From the
Macrophage-colony stimulating factor (M-CSF) contributes to
atherogenesis by regulating macrophage-derived foam cells in
atherosclerotic lesions. Here we report that nitric oxide (NO) inhibits
the expression of M-CSF in human vascular endothelial cells independent
of guanylyl cyclase activation. The induction of M-CSF mRNA expression
by either oxidized low density lipoprotein (ox-LDL) or tumor necrosis
factor-
The (
Nitric
oxide exerts many antiatherogenic actions via stimulation of guanylyl
cyclase activity(9, 10, 11) . Abnormal
endothelial-derived nitric oxide activity contributes to impaired
vascular responses in atherosclerotic vessels of humans and
animals(12, 13) . Inhibition of endogenous NO production
by N
The induction of
various inflammatory cytokines important in atherogenesis requires
activation of NF-
Since
cellular adhesion molecules and proinflammatory cytokines participate
in atherogenesis and share common
Seventy-two hours after
transfection, cellular extracts were prepared 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. CAT activity was determined by incubating the
cellular extracts (100 µl) with
[
We have shown that both endogenous and exogenous nitric oxide
(NO) can limit the expression of a proatherogenic cytokine,
macrophage-colony stimulating factor (M-CSF) induced by two
pro-atherogenic mediators, TNF
Actinomycin D studies and nuclear run-on assays
indicated that the regulation of M-CSF expression occurred at the level
of M-CSF gene transcription. Analyses of the M-CSF promoter revealed
that two tandem
Interestingly, co-transfection with p65 alone resulted in a greater
increase in M-CSF promoter activity compared to that achieved with the
combination of p65 and p50. Since the p50 homodimer can bind
NF-
The activation of NF-
Our
findings provide a novel antiatherogenic effect of NO which is
independent of its classically recognized effect on soluble guanylyl
cyclase. Although we report here the inhibitory effects of NO on
NF-
We thank Drs. G. Nabel for RSVp65 and RSVp50
expression vectors and H. Yamada and D. Kufe for M-CSF promoter
constructs.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(TNF
) was attenuated by NO donors, S-nitrosoglutathione (GSNO), sodium nitroprusside (SNP), and
3-morpholinosydnonimine, but not by cGMP analogues, glutathione, or
nitrite. Inhibition of endogenous NO production by N-monomethyl-L-arginine (L-NMA) also
increased M-CSF expression in control and TNF
-stimulated cells.
Nuclear run-on assays and transfection studies using M-CSF promoter
constructs linked to chloramphenicol acetyltransferase reporter gene
indicated that NO repressed M-CSF gene transcription through nuclear
factor-
B (NF-
B). Electrophoretic mobility shift assays
demonstrated that activation of NF-
B by L-NMA, ox-LDL,
and TNF
was attenuated by GSNO and SNP, but not by glutathione or
cGMP analogues. Since the induction of M-CSF expression depends upon
NF-
B activation, the ability of NO to inhibit NF-
B activation
and M-CSF expression may contribute to some of NO's
antiatherogenic properties.
)activation of mononuclear phagocytes in the
vessel wall is an important event in atherogenesis(1) .
Macrophage-colony stimulating factor (M-CSF)
regulates
macrophage growth (2) and differentiation (3) and may
contribute to the development of macrophage-derived foam cells in
atherosclerotic lesions (4). Expression of M-CSF in vascular
endothelial cells is induced by minimally modified low density
lipoprotein (LDL) (5) and various cytokines such as
interleukin-1 and TNF
(6) . Atherosclerotic lesions contain
both oxidized lipids (7) and inflammatory cytokines (8) which may induce the local expression of M-CSF. Indeed,
human and rabbit atherosclerotic lesions contain increased levels of
M-CSF compared to normal arterial
tissues(5, 6, 7) . Consequently, factors which
regulate the expression of M-CSF may modulate atherogenesis.
-nitro-L-arginine methyl ester
promotes vasoconstriction and endothelial-leukocyte adhesion, processes
which are mitigated, to some extent, by addition of cGMP
analogues(14, 15) . Furthermore, enriching the diets of
cholesterol-fed rabbits with L-arginine, the precursor of NO,
improves endothelial-dependent relaxation, reduces leukocyte attachment
to the endothelial surface, and limits the extent of atherosclerotic
lesions(16) . Although many effects of nitric oxide are
attributed to its stimulation of guanylyl cyclase, little is known
regarding other cellular pathway(s) mediated by nitric oxide. The
findings of our recent study indicate that the regulation of
endothelial vascular cell adhesion molecule-1 expression by NO is not
mediated by cGMP, but rather is associated with the inhibition of
nuclear binding protein, NF-
B(17) .
B(18, 19, 20) . NF-
B
was originally described as a heterodimeric cytosolic protein in
B-cells which, upon activation, translocated into the nucleus where it
binds to specific decameric sequences in the IgG
light chain
enhancer(21) . Subsequent studies have shown that this
pleiotropic binding protein can also activate viral enhancer elements
as well as transcriptionally induce the expression of many
proinflammatory cytokines and cellular adhesion
molecules(22, 23, 24) . The NF-
B family
includes p65, p105/p50, p100/p52, c-rel, and relB
which bind as homo- or heterodimers to promoter regions of target
genes(23, 24) . In endothelial cells, NF-
B consists
predominantly of the p65 and p50 heterodimer(25) .
B binding motifs in their
transcriptional promoters, we hypothesized that NO may regulate their
gene expression through NF-
B. This study, therefore, tested
whether NO could regulate the expression of an important proatherogenic
molecule, M-CSF, through NF-
B.
Materials
All standard culture reagents were
obtained from JRH Bioscience (Lenexa, KS). Glutathione, nitrite, sodium
nitroprusside, dimethyl sulfoxide, dithiothreitol, L-arginine,
heparin sulfate, cupric sulfate (CuSO), polymyxin B,
butylated hydroxytoluene, thiobarbituric acid, and
1,1,3,3-tetramethoxypropane, phenylmethylsulfonyl fluoride, and cGMP
analogues, 8-bromo-cGMP and dibutyryl cGMP, were purchased from Sigma.
GSNO was synthesized from glutathione and nitrite as described
previously(26) . Purified human low density lipoprotein (LDL,
Lot No. 730793) and N-monomethyl-L-arginine (L-NMA) were obtained from Calbiochem. The Limulus amebocyte lysate kinetic assay was performed by BioWhittaker
(Walkersville, MD). Recombinant human TNF
was purchased from
Endogen, Inc. (Boston, MA). [
-
P]CTP (3000
Ci/mmol), [
-
P]ATP (3000 Ci/mmol),
P
(1000 Ci/mmol), and
[
H]chloramphenicol (37 Ci/mmol) were supplied by
DuPont NEN. The oligonucleotide corresponding to the two tandem
B
sequences in the M-CSF promoter was synthesized by Genosys
Biotechnologies, Inc. (The Woodlands, TX). Rabbit polyclonal antisera
to NF-
B subunits, p65 and p50, were obtained from Santa Cruz
Biotechnologies (Santa Cruz, CA). Nylon transfer membranes were
purchased from Schleicher and Schuell. The expression vectors
containing the RSV promoter linked to NF-
B subunits, p65 and p50,
were kindly provided by G. Nabel (University of Michigan). The human
M-CSF promoter constructs linked to the chloramphenicol
acetyltransferase (CAT) reporter gene were generously provided by D.
Kufe (Dana Farber Cancer Institute, Boston, MA).
Cell Culture
Human saphenous vein and bovine
aortic endothelial cells were cultured and characterized as described
previously(27) . Only endothelial cells of less than three
passages were used. Cells were pretreated with NO donors for 30 min
prior to addition of LDL or TNF. Cellular viability was determined
by morphology and trypan blue exclusion.
Characterization of LDL
Native LDL (density
1.02-1.06 g/ml) from a single donor was isolated using a
sequential ultracentrifugation method in the presence of butylated
hydroxytoluene and polymyxin B as described previously(28) . Its
identity was confirmed by SDS-polyacrylamide gel electrophoresis.
Cholesterol, triglyceride, and protein content were determined as
described previously(27) . Oxidized LDL (80% lipid, 20% protein)
was prepared by exposing samples of native LDL to CuSO (5
µM) at 37 °C for 2 to 24 h. Both native and oxidized
LDL were dialyzed with three changes of sterile buffer (150 mM NaCl, 0.01% EDTA, and 100 µg/ml polymyxin B, pH 7.4) before
filtering through a 0.2-µm membrane. The degree of LDL oxidation
was estimated by measuring the amounts of thiobarbituric acid reactive
substances (TBARS) produced using a colorimetric assay standardized
with malondialdehyde(29) . The TBARS value is expressed as
nanomoles of malondialdehyde per mg of LDL protein.
Northern Blotting
RNA was extracted using
guanidinium isothiocyanate and purified by cesium chloride
ultracentrifugation (30). Equal amounts of total RNA (20 µg/lane)
were separated by 1.2% formaldehyde-agarose gel electrophoresis,
transferred overnight onto nitrocellulose membrane by capillary action,
and baked (72 °C) for 2 h prior to prehybridization. Radiolabeling
of a 1.8-kilobase human M-CSF cDNA probe was performed using random
hexamer priming with [-
P]CTP and a Klenow
fragment of DNA polymerase I (Pharmacia Biotech). The membranes and
probe were hybridized overnight at 52 °C in a buffer 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 and washed in 0.2
SSC, 0.1% SDS at
65 °C before autoradiography at -80 °C for 24-72 h.
All blots were subsequently rehybridized with
-actin cDNA probe as
an internal control (ATCC 37997, Rockville, MD).
Stimulation of cGMP-dependent Kinases
Confluent
endothelial cells (5 10
) were incubated with
P
(500 µCi) for 1 h prior to the addition
of 8-bromo-cGMP at the indicated concentrations and incubated for an
additional 1 h. The study was terminated by the addition of sodium
phosphate (50 mM), trichloroacetic acid (20%), and sodium
vanadate (1 mM). Cells were scraped and lysed by a Dounce
homogenizer. Protein concentrations from cellular extracts were
determined by the method of Lowry et al.(31) . Proteins
(50 µg) were suspended in denaturing buffer containing Tris-HCl
(125 mM, pH 6.8), SDS (4%), glycerol (20%), and
2-mercaptoethanol (10%) and centrifuged at 12,000
g for 10 min. The supernatants and known molecular weight markers
(Bethesda Research Laboratory) were separated by SDS-polyacrylamide gel
electrophoresis (10% running, 4% stacking gel). The gels were then
fixed with Coomassie Blue (0.4%), methanol (20%), and glacial acetic
acid (10%) and dried by a gel dryer before autoradiography at -70
°C for 12-24 h.
In Vitro Transcription Studies
Nuclei from
10 endothelial cells were prepared, and in vitro transcription with [
P]UTP was performed as
described(27) . Linearized plasmids (1 µg) containing M-CSF,
pGEM (Promega), and rat
-tubulin cDNAs were immobilized on nylon
membranes using a vacuum-transfer slot blot apparatus (Schleicher &
Schuell), and the membranes were hybridized to radiolabeled transcripts
(
5-8
10
cpm/ml) at 45 °C for 48 h in
a buffer 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. The membranes were
then washed with 1
SSC, 0.1% SDS for 1 h at 65 °C before
autoradiography for 72 h at -80 °C.
Transfection CAT Assays
For transient
transfections, bovine rather than human endothelial cells were used
because of their higher transfectional efficiency by the
calcium-phosphate precipitation method (32). Two different M-CSF
promoter constructs, [-565]M1 and
[-248]M4, linked to the chloramphenicol
acetyltransferase (CAT) gene were used(33) . Cells were
transfected with the indicated promoter constructs (30 µg): p.CAT
(no promoter), pSV2.CAT (SV40 early promoter), M1, or M4. Approximately
60 h after transfection, cells were treated with ox-LDL (50 µg/ml)
or TNF (10 ng/ml). For co-transfection studies with RSVp65 and
RSVp50, GSNO (0.2 mM) was added 12 h after transfection, and
media were changed and GSNO was renewed every 12 h. As an internal
control for transfection efficiency, pRSV.
GAL plasmid (10 µg)
was co-transfected in all experiments.
H]chloramphenicol (50 µCi/ml) and n-butyryl coenzyme A (250 µg/ml) for 20 h at 37 °C as
described previously(34) . The relative CAT activity was
calculated as the ratio of CAT to
-galactosidase activity. M-CSF
promoter activity (-fold induction) was expressed as the ratio of
relative CAT activity to the relative basal CAT activity of
[-565]M1.CAT. Each experiment was performed three times
in duplicate.
Electrophoretic Mobility Shift Assay
Nuclear
extracts were prepared as described(35) . The NF-B
oligonucleotide corresponding to the two tandem
B sites in the
M-CSF promoter (GGGGATTTTCAGGGCC TGGAGGGAAAGTCCCTT) was end-labeled
with [
-
P]ATP and T4 polynucleotide kinase
(New England Biolabs) and purified by Sephadex G-50 columns (Pharmacia
Biotech). Nuclear extracts (10 µg) were added to
P-labeled NF-
B oligonucleotide (
20,000 cpm, 0.2
ng) in buffer containing 2 µg of poly[dI
dC], 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. 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 for 10 min before addition of radiolabeled probe. In some
studies, GSNO or unlabeled NF-
B oligonucleotide (20 ng) was added
directly to the nuclear extracts 10 min prior to addition of
radiolabeled probe.
Data Analysis
Band intensities from Northern and in vitro transcription assay blots were analyzed
densitometrically by the National Institutes of Health IMAGE
program(36) . 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 and densitometric
measurements. A significant difference was considered for p values of less than 0.05.
Cell Culture
There were no observable adverse
effects of oxidized LDL, GSNO, or SNP on cellular morphology, and
cellular confluency (7
10
cells/T-150 cm
flask) and viability were maintained for all treatment conditions
described. LDL samples, L-NMA, and 8-bromo-cGMP had no
detectable levels of endotoxin (<0.10 unit/ml).
Characterization of LDL
The native LDL was
comprised of protein (6.2 ± 0.17 mg/ml), cholesterol (22
± 2.0 mg/ml), and triglyceride (1.6 ± 0.14 mg/ml). The
initial TBARS value was 0.2 ± 0.1 nmol/mg which increased to 1.6
± 0.4 nmol/mg and 2.3 ± 0.6 nmol/mg after 6 h and 24 h of
incubation with endothelial cells in Medium 199, respectively. LDL
samples which have been oxidized by exposure to CuSO (5
µM) exhibited TBARS values ranging from 2.6 ± 1.2
nmol/mg after 2 h to 24.2 ± 5.1 nmol/mg after 24 h.
Induction of M-CSF Expression
Northern analyses
revealed that TNF induced the mRNA expression of M-CSF in a
time-dependent manner with maximum induction occurring between 2 and 6
h after TNF
stimulation (Fig. 1A). Similarly,
oxidized LDL (50 µg/ml, TBARS 13.4 nmol/mg) also induced the mRNA
expression of M-CSF, but, compared to that of TNF
stimulation,
maximum induction was 2.7-fold lower and occurred later at 24 h (Fig. 1B). The induction of M-CSF depended upon the
degree of LDL oxidation as measured by the presence of thiobarbituric
acid reactive substances (TBARS) (Fig. 2). Mild to moderately
oxidized LDL (TBARS 7.3 to 13.4 nmol/mg) more potently stimulated M-CSF
expression than native LDL (TBARS 0.2 nmol/mg) or highly oxidized LDL
(TBARS 24.2 nmol/mg) after 12 h.
Figure 1:
Northern analyses (20 µg of total
RNA/lane) showing the time course of M-CSF mRNA expression in response
to TNF (10 ng/ml) (A) and oxidized LDL (50 µg/ml,
TBARS 13.4 nmol/mg) (B). RNA loading was determined by
hybridization to human
-actin. Each blot is representative of
three separate experiments.
Figure 2:
Northern analyses (20 µg of total
RNA/lane) showing the effects of native (50 µg/ml, TBARS 0.2
nmol/mg) and oxidized LDL (50 µg/ml) on M-CSF mRNA expression at 24
h with respect to oxidative modification (TBARS). The effect of
TNF on M-CSF mRNA expression at 6 h is shown for comparison. Equal
RNA loading for each experiment was verified by hybridization to
-actin. Experiments were performed
twice.
Effect of NO on M-CSF Expression
The induction of
M-CSF mRNA expression by oxidized LDL (50 µg/ml, TBARS 13.4
nmol/mg) or TNF (10 ng/ml) was attenuated by S-nitrosoglutathione (GSNO, 0.2 mM) in a
time-dependent manner (Fig. 3A). Two other structurally
different NO donors, sodium nitroprusside (SNP) and
3-morpholinosydnonimine, but not glutathione (GSH) or nitrite,
inhibited M-CSF mRNA expression induced by oxidized LDL (50 µg/ml,
TBARS 13.4 nmol/mg) indicating that this effect was likely due to NO (Fig. 3B).
Figure 3:
A,
Northern analyses (20 µg of total RNA/lane) showing the
time-dependent effects of GSNO (0.2 mM) on M-CSF mRNA
expression induced by ox-LDL (50 µg/ml, TBARS 13.4 nmol/mg) and
TNF (10 ng/ml). B, Northern analyses (20 µg of total
RNA/lane) showing the effects of glutathione (GSH, 0.2 mM),
sodium nitrite (NO
, 0.2 mM),
GSNO (0.2 mM), SNP (1 mM), and
3-morpholinosydnonimine (1 mM) on M-CSF mRNA expression
induced by ox-LDL (50 µg/ml, TBARS 13.4 nmol/mg) at 12 h. Equal RNA
loading was verified by hybridization to
-actin. Experiments were
performed twice with similar results.
Inhibition of endogenous NO production by L-NMA (1 mM) caused a 3.2-fold increase in M-CSF mRNA
expression compared to basal levels (Fig. 4A). Addition
of L-NMA to TNF-stimulated cells augmented M-CSF mRNA
expression by 3.3-fold compared to that of TNF
stimulation alone.
GSNO was effective in reducing both basal and TNF
-stimulated M-CSF
mRNA expression by 92% ± 12% and 68% ± 10%, respectively.
Treatment of control and TNF
-stimulated endothelial cells with the
cGMP analogue, 8-bromo-cGMP (1 mM), affected M-CSF mRNA
expression only minimally suggesting that the activation of endothelial
guanylyl cyclase does not mediate this inhibitory effect of NO. The
cGMP analogue, 8-bromo-cGMP, however, did stimulate cGMP- and,
possibly, cAMP-dependent protein kinase activity in a
concentration-dependent manner as demonstrated by increases in protein
phosphorylation in endothelial cells (Fig. 4B).
Figure 4:
A, Northern analyses (20 µg of total
RNA/lane) showing the effects of N-monomethyl-L-arginine (1 mM), 8-bromo-cGMP
(1 mM), and GSNO (0.2 mM) on unstimulated (Control) and TNF-stimulated endothelial cells after 6 h.
Equal RNA loading for each experiment was verified by hybridization to
-actin. Experiments were performed twice with similar results. B, SDS-polyacrylamide gel electrophoresis analysis (50
µg/lane) showing the effects of 8-bromo-cGMP on
P
labeling of cellular
proteins.
Effect of NO on M-CSF Gene Transcription
In the
presence of actinomycin D, GSNO (0.2 mM) did not alter the
post-transcriptional stability of M-CSF mRNA induced by TNF (Fig. 5A). The calculated half-life of M-CSF mRNA in the
presence and absence of GSNO (0.2 mM) was not significantly
different (7.5 ± 2.7 h versus 8.2 ± 2.4 h, p > 0.05). In vitro transcription studies showed a
moderate basal transcriptional activity of the M-CSF gene under
standard tissue culture conditions (Fig. 5B). Treatment
with oxidized LDL (50 µg/ml, TBARS 13.4 nmol/mg) or TNF
(10
ng/ml) increased M-CSF gene transcription 7.8- and 18-fold relative to
-tubulin gene transcription, respectively. NO essentially
abolished all transcriptional activity of the M-CSF gene induced by
oxidized LDL or TNF
, but did not substantially affect
-tubulin gene transcription. Preliminary studies using different
amounts of radiolabeled RNA transcripts demonstrate that under our
experimental conditions, hybridization was linear and nonsaturable. The
density of each M-CSF band was standardized to the density of its
corresponding
-tubulin band. The specificity of each band was
determined by the lack of hybridization to the nonspecific pGEM cDNA
vector.
Figure 5:
A,
densitometric analysis of Northern blots (20 µg of total RNA/lane)
showing the effects of TNF (10 ng/ml) alone or in combination with
GSNO (0.2 mM) on M-CSF 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 TNF
± GSNO. B, nuclear run-on assay showing
the effects of NO (GSNO, 0.2 mM) on M-CSF gene transcription
by ox-LDL (50 µg/ml, TBARS 13.4 nmol/mg) or TNF
(10 ng/ml) at
6 h. The pGEM and
-tubulin gene transcription served as internal
controls for nonspecific binding and standardization,
respectively.
Effect of NO on M-CSF Promoter Activity
To
characterize further the effects of NO on M-CSF gene transcription, we
transfected bovine aortic endothelial cells using two M-CSF promoter
constructs, M1 and M4, linked to the chloramphenicol acetyltransferase
(CAT) reporter gene(33) . Analyses of the M-CSF promoter
revealed putative DNA binding sequences for NF-B, SP1, SSRE
(shear-stress responsive element), ``CAT'' and
``TTAAA'' boxes, and initiation start site (Fig. 6A). [-565]M1 promoter contains
two tandem
B sites, while the deletional [-248]M4
promoter lacks these
B sites.
Figure 6:
A, M-CSF promoter CAT gene reporter
constructs, M1 and M4, showing putative DNA binding sequences for
NF-B, Sp1, SSRE (shear-stress responsive element), CAT and TATA
boxes, and initiation start site (arrow). B, M-CSF
promoter activity was assessed by CAT assays on bovine aortic
endothelial cells transfected with plasmid vectors containing no
promoter (p.CAT), the SV40 promoter (pSV2.CAT), and M-CSF promoter
constructs, M1 and M4. Cells were stimulated with ox-LDL (50 µg/ml,
TBARS 13.4 nmol/mg) and TNF
(10 ng/ml) or co-transfected with
RSVp65 and/or RSVp50 in the absence (Control) or presence of
GSNO (0.2 mM). The promoter activity for each condition was
standardized by
-galactosidase activity and expressed relative to
the basal (None) transcriptional activity of M1 (Fold
induction). The * represented a significant change in promoter
activity between control and GSNO (p <
0.05).
Stimulation with TNF (10
ng/ml) or oxidized LDL (50 µg/ml, TBARS 13.4 nmol/mg) increased M1
promoter activity by 9.2- and 7.5-fold, respectively (Fig. 6B). Co-transfection of M1.CAT with the expression
vector RSVp65 alone resulted in a 13-fold induction in M1 promoter
activity compared to a 5.7-fold induction with a combination of RSVp65
and RSVp50 and 2.2-fold induction with RSVp50 alone. Treatment with
GSNO (0.2 mM) was effective in decreasing M1 promoter activity
induced by TNF
(65% reduction), oxidized LDL (72% reduction), and
co-transfections with p65 alone (61% reduction) or in combination with
p50 (52% reduction). Basal M4 promoter activity was 1.8-fold lower than
that of basal M1. Co-transfection with RSVp65 with M4.CAT produced
essentially no promoter activity. Stimulation with TNF
or oxidized
LDL produced only a 2.5-fold induction of the non-
B containing M4
promoter activity. GSNO did not significantly affect M4 promoter
activity induced by TNF
, ox-LDL, or co-transfection with RSVp65.
Transcriptional repression was not due to general toxicity since GSNO
did not affect basal M1 and M4 or the SV40 promoter activity.
Effect of NO on NF-
Electrophoretic mobility shift assays demonstrated
rapid and near-maximal activation of NF-B
Activation
B by TNF
after 30 min (Fig. 7). In contrast, the degree of NF-
B activation by
native LDL (50 µg/ml, TBARS 0.2 nmol/mg) was smaller compared to
TNF
and occurred only after 6 h when the measured TBARS value was
1.6 nmol/mg, presumably secondary to endothelial cell modification of
native LDL. Activation of NF-
B by oxidized LDL (50 µg/ml,
TBARS 13.4 nmol/mg) occurred in a time-dependent manner, and, after 6
h, resemble that of TNF
after 30 min. GSNO (0.2 mM) and
sodium nitroprusside (SNP) attenuated the activation of NF-
B by
both oxidized LDL and TNF
( Fig. 7and Fig. 8).
Addition of 8-bromo-cGMP (1 mM) did not affect
TNF
-induced activation of NF-
B suggesting that NO's
inhibitory effect was not due to guanylyl cyclase activation.
Figure 7:
Electrophoretic mobility shift assay
showing the time-dependent effects of GSNO (0.2 mM) on
NF-B activation by TNF
(10 ng/ml), native (n) LDL
(50 µg/ml, TBARS 1.6 nmol/mg), or ox-LDL (50 µg/ml, TBARS 13.4
nmol/mg). Three separate experiments yielded similar
results.
Figure 8:
Electrophoretic mobility shift assay
showing the effects of 8-bromo-cGMP (1 mM), GSNO (0.2
mM), and SNP (1 mM) on NF-B activation by
TNF
(10 ng/ml) and L-NMA (1 mM) at 30 min. 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. These experiments were repeated three times with
similar results.
GSNO
was effective only when added to whole cells rather than directly to
nuclear extracts suggesting that NO does not interfere with NF-B
binding to DNA (Fig. 8). Activation of NF-
B was also
observed when endogenous NO production was inhibited by L-NMA
(1 mM). However, L-NMA produced NF-
B activation
to a lesser extent than did TNF
. Preliminary studies indicate that
treatment with L-NMA (1 mM) reduced basal NO synthase
activity by 80% (data not shown). Although the level of NF-
B
activation by L-NMA treatment was much less compared to that
caused by TNF
, L-NMA-induced NF-
B activation was
more completely abolished by treatment with GSNO (0.2 mM). The
indicated band was specific for NF-
B since, in the presence of
antibodies to p50 and p65, this band was ``supershifted'' and
attenuated.
and oxidized LDL. Since M-CSF may
contribute to the development of macrophage-derived foam
cells(4, 6) , inhibition of M-CSF expression by NO may
be one mechanism by which NO can attenuate atherogenesis. NO's
inhibitory effect on M-CSF mRNA expression was not mediated by
stimulation of guanylyl cyclase since cGMP analogues did not inhibit
M-CSF expression. This is in contrast to other antiatherogenic effects
of NO which are mediated by cGMP such as vascular smooth muscle
relaxation (12) and inhibition of platelet
aggregation(10) . Thus, our findings provide a novel mechanism
by which NO can modulate the expression of an important atherogenic
cytokine, M-CSF.
B binding sites located approximately 400 bp
upstream from the initiation start site were necessary for full
transcriptional induction by TNF
and oxidized LDL. These results
agree with previous studies showing the obligatory role of nuclear
binding protein NF-
B in transcriptionally activating the M-CSF
promoter(33) . However, the deletional construct lacking the
B sites still exhibits substantial promoter activity in response
to TNF
and oxidized LDL suggesting that other non-NF-
B
binding proteins could also participate and perhaps act synergistically
in transactivating the M-CSF promoter. Electrophoretic mobility shift
assays demonstrated that the transcriptional repression of the M-CSF
gene by NO was due principally to the inhibition of NF-
B
activation. NO did not physically inhibit the binding of NF-
B to
its cognate DNA since the addition of NO directly to nuclear extracts
of TNF
-stimulated cells did not affect the activation of
NF-
B. Thus, cellular factor(s) must be present in the intact cell
which mediate NO's inhibitory effect on NF-
B activation.
B
sites, but is a relatively weak transactivator(37) ,
co-transfection with p65 and p50 presumably leads to a competition
between the p65 homodimer with the p65/p50 heterodimer and the p50
homodimer for
B sites, resulting overall in less promoter activity
compared to that of the p65 homodimer. Furthermore, the ability of NO
to attenuate M-CSF promoter activity in cells co-transfected with
either p65 alone or in combination with p50 suggests that NO's
inhibitory effects are likely mediated through p65.
B
mediates transcriptional activation of the M-CSF gene in response to
TNF
(33) . Recent studies indicate that the activation of
NF-
B by TNF
and bacterial lipopolysaccharide involves the
generation of reactive oxygen species such as superoxide
anion(38) . Indeed, antioxidants such as N-acetylcysteine or pyrrolidine dithiocarbamate attenuate the
activation of NF-
B(38, 39) . NF-
B, therefore,
is an attractive candidate for inhibition by nitric oxide (NO) since,
under certain conditions, NO can function as an antioxidant through its
scavenging effects on superoxide anion(40, 41) . NO
interacts with superoxide anion to form peroxynitrite, thereby
diverting superoxide anion away from its dismutation product, hydrogen
peroxide(41) . Thus, less hydrogen peroxide would be available
to activate NF-
B(38, 39, 40) . However, in
the presence of NO, the consequence of peroxynitrite formation remains
to be determined, but can lead to tyrosine phosphorylation(42) .
B and increase in M-CSF expression also
occurred in the presence of the NO synthase inhibitor, L-NMA.
Endogenous NO production by the constitutive NO synthase may therefore
tonically inhibit the expression of proinflammatory genes through
suppression of NF-
B. Interestingly, both TNF
and oxidized LDL
decrease the expression of endothelial NO synthase (27, 43) which, in turn, may serve to augment the
activation of NF-
B. Treatment with NO donors produced further
inhibition of basal and TNF
-stimulated expression of M-CSF. Such
higher levels of NO may be encountered by endothelial cells at sites of
inflammation where induction of NO synthase activities in macrophages
and vascular smooth muscle cells could generate concentrations of NO
comparable to that given exogenously in this study(44) . In
solution, NO has a very short half-life(42) . However, in the
local environment of an inflammatory atherosclerotic lesion, NO can act
at short distances, given its intracellular origin and the close
proximity of endothelial cells to macrophages and vascular smooth
muscle cells and, thus, be less subject to inactivation.
B activation and M-CSF expression, these effects may extend to
other inflammatory cytokines and adhesion molecules which contain
functional
B sites in their transcriptional promoters. We propose
that NO is an important physiological mediator of both homeostasis and
inflammation.
, tumor necrosis factor
;
ox-LDL, oxidized low density lipoprotein; RSV, Rous sarcoma virus;
TBARS, thiobarbituric acid reactive substances; CAT, chloramphenicol
acetyltransferase; SNP, sodium nitroprusside; L-NMA, N-monomethyl-L-arginine.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.