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Division of Endocrinology and Metabolism, the Second Department of Internal Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519, Japan
Address all correspondence and requests for reprints to: Dr. Yukio Hirata, Division of Endocrinology and Metabolism, Second Department of Internal Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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potently
stimulated nitrite/nitrate (NOx) production with a concomitant
expression of iNOS mRNA and protein as demonstrated by Northern and
Western blot analysis, respectively. Both IL-1ß and TNF- activated
nuclear factor (NF)-
B as demonstrated by electrophoretic mobility
shift assay. Dexamethasone, salicylate and aspirin, but not
indomethacin, dose dependently inhibited cytokine-stimulated NOx
production and iNOS protein expression. Dexamethasone decreased
cytokine-induced NF-B activation and iNOS mRNA expression, but
neither salicylate nor aspirin affected NF-B activation or iNOS mRNA
expression. IL-1ß caused a rapid increase in phosphorylated IB-
levels and subsequent transient decrease in IB- levels, an
inhibitor of NF-B, as revealed by Western blot analysis using
specific antibodies for phosphorylated and nonphosphorylated IB-.
These effects were blocked by pretreatment with dexamethasone. Aspirin
dose dependently inhibited iNOS enzymatic activity, whereas salicylate
and dexamethasone had limited effect. The present study demonstrates
that 1) inhibitory effect of dexamethasone on cytokine-induced iNOS
expression and NO production in rat VSMCs, although potentially acting
at multiple levels, is partly mediated by inhibition of NF-B
activation resulting from decreased phosphorylation and degradation of
IB-, 2) both salicylate and aspirin inhibit cytokine-stimulated
NO production at translational and/or posttranslational levels without
affecting NF-B- mediated iNOS gene expression, and 3) aspirin
directly inhibits iNOS enzyme activity. These data suggest the
differential inhibitory mechanisms of iNOS-mediated NO synthesis by
glucocorticoids and NSAIDs in the vasculature. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The promoter region of the murine iNOS gene contains several potential
cis-elements for the binding of different transcription factors,
including two putative binding sites for nuclear factor-kappa B
(NF-B) (6, 7). NF-B, a key regulator of immune system and
inflammatory genes, is a heterodimeric complex, usually consisting of
p50/p65 subunit. p50/p65 heterodimer associates with a cytoplasmic
inhibitor, IB, to form an inactive ternary complex. p65 subunit may
also associate with precursor protein (p105) of p50 to form an inactive
complex. Activation of NF-B by LPS or cytokines requires either the
degradation of IB- (8), or proteolytic cleavage of p105 through
the ubiquitin-proteasome pathway after phosphorylation (9). Following
degradation of IB-, an active heterodimer NF-B translocates to
the nucleus, binds to the consensus sequences of B site, and
activates gene expression. However, the exact role of IB- in
mediation of NF-B-dependent iNOS expression in response to cytokines
in VSMCs has not been elucidated.
Glucocorticoids are potent inhibitors of immune responses and
inflammatory process. NF-B may be a key target for
glucocorticoid-mediated immunosuppression because glucocorticoids have
been reported to induce the synthesis of IB- and prevent NF-B
activation in monocytes and lymphocytes (10, 11). However, it has been
reported that induction of IB- cannot account for
glucocorticoid-mediated repression of NF-B-dependent IB-
expression in endothelial cells (12). Nonsteroidal antiinflammatory
drugs (NSAIDs), such as sodium salicylate and aspirin, have also been
shown to inhibit NF-B activation by preventing phosphorylation and
degradation of IB in certain cells (13). Therefore, it is intriguing
whether glucocorticoids and NSAIDs may inhibit cytokine-induced iNOS
expression by modulation of NF-B in VSMCs.
These observations led us to study whether 1) glucocorticoid
(dexamethasone) and NSAIDs (salicylate and aspirin) inhibit
cytokine-induced NO production through inhibition of NF-B-mediated
iNOS expression in rat VSMCs, 2) glucocorticoid affects
cytokine-induced phosphorylation and degradation of IB-, and 3)
glucocorticoid and NSAIDs affect iNOS enzymatic activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was kindly provided by Otsuka Pharmaceutical (Osaka,
Japan). Aspirin, sodium salicylate, indomethacin, calmodulin, FAD,
sodium dodecyl sulfate (SDS), and EDTA were from Wako Pure Chemical Industries Ltd. (Osaka, Japan), NADPH from
Oriental Chemical (Tokyo, Japan), tetrahydrobiopterin from
Research Biochemical International (Natick, MA),
phenylmethylsulphonyl fluoride (PMSF), dithiothreitol (DTT), poly
(dI-dC), and L-arginine from Sigma Chemical Co. (St. Louis, MO), MG115 from Peptide Institute
(Osaka, Japan), [-32P] dCTP from Amersham International (Tokyo, Japan), dNTP and a Klenow fragment of DNA
polymerase I from Takara Shuzo (Shiga, Japan).
Cell culture
VSMCs prepared by the explant method from the thoracic aorta of
male Sprague Dawley rats (The Jackson Laboratory,
Bar Harbor, ME) were cultured in DMEM containing 10% FCS at 37 C in a
humidified atmosphere of 95% air-5% CO2 as described
(14). Subcultured VSMCs (10–15th passages) were used in the
experiments.
Determination of nitrite/nitrate (NOx)
Confluent VSMCs (106 cells/well), deprived of serum
for 24 h and pretreated with or without dexamethasone or NSAIDs
for 6 h, were stimulated with IL-1ß (10 ng/ml) or TNF- (100
ng/ml) in serum-free DMEM for 15 h; NOx concentrations in the
conditioned media were measured by an autoanalyzer (TCI-NOX 100, Tokyo
Kasei Kogyo, Tokyo) as described (15). In brief, samples premixed
with the carrier solution (0.07% EDTA and 0.3% NH4Cl) was
passed through a copperized cadmium reduction column to reduce
NO3- to NO2-, which reacts with Griess reagent
(1% sulfonamide/0.1% N-1-naphtylethylenediamine
dihydrochloride/5% HCl). Absorbance at 540 nm was measured by a
flow-through visible spectrophotometer (model S/3250, Soma-Kogaku,
Tokyo). NO3- was used as a standard.
Electrophoretic mobility shift assay (EMSA)
Confluent VSMCs (5 x 106 cells/dish), deprived
of serum for 24 h and preincubated with or without dexamethasone
or NSAIDs for 6 h, were treated with cytokines for 2 h,
washed with ice-cold PBS and harvested in 0.4 ml ice-cold hypotonic
lysis buffer (10 mM HEPES, pH 7.8, 10 mM KCl, 2
mM MgCl2, 1 mM DTT, 0.1
mM EDTA, 0.1 mM PMSF, 5 µg/ml leupeptin).
After 15 min incubation, 25 µl 10% Nonidet P-40 was added and
centrifuged at 10,000 x g for 1 min. The nuclei
pellets from a single dish were collected, resuspended in 30 µl
hypertonic extraction buffer (50 mM HEPES, pH 7.8, 50
mM KCl, 300 mM NaCl, 0.1 mM PMSF),
centrifuged at 10,000 x g for 10 min, and the
supernatant was subjected to EMSA as described (16). The
single-stranded oligonucleotides (forward: 5'-TGGGGACTCTCC-3',
complement: 5'-AAGGGAGAGTCC-3') corresponding to NF-B binding site
(-107 to -97) in the 5'-flanking region of the rat iNOS gene (17)
were annealed at 65 C for 15 min and filled with
[-32P] dCTP (111 TBq/mmol), dNTP, and a Klenow
fragment of DNA polymerase I. Nuclear proteins (10 µg) were incubated
with 2000 cpm [32P]-labeled NF-B double-stranded
oligonucleotide and 1 µg poly (dI-dC) in EMSA buffer (10
mM Tris-HCl pH 7.5, 2% glycerol, 0.2 mM EDTA,
0.5 mM DTT, 50 mM NaCl) for 30 min, loaded into
a 5% polyacrylamide gel and run in TGE buffer (50 mM Tris,
0.38 M glycine, 2 mM EDTA, pH 8.5) at 150 V for
3 h. The gel was then dried and autoradiographed. To examine the
specificity of the NF-B binding protein, the gel shift assay was
performed in parallel in the presence of a 100-fold excess of unlabeled
oligonucleotide as a competitor. For supershift assay, nuclear protein
was incubated for 30 min with goat polyclonal antibodies (2 µg IgG)
against human NF-B p50 or p65 subunit (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) before EMSA.
Northern blot analysis
Confluent VSMCs (5 x 106 cells/dish), deprived
of serum for 24 h, were stimulated with cytokines for 6 h
unless otherwise stated, and total RNAs were extracted from a single
dish by the acid guanidinium thiocyanate-phenol-chloroform methods
(18). Total RNAs (20 µg) from a single dish were separated by
formaldehyde/1.1% agarose gel electrophoresis and transferred to a
Magna Graph nylon membrane (Micron Separations Inc., Westboro,
MA) by capillary elution. The probe for rat iNOS cDNA recently
cloned from rat endothelial cells (19) was labeled with
[-32P] dCTP (111 TBq/mmol) by random-primed labeling
method. RNA immobilized on the membrane was hybridized with the labeled
probes, washed in 0.1 x SSPE/0.5% SDS, autoradiographed, and
signals were quantitated using a BAS2000 Imaging Analyzer (Fuji Photo Film, Inc., Tokyo, Japan) as described (20).
Western blot analysis
After confluent VSMCs (5 x 106 cells/dish)
were stimulated with cytokines for the indicated times, cells were
lysed in 50 mM Tris-HCl, pH 6.8 (10% glycerol, 1% SDS, 1
µg/ml pepstatin, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 1
mM PMSF). After boiling cell lyates the protein content was
determined with a BCA assay kit (Pierce Chemical Co.,
Rockford, IL). Extracted proteins (10 µg) from a single dish were
separated on a 7.5% SDS-polyacrylamide gel and transferred to a Hybond
ECL nitrocellulose membrane (Amersham International).
Mouse monoclonal antibody for murine iNOS (Transduction Laboratories, Inc., Lexington, KY), rabbit polyclonal antibody
for human IB- (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA), and rabbit polyclonal antibody for phospho-specific
IB- (New England Biolabs, Inc., Beverly, MA) was
applied at a 1:1000 dilution at 4 C overnight. After extensive washing,
the secondary antibody (sheep antimouse Ig conjugated to horseradish
peroxidase, Amersham) was applied at 1:500 dilution for
1 h, and exposure was performed by an ECL kit
(Amersham) as described (16).
NOS enzymatic assay
NOS activity was determined by essentially the same method of
citrulline assay (21) except for measurement of NO. Rat VSMCs
stimulated with IL-1ß (10 ng/ml) for 15 h were homogenized in 50
mM Tris-HCl, pH 7.4 (0.5 mM EDTA, 0.5
mM EGTA, 1 µg/ml pepstatin, 2 µg/ml leupeptin, and 0.1
mM PMSF), centrifuged for 30 min at 10,000 x
g, and the supernatant was used as crude enzyme
preparations; protein concentration was measured with a BCA assay kit.
Enzymatic reaction was performed at 37 C for 1 h in a final volume
of 100 µl assay buffer (12.5 mM Tris-HCl, pH 7.4, 1
mM L-arginine, 1 mM NADPH, 10
µg/ml calmodulin, 9 µM tetrahydrobiopterin, 9
µM FAD, and 0.5 mM EGTA) containing cytosolic
protein (100 µg) and test compounds. The reaction was terminated by
adding 0.2 ml 20 mM HEPES buffer, pH 5.5, containing 2
mM EDTA, and the reaction mixtures were centrifuged for 5
min at 10,000 x g; concentrations of NOx in the
supernatant were measured by an autoanalyzer as described (15).
Statistical analysis
All values of NOx concentrations were given as means ±
SE of 3–4 dishes. Statistical analysis was performed by
using analyses of variance for repeated measures. A P value
less than 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(100 ng/ml)
time-dependently (6–24 h) stimulated NOx production in rat VSMCs
(Fig. 1A
). Both cytokines induced iNOS
mRNA expression which peaked after 6–12 h and then decreased after
24 h (Fig. 1B), whereas maximal iNOS protein expression by these
cytokines was induced after 12 h (data not shown). Both IL-1ß
(0.1–10 ng/ml) and TNF- (1–100 ng/ml) dose dependently stimulated
NOx production (15 h) as well as the expressions of iNOS mRNA (6 h) and
protein (12 h) (data not shown). Therefore, subsequent studies were
performed using maximal doses of IL-1ß (10 ng/ml) and TNF- (100
ng/ml) for the indicated times.
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); a maximal inhibition (
75%) was
induced with 1 µM. Dexamethasone (1 µM)
reduced iNOS messenger RNA (mRNA) and protein expression induced by
IL-1ß (Fig. 2B); the IL-1ß-induced iNOS mRNA levels in
dexamethasone-treated cells were significantly (P <
0.01) reduced by 58.7 ± 5.1% compared with those in nontreated
cells (n = 3) as measured by the relative ratio of mRNA/28S
ribosomal RNA with a densitometer. The same inhibitory effects
by dexamethasone were observed when TNF- (100 ng/ml) was used as an
inducer (data not shown).
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); a maximal inhibition (5070%) was
induced with 20 mM. However, indomethacine (0.01–0.1
mM) did not affect IL-1ß-stimulated NOx production.
Neither aspirin nor salicylate affected iNOS mRNA expression induced by
IL-1ß, whereas both compounds dose dependently (10–20
mM) reduced IL-1ß-induced iNOS protein expression (Fig. 3B). Similar results were obtained when TNF- was used as an inducer
(data not shown). Neither cell viability nor cell morphology was
affected by any of the above compounds at concentrations used in the
experiments; the buffered-culture media containing even 20
mM aspirin remained neutral.
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B activation by
cytokines induced NF-B activation in rat VSMCs
as early as 30 min, which persisted during 24 h as revealed by
EMSA using synthetic oligonucleotides corresponding to the NF-B site
(-107 to -97) in the 5'-flanking region of the rat iNOS gene as a
probe (Fig. 4). Addition of anti-p50
antibody resulted in the appearance of supershifted band, whereas
addition of anti-p65 antibody caused a supershifted band in addition to
the reduced shifted band (Fig. 4A), suggesting that NF-B binding
protein comprises p50/p65 heterodimer subunit immunologically similar,
if not identical, to its human counterparts. Furthermore, the
IL-1ß-induced NF-B protein-DNA complex was completely eliminated
by a 100-fold excess unlabeled oligomers (Fig. 4A), indicating the
specificity of the NF-B binding gel shift assay. The shifted band
was markedly reduced by dexamethasone (1 µM) (Fig. 4B)
but not affected by either aspirin or salicylate (20 mM)
(Fig. 4C).
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B- degradation
and phosphorylationB as demonstrated by EMSA, we
studied whether cytokines affect degradation and phosphorylation of
IB- in rat VSMCs by Western blot analysis using anti-IB-
and antiphospho IB- antibodies, respectively. Addition of IL-1ß
(10 ng/ml) resulted in a rapid (10–15 min) decrease in IB-
protein levels, which then returned to baseline levels within 1 h
(Fig. 5A). IL-1ß (10 ng/ml) also caused
an immediate (within 5 min) increase in phosphorylated IB-
levels, which then returned to the undetected level by 15 min (Fig. 5B). A proteasome inhibitor (MG115) completely prevented the transient
decrease in IB-, but not the rapid increase in phosphorylated
IB- induced by IL-1ß (data not shown). Similar results were
obtained when TNF- was used as a stimulant. These data suggest that
both IL-1ß and TNF- cause a rapid phosphorylation of
Ser32 residue of IB- and its subsequent degradation
via the ubiquitine/proteasome pathway in rat VSMCs.
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B- levels (Fig. 6A); dexamethasone added alone was
without effect. Pretreatment with dexamethasone decreased the
IL-1ß-induced rapid increase in phosphorylated IB- levels (Fig. 6B); dexamethasone added alone was without effect. These data suggest
that dexamethasone inhibits cytokine-stimulated phosphorylated
IB- before its degradation in rat VSMCs.
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B
activation, we studied whether NSAIDs directly affect iNOS catalytic
activity (Fig. 7). Aspirin dose
dependently (1–20 mM) inhibited iNOS enzymatic activity; a
marked inhibition (40%) was induced at 1 mM and a
maximal inhibition (75%) at 20 mM. In contrast, the
inhibitory effect of salicylate (20 mM) was minimal
(10%) and dexamethasone (1 µM) was without
effect.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and NF/IL-6 sites is critical for inducibility by
LPS, whereas the region between -1029 and -913 bp containing NF-B,
IRF-1, and ISRE/GAS sites mediates IFN-
-potentiation of LPS
induction in mouse macrophages (6, 7). Of the two putative NF-B
binding sites, the consensus sequence between -965 and -955 bp is the
most common in many genes, whereas that between -107 and -97 is
unique, found in only iNOS gene of mouse, rat, and human (6, 7, 17, 22). Therefore, we performed EMSA using synthetic oligomer
corresponding to the putative NF-B site (-107 to -97) of the rat
iNOS gene promoter region (17) as a probe to determine whether
cytokines stimulate NF-B-DNA binding activity in rat VSMCs. Both
IL-1ß and TNF- potently induced NF-B activation in rat VSMCs;
the specificity of NF--binding gel shift assay was confirmed by the
complete disappearance of the shifted band in the presence of excess
unlabeled oligomers as well as by the appearance of the supershifted
band in the presence of specific antibodies for p50 and p65 subunits
(16). The present study also confirmed that both IL-1ß and TNF-
time dependently and potently stimulated NO production in rat VSMCs
with a concomitant iNOS mRNA and protein expression as demonstrated by
Northern and Western blot analyses, respectively. These results are
consistent with the previous studies showing that cytokines induce iNOS
expression via activation of NF-B in different cell types
(23, 24, 25). The present study showed that dexamethasone dose dependently inhibited IL-1ß-stimulated NO production in rat VSMCs with a maximal inhibition (-75%) at 1 µM, which also caused a modest inhibition (-60%) on IL-1ß-induced iNOS mRNA and protein expression as evaluated by Northern and Western blot analysis, respectively. Our data are consistent with those of previous studies using several cell types including VSMCs (25, 26, 27). Dexamethasone has been shown to be a potent inhibitor of iNOS but has transcriptional and translational effects on iNOS expression. For example, in rat mesangial cells, dexamethasone inhibited IL-1ß-induced NO production and iNOS expression mainly due to reduced translation and increased degradation, although it decreased transcriptional rate and increased mRNA half-life (28). In rat insulin-secreting islet cell line (RIN m5F), dexamethasone has been shown to inhibit IL-1ß-stimulated NO production without affecting iNOS mRNA levels (29). In rat VSMCs, however, it has been shown that dexamethasone did not inhibit IL-1ß-stimulated NO production, although it modestly decreased iNOS mRNA expression resulting from decreased transcriptional activity despite increased mRNA stability (30). In contrast, dexamethasone has been shown to completely inhibit cytokine-stimulated NO production with only a modest decrease in iNOS expression, mainly due to inhibition of tetrahydrobiopterin synthesis and L-arginine transport in rat microvessel endothelial cells, suggesting its posttranslational effects (31). Collectively, it is suggested that glucocorticoids act at multiple levels to regulate iNOS expression depending on cell types and species differences.
The present study further showed that dexamethasone (1
µM) also modestly inhibited NF-B-DNA binding activity
stimulated by IL-1ß, suggesting that glucocorticoids inhibit iNOS
expression partly via its interference with NF-B activation pathway.
It is well known that glucocorticoids mediate immunosuppressive and
antiinflammatory effects through a cytoplasmic receptor, glucocorticoid
receptor (GR). Upon hormone binding, activated GR enters the nucleus,
dimerizes, binds to specific DNA sequences, termed glucocorticoid
response elements (GRE), and activates gene transcription (32).
However, many genes involved in the inflammatory response, including
iNOS gene, do not contain GRE in their promoters. Recent studies
suggested that transcriptional repression by glucocorticoids is
mediated by interference with interaction of GR with other
transcription factors, such as AP-1 and NF-B, through cross-coupling
mechanism (33, 34).
The present study revealed that stimulation with IL-1ß caused a
transient decrease in IB- levels preceded by a rapid (within 5
min) increase in phosphorylated IB- levels as demonstrated by
Western blot analyses using anti-IB- antibody and anti-phospho
Ser32 of IB- antibody, respectively.
Cytokine-responsive IB- kinases recently identified (35, 36)
phosphorylate two serine residues (Ser32 and
Ser36) of IB- (37), whose phosphorylation is a
prerequisite for polyubiquitination and subsequent degradation of
IB- by 26S proteasome (38). It has been shown that
glucocorticoids induce up-regulation of IB- that renders active
NF-B heterodimer into an inactive cytoplasmic ternary complex (Fig. 8) in certain cells, such as monocytes,
lymphocytes (10, 11), and rat hepatocytes (39). In contrast, induction
of IB- synthesis is not involved in dexamethasone-mediated
inhibition of NF-B activity in human endothelial cells (12) and
alveolar epithelial cells (40), suggesting the importance of
protein-protein interaction between NF-B and GR in repression of
NF-B activity (Fig. 8). In the present study, dexamethasone did not
up-regulate IB- expression but prevented IL-1ß-induced increase
in phosphorylated IB- and subsequent decrease in IB- in rat
VSMCs. Taken together, it is suggested that IL-1ß rapidly stimulates
IB- kinase to phosphorylate IB- and subsequent
proteasome-mediated degradation of IB-, thereby leading to
NF-B activation, and that inhibitory effect of dexamethasone on the
IL-1ß-induced NF-B activation is in part due to inhibition of
IB- phosphorylation and its subsequent degradation in rat VSMCs
(Fig. 8). However, the question as to whether glucocorticoids directly
affect IB- kinase activity and/or the upstream signals following
cytokine stimulation remains unknown.
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B activation in hematopoietic progenitor cells
(42), cerebellar granule cells (43), and human Jurkat T cells and mouse
pre-B cell line (13), by preventing phosphorylation and degradation of
IB-. The present study clearly revealed that cytokine-stimulated
NO production in rat VSMCs was dose dependently inhibited by salicylate
and aspirin but not by indomethacin. However, neither salicylate nor
aspirin affected the cytokine-induced NF-B activation or iNOS mRNA
expression in rat VSMCs. Our results appear to be consistent with those
of most studies in which salicylate and aspirin inhibited
cytokine-induced NO production without affecting iNOS mRNA expression,
such as in mouse macrophages (44), rat hepatocytes, (45) and rat
pancreatic ß-cells (RIN m5F) (46). In contrast, however, salicylate
and aspirin have been shown to suppress cytokine-induced iNOS mRNA
expression and NO production without affecting NF-B activation in
rat cardiac fibroblasts (47, 48). The apparent discrepancies between
the several results including ours suggest that NF-B activation
pathway may be differentially modulated by NSAIDs in a cell- or
tissue-specific manner.
Both salicylate and aspirin dose dependently (10–20 mM)
decreased iNOS protein expression in rat VSMCs. Although the
concentrations of both compounds used in this study were
supraphysiological compared with the therapeutic plasma concentrations
(1–3 mM) such as in patients with rheumatoid arthritis,
they did not cause nonspecific or cytotoxic effect in our study, and pH
of the media containing aspirin remained neutral even after 15 h
incubation. Our data are consistent with those of mouse macrophages
(44), rat hepatocyte (45), and rat pancreatic ß-cells (46) in which
aspirin and salicylate in supraphysiological concentrations (2–20
mM) markedly reduced cytokine-induced iNOS protein
expression. Collectively, these data suggest that NSAIDs may decrease
the translational rate and/or promote the degradation of iNOS protein
in rat VSMCs (Fig. 8
).
By measuring enzymatic activity, we found that aspirin directly
inhibits iNOS catalytic activity in a dose-dependent manner, whereas
salicylate and dexamethasone were without effect. It should be noted
that aspirin as low as 1 mM markedly (-40%) decreased
iNOS enzyme activity. Our data are consistent with those of mouse
macrophages showing that aspirin and N-acetylimidazole, an
acetylating agent, but not salicylate or indomethacin, directly
interfered with the catalytic activity of iNOS (Fig. 8) (44). Although
aspirin has been shown to suppress the catalytic activity of COX by
acetylating a Ser residue (49), it remains unknown whether the
inhibitory effect by aspirin on iNOS catalytic activity involves
acetylating functional components of the enzyme and/or interference
with interaction with its essential cofactors, such as
tetrahydrobiopterin and heme moiety.
The possible mechanisms underlying the inhibitory effects of
glucocorticoids and NSAIDs on cytokine-induced NO production in rat
VSMCs as revealed from the present study are summarized in Fig. 8. The
inhibitory effect by glucocorticoids on cytokine-stimulated NO
production and iNOS expression in rat VSMCs, although potentially
acting at multiple levels, is partly mediated by decreasing
phosphorylation/degradation of IB- and subsequent inhibition of
NF-B activation. In contrast, aspirin suppresses cytokine-stimulated
NO production by inhibiting iNOS protein expression and catalytic
activity without affecting NF-B-mediated iNOS mRNA expression,
whereas the inhibitory effect by salicylate is mainly due to decreased
iNOS protein expression.
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Footnotes |
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Received June 25, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and
the initiation of the atherosclerotic lesion. Lab Invest 68:499–508[Medline]
and
lipopolysaccharide. Proc Natl Acad Sci USA 90:9730–9734 and bacterial lipopolysaccharide. J Exp
Med 177:1779–1784B-: a mechanism for NF-B activation. Mol Cell Biol 13:3301–3310B1 precursor protein and the activation of NF-B. Cell 78:773–785[CrossRef][Medline]
B
activity through induction of IB synthesis. Science 270:286–290B- in mediation of
immunosuppression by glucocorticoids. Science 270:283–286B activity in endothelial cells does not involve induction of
IB- synthesis. J Biol Chem 271:19612–19616B by
sodium salicylate and aspirin. Science 265:956–959B/Rel in induction of nitric oxide synthase.
J Biol Chem 269:4705–4708B- by nitric oxide mediates inhibition of
NF-B. J Biol Chem 270:14214–14219B. J Biol Chem 271:6039–6044B kinase that activates the
transcription factor NF-B. Nature 388:548–554[CrossRef][Medline]
B kinases essential for NF-B
activation. Science 278:860–866B
phosphorylation sites that signal its ubiquitination and degradation.
Mol Cell Biol 16:1295–1304[Abstract]
B- to the ubiquitin-proteasome pathway.
Genes Dev 9:1586–1597B- and inhibiting NF-B. Am J Physiol
273:G1290–G1296
B. Mol Pharmacol 49:15–21[Abstract]
B activation. Science 274:1383–1385This article has been cited by other articles:
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