF-
B
and C/EBP. IFN-
does not induce or enhance the activation of NF-
B
and C/EBP; however, it induces IRF-1 and STAT1 activation and may
enhance iNOS transcription through cooperation among IRF-1, STAT1,
NF-
B, and C/EBP. In RASMC, like IL-1
, TNF-
strongly induces
NF-
B; however, unlike IL-1
, TNF-
alone does not induce iNOS
(41). This finding suggests that iNOS transcription in
RASMC normally requires the cooperation of multiple transcription
activation.
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ACKNOWLEDGEMENTS |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-52958.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. D. Catravas, Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912-2500 (E-mail: jcatrava{at}mail.mcg.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 4 December 2000; accepted in final form 17 September 2001.
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