©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Induction and Stabilization of IB by Nitric Oxide Mediates Inhibition of NF-B (*)

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

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To determine the mechanism(s) by which the endogenous mediator nitric oxide (NO) inhibits the activation of transcription factor NF-B, we stimulated human vascular endothelial cells with tumor necrosis factor- in the presence of two NO donors, sodium nitroprusside and S-nitrosoglutathione. Electrophoretic mobility shift assays demonstrated that both NO donors inhibited NF-B activation by tumor necrosis factor-. This effect was not mediated by guanylyl cyclase activation since the cGMP analogue 8-bromo-cGMP had no similar effect. Inhibition of endogenous constitutive NO production by L-N-monomethylarginine, however, activated NF-B, suggesting tonic inhibition of NF-B under basal conditions. NO had little or no effects on other nuclear binding proteins such as AP-1 and GATA. Immunoprecipitation studies showed that NO stabilized the NF-B inhibitor, IB, by preventing its degradation from NF-B. NO also increased the mRNA expression of IB, but not NF-B subunits, p65 or p50, and transfection experiments with a chloramphenicol acetyltransferase reporter gene linked to the IB promoter suggested transcriptional induction of IB by NO. We propose that the induction and stabilization of IB by NO are important mechanisms by which NO inhibits NF-B and attenuate atherogenesis.


INTRODUCTION

Nitric oxide (NO) possesses many anti-atherogenic properties including its ability to inhibit vascular smooth muscle cell proliferation (1) , reduce platelet aggregation (2) , and prevent monocyte chemotaxis and adhesion (3) . Recent in vivo studies have shown that NO can attenuate endothelium-leukocyte interactions and limit the extent of atherosclerotic lesions (4, 5) . Although many of the effects of NO are attributed to cGMP-dependent pathways, the precise mechanism(s) by which NO attenuates atherogenesis remain largely unknown. We have recently demonstrated that NO reduces leukocyte attachment to the endothelial surface by decreasing cytokine-induced expression of endothelial vascular cell adhesion molecule-1 (designated VCAM-1), endothelial leukocyte adhesion molecule-1 (ELAM-1 or E-selectin), and to a lesser extent, intercellular adhesion molecule-1 (ICAM-1) (6) . Furthermore, the expression of other pro-inflammatory mediators such as interleukin-6 and interleukin-8 were similarly inhibited by NO. All of these genes share specific DNA binding motifs in their promoters for interaction with the transcription factor, NF-B. Indeed, we found that NO's inhibitory effects were mediated not via cGMP-dependent pathways but instead via inhibition of NF-B (6) .

NF-B was initially described as a heterodimeric complex, which binds to specific decameric sequences in the immunoglogulin light chain enhancer (7) . Members of the mammalian NF-B family possess Rel homology domains necessary for dimerization, nuclear translocation, and DNA binding. They can be divided into two groups based upon their structure and function (8) . The first group consists of p65 (Rel A), c-Rel, and RelB, which contain transcriptional activation domains necessary for gene induction (9) . The second group consists of p105 and p100, which upon proteolytic processing give rise to p50 (NF-B1) and p52 (NF-B2), respectively (10) . The carboxyl-terminal region of p105 and p100 share structural features with the NF-B inhibitor, IB, and thus, functions to retain NF-B in the cytoplasm (11, 12) . The mature proteins, p50 and p52, however, can form functional dimers with members of both groups (8) . With the exception of RelB, which cannot form homodimers, members of both groups can bind in a tissue-specific manner as homo- or heterodimers to enhancer elements of target genes (13). In vascular endothelial cells, the transcriptional induction of vascular cell adhesion molecule-1 depends upon the activation of the p65/p50 heterodimer (14) .

NF-B is sequestered in the cytoplasm through its association with its inhibitors, p105 or IB-like proteins (8, 10) . Although several different IB-like proteins have been identified such as IB (MAD3) (15) , IB (16) , IB, (17) , Bcl-3 (18) , and unprocessed p105 (11, 12) , the most well characterized is the 37-kDa protein, IB (10) . Activation of NF-B by cytokines or oxidative stress requires either the degradation of its cytoplasmic inhibitor IB or proteolytic cleavage of p105 through the ubiquitin-proteasome pathway (8, 19) . Recent studies indicate that phosphorylation of IB or p105 is a key step in targeting these inhibitors for degradation (20, 21) . Thus, factors that affect the phosphorylation and/or expression of IB could modulate the activation of NF-B.

Several lines of evidence suggest that NO may modulate IB. First, NO has been shown to activate protein phosphatases in peripheral blood mononuclear cells (22) , leading to the possibility that NO may inhibit NF-B via dephosphorylation of IB. Second, oxidants such as hydrogen peroxide have been shown to stimulate protein kinase activity and activate NF-B (23, 24) . Because NO can avidly scavenge superoxide anion, it can prevent superoxide anion from forming its dimutation product, hydrogen peroxide (25) . Furthermore, under certain conditions, NO can act as an electron donor or antioxidant (26) . Antioxidants such as N-acetylcysteine or pyrrolidine dithiocarbamate (PDTC)() have been shown to inhibit the activation of NF-B by preventing the dissociation of the NF-B/IB complex (27, 28) . Thus, we hypothesized that NO inhibits NF-B by modulating IB. The purpose of this study was to determine the mechanism(s) by which this occurs.


EXPERIMENTAL PROCEDURES

Materials

All standard culture reagents were obtained from JRH Bioscience (Lenexa, KS). Glutathione, sodium nitrite, sodium nitroprusside PDTC, 8-bromo-cGMP, and phenylmethylsulfonyl fluoride were purchased from Sigma. S-Nitrosoglutathione (GSNO) was synthesized from glutathione and sodium nitrite as described (29) . L-N-Monomethylarginine was obtained from Calbiochem (San Diego, CA). Recombinant human TNF was purchased from Endogen, Inc. (Boston, MA). The Limulus amebocyte lysate kinetic chromogenic assay for endotoxin was performed by BioWhittaker (Walkersville, MD). [-P]CTP (3000 Ci/mmol), [-P]ATP (3000 Ci/mmol), and [H]chloramphenicol (37 Ci/mmol) were supplied by DuPont NEN. The oligonucleotides corresponding to the palindromic NF-B, AP-1, and GATA consensus sequence and affinity-purified rabbit polyclonal antisera to p65, p50, IB, c-Fos, c-Jun, GATA-2, and GATA-3 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Nucleic acid and protein molecular weight markers were purchased from Life Technologies, Inc. The antibody detection kit (Enhanced Chemiluminescence) using horseradish peroxidase and luminol was obtained from Amersham Corp. Nylon transfer membranes were purchased from Schleicher & Schuell. The full-length human cDNA probes for NF-B subunits, p65 and p50, and IB were generously provided by Gary Nabel (University of Michigan, Ann Arbor, MI) and Stephen Haskill (University of North Carolina, Chapel Hill, NC), respectively. The murine IB promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene was generously provided by P. Chiao and I. Verma (Salk Institute, La Jolla, CA).

Cell Culture

Human endothelial cells were harvested from saphenous veins using Type II collagenase (Worthington Biochemical Corp., Freehold, NJ) and grown to confluence in a culture medium containing Medium 199, 20 mM HEPES, 50 µg/ml endothelial cell growth serum (Collaborative Research Inc., Bedford, MA), 100 µg/ml heparin sulfate, 5 mML-glutamine (Life Technologies, Inc.), 5% fetal calf serum (HyClone, Logan, UT), and antibiotic mixture of penicillin (100 units/ml)/streptomycin (100 µg/ml)/fungizone (1.25 µg/ml) as described previously (30) . They were characterized by Nomarski optical microscopy (Zeiss ICM 405, 40 objective) and staining for Factor VIII-related antigen (31) . Only endothelial cells of less than three passages were used. Cells were pretreated with NO donors for 30 min prior to stimulation with TNF. Cellular viability was determined by trypan blue exclusion.

Electrophoretic Mobility Shift Assay

Nuclear extracts were prepared as described (32) . The NF-B oligonucleotide corresponding to the palindromic NF-B consensus sequence (AGTTGAGGGGACTTTCCCAGG) was end-labeled with [-P]ATP and T4 polynucleotide kinase (New England Biolabs), and purified by G-50 Sephadex columns (Pharmacia Biotech Inc.). Nuclear extracts (10 µg) were added to P-labeled NF-B oligonucleotide (20,000 cpm, 0.2 ng) in a buffer containing 2 µg of poly(dIdC) (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 TBE) at 4 °C. For supershift assays, the indicated antibody (15 µg/ml) was added to the nuclear extracts from TNF-stimulated cells for 10 min prior to the addition of radiolabeled probe. In some studies, GSNO or unlabeled NF-B oligonucleotide (20 ng) was added directly to the nuclear extracts from TNF-stimulated cells for 10 min prior to addition of radiolabeled probe. The binding conditions for AP-1 (CGCTTGATGACT-CAGCCGGAA) and GATA (CACTTGATAACAGAAAGTGATAACTCT) oligonucleotides were the same as that for NF-B except 50 mM KCl and 10 mM MgCl were substituted for 50 mM NaCl.

Immunoprecipitation of IB

Agarose-conjugated anti-p65 antibody (100 µg of IgG/ml) was incubated with whole cell lysates (200 µg) or nuclear extracts (100 µg) in 100 µl of immunoprecipitation buffer containing NaCl (150 mM), Tris-HCl (50 mM, pH 7.4), SDS (0.2%), and Triton X-100 (1%) for 16 h at 4°C with gentle rotation. Preliminary studies indicated that all p65 was completely precipitated by this procedure, since immunoblotting analysis of the supernatant using the anti-p65 antibody did not reveal the presence of 65-kDa proteins. The immunoprecipitate was collected by centrifugation at 12,000 g, washed twice with immunoprecipitation buffer (pH 8.3) and once with NaCl (150 mM), Tris-HCl (50 mM, pH 7.4), and EDTA (5 mM), and then resuspended in denaturing buffer containing Tris-HCl (125 mM, pH 6.8), SDS (4%), glycerol (20%), and 2-mercaptoethanol (10%). The mixture was placed briefly (1 min) in boiling water prior to centrifugation at 12,000 g for 10 min. The supernatants and known molecular size markers (Life Technologies, Inc.) were separated by SDS-polyacrylamide gel electrophoresis (12% running, 4% stacking gel).

Western Blotting

Proteins were electrophoretically transferred onto Westran polyvinylidene difluoride membranes and incubated overnight at 4 °C with blocking solution (5% skim milk in PBS). Affinity-purified rabbit antibodies (0.4 µg of IgG/ml) to NF-B subunits and IB were incubated with the blots overnight at 4 °C in PBS buffer containing 0.1% Tween 20. The blots were washed twice with PBS buffer and then treated with donkey anti-rabbit antibody (1:4000 dilution) coupled to horseradish peroxidase. Immunodetection was accomplished using the Enhanced Chemiluminescence kit as described previously (31) .

Northern Blotting

Total RNA was extracted by guanidinium isothiocyanate and isolated by CsCl equilibrium centrifugation as described (30) . Equal amounts of total RNA (20 µg/lane) were separated by 1% formaldehyde-agarose gel electrophoresis, transferred overnight onto nylon membranes by capillary action, and baked for 2 h at 80 °C prior to prehybridization for at least 4 h in a solution containing 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. Radiolabeling of p65, p50, IB, and human -actin cDNA probe (ATCC 37997, Rockville, MD) was performed using random hexamer priming with [-P]CTP and Klenow fragment of DNA polymerase I (Pharmacia). The membranes were hybridized separately with individual probes overnight at 45 °C in a solution 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. All Northern blots were subjected to stringent washing conditions (0.2 SSC, 0.1% SDS at 65 °C) prior to autoradiography with intensifying screen at -80 °C for 24-72 h.

Transient Transfections

The functional murine IB promoter (-1.6 kb upstream from transcriptional initiation start site) linked to the CAT reporter gene (pBS[-1.6 kb]CAT) was described previously by Chiao and Verma (33) . Bovine rather than human endothelial cells were used because of their higher transfection efficiency. Cells (2 10, 70% confluent) were transfected with 25 µg of either the IB promoter construct, pSV2.CAT (SV40 early promoter), or p.CAT (no promoter) using the calcium phosphate precipitation method (34) . As an internal control for tranfection efficiency, pRSV.GAL plasmid (10 µg) was co-transfected in all experiments. Preliminary results using -galactosidase staining indicate that cellular transfection efficiency was approximately 12-15%. After 72 h, cells were treated with 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 supernatant was obtained after centrifuging the extracts at 12,000 g for 10 min.

CAT Assays

CAT activity was determined by incubating the supernatant (100 µl) with [H]chloramphenicol (50 µCi/ml) and n-butyryl coenzyme A (250 µg/ml) for 20 h at 37 °C (35) . The n-butyryl [H]chloramphenicol was then separated from unmodified chloramphenicol by xylene phase extraction and counted for 2 min in a liquid scintillation counter (Beckman LS1800). CAT activity was calculated from a standard curve using various concentrations of purified CAT (Promega). -Galactosidase activity was assayed spectophometrically (absorption at 410 nm) and compared to a standard curve using known amounts of purified -galactosidase (Sigma) as described previously (36) . The relative CAT activity was calculated as the ratio of CAT to -galactosidase activity. Each experiment was performed three times in duplicate.

Data Analysis

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. A significant difference was taken for p values less than 0.05.


RESULTS

Cell Culture

Human saphenous vein endothelial cells were confirmed by their morphological features (i.e. cuboidal, cobblestone, contact-inhibited) using phase-contrast microscopy and immunofluorescent-staining with antibodies to Factor VIII-related antigen. With the exception of transient transfections, there were no observable adverse effects of any treatment modalities on cellular confluence, morphology, and viability. L-N-Monomethylarginine (L-NMA), 8-bromo-cGMP, and PDTC contain no detectable levels of endotoxin (<0.01 ng/ml). GSNO had an endotoxin level of 0.02 ± 0.1 ng/ml.

Inhibition of NF-B Activation by Exogenous NO

Electrophoretic mobility shift assay demonstrated that activation of NF-B by TNF is attenuated by NO in a time- and concentration-dependent manner (Fig. 1). Higher concentration of GSNO (0.5 mM) was required to inhibit NF-B after 2 h of stimulation compared to that after 30 min, probably due to relatively lower levels of NO released from GSNO after 2 h. GSNO was effective only when added to whole cells rather than to nuclear extracts, suggesting that NO did not interfere directly with NF-B binding to DNA and that mediators present in the intact cells were required to mediate NO's inhibitory effect on NF-B. The shifted bands were specific for NF-B since the addition of antibodies to NF-B subunits, p65 and p50, abolished the NF-B band and caused further gel retardation (supershift). Furthermore, the addition of 100-fold excess unlabeled (cold) NF-B oligonucleotide to the nuclear extract specifically abolished the NF-B band.


Figure 1: Electrophoretic mobility shift assay (EMSA) showing the effects of GSNO on NF-B activation by TNF (10 ng/ml). 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 (Supershift) or excess unlabeled (Cold) NF-B oligonucleotide. This is a representative assay from four separate experiments.



Inhibition of NF-B Activation by Endogenous NO

Vascular endothelial cells possess constitutive NO synthase activity (30) . Inhibition of endogenous endothelial NO production by L-NMA (1 mM) caused the activation of NF-B without addition of cytokine, but did not further augment NF-B activation in TNF-stimulated cells (Fig. 2A). L-NMA reduced endogenous NO production by more than 80%, as measured by the conversion of [H]arginine to [H]citrulline (data not shown). Activation of NF-B by L-NMA was not due to contamination of L-NMA samples with bacterial LPS since endotoxin level was undetectable using the Limulus amebocyte lysate chromogenic assay (<0.01 ng/ml). Addition of exogenous NO (GSNO, 0.2 mM) to L-NMA-treated endothelial cells reduced NF-B activation, indicating that endogenous NO is physiologically important in inhibiting NF-B activation under basal conditions. The permeable cGMP analogue 8-bromo-cGMP (1 mM) had no appreciable effect on NF-B activation, indicating that inhibition of NF-B by NO is mediated via a cGMP-independent pathway. Although 8-bromo-cGMP did not affect NF-B, it was still able to stimulate cGMP- and cAMP-dependent protein phosphorylation in endothelial cells (data not shown).


Figure 2: A, EMSA showing the effects of L-NMA (1 mM) and 8-bromo-cGMP (cGMP, 1 mM) on NF-B activation in unstimulated and TNF (10 ng/ml)-stimulated cells. This is a representative assay from 3 separate experiments. B, EMSA showing NF-B activation by TNF (10 ng/ml, 2 h) in the absence (Control) and presence of glutathione (GSH), GSNO, sodium nitroprusside (SNP, 1 mM), nitrite, or PDTC (0.2 mM). This is a representative assay from three separate experiments.



Activation of NF-B by TNF was not altered by glutathione or nitrite, the parent compounds used to synthesize GSNO (29) , suggesting that inhibition of NF-B was actually due to the NO released by our NO donors (Fig. 2B). Both GSNO and another structurally different NO-generating compound, sodium nitroprusside (1 mM), have similar inhibitory effects on NF-B. These findings make it relatively unlikely that inhibition of NF-B by the NO donors was due to any metabolite other than NO. As a positive control, the antioxidant and metal chelator, pyrrolidine dithiocarbamate (0.2 mM) also inhibited the activation of NF-B as demonstrated previously (27) . The relative specificity of NO for NF-B compared to other transcription factors was demonstrated by the fact that GSNO only minimally inhibited the activation of nuclear binding protein AP-1 and did not affect the activation of nuclear binding proteins GATA-2 or GATA-3 (Fig. 3).


Figure 3: EMSA showing the effects of GSNO on AP-1 and GATA activation by TNF (10 ng/ml). Control (lanes1 and 6), TNF (30 min) (lanes2 and 7), and TNF + GSNO (0.2 mM, 30 min) (lanes3 and 8). Specificity was determined by antibodies (15 µg of IgG/ml) to c-Fos (lane4), c-Jun (lane5), GATA-2 (lane9), and GATA-3 (lane10). This is a representative assay from two separate experiments.



Stabilization of IB

Immunoprecipitation studies with agarose-conjugated anti-p65 antibody followed by immunoblotting demonstrated that stimulation by TNF resulted in the loss of IB from NF-B complex (p65 and p50 subunits) after 30 min (Fig. 4). This loss of IB, however, was not observed in the presence of GSNO. Similarly, stimulation with TNF caused a rapid disappearance of IB from whole cell lysates at 30 min followed by reappearance after 2 h (Fig. 5). In the presence of GSNO, there was no disappearance of IB but, instead, an induction of IB in a time-dependent manner. These results indicate that NO inhibited NF-B by either stabilizing and/or reducing IB degradation.


Figure 4: Whole cell lysates (200 µg) immunoprecipitated with agarose-conjugated p65 antibody, followed by immunoblotting using combined p65, p50, and IB antibodies. Cells were stimulated for 30 min with TNF (10 ng/ml) ± GSNO (0.2 mM). Three separate studies yielded similar results.




Figure 5: Immunoblotting of whole cell lysates (100 µg) using IB antibody showing the fate of IB after stimulation with TNF (10 ng/ml) in the absence (Control) or presence of GSNO (0.2 mM). This is representative of three separate studies.



Induction of IB

In a concentration-dependent manner, NO increased the steady-state mRNA expression of IB without affecting the expression of NF-B subunits, p65 and p50, or -actin at 24 h (Fig. 6). Activation of NF-B by TNF has been shown previously to induce the expression of IB (14) . The addition of GSNO produced a smaller increase in IB expression at 30 min, consistent with our hypothesis that NO inhibits NF-B-mediated gene transcription (Fig. 7). GSNO, however, produced a greater increase in IB expression at subsequent time points compared to TNF alone. This is because activation of NF-B induces the expression of IB, which in turn, inhibits NF-B and decreases IB's own subsequent expression. Since the induction of IB expression by NO is not mediated by NF-B, IB expression is not subjected to its own negative autoregulatory control.


Figure 6: Northern analyses (20 µg of total RNA/lane) showing the concentration-dependent effects of GSNO (0.2 mM) on p50, p65, and IB mRNA expression at 24 h. The same blot was re-hybridized separately to each cDNA probe. Hybridization to -actin served as an internal control. Results of three separate blots yielded similar results.




Figure 7: Northern analyses (20 µg of total RNA/lane) showing the time-course of IB mRNA expression after stimulation with TNF (10 ng/ml) in the absence (Control) or presence of GSNO (0.2 mM). Three separate experiments yielded similar results.



Transcriptional Activation of IB

The murine IB promoter linked to the CAT reporter gene, pBS[-1.6 kb]CAT, was used in transient transfection studies. These studies were performed with bovine rather than human endothelial cells due to higher transfectional effeciency with bovine cells using the calcium phosphate precipitation method (data not shown). The promoterless p.CAT produced essentially no relative CAT activity either basally (50 ± 35) or after treatment with TNF (38 ± 34) or GSNO (25 ± 18) (Fig. 8). The highly expressed pSV2.CAT containing the SV40 early promoter exhibited a high level of relative CAT activity (1380 ± 258), which was also unaffected by TNF (1240 ± 235) or GSNO (1334 ± 302). The pBS[-1.6 kb]CAT had a basal relative CAT activity of 206 ± 35. Treatment with GSNO caused a greater increase in relative CAT activity compared to TNF (2546 ± 383 versus 1112 ± 232, p < 0.05). The combination of TNF and GSNO produced a relative CAT activity of 3446 ± 383, which was significantly higher than that of GSNO alone (p < 0.05), indicating that transcriptional induction of IB by GSNO most likely occurred through transcription factor(s) other than NF-B.


Figure 8: IB promoter activity in bovine aortic endothelial cells transfected with plasmid vectors containing the CAT reporter gene linked to no promoter (p.CAT), the SV40 early promoter (pSV2.CAT), and IB promoter (pBS[-1.6]CAT). Cells were stimulated with TNF (10 ng/ml) or GSNO (0.2 mM) for 12 h. CAT activity was standardized to -galactosidase activity (relative CAT activity). Assays were performed three separate times in duplicate.




DISCUSSION

We have shown that NO inhibits the activation of NF-B through the induction and stabilization of the NF-B inhibitor, IB. The mechanism for NO's effect is independent of guanylyl cyclase activation since treatment with cGMP analogue 8-bromo-cGMP did not affect NF-B activation. Interestingly, GSNO is effective only when added to whole cells rather than to nuclear extracts, suggesting that NO does not interfere directly with the physical binding of NF-B to its cognate DNA and that NO requires cellular mediator(s) for its inhibitory effect(s) on NF-B. These results are consistent with our finding that NO's inhibitory effects on NF-B is mediated by IB. The relative selectivity of NO for NF-B compared to AP-1, GATA-2, and GATA-3 also agrees with this conclusion since the activation of these other nuclear binding factors are presumably not regulated by IB. These results, therefore, provide a novel mechanism whereby NO could down-regulate the expression of NF-B-dependent pro-inflammatory genes through induction and stabilization of IB.

Although NF-B has been extensively studied in cells of the immune system (8) , recent evidence indicates that this pleiotropic transcription factor also has importance in vascular biology, especially in the area of transplantation arteriosclerosis in which immunological regulation of NF-B may modulate the expression of pro-inflammatory mediators in monocytes/macrophages and vascular endothelial and smooth muscle cells (37) . A recent study by Lander et al. (22) reportedly shows that NO activates rather than inhibits NF-B in human peripheral blood mononuclear cells. The discrepancy between their results and ours may be due to differences in the response of mononuclear cells to NO-generating compounds compared to that of vascular endothelial cells. Although NO itself may inhibit NF-B, NO may activate other signaling pathways in mononuclear cells which may ultimately lead to the activation rather than to the inhibition of NF-B. Furthermore, we studied the effects of NO-generating compounds on NF-B in cytokine-stimulated cells, whereas their findings were based upon the effects of NO-generating compounds in unstimulated cells. Thus, it is possible that depending on the conditions, NO may have dual regulatory effects on NF-B.

NF-B is sequestered in the cytoplasm when complexed to its inhibitor, IB, or as dimers with unprocessed p105 or p100 (8, 10) . The p65 subunit serves to bind IB to the NF-B complex (38). Activation of NF-B involves phosphorylation of IB, followed by rapid degradation of the inhibitory subunit via the ubiquitin-proteasome pathway (19, 39) . This is consistent with our finding that stimulation by TNF resulted in the loss of IB from p65 and its initial disappearance from the cytosolic pool. Reappearance of IB resulted from the induction of IB expression by NF-B (40) . The loss of IB was prevented by NO, which stabilizes the NF-B/IB complex either through replacement of lost IB or inhibition of IB phosphorylation and/or degradation.

Interestingly, vascular endothelial cells can produce NO constitutively and both endothelial and smooth muscle cells can express the inducible isoform of NO synthase in response to cytokine stimulation (41) . Thus, the net activation of NF-B in vascular wall cells during inflammation probably depends on a complex balance between stimulatory and inhibitory factors. The finding that inhibition of endogenous endothelial NO production by L-NMA could activate NF-B suggests that constitutively produced NO may play an important physiologic role in tonically inhibiting the activation of NF-B under basal conditions. This is supported by in vivo findings showing that inhibition of endogenous NO production by L-nitroarginine methylester promotes endothelium-leukocyte interactions, probably through the expression of NF-B-dependent adhesion molecules (3, 4, 6) . Furthermore, chronic supplementation of the nitric oxide precursor, L-arginine, in diets of hypercholesterolemic rabbits improves endothelial-dependent vasodilation and limits the extent of atherosclerotic lesions (5) .

Endogenous levels of constitutive NO production, however, are not sufficiently high to inhibit TNF-induced activation of NF-B. Higher levels of NO such as those encountered by endothelial cells at sites of inflammation or the amount of NO achieved by our NO donors may be required to suppress the activation of NF-B by cytokines. Exposure of macrophages and vascular smooth muscle cells to cytokines leads to higher levels of NO generated by the inducible form of NO synthase compared to that produced by endothelial cells under basal condition (41) . Furthermore, higher NO activity could be achieved locally since endothelial cells are in close proximity to these endogenous sources of inducible NO in vivo and the possibility that NO could be modified into more stable and potent adducts such as nitrosothiols (42) . It is interesting to speculate that higher levels of NO produced by macrophages and vascular smooth muscle cells may be the mechanism by which NO production is ultimately terminated since the induction of NO synthase in these cells requires the activation of NF-B (41) .

Antioxidants such as N-acetylcysteine or PDTC have also been shown to stabilize the NF-B/IB complex through scavenging reactive oxygen species such as superoxide anion, which may activate NF-B (24, 42, 43) . Stimulation of NADPH oxidase activity in leukocytes generates hydrogen peroxide from superoxide anion and induces protein phosphorylation (23) . It is not known whether NF-B activation induced by oxidative stress is due to hydrogen peroxide-mediated IB phosphorylation. Nevertheless, NO can bind superoxide anion with extremely high affinity (25) . Thus, one mechanism by which NO may stabilize IB and inhibit NF-B activation is through scavenging superoxide anion, thereby decreasing its dimutation product, hydrogen peroxide. In addition, NO itself may directly affect protein kinases and/or phosphatases that regulate IB phosphorylation. Indeed, recent studies have shown that NO can stimulate the activity of protein tyrosine phosphatase(s) (22) . It remains to be determined whether this increase in phosphatase activity could lead to the dephosphorylation and stabilization of IB.

A novel finding in this study is that a free radical such as NO can induce the expression of a transcription factor inhibitor, IB. Transfection studies using the IB promoter linked to the CAT reporter gene suggests that this effect occurs at the transcriptional level. NO had no effect on the expression of NF-B subunits, p65 and p50. Previous analyses of the IB promoter have revealed multiple functional B sites necessary for transcriptional induction by NF-B (44, 45) . This provides for an inducible autoregulatory pathway for terminating the activation of NF-B (33, 40) . However, the induction of IB by NO is probably not mediated by NF-B since NO inhibits NF-B. Further analyses of IB promoter will be necessary to determine which DNA binding domain(s) constitute NO's cis-regulatory element(s).

In summary, we have shown that NO can inhibit the activation of NF-B through the induction and stabilization of its inhibitor, IB. The ability of endogenous NO to inhibit NF-B provides new insight into the mechanisms of NO's anti-inflammatory and anti-atherogenic properties.


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 (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 should be addressed: Cardiovascular Division, Dept. of Medicine, 221 Longwood Ave., LMRC-307, Boston, MA 02115. Tel.: 617-732-6538; Fax: 617-732-6961.

The abbreviations used are: PDTC, pyrrolidine dithiocarbamate; EMSA, electrophoretic mobility shift assay; GSNO, S-nitrosoglutathione; CAT, chloramphenicol acetyltransferase; TNF, tumor necrosis factor; kb, kilobase pair(s); PBS, phosphate-buffered saline; L-NMA, L-N-monomethylarginine.


ACKNOWLEDGEMENTS

We are grateful to Gary Nabel (p65 and p50 cDNA), Stephen Haskill (IB cDNA), and I. Verma (IB promoter).


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