Department of Surgery, Georgetown University Medical Center, Washington, District of Columbia 20007
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ABSTRACT |
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On the basis of
previous work demonstrating nitric oxide (NO)-mediated inhibition of
nuclear factor-B (NF-
B) DNA binding, we hypothesized that NO
downregulates NF-
B-dependent interleukin-1
(IL-1
) production
in an ANA-1 macrophage model of lipopolysaccharide (LPS)
stimulation. In the presence of LPS (100 ng/ml), levels of
IL-1
protein and mRNA were significantly upregulated with NO
synthase inhibition. Using nuclear run-on analysis and transient transfection studies, IL-1
gene transcription and IL-1
promoter activity were also found to be increased with inhibition of NO production. Parallel transfection studies using an NF-
B long terminal repeat-reporter plasmid exhibited similar
findings, suggesting an NO-mediated effect on NF-
B activity. Gel
shift studies showed that LPS-associated NF-
B DNA binding was
increased, both in the setting of NO synthase inhibition and in a
reducing environment. Repletion of NO by addition of an
S-nitrosothiol restored IL-1
protein synthesis, mRNA levels, gene transcription, promoter activity, and NF-
B DNA binding to levels noted in the presence of LPS alone. Our studies indicate that NO may regulate LPS-associated inflammation by downregulating IL-1
gene transcription through
S-nitrosation of NF-
B.
S-nitrosation; inducible nitric
oxide synthase; cytokine; nuclear factor-B; lipopolysaccharide; interleukin-1
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INTRODUCTION |
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IN PATHOPHYSIOLOGICAL STATES that are characterized by
the elaboration of proinflammatory cytokines, inducible nitric oxide synthase (iNOS) is expressed and nitric oxide (NO) is produced (20).
This multifunctional free radical alters numerous biochemical functions, such as mitochondrial electron transport, activation of
guanylyl cyclase, and expression of adhesion molecules. However, although it is apparent that NO production frequently accompanies inflammatory states, it is unclear whether NO is indifferent, promotes,
or inhibits the inflammatory process. In this regard, we have
demonstrated that NO S-nitrosylates a
key thiol group in the DNA binding domain of nuclear factor-B
(NF-
B), a key transcription factor for the elaboration of multiple
proinflammatory cytokines, such as interleukin-1
(IL-1
) and tumor
necrosis factor-
(TNF-
) (1, 2, 6, 7).
S-nitrosation of NF-
B at the redox-sensitive C62 residue of p50 is associated with inhibition of
NF-
B DNA binding. Given the central role of NF-
B in
proinflammatory gene transcription,
S-nitrosation-associated inhibition of
NF-
B-dependent promoter activity suggests a potential role for NO as
a feedback inhibitor of the inflammatory process.
Excessive macrophage elaboration of IL-1 plays a fundamental role in
the pathogenesis of the sepsis syndrome (8). Transcriptional regulation
is a major determinant of IL-1
protein synthesis, and maximal
transcription of the IL-1
gene is NF-
B dependent (1, 2, 5, 8,
10). NF-
B therefore plays a central role in regulating IL-1
production and subsequent IL-1
-dependent inflammatory processes.
Using a murine macrophage model of endotoxin [lipopolysaccharide
(LPS)]-mediated NO production, we sought to test the hypothesis
that endogenous synthesis of NO inhibits the synthesis of IL-1
, an
NF-
B-dependent proinflammatory protein, by
S-nitrosation of NF-
B. Our results
suggest a novel mechanism in which NO production feedback inhibits
IL-1
gene transcription in the context of LPS stimulation.
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MATERIALS AND METHODS |
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Materials.
ANA-1 macrophages were a gift from Dr. George Cox (National Cancer
Institute, Frederick, MD). The murine IL-1 promoter-chloramphenicol acetyltransferase (CAT) reporter gene plasmid construct
and NF-
B-TNF-CAT reporter plasmid were gifts from Dr. Clifford J. Bellone (St. Louis University School of Medicine, St. Louis, MO). Human
recombinant NF-
B p50 and the Gel Shift assay system, containing the
NF-
B consensus oligonucleotide
5'-CGCTTGATGAGTCAGCCGGAA-3', were obtained from Promega,
(Madison, WI). The rabbit anti-human NF-
B p50 monoclonal antibody
(MAb) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The
DIG gel shift kit was purchased from Boehringer Mannheim (Indianapolis,
IN). LPS (Escherichia coli serotype
0111:B4), L-arginine, sodium
nitrite, and nitrate reductase were obtained from Sigma (St. Louis,
MO). DMEM, glutamine, penicillin-streptomycin, trypsin-EDTA, and endotoxin-free FCS were purchased from
GIBCO BRL (Grand Island, NY). All other chemicals were of reagent grade.
Induction of NO synthesis in ANA-1 macrophages. ANA-1 macrophages were maintained in DMEM with 0.4 mM L-glutamine, pyridoxine hydrochloride, 5% heat-inactivated, endotoxin-free FCS, and 1 mM L-arginine. LPS (0-1,000 ng/ml) was then added in the presence and absence of FCS (5%) to induce NO synthesis. In selected instances, the competitive substrate inhibitor of NO synthase NG-nitro-L-arginine methyl ester (L-NAME; 100 µM), superoxide dismutase (SOD; 100 U/ml), or the NO donor S-nitroso-N-acetylcysteine (SNAC; 100 µM), or a combination of these compounds, was added. After incubation for 6 h at 37°C in 95% O2-5% CO2, the supernatant was aspirated, and the cells washed twice with Dulbecco's PBS. After treatment with trypsin-EDTA, the macrophages were harvested for biochemical assays.
Measurement of NO. NO release from cells in culture was quantified by measurement of the NO metabolite nitrite by the technique of Snell and Snell (27). To reduce nitrate to nitrite, conditioned medium (200 µl) was incubated in the presence of 1.0 unit of nitrate reductase, 50 µM NADPH, and 5 µM FAD for 15 min at 37°C in the dark. Sulfanilamide (1%, 50 µl) in 0.5 N HCl (50% vol/vol) was then added to 50 µl of the treated medium. After a 5-min incubation at room temperature, an equal volume of 0.02% N-(1-naphthyl)ethylenediamine was added; after incubation at room temperature for 10 min, the absorbance at 570 nm was compared with that of an NaNO2 standard.
Determination of IL-1 and TNF-
concentration.
Cell culture medium levels of IL-1
and tumor necrosis factor-
(TNF-
) were determined by the University of Maryland Cytokine Core
Laboratory with an ELISA technique utilizing murine MAbs.
Immunoblot analysis of IL-1 and iNOS proteins.
Isolated murine macrophages were washed three times in PBS and
incubated with boiling 2× nonreducing electrophoresis sample buffer for 2 min. Separation was performed by SDS-12% PAGE, and then
the products were electrotransferred to a polyester-supported nitrocellulose membrane for 90 min at 150 mA. The membrane
was blocked overnight at 4°C in Tris-buffered saline (TBS)
containing 3% BSA. Blocked membranes were incubated with the
anti-mouse IL-1
MAb or anti-rat iNOS MAb (Transduction Laboratories,
Lexington, KY), washed three times in TBS-0.1% Tween, and incubated
with biotinylated sheep anti-mouse IgG (Amersham, Arlington Heights, IL) for 1 h. After being washed three additional times, membranes were
incubated with a strepavidin-horseradish peroxidase conjugate. After an
additional washing, bound antibodies were detected by the ECL detection
system (Amersham, Arlington Heights, IL). Blots were scanned, and the
area under the curve was normalized to the murine IL-1
or iNOS standard.
RNA extraction and RT-PCR.
Total cellular RNA was extracted by a commercial modification of the
phenol-chloroform-isoamyl alcohol extraction method (RNAgents total RNA
isolation system; Promega) (14). On completion of RNA extraction, all
samples were treated with 10 U/µl RNase-free DNase (Stratagene
Cloning Systems, La Jolla, CA). Cellular RNA (0.5 µg) was reverse
transcribed, and PCR was performed as previously described (14, 15).
The sequences of primer pairs used to amplify mouse IL-1 and TNF-
were 5'-CAGGATGAGGACATGAGCACC-3' and
5'-CTCTGCAGACTCAAACTCCAC-3' and
5'-ATGAGCACAGAAAGCATGATC-3' and
5'-TACAGGCTTGTCACTCGAATT-3', respectively. The primer
sequences for
-actin mRNA, used to determine constitutive mRNA
expression, were 5'-CATCGTGGGCCGCTCTAGGCAC-3' and
5'-CCGGCCAGCCAAGTCCAGACGC-3'. A log-linear dose-response
curve was determined for each set of primers to determine the
appropriate number of amplification cycles. Verification of the
amplified PCR products was performed by automated DNA sequencing
(Applied Biosystems, Foster City, CA) for rat iNOS and
-actin mRNA.
PCR products for TNF-
, IL-1
, and interferon-
(IFN-
) have
been previously verified (Stratagene). The PCR products were visualized
by ultraviolet (UV) illumination after electrophoresis through 1.0%
agarose (UltraPure; Sigma) and staining in Tris-borate-EDTA buffer
containing ethidium bromide. DNA was visualized on a UV illuminator;
gel photographs were scanned, and the area under the curve was
normalized for
-actin mRNA content.
NF-B DNA binding analysis.
Nuclear extracts from ANA-1 macrophages were incubated with boiling
2× nonreducing electrophoresis sample buffer for 2 min. In
certain instances, dithiothreitol (DTT; 20 mM) was added before boiling
to reduce any S-nitrosothiol bonds.
Separation was performed on 5-15% gradient SDS-PAGE gels, and
then the products were electrotransferred to a polyester-supported
nitrocellulose membrane for 90 min at 150 mA. The membrane
was blocked overnight at 4°C in TBS (10 mM Tris · HCl, pH 7.5, 150 mM NaCI) containing 3% BSA.
Blocked membranes were incubated with a p50 MAb with known
cross-reactivity to murine NF-
B p50 (primary anti-human NF-
B p50
MAb; Santa Cruz Biotechnology), washed three times in TBS-0.1% Tween,
and incubated with biotinylated sheep anti-rabbit IgG (Amersham) for 1 h. After being washed three additional times, membranes were incubated
with strepavidin-horseradish peroxidase conjugate. After an additional
washing, bound antibodies were detected by the ECL detection system
(Amersham). Blots were scanned with a computerized laser densitometer
(Hoeffer Scientific Instruments, San Francisco, CA), and the area under
the curve was normalized to the human NF-
B p50 standard.
Synthesis of SNAC. SNAC was synthesized by combining equimolar NaNO2 and N-acetylcysteine in 0.5 N HCl for 30 min at room temperature as previously described. Before use, the S-nitrosoprotein solution was neutralized to pH 7.0 with 0.1 N NaOH. Previous work has confirmed the presence of S-nitrosothiol bonds in the above species by 15N-NMR spectroscopy (28).
Transient transfection with murine IL-1 promoter
and reporter gene constructs.
Transient transfection of ANA-1 macrophages was performed
by electroporation with the Bio-Rad Gene Pulser (9). After cells were
washed twice with media, 20 µg of plasmid DNA containing the IL-1
promoter construct were added with 20 µg of
-galactosidase reference plasmid to 107 cells in
1 ml of medium and the medium was transferred to two 0.4-cm
cuvettes. After a single 250-V, 960-µF pulse, the cells were combined into a 60-mm dish containing 5 ml of complete medium. At
least 24 h later, the medium was changed, and LPS,
LPS-L-NAME, or
LPS-L-NAME-SNAC was
added. Approximately 24 h later, the cells were washed
with ice-cold PBS, resuspended in 0.25 mM Tris (pH 7.8), and subjected
to three cycles of freezing and thawing. Lysates were centrifuged
(11,700 g for 10 min at 4°C); the
supernatant was heated at 65°C for 10 min to inactivate CAT
inhibitors and then centrifuged as described above. The supernatant was
assayed for CAT activity by a CAT ELISA technique (Boehringer
Mannheim). Transfection efficiency was normalized by cotransfection of
a
-galactosidase reporter gene with a constitutively active early simian virus 40 promoter. All values are expressed as picograms of CAT
per milliliter.
Nuclear run-on assays.
Nuclear run-on analysis was performed as previously described (14).
Briefly, 100 µl of hepatocyte nuclei were incubated for 5 min at
30°C with 150 µCi of
[-32P]rUTP (800 Ci/mmol) in 100 µl of 10 mM Tris · HCl (pH 8.0), 5 mM MgCl2, 300 mM KCl, and 5.0 mM
(each) ATP, CTP, and GTP. Labeled RNA was isolated by the
acid-guanidinium thiocyanate method. Before ethanol precipitation,
labeled RNA was treated with 0.2 M NaOH for 10 min on ice. The solution
was neutralized by the addition of HEPES (acid free) to a final
concentration of 0.24 M. After ethanol precipitation, the RNA pellet
was resuspended in 10 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (pH 7.4), 0.2%
SDS, and 10 mM EDTA. Target DNA was spotted onto nylon membranes with a
slot blot apparatus. Target DNA for murine IL-1
was amplified by PCR
based on the published sequence (1,800 bp;
1021 to
2821; GenBank X04964).
-Actin and
-bacteriophage DNA served as positive
and negative controls, respectively. Hybridization was performed at
42°C for 48 h with 5 × 106 cpm of labeled RNA in
hybridization buffer [50% formamide, 4× sodium
chloride-sodium citrate (SSC), 0.1% SDS, 5× Denhardt's solution, 0.1 M sodium phosphate (pH 7.2), and 10 µg/ml salmon sperm
DNA]. After hybridization, the membranes were
washed twice at room temperature in 2× SSC and 0.1% SDS, and
three times at 56°C in 0.1× SSC and 0.1% SDS. The membranes
were then exposed to X-ray film and scanned on a laser densitometer.
Statistical analysis. All values are presented as means ± SD of three or four experiments. Data were analyzed by one-way ANOVA, Student's t-test, or the Wilcoxon rank sum test, as appropriate. P values < 0.05 were considered significant.
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RESULTS |
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The NO dose-response relationship with concentrations of LPS
ranging from 1 to 1,000 ng/ml is depicted as a
semilogarithmic plot in Fig.
1A.
Increasing concentrations of LPS were associated with significantly
increased NO production (P = 0.0001 by
ANOVA). When L-NAME (100 µM)
was added with LPS, NO production at all concentrations of LPS was
ablated to a level that was no different from that noted in the absence
of LPS, 22.3 ± 2.1 nmol/mg protein (P < 0.01 for LPS vs.
LPS-L-NAME). The addition of SOD
did not alter NO synthesis in a significant fashion. The concentration of IL-1 in the cell culture medium was then determined (Fig. 1B). In the absence of LPS, the
IL-1
concentration was 1.5 ± 3.2 pg/mg protein. Logarithmically
increasing concentrations of LPS (1-1,000 ng/ml) were associated
with significant increases in IL-1
production
(P = 0.0001 by ANOVA). In the presence
of L-NAME (100 µM),
LPS-induced IL-1
synthesis was increased two- to threefold
(P < 0.01). NO was then made replete
by the addition of the S-nitrosothiol
SNAC (100 µM). In
LPS-L-NAME-treated cells, the
addition of SNAC restored IL-1
production to levels that were not
different from that noted with LPS alone. Similarly, the addition of
SNAC to LPS-treated cells did not alter the extent of IL-1
synthesis. IL-1
production in the
LPS-L-NAME-SOD-treated cells was
not altered. These data indicate that inhibition of NO production
increases IL-1
levels in cell culture media after LPS stimulation.
The effect of LPS-mediated NO synthesis on TNF-
was then analyzed
(Fig. 1C). In the absence of LPS,
TNF-
levels in the culture medium were 6.7 ± 10.1 pg/mg protein.
The addition of LPS (1 ng/ml) resulted in a dramatic increase in
TNF-
production. Subsequent concentrations of LPS (10, 100, and
1,000 ng/ml) were associated with incremental but significant increases
in TNF-
levels (P = 0.01 by ANOVA).
The addition of L-NAME (100 µM), SOD (100 U/ml), and/or SNAC (100 µM) with LPS did not alter
the TNF-
production profile, indicating that NO synthesis does not
alter the extent of TNF-
synthesis in ANA-1 murine macrophages.
Subsequent studies were performed at an LPS concentration of 100 ng/ml.
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Immunoblot analysis was performed on cellular protein extracts (Fig.
2). TNF- protein expression in cellular
extracts reflected that seen in the cell culture medium (data not
shown). In the absence of LPS, no immunoreactive protein was detected.
In contrast, in LPS-,
LPS-L-NAME-, and
LPS-L-NAME-SNAC-treated
macrophages, immunoreactive TNF-
protein was readily detected.
However, the presence or absence of NO, whether endogenous or exogenous
in origin, did not alter the amount of TNF-
detected. In contrast, IL-1
protein expression was noted to vary significantly with NO
production. In the absence of LPS (and NO), no IL-1
immunoreactive protein was detected. In LPS-treated cells, IL-1
was detected. In
the presence of both LPS and
L-NAME (100 µM), the amount of IL-1
synthesized was increased by approximately three- to fourfold over that found in the presence of LPS alone
(P < 0.01). When NO was made replete
in LPS-L-NAME-treated cells by
the addition of SNAC (100 µM), IL-1
production decreased to a
level that was equivalent to that of LPS-treated cells. When SNAC (100 µM) was added to LPS-treated cells, the immunoreactive IL-1
protein level did not change, reflecting that noted in the cell culture
medium (data not shown). These data corroborate the cell culture medium findings regarding the influence of NO on IL-1
protein production. An immunoblot analysis of iNOS protein expression was performed under
these conditions. iNOS protein was detected in the LPS
cells. Inhibition of iNOS by the addition of
L-NAME resulted in a 35% increase in iNOS protein expression (P < 0.01 vs. LPS or
LPS-L-NAME-SNAC). Again, the
addition of SNAC to LPS-treated cells returned iNOS protein expression
to a level equivalent to that for LPS treatment alone.
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Steady-state levels of IL-1 mRNA were then analyzed
semiquantitatively by RT-PCR (Fig. 3). In
control cells, no iNOS or IL-1
mRNA was detected. In the presence of
LPS, iNOS and IL-1
mRNA was expressed. Inhibition of NO synthesis by
the addition of L-NAME (100 µM) was associated with a four- to fivefold increase in the normalized density of the IL-1
mRNA band
(P < 0.01 vs. LPS alone) and a two-
to threefold increase in the iNOS density
(P < 0.01 vs. LPS alone). When NO
was made replete in
LPS-L-NAME-treated cells by the
addition of SNAC (100 µM), steady-state levels of IL-1
mRNA
production decreased to a level that was equivalent to that for
LPS-treated cells. To determine the effect of NO on IL-1
gene
transcription, nuclear run-on studies were performed (Fig.
4). Inhibition of NO synthesis by the
addition of L-NAME (100 µM)
with LPS resulted in a 2.5-fold increase in IL-1
gene transcription
(P < 0.01). Again, repletion of NO
by the addition of SNAC (100 µM) to LPS-
L-NAME-treated cells was
associated with IL-1
gene transcription that was not different from
that for cells exposed to LPS alone. The addition of SNAC to
LPS-treated cells did not alter the extent of IL-1
gene
transcription as determined by the nuclear run-on assay (data not
shown). These data indicate that NO synthesis inhibits IL-1
gene
transcription in LPS-treated macrophages.
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Transient transfection studies were performed with the murine IL-1
promoter to determine whether the effect of NO was dependent on the
IL-1
promoter region or a noncontiguous enhancer/repressor region
(Fig. 5). The IL-1
murine promoter
plasmid contains 4,093 bp of the 5'-flanking sequence containing
the entire first exon, first intron, and untranslated portion of the
second exon. This has been previously shown to have strong inducibility
by LPS (over 30-fold) (9). As a positive control for LPS induction of
NF-
B-dependent transcriptional activity, parallel experiments were
performed by transfection of a CAT plasmid containing three
multimerized NF-
B sites upstream from a TNF-
minimal promoter
(9). After exposure to LPS (100 ng/ml),
LPS-L-NAME (100 µM), LPS-SNAC
(100 µM), or LPS-L-NAME-SNAC,
levels of IL-1
promoter and NF-
B inducibility were determined by
CAT expression. Transfection of a mock plasmid containing only a CAT
reporter revealed no detectable CAT protein in the presence of LPS
stimulation. Full-length IL-1
promoter transfection was associated
with minimal CAT expression under unstimulated control conditions.
Stimulation with LPS (100 ng/ml) resulted in significantly increased
CAT expression, over sixfold greater than that of the control
(P < 0.01 vs. control). Compared with that resulting from LPS treatment, CAT expression was increased an
additional fivefold in
LPS-L-NAME-treated cells
(P < 0.01 vs. LPS alone). Repletion
of NO by exogenous administration of the S-nitrosothiol SNAC decreased IL-1
promoter activity to a level that was not different from that for
LPS-treated cells. Last, IL-1
promoter activity in LPS-SNAC-treated
cells was not different from that in ANA-1 macrophages treated with LPS
alone. Results from the parallel experiments with the NF-
B long
terminal repeat (LTR) CAT reporter plasmid mirrored those
for the IL-1
promoter. Inhibition of LPS-mediated NO synthesis with
L-NAME significantly increased
NF-
B LTR activity. This activity was decreased to baseline levels
with repletion of NO in
LPS-L-NAME-SNAC-treated cells. These data suggest that inhibition of NO synthesis increases
LPS-induced IL-1
promoter activation and NF-
B LTR activation.
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To determine whether the effect of NO occurs at the level of NF-B
DNA binding, gel shift analysis was performed with ANA-1 macrophages
(Fig. 6). In certain instances, DTT (20 mM)
was added to the nuclear extracts to reduce any potential sulfur-NO
(S-NO) bonds. In control cells, no NF-
B DNA binding was found. The
addition of LPS (100 ng/ml) resulted in NF-
B DNA binding, which was
confirmed in supershift studies with a MAb to NF-
B
p50. NF-
B binding in LPS-L-NAME was significantly
increased over that found in LPS-treated cells; the further addition of
SNAC to LPS-L-NAME-treated cells restored NF-
B DNA binding to a level equivalent to that for
LPS-treated cells. The addition of the reducing agent DTT (20 mM) to
LPS-treated cells significantly augmented NF-
B binding to its
consensus DNA binding element. These data indicate that inhibition of
NO synthesis significantly increases NF-
B DNA binding. The addition
of DTT in the setting of NO synthesis augments NF-
B binding,
suggesting that inhibition of NF-
B DNA binding may be the result of
S-nitrosation of NF-
B, as we and
others have previously demonstrated.(6, 7, 16)
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DISCUSSION |
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In this study, we hypothesized that LPS-mediated NO production inhibits
production of the NF-B-dependent protein IL-1
. Our results
indicate that inhibition of NO production in the setting of LPS
stimulation 1) increases IL-1
protein expression in the cell culture supernatant and cell lysates,
2) increases steady-state levels of
IL-1
mRNA, 3) augments IL-1
gene transcription, 4) increases
IL-1
promoter activity, and 5)
promotes NF-
B DNA binding. The addition of the
S-nitrosothiol SNAC as an exogenous NO
donor restores IL-1
protein expression, gene transcription, and
NF-
B DNA binding to levels noted with LPS alone. Finally, gel shift analysis showed that NF-
B DNA binding in LPS-treated cells is significantly increased in the presence of the reducing agent DTT. This
would suggest that S-nitrosation of
NF-
B with resulting inhibition of DNA binding is a plausible
mechanism underlying decreased expression of the NF-
B-dependent
protein IL-1
. Given the central position of NF-
B in
proinflammatory gene transcription, S-nitrosation-associated inhibition of
NF-
B-dependent promoter activity suggests a potential role for NO as
a feedback inhibitor of specific components in the inflammatory
process, such as IL-1
.
We and others have previously demonstrated that ex vivo biochemical
modification of NF-B p50 inhibits DNA binding as determined by the
gel shift assay. In particular, Matthews and co-workers (16) showed
that NO donors inhibit the DNA binding activity of p50 and that a p50
mutant with a serine substitution (C62S) was significantly resistant to
DNA binding inhibition; they also showed, by electron spray ionization
mass spectrometry, that the C62 residue in p50 was
S-nitrosylated by NO. We characterized the biochemical kinetic effects of p50
S-nitrosation and found that NO
decreased the dissociation constant by fourfold, from 1.0 × 1010
M
1 to 0.29 × 1010
M
1 (6). Using the ANA-1
macrophage model, we have subsequently demonstrated that inhibition of
the endotoxin-mediated synthesis of NO enhances NF-
B p50 DNA binding
without altering NF-
B activation and nuclear translocation (7).
Immunoprecipitation studies confirm the presence of S-NO bonds in p50
isolated from endotoxin-stimulated cells and, conversely, the absence
of S-NO bonds in the presence of the iNOS inhibitor
L-NAME. The p50 isolated in this
fashion retains functional DNA binding properties, mirroring that found in our ex vivo kinetic studies. Transcription of the NF-
B-dependent genes for iNOS and macrophage colony-stimulating factor (M-CSF) was
enhanced in the setting of NO inhibition. In total, these results
strongly suggest a role for
S-nitrosation as a specific modulator
of NF-
B DNA binding, with its associated effects on NF-
B-dependent gene transcription. In this study, using ANA-1 macrophages we demonstrate that NO alters IL-1
gene transcription and, ultimately, IL-1
protein expression.
These observations correlate well with others relating NO synthesis and
the functional results of NF-B inhibition. NF-
B activation is a
prerequisite for the transcription of a variety of proinflammatory
mediators and markers, including IL-1
, iNOS, M-CSF, TNF-
, and
vascular cell adhesion molecule (VCAM) (21, 25, 26). In particular, a
number of previous studies have documented the regulatory interplay
between NO and IL-1
synthesis in a variety of experimental models
(4, 12, 13, 17, 18, 24, 29). Inhibition of NO synthesis is associated
with increased IL-1
protein expression. Until recently, an
underlying mechanism has not been elucidated. In models of macrophage
synthesis of NO, Kim and colleagues (12) have demonstrated that NO
suppresses IL-1
protein processing by inhibition of caspase-1, a key
enzyme required for IL-1
maturation and release. The inhibition of
caspase-1 activity is reversed in the presence of DTT. This, in
combination with previous work by the same group, indicates
S-nitrosation of caspase proteases as
a potential mechanism (11). These results notwithstanding, Kim and
colleagues also demonstrate that inhibition of NO synthesis is
associated with 1) increased IL-1
and TNF-
mRNA expression and 2)
increased IL-1
protein expression but unchanged TNF-
protein
expression. Our findings certainly corroborate the
results of Kim and coworkers and indicate that
S-nitrosation may regulate the
activity of key regulatory proteins involved at multiple levels of
protein synthesis, including transcription and posttranslational processing.
Previous work has demonstrated that
S-nitrosation of NF-B p50 is
associated with decreased gene transcription and synthesis of the
NF-
B-dependent proteins iNOS and M-CSF (7). However, the effects of
S-nitrosation are not generalized to
all NF-
B-dependent proteins, as evidenced by our observation that
LPS-stimulated TNF-
elaboration is not altered by inhibition of NO
synthesis. Posttranscriptional regulatory mechanisms may obviate the
effects of transcriptional inhibition.
Although TNF- mRNA levels are increased in the presence of NO
inhibition, as shown by Kim et al. (12), additional posttranscriptional mechanisms in the macrophage may obviate these effects. In contrast, other investigators have demonstrated that inhibition of NO synthesis is associated with increased expression of both IL-1
and TNF-
proteins (17, 18, 29). Although these differences in the effect of NO
on TNF-
biology may be the result of differences in the experimental
models, inhibition of IL-1
synthesis by NO is a recurring theme in
the work of many groups. The results from the present study suggest
that NF-
B DNA binding properties, IL-1
gene transcription, and
IL-1
protein expression are significantly decreased in the presence
of NO. The underlying mechanism is consistent with our previous
observations concerning S-nitrosation
of NF-
B and its effects on DNA binding and gene transcription (7).
As a result of its participation in redox chemistry, NO is a
pluripotent regulator of multiple cellular functions (19). The
formation of S-nitrosothiols
exemplifies these pathways of NO oxidation, which lead to surrogate
NO-like bioactivity and result in allosteric receptor modification,
inhibition of sulfhydryl enzyme activities, and downregulation of
transcriptional activators. With respect to NF-B activity, studies
utilizing exogenous NO donors have shown that NF-
B DNA binding is
inhibited (3, 21, 22). In a system of human vascular endothelial cells,
Peng and colleagues (21, 22) showed that exogenous NO
stabilized NF-
B by inhibiting the dissociation of the I
B
inhibitor while simultaneously increasing I
B mRNA levels. These
investigators also found that NO minimally altered activation of
nuclear binding proteins AP-1, GATA-2, and GATA-3 (21,
22). Recently, Calmels and colleagues (3) also
demonstrated NO-mediated inhibition of p53 DNA binding resulting from
NO-induced conformational and functional modifications, suggesting
S-nitrosation, given the absence of an
I
B correlate for p53. Of added interest is the relative specificity
of S-nitrosation for NF-
B- and,
perhaps, p53-associated activity. Nuclear proteins, such as IRF, GATA,
AP-1, oct-2, and STAT, do not exhibit NO-induced alteration in activity
(6, 21, 22). Future identification of a pool of NO-modulated nuclear
protein transcription factors would allow elucidation of the pathways
of NO-mediated transcriptional regulation.
These results indicate that NO may play a direct regulatory role in the
inflammatory response. Our study suggests a novel mechanism in which NO
production feedback inhibits IL-1 gene transcription in the context
of LPS stimulation. Although initial studies in the field suggested
that iNOS expression and NO production were associated with deleterious
biological effects, more recent studies have elucidated a feedback
inhibitory role for iNOS and NO in the elaboration of IL-1
, IFN-
inducing factor, IL-6, and VCAM-1 (12, 23, 24). Under certain
inflammatory states, we propose that NO may function to inhibit by
feedback the expression of specific NF-
B-dependent proteins in an
autoregulatory inducible fashion.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. C. Kuo, Dept. of Surgery, Georgetown Univ. Medical Center, 4 PHC; 3800 Reservoir Rd. NW, Washington, DC 20007 (E-mail: kuop{at}gunet.georgetown.edu).
Received 11 January 1999; accepted in final form 9 May 1999.
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