©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Nitric Oxide Attenuates Vascular Smooth Muscle Cell Activation by Interferon-
THE ROLE OF CONSTITUTIVE NF-kappaB ACTIVITY (*)

(Received for publication, January 22, 1996)

Wee Soo Shin (§) Yi-Hui Hong Hai-Bing Peng Raffaele De Caterina (¶) Peter Libby James K. Liao (**)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Atherogenesis involves cellular immune responses and altered vascular smooth muscle cell (SMC) function. Cytokines such as interleukin (IL)-1alpha 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-1alpha 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-kappa B (NF-kappaB). Electrophoretic mobility shift assay revealed that SMC possess basal constitutive NF-kappaB activity, which was augmented by treatment with IL-1alpha. In contrast, IFN- induced and activated interferon regulatory factor (IRF)-1 but had little effect on basal constitutive NF-kappaB activity. NO donors had no inhibitory effect on IRF-1 activation but did inhibit basal and IL-1alpha-stimulated NF-kappaB activation. These findings suggest that the induction of ICAM-1 and VCAM-1 expression requires NF-kappaB activation and that NO attenuates IFN--induced VCAM-1 expression primarily by inhibiting basal constitutive NF-kappaB activity in SMC.


INTRODUCTION

Atherosclerotic lesions contain proliferating intimal smooth muscle cells (SMC) (^1)and cytokines such as tumor necrosis factor (TNF)-alpha 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-kappa B (NF-kappaB)(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-1alpha and TNF-alpha can activate NF-kappaB 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-kappaB 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-kappaB 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-kappaB 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-alpha and IL-1alpha, IFN- is not known to activate NF-kappaB 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 kappaB sites located in close promixity to an ISRE site(28, 29) . Recent studies have shown that IRF-1 synergizes with NF-kappaB in transactivating the VCAM-1 gene(30) .

Since SMC, but not endothelial cells, possess basal constitutive NF-kappaB/Rel-like activity(6, 7, 31) , we hypothesized that the presence of this basal constitutive NF-kappaB 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-kappaB 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-kappaB activity.


EXPERIMENTAL PROCEDURES

Materials

All standard culture reagents were obtained from JRH Bioscience (Lenexa, KS). Actinomycin D and cycloheximide were obtained from Calbiochem. [alpha-P]CTP (3000 Ci/mmol), [-P]ATP (3000 Ci/mmol), and [alpha-P]UTP (800 Ci/mmol) were supplied by DuPont NEN. Human IL-1alpha was kindly donated by Hoffmann-La Roche (Nutley, NJ), and human IFN- was purchased from Genzyme (Cambridge, MA). The NO donors, 3-morpholino sydnonimine (SIN-1) and diethylamine NONOate were obtained from Cayman Chemical Co. (Ann Arbor, MI). Sodium nitroprusside (SNP) was purchased from Elkins-Sinn (Cherry Hill, NJ). S-Nitrosoglutathione (GSNO) was synthesized as described previously(10) . All other agents, where not specified in the text, were purchased from Sigma. Oligonucleotides corresponding to kappaB and ISRE sites of the VCAM-1 promoter were synthesized by Genosys (Woodland Hills, TX). Antibodies to NF-kappaB subunits, p65 and p50, and IRF-1 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Murine monoclonal antibodies directed against human ICAM-1 (Hu5/3) and VCAM-1 (E1/6) were provided by M. A. Gimbrone, Jr. (Brigham & Women's Hospital, Boston, MA). The cDNA probes for human VCAM-1 and ICAM-1 were obtained from T. Collins (Brigham & Women's Hospital, Boston, MA) and T. Springer (Harvard Medical School, Boston, MA), respectively.

Cell Culture

SMC were isolated from outgrowths of tunica media explants derived from human aortic and saphenous vein tissues as described previously(32) . Cells were grown to confluence in Dulbecco's modified Eagle's medium containing 20 mM HEPES, 5 mML-glutamine (Life Technologies, Inc.), 10% fetal calf serum (Hyclone lot 1112288, Logan, UT), penicillin (100 units/ml), streptomycin (100 µg/ml), and Fungizone (1.25 µg/ml). The cells were characterized by phase contrast microscopy (Zeiss ICM 405, times 40 objective) and staining for SMC-specific alpha-actin. Only SMC of passage level of less than 6 were used. Before any treatment with cytokines or NO donors, SMC were rendered quiescent by incubating in insulin-transferrin media for 24 h(32) .

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).

Cell Surface Enzyme Immunoassay

Cytokine-stimulated SMC were cultured on 96-well Falcon plates (Lincoln Park, NJ), rinsed with phosphate-buffered saline and 2% fetal calf serum, and incubated with the indicated murine monoclonal antibody to human ICAM-1 and VCAM-1 for 2 h. After rinsing three times with phosphate-buffered saline, cells were incubated with biotinylated secondary antibody (horse anti-mouse IgG, Vector Labs, Inc., Burlingame, CA, 1:1000) for 1 h before incubation with streptavidin-alkaline phosphatase (Zymed, South San Francisco, CA) for 30 min. Cells were then treated with p-nitrophenylphosphate (1 µg/ml) for 30 min at room temperature. Light absorbance was measured in a plate reader (Dynatech Laboratories) at 410 nm, using cells without primary antibody as a blank.

Immunocytochemistry

SMC were cultured on 4-well Nunc culture plate (Naperville, IL) before treatment with IFN- (1000 units/ml) in the presence and absence of NO donors, SNP (10M) or SIN-1 (10M), for 24 h. After fixation with cold acetone, cells were incubated with the primary antibody (anti-human VCAM-1) at room temperature for 2 h, washed, and then incubated with biotinylated secondary antibody (horse anti-mouse IgG) for 1.5 h. Antibody detection was accomplished with avidin-biotin peroxidase complex (Vectorstain ABC kit, PK 6100, Vector Labs, Inc.), 3-amino-9-ethyl carbazole, and Gill's hematoxylin.

Stimulation of cGMP-dependent Kinases

Confluent SMC (5 times 10^6) were incubated with [P(i)] (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(33) . 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 times g for 10 min. The supernatants and known molecular weight markers (Life Technologies, Inc.) 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.

Northern Blotting

Equal amounts of total RNA (20 µg) from approximately 5 times 10^5 SMC were separated by 1% formaldehyde-agarose gel electrophoresis, transferred overnight onto nylon membranes by capillary action, and baked for 2 h at 80 °C. Radiolabeling of ICAM-1 (1.4-kilobase pair fragment from SalI/BglII digest), VCAM-1 (2.1-kilobase pair fragment from HindIII/SphI digest), or full-length alpha-actin cDNA probe was performed using random hexamer priming, [alpha-P]CTP, and DNA polymerase I (Klenow fragment, Pharmacia Biotech. Inc.). The membranes were hybridized with the probes overnight at 45 °C in a solution containing 50% formamide, 5 times SSC, 2.5 times 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 times SSC, 0.1% SDS at 65 °C) before autoradiography with an intensifying screen for 24-72 h at -80 °C.

Nuclear Run-on Assay

Confluent SMC (10^8 cells) were stimulated with IFN- (1000 units/ml) alone or in combination with GSNO (0.2 mM) for 4 h. Cells were subsequently washed twice with phosphate-buffered saline, trypsinized, and centrifuged at 300 times g for 5 min at 4 °C. The cellular pellet was gently resuspended in a buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl(2), and 0.5% Nonidet P-40, allowed to swell on ice for 15 min, and lysed by a Dounce homogenizer (60-70 strokes) with intermittent inspection of nuclei. The lysate was recentrifuged at 300 times g, and the resulting nuclear pellet was resuspended in 100 ml of buffer containing 20 mM Tris-HCl (pH 8.1), 75 mM NaCl, 0.5 mM EDTA, 1 mM dithiothreitol, and 50% glycerol. In vitro transcription using the nuclear pellet (100 µl) was performed in a shaking water bath at 30 °C for 30 min in a buffer containing 10 mM Tris-HCl (pH 8.0), 5 mM MgCl(2), 300 mM KCl, 50 mM EDTA, 1 mM dithiothreitol, 0.5 units of RNAsin (Promega, Madison, WI), 0.5 mM CTP, ATP, GTP, and 250 µCi [alpha-P]UTP as described previously(17) .

Equal amounts (1 µg) of purified, denatured full-length VCAM-1, human beta-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 times 10^7 cpm) were resuspended in 2 ml of hybridization buffer containing 50% formamide, 5 times SSC, 2.5 times 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 times SSC, 0.1% SDS for 1 h at 65 °C prior to autoradiography for 72 h at -80 °C.

Electrophoretic Mobility Shift Assay

Nuclear extracts were prepared as described(34) . Oigonucleotides corresponding to the kappaB (5`-TGCCCTGGGTTTCCCCTTGAAGGGATTTCCCTCC-3`) and ISRE (5`-GGAGTGAAATAGAAAGTCTGTG-3`) sites in the VCAM-1 promoter were radiolabeled with [-P]ATP and T(4) polynucleotide kinase (New England Biolabs) and purified by G-50 Sephadex columns (Pharmacia). Nuclear extracts (10 µg) were added to P-labeled oligonucleotides (20,000 cpm, 0.2 ng) in a buffer containing 4 µg of poly(dIbulletdC) (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 times TBE) at 4 °C. For supershift assays, the indicated antibody (15 µg/ml) was added to the nuclear extracts for 10 min before the addition of radiolabeled probe. To determine the specificity of shifted bands, excess unlabeled oligonucleotide (20 ng) was added directly to the nuclear extracts for 10 min before the addition of corresponding radiolabeled probe.

Transfection and Chloramphenicol Acetyltransferase (CAT) Assay

The human VCAM-1 promoter constructs containing the CAT reporter were previously described by Neish et al.(28) . Human SMC were transfected with each reporter plasmid (25 µg) using the calcium phosphate precipitation method(10) . As an internal control for transfection efficiency, pRSV.betaGAL plasmid (10 µg) was co-transfected in all experiments. Preliminary results using beta-galactosidase staining indicate that cellular transfection efficiency was approximately 15%. Cells (60-70% confluent) were stimulated 48 h after transfection with IFN- (1000 units/ml) in the presence and absence of 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 cellular extracts were centrifuged at 12,000 times g for 10 min, and the supernatant was subjected to CAT and beta-galactosidase assay as described previously(10, 17) . The relative CAT activity was calculated as the ratio of CAT to beta-galactosidase activity. Each experiment was performed three times in duplicate, and all experiments included both positive (highly expressed pSV40.CAT) and negative (promoterless p.CAT) controls.

Data Analysis

Band intensities from Northern blots, nuclear run-on assays, and electrophoretic mobility shift assay (EMSA) blots were analyzed densitometrically by the NIH Image program(35) . All values are expressed as mean ± S.E. compared with controls and among separate experiments. Paired and unpaired Student's t tests were employed to determine the significance of changes in absorbance values and densitometric measurements. p values of less than 0.05 were considered significant.


RESULTS

NO Donors Inhibit Cytokine-induced Expression of ICAM-1 and VCAM-1

IL-1alpha (10 pg/ml) produced a 4.2-fold increase in the surface expression of ICAM-1 on vascular SMC as determined by enzyme-linked immunoassay (data not shown). In a concentration-dependent manner, co-treatment with either SNP or SIN-1 decreased IL-1alpha-induced ICAM-1 expression by 94 ± 5% and 76 ± 6% after 24 h, respectively (Fig. 1A). Because IL-1alpha only marginally increased surface expression of VCAM-1 (1.8-fold increase), we used IFN- (1000 units/ml) to induce VCAM-1 expression more robustly (8.6-fold increase). Both SNP and SIN-1 attenuated IFN--induced VCAM-1 expression in a concentration-dependent manner resulting in a 52 ± 4% and 62 ± 5% reduction after 24 h, respectively (Fig. 1B). To exclude possible nonspecific effects of SNP and SIN-1, two other NO donors, GSNO and diethylamine NONOate, were used, which similarly decreased IFN--induced VCAM-1 expression after 24 h (data not shown). These results correlated with attenuated IFN--induced VCAM-1 surface expression as assessed by immunohistochemistry (Fig. 2). Interestingly, preincubation with SNP (1 mM) or SIN-1 (1 mM) for 1 h inhibited VCAM-1 expression (53 ± 7% and 51 ± 4%) more than did co-incubation (38 ± 6% and 38 ± 4%) (p < 0.01). These findings indicate that the regulation of VCAM-1 expression by NO occurs very early during IFN- induction and suggest that NO may affect VCAM-1 expression at the transcriptional level.


Figure 1: The concentration-dependent effects of NO donors, SNP and SIN-1, on IL-1alpha (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 (10M) and SIN-1 (10M), 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-1alpha 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(i) labeling of SMC cellular proteins. Two separate experiments yielded similar results.



NO Donors Decrease Cytokine-induced mRNA Expression of ICAM-1 and VCAM-1

Vascular SMC under basal culture conditions express low levels of ICAM-1. Treatment with IL-1alpha (10 pg/ml) augmented steady-state ICAM-1 mRNA levels by 5.1-fold (Fig. 4). In a concentration-dependent manner, SNP and SIN-1 inhibited both basal and IL-1alpha-induced ICAM-1 mRNA levels. After 6 h, SNP and SIN-1 reduced IL-1alpha-induced ICAM-1 mRNA levels by 98 ± 5% and 76 ± 5%, respectively.


Figure 4: Northern analyses (20 µg total RNA/lane) showing the concentration-dependent effects of SNP and SIN-1 on basal and IL-1alpha- (10 pg/ml) stimulated ICAM-1 steady-state mRNA levels at 6 h. RNA loading was determined by hybridization to SMC alpha-actin. Each blot is representative of three separate experiments



Under basal culture conditions, SMC express little or no VCAM-1. Exposure to IL-1alpha 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 10M, 10M, and 10M, 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 alpha-actin. Experiments were performed three times.



NO Represses IFN--induced VCAM-1 Gene Transcription

Actinomycin D studies showed that GSNO (0.2 mM) did not significantly affect VCAM-1 mRNA stability (half-life of 6.5 ± 2.1 h versus 7.1 ± 1.8 h, p > 0.05) (Fig. 6A). To confirm that GSNO decreases IFN--induced steady-state VCAM-1 mRNA levels by transcriptional repression, we performed nuclear run-on experiments using SMC stimulated with IFN- (1000 units/ml) for 4 h in the presence or absence of GSNO (0.2 mM) (Fig. 6B). Preliminary studies using different amounts of radiolabeled RNA transcripts demonstrate that under our experimental conditions, hybridization was linear and nonsaturable. The density of each VCAM-1 band was standardized to the density of its corresponding beta-tubulin band. The specificity of each band was determined by the lack of hybridization to the nonspecific pGEM cDNA vector. In unstimulated SMC (control), there was little basal VCAM-1 transcriptional activity. IFN- augmented VCAM-1 gene transcription by 20-fold. Co-treatment with GSNO (0.2 mM) resulted in only a 3-fold induction, indicating repression of VCAM-1 gene transcription by NO.


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 beta-tubulin gene transcription served as internal controls for nonspecific binding and standardization, respectively.



NO Inhibits Activation of NF-kappaB, but Not IRF-1

EMSA showed that under our basal culture conditions, there were two constitutive bands corresponding to NF-kappaB that were both ``supershifted'' in the presence of antibody to p65, whereas only the lower band was supershifted in the presence of antibody to p50 (Fig. 7A). These findings suggest that the composition of NF-kappaB binding to the tandem kappaB sites of the VCAM-1 promoter probably consists of the p65 homodimer (top band) and p50-p65 heterodimer (lower band). The anti-c-Rel antibody neither obliterated nor supershifted these basally active NF-kappaB bands (data not shown). IFN- (1000 units/ml) slightly augmented, while IL-1alpha (10 pg/ml) caused an increase in, basal constitutive NF-kappaB activation. Higher concentrations of IL-1alpha (0.1-10 ng/ml) produced an even greater activation of NF-kappaB (data not shown). Treatment with NO donors inhibited both basal constitutive and IL-1alpha- (10 pg/ml) stimulated NF-kappaB activation.


Figure 7: A, EMSA showing NF-kappaB activity in SMC under basal conditions (Control) and stimulated with IFN- (1000 units/ml) or IL-1alpha (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-1alpha) 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).

Induction of VCAM-1 Gene Transcription by IFN- Requires kappaB Enhancer Element

Transient transfection studies using various VCAM-1 promoter constructs (F(0), F(3), and F(4)) linked to the CAT reporter gene demonstrated that the two tandem kappaB enhancer elements in the VCAM-1 promoter are required for transcriptional induction and repression by IFN- and NO (Fig. 8). The promoterless p.CAT showed no basal or IFN--stimulated relative CAT activity (32 ± 21 and 34 ± 20). The highly expressed constitutive SV40 promoter (basal relative CAT activity of 1350 ± 210) showed no response to IFN- and NO (relative CAT activity of 1360 ± 198 and 1300 ± 254, p > 0.05). The induction and activation of IRF-1 by IFN- and its subsequent binding to the ISRE is not sufficient to transactivate [-44]F(4) (basal relative CAT activity of 54 ± 23 and IFN--stimulated CAT activity of 51 ± 22, p > 0.05). However, [-98]F(3) (basal relative CAT activity of 64 ± 28), which contains the kappaB enhancer elements exhibits responsiveness to IFN- and NO (relative CAT activity of 524 ± 34 and 204 ± 27, p < 0.05). A greater transcriptional response to IFN- and NO (relative CAT activity of 698 ± 60 and 273 ± 33, p < 0.05) may require other response elements contained in [-755]F(0) (basal relative CAT activity of 99 ± 11) such as AP-1 and GATA. Since IFN- does not activate NF-kappaB (Fig. 7A), these findings indicate that IRF-1 and basal constitutive NF-kappaB activity are necessary for the transcriptional induction of the VCAM-1 gene by IFN-. Furthermore, since NO does not affect IFN--induced IRF-1 activity (Fig. 7B), NO attenuates IFN--induced VCAM-1 expression via inhibition of basal constitutive NF-kappaB activity.


Figure 8: VCAM-1 promoter constructs, F(0), F(3), and F(4), 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 beta-galactosidase activity (relative CAT activity). The asterisk represented a significant change in promoter activity between IFN- alone and IFN- with NO (p < 0.05).




DISCUSSION

We have shown that NO can attenuate the surface expression of ICAM-1 and VCAM-1 on SMC in response to stimulation with IL-1alpha 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-1alpha-stimulated NF-kappaB 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-kappaB activation (10) . However, SMC differ from endothelial cells in exhibiting basal constitutive NF-kappaB 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-kappaB 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-kappaB 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-alpha(27, 30) . IRF-1 binds to ISRE sites in the promoters of IFN-alpha/beta, 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 kappaB motifs in the VCAM-1 promoter constructs, F(0) and F(3), 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-kappaB activity. These results indicate that basal constitutive NF-kappaB 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-kappaB and IFN--stimulated IRF-1. These results are in agreement with a previous study showing that cooperativity between IRF-1 and NF-kappaB 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-kappaB activity in endothelial cells(18, 31) . Interestingly, endothelial cells, but not SMC, have basal constitutive NO production that may render NF-kappaB 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-kappaB(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-kappaB activation, but also on basal NF-kappaB 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL-05280 and HL-52233 (to J. K. L.) and Grant HL-34636 (to P. L.) and by 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.

Presented in abstract form at the 1993 Annual Scientific Meeting of the American Heart Association, Atlanta, GA, November 11, 1993.

§
Present address: The Second Department of Medicine, University of Tokyo, Tokyo 113, Japan.

Present address: Consiglio Nazionale delle Ricerche Institute of Clinical Physiology, I-56100 Pisa, Italy.

**
To whom correspondence should be addressed: Cardiovascular Division, Brigham & Women's Hospital, 221 Longwood Ave., LMRC-307, Boston, MA 02115. Tel.: 617-732-6538; Fax: 617-264-6336; jkliao{at}bics.bwh.harvard.edu.

(^1)
The abbreviations used are: SMC, smooth muscle cell(s); TNF-alpha, tumor necrosis factor-alpha; IL, interleukin; ICAM, intercellular adhesion molecule; VCAM, vascular cell adhesion molecule; NO, nitric oxide; IFN-, interferon-; NF-kappaB, nuclear factor-kappaB; STAT, signal transducers and activators of transcription; GAF, -activating factor; IRF, interferon regulatory factor; ISRE, interferon-stimulated response element; SIN-1, 3-morpholino sydnonimine; SNP, sodium nitroprusside; GSNO, S-nitrosoglutathione; EMSA, electrophoretic mobility shift assay; ISGF, interferon-stimulated gene factor.


ACKNOWLEDGEMENTS

We thank Tucker Collins for VCAM-1 cDNA and promoter CAT constructs, Timothy A. Springer for ICAM-1 cDNA, and Michael A. Gimbrone Jr. for antibodies to ICAM-1 and VCAM-1.


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