©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Nitric Oxide Inhibits Macrophage-Colony Stimulating Factor Gene Transcription in Vascular Endothelial Cells (*)

Hai-Bing Peng , Tripathi B. Rajavashisth , Peter Libby , James K. Liao (§)

From the (1)Cardiovascular Division, Brigham & Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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- (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.


INTRODUCTION

The ()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.

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-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) .

The induction of various inflammatory cytokines important in atherogenesis requires activation of NF-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) .

Since cellular adhesion molecules and proinflammatory cytokines participate in atherogenesis and share common 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.


EXPERIMENTAL PROCEDURES

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.

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 [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[dIdC], 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. DNAprotein 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.


RESULTS

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-B Activation

Electrophoretic mobility shift assays demonstrated rapid and near-maximal activation of NF-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.


DISCUSSION

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 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.

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 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.

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 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.

NF-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) .

The activation of NF-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.

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-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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL02508 (to J. K. L.) and HL34636 (to P. L.) and an American Heart Association grant-in-aid award (to J. K. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Cardiovascular Division, Department of Medicine, 221 Longwood Ave., LMRC-307, Boston, MA 02115. Tel.: 617-732-6538; Fax: 617-732-6961.

The abbreviations used are: M-CSF, macrophage colony stimulating factor; NO, nitric oxide; GSNO, S-nitrosoglutathione; TNF, 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.


ACKNOWLEDGEMENTS

We thank Drs. G. Nabel for RSVp65 and RSVp50 expression vectors and H. Yamada and D. Kufe for M-CSF promoter constructs.


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