(Received for publication, January 22, 1996)
From the
Atherogenesis involves cellular immune responses and altered
vascular smooth muscle cell (SMC) function. Cytokines such as
interleukin (IL)-1 and interferon-
(IFN-
) may contribute
to this process by activating SMC. To determine whether the
anti-atherogenic mediator, nitric oxide (
NO), can
modulate cytokine-induced SMC activation, we investigated the effects
of various
NO-generating compounds on the expression
of intercellular and vascular cell adhesion molecules (ICAM-1 and
VCAM-1). Induction of ICAM-1 expression by IL-1
and VCAM-1
expression by IFN-
was attenuated by
NO donors
but not by cGMP analogues. Nuclear run-on assays and transfection
studies using various VCAM-1 promoter constructs linked to the
chloramphenicol acetyltransferase reporter gene showed that
NO repressed IFN-
-induced VCAM-1 gene
transcription, in part, through inhibition of nuclear factor-
B
(NF-
B). Electrophoretic mobility shift assay revealed that SMC
possess basal constitutive NF-
B activity, which was augmented by
treatment with IL-1
. In contrast, IFN-
induced and activated
interferon regulatory factor (IRF)-1 but had little effect on basal
constitutive NF-
B activity.
NO donors had no
inhibitory effect on IRF-1 activation but did inhibit basal and
IL-1
-stimulated NF-
B activation. These findings suggest that
the induction of ICAM-1 and VCAM-1 expression requires NF-
B
activation and that
NO attenuates IFN-
-induced
VCAM-1 expression primarily by inhibiting basal constitutive NF-
B
activity in SMC.
Atherosclerotic lesions contain proliferating intimal smooth
muscle cells (SMC) ()and cytokines such as tumor necrosis
factor (TNF)-
and interleukin
(IL)-1(1, 2, 3) . Although the involvement of
cytokines in atherogenesis is well established, their signaling events
leading to SMC activation and proliferation are still poorly
understood. Recent studies have suggested that many cytokines activate
the oxidant-sensitive transcription factor, nuclear factor-
B
(NF-
B)(4, 5) , which may be important in
mediating SMC activation and proliferation(6, 7) .
Activated SMC express proinflammatory genes such as intercellular and
vascular cell adhesion molecules (ICAM-1 and
VCAM-1)(8, 9) . Indeed, we have shown that cytokines
such as IL-1
and TNF-
can activate NF-
B and induce the
expression of VCAM-1 in human vascular endothelial cells(10) .
It is not known, however, whether
NO can similarly
modulate cytokine-induced NF-
B activity in SMC.
SMC responds to
endothelium-derived nitric oxide (NO), which has
emerged as an important modulator of vascular tone via stimulation of
soluble guanylyl cyclase(11, 12) . However,
NO may have other important effects on SMC such as
inhibition of SMC activation and
proliferation(13, 14) . Supplementation of L-arginine, the precursor of
NO, lessens the
extent of atherosclerosis in diet-induced hypercholesterolemic
rabbits(15) . In vivo transfer of the type III
NO synthase gene into balloon-injured vessels
decreases intimal SMC proliferation in rat carotid arteries (16) . These studies demonstrate that
NO can
antagonize the effects of cytokines and growth factors, in part, by
attenuating SMC activation and proliferation. Although the mechanism(s)
by which
NO exerts its inhibitory effect(s) on SMC is
not presently known, recent studies from our laboratory have indicated
that
NO can modulate endothelial activation via
cGMP-independent inhibition of cytokine-induced NF-
B
activation(17, 18) . Thus,
NO
production in the vessel wall may influence SMC not only in their
vasomotor functions, but also perhaps in their more prolonged
transcriptional responses to NF-
B activation by cytokines.
The
cellular immune response in atherosclerotic lesions is evidenced by the
marked infiltration of T-lymphocytes(19, 20) .
Although the precise role of T-lymphocytes in the vessel wall has not
been established, recent findings suggest that T-lymphocytes can
modulate SMC activation, in part, through the lymphokine,
interferon-gamma (IFN-)(21) . In contrast to cytokines
such as TNF-
and IL-1
, IFN-
is not known to activate
NF-
B or induce VCAM-1 expression in endothelial cells (22) . IFN-
, however, can potently induce the expression
of VCAM-1 and major histocompatability complex class II antigens in
SMC(21, 23, 24) . The signaling pathway for
IFN-
-stimulated responses involves the protein tyrosine
phosphorylation of signal transducers and activators of transcription
(STATs) and
-activating factor (GAF) (25, 26) .
Activation of GAF, in turn, induces the expression of another
transcription factor, interferon regulatory factor (IRF)-1, which binds
to the promoters of target genes containing the interferon-stimulated
response element (ISRE)(27) . The VCAM-1 promoter contains two
tandem
B sites located in close promixity to an ISRE
site(28, 29) . Recent studies have shown that IRF-1
synergizes with NF-
B in transactivating the VCAM-1
gene(30) .
Since SMC, but not endothelial cells, possess
basal constitutive NF-B/Rel-like
activity(6, 7, 31) , we hypothesized that the
presence of this basal constitutive NF-
B activity may contribute
to the differential responses of vascular wall cells to IFN-
. The
purpose of this study, therefore, was to determine the role of basal
constitutive NF-
B activity in mediating the effects of IFN-
and
NO on VCAM-1 expression in SMC. We found that
NO can modulate IFN-
-induced SMC activation
through its effects on basal constitutive NF-
B activity.
Cellular confluence was maintained for all treatment conditions. Cellular viability was assessed by morphology, cell number, DNA content, and trypan blue exclusion. The amount of DNA was measured by a Microfluor reader (Dynatech Laboratories, Inc., Chantilly, VA) using a fluorescent dye (Hoechst 33258) that binds specifically to DNA (Calbiochem).
Equal amounts (1 µg) of purified,
denatured full-length VCAM-1, human -tubulin (ATCC number 37855),
and linearized pGEM-3z cDNA were vacuum-transferred onto nylon
membranes using a slot blot apparatus (Schleicher & Schuell). The
membranes were baked and prehybridized as described for Northern blots.
The precipitated radiolabeled transcripts (
8
10
cpm) were resuspended in 2 ml of hybridization buffer containing
50% formamide, 5
SSC, 2.5
Denhardt's solution, 25
mM sodium phosphate buffer (pH 6.5), 0.1% SDS, and 250 mg/ml
salmon sperm DNA. Hybridization of radiolabeled transcripts to the
nylon membranes was carried out at 45 °C for 48 h. The membranes
were then washed with 1
SSC, 0.1% SDS for 1 h at 65 °C
prior to autoradiography for 72 h at -80 °C.
Figure 1:
The concentration-dependent effects of NO donors, SNP and SIN-1, on IL-1
(10
pg/ml)-induced ICAM-1 (A) and IFN-
- (1000 units/ml)
induced VCAM-1 surface expression (B) after 24 h as measured
by an enzyme immunofluorescent assay (percentage of expression relative
to cytokines alone). All experiments were performed three different
times with at least six replicates.
Figure 2:
Immunostaining of cultured SMC monolayer
showing surface expression of VCAM-1 in unstimulated SMC (control) or
SMC stimulated with IFN- (1000 units/ml) in the presence or
absence of
NO donors, SNP (10
M) and SIN-1 (10
M), at 24
h. A, control; B, IFN-
alone, C,
IFN-
and SNP; D, IFN-
and
SIN-1.
To
exclude possible cellular toxicity produced by the NO
donors, we examined their effects on cell number, DNA content, and
trypan blue exclusion. We found that neither SNP nor SIN-1, at
concentrations used in our study, significantly affected cellular
viability with respect to cell number, DNA content, and trypan blue
exclusion (Table 1). This result agrees with immunohistochemical
analyses showing that treatment with
NO donors did not
appreciably affect SMC morphology (Fig. 2).
Stimulation of
SMC with either IL-1 or IFN-
did not induce type II
NO synthase expression by Northern analyses or result
in increased
NO production by SMC as measured by
nitrite production (data not shown). In addition, activation of soluble
guanylyl cyclase by exogenous
NO did not contribute to
the observed decrease in cytokine-induced ICAM-1 and VCAM-1 expression,
since neither 8-bromo-cGMP (1 mM) nor dibutyryl-cGMP (1
mM) inhibited ICAM-1 or VCAM-1 surface expression (Table 2). In fact, there was a slight increase in ICAM-1 and
VCAM-1 expression with higher concentrations of 8-bromo-cGMP (0.1
mM to 1.0 mM). 8-bromo-cGMP (1 mM), however,
did stimulate cGMP- and probably cAMP-dependent protein kinase activity (Fig. 3).
Figure 3:
SDS-polyacrylamide gel electrophoresis
analysis (50 µg/lane) showing the concentration-dependent effects
of 8-bromo-cGMP on P
labeling of SMC cellular
proteins. Two separate experiments yielded similar
results.
Figure 4:
Northern analyses (20 µg total
RNA/lane) showing the concentration-dependent effects of SNP and SIN-1
on basal and IL-1- (10 pg/ml) stimulated ICAM-1 steady-state mRNA
levels at 6 h. RNA loading was determined by hybridization to SMC
-actin. Each blot is representative of three separate
experiments
Under basal culture
conditions, SMC express little or no VCAM-1. Exposure to IL-1
weakly induces and exposure to IFN-
strongly induces VCAM-1
(3.7-fold and 17.4-fold, respectively). In a time- and
concentration-dependent manner, both SNP (1 mM) and SIN-1 (1
mM) decreased IFN-
- (1000 units/ml) induced VCAM-1 mRNA
level, resulting in 83 ± 6% and 70 ± 5% reduction after 6
h, respectively (Fig. 5, A and B). Another
NO donor, GSNO, also decreased IFN-
-induced
VCAM-1 mRNA levels in a concentration-dependent manner (73, 53, and 35%
reduction with GSNO concentrations of 10
M,
10
M, and 10
M,
respectively). Neither glutathione (0.2 mM) nor sodium nitrite
(0.2 mM) alone significantly affected IFN-
-induced VCAM-1
mRNA levels (4 ± 3% and 8 ± 6% reduction, respectively.
Figure 5:
Northern analyses (20 µg total
RNA/lane) showing the concentration-dependent (A) and
time-dependent (B) effects of SNP and SIN-1 on IFN--
(1000 units/ml) induced VCAM-1 steady-state mRNA levels at 6 h. Equal
RNA loading for each experiment was verified by hybridization to
-actin. Experiments were performed three
times.
Figure 6:
A,
densitometric analyses of Northern blots (20 µg total RNA/lane)
showing the effects of IFN- (1000 units/ml) alone or in
combination with GSNO (0.2 mM) on VCAM-1 mRNA levels (relative
intensity) plotted logarithmically as a function of time. Time 0
represented the time actinomycin D was added and corresponded to 6 h
after treatment with IFN-
± GSNO. B, nuclear
run-on assay showing the effects of
NO (GSNO, 0.2
mM) on VCAM-1 gene transcription by IFN-
(1000 units/ml)
at 6 h. The pGEM and
-tubulin gene transcription served as
internal controls for nonspecific binding and standardization,
respectively.
Figure 7:
A, EMSA showing NF-B activity in SMC
under basal conditions (Control) and stimulated with IFN-
(1000 units/ml) or IL-1
(10 pg/ml) in the presence and absence of
NO (GSNO, 0.2 mM) at 2 h. Specificity was
determined by prior incubation with antibodies (15 µg of IgG/ml) to
p65 or p50. NS, nonspecific shifted bands. These experiments
were repeated three times with similar results. B, EMSA
showing induction of IRF-1 activity by IFN-
(1000 units/ml) after
2 h in the presence and absence of
NO (GSNO, 0.2
mM). Specificity was determined by an antibody (15 µg/ml
IgG) to IRF-1 and by excess unlabeled (cold) ISRE oligonucleotide (20
ng). Three separate experiments yielded similar
results.
Using the VCAM-1 ISRE oligonucleotide, several different antibodies
to p91 (STAT-1) failed to supershift any bands induced by
IFN-
(data not shown), suggesting that interferon-stimulated gene
factor-3 (ISGF-3) does not bind to the ISRE of VCAM-1 promoter and,
therefore, may play only a limited role in the transcriptional
activation of the VCAM-1 promoter by IFN-
. However, IFN-
did
induce IRF-1 in a cycloheximide-sensitive, time-dependent manner (data
not shown). The induction and activation of IRF-1 appeared no sooner
than 2 h after stimulation with IFN-
and was not inhibited by
treatment with
NO (Fig. 7B).
Figure 8:
VCAM-1 promoter constructs, F,
F
, and F
, showing putative cis-acting
elements. VCAM-1 promoter activity was assessed by CAT assays in human
SMC transfected with plasmid vectors containing no promoter (p.CAT),
the SV40 promoter (pSV2.CAT), and the indicated VCAM-1 promoter
constructs. Cells were then stimulated with IFN-
(1000 units/ml)
in the absence (control) or presence of GSNO (0.2 mM). The
promoter activity for each condition was standardized to
-galactosidase activity (relative CAT activity). The asterisk represented a significant change in promoter activity between
IFN-
alone and IFN-
with
NO (p <
0.05).
We have shown that NO can attenuate the
surface expression of ICAM-1 and VCAM-1 on SMC in response to
stimulation with IL-1
and IFN-
, respectively. The mechanism
for
NO's effect is independent of cGMP
production, occurs at the transcriptional level, and involves
inhibition of both basal constitutive and IL-1
-stimulated
NF-
B activity. These findings agree with our earlier findings that
NO decreases cytokine-induced endothelial expression
of VCAM-1 and ICAM-1 via inhibition of NF-
B activation (10) . However, SMC differ from endothelial cells in exhibiting
basal constitutive NF-
B activity(6, 18) . Indeed,
we observed a small amount of SMC activation under basal culture
conditions as exhibited by low levels of VCAM-1 mRNA expression, gene
transcription, and promoter activity. The presence of basal
constitutive NF-
B activity has also been shown to be important in
mediating SMC proliferation(7) .
Previous studies have shown
that NO inhibits SMC proliferation via a
cGMP-dependent mechanism(13, 14) . However, the
expression of ICAM-1 and VCAM-1 were not affected by increasing
concentrations of two different cGMP analogues that are able to
stimulate protein kinase activity. Indeed, several groups have shown
that
NO can exert non-cGMP-dependent effects on other
cell types such as platelets(36) , fibroblasts(37) ,
and macrophages(38) . Interestingly, the inhibitory effects of
NO on basal and stimulated NF-
B activation
resemble those of antioxidants such as N-acetylcysteine and
pyrrolidine dithiocarbamate(39, 40) . Antioxidants
have been shown to inhibit SMC proliferation, and at higher
concentrations they appear to induce SMC apoptosis(41) . SMC
did not exhibit any signs of cellular toxicity with the concentrations
of
NO donors used. Furthermore, the actual amount of
NO released was probably comparable with the levels
achieved by the continuous release of
NO from
cytokine-induced type II
NO synthase(42) .
Such localized high concentrations of
NO are readily
achieved within the vicinity of cytokine-activated SMC, endothelial
cells, or macrophages in atherosclerotic lesions.
Atherosclerotic
plaques contain a variety of cell types including SMC, macrophages, and
lymphocytes(1, 2, 20) . Immunohistochemical
analyses of cellular subtypes in plaques have revealed that most of the
lymphocytes are T-cells(19, 20) . IFN-, a major
product of activated T-cells, exerts a variety of paracrine effects on
neighboring cells and, thus, may modulate the evolution of
atherosclerotic lesions. For example, IFN-
can inhibit collagen
production by SMC(43) , augment the expression of major
histocompatability complex class I, and induce the expression of major
histocompatability complex class II antigens on endothelial cells and
SMC(24, 44) , and in combination with other
proinflammatory cytokines, it can induce apoptotic death of
SMC(45, 46) . Consequently, SMC within human and
experimental atheroma can express increased levels of ICAM-1 and
VCAM-1, indicating a state of activation compared with those in normal
vessels(47) . However, the expression of these adhesion
molecules on SMC in atheroma is quite heterogeneous(48) . This
may be attributed to the locally produced effects of cytokines and
endogenously released
NO or to a heterogeneous
population of intimal SMC that responds differently to external
stimuli. In any case, factors such as cytokines,
NO,
and antioxidants that can regulate the expression of ICAM-1 and VCAM-1
may modulate the course of atherogenesis.
IFN- activates at
least two transcription factors, ISGF-3 and IRF-1, which are capable of
binding to the ISRE(25, 27) . ISGF-3 is a multicomplex
DNA binding protein that contains the Janus kinase substrates, STATs
(p91/84, p113)(25) . Upon phosphorylation, ISGF-3 translocates
into the nucleus, where it can bind to the ISRE of target genes.
However, phosphorylation of p91 or GAF, but not p113, allows GAF to
migrate to the nucleus by itself and participate in DNA-binding
complexes that recognize a different DNA binding motif, the
-activated sequence(26) . The IRF-1 gene contains
-activated sequence elements in its promoter, and the expression
of IRF-1 is induced by activated GAF in response to IFN-
or
TNF-
(27, 30) . IRF-1 binds to ISRE sites in the
promoters of IFN-
/
, inducible type II
NO
synthase, and IFN-inducible genes such as VCAM-1 (27, 30) . The induction and activation of IRF-1 is
linked to tumor-suppressive properties and, in some instances, to the
induction of apoptosis following DNA damage or in response to
serum-depriving conditions(49) . In our study, we do not find
evidence of ISGF-3 binding to ISRE of the VCAM-1 promoter. However, the
induction and binding of IRF-1 to ISRE, although not sufficient by
itself, was necessary for the induction of VCAM-1 in response to
IFN-
.
The induction of VCAM-1 expression by IFN- also
required the two tandem
B motifs in the VCAM-1 promoter
constructs, F
and F
, and
NO's inhibitory effect on IFN-
-induced
VCAM-1 expression in SMC depends not on inhibition of IRF-1 induction
or activity but on inhibition of basal constitutive NF-
B activity.
These results indicate that basal constitutive NF-
B is necessary,
but by itself is only modestly sufficient to transactivate the VCAM-1
gene in SMC. A more robust transcriptional induction of the VCAM-1 gene
by IFN-
is mediated by the synergistic effects of basal
constitutive NF-
B and IFN-
-stimulated IRF-1. These results
are in agreement with a previous study showing that cooperativity
between IRF-1 and NF-
B is necessary and sufficient in
transactivating the VCAM-1 gene in vascular endothelial
cells(30) . Consequently, the inability of IFN-
to
stimulate VCAM-1 expression in endothelial cells may result from the
lack of basal constitutive NF-
B activity in endothelial
cells(18, 31) . Interestingly, endothelial cells, but
not SMC, have basal constitutive
NO production that
may render NF-
B inactive under basal conditions. Indeed, treatment
with the type III
NO synthase inhibitor, N
-arginine methyl ester, inhibits basal
NO production in endothelial cells and leads to the
activation of NF-
B(10, 17) .
In summary, we
have identified an important mechanism by which NO
inhibits IFN-
-induced VCAM-1 expression in SMC. Our findings add
to the evidence that
NO may be anti-atherogenic
through its inhibitory effects on not only cytokine-stimulated
NF-
B activation, but also on basal NF-
B activity. These
results provide new insights into how
NO may modulate
SMC inflammatory activation in a manner highly relevant to the
evolution of human atheroma.
Presented in abstract form at the 1993 Annual Scientific Meeting of the American Heart Association, Atlanta, GA, November 11, 1993.