© 1998 American Heart Association, Inc.
Original Contributions |
NO Inhibits Cytokine-Induced iNOS Expression and NF-
B Activation by Interfering With Phosphorylation and Degradation of IB-
From the Endocrine-Hypertension Division, Second Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo, Japan.
Correspondence to Yukio Hirata, MD, Endocrine-Hypertension Division, 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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Abstract—Nitric oxide (NO) is known to have antiatherogenic and anti-inflammatory properties, but its effects on the cytokine-induced nuclear factor-kappa B (NF-
B) activation pathway in relation to the regulation of inducible
nitric oxide synthase (iNOS) gene in vascular smooth muscle cells
(VSMCs) remain elusive. To elucidate the roles of NO in the regulation
of cytokine-induced NF-B activation and consequent iNOS
gene expression, we studied the effects of NO donors
[(±)-(E)-ethyl-2-[(E)-hydroxyamino]-5-nitro-3-hexeneamide
(NOR3) and sodium nitroprusside] on interleukin (IL)-1ß–induced
NF-B activation and IB- degradation and subsequent iNOS
expression in rat VSMCs. Northern blot and Western blot
analyses demonstrated that NO donors decreased IL-1ß–induced
iNOS mRNA and protein expression. Electrophoretic mobility shift assay
using synthetic oligonucleotide corresponding to the
downstream NF-B site of rat iNOS promoter as a probe showed that
NOR3 inhibited IL-1ß–induced NF-B activation and its nuclear
translocation, as demonstrated with immunocytochemical study. These
effects were independent of guanylate cyclase activation;
an inhibitor of soluble guanylate cyclase
(1H-oxadiazolo-1,2,4-[4,3-]quinoxaline-1-one) had
no effect on NOR3-induced inhibition of NF-B activation or iNOS mRNA
expression by IL-1ß, and a cGMP derivative (8-bromo-cGMP) failed to
mimic the effects of NO donors. Western blot analysis using
anti–IB- and anti–phospho-IB- antibodies revealed that
IL-1ß induced a transient degradation of IB- preceded by a
rapid appearance of phosphorylated IB-, both of
which were completely blocked by NOR3. A proteasome
inhibitor (MG115) blocked IL-1ß–induced transient
degradation of IB- and stabilized the appearance of
phosphorylated IB- stimulated by IL-1ß. NOR3
inhibited the appearance of IL-1ß–induced
phosphorylated IB- even in the presence of MG115.
Our results indicate that an inhibitory action by NO on
cytokine-induced NF-B activation and iNOS gene
expression is due to its direct blockade on
phosphorylation and subsequent degradation of IB-
via the cGMP-independent pathway in rat VSMCs.
Key Words: NF-B • IL-1ß • inducible nitric oxide synthase • IB-
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Introduction |
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Nitric oxide (NO) is synthesized from L-arginine by NO synthases (NOSs). Three distinct isozymes of NOS have been identified to date: 2 Ca2+/calmodulin-dependent constitutive isozymes dominantly expressed in the brain and endothelium, and a Ca2+-independent, cytokine-inducible isozyme (iNOS).1 iNOS produces large amounts of NO in response to bacterial lipopolysaccharides (LPS) and certain cytokines in a variety of cells, including vascular smooth muscle cells (VSMCs).2 NO possesses diverse physiological properties, such as vasodilation, neurotransmission, and mediation of immune responses.1 High-output NO produced by iNOS in VSMCs not only causes inhibition of cell proliferation but apoptosis of VSMCs as well.3 4 Therefore, regulation of iNOS gene expression has been implicated in the pathogenesis of vascular remodeling and atherosclerosis.5 Many of the biological effects of NO have been attributed to cGMP generation via the stimulation of soluble guanylate cyclase, although a cGMP-independent mechanism is also involved in its diverse actions.
The promoter region of the rat and mouse iNOS gene contains several
potential cis-elements for the binding of different
transcription factors, among which 2 putative binding sites for nuclear
factor-kappa B (NF-B) exist in the upstream (GGGGATTTTCC,
nucleotides -965 to -955: NF-Bu) and downstream
(GGGGACTCTCC, nucleotides -107 to -97: NF-Bd)
regions.6 7 8 The sequence of NF-Bd is unique
in that it is found only in murine and human iNOS genes. It has been
shown that a key region of the promoter activity in mediation of LPS
inducibility resides in the NF-Bd region in mouse
macrophages.6 7 However, its role in
mediation of iNOS expression in response to cytokines in VSMCs
remains largely unknown.
NF-B complexes function as a pleiotropic regulator of many genes
modulating immunologic and inflammatory processes. NF-B contains
heterodimeric complexes, usually consisting of p50 and p65 (Rel-A)
subunits and p50/p50 homodimers in human VSMCs.9
p50/p65 heterodimer associates with its cytoplasmic
inhibitor, IB-, to form an inactive cytoplasmic
ternary complex. p65 subunit may also complex with p105, a precursor
protein of p50, as an inactive form. Activation of NF-B by LPS or
cytokines requires either degradation of
IB-10 or proteolytic cleavage of p105
through a common ubiquitin-proteasome pathway after
phosphorylation.11 After
degradation of IB-, an active NF-B translocates to the nucleus
and activates the expression of a plethora of genes. Recently,
a cytokine-responsive IB- kinase (IKK) that
activates NF-B by phosphorylation of
Ser32 and Ser36 residue in
IB- has been identified.12 13 14 15 16 It has been
reported that NO inhibits NF-B by induction and stabilization of
IB- in human endothelial
cells.17 However, it remains unknown whether NO
inhibits cytokine-induced NF-B activation via inhibition
of phosphorylation and degradation of IB-,
thereby blocking iNOS gene expression in VSMCs.
These observations led us to examine (1) whether interleukin
(IL)-1ß–induced iNOS gene expression is mediated by the NF-B
activation pathway involving phosphorylation and
subsequent degradation of IB- in cultured rat VSMCs and (2)
whether NO inhibits IL-1ß–induced iNOS gene expression by
interfering with phosphorylation and subsequent
degradation of IB-.
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Methods |
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Materials
Human recombinant IL-1ß was kindly provided by Otsuka Pharmaceutical. Murine recombinant tumor necrosis factor (TNF)-
was
purchased from Gibco BRL;
(±)-(E)-ethyl-2-[(E)-hydroxyamino]-5-nitro-3-hexeneamide
(NOR3), SDS, and EDTA were purchased from Wako Pure Chemical;
8-bromo-cGMP, N-acetyl-L-cysteine,
1H-oxadiazolo-1,2,4-[4,3,-]quinoxaline-1-one (ODQ),
PMSF, DTT, poly(dI-dC), and EGTA were purchased from Sigma Chemical;
pyrrolidinedithiocarbamate was purchased from Katayama Chemical; sodium
nitroprusside (SNP) was purchased from Calbiochem Novabiochem; MG115
was purchased from Peptide Institute;
[-32P]dCTP was purchased from Amersham
International; and dNTP and a Klenow fragment of DNA polymerase I were
purchased from Takara Shuzo.
Cell Culture
VSMCs from the thoracic aorta of 15-week-old male Wistar rats
were prepared by the explant method and cultured in Dulbecco's
modified Eagle's medium containing 10% FBS at 37°C in a humidified
atmosphere of 95% air and 5% CO2 as described
previously.18 Subcultured VSMCs (15th to 20th
passages) from 2 independent isolates were used in the experiments.
Electrophoretic Mobility Shift Assay (EMSA)
Confluent VSMCs (5x106 cells per dish)
pretreated with or without NO donors (NOR3, SNP) for 60 minutes were
stimulated with IL-1ß for 2 hours, washed with ice-cold PBS, and
harvested in 0.4 mL ice-cold hypotonic lysis buffer (10 mmol/L
HEPES, pH 7.8; 10 mmol/L KCl; 2 mmol/L
MgCl2; 1 mmol/L DTT; 0.1 mmol/L EDTA;
0.1 mmol/L PMSF; and 5 µg/mL leupeptin). After 15 minutes of
incubation, 25 µL 10% Nonidet P-40 was added. The nuclei pellets
were collected, resuspended in 30 µL hypertonic extraction buffer
(50 mmol/L HEPES, pH 7.8; 50 mmol/L KCl; 300 mmol/L
NaCl; 0.1 mmol/L EDTA; 1 mmol/L DTT; 10% glycerol; and
0.1 mmol/L PMSF), and centrifuged, and the supernatant was
subjected to EMSA. The single-stranded oligonucleotides
(forward: 5'-TGGGGACTCTCC-3', complement: 5'-AAGGGAG- AGTCC-3')
corresponding to the NF-B binding sequence of the downstream region
(-107 to -97) of rat iNOS gene promoter8 were
annealed at 65°C for 15 minutes and filled with
[-32P]dCTP (111 TBq/mmol), dNTP, and a
Klenow fragment of DNA polymerase I. Nuclear proteins (10 µg) were
incubated with 20 000 cpm 32P-labeled NF-B
double-stranded oligonucleotide and 1 µg poly(dI-dC)
in EMSA buffer (10 mmol/L Tris-HCl, pH 7.5; 2% glycerol; 0.2
mmol/L EDTA; 0.5 mmol/L DTT; 50 mmol/L NaCl) for 30 minutes
and subjected to polyacrylamide gel electrophoresis. To examine
the specificity of the NF-B binding protein, the gel shift assay was
performed in parallel with the same samples in the presence of a
100-fold excess unlabeled oligonucleotide as a
competitor. For gel supershift assay, nuclear protein was preincubated
for 30 minutes with goat polyclonal antibodies against human NF-B
p50 or p65 subunit (Santa Cruz Biotechnology).
Northern Blot Analysis
Confluent VSMCs (5x106 cells per dish)
pretreated with or without NO donors (NOR3, SNP) for 60 minutes were
stimulated with IL-1ß for 6 hours, and total RNAs were extracted by
the acid guanidinium thiocyanate-phenol-chloroform
methods.19 Total RNAs (20 µg), separated by
formaldehyde/1.1% agarose gel electrophoresis, were transferred to a
Magna Graph nylon membrane (Micron Separations Inc). The cDNA probe for
rat iNOS recently cloned from rat endothelial
cells20 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 0.1xSSPE/0.5% SDS, and
autoradiographed.
Western Blot Analysis
Western blot analyses were performed essentially as
described.21 Confluent cells
(5x106 cells per dish), pretreated with or
without NO donors (NOR3, SNP) or MG115 for 60 minutes, were stimulated
with IL-1ß for the indicated times for IB- and
phospho-IB-, or for 15 hours for iNOS. Cells were lysed in
50 mmol/L Tris-HCl, pH 6.8 (10% glycerol, 1% SDS, 1 µg/mL
pepstatin, 2 µg/mL leupeptin, 2 µg/mL aprotinin, and 1 mmol/L
PMSF). Whole-cell lysates were boiled, and extracted proteins were
separated on 12% (for IB- and phospho-IB-) or 7.5% (for
iNOS) SDS-polyacrylamide gel and transferred to Hybond ECL
nitrocellulose membranes (Amersham), which were incubated overnight
with rabbit polyclonal antibody for human IB- (1:500; Santa Cruz
Biotechnology), rabbit polyclonal antibody for human phospho-IB-
(Ser32) (1:1000; New England Biolabs), or mouse
monoclonal antibody for murine iNOS (1:1000; Transduction Laboratories)
at 4°C. Anti–phospho-IB- antibody detects IB- only when
phosphorylated at Ser32 and has
no cross-reactivities with the corresponding
phosphorylated Ser of IB-ß or IB-
. After
extensive washing, the secondary antibody (donkey anti-rabbit IgG or
sheep anti-mouse IgG horseradish peroxidase; 1:500, Amersham) was
applied for 1 hour, and exposure was performed by using an ECL kit
(Amersham).
Immunocytochemical Staining
Subconfluent cells grown on LAB-TEK Chamber Slide (Nalge Nunc
Int) were treated with IL-1ß in the absence or presence of NOR3 for 2
hours, fixed with 70% acetone for 20 minutes at room temperature, and
then washed with PBS for 10 minutes. Goat polyclonal antibody specific
for NF-B p50 subunit (Santa Cruz Biotechnology) was used; the
antibody did not show any cross-reactivities with p105, p52, or p100.
Immunostaining was visualized with the indirect
immunoperoxidase avidin-biotin-peroxidase kit (Vector Laboratory).
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Results |
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NF-
B Activation by IL-1ßTo determine whether NF-
B activation was induced with IL-1ß
in rat VSMCs, EMSA was performed using synthetic
oligonucleotides corresponding to the NF-Bd site
(-107 to -97) of the rat iNOS promoter as a probe (Figure 1
). IL-1ß (10 ng/mL) caused a distinct
shifted band, whereas there was no distinct band in the control
cells. The specificity of the NF-B binding gel shift assay was
examined using coincubation with excess unlabeled probe as a
competitor. The band was completely eliminated in the presence of a
100-fold molar excess of unlabeled oligomers (Figure 1A). For
characterization of NF-B subunits, specific antibodies for human p50
and p65 subunits were examined. The IL-1ß–induced NF-B
protein-DNA complexes were supershifted by anti-p50 antibody, whereas
anti-p65 antibody caused a supershifted band in addition to the reduced
shifted band (Figure 1B).
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NO Inhibits IL-1ß–Induced NF-B Activation and iNOS mRNA and
Protein Expression
We studied whether NO donors affect NF-B activation and iNOS
expression induced by IL-1ß in rat VSMCs (Figure 2). Northern blot analysis using
rat iNOS cDNA as a probe revealed that IL-1ß (10 ng/mL) induced iNOS
mRNA expression (4.5 kb), the effects of which were dose-dependently
(10-5 to 10-3 mol/L)
suppressed by NOR3 (Figure 2A; top panel). EMSA showed that NOR3
similarly blocked the IL-1ß–induced NF-B activation in a
dose-dependent manner (10-5 to
10-3 mol/L) (Figure 2A; bottom panel). NOR3
(10-3 mol/L) completely blocked both NF-B
activation and iNOS mRNA expression stimulated by IL-1ß. SNP
(10-3 mol/L) also attenuated the
IL-1ß–induced iNOS mRNA expression and NF-B activation (Figure 2B).
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To determine whether the inhibitory effect of NO donors is
mediated via a cGMP-dependent mechanism, the effects of a selective
inhibitor of soluble guanylate cyclase (ODQ)
and a cell-permeable cGMP analogue (8-bromo-cGMP) were tested (Figure 3). ODQ (10-5
mol/L) did not affect the inhibitory effect of NOR3
(10-4 mol/L) on IL-1ß–induced iNOS mRNA
expression or NF-B activation (Figure 3A). 8-Bromo-cGMP
(10-3 mol/L) did not inhibit the
IL-1ß–induced iNOS mRNA expression or NF-B activation (Figure 3B). Western blot analysis using specific anti-murine iNOS
antibody demonstrated a distinct band of 130-kDa iNOS protein after
stimulation with IL-1ß (10 ng/mL), which was completely abolished in
the presence of NOR3 (10-4 mol/L) (Figure 4). These data indicate that NO blocks
IL-1ß–induced NF-B activation as well as iNOS mRNA and protein
expression via a cGMP-independent mechanism.
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NO Prevents IL-1ß–Induced NF-B Nuclear Translocation
To confirm that NO donor prevents nuclear translocation of active
NF-B in rat VSMCs after cytokine stimulation,
immunohistochemical staining using anti-p50 antibody was performed.
Nonstimulated cells revealed a diffuse but faint distribution of
immunoreactive p50 within the cytoplasm (Figure 5A). In contrast, exposure of rat VSMCs
to IL-1ß (10 ng/mL) resulted in dense accumulations of immunoreactive
p50 within the nucleus (Figure 5B), the effect of which was prevented
by pretreatment with NOR3 (10-3 mol/L) (Figure 5D); NOR3 added alone was without effect (Figure 5C).
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NO Prevents IL-1ß–Induced IB- Degradation
To determine whether IL-1ß causes IB- degradation in rat
VSMCs, Western blot analysis using anti–IB- antibody was
performed. Addition of IL-1ß (10 ng/mL) resulted in a rapid (within
15 to 30 minutes) decrease in IB- protein, which then returned to
baseline levels within 1 to 2 hours (Figure 6A). Pretreatment with NOR3
(10-3 mol/L) completely prevented the
IL-1ß–induced transient decrease in IB- levels (Figure 6B);
NOR3 added alone was without effect. A proteasome
inhibitor, MG115 (10-5 mol/L), also
prevented the transient decrease in IB- levels induced by IL-1ß
(Figure 6B). These data suggest that both NO and MG115 interfere with
the transient degradation of IB- induced by IL-1ß in rat
VSMCs.
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NO, but Not Proteasome Inhibitor, Prevents
IL-1ß–Induced IB- Phosphorylation
To determine whether IL-1ß causes IB-
phosphorylation in rat VSMCs, Western blot
analysis using anti–phospho-Ser32 of
IB- antibody was performed. Addition of IL-1ß (10 ng/mL)
resulted in a rapid (within 3 to 5 minutes) appearance of
phosphorylated IB-, which peaked at 5 minutes and
then decreased by 30 minutes (Figure 7A;
top panel). Pretreatment with MG115 (10-5 mol/L)
stabilized the IL-1ß–induced phosphorylated
IB- during a 30-minute incubation period (Figure 7A; bottom
panel). NOR3 (10-3 mol/L) prevented the
IL-1ß-stimulated transient increase in phosphorylated
IB- with or without pretreatment with MG115 (Figure 7B); NOR3 or
MG115 added alone was without effect. These data suggest that NO
inhibits rapid phosphorylation of
Ser32 of IB- induced by IL-1ß before its
degradation.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The present study clearly demonstrates that IL-1ß–induced iNOS mRNA and protein expression is associated with NF-
B activation
in rat VSMCs. By EMSA using a synthetic oligomer corresponding to the
unique downstream NF-B site of rat iNOS promoter as a probe, we have
shown that the IL-1ß specifically activated NF-B composed
of p50/p65 heterodimer that is immunologically similar, if not
identical, to its human counterpart. These results are in agreement
with a recent report demonstrating that NF-B complexes were mainly
composed of p50/p65 heterodimers in human cultured
VSMCs.9
Transcriptional activity of p50/p65 heterodimer of NF-B can be
regulated by at least 2 pathways.11 The p50/p65
heterodimer constitutes an inactive cytoplasmic ternary complex with
the inhibitor protein, IB-, which masks the nuclear
localization sequences of p50/p65 heterodimer. IB- can be rapidly
phosphorylated and degraded after stimulation with LPS
or cytokines, allowing transcriptionally active p50/p65
heterodimers to translocate to the nucleus to activate a set of
genes related to inflammation and proliferation. An alternative pathway
to regulate p65 (Rel-A) is derived from its association with the
unprocessed p105, the C-terminal portion of which bears a
striking resemblance to IB- to form an inactive p105/p65
cytoplasmic complex. Processing of p105 results in a rapid degradation
of the IB- homologous sequence and formation of transcriptionally
active p50/p65 heterodimer. In both pathways,
phosphorylation of IB- and p105 by a protein
kinase(s) is essential for the subsequent degradation of IB- and
the processing of p105, respectively. Resynthesis of IB- after
its rapid degradation depends on the induction of IB- expression
after NF-B activation.22
Cellular responses to cytokine signaling, such as activation of
several protein kinases, including ceramide-activated protein
kinase, p42/p44 mitogen-activated protein kinase, p38
mitogen-activated protein kinase, and Jun N-terminal
kinase, have been suggested to exert their diverse biological
consequences.23 However, little information is
yet available as to how immediate signals by cytokines cause
NF-B activation in VSMCs. The present experiments using cultured
rat VSMCs clearly demonstrated that stimulation with IL-1ß caused a
rapid phosphorylation of Ser32 of
IB- and subsequent transient degradation of IB-, followed
by NF-B activation and its nuclear translocation. A
cytokine-responsive IB- kinase recently identified
phosphorylates two serine residues
(Ser32 and Ser36) of
IB-.24 Phosphorylation of
these residues is a prerequisite for polyubiquitination and subsequent
degradation of IB- by 20S proteasome.25
TNF- rapidly activates IB- kinase, peaking at 5 to 10
minutes in HeLa cells.24 In the present
study, IL-1ß induced a rapid (3 to 5 minutes)
phosphorylation of Ser32 residue
of IB-, followed by a transient (15 minutes) degradation and
subsequent resynthesis of IB- in rat VSMCs. In the present
study, a proteasome inhibitor, MG115, completely prevented
the IL-1ß–induced transient degradation of IB- and also
stabilized the phosphorylated IB- induced by
IL-1ß in rat VSMCs. These data suggest that IL-1ß rapidly
stimulates IB- kinase to phosphorylate
Ser32 and possibly Ser36
residues of IB-, which in turn is degraded via the
ubiquitin/proteasome pathway, thereby leading to NF-B activation in
rat VSMCs. In fact, we have shown that the IL-1ß–induced NF-B
activation and its nuclear translocation in rat VSMCs were completely
prevented by pretreatment with MG115 (K.K. et al, unpublished data,
1998).
Our present study has clearly shown that NO donors (NOR3, SNP)
inhibited the IL-1ß–induced NF-B activation and its nuclear
translocation, accompanied by inhibition of both iNOS mRNA and protein
expression. The present study has further shown that NO donor
blocked both the transient decrease in IB- levels as well as its
rapid phosphorylation induced by IL-1ß, whereas NO
donor added alone had no effect on basal IB- levels. Furthermore,
NO donor abolished the appearance of IL-1ß–induced
phosphorylated IB-, even in the presence of a
proteasome inhibitor, which stabilized IB-. These
results suggest that the inhibitory effect of NO on the
IL-1ß–induced iNOS expression is mediated via inhibition of NF-B
activation primarily due to its inhibition of IB-
phosphorylation rather than the proteasome-mediated
IB- degradation. Our data are in agreement with those of previous
reports showing the inhibitory effects of NO on
TNF-–induced NF-B activation in endothelial and
neuronal cells.26 27 However, our results appear
to be in contrast to 2 recent (though contradictory) reports from the
same laboratory.17 28 Liao and his associates
have shown that NO donors inhibited TNF-–induced NF-B activation
by induction and stabilization of IB- in human
endothelial cells; S-nitrosoglutathione
(GSNO) prevented IB- degradation 30 minutes after stimulation
with TNF-.17 The same group has subsequently
reported that GSNO did not prevent phosphorylation and
degradation of IB- 15 minutes after stimulation with TNF- in
the same cells, suggesting that NO inhibits NF-B activation by the
late (2 hours) induction and nuclear translocation of
IB-.28 In the present study, however,
NOR3 itself had no effect on basal IB- expression during a
75-minute incubation period. The exact reasons for the apparent
discrepancy between their reports and ours are unknown. This may be
accounted for by the different cell types, species, cytokines,
and NO donors used in the experiments. Among these, caution must be
paid to the physicochemical nature of NO donors used, because they
release NO with different kinetics and generate various metabolites
that may affect cell functions. NOR3,
(±)-(E)-ethyl-2-[(E)-hydroxyamino]-5-nitro-3-hexeneamide
(FK409), is a novel and potent NO donor that spontaneously releases NO
under neutral aqueous conditions with a half-life of 46
minutes.29 Although the possible involvement of
the metabolite cannot be excluded, concentration-dependent inhibition
of NF-B activation by NOR3, with its greater potency compared with
other NO donors (such as SNP and GSNO) as demonstrated in this study,
strongly suggests that the effect of NOR3 is due to the biologically
active NO molecule.
The exact intracellular signaling mechanism by which NO blocks
cytokine-induced NF-B activation remains unknown. To
address the question of whether the effect of NO is mediated by
guanylate cyclase activation and subsequent cGMP
generation, a cell-permeable cGMP analogue (8-bromo-cGMP) and a soluble
guanylate cyclase inhibitor (ODQ) were tested.
8-Bromo-cGMP did not inhibit IL-1ß–induced NF-B activation or
iNOS mRNA expression, whereas ODQ did not affect the NO-induced
inhibition of either NF-B activation or iNOS mRNA expression. These
results argue against an intermediate role of cGMP and the involvement
of cGMP-dependent protein kinase in the mechanism of NO inhibition of
both NF-B activation and iNOS expression.
NO modifies activities of several heme- and nonheme-containing enzymes
by direct nitrosylation. For example, NO interacts with the heme moiety
of soluble guanylate cyclase to stimulate its enzyme
activity, whereas NO inhibits enzyme activity of heme-containing
iNOS.30 NO also binds to nonheme-containing
enzymes, particularly to iron-sulfur clusters
(cis-aconitase, mitochondrial complexes I and II,
ribonucleotide reductase), to inhibit their enzymatic
activities.31 32 33 NO can also modify proteins by
nitrosylation of Cys residue to form
S-nitrosothiols.34 Therefore, it is
possible to speculate that the inhibitory effect of NO on
cytokine-induced NF-B activation may be due to its
direct nitrosylation of Cys residue(s) of IB- kinase to decrease
its enzymatic activity. It is also possible that NO may inhibit NF-B
by dephosphorylation of IB-, because NO has been
shown to activate protein phosphatases in
monocytes.35 In fact, okadaic acid, an
inhibitor of protein phosphatase 2A, has been shown to
activate NF-B and induce IB-
phosphorylation.36 NO may
function as an antioxidant to scavenge pro-oxidants, such as superoxide
anion and hydrogen peroxide, which in turn stimulate redox-sensitive
protein kinase(s) to activate NF-B.37
However, antioxidants, such as
N-acetyl-L-cysteine and
pyrrolidinedithiocarbamate, failed to block IL-1ß–induced NF-B
activation and IB- degradation in our cell culture (K.K. et al,
unpublished data, 1998).
The NO-induced suppression of cytokine-induced IB-
phosphorylation and degradation in rat VSMCs as
demonstrated in the present study, along with the direct inhibition
by NO on the enzyme activity of iNOS as recently
reported,30 may constitute an
auto-inhibitory mechanism to lessen the magnitude of
NO-induced deleterious effects by high-output iNOS. Excessive NO
production by augmented iNOS expression in the blood vessel,
such as in inflammation, atherosclerosis, and septic
shock, could be terminated not only via inhibition of iNOS enzyme
activity, but also via transcriptional inhibition of iNOS gene by NO
per se. Because NO inhibits cytokine-induced
endothelial expression of adhesion molecules (vascular
cell adhesion molecule-1, E-selectin, intercellular adhesion
molecule-1) and proinflammatory cytokines (IL-1ß, IL-8) via
inhibition of NF-B,38 the ability of NO to
inhibit NF-B to decrease iNOS gene expression in VSMCs may also
contribute to the prevention of atherogenesis and inflammation in the
vessel walls.
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Acknowledgments |
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This work was supported in part by grants-in-aid (Y.H.) from the Ministry of Education, Science and Culture, Japan, and from the Ministry of Health and Welfare, Japan.
Received December 11, 1997; accepted June 21, 1998.
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