 |
INTRODUCTION |
Inducible expression of a variety of genes is mediated through
mitogen-activated protein
(MAP)1 kinase signaling
pathways. MAP kinases regulate the phosphorylation and activation of
transcription factors, including c-Jun, activating transcription
factor-2, and Elk-1. The activation of MAP kinase pathways is
critical in the induced expression of inflammatory cytokines. For
example, MAP kinases regulate the transcriptional expression of the
genes encoding tumor necrosis factor-
, and interferon-
(IFN-
)
(1-3). In addition, MAP kinase pathways have been demonstrated to
contribute to the activation of the ubiquitous transcription factor,
nuclear factor-
B (NF-
B) (4, 5). The NF-
B pathway also has a
critical role in the immune system, regulating both innate and adaptive
responses. NF-
B is essential for the inducible expression of
cytokines, receptors for immune recognition, cell adhesion proteins,
and the genes encoding inducible nitric-oxide synthase (iNOS) and
cyclooxygenase-2 (6).
Although NF-
B is comprised of a family of homologous DNA-binding
proteins, it is prototypically represented as a heterodimer of p50 and
p65 (RelA) subunits. Each subunit contains a conserved Rel homology
domain, which encompasses the dimerization and DNA-binding motifs, and
a nuclear localization signal (7, 8). Transcriptional enhancement is
conferred by the transactivation domain of NF-
B, which is encoded at
the C-terminal end of the p65 subunit (9). In nonstimulated cells,
NF-
B is retained in the cytoplasm in an inactive complex through
interactions with the inhibitory protein I
B (10, 11). NF-
B can be
activated by a number of immunological stimuli, including the bacterial
product, lipopolysaccharide (LPS), cytokines, and double-stranded RNA.
Treatment with an appropriate stimulus induces rapid phosphorylation of
I
B on two specific serine residues (Ser-32 and Ser-36 for I
B-
)
(12-14). Subsequent ubiquitination of I
B and degradation by the 26 S proteasome releases the inhibition on NF-
B, which then
translocates to the nucleus and activates transcription of target genes
(15-18).
Transcriptional expression of IFN-
is regulated by an enhancer
region located 55-105 bp upstream of the transcription start site
(+1), and which is highly conserved between human and mouse (19).
Induction of IFN-
mRNA does not require de novo
protein synthesis, but depends on existing transcription factors, which assemble into an enhanceosome complex at specific positive regulatory domains within the IFN-
promoter. The activation of NF-
B and MAP
kinase signaling pathways are critical for the formation of the IFN-
enhanceosome, because NF-
B and a heterodimer of c-Jun/activating transcription factor-2 bind to positive regulatory domains II and IV,
respectively (20-23).
Nitric oxide (NO) is an endogenously synthesized free radical species
that has been demonstrated to modulate a variety of cellular and
physiological processes. Elevated levels of NO, as are generated by
macrophages at sites of inflammation, infection, or during septic shock
can potentially lead to nitrosative stress. The effects of nitrosative
stress relate to the ability of NO and NO-derived species to react with
biological molecules and thereby alter normal cellular functions (24).
Alterations in gene expression have been shown to occur in cells
subjected to nitrosative stress, because of both direct and indirect
effects on various signaling pathways and transcription factors (25, 26). In this study we investigate the effect of NO on NF-
B activation and MAP kinase signaling pathways, and demonstrate both
time- and concentration-dependent effects. Furthermore, we correlate the effects of NO on signal transduction pathways to the
transcriptional expression of two LPS-inducible genes, IFN-
and
I
B-
. In conclusion, we propose a model for the actions of NO in
enhancing and propagating pathological inflammatory conditions and
mediating immune responses.
 |
MATERIALS AND METHODS |
Reagents and Tissue Culture--
RAW 264.7 macrophages were
obtained from American Type Culture Collection and grown in 10-cm
culture dishes containing Dulbecco's modified Eagle's medium
(Mediatech) supplemented with 50 mM HEPES, 4 mM
L-glutamine, 1.2 mM L-arginine, 10 IU/ml penicillin, 10 µg/ml streptomycin, 250 ng/ml amphotericin B,
and 10% fetal bovine serum. Cells were grown to confluence, then split
using 0.05% trypsin, 0.53 mM EDTA and plated at a
density of 3 × 106 cells per 10-cm dish for
propagation, or as indicated for experiments. Cells were counted using
a hemacytometer, and viability assayed using trypan blue dye exclusion
was typically greater than 95%. Cultures were maintained until passage
20, and then discarded. LPS (Escherichia coli serotype
0128:B12, Sigma) was reconstituted in sterile water and added to obtain
the desired concentration for experiments. The nitric oxide donors,
diethylammonium
(Z)-1-(N,N'-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO; Cayman Chemical) and
(Z)-1-[N-methyl-N-[6-(N-methylaminohexyl)amino]]diazen-1-ium-1,2-diolate (MAHMA/NO; Cayman Chemical) were dissolved in slightly basic (pH 10.0),
ice-cold sterile water immediately prior to addition to cell culture.
The compound
2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxyl-3-oxide (carboxy-PTIO; Cayman Chemical) was dissolved in water immediately prior to use. Both DEA/NO and MAHMA/NO were examined for their effect
on macrophage viability using the trypan blue dye exclusion assay, and
each donor was determined to be negligible under the conditions
used in the experiments.
Protein Extraction--
Following treatment with experimental
agents, cells were washed with cold phosphate-buffered saline, scraped,
and collected by centrifugation. For total proteins, cell pellets were
resuspended in lysis buffer (10 mM Tris, pH 7.05, 50 mM NaCl, 30 mM sodium pyrophosphate, 2 mM EDTA, 50 mM NaF, 1% (v/v) Triton X-100; 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotonin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A). Samples were incubated on
ice for 15 min then centrifuged at 14,000 × g for 15 min at 4 °C to remove debris, and supernatant was stored at
80 °C. Protein concentrations were determined by the Bio-Rad
protein assay, and samples were diluted to 2 µg/µl. Nuclear
extracts were prepared as previously described, with slight
modifications. Briefly, fresh cell pellets were resuspended in 800 µl
of ice-cold buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA,
0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride) and 50 µl of Igepal CA-630 was added. Nuclei were pelleted by centrifugation at 1000 × g for 5 min at 4 °C,
and washed twice with buffer A. Nuclear proteins were extracted by
addition of buffer B (10 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 25% glycerol, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride),
and centrifuged at 14,000 × g for 10 min at 4 °C to remove debris. Nuclear protein concentrations were determined by the
Bio-Rad protein assay, and samples were diluted to 1 µg/µl.
Protein Immunoblots--
An equal amount of protein per sample
(as indicated in figure legends) was resolved by polyacrylamide gel
electrophoresis, and electroblotted onto a 0.2-µm nitrocellulose
membrane using a Mini Trans-Blot apparatus (Bio-Rad). Membranes were
incubated with blocking buffer (20 mM Tris, pH 7.6, 140 mM NaCl, 0.05% Tween 20, 5% nonfat dry milk) prior to
incubation with primary antibody diluted in blocking buffer. After
washing 3 times in Tris-buffered saline containing 0.1% Tween 20, membranes were incubated with the appropriate horseradish
peroxidase-conjugated secondary antibody. Membranes were subsequently
washed 3 times in Tris-buffered saline containing 0.1% Tween 20, and a
chemiluminescence-based detection with luminol reagent was performed
according to the manufacturer's protocol (Santa Cruz Biotechnology,
Inc). Antibodies were obtained from the following sources: anti-p38,
anti-phospho-p38, anti-JNK, anti-phospho-JNK, and anti-I
B-
from
Cell Signaling Technology; anti-p50/p105, anti-p65, anti-actin, and all
secondary antibodies were purchased from Santa Cruz Biotechnology, Inc.
Electrophoretic Mobility Shift Assay (EMSA)--
Double
stranded oligonucleotide encompassing the
B element
5'-GGGGATTTCCC-3' (Santa Cruz Biotechnology, Inc.) was
end-labeled using [
-32P]ATP, 7,000 Ci/mmol (ICN), and
T4 polynucleotide kinase (Promega). Assays were performed according to
an established method, with slight variations (27). Briefly, 5 µg of
nuclear protein extract was incubated for 30 min at room temperature in
binding buffer (25 mM HEPES, pH 7.9, 100 mM
KCl, 5% Ficoll-400, 2% glycerol, 0.025% Igepal CA-630, 0.05 mM EDTA, 0.05 mM EGTA, 2 mM DTT,
0.5 mM phenylmethylsulfonyl fluoride) containing 10 µg of
bovine serum albumin and 1 µg of double-stranded poly(dI·dC).
100,000 cpm of radiolabeled consensus oligonucleotide (~0.1 pmol) was
subsequently added to each sample, and incubated at room temperature
for an additional 30 min. Samples labeled cold competitor indicate the addition of a 100-fold excess of unlabeled
B oligonucleotide. Protein-DNA complexes were subsequently resolved in a 5% native Tris/taurine-buffered gel. Gels were dried and exposed to
autoradiographic film at
80 °C.
Ribonuclease Protection Assay--
Following treatment with
experimental agents, cells were washed with cold phosphate-buffered
saline, harvested using a cell scraper and collected by centrifugation.
Total RNA was extracted using the RNeasy Mini kit (Qiagen). RNA was
quantified by absorbance at 260 and 280 nm. 15 µg of RNA was dried
using a Speed-Vac (Savant) and stored at
80 °C. Radiolabeled
antisense RNA was synthesized using the Riboprobe transcription kit
(Promega) and [
-32P]CTP, 800 Ci/mmol (ICN). The
antisense templates for murine IFN-
and I
B-
were constructed
using a reverse transcriptase-PCR method. Briefly, total RNA collected
from LPS-treated RAW 264.7 macrophages was reverse transcribed using
the SuperScript preamplification system (Invitrogen). First strand
cDNA was subsequently amplified by PCR using the puReTaq
Ready-To-Go PCR system (Amersham Biosciences). For
IFN-
, the primers 5'-AAACAATTTCTCCAGCACTG-3' and
5'-ATTCTGAGGCATCAACTGAC-3' were used, and for I
B-
the primers
5'-TGGCCTTCCTCAACTTCC-3' and 5'-CTGCGTCAAGACTGCTACAC-3' were
used. The resulting PCR products were annealed into the pGEM-T Easy
vector (Promega), amplified in competent E. coli, and
linearized using the restriction enzymes NcoI and
SpeI, respectively. The probe for IFN-
yields a protected fragment of 312 nucleotides, and the probe for I
B-
yields a protected fragment of 194 nucleotides. The template for murine
-actin was purchased from Ambion and yields a protected fragment of
245 nucleotides. Ribonuclease protection assay was performed using the
RPA-III kit (Ambion), but substituting RNase ONE (Promega) for the
supplied RNases. Protected fragments were resolved using a 5%
polyacrylamide:bisacrylamide (19:1) 25 mM Tris borate, 2.5 mM EDTA, 8 M urea gel. Gels were dried, and
exposed to both autoradiographic film and a PhosphorImager cassette
(Amersham Biosciences). The radiographic intensities of
individual bands on the gels were determined using the program
ImageQuant (Amersham Biosciences).
 |
RESULTS |
Macrophages Treated with Nitric Oxide Exhibit LPS-independent
Phosphorylation of p38 and JNK MAP Kinases--
Treatment of RAW 264.7 macrophages LPS (100 ng/ml) induced a rapid phosphorylation of p38 MAP
kinase on amino acid residues threonine 180 (Thr-180) and tyrosine 182 (Tyr-182). Phosphorylation of JNK (p46 and p54) was also induced on
amino acid residues threonine 183 (Thr-183) and tyrosine 185 (Tyr-185)
(Fig. 1). Nonstimulated macrophages did
not have detectable levels of phosphorylated p38 or JNK. At 15 min
after LPS addition, phosphorylation of both p38 and JNK was noticeably
up-regulated. However, by 45 min levels of phospho-p38 and phospho-JNK
had decreased significantly, and by 75 min had returned to an almost
undetectable level, as in nonstimulated cells.

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Fig. 1.
DEA/NO stimulates phosphorylation of p38 and
JNK MAP kinases. A, RAW 264.7 macrophages were
untreated, treated with DEA/NO (1 mM) ± LPS (100 ng/ml), or treated with decomposed DEA/NO (D, 1 mM) as indicated. Total protein extracts were made at the
indicated times after treatment and 20 µg of total protein was
resolved by polyacrylamide gel electrophoresis. Nitrocellulose blots
were probed with antibodies specific for p38 or
phospho-(Thr-180/Tyr-182)-p38 (P-p38). B,
nitrocellulose blots were probed with antibodies specific for JNK
(p46/p54) or phospho-(Thr-183/Tyr-185)-JNK (P-p46/p54). The
immunoblots shown are representative of three independent
experiments.
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Simultaneous treatment of macrophages with LPS and the NO donor,
DEA/NO, significantly enhanced phosphorylation of both p38 and JNK. In
contrast to macrophages treated with LPS alone, when treated with LPS
plus DEA/NO, the levels of phosphorylated p38 and JNK remained elevated
at the 45- and 75-min time points after addition. This is despite our
calculations that the NO donor was fully degraded at these time points,
because of its relatively short half-life (~2 min at 37 °C, pH
7.4).
The effect of adding DEA/NO alone on p38 and JNK phosphorylation was
also investigated. DEA/NO was capable of inducing a rapid and sustained
phosphorylation of both MAP kinases, which was significantly stronger
and of greater duration than in cells treated with LPS alone. As a
control, the products of DEA/NO decomposition were tested for their
ability to activate p38 and JNK phosphorylation. In contrast to freshly
prepared DEA/NO, decomposed DEA/NO (obtained by allowing DEA/NO to
decay in 0.1 M Tris, pH 7.4, at 25 °C for several days),
which contains mostly nitrite and diethylamine, had no observable
effect on either p38 or JNK phosphorylation.
Nitric Oxide Induces a Sustained Degradation of I
B-
in
LPS-stimulated Macrophages--
MAP kinase pathways have been
demonstrated to contribute to the activation of NF-
B. Therefore, the
effect of DEA/NO on the degradation of the NF-
B inhibitory protein,
I
B-
, was investigated. Accordingly, RAW macrophages were treated
with LPS (100 ng/ml), DEA/NO (1 mM), or LPS plus DEA/NO for
various amounts of time, and total protein samples were analyzed by
immunoblot for I
B-
expression. Treatment of macrophages with LPS
alone induced a rapid degradation of I
B-
(Fig.
2). A total loss of detectable I
B-
protein was observed 15 min after LPS addition, but by 45 min the
levels of I
B-
had recovered to the basal level. In contrast, when
macrophages were treated simultaneously with a combination of LPS (100 ng/ml) and DEA/NO (1 mM), I
B-
levels remained low at
45 and 75 min after treatment. The ability of DEA/NO to inhibit the
restoration of I
B-
levels in LPS-stimulated macrophages was
both concentration- and time-dependent. Higher
concentrations of DEA/NO (100 µM to 1 mM)
were most effective at inhibiting I
B-
levels, and at maintaining
this inhibition at later time points (Fig.
3). Because DEA/NO alone was capable of
inducing p38 and JNK phosphorylation, it was hypothesized that DEA/NO
might also induce I
B-
degradation in the absence of LPS. However,
I
B-
levels in cells not treated with LPS were unaffected by the
addition of DEA/NO, indicating that NO alone and the resulting
activation of p38 and JNK pathways is insufficient to cause NF-
B
activation (data not shown).

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Fig. 2.
DEA/NO enhances LPS-induced degradation of
I B- . RAW 264.7 macrophages
were untreated or treated with DEA/NO (1 mM), ± LPS (100 ng/ml) as indicated. 20 µg of total protein per sample was resolved
by polyacrylamide gel electrophoresis. Nitrocellulose blots were probed
with antibodies specific for I B- or actin (loading control). The
immunoblots shown are representative of three independent
experiments.
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Fig. 3.
Concentration-dependent
enhancement of LPS-induced degradation of
I B- by DEA/NO.
RAW 264.7 macrophages were untreated, treated with LPS (100 ng/ml) or
LPS + DEA/NO (100 µM to 1 mM) for 45 (A) min, or 75 min (B). 20 µg of total protein
per sample was resolved by polyacrylamide gel electrophoresis.
Nitrocellulose blots were probed with antibodies specific for I B-
or actin (loading control). The immunoblots shown are representative of
three independent experiments.
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Nitric Oxide Stimulates Nuclear Accumulation of p50 and p65 and
Enhances p105 Turnover--
Activation of the transcription factor
NF-
B is regulated by the degradation of the inhibitor, I
B, and
the resulting translocation of the p50/p65 heterodimer into the
nucleus. Nuclear p50/p65 then mediates the transcription of target
genes. Because DEA/NO enhanced the magnitude and duration of
LPS-induced I
B-
degradation, we decided to examine the effect of
DEA/NO on LPS-induced NF-
B translocation. A modest level of NF-
B
exists in the nucleus of nonstimulated RAW 264.7 macrophages. When
treated with LPS (100 ng/ml) for 75 min, the nuclear levels of p50 and
p65 were moderately increased. However, when treated with a combination
of LPS and DEA/NO (100 µM to 1 mM), the
nuclear levels of p50 and p65 increased in a concentration-dependent manner (Fig.
4).

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Fig. 4.
DEA/NO enhances LPS-stimulated nuclear
expression of NF- B. RAW 264.7 macrophages
were untreated, treated with LPS (100 ng/ml) or LPS + DEA/NO (100 µM to 1 mM) for 75 min. Nuclear extracts were
made and 10 µg of protein was resolved by polyacrylamide gel
electrophoresis. Nitrocellulose blots were probed with antibodies
specific for p50/p105, p65, or actin, which was used as a loading
control. The immunoblots shown are representative of four individual
experiments.
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The activity of NF-
B in the nucleus is also regulated by the
inhibitory actions of I
B, which has been reported to enter the
nucleus and down-regulate NF-
B-mediated gene expression (28). Therefore, the effect of LPS and DEA/NO on nuclear I
B-
expression was also investigated. Whereas nuclear I
B-
expression by
immunoblot analysis could not be detected, significant levels of the
p50 precursor protein, p105 were observed. Like I
B, p105 is able to
repress NF-
B through its inhibitory associations with the transcription factor, masking the nuclear translocation and DNA-binding domains (29). LPS alone did not cause a significant reduction in p105
levels at 45 min following treatment (Fig. 4). However, simultaneous
addition of LPS and DEA/NO induced a marked,
concentration-dependent decrease in nuclear p105
expression, suggesting that less nuclear NF-
B is in an inactive
state, and able to promote the transcription of target genes.
Other investigators have reported that NO may inhibit DNA
binding of NF-
B because of the formation of an
S-nitrosothiol on residue Cys-62 of the p50 subunit.
Therefore, nuclear protein extracts from DEA/NO-treated macrophages
were tested for their ability to bind to a
B oligonucleotide. As
determined by EMSA, a dramatic increase in NF-
B binding was seen at
higher DEA/NO concentrations (Fig. 5),
consistent with the enhanced p50 and p65 levels in the nucleus (Fig.
4). Nuclear extracts from macrophages were also treated in
vitro with DEA/NO to verify that NO could inhibit the DNA binding
of NF-
B, which has been reported by other investigators (30, 31).
Nuclear protein extracts from LPS-induced macrophages, or recombinant
human p50 were treated with DEA/NO (1 mM final
concentration) and analyzed by EMSA in the presence or absence of DTT
(1 mM). DTT was added 1 h subsequent to the NO donor
to limit the production of HNO from the reaction of NO with DTT.
Consistent with the observations in these earlier reports, the addition
of NO to NF-
B in the absence of reducing thiols completely inhibited
NF-
B binding to DNA (data not shown). When DTT was added to the
reaction after DEA/NO, the inhibition was reversed, and the full level
of DNA binding was restored. The results of this experiment are
consistent with results from the exposure of NO to cultured cells (Fig.
5). Thus, the reason that the inhibition of NF-
B was not observed
when cultured cells were treated with DEA/NO is likely because of the
high intracellular thiol content, which limits
S-nitrosation.

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Fig. 5.
DEA/NO enhances LPS-stimulated
NF- B activation. RAW 264.7 macrophages
were untreated, treated with LPS (100 ng/ml), or LPS plus DEA/NO (100 µM to 1 mM) for 75 min. Nuclear extracts were
made and 5 µg of nuclear protein was assayed by electrophoretic
mobility shift assay for binding to a consensus B oligonucleotide.
Preincubation with unlabeled, cold oligo (C) significantly
attenuated binding. Results are representative of five independent
experiments.
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The increase in NF-
B activation caused by DEA/NO was significantly
greater at higher concentrations (500 µM to 1 mM). Therefore, we hypothesized that the effect on NF-
B
was related to nitrosation reactions associated with the generation of
NO2 and N2O3, which is facilitated
by high NO levels. To examine this possibility, the compound
carboxy-PTIO was used. In combination with NO added in the form of
DEA/NO, carboxy-PTIO can be utilized to generate NO2 and
N2O3 (32). Thus, carboxy-PTIO enhances the
nitrosative reactions caused by the addition of DEA/NO. Accordingly,
the addition of DEA/NO (1 mM) alone caused a significant
increase in NF-
B binding, but the simultaneous addition of DEA/NO
and carboxy-PTIO (100 µM) caused a further increase in
NF-
B (Fig. 6). Therefore, we speculate
that the biochemical mechanism by which NO increases NF-
B activation
is likely correlated with a nitrosation event.

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Fig. 6.
Carboxy-PTIO potentiates the DEA/NO-enhanced
activation of NF- B. RAW 264.7 macrophages
were untreated, or treated with LPS (100 ng/ml). DEA/NO (1 mM) and carboxy-PTIO (c-PTIO, 100 µM) were added as indicated. Nuclear extracts were made
after 75 min, and 5 µg of nuclear protein was assayed by
electrophoretic mobility shift assay for binding to a consensus B
oligonucleotide. Preincubation with unlabeled, cold oligo
(C) significantly attenuated binding. Results are
representative of three independent experiments.
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Nitric Oxide Enhances the Transcriptional Expression of IFN-
and
I
B-
--
NF-
B and the MAP kinase-activated transcription
factors c-Jun and activating transcription factor-2 are part of an
enhanceosome complex that is responsible for the induction of IFN-
in LPS-stimulated or virus-challenged cells. Because DEA/NO enhanced
NF-
B activation in LPS-treated macrophages and stimulated
phosphorylation of p38 and JNK MAP kinases, we decided to investigate
the effect of DEA/NO on IFN-
gene expression.
Addition of LPS (100 ng/ml) induced a modest expression of IFN-
mRNA, measured 3 h later (Fig.
7). Simultaneous treatment with LPS and
DEA/NO (100 µM to 1 mM) caused a dramatic
concentration-dependent increase in IFN-
mRNA
levels. To examine the contribution of NO-derived nitrosating species
(NO2 and N2O3) to the enhanced expression of IFN-
mRNA, carboxy-PTIO (100 µM) was
added in combination with DEA/NO. Accordingly, the addition of
carboxy-PTIO caused a marked increase in IFN-
mRNA levels
compared with treatment with DEA/NO alone (Fig. 7). This data supports
the hypothesis that the stimulatory effect of DEA/NO correlates with
the nitrosation of some biochemical species, which promotes the
enhanced activation of NF-
B and transcription of NF-
B regulated
genes.

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Fig. 7.
Concentration-dependent
enhancement of IFN- mRNA levels by DEA/NO
and DEA/NO plus carboxy-PTIO. RAW 264.7 macrophages were untreated
or treated with LPS (100 ng/ml). DEA/NO (100 µM to 1 mM) and carboxy-PTIO (c-PTIO, 100 µM) were added as indicated. Total RNA was extracted
after 3 h, and 15 µg of RNA was probed for IFN- and -actin
(loading control) by ribonuclease protection assay. Relative values for
IFN- mRNA levels normalized to -actin are indicated
below the gel, and were obtained using PhosphorImager
analysis. Results are representative of five independent
experiments.
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The expression of I
B-
is also mediated by NF-
B,
evidenced by the presence of
B regulatory elements in its promoter
region (33, 34). Therefore, the effect of DEA/NO on LPS-stimulated I
B-
message expression was also examined. Similar to the effect on IFN-
, the addition of LPS (100 ng/ml) induced a modest expression of I
B-
mRNA (Fig. 8).
Furthermore, simultaneous treatment with LPS and DEA/NO (100 µM to 1 mM) caused a dramatic
concentration-dependent increase in I
B-
message
expression. However, the magnitude of the effect was not as substantial
as the increase in IFN-
expression caused by DEA/NO. We currently
speculate that the reason IFN-
expression is more enhanced by DEA/NO
than I
B-
is because of the additional contribution of MAP
kinase-stimulated transcription factors in the IFN-
promoter.

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Fig. 8.
Concentration-dependent
enhancement of I B-
mRNA levels by DEA/NO. RAW 264.7 macrophages were
untreated or treated with LPS (100 ng/ml). DEA/NO (100 µM
to 1 mM) and carboxy-PTIO (c-PTIO) were added as
indicated. Total RNA was extracted after 3 h, and 15 µg of RNA
was probed for I B- and -actin (loading control) by
ribonuclease protection assay. Relative values for I B-
mRNA levels normalized to -actin (loading control) are
indicated below the gel and were obtained using
PhosphorImager analysis. Results are representative of five
independent experiments.
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|
The NO donor, DEA/NO, decomposes spontaneously at
physiological pH to NO and other products. To confirm that our
observations were because of an effect of NO, and not other
decomposition products, an additional NO donor was compared for its
effects on IFN-
and I
B-
mRNA expression. Thus, the NO
donor, MAHMA/NO (500 µM, 1 mM) was added to
macrophages in combination with LPS (100 ng/ml), and total RNA was
extracted 3 h later. RNA was then analyzed by ribonuclease
protection assay for the levels of IFN-
and I
B-
transcripts.
The addition of MAHMA/NO caused a similar,
concentrationdependent enhancement in the expression of IFN-
and I
B-
(Fig. 9). Moreover, the
magnitude of the increase in mRNA expression was comparable with
that caused by DEA/NO under the same range of concentrations.

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Fig. 9.
Concentration-dependent
enhancement of IFN- and
I B- mRNA levels
by MAHMA/NO. RAW 264.7 macrophages were untreated or treated
with LPS (100 ng/ml). MAHMA/NO (500 µM or 1 mM) and carboxy-PTIO (c-PTIO, 100 µM) were added as indicated. Total RNA was extracted
after 3 h, and 15 µg of RNA was probed for IFN- , I B- ,
and -actin (loading control) by ribonuclease protection assay.
Relative values for IFN- and I B- mRNA levels normalized to
-actin (loading control) are indicated below the gel, and
were obtained using PhosphorImager analysis. Results are representative
of two independent experiments.
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|
We also examined the possibility that an increase in mRNA stability
was accounting for the observed elevation in IFN-
message levels.
However, the addition of actinomycin D (10 µg/ml) to inhibit mRNA
transcription indicated no differences in IFN-
mRNA lifetime between LPS-treated and LPS plus DEA/NO-treated cells (Fig.
10). This data implies that the
enhancement of IFN-
mRNA levels caused by DEA/NO is not because
of a change in message stability, but rather to an increase in the
efficiency of transcription.

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Fig. 10.
IFN- mRNA
stability is not affected by DEA/NO. RAW 264.7 macrophages were
treated with LPS (100 ng/ml), or LPS + DEA/NO (1 mM). After
3 h of activation, actinomycin D (act-D, 10 µg/ml)
was added. At various times after the addition of act-D, total RNA was
extracted and 15 µg of RNA was probed for IFN- and
-actin (loading control) by ribonuclease protection assay. Relative
values for IFN- mRNA levels normalized to -actin are
indicated in the graph below the gel, and were obtained
using PhosphorImager analysis. Results are representative of two
independent experiments.
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|
 |
DISCUSSION |
NO is an endogenous free radical species that is both a signaling
molecule and a cytotoxic agent (35, 36). The function of NO in the
immune system is principally to facilitate the killing of invading
microorganisms. However, elevated levels of NO can also contribute to
various pathological conditions, such as rheumatoid and osteoarthritis,
and inflammatory bowel disease (37-39). In this report we illustrate a
mechanism for the enhanced expression of genes by NO because of effects
on MAP kinase signaling pathways and NF-
B activation. We also
provide evidence that NO stimulates the transcriptional expression of
the gene encoding the proinflammatory cytokine, IFN-
.
MAP kinases function in nearly all aspects of the immune response,
including the initiation of innate immunity, the activation of adaptive
immunity, and in the apoptosis of immune cells (40). In the initiation
of a macrophage response to inflammation or infection, MAP
kinase-activated transcription factors are critical in triggering the
expression of genes encoding cytokines, cell adhesion proteins, and
enzymes that mediate the cytotoxic response, including iNOS (41-45).
The three main groups of MAP kinases found in mammalian cells include
extracellular signal-regulated kinases, p38 MAP kinases, and JNKs. The
p38 and JNK pathways are also referred to as the stress-activated MAP
kinase pathways because of their more potent activation by stimuli such
as inflammatory cytokines and environmental stress (46).
In this report we provide evidence for the activation of p38 MAP kinase
and JNK by the NO donor, DEA/NO. The role of NO in regulating MAP
kinases has been previously examined in various cell types and
iNOS(
/
) knockout mice. For example, treatment of Jurkat human T
lymphocytes with genuine NO or the iron-nitrosyl compound, sodium
nitroprusside (SNP, 0.1-1000 µM) was shown to rapidly
activate p38 and JNK (47). Adding SNP (1 mM) to RAW 264.7 macrophages also stimulated p38 and JNK MAP kinases (48). Activation of
p38 MAP kinase has been reported to mediate the pro-apoptotic effects
of NO in several cell types, including human PC-12 cells, rat neural
progenitor cells, and rabbit articular chondrocytes (49-51).
Activation of JNK has been demonstrated in HEK293 cells treated with
SNP (500 µM), as well as in response to nNOS activation
with the ionophore, A23187 (20 µM) (52). Also, bovine
chondrocytes exhibited an increase in JNK activity when treated with
the nitrosothiol, S-nitroso-N-acetylpenicillamine (100 µM) (53). In vivo work using iNOS(
/
)
knockout mice has demonstrated a physiological role for NO in promoting
MAP kinase activation. Accordingly, following the induction of septic
shock, T-cells and macrophages from iNOS knockouts showed a significant reduction in phospho-p38 levels relative to their wild-type
counterparts (54). However, other findings suggest differently, that NO
may suppress MAP kinase activation. Accordingly, the nitrosothiol species S-nitroso-N-acetylpenicillamine (100 µM) and S-nitrosoglutathione (100 µM) were demonstrated to selectively inhibit JNK activity through S-nitrosation of cysteine 116 (55, 56). This
suggests that NO may have multiple regulatory roles on MAP kinase
activities, depending on cell type, donor species, and concentration.
The transcription factor NF-
B has a critical role in the immune
system, regulating both innate and adaptive responses (57, 58). The
activation of NF-
B is associated with increased transcription of a
number of genes involved in immune responses, including those encoding
adhesion molecules, cytokines, and iNOS. Expression of these proteins
by macrophages enhances the migration of inflammatory cells into areas
of infection or inflammation, and contributes to the cytotoxic response.
In this paper we describe a time- and
concentration-dependent enhancement of LPS-stimulated
NF-
B activation by an NO donor. Two mechanisms for the increase in
NF-
B activation are presented. NO enhanced and prolonged the
degradation of the inhibitory protein I
B-
. The addition of NO
also stimulated a decrease in the nuclear levels of the inhibitory
protein, p105. The combination of these effects we believe contributed
to the enhanced nuclear translocation of p50/p65, and the increase in
DNA-binding activity determined by EMSA. Interestingly, DEA/NO and
MAHMA/NO also stimulated an increase in I
B-
mRNA expression,
likely because of its regulation by NF-
B. Therefore, we speculate
that the decrease in I
B-
levels caused by NO donor is not because
of an effect of NO on I
B-
transcription, but rather to a
stimulation of I
B-
protein degradation.
The consequences of NO exposure on NF-
B activation have been
investigated in several cell types under a variety of conditions. In vitro treatment of recombinant NF-
B with nitrosothiols
or acidified nitrite has been shown to inhibit DNA binding because of
nitrosation of cysteine residue 62 of the p50 subunit (30, 31, 59, 60).
Inhibition of NF-
B activity by NO has also been attributed to
stabilization of I
B-
in Jurkat T cells and human vascular
endothelial cells (61, 62).
However, other reports indicate that NO and NO reaction
products enhance induction of NF-
B. For example, cytokine-stimulated nuclear translocation of p50 in rat astroglial cells was enhanced by
spermine NONOate (100 µM) (63). When also stimulated with IL-1
(0.5 ng/ml) and IFN-
(50 units/ml), spermine NONOate
enhanced both p50 and p65 nuclear translocation. In another study,
S-nitrosoglutathione was reported to stimulate NF-
B
activation by various mechanisms in RAW macrophages. Accordingly,
treatment of RAW cells with S-nitrosoglutathione (200 µM) alone induced I
B-
degradation, NF-
B DNA
binding, and enhanced NF-
B-dependent reporter gene
expression. The effect of S-nitrosoglutathione on NF-
B
was also correlated to the expression of cyclooxygenase-2, which was
dramatically elevated in comparison to untreated cells (64). In
vivo studies using iNOS(
/
) knockout mice have also supported a
role for NO in promoting NF-
B activation. Accordingly, the
activation of NF-
B caused by induction of hemorrhagic shock was
markedly attenuated in the iNOS knockout strain (65).
Interestingly, peroxynitrite, the reaction product of NO and superoxide
(O
), has been shown in several cases to enhance NF-
B
activation and stimulate the transcriptional expression of
NF-
B-dependent genes (66-68). As with the effects of NO
on MAP kinase pathways, current data on the inhibition and activation
of NF-
B suggests multiple modes of regulation by NO, depending on
cell type, donor species, and concentration. Accordingly, other
investigators have provided evidence for the multifunctional role for
NO in the regulation of NF-
B (69). These results have indicated a
potential physiological role for NO-derived oxidants in promoting the
release of proinflammatory cytokines in the human vasculature. They
also concur with the findings presented here, that NO enhanced NF-
B
activation and stimulated IFN-
production.
Along with interferon regulatory factors, NF-
B and c-Jun/activating
transcription factor-2 are responsible for the induction of IFN-
in
LPS-stimulated or virus-challenged cells (20-23) (Fig. 11). Our finding that the NO donors,
DEA/NO and MAHMA/NO, increase IFN-
mRNA levels in LPS-treated
macrophages correlates with the enhanced activation of NF-
B and MAP
kinase signaling cascades. The biochemical mechanism underlying the
effects of NO donors reported here remain to be determined. However,
the data agree with earlier studies in Jurkat T cells, where SNP,
S-nitroso-N-acetylpenicillamine, and genuine NO
were shown to enhance p21ras activity via
S-nitrosation of cysteine 118. The NO-induced activation of
p21ras was further correlated to an increase in NF-
B and MAP
kinase activities (70, 71). In conclusion, NF-
B and MAP kinase
signaling are vital in the regulation of immune functions. Their
enhancement by NO, particularly under conditions of sepsis or because
of the localized generation of NO by activated macrophages, may promote or maintain an activated or inflammatory state.

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Fig. 11.
Model for NO-enhanced induction
of IFN- mRNA levels. In cell medium,
DEA/NO decomposes spontaneously to release NO, which is freely
permeable through biological membranes. NO stimulates phosphorylation
of JNK and p38 MAP kinases, and enhances LPS-induced degradation of
I B- through unidentified mechanisms. NO also correlates with loss
of nuclear p105 levels. Enhanced NF- B and MAP kinase pathways
facilitate the assembly of an IFN- enhanceosome, promoting the
enhanced expression of the IFN- gene.
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