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Endocrinology Vol. 140, No. 5 2183-2190
Copyright © 1999 by The Endocrine Society

ARTICLES

Differential Inhibitory Actions by Glucocorticoid and Aspirin on Cytokine-Induced Nitric Oxide Production in Vascular Smooth Muscle Cells1

Koichi Katsuyama, Masayoshi Shichiri, Hiroki Kato, Taihei Imai, Fumiaki Marumo and Yukio Hirata

Division of Endocrinology and Metabolism, the Second Department of Internal Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519, Japan

Address all correspondence and requests for reprints to: Dr. Yukio Hirata, Division of Endocrinology and Metabolism, Second Department of Internal Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoids and nonsteroidal antiinflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory and immune diseases. Nitric oxide (NO) has a diversity of physiological functions, but its excess production has been implicated in the inflammatory process. The present study was designed to elucidate the mechanisms by which glucocorticoids and NSAIDs affect inducible nitric oxide synthase (iNOS) expression in cultured rat vascular smooth muscle cells (VSMCs). Both interleukin (IL)-1ß and tumor necrosis factor (TNF)-{alpha} potently stimulated nitrite/nitrate (NOx) production with a concomitant expression of iNOS mRNA and protein as demonstrated by Northern and Western blot analysis, respectively. Both IL-1ß and TNF-{alpha} activated nuclear factor (NF)-{kappa}B as demonstrated by electrophoretic mobility shift assay. Dexamethasone, salicylate and aspirin, but not indomethacin, dose dependently inhibited cytokine-stimulated NOx production and iNOS protein expression. Dexamethasone decreased cytokine-induced NF-{kappa}B activation and iNOS mRNA expression, but neither salicylate nor aspirin affected NF-{kappa}B activation or iNOS mRNA expression. IL-1ß caused a rapid increase in phosphorylated I{kappa}B-{alpha} levels and subsequent transient decrease in I{kappa}B-{alpha} levels, an inhibitor of NF-{kappa}B, as revealed by Western blot analysis using specific antibodies for phosphorylated and nonphosphorylated I{kappa}B-{alpha}. These effects were blocked by pretreatment with dexamethasone. Aspirin dose dependently inhibited iNOS enzymatic activity, whereas salicylate and dexamethasone had limited effect. The present study demonstrates that 1) inhibitory effect of dexamethasone on cytokine-induced iNOS expression and NO production in rat VSMCs, although potentially acting at multiple levels, is partly mediated by inhibition of NF-{kappa}B activation resulting from decreased phosphorylation and degradation of I{kappa}B-{alpha}, 2) both salicylate and aspirin inhibit cytokine-stimulated NO production at translational and/or posttranslational levels without affecting NF-{kappa}B- mediated iNOS gene expression, and 3) aspirin directly inhibits iNOS enzyme activity. These data suggest the differential inhibitory mechanisms of iNOS-mediated NO synthesis by glucocorticoids and NSAIDs in the vasculature.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NITRIC OXIDE (NO) is synthesized from L-arginine by the catalytic action of NO synthases (NOS) (1, 2). NO appears to play diverse physiological roles, including vasodilation, neurotransmission, and mediation of immune responses. Two distinct types of NOS have been identified. One is a Ca2+/calmodulin-dependent and constitutive form and the other is a Ca2+- independent and cytokine-inducible form (1). Two constitutive NOS isoforms, neuronal (n)NOS and endothelial (e)NOS, can be rapidly activated by several Ca2+-mobilizing agonists and shear stress, whereas inducible NOS (iNOS) can be induced by bacterial lipopolysaccharide (LPS) and certain cytokines in a variety of cells. Vascular smooth muscle cells (VSMCs) do not produce NO under normal conditions. However, following stimulation with cytokines and LPS, augmented iNOS expression in VSMCs occurs, leading to excess NO production that causes a profound and intractable hypotension, a hallmark of endotoxic shock. High output NO also results in inhibition of cell proliferation and induction of apoptosis in VSMCs (3, 4), suggesting its role in the process of vascular remodeling and atherosclerosis (5).

The promoter region of the murine iNOS gene contains several potential cis-elements for the binding of different transcription factors, including two putative binding sites for nuclear factor-kappa B (NF-{kappa}B) (6, 7). NF-{kappa}B, a key regulator of immune system and inflammatory genes, is a heterodimeric complex, usually consisting of p50/p65 subunit. p50/p65 heterodimer associates with a cytoplasmic inhibitor, I{kappa}B, to form an inactive ternary complex. p65 subunit may also associate with precursor protein (p105) of p50 to form an inactive complex. Activation of NF-{kappa}B by LPS or cytokines requires either the degradation of I{kappa}B-{alpha} (8), or proteolytic cleavage of p105 through the ubiquitin-proteasome pathway after phosphorylation (9). Following degradation of I{kappa}B-{alpha}, an active heterodimer NF-{kappa}B translocates to the nucleus, binds to the consensus sequences of {kappa}B site, and activates gene expression. However, the exact role of I{kappa}B-{alpha} in mediation of NF-{kappa}B-dependent iNOS expression in response to cytokines in VSMCs has not been elucidated.

Glucocorticoids are potent inhibitors of immune responses and inflammatory process. NF-{kappa}B may be a key target for glucocorticoid-mediated immunosuppression because glucocorticoids have been reported to induce the synthesis of I{kappa}B-{alpha} and prevent NF-{kappa}B activation in monocytes and lymphocytes (10, 11). However, it has been reported that induction of I{kappa}B-{alpha} cannot account for glucocorticoid-mediated repression of NF-{kappa}B-dependent I{kappa}B-{alpha} expression in endothelial cells (12). Nonsteroidal antiinflammatory drugs (NSAIDs), such as sodium salicylate and aspirin, have also been shown to inhibit NF-{kappa}B activation by preventing phosphorylation and degradation of I{kappa}B in certain cells (13). Therefore, it is intriguing whether glucocorticoids and NSAIDs may inhibit cytokine-induced iNOS expression by modulation of NF-{kappa}B in VSMCs.

These observations led us to study whether 1) glucocorticoid (dexamethasone) and NSAIDs (salicylate and aspirin) inhibit cytokine-induced NO production through inhibition of NF-{kappa}B-mediated iNOS expression in rat VSMCs, 2) glucocorticoid affects cytokine-induced phosphorylation and degradation of I{kappa}B-{alpha}, and 3) glucocorticoid and NSAIDs affect iNOS enzymatic activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Human recombinant interleukin (IL)-1ß and tumor necrosis factor (TNF)-{alpha} was kindly provided by Otsuka Pharmaceutical (Osaka, Japan). Aspirin, sodium salicylate, indomethacin, calmodulin, FAD, sodium dodecyl sulfate (SDS), and EDTA were from Wako Pure Chemical Industries Ltd. (Osaka, Japan), NADPH from Oriental Chemical (Tokyo, Japan), tetrahydrobiopterin from Research Biochemical International (Natick, MA), phenylmethylsulphonyl fluoride (PMSF), dithiothreitol (DTT), poly (dI-dC), and L-arginine from Sigma Chemical Co. (St. Louis, MO), MG115 from Peptide Institute (Osaka, Japan), [{alpha}-32P] dCTP from Amersham International (Tokyo, Japan), dNTP and a Klenow fragment of DNA polymerase I from Takara Shuzo (Shiga, Japan).

Cell culture
VSMCs prepared by the explant method from the thoracic aorta of male Sprague Dawley rats (The Jackson Laboratory, Bar Harbor, ME) were cultured in DMEM containing 10% FCS at 37 C in a humidified atmosphere of 95% air-5% CO2 as described (14). Subcultured VSMCs (10–15th passages) were used in the experiments.

Determination of nitrite/nitrate (NOx)
Confluent VSMCs (106 cells/well), deprived of serum for 24 h and pretreated with or without dexamethasone or NSAIDs for 6 h, were stimulated with IL-1ß (10 ng/ml) or TNF-{alpha} (100 ng/ml) in serum-free DMEM for 15 h; NOx concentrations in the conditioned media were measured by an autoanalyzer (TCI-NOX 100, Tokyo Kasei Kogyo, Tokyo) as described (15). In brief, samples premixed with the carrier solution (0.07% EDTA and 0.3% NH4Cl) was passed through a copperized cadmium reduction column to reduce NO3- to NO2-, which reacts with Griess reagent (1% sulfonamide/0.1% N-1-naphtylethylenediamine dihydrochloride/5% HCl). Absorbance at 540 nm was measured by a flow-through visible spectrophotometer (model S/3250, Soma-Kogaku, Tokyo). NO3- was used as a standard.

Electrophoretic mobility shift assay (EMSA)
Confluent VSMCs (5 x 106 cells/dish), deprived of serum for 24 h and preincubated with or without dexamethasone or NSAIDs for 6 h, were treated with cytokines for 2 h, washed with ice-cold PBS and harvested in 0.4 ml ice-cold hypotonic lysis buffer (10 mM HEPES, pH 7.8, 10 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, 5 µg/ml leupeptin). After 15 min incubation, 25 µl 10% Nonidet P-40 was added and centrifuged at 10,000 x g for 1 min. The nuclei pellets from a single dish were collected, resuspended in 30 µl hypertonic extraction buffer (50 mM HEPES, pH 7.8, 50 mM KCl, 300 mM NaCl, 0.1 mM PMSF), centrifuged at 10,000 x g for 10 min, and the supernatant was subjected to EMSA as described (16). The single-stranded oligonucleotides (forward: 5'-TGGGGACTCTCC-3', complement: 5'-AAGGGAGAGTCC-3') corresponding to NF-{kappa}B binding site (-107 to -97) in the 5'-flanking region of the rat iNOS gene (17) were annealed at 65 C for 15 min and filled with [{alpha}-32P] dCTP (111 TBq/mmol), dNTP, and a Klenow fragment of DNA polymerase I. Nuclear proteins (10 µg) were incubated with 2000 cpm [32P]-labeled NF-{kappa}B double-stranded oligonucleotide and 1 µg poly (dI-dC) in EMSA buffer (10 mM Tris-HCl pH 7.5, 2% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 50 mM NaCl) for 30 min, loaded into a 5% polyacrylamide gel and run in TGE buffer (50 mM Tris, 0.38 M glycine, 2 mM EDTA, pH 8.5) at 150 V for 3 h. The gel was then dried and autoradiographed. To examine the specificity of the NF-{kappa}B binding protein, the gel shift assay was performed in parallel in the presence of a 100-fold excess of unlabeled oligonucleotide as a competitor. For supershift assay, nuclear protein was incubated for 30 min with goat polyclonal antibodies (2 µg IgG) against human NF-{kappa}B p50 or p65 subunit (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) before EMSA.

Northern blot analysis
Confluent VSMCs (5 x 106 cells/dish), deprived of serum for 24 h, were stimulated with cytokines for 6 h unless otherwise stated, and total RNAs were extracted from a single dish by the acid guanidinium thiocyanate-phenol-chloroform methods (18). Total RNAs (20 µg) from a single dish were separated by formaldehyde/1.1% agarose gel electrophoresis and transferred to a Magna Graph nylon membrane (Micron Separations Inc., Westboro, MA) by capillary elution. The probe for rat iNOS cDNA recently cloned from rat endothelial cells (19) was labeled with [{alpha}-32P] dCTP (111 TBq/mmol) by random-primed labeling method. RNA immobilized on the membrane was hybridized with the labeled probes, washed in 0.1 x SSPE/0.5% SDS, autoradiographed, and signals were quantitated using a BAS2000 Imaging Analyzer (Fuji Photo Film, Inc., Tokyo, Japan) as described (20).

Western blot analysis
After confluent VSMCs (5 x 106 cells/dish) were stimulated with cytokines for the indicated times, cells were lysed in 50 mM Tris-HCl, pH 6.8 (10% glycerol, 1% SDS, 1 µg/ml pepstatin, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 mM PMSF). After boiling cell lyates the protein content was determined with a BCA assay kit (Pierce Chemical Co., Rockford, IL). Extracted proteins (10 µg) from a single dish were separated on a 7.5% SDS-polyacrylamide gel and transferred to a Hybond ECL nitrocellulose membrane (Amersham International). Mouse monoclonal antibody for murine iNOS (Transduction Laboratories, Inc., Lexington, KY), rabbit polyclonal antibody for human I{kappa}B-{alpha} (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and rabbit polyclonal antibody for phospho-specific I{kappa}B-{alpha} (New England Biolabs, Inc., Beverly, MA) was applied at a 1:1000 dilution at 4 C overnight. After extensive washing, the secondary antibody (sheep antimouse Ig conjugated to horseradish peroxidase, Amersham) was applied at 1:500 dilution for 1 h, and exposure was performed by an ECL kit (Amersham) as described (16).

NOS enzymatic assay
NOS activity was determined by essentially the same method of citrulline assay (21) except for measurement of NO. Rat VSMCs stimulated with IL-1ß (10 ng/ml) for 15 h were homogenized in 50 mM Tris-HCl, pH 7.4 (0.5 mM EDTA, 0.5 mM EGTA, 1 µg/ml pepstatin, 2 µg/ml leupeptin, and 0.1 mM PMSF), centrifuged for 30 min at 10,000 x g, and the supernatant was used as crude enzyme preparations; protein concentration was measured with a BCA assay kit. Enzymatic reaction was performed at 37 C for 1 h in a final volume of 100 µl assay buffer (12.5 mM Tris-HCl, pH 7.4, 1 mM L-arginine, 1 mM NADPH, 10 µg/ml calmodulin, 9 µM tetrahydrobiopterin, 9 µM FAD, and 0.5 mM EGTA) containing cytosolic protein (100 µg) and test compounds. The reaction was terminated by adding 0.2 ml 20 mM HEPES buffer, pH 5.5, containing 2 mM EDTA, and the reaction mixtures were centrifuged for 5 min at 10,000 x g; concentrations of NOx in the supernatant were measured by an autoanalyzer as described (15).

Statistical analysis
All values of NOx concentrations were given as means ± SE of 3–4 dishes. Statistical analysis was performed by using analyses of variance for repeated measures. A P value less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of cytokines on NO production and iNOS expression in rat VSMCs
Both IL-1ß (10 ng/ml) and TNF-{alpha} (100 ng/ml) time-dependently (6–24 h) stimulated NOx production in rat VSMCs (Fig. 1AGo). Both cytokines induced iNOS mRNA expression which peaked after 6–12 h and then decreased after 24 h (Fig. 1BGo), whereas maximal iNOS protein expression by these cytokines was induced after 12 h (data not shown). Both IL-1ß (0.1–10 ng/ml) and TNF-{alpha} (1–100 ng/ml) dose dependently stimulated NOx production (15 h) as well as the expressions of iNOS mRNA (6 h) and protein (12 h) (data not shown). Therefore, subsequent studies were performed using maximal doses of IL-1ß (10 ng/ml) and TNF-{alpha} (100 ng/ml) for the indicated times.



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  		Figure 1. Effects of cytokines on NO production and iNOS mRNA expression in rat VSMCs. Confluent cells (106 cells/dish) were stimulated with IL-1ß (10 ng/ml) or TNF-{alpha} (100 ng/ml) for the indicated times. A, Concentrations of NOx released into media were measured. Each point represents the mean ± SE (n = 4); SE values are within each point. B, Northern blot analysis of total RNAs (10 µg) with rat iNOS cDNA as a probe; iNOS mRNA (upper panel); 28S ribosomal RNA (lower panel). Each lane is from a single dish. The blot is a representative of three essentially similar experiments.

 
Effects of glucocorticoid and NSAIDs on cytokine-induced NO production and iNOS expression
To determine whether glucocorticoids and NSAIDs suppress cytokine-stimulated NO production in rat VSMCs, the effects of a potent synthetic glucocorticoid (dexamethasone) and various NSAIDs (salicylate, aspirin, indomethacin) were tested. Dexamethasone dose dependently (0.01–1 µM) inhibited NOx production stimulated with IL-1ß (10 ng/ml) (Fig. 2AGo); a maximal inhibition (~75%) was induced with 1 µM. Dexamethasone (1 µM) reduced iNOS messenger RNA (mRNA) and protein expression induced by IL-1ß (Fig. 2BGo); the IL-1ß-induced iNOS mRNA levels in dexamethasone-treated cells were significantly (P < 0.01) reduced by 58.7 ± 5.1% compared with those in nontreated cells (n = 3) as measured by the relative ratio of mRNA/28S ribosomal RNA with a densitometer. The same inhibitory effects by dexamethasone were observed when TNF-{alpha} (100 ng/ml) was used as an inducer (data not shown).



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  		Figure 2. Effect of dexamethasone on IL-1ß-induced NO production and iNOS expression in rat VSMCs. A, Confluent cells pretreated with or without dexamethasone (DEX) in the indicated doses were stimulated with IL-1ß (10 ng/ml) for 15 h; concentrations of NOx released into media were measured. Each column with bar represents mean ± SE (n = 3). *P < 0.05 and **P < 0.01 vs. IL-1ß stimulation in the absence of DEX. B, Cells pretreated with DEX (1 µM) were stimulated with IL-1ß (10 ng/ml) for 6 h for Northern blotting and 12 h for Western blotting, respectively. Northern blot analysis for iNOS mRNA (top), 28S ribosomal RNA (middle), and Western blot analysis (bottom) for iNOS protein are shown. Each lane is from a single dish. Northern and Western blottings were performed two to three times with essentially similar results.

 
Both salicylate and aspirin dose dependently (1–20 mM) inhibited IL-1ß-stimulated NOx production (Fig. 3AGo); a maximal inhibition (50~70%) was induced with 20 mM. However, indomethacine (0.01–0.1 mM) did not affect IL-1ß-stimulated NOx production. Neither aspirin nor salicylate affected iNOS mRNA expression induced by IL-1ß, whereas both compounds dose dependently (10–20 mM) reduced IL-1ß-induced iNOS protein expression (Fig. 3BGo). Similar results were obtained when TNF-{alpha} was used as an inducer (data not shown). Neither cell viability nor cell morphology was affected by any of the above compounds at concentrations used in the experiments; the buffered-culture media containing even 20 mM aspirin remained neutral.



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  		Figure 3. Effects of NSAIDs on IL-1ß-induced NO production and iNOS expression in rat VSMCs. A, Confluent cells pretreated with or without indicated doses of sodium salicylate (NaSA), aspirin, or indomethacin (Indo) were stimulated with IL-1ß (10 ng/ml) for 15 h; concentrations of NOx released into media were measured. Each column with bar represents the mean ± SE (n = 3). *P < 0.05 and **P < 0.01 vs. IL-1ß stimulation in the absence of NSAIDs. B, Cells pretreated with NaSA (10–20 mM) or aspirin (10–20 mM) were stimulated with IL-1ß (10 ng/ml) for 6 h for Northern blotting and 12 h for Western blotting, respectively; each lane is from a single dish. Northern blot analysis for iNOS mRNA (top), 28S ribosomal RNA (middle), and Western blot analysis for iNOS protein (bottom) are shown. These Northern and Western blottings are representatives of two to three similar experiments.

 
Effects of glucocorticoid and NSAIDs on NF-{kappa}B activation by cytokines
Both IL-1ß and TNF-{alpha} induced NF-{kappa}B activation in rat VSMCs as early as 30 min, which persisted during 24 h as revealed by EMSA using synthetic oligonucleotides corresponding to the NF-{kappa}B site (-107 to -97) in the 5'-flanking region of the rat iNOS gene as a probe (Fig. 4Go). Addition of anti-p50 antibody resulted in the appearance of supershifted band, whereas addition of anti-p65 antibody caused a supershifted band in addition to the reduced shifted band (Fig. 4AGo), suggesting that NF-{kappa}B binding protein comprises p50/p65 heterodimer subunit immunologically similar, if not identical, to its human counterparts. Furthermore, the IL-1ß-induced NF-{kappa}B protein-DNA complex was completely eliminated by a 100-fold excess unlabeled oligomers (Fig. 4AGo), indicating the specificity of the NF-{kappa}B binding gel shift assay. The shifted band was markedly reduced by dexamethasone (1 µM) (Fig. 4BGo) but not affected by either aspirin or salicylate (20 mM) (Fig. 4CGo).



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  		Figure 4. Effects of dexamethasone and NSAIDs on IL-1ß-induced NF-{kappa}B activation in rat VSMCs. A, Confluent cells were stimulated with or without IL-1ß (10 ng/ml) for 2 h; the nuclear proteins were preincubated with anti-p50 and anti-p65 antibodies or excess unlabeled oligomers before EMSA. Cells pretreated with or without (B) dexamethasone (DEX, 1 µM), (C) sodium salicylate (NaSA, 20 mM) or aspirin (20 mM) were stimulated with IL-1ß (10 ng/ml) for 2 h, and the nuclear proteins were subjected to EMSA; each lane is from a single dish. EMSA was performed three times with essentially similar results. An arrow with a straight line shows the position of the shifted band; an arrow with a dotted line the supershifted band.

 
Effect of glucocorticoid on cytokine-induced I{kappa}B-{alpha} degradation and phosphorylation
Because cytokines activated NF-{kappa}B as demonstrated by EMSA, we studied whether cytokines affect degradation and phosphorylation of I{kappa}B-{alpha} in rat VSMCs by Western blot analysis using anti-I{kappa}B-{alpha} and antiphospho I{kappa}B-{alpha} antibodies, respectively. Addition of IL-1ß (10 ng/ml) resulted in a rapid (10–15 min) decrease in I{kappa}B-{alpha} protein levels, which then returned to baseline levels within 1 h (Fig. 5AGo). IL-1ß (10 ng/ml) also caused an immediate (within 5 min) increase in phosphorylated I{kappa}B-{alpha} levels, which then returned to the undetected level by 15 min (Fig. 5BGo). A proteasome inhibitor (MG115) completely prevented the transient decrease in I{kappa}B-{alpha}, but not the rapid increase in phosphorylated I{kappa}B-{alpha} induced by IL-1ß (data not shown). Similar results were obtained when TNF-{alpha} was used as a stimulant. These data suggest that both IL-1ß and TNF-{alpha} cause a rapid phosphorylation of Ser32 residue of I{kappa}B-{alpha} and its subsequent degradation via the ubiquitine/proteasome pathway in rat VSMCs.



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  		Figure 5. Effect of IL-1ß on degradation and phosphorylation of I{kappa}B-{alpha} in rat VSMCs. Confluent cells were incubated with IL-1ß (10 ng/ml) for the indicated time and the extract proteins were subjected to Western blot analysis using (A) anti-I{kappa}B-{alpha} and (B) antiphospho I{kappa}B-{alpha} antibodies, respectively; each lane is from a single dish. These Western blottings are representatives of two similar experiments.

 
Pretreatment with dexamethasone (1 µM) prevented the IL-1ß-induced transient decrease in I{kappa}B-{alpha} levels (Fig. 6AGo); dexamethasone added alone was without effect. Pretreatment with dexamethasone decreased the IL-1ß-induced rapid increase in phosphorylated I{kappa}B-{alpha} levels (Fig. 6BGo); dexamethasone added alone was without effect. These data suggest that dexamethasone inhibits cytokine-stimulated phosphorylated I{kappa}B-{alpha} before its degradation in rat VSMCs.



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  		Figure 6. Effect of dexamethasone on IL-1ß-induced degradation and phosphorylation of I{kappa}B-{alpha} in rat VSMCs. Confluent cells pretreated with or without dexamethasone (DEX, 1 µM) for 15 min were incubated with or without IL-1ß (10 ng/ml) (A) for 15 min for I{kappa}B-{alpha} protein and (B) for 5 min for phospho I{kappa}B-{alpha} protein as evaluated by Western blot analysis as in Fig. 5Go. These Western blottings are representatives of two similar experiments.

 
Effects of NSAIDs on enzymatic activity
Because salicylate and aspirin inhibit cytokine-stimulated NO production without affecting iNOS mRNA expression or NF-{kappa}B activation, we studied whether NSAIDs directly affect iNOS catalytic activity (Fig. 7Go). Aspirin dose dependently (1–20 mM) inhibited iNOS enzymatic activity; a marked inhibition (~40%) was induced at 1 mM and a maximal inhibition (~75%) at 20 mM. In contrast, the inhibitory effect of salicylate (20 mM) was minimal (~10%) and dexamethasone (1 µM) was without effect.



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  		Figure 7. Effects of NSAIDs and dexamethasone on iNOS enzymatic activity. Cytosolic fractions prepared from IL-1ß-treated VSMCs were used as a crude enzyme. NOS activity was measured as described in Materials and Methods. Each column with bar represents the mean ± SE (n = 3). *, P < 0.05, and **, P < 0.01 vs. control. NaSA, Sodium salicylate; DEX, dexamethasone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Functional analysis of the 5'-flanking region of murine iNOS promoter (-1.7 kb) revealed that the region between -48 and -209 bp containing NF-{kappa}B and NF/IL-6 sites is critical for inducibility by LPS, whereas the region between -1029 and -913 bp containing NF-{kappa}B, IRF-1, and ISRE/GAS sites mediates IFN-{gamma}-potentiation of LPS induction in mouse macrophages (6, 7). Of the two putative NF-{kappa}B binding sites, the consensus sequence between -965 and -955 bp is the most common in many genes, whereas that between -107 and -97 is unique, found in only iNOS gene of mouse, rat, and human (6, 7, 17, 22). Therefore, we performed EMSA using synthetic oligomer corresponding to the putative NF-{kappa}B site (-107 to -97) of the rat iNOS gene promoter region (17) as a probe to determine whether cytokines stimulate NF-{kappa}B-DNA binding activity in rat VSMCs. Both IL-1ß and TNF-{alpha} potently induced NF-{kappa}B activation in rat VSMCs; the specificity of NF-{kappa}-binding gel shift assay was confirmed by the complete disappearance of the shifted band in the presence of excess unlabeled oligomers as well as by the appearance of the supershifted band in the presence of specific antibodies for p50 and p65 subunits (16). The present study also confirmed that both IL-1ß and TNF-{alpha} time dependently and potently stimulated NO production in rat VSMCs with a concomitant iNOS mRNA and protein expression as demonstrated by Northern and Western blot analyses, respectively. These results are consistent with the previous studies showing that cytokines induce iNOS expression via activation of NF-{kappa}B in different cell types (23, 24, 25).

The present study showed that dexamethasone dose dependently inhibited IL-1ß-stimulated NO production in rat VSMCs with a maximal inhibition (-75%) at 1 µM, which also caused a modest inhibition (-60%) on IL-1ß-induced iNOS mRNA and protein expression as evaluated by Northern and Western blot analysis, respectively. Our data are consistent with those of previous studies using several cell types including VSMCs (25, 26, 27). Dexamethasone has been shown to be a potent inhibitor of iNOS but has transcriptional and translational effects on iNOS expression. For example, in rat mesangial cells, dexamethasone inhibited IL-1ß-induced NO production and iNOS expression mainly due to reduced translation and increased degradation, although it decreased transcriptional rate and increased mRNA half-life (28). In rat insulin-secreting islet cell line (RIN m5F), dexamethasone has been shown to inhibit IL-1ß-stimulated NO production without affecting iNOS mRNA levels (29). In rat VSMCs, however, it has been shown that dexamethasone did not inhibit IL-1ß-stimulated NO production, although it modestly decreased iNOS mRNA expression resulting from decreased transcriptional activity despite increased mRNA stability (30). In contrast, dexamethasone has been shown to completely inhibit cytokine-stimulated NO production with only a modest decrease in iNOS expression, mainly due to inhibition of tetrahydrobiopterin synthesis and L-arginine transport in rat microvessel endothelial cells, suggesting its posttranslational effects (31). Collectively, it is suggested that glucocorticoids act at multiple levels to regulate iNOS expression depending on cell types and species differences.

The present study further showed that dexamethasone (1 µM) also modestly inhibited NF-{kappa}B-DNA binding activity stimulated by IL-1ß, suggesting that glucocorticoids inhibit iNOS expression partly via its interference with NF-{kappa}B activation pathway. It is well known that glucocorticoids mediate immunosuppressive and antiinflammatory effects through a cytoplasmic receptor, glucocorticoid receptor (GR). Upon hormone binding, activated GR enters the nucleus, dimerizes, binds to specific DNA sequences, termed glucocorticoid response elements (GRE), and activates gene transcription (32). However, many genes involved in the inflammatory response, including iNOS gene, do not contain GRE in their promoters. Recent studies suggested that transcriptional repression by glucocorticoids is mediated by interference with interaction of GR with other transcription factors, such as AP-1 and NF-{kappa}B, through cross-coupling mechanism (33, 34).

The present study revealed that stimulation with IL-1ß caused a transient decrease in I{kappa}B-{alpha} levels preceded by a rapid (within 5 min) increase in phosphorylated I{kappa}B-{alpha} levels as demonstrated by Western blot analyses using anti-I{kappa}B-{alpha} antibody and anti-phospho Ser32 of I{kappa}B-{alpha} antibody, respectively. Cytokine-responsive I{kappa}B-{alpha} kinases recently identified (35, 36) phosphorylate two serine residues (Ser32 and Ser36) of I{kappa}B-{alpha} (37), whose phosphorylation is a prerequisite for polyubiquitination and subsequent degradation of I{kappa}B-{alpha} by 26S proteasome (38). It has been shown that glucocorticoids induce up-regulation of I{kappa}B-{alpha} that renders active NF-{kappa}B heterodimer into an inactive cytoplasmic ternary complex (Fig. 8Go) in certain cells, such as monocytes, lymphocytes (10, 11), and rat hepatocytes (39). In contrast, induction of I{kappa}B-{alpha} synthesis is not involved in dexamethasone-mediated inhibition of NF-{kappa}B activity in human endothelial cells (12) and alveolar epithelial cells (40), suggesting the importance of protein-protein interaction between NF-{kappa}B and GR in repression of NF-{kappa}B activity (Fig. 8Go). In the present study, dexamethasone did not up-regulate I{kappa}B-{alpha} expression but prevented IL-1ß-induced increase in phosphorylated I{kappa}B-{alpha} and subsequent decrease in I{kappa}B-{alpha} in rat VSMCs. Taken together, it is suggested that IL-1ß rapidly stimulates I{kappa}B-{alpha} kinase to phosphorylate I{kappa}B-{alpha} and subsequent proteasome-mediated degradation of I{kappa}B-{alpha}, thereby leading to NF-{kappa}B activation, and that inhibitory effect of dexamethasone on the IL-1ß-induced NF-{kappa}B activation is in part due to inhibition of I{kappa}B-{alpha} phosphorylation and its subsequent degradation in rat VSMCs (Fig. 8Go). However, the question as to whether glucocorticoids directly affect I{kappa}B-{alpha} kinase activity and/or the upstream signals following cytokine stimulation remains unknown.



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  		Figure 8. The possible multiple sites of inhibitory actions by glucocorticoids and NSAIDs on cytokine-stimulated NO production in rat VSMC.

 
NSAIDs, such as salicylate and aspirin, inhibit production of prostaglandin E2 and thromboxan A2, two major mediators of inflammatory and immune responses, by blocking cyclooxygenases (COX) (41). This explains the mechanism of action by NSAIDs for treatment of immunological and inflammatory diseases. It has recently been reported that salicylate and aspirin inhibit cytokine-induced NF-{kappa}B activation in hematopoietic progenitor cells (42), cerebellar granule cells (43), and human Jurkat T cells and mouse pre-B cell line (13), by preventing phosphorylation and degradation of I{kappa}B-{alpha}. The present study clearly revealed that cytokine-stimulated NO production in rat VSMCs was dose dependently inhibited by salicylate and aspirin but not by indomethacin. However, neither salicylate nor aspirin affected the cytokine-induced NF-{kappa}B activation or iNOS mRNA expression in rat VSMCs. Our results appear to be consistent with those of most studies in which salicylate and aspirin inhibited cytokine-induced NO production without affecting iNOS mRNA expression, such as in mouse macrophages (44), rat hepatocytes, (45) and rat pancreatic ß-cells (RIN m5F) (46). In contrast, however, salicylate and aspirin have been shown to suppress cytokine-induced iNOS mRNA expression and NO production without affecting NF-{kappa}B activation in rat cardiac fibroblasts (47, 48). The apparent discrepancies between the several results including ours suggest that NF-{kappa}B activation pathway may be differentially modulated by NSAIDs in a cell- or tissue-specific manner.

Both salicylate and aspirin dose dependently (10–20 mM) decreased iNOS protein expression in rat VSMCs. Although the concentrations of both compounds used in this study were supraphysiological compared with the therapeutic plasma concentrations (1–3 mM) such as in patients with rheumatoid arthritis, they did not cause nonspecific or cytotoxic effect in our study, and pH of the media containing aspirin remained neutral even after 15 h incubation. Our data are consistent with those of mouse macrophages (44), rat hepatocyte (45), and rat pancreatic ß-cells (46) in which aspirin and salicylate in supraphysiological concentrations (2–20 mM) markedly reduced cytokine-induced iNOS protein expression. Collectively, these data suggest that NSAIDs may decrease the translational rate and/or promote the degradation of iNOS protein in rat VSMCs (Fig. 8Go).

By measuring enzymatic activity, we found that aspirin directly inhibits iNOS catalytic activity in a dose-dependent manner, whereas salicylate and dexamethasone were without effect. It should be noted that aspirin as low as 1 mM markedly (-40%) decreased iNOS enzyme activity. Our data are consistent with those of mouse macrophages showing that aspirin and N-acetylimidazole, an acetylating agent, but not salicylate or indomethacin, directly interfered with the catalytic activity of iNOS (Fig. 8Go) (44). Although aspirin has been shown to suppress the catalytic activity of COX by acetylating a Ser residue (49), it remains unknown whether the inhibitory effect by aspirin on iNOS catalytic activity involves acetylating functional components of the enzyme and/or interference with interaction with its essential cofactors, such as tetrahydrobiopterin and heme moiety.

The possible mechanisms underlying the inhibitory effects of glucocorticoids and NSAIDs on cytokine-induced NO production in rat VSMCs as revealed from the present study are summarized in Fig. 8Go. The inhibitory effect by glucocorticoids on cytokine-stimulated NO production and iNOS expression in rat VSMCs, although potentially acting at multiple levels, is partly mediated by decreasing phosphorylation/degradation of I{kappa}B-{alpha} and subsequent inhibition of NF-{kappa}B activation. In contrast, aspirin suppresses cytokine-stimulated NO production by inhibiting iNOS protein expression and catalytic activity without affecting NF-{kappa}B-mediated iNOS mRNA expression, whereas the inhibitory effect by salicylate is mainly due to decreased iNOS protein expression.


   Footnotes
 
1 This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture, Japan, and from the Ministry of Health and Welfare, Japan. Back

Received June 25, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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