From the
To determine the mechanism(s) by which the endogenous mediator
nitric oxide (NO) inhibits the activation of transcription factor
NF-
Nitric oxide (NO) possesses many anti-atherogenic properties
including its ability to inhibit vascular smooth muscle cell
proliferation
(1) , reduce platelet aggregation
(2) , and
prevent monocyte chemotaxis and adhesion
(3) . Recent in vivo studies have shown that NO can attenuate endothelium-leukocyte
interactions and limit the extent of atherosclerotic
lesions
(4, 5) . Although many of the effects of NO are
attributed to cGMP-dependent pathways, the precise mechanism(s) by
which NO attenuates atherogenesis remain largely unknown. We have
recently demonstrated that NO reduces leukocyte attachment to the
endothelial surface by decreasing cytokine-induced expression of
endothelial vascular cell adhesion molecule-1 (designated VCAM-1),
endothelial leukocyte adhesion molecule-1 (ELAM-1 or E-selectin), and
to a lesser extent, intercellular adhesion molecule-1
(ICAM-1)
(6) . Furthermore, the expression of other
pro-inflammatory mediators such as interleukin-6 and interleukin-8 were
similarly inhibited by NO. All of these genes share specific DNA
binding motifs in their promoters for interaction with the
transcription factor, NF-
NF-
NF-
Several
lines of evidence suggest that NO may modulate I
We have shown that NO inhibits the activation of NF-
Although
NF-
NF-
Interestingly, vascular
endothelial cells can produce NO constitutively and both endothelial
and smooth muscle cells can express the inducible isoform of NO
synthase in response to cytokine stimulation
(41) . Thus, the net
activation of NF-
Endogenous levels of
constitutive NO production, however, are not sufficiently high to
inhibit TNF
Antioxidants such
as N-acetylcysteine or PDTC have also been shown to stabilize
the NF-
A novel finding
in this study is that a free radical such as NO can induce the
expression of a transcription factor inhibitor, I
In summary, we have
shown that NO can inhibit the activation of NF-
We are grateful to Gary Nabel (p65 and p50 cDNA),
Stephen Haskill (I
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
B, we stimulated human vascular endothelial cells with tumor
necrosis factor-
in the presence of two NO donors, sodium
nitroprusside and S-nitrosoglutathione. Electrophoretic
mobility shift assays demonstrated that both NO donors inhibited
NF-
B activation by tumor necrosis factor-
. This effect was
not mediated by guanylyl cyclase activation since the cGMP analogue
8-bromo-cGMP had no similar effect. Inhibition of endogenous
constitutive NO production by L-N-monomethylarginine,
however, activated NF-
B, suggesting tonic inhibition of NF-
B
under basal conditions. NO had little or no effects on other nuclear
binding proteins such as AP-1 and GATA. Immunoprecipitation studies
showed that NO stabilized the NF-
B inhibitor, I
B
, by
preventing its degradation from NF-
B. NO also increased the mRNA
expression of I
B
, but not NF-
B subunits, p65 or p50, and
transfection experiments with a chloramphenicol acetyltransferase
reporter gene linked to the I
B
promoter suggested
transcriptional induction of I
B
by NO. We propose that the
induction and stabilization of I
B
by NO are important
mechanisms by which NO inhibits NF-
B and attenuate atherogenesis.
B. Indeed, we found that NO's
inhibitory effects were mediated not via cGMP-dependent pathways but
instead via inhibition of NF-
B
(6) .
B was
initially described as a heterodimeric complex, which binds to specific
decameric sequences in the immunoglogulin
light chain
enhancer
(7) . Members of the mammalian NF-
B family possess
Rel homology domains necessary for dimerization, nuclear translocation,
and DNA binding. They can be divided into two groups based upon their
structure and function
(8) . The first group consists of p65 (Rel
A), c-Rel, and RelB, which contain transcriptional activation domains
necessary for gene induction
(9) . The second group consists of
p105 and p100, which upon proteolytic processing give rise to p50
(NF-
B1) and p52 (NF-
B2), respectively
(10) . The
carboxyl-terminal region of p105 and p100 share structural features
with the NF-
B inhibitor, I
B, and thus, functions to retain
NF-
B in the cytoplasm
(11, 12) . The mature
proteins, p50 and p52, however, can form functional dimers with members
of both groups
(8) . With the exception of RelB, which cannot
form homodimers, members of both groups can bind in a tissue-specific
manner as homo- or heterodimers to enhancer elements of target genes
(13). In vascular endothelial cells, the transcriptional induction of
vascular cell adhesion molecule-1 depends upon the activation of the
p65/p50 heterodimer
(14) .
B is sequestered in the
cytoplasm through its association with its inhibitors, p105 or
I
B-like proteins
(8, 10) . Although several
different I
B-like proteins have been identified such as
I
B
(MAD3)
(15) , I
B
(16) ,
I
B
,
(17) , Bcl-3
(18) , and unprocessed
p105
(11, 12) , the most well characterized is the 37-kDa
protein, I
B
(10) . Activation of NF-
B by cytokines
or oxidative stress requires either the degradation of its cytoplasmic
inhibitor I
B
or proteolytic cleavage of p105 through the
ubiquitin-proteasome pathway
(8, 19) . Recent studies
indicate that phosphorylation of I
B
or p105 is a key step in
targeting these inhibitors for degradation
(20, 21) .
Thus, factors that affect the phosphorylation and/or expression of
I
B
could modulate the activation of NF-
B.
B
. First, NO
has been shown to activate protein phosphatases in peripheral blood
mononuclear cells
(22) , leading to the possibility that NO may
inhibit NF-
B via dephosphorylation of I
B
. Second,
oxidants such as hydrogen peroxide have been shown to stimulate protein
kinase activity and activate NF-
B
(23, 24) . Because
NO can avidly scavenge superoxide anion, it can prevent superoxide
anion from forming its dimutation product, hydrogen
peroxide
(25) . Furthermore, under certain conditions, NO can act
as an electron donor or antioxidant
(26) . Antioxidants such as
N-acetylcysteine or pyrrolidine dithiocarbamate
(PDTC)
(
)
have been shown to inhibit the
activation of NF-
B by preventing the dissociation of the
NF-
B/I
B
complex
(27, 28) . Thus, we
hypothesized that NO inhibits NF-
B by modulating I
B
. The
purpose of this study was to determine the mechanism(s) by which this
occurs.
Materials
All standard culture reagents were
obtained from JRH Bioscience (Lenexa, KS). Glutathione, sodium nitrite,
sodium nitroprusside PDTC, 8-bromo-cGMP, and phenylmethylsulfonyl
fluoride were purchased from Sigma. S-Nitrosoglutathione
(GSNO) was synthesized from glutathione and sodium nitrite as
described
(29) . L-N-Monomethylarginine was
obtained from Calbiochem (San Diego, CA). Recombinant human TNF
was purchased from Endogen, Inc. (Boston, MA). The Limulus amebocyte lysate kinetic chromogenic assay for endotoxin was
performed by BioWhittaker (Walkersville, MD).
[
-
P]CTP (3000 Ci/mmol),
[
-
P]ATP (3000 Ci/mmol), and
[
H]chloramphenicol (37 Ci/mmol) were supplied by
DuPont NEN. The oligonucleotides corresponding to the palindromic
NF-
B, AP-1, and GATA consensus sequence and affinity-purified
rabbit polyclonal antisera to p65, p50, I
B
, c-Fos, c-Jun,
GATA-2, and GATA-3 were obtained from Santa Cruz Biotechnology (Santa
Cruz, CA). Nucleic acid and protein molecular weight markers were
purchased from Life Technologies, Inc. The antibody detection kit
(Enhanced Chemiluminescence) using horseradish peroxidase and luminol
was obtained from Amersham Corp. Nylon transfer membranes were
purchased from Schleicher & Schuell. The full-length human cDNA
probes for NF-
B subunits, p65 and p50, and I
B
were
generously provided by Gary Nabel (University of Michigan, Ann Arbor,
MI) and Stephen Haskill (University of North Carolina, Chapel Hill,
NC), respectively. The murine I
B
promoter linked to the
chloramphenicol acetyltransferase (CAT) reporter gene was generously
provided by P. Chiao and I. Verma (Salk Institute, La Jolla, CA).
Cell Culture
Human endothelial cells were
harvested from saphenous veins using Type II collagenase (Worthington
Biochemical Corp., Freehold, NJ) and grown to confluence in a culture
medium containing Medium 199, 20 mM HEPES, 50 µg/ml
endothelial cell growth serum (Collaborative Research Inc., Bedford,
MA), 100 µg/ml heparin sulfate, 5 mML-glutamine
(Life Technologies, Inc.), 5% fetal calf serum (HyClone, Logan, UT),
and antibiotic mixture of penicillin (100 units/ml)/streptomycin (100
µg/ml)/fungizone (1.25 µg/ml) as described
previously
(30) . They were characterized by Nomarski optical
microscopy (Zeiss ICM 405, 40 objective) and staining for
Factor VIII-related antigen
(31) . Only endothelial cells of less
than three passages were used. Cells were pretreated with NO donors for
30 min prior to stimulation with TNF
. Cellular viability was
determined by trypan blue exclusion.
Electrophoretic Mobility Shift Assay
Nuclear
extracts were prepared as described
(32) . The NF-B
oligonucleotide corresponding to the palindromic NF-
B consensus
sequence (AGTTGAGGGGACTTTCCCAGG) was end-labeled with
[
-
P]ATP and T4 polynucleotide kinase (New
England Biolabs), and purified by G-50 Sephadex columns (Pharmacia
Biotech Inc.). Nuclear extracts (10 µg) were added to
P-labeled NF-
B oligonucleotide (
20,000 cpm, 0.2
ng) in a buffer containing 2 µg of poly(dI
dC) (Boehringer
Mannheim), 10 µg of bovine serum albumin, 10 mM Tris-HCl
(pH 7.5), 50 mM NaCl, 1 mM dithiothreitol, 1
mM EDTA, and 5% glycerol (total volume of 20 µl).
DNA-protein complexes were resolved on 4% nondenaturing polyacrylamide
gel electrophoresed at 12 V/cm for 3 h in low ionic strength buffer
(0.5
TBE) at 4 °C. For supershift assays, the indicated
antibody (15 µg/ml) was added to the nuclear extracts from
TNF
-stimulated cells for 10 min prior to the addition of
radiolabeled probe. In some studies, GSNO or unlabeled NF-
B
oligonucleotide (20 ng) was added directly to the nuclear extracts from
TNF
-stimulated cells for 10 min prior to addition of radiolabeled
probe. The binding conditions for AP-1 (CGCTTGATGACT-CAGCCGGAA) and
GATA (CACTTGATAACAGAAAGTGATAACTCT) oligonucleotides were the same as
that for NF-
B except 50 mM KCl and 10 mM
MgCl
were substituted for 50 mM NaCl.
Immunoprecipitation of
I
Agarose-conjugated anti-p65 antibody (100 µg of
IgG/ml) was incubated with whole cell lysates (200 µg) or nuclear
extracts (100 µg) in 100 µl of immunoprecipitation buffer
containing NaCl (150 mM), Tris-HCl (50 mM, pH 7.4),
SDS (0.2%), and Triton X-100 (1%) for 16 h at 4°C with gentle
rotation. Preliminary studies indicated that all p65 was completely
precipitated by this procedure, since immunoblotting analysis of the
supernatant using the anti-p65 antibody did not reveal the presence of
65-kDa proteins. The immunoprecipitate was collected by centrifugation
at 12,000 B
g, washed twice with immunoprecipitation
buffer (pH 8.3) and once with NaCl (150 mM), Tris-HCl (50
mM, pH 7.4), and EDTA (5 mM), and then resuspended in
denaturing buffer containing Tris-HCl (125 mM, pH 6.8), SDS
(4%), glycerol (20%), and 2-mercaptoethanol (10%). The mixture was
placed briefly (1 min) in boiling water prior to centrifugation at
12,000
g for 10 min. The supernatants and known
molecular size markers (Life Technologies, Inc.) were separated by
SDS-polyacrylamide gel electrophoresis (12% running, 4% stacking gel).
Western Blotting
Proteins were electrophoretically
transferred onto Westran polyvinylidene difluoride membranes and
incubated overnight at 4 °C with blocking solution (5% skim milk in
PBS). Affinity-purified rabbit antibodies (0.4 µg of IgG/ml) to
NF-B subunits and I
B
were incubated with the blots
overnight at 4 °C in PBS buffer containing 0.1% Tween 20. The blots
were washed twice with PBS buffer and then treated with donkey
anti-rabbit antibody (1:4000 dilution) coupled to horseradish
peroxidase. Immunodetection was accomplished using the Enhanced
Chemiluminescence kit as described previously
(31) .
Northern Blotting
Total RNA was extracted by
guanidinium isothiocyanate and isolated by CsCl equilibrium
centrifugation as described
(30) . Equal amounts of total RNA (20
µg/lane) were separated by 1% formaldehyde-agarose gel
electrophoresis, transferred overnight onto nylon membranes by
capillary action, and baked for 2 h at 80 °C prior to
prehybridization for at least 4 h in a solution containing 5
SSC, 2.5
Denhardt's solution, 25 mM sodium
phosphate buffer (pH 6.5), 0.1% SDS, and 250 µg/ml salmon sperm
DNA. Radiolabeling of p65, p50, I
B
, and human
-actin
cDNA probe (ATCC 37997, Rockville, MD) was performed using random
hexamer priming with [
-
P]CTP and Klenow
fragment of DNA polymerase I (Pharmacia). The membranes were hybridized
separately with individual probes overnight at 45 °C in a solution
containing 50% formamide, 5
SSC, 2.5
Denhardt's
solution, 25 mM sodium phosphate buffer (pH 6.5), 0.1% SDS,
and 250 µg/ml salmon sperm DNA. All Northern blots were subjected
to stringent washing conditions (0.2
SSC, 0.1% SDS at 65
°C) prior to autoradiography with intensifying screen at -80
°C for 24-72 h.
Transient Transfections
The functional murine
IB
promoter (-1.6 kb upstream from transcriptional
initiation start site) linked to the CAT reporter gene
(pBS[-1.6 kb]CAT) was described previously by Chiao and
Verma
(33) . Bovine rather than human endothelial cells were used
because of their higher transfection efficiency. Cells (2
10
, 70% confluent) were transfected with 25 µg of
either the I
B
promoter construct, pSV2.CAT (SV40 early
promoter), or p.CAT (no promoter) using the calcium phosphate
precipitation method
(34) . As an internal control for
tranfection efficiency, pRSV.
GAL plasmid (10 µg) was
co-transfected in all experiments. Preliminary results using
-galactosidase staining indicate that cellular transfection
efficiency was approximately 12-15%. After 72 h, cells were
treated with GSNO (0.2 mM) and cellular extracts were prepared
12 h later using lysis buffer (100 µg/ml leupeptin, 50 µg/ml
aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM
EDTA, 5 mM EGTA, 100 mM NaCl, 5 mM Tris-HCl,
pH 7.4) and one freeze-thaw cycle. The supernatant was obtained after
centrifuging the extracts at 12,000
g for 10 min.
CAT Assays
CAT activity was determined by
incubating the supernatant (100 µl) with
[H]chloramphenicol (50 µCi/ml) and
n-butyryl coenzyme A (250 µg/ml) for 20 h at 37
°C
(35) . The n-butyryl
[
H]chloramphenicol was then separated from
unmodified chloramphenicol by xylene phase extraction and counted for 2
min in a liquid scintillation counter (Beckman LS1800). CAT activity
was calculated from a standard curve using various concentrations of
purified CAT (Promega).
-Galactosidase activity was assayed
spectophometrically (absorption at 410 nm) and compared to a standard
curve using known amounts of purified
-galactosidase (Sigma) as
described previously
(36) . The relative CAT activity was
calculated as the ratio of CAT to
-galactosidase activity. Each
experiment was performed three times in duplicate.
Data Analysis
All values are expressed as mean
± S.E. compared to controls and among separate experiments.
Paired and unpaired Student's t tests were employed to
determine the significance of changes in CAT activity. A significant
difference was taken for p values less than 0.05.
Cell Culture
Human saphenous vein endothelial
cells were confirmed by their morphological features (i.e. cuboidal, cobblestone, contact-inhibited) using phase-contrast
microscopy and immunofluorescent-staining with antibodies to Factor
VIII-related antigen. With the exception of transient transfections,
there were no observable adverse effects of any treatment modalities on
cellular confluence, morphology, and viability.
L-N-Monomethylarginine (L-NMA),
8-bromo-cGMP, and PDTC contain no detectable levels of endotoxin
(<0.01 ng/ml). GSNO had an endotoxin level of 0.02 ± 0.1
ng/ml.
Inhibition of NF-
Electrophoretic mobility shift assay demonstrated that
activation of NF-B Activation by Exogenous
NO
B by TNF
is attenuated by NO in a time- and
concentration-dependent manner (Fig. 1). Higher concentration of
GSNO (0.5 mM) was required to inhibit NF-
B after 2 h of
stimulation compared to that after 30 min, probably due to relatively
lower levels of NO released from GSNO after 2 h. GSNO was effective
only when added to whole cells rather than to nuclear extracts,
suggesting that NO did not interfere directly with NF-
B binding to
DNA and that mediators present in the intact cells were required to
mediate NO's inhibitory effect on NF-
B. The shifted bands
were specific for NF-
B since the addition of antibodies to
NF-
B subunits, p65 and p50, abolished the NF-
B band and
caused further gel retardation (supershift). Furthermore, the addition
of 100-fold excess unlabeled (cold) NF-
B oligonucleotide to the
nuclear extract specifically abolished the NF-
B band.
Figure 1:
Electrophoretic mobility shift assay
(EMSA) showing the effects of GSNO on NF-B activation by TNF
(10 ng/ml). GSNO was added either to whole cells or directly to nuclear
extracts (NE). Specificity was determined by antibodies (15
µg of IgG/ml) to p65 or p50 (Supershift) or excess
unlabeled (Cold) NF-
B oligonucleotide. This is a
representative assay from four separate
experiments.
Inhibition of NF-
Vascular endothelial cells possess constitutive NO synthase
activity
(30) . Inhibition of endogenous endothelial NO
production by L-NMA (1 mM) caused the activation of
NF-B Activation by Endogenous
NO
B without addition of cytokine, but did not further augment
NF-
B activation in TNF
-stimulated cells
(Fig. 2A). L-NMA reduced endogenous NO
production by more than 80%, as measured by the conversion of
[
H]arginine to
[
H]citrulline (data not shown). Activation of
NF-
B by L-NMA was not due to contamination of
L-NMA samples with bacterial LPS since endotoxin level was
undetectable using the Limulus amebocyte lysate chromogenic
assay (<0.01 ng/ml). Addition of exogenous NO (GSNO, 0.2
mM) to L-NMA-treated endothelial cells reduced
NF-
B activation, indicating that endogenous NO is physiologically
important in inhibiting NF-
B activation under basal conditions.
The permeable cGMP analogue 8-bromo-cGMP (1 mM) had no
appreciable effect on NF-
B activation, indicating that inhibition
of NF-
B by NO is mediated via a cGMP-independent pathway. Although
8-bromo-cGMP did not affect NF-
B, it was still able to stimulate
cGMP- and cAMP-dependent protein phosphorylation in endothelial cells
(data not shown).
Figure 2:
A, EMSA showing the effects of
L-NMA (1 mM) and 8-bromo-cGMP (cGMP, 1
mM) on NF-B activation in unstimulated and TNF
(10
ng/ml)-stimulated cells. This is a representative assay from 3 separate
experiments. B, EMSA showing NF-
B activation by TNF
(10 ng/ml, 2 h) in the absence (Control) and presence of
glutathione (GSH), GSNO, sodium nitroprusside (SNP, 1
mM), nitrite, or PDTC (0.2 mM). This is a
representative assay from three separate
experiments.
Activation of NF-B by TNF
was not
altered by glutathione or nitrite, the parent compounds used to
synthesize GSNO
(29) , suggesting that inhibition of NF-
B
was actually due to the NO released by our NO donors
(Fig. 2B). Both GSNO and another structurally different
NO-generating compound, sodium nitroprusside (1 mM), have
similar inhibitory effects on NF-
B. These findings make it
relatively unlikely that inhibition of NF-
B by the NO donors was
due to any metabolite other than NO. As a positive control, the
antioxidant and metal chelator, pyrrolidine dithiocarbamate (0.2
mM) also inhibited the activation of NF-
B as demonstrated
previously
(27) . The relative specificity of NO for NF-
B
compared to other transcription factors was demonstrated by the fact
that GSNO only minimally inhibited the activation of nuclear binding
protein AP-1 and did not affect the activation of nuclear binding
proteins GATA-2 or GATA-3 (Fig. 3).
Figure 3:
EMSA showing the effects of GSNO on AP-1
and GATA activation by TNF (10 ng/ml). Control (lanes1 and 6), TNF
(30 min) (lanes2 and 7), and TNF
+ GSNO (0.2
mM, 30 min) (lanes3 and
8). Specificity was determined by antibodies (15 µg of
IgG/ml) to c-Fos (lane4), c-Jun
(lane5), GATA-2 (lane9), and GATA-3 (lane10). This
is a representative assay from two separate
experiments.
Stabilization of I
Immunoprecipitation
studies with agarose-conjugated anti-p65 antibody followed by
immunoblotting demonstrated that stimulation by TNFB
resulted in
the loss of I
B
from NF-
B complex (p65 and p50 subunits)
after 30 min (Fig. 4). This loss of I
B
, however, was
not observed in the presence of GSNO. Similarly, stimulation with
TNF
caused a rapid disappearance of I
B
from whole cell
lysates at 30 min followed by reappearance after 2 h (Fig. 5). In
the presence of GSNO, there was no disappearance of I
B
but,
instead, an induction of I
B
in a time-dependent manner. These
results indicate that NO inhibited NF-
B by either stabilizing
and/or reducing I
B
degradation.
Figure 4:
Whole cell lysates (200 µg)
immunoprecipitated with agarose-conjugated p65 antibody, followed by
immunoblotting using combined p65, p50, and IB
antibodies.
Cells were stimulated for 30 min with TNF
(10 ng/ml) ± GSNO
(0.2 mM). Three separate studies yielded similar
results.
Figure 5:
Immunoblotting of whole cell lysates (100
µg) using IB
antibody showing the fate of I
B
after stimulation with TNF
(10 ng/ml) in the absence
(Control) or presence of GSNO (0.2 mM). This is
representative of three separate studies.
Induction of I
In a
concentration-dependent manner, NO increased the steady-state mRNA
expression of IB
B
without affecting the expression of
NF-
B subunits, p65 and p50, or
-actin at 24 h (Fig. 6).
Activation of NF-
B by TNF
has been shown previously to induce
the expression of I
B
(14) . The addition of GSNO
produced a smaller increase in I
B
expression at 30 min,
consistent with our hypothesis that NO inhibits NF-
B-mediated gene
transcription (Fig. 7). GSNO, however, produced a greater
increase in I
B
expression at subsequent time points compared
to TNF
alone. This is because activation of NF-
B induces the
expression of I
B
, which in turn, inhibits NF-
B and
decreases I
B
's own subsequent expression. Since the
induction of I
B
expression by NO is not mediated by
NF-
B, I
B
expression is not subjected to its own negative
autoregulatory control.
Figure 6:
Northern analyses (20 µg of total
RNA/lane) showing the concentration-dependent effects of GSNO (0.2
mM) on p50, p65, and IB
mRNA expression at 24 h. The
same blot was re-hybridized separately to each cDNA probe.
Hybridization to
-actin served as an internal control. Results of
three separate blots yielded similar
results.
Figure 7:
Northern analyses (20 µg of total
RNA/lane) showing the time-course of IB
mRNA expression after
stimulation with TNF
(10 ng/ml) in the absence (Control)
or presence of GSNO (0.2 mM). Three separate experiments
yielded similar results.
Transcriptional Activation of I
The
murine IB
B
promoter linked to the CAT reporter gene,
pBS[-1.6 kb]CAT, was used in transient transfection
studies. These studies were performed with bovine rather than human
endothelial cells due to higher transfectional effeciency with bovine
cells using the calcium phosphate precipitation method (data not
shown). The promoterless p.CAT produced essentially no relative CAT
activity either basally (50 ± 35) or after treatment with
TNF
(38 ± 34) or GSNO (25 ± 18) (Fig. 8). The
highly expressed pSV2.CAT containing the SV40 early promoter exhibited
a high level of relative CAT activity (1380 ± 258), which was
also unaffected by TNF
(1240 ± 235) or GSNO (1334 ±
302). The pBS[-1.6 kb]CAT had a basal relative CAT
activity of 206 ± 35. Treatment with GSNO caused a greater
increase in relative CAT activity compared to TNF
(2546 ±
383 versus 1112 ± 232, p < 0.05). The
combination of TNF
and GSNO produced a relative CAT activity of
3446 ± 383, which was significantly higher than that of GSNO
alone (p < 0.05), indicating that transcriptional induction
of I
B
by GSNO most likely occurred through transcription
factor(s) other than NF-
B.
Figure 8:
IB
promoter activity in bovine
aortic endothelial cells transfected with plasmid vectors containing
the CAT reporter gene linked to no promoter (p.CAT), the SV40 early
promoter (pSV2.CAT), and I
B
promoter
(pBS[-1.6]CAT). Cells were stimulated with TNF
(10
ng/ml) or GSNO (0.2 mM) for 12 h. CAT activity was
standardized to
-galactosidase activity (relative CAT activity).
Assays were performed three separate times in
duplicate.
B
through the induction and stabilization of the NF-
B inhibitor,
I
B
. The mechanism for NO's effect is independent of
guanylyl cyclase activation since treatment with cGMP analogue
8-bromo-cGMP did not affect NF-
B activation. Interestingly, GSNO
is effective only when added to whole cells rather than to nuclear
extracts, suggesting that NO does not interfere directly with the
physical binding of NF-
B to its cognate DNA and that NO requires
cellular mediator(s) for its inhibitory effect(s) on NF-
B. These
results are consistent with our finding that NO's inhibitory
effects on NF-
B is mediated by I
B
. The relative
selectivity of NO for NF-
B compared to AP-1, GATA-2, and GATA-3
also agrees with this conclusion since the activation of these other
nuclear binding factors are presumably not regulated by I
B
.
These results, therefore, provide a novel mechanism whereby NO could
down-regulate the expression of NF-
B-dependent pro-inflammatory
genes through induction and stabilization of I
B
.
B has been extensively studied in cells of the immune
system
(8) , recent evidence indicates that this pleiotropic
transcription factor also has importance in vascular biology,
especially in the area of transplantation arteriosclerosis in which
immunological regulation of NF-
B may modulate the expression of
pro-inflammatory mediators in monocytes/macrophages and vascular
endothelial and smooth muscle cells
(37) . A recent study by
Lander et al.
(22) reportedly shows that NO activates
rather than inhibits NF-
B in human peripheral blood mononuclear
cells. The discrepancy between their results and ours may be due to
differences in the response of mononuclear cells to NO-generating
compounds compared to that of vascular endothelial cells. Although NO
itself may inhibit NF-
B, NO may activate other signaling pathways
in mononuclear cells which may ultimately lead to the activation rather
than to the inhibition of NF-
B. Furthermore, we studied the
effects of NO-generating compounds on NF-
B in cytokine-stimulated
cells, whereas their findings were based upon the effects of
NO-generating compounds in unstimulated cells. Thus, it is possible
that depending on the conditions, NO may have dual regulatory effects
on NF-
B.
B is sequestered in the cytoplasm when
complexed to its inhibitor, I
B
, or as dimers with unprocessed
p105 or p100
(8, 10) . The p65 subunit serves to bind
I
B
to the NF-
B complex (38). Activation of NF-
B
involves phosphorylation of I
B
, followed by rapid degradation
of the inhibitory subunit via the ubiquitin-proteasome
pathway
(19, 39) . This is consistent with our finding
that stimulation by TNF
resulted in the loss of I
B
from
p65 and its initial disappearance from the cytosolic pool. Reappearance
of I
B
resulted from the induction of I
B
expression
by NF-
B
(40) . The loss of I
B
was prevented by NO,
which stabilizes the NF-
B/I
B
complex either through
replacement of lost I
B
or inhibition of I
B
phosphorylation and/or degradation.
B in vascular wall cells during inflammation
probably depends on a complex balance between stimulatory and
inhibitory factors. The finding that inhibition of endogenous
endothelial NO production by L-NMA could activate NF-
B
suggests that constitutively produced NO may play an important
physiologic role in tonically inhibiting the activation of NF-
B
under basal conditions. This is supported by in vivo findings
showing that inhibition of endogenous NO production by
L-nitroarginine methylester promotes endothelium-leukocyte
interactions, probably through the expression of NF-
B-dependent
adhesion molecules
(3, 4, 6) . Furthermore,
chronic supplementation of the nitric oxide precursor,
L-arginine, in diets of hypercholesterolemic rabbits improves
endothelial-dependent vasodilation and limits the extent of
atherosclerotic lesions
(5) .
-induced activation of NF-
B. Higher levels of NO
such as those encountered by endothelial cells at sites of inflammation
or the amount of NO achieved by our NO donors may be required to
suppress the activation of NF-
B by cytokines. Exposure of
macrophages and vascular smooth muscle cells to cytokines leads to
higher levels of NO generated by the inducible form of NO synthase
compared to that produced by endothelial cells under basal
condition
(41) . Furthermore, higher NO activity could be
achieved locally since endothelial cells are in close proximity to
these endogenous sources of inducible NO in vivo and the
possibility that NO could be modified into more stable and potent
adducts such as nitrosothiols
(42) . It is interesting to
speculate that higher levels of NO produced by macrophages and vascular
smooth muscle cells may be the mechanism by which NO production is
ultimately terminated since the induction of NO synthase in these cells
requires the activation of NF-
B
(41) .
B/I
B
complex through scavenging reactive oxygen
species such as superoxide anion, which may activate
NF-
B
(24, 42, 43) . Stimulation of NADPH
oxidase activity in leukocytes generates hydrogen peroxide from
superoxide anion and induces protein phosphorylation
(23) . It is
not known whether NF-
B activation induced by oxidative stress is
due to hydrogen peroxide-mediated I
B
phosphorylation.
Nevertheless, NO can bind superoxide anion with extremely high
affinity
(25) . Thus, one mechanism by which NO may stabilize
I
B
and inhibit NF-
B activation is through scavenging
superoxide anion, thereby decreasing its dimutation product, hydrogen
peroxide. In addition, NO itself may directly affect protein kinases
and/or phosphatases that regulate I
B
phosphorylation. Indeed,
recent studies have shown that NO can stimulate the activity of protein
tyrosine phosphatase(s)
(22) . It remains to be determined
whether this increase in phosphatase activity could lead to the
dephosphorylation and stabilization of I
B
.
B
.
Transfection studies using the I
B
promoter linked to the CAT
reporter gene suggests that this effect occurs at the transcriptional
level. NO had no effect on the expression of NF-
B subunits, p65
and p50. Previous analyses of the I
B
promoter have revealed
multiple functional
B sites necessary for transcriptional
induction by NF-
B
(44, 45) . This provides for an
inducible autoregulatory pathway for terminating the activation of
NF-
B
(33, 40) . However, the induction of
I
B
by NO is probably not mediated by NF-
B since NO
inhibits NF-
B. Further analyses of I
B
promoter will be
necessary to determine which DNA binding domain(s) constitute
NO's cis-regulatory element(s).
B through the
induction and stabilization of its inhibitor, I
B
. The ability
of endogenous NO to inhibit NF-
B provides new insight into the
mechanisms of NO's anti-inflammatory and anti-atherogenic
properties.
, tumor necrosis factor; kb, kilobase pair(s); PBS,
phosphate-buffered saline; L-NMA,
L-N-monomethylarginine.
B
cDNA), and I. Verma (I
B
promoter).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.