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Endocrinology Vol. 139, No. 11 4506-4512
Copyright © 1998 by The Endocrine Society

ARTICLES

Role of Nuclear Factor-{kappa}B Activation in Cytokine- and Sphingomyelinase-Stimulated Inducible Nitric Oxide Synthase Gene Expression in Vascular Smooth Muscle Cells1

Koichi Katsuyama, Masayoshi Shichiri, Fumiaki Marumo and Yukio Hirata

Endocrine-Hypertension Division, Second Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo 113, Japan

Address all correspondence and requests for reprints to: Yukio Hirata, M.D., Endocrine-Hypertension Division, 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
 Note Added in Proof
 References
 
Inflammatory cytokines, such as interleukin-1ß (IL-1ß) and tumor necrosis factor-{alpha} (TNF{alpha}), are known to activate sphingomyelinase (SMase) and nuclear factor-{kappa}B (NF-{kappa}B) in certain cell types, which also stimulate inducible nitric oxide synthase (iNOS) gene in vascular smooth muscle cells (VSMCs). However, it remains unknown whether the SMase pathway is involved in iNOS gene expression in VSMCs. Therefore, the present study was designed to examine whether SMase induces iNOS gene expression via the NF-{kappa}B activation pathway similar to that of IL-1ß and TNF{alpha} in cultured rat VSMCs. Neutral SMase, although less potently than IL-1ß and TNF{alpha}, stimulated nitrite/nitrate (NOx) production, and iNOS messenger RNA and protein expression, as assessed by Northern and Western blot analyses, respectively. Neutral SMase, IL-1ß, and TNF{alpha} activated NF-{kappa}B, as revealed by electrophoretic mobility shift assay, and its nuclear translocation, as demonstrated by immunocytochemical study. Neutral SMase potentiated NOx production, iNOS expression, and NF-{kappa}B activation stimulated by TNF{alpha}, but not by IL-1ß. Aldehyde peptide proteasome inhibitors completely blocked NOx production, iNOS expression, NF-{kappa}B activation, and its nuclear translocation induced by cytokines and neutral SMase. IL-1ß and TNF{alpha}, but not neutral SMase, caused a transient decrease in I{kappa}B-{alpha} protein levels, whereas I{kappa}B-ß protein expression was not affected by either agent. Proteasome inhibitors prevented cytokine-mediated I{kappa}B-{alpha} degradation. Several cell-permeable ceramide analogs (C2, C6, and C8), hydrolysis products of sphingomyelin, activated NF-{kappa}B less potently than neutral SMase, but had no effect on NOx production. These results demonstrate an essential role of NF-{kappa}B activation in mediation of neutral SMase-induced iNOS expression, but distinct from the proteasome-mediated I{kappa}B-{alpha} degradation by cytokines, suggesting the possible involvement of an additional signaling pathway(s).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
NITRIC oxide (NO), synthesized from L-arginine by NO synthases (NOS), possesses diverse physiological properties, such as vasodilation, neurotransmission, and mediation of immune responses (1). Three distinct isozymes have been identified to date. Two constitutive isozymes dominantly expressed in brain (NOS1) and endothelium (NOS3) are Ca2+/calmodulin dependent. Cytokine-inducible isozyme (iNOS2) is Ca2+ independent and produces excess amounts of NO by bacterial lipopolysaccharides (LPS) and certain cytokines in a variety of cells, including vascular smooth muscle cells (VSMCs).

VSMCs do not produce NO under normal conditions. However, after stimulation with LPS and certain cytokines, such as interleukin-1ß (IL-1ß) and tumor necrosis factor-{alpha} (TNF{alpha}), augmented iNOS expression causes excessive NO production, thereby leading to a profound and intractable hypotension, i.e. endotoxic shock. As VSMCs play a pivotal role in the regulation of vascular tonus, elucidation of iNOS gene regulation in VSMCs is critical for understanding the pathogenesis of endotoxic shock. Recent studies have suggested that iNOS gene expression has also been implicated in the pathogenesis of vascular remodeling and atherosclerosis (2), because excess NO production by iNOS in the blood vessels causes inhibition of cell proliferation and apoptosis of VSMCs (3, 4).

Nuclear factor-{kappa}B (NF-{kappa}B) is a heterodimeric complex, usually consisting of p50 and p65 (RelA) subunits, and functions as a pleiotropic regulator of many genes modulating immunological and inflammatory processes (5). p50/p65 heterodimer associates with I{kappa}B to form an inactive cytoplasmic ternary complex. p65 subunit may also associate with a precursor protein (p105) of p50 to form an inactive complex. Activation of NF-{kappa}B by LPS or cytokines requires either the degradation of its cytoplasmic inhibitor, I{kappa}B-{alpha} (6), or proteolytic cleavage of p105 through the ubiquitin- proteasome pathway after phosphorylation (7). After degradation of I{kappa}B-{alpha}, an active heterodimer, NF-{kappa}B translocates into the nucleus and activates gene expression. However, the degradative process of I{kappa}B-{alpha} in mediation of cytokine-stimulated iNOS gene expression in VSMCs remains unknown.

One of the pivotal signal transduction pathways by cytokines is a sequential activation of phosphatidylcholine- specific phospholipase C (PC-PLC) and acidic sphingomyelinase (SMase), causing hydrolysis of sphingomyelin to generate ceramide (8). Ceramide can trigger the activation of several transcription factors, including NF-{kappa}B (9). By contrast, a functionally and topologically distinct neutral SMase in the plasma membrane also initiates the hydrolysis of sphingomyelin to generate ceramide, which, in turn, stimulates several proline-directed protein kinases, such as ceramide-activated protein (CAP) kinase and Jun N-terminal kinase (JNK) (10). However, it remains unknown whether the sphingomyelin/ceramide pathway is involved in iNOS gene expression.

These observations led us to examine 1) whether cytokine-induced iNOS gene expression in VSMCs is mediated by activation of NF-{kappa}B via the ubiquitin-proteasome pathway, 2) whether SMase induces iNOS gene expression by activating NF-{kappa}B in the same manner as cytokines, and 3) whether cytokines and SMase trigger degradation of I{kappa}B-{alpha} to translocate NF-{kappa}B into the nucleus.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Note Added in Proof
 References
 
Materials
Human recombinant IL-1ß was provided by Otsuka Pharmaceutical (Tokushima, Japan). Murine recombinant TNF{alpha} was purchased from Life Technologies (Grand Island, NY); SDS and EDTA were obtained from Wako Pure Chemical (Osaka, Japan); neutral SMase (Bacillus cereus), N-acetyl-D-sphingosine (C2-ceramide), PC-PLC (Clostridium perfringens), phenylmethylsulfonylfluoride (PMSF), dithiothreitol (DTT), poly(dI-dC), and EGTA were obtained from Sigma Chemical Co. (St. Louis, MO), [{alpha}-32P]Deoxy (d)-CTP was purchased from Amersham International (Aylesbury, UK), dNTP and a Klenow fragment of DNA polymerase I were obtained from Takara Shuzo (Shiga, Japan), MG115 and MG132 were purchased from Peptide Institute, and N-hexanoylsphingosine (C6 ceramide) and N-octanoylsphingosine (C8 ceramide) were obtained from Biomol Research Laboratories, Inc. (Plymouth Meeting, PA).

Cell culture
VSMCs from the thoracic aorta of 15-week-old male Wistar rats were prepared by the explant method and cultured in DMEM containing 10% FCS at 37 C in a humidified atmosphere of 95% air-5% CO2 as described previously (11). Subcultured VSMCs (15–30th passages) were used in the experiments.

Determination of nitrite/nitrate (NOx)
Confluent VSMCs (106 cells/well) preincubated with serum-free DMEM in the absence or presence of the indicated drugs for 30 min were stimulated with IL-1ß (10 ng/ml), TNF{alpha} (100 ng/ml), or SMase (0.5–2 U/ml) for 15 h; NOx concentrations in the conditioned media were measured by an autoanalyzer (TCI-NOX 100, Tokyo Kasei Kogyo) as previously described (12). 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, and 5% HCl). Absorbance at 540 nm was measured by a flow-through visible spectrophotometer (model S/3250, Soma-Kogaku, Tokyo, Japan). NO3- was used as a standard.

Electrophoretic mobility shift assay (EMSA)
After pretreatment with test compounds for 30 min, confluent cells (5 x 106 cells/dish) were treated with IL-1ß (10 ng/ml), TNF{alpha} (100 ng/ml), or SMase (1 U/ml) 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, and 5 µg/ml leupeptin]. After 15-min incubation, 25 µl 10% Nonidet P-40 were added and centrifuged at 10,000 x g for 1 min. The nuclei pellets were collected, resuspended in 30 µl hypertonic extraction buffer [50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 10% glycerol, and 0.1 mM PMSF], and centrifuged at 10,000 x g for 10 min, and the supernatant was subjected to EMSA. The single stranded oligonucleotides (forward, 5'-TGGGGACTCTCC-3'; complement, 5'-AAGGGAGAGTCC-3') corresponding to the NF-{kappa}B-binding sequence of the downstream region (-107 to -97) of the rat iNOS gene promoter (13) 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 20,000 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, and 50 mM NaCl] for 30 min, loaded into a 5% polyacrylamide gel, and run in 50 mM Tris, 0.38 M glycine, and 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 gel assay, nuclear protein was preincubated for 30 min with goat polyclonal antibodies against human NF-{kappa}B p50 or p65 subunit (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) before EMSA.

Northern blot analysis
Confluent cells (5 x 106 cells/dish) were stimulated with cytokines and/or SMase for 6 h, and total RNAs were extracted by the acid-guanidinium thiocyanate-phenol-chloroform method (14). Total RNAs (20 µg), separated by formaldehyde-1.1% agarose gel electrophoresis, were transferred to a Magna Graph nylon membrane (Micron Separations, Inc., Westboro, MA) that was hybridized with rat iNOS complementary DNA (15) labeled with [{alpha}-32P]dCTP (111 TBq/mmol) by the random primed labeling method, washed in 0.1 x SSPE(15 mM NaCl, 1 mM NaH2PO4, 0.1 mM EDTA)-0.5% SDS, autoradiographed.

Western blot analysis
Western blot analyses were performed essentially as previously described (16). Confluent cells (5 x 106 cells/dish) were stimulated with cytokines and/or SMase for the indicated times for I{kappa}B-{alpha} and I{kappa}B-ß or for 15 h for iNOS. After stimulation, 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). Whole cell lysates were boiled, and extracted proteins were separated on 12% (for I{kappa}B-{alpha} and I{kappa}B-ß) or 7.5% (for iNOS) SDS-polyacrylamide gel and transferred to Hybond enhanced chemiluminescence nitrocellulose membranes (Amersham), which were incubated with either rabbit polyclonal antibody for human I{kappa}B-{alpha} (1:500) and I{kappa}B-ß (1:500; Santa Cruz Biotechnology) or mouse monoclonal antibody for murine iNOS (1:1000; Transduction Laboratories, Inc., Lexington, KY) at 4 C overnight. After extensive washing, the secondary antibody (donkey antirabbit IgG or sheep antimouse Ig horseradish peroxidase; 1:500; Amersham) was incubated for 1 h, and exposure was performed using an enhanced chemiluminescence kit (Amersham).

Immunocytochemical staining
Subconfluent cells grown on Lab-Tek chamber slides (Nalge Nunc International, Chicago, IL) were treated with cytokines or SMase for 2 h and fixed with 70% acetone for 20 min at room temperature, then washed with PBS for 10 min. Goat polyclonal antibody specific for NF-{kappa}B p50 subunit (Santa Cruz Biotechnology) was used; the antibody did not show any cross-reactivity with p105, p52, or p100. Immunostaining was visualized with the indirect immunoperoxidase avidin-biotin-peroxidase kit (Vector Laboratories, Inc., Burlingame, CA) as previously described (17).

Statistical analysis
All values are given as the mean ± SE. Statistical analysis was performed using Student’s t test. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Note Added in Proof
 References
 
Cytokines stimulated NO production and iNOS expression via the NF-{kappa}B pathway
We studied whether inhibition of NF-{kappa}B activation by proteasome inhibitors affects NO production, iNOS messenger RNA (mRNA), and protein levels in rat VSMCs. As both IL-1ß (1–10 ng/ml) and TNF{alpha} (10–100 ng/ml) dose dependently stimulated NOx production in rat VSMCs, we used the maximal concentrations of IL-1ß (10 ng/ml) and TNF{alpha} (100 ng/ml) in subsequent experiments. As shown in Fig. 1Go, IL-1ß and TNF{alpha} increased NOx production during 15-h incubation by 10- and 8-fold over that in control cells, respectively. These effects were suppressed with a proteasome inhibitor, MG115, in a dose-dependent fashion (0.5–10 µM); almost complete inhibition was induced with 5 µM (TNF{alpha}) and 10 µM (IL-1ß), respectively. As the apparent Ki value of MG115 was 20 µM (7), and the maximal inhibition required to block IL-1ß-stimulated NOx production was 10–40 µM, we chose 40 µM MG115 in subsequent experiments. Neither cell viability nor cell morphology was affected by 40 µM. Both IL-1ß and TNF{alpha} induced iNOS mRNA expression by Northern blot analysis (Fig 2Go, upper panel) and iNOS protein expression by Western blot analysis (Fig 2Go, middle panel); the effects of IL-1ß were greater than those of TNF{alpha}. These effects were completely blocked with MG115 (40 µM).



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  		Figure 1. Effect of proteasome inhibitor on cytokine-induced NO production in rat VSMCs. Confluent cells pretreated for 30 min with MG115 in the indicated concentrations were stimulated with IL-1ß (10 ng/ml) or TNF{alpha} (100 ng/ml) for 15 h; concentrations of NOx released into media were measured. Each point represents the mean value (n = 3), and the SE is within each point.

 


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  		Figure 2. Effect of proteasome inhibitor on cytokine-induced iNOS expression and NF-{kappa}B activation in rat VSMCs. Confluent cells pretreated with (+) or without (-) MG115 (40 µM) for 30 min were stimulated with IL-1ß (10 ng/ml) or TNF{alpha} (100 ng/ml) for 6 h for Northern blotting (upper panel) of iNOS mRNA (28S ribosomal RNA), for 15 h for Western blotting (middle panel) of 130-kDa iNOS protein, and for 2 h for EMSA (lower panel) of NF-{kappa}B activity, respectively. A representative from two or three separate experiments is shown.

 
EMSA using synthetic oligonucleotides for the downstream NF-{kappa}B-binding site as a probe revealed that both IL-1ß and TNF{alpha}, although TNF{alpha} was less potent than IL-1ß, caused a distinct shifted band, whereas there was no distinct band in the control cells (Fig. 2Go, bottom panel). MG115 (40 µM) also completely blocked the appearance of the shifted band induced by IL-1ß or TNF{alpha}. The shifted band was abolished in the presence of excess unlabeled oligonucleotide as a competitor and was supershifted after preincubation with anti-p50 and anti-p65 antibodies (data not shown). Identical results were obtained when MG132 was used (data not shown). These data demonstrate that the IL-1ß- and TNF{alpha}-induced iNOS expression and subsequent NOx production are blocked with proteasome inhibitors by inhibiting the NF-{kappa}B pathway.

Neutral SMase, but not PC-PLC, stimulated NO production and iNOS expression via the NF-{kappa}B pathway
To determine whether SMase directly affects iNOS gene expression in VSMCs, the effects of neutral SMase from bacterial origin on NOx production, iNOS mRNA and protein expression, and NF-{kappa}B activation were examined. Neutral SMase dose dependently (0.5–2 U/ml) stimulated NOx production, whose effect was potentiated in the presence of TNF{alpha} (100 ng/ml; Fig. 3Go), but not in the presence of IL-1ß (10 ng/ml; data not shown). SMase (1 U/ml) induced the expression of iNOS mRNA and protein, whose effects were similarly potentiated by the addition of TNF{alpha} (100 ng/ml; Fig. 4Go, upper and middle panels), but not by IL-1ß (data not shown). SMase also induced activation of NF-{kappa}B, which was similarly potentiated in the presence of TNF{alpha} (Fig. 4Go, lower panel).



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  		Figure 3. Effect of neutral SMase on TNF{alpha}-induced NOx production in rat VSMCs. Confluent cells were stimulated with neutral SMase in the indicated concentrations in the presence ({blacksquare}) and absence ({square}) of TNF{alpha} (100 ng/ml) for 15 h, and concentrations of NOx released into media were measured. Each column with bar represents the mean ± SE (n = 4).

 


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  		Figure 4. Effects of neutral SMase on TNF{alpha}-induced iNOS expression and NF-{kappa}B activation in rat VSMCs. Confluent cells were stimulated with (+) or without (-) SMase (1 U/ml) in the presence (+) or absence (-) of TNF{alpha} (100 ng/ml) for Northern blotting of iNOS mRNA (upper panel), Western blotting of iNOS protein (middle panel), and EMSA of NF-{kappa}B activity (lower panel), respectively. A representative from two or three separate experiments is shown.

 
To determine whether PC-PLC-mediated acidic SMase activation is involved in iNOS induction, we tested the effect of PC-PLC from bacterial origin on NOx production. PC-PLC (0.25–2.0 U/ml) had no effect on NOx production (data not shown), mitigating against a role for the PC-PLC/acidic SMase pathway in the mediation of iNOS induction.

To determine whether activation of NF-{kappa}B by neutral SMase is mediated via proteasome, we tested the effects of MG115 on SMase-induced NOx production, iNOS expression, and NF-{kappa}B activation (Fig. 5Go). MG115 (40 µM) completely blocked NOx production (Fig. 5Go, left panel), expression of iNOS mRNA and protein, and NF-{kappa}B activation (Fig. 5Go, right panel) stimulated by neutral SMase (1 U/ml). However, cell-permeable ceramide analogs (C2, C6, and C8; 100–500 µM) had no effect on NOx production; C2 ceramide (100–500 µM) caused NF-{kappa}B activation far less potently than neutral SMase (data not shown).



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  		Figure 5. Effects of proteasome inhibitor on neutral SMase-induced NOx production, iNOS expression, and NF-{kappa}B activation in rat VSMCs. Confluent cells pretreated (A) with or without MG115 (40 µM) for 30 min were stimulated with neutral SMase (2 U/ml) at 37 C for NOx production, or (B) with neutral SMase (1 U/ml) for Northern blotting of iNOS mRNA (upper panel), for Western blotting of 130-kDa iNOS protein (middle panel), and for EMSA of NF-{kappa}B activity (lower panel).

 
Cytokines and neutral SMase induced NF-{kappa}B nuclear translocation
To confirm that proteasome inhibitors in fact prevent nuclear translocation of NF-{kappa}B in rat VSMCs, immunohistochemical staining using anti-p50 antibody was performed. Nonstimulated cells revealed a diffuse, but faint, distribution of immunoreactive p50 within cytoplasm (Fig. 6Go, A and E). In contrast, exposure of rat VSMCs to IL-1ß (Fig. 6BGo), TNF{alpha} (Fig. 6CGo), and neutral SMase (Fig. 6DGo) resulted in dense accumulations of immunoreactive p50 within the nucleus, whose effects were similarly prevented by pretreatment with MG115 (Fig. 6Go, F–H). These data indicate that cytokines as well as neutral SMase cause proteasome-mediated nuclear translocation of p50 subunit of NF-{kappa}B.



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  		Figure 6. Effect of proteasome inhibitor on cytokine- and neutral SMase-induced nuclear translocation of NF-{kappa}B by immunohistochemical staining. Cells pretreated with or without MG115 (40 µM) for 30 min were stimulated with IL-1ß (10 ng/ml), TNF{alpha} (100 ng/ml), or neutral SMase (1 U/ml) for 2 h, fixed with 70% acetone, and stained with goat polyclonal antibody specific for the NF-{kappa}B p50 subunit. A, Control; B, IL-1ß; C, TNF{alpha}; D, SMase; E, MG115; F, IL-1ß plus MG115; G, TNF{alpha} plus MG115; H, SMase plus MG115.

 
Cytokines, but not neutral SMase, induced I{kappa}B-{alpha} degradation
To determine whether cytokines and neutral SMase cause degradation of I{kappa}B-{alpha} and I{kappa}B-ß in rat VSMCs, Western blot analyses using anti-I{kappa}B-{alpha} and anti-I{kappa}B-ß antibodies were performed (Fig. 7Go). Addition of IL-1ß (10 ng/ml) or TNF{alpha} (100 ng/ml) resulted in a rapid (within 15–30 min) degradation of I{kappa}B-{alpha}, which then returned to baseline levels within 1 h (Fig. 7AGo). However, neutral SMase (1 U/ml) did not induce the loss of I{kappa}B-{alpha} during a 2-h observation period. In contrast, I{kappa}B-ß protein levels were not affected by IL-1ß, TNF{alpha}, or SMase during 2-h incubation (Fig. 7BGo). Pretreatment with MG115 (40 µM) prevented transient degradation of I{kappa}B-{alpha} induced by IL-1ß and TNF{alpha} (Fig. 8Go). These data suggest that the proteasome-mediated degradation of I{kappa}B-{alpha} and subsequent resynthesis occur by stimulation with cytokines, but not with neutral SMase, whereas I{kappa}B-ß is unaltered.



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  		Figure 7. Effects of cytokines and neutral SMase on I{kappa}B-{alpha} and I{kappa}B-ß expression in rat VSMCs. Confluent cells were incubated with IL-1ß (10 ng/ml), TNF{alpha} (100 ng/ml), and SMase (1 U/ml) for the indicated times, and subjected to Western blot analysis using anti-I{kappa}B-{alpha} antibody (A) and anti-I{kappa}B-ß antibody (B).

 


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  		Figure 8. Effect of proteasome inhibitor on cytokine-induced I{kappa}B{alpha} degradation in rat VSMCs. Confluent cells pretreated with or without MG115 (40 µM) were stimulated with (A) IL-1ß (10 ng/ml) or (B) TNF{alpha} (100 ng/ml) for 15 min and subjected to Western blot analysis using anti-I{kappa}B-{alpha} antibody.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Note Added in Proof
 References
 
The transcriptional activity of the p50/p65 heterodimer can be regulated by at least two pathways (7). First, the p50/p65 heterodimer of NF-{kappa}B forms an inactive cytoplasmic ternary complex with the inhibitor protein, I{kappa}B-{alpha}, thereby masking the nuclear localization sequences of p50/p65 heterodimer. Stimulation by LPS or TNF{alpha} causes rapid phosphorylation and degradation of I{kappa}B-{alpha}, which allows transcriptionally active p50/p65 heterodimer to translocate into the nucleus to activate a set of genes. Second, p65 (Rel A) or other Rel family proteins (c-Rel and Rel B) can associate with the unprocessed p105, whose C-terminal portion bears a striking resemblance to I{kappa}B-{alpha}, to form an inactive p105/p65 cytoplasmic complex. Processing of p105 results in a rapid degradation of I{kappa}B-{alpha} homologous sequence and formation of transcriptionally active p50/p65 heterodimer. In both pathways, phosphorylation of I{kappa}B-{alpha} and p105 by a protein kinase(s) is essential for the subsequent degradation of I{kappa}B-{alpha} and processing of p105, respectively. The present experiments using rat VSMCs have demonstrated that stimulation with IL-1ß and TNF{alpha} resulted in a transient loss of I{kappa}B-{alpha}, followed by NF-{kappa}B activation, as assessed by EMSA, and its nuclear translocation, as demonstrated by immunocytochemical analysis.

It has recently been demonstrated that the ubiquitin- proteasome pathway is required for NF-{kappa}B activation by the degradation of I{kappa}B-{alpha} and/or by processing of the p105 precursor to p50 subunit after phosphorylation (7). Cell-permeable aldehyde peptide inhibitors have been shown to potently block TNF{alpha}-induced degradation of I{kappa}B-{alpha} and activation of NF-{kappa}B in Jurkat cells and HeLa cells (18, 19). In the present study, we have demonstrated that potent proteasome inhibitors (MG115 and MG132) completely blocked the degradation of I{kappa}B-{alpha} and the activation of NF-{kappa}B induced by IL-1ß and TNF{alpha} in rat VSMCs. These data are corroborated by the immunocytochemical study showing that MG115 prevented the nuclear translocation of p50 subunit induced by IL-1ß and TNF{alpha}. The present study has clearly demonstrated that MG115 completely blocked the IL-1ß- and TNF{alpha}-induced iNOS mRNA and protein expression and subsequent NO production in rat VSMCs. These results demonstrate for the first time that the proteasome-mediated degradation of I{kappa}B-{alpha} and subsequent NF-{kappa}B activation are essential for the mechanism of cytokine-induced iNOS expression in rat VSMCs.

Recent evidence suggests that TNF{alpha} and IL-1ß employ two sphingomyelin pathways to effect signal transduction by their receptors (10). One pathway is initiated by hydrolysis of plasma membrane sphingomyelin by neutral SMase and another by PC-PLC-mediated acidic SMase via diacylglycerol generation. Both neutral and acidic SMases generate ceramide, which serves as a second messenger to stimulate several protein kinases, such as CAP kinase and mitogen-activated protein (MAP) kinase. For example, it has been shown that TNF{alpha} activates c-raf-1 kinase, which activates MAP kinase via neutral SMase (20), and that raf-1 induces NF-{kappa}B activation (21).

Of particular interest in our study is that exogenous neutral SMase mimicked the effects of IL-1ß or TNF{alpha} in rat VSMCs; neutral SMase activated NF-{kappa}B and stimulated iNOS mRNA and protein expression and subsequent NO production. However, SMase alone was less effective than TNF{alpha} in stimulating NO production, but was more effective in stimulating iNOS mRNA and protein expression and NF-{kappa}B activation. The reasons for the apparent discrepancy between the effects of SMase and TNF{alpha} remain unknown, but several possibilities could be speculated. First, this may be simply due to the different experimental conditions employed (incubation times, extraction procedures, etc.) and/or the stability of the final products measured. Second, the apparently lesser effect of TNF{alpha} on NF-{kappa}B activation and iNOS expression than on NO production may be secondary to the direct inhibition by massive NO generation on NF-{kappa}B-DNA binding because NO donor has been shown to inhibit formation of the NF-{kappa}B-DNA complex (22). Finally, the apparently lesser effect of SMase on NO production than on NF-{kappa}B activation and iNOS expression may be consequent to the posttranslational modification of iNOS enzyme and/or cofactor availability resulting from phosphorylation by ceramide-mediated protein kinases.

It should be noted that the effects by neutral SMase were potentiated in the presence of TNF{alpha}, but not of IL-1ß, and the effects of NF-{kappa}B activation, iNOS mRNA and protein expression, and NO production induced by IL-1ß were consistently greater than those by TNF{alpha} in rat VSMCs. These results suggest that the mechanisms involved in neutral SMase-induced NF-{kappa}B activation and iNOS expression are similar, if not identical, to that of IL-1ß, but are distinct from that of TNF{alpha}. I{kappa}B-ß, another I{kappa}B isoform that interacts with the same Rel protein dimers, displays distinct responses to NF-{kappa}B inducers; TNF{alpha} or phorbol ester causes a transient loss of I{kappa}B-{alpha} with a transient activation of NF-{kappa}B, whereas IL-1 or LPS causes a transient loss of I{kappa}B-{alpha}, but persistent loss of I{kappa}B-ß with continued activation of NF-{kappa}B (23). Thus, the continued activation of NF-{kappa}B in the absence of I{kappa}B-ß may lead to the persistent expression of the genes induced by IL-1 and LPS. However, the present study did not show any changes in I{kappa}B-ß protein expression by IL-1ß, TNF{alpha}, or neutral SMase in rat VSMCs. Therefore, the possibility of different signaling pathways by cytokines and neutral SMase needs to be considered.

Initially, it has been suggested that activation of acidic SMase by cytokine-stimulated PC-PLC induces NF-{kappa}B activation via ceramide (24, 25). However, recent studies have shown that TNF{alpha} activates NF-{kappa}B in fibroblasts derived from acidic SMase-deficient mice (26) and from patients with Niemann-Pick disease (27). In the present study, exogenous PC-PLC had no effect on NO production in rat VSMCs. Our results are in agreement with those of recent studies showing a limited role of the PC-PLC/acidic SMase/ceramide pathway in mediation of NF-{kappa}B activation by cytokines in several cell types (28, 29, 30).

The present study has clearly demonstrated that NF-{kappa}B activation and its nuclear translocation as well as the expression of iNOS mRNA and protein and subsequent NO production induced by neutral SMase were equally blocked by a proteasome inhibitor, MG115. It should be noted, however, that neutral SMase did not degrade I{kappa}B-{alpha}. These data are consistent with the idea that the effect of neutral SMase to induce NF-{kappa}B activation is mediated through a mechanism other than I{kappa}B-{alpha} degradation, such as proteasome-mediated processing of p105. Taken together, it is suggested that neutral SMase partly shares a signaling pathway(s) in common with cytokines via the NF-{kappa}B pathway, but distinct from the degradative process of I{kappa}B-{alpha}.

In this study, exogenous ceramide analogs caused NF-{kappa}B activation far less potently than neutral SMase, but had no effect on NO production. These data suggest that ceramide plays some role in NF-{kappa}B activation, but appears insufficient to induce full activation of NF-{kappa}B-mediated iNOS expression. Similar dissociation of NF-{kappa}B activation and cellular responses by ceramide and neutral SMase has been reported in other cell types. For example, ceramide and neutral SMase have been shown to be insufficient to induce NF-{kappa}B activation, but enhance the cytokine-induced cytotoxic effect in T cells (31) and E-selectin expression in endothelial cells (32). Collectively, our data suggest that a signaling pathway(s) to NF-{kappa}B activation by neutral SMase/ceramide is different from that of cytokines in rat VSMCs.

Recently, two closely related I{kappa}B-{alpha} kinases that directly phosphorylate I{kappa}B-{alpha} (33, 34, 35, 36, 37) as well as the upstream kinases, such as NF-{kappa}B-inducing kinase (38) and a MAP kinase kinase kinase 1 (MEKK1) (39), have been identified that integrate IL-1- and TNF{alpha}-induced NF-{kappa}B activation (Fig. 9Go). However, an additional pathway(s) other than I{kappa}B kinase-mediated phosphorylation by cytokines and SMase should be considered. In fact, it has been reported that TNF{alpha}-induced E-selectin expression is activated by NF-{kappa}B and the JNK/p38 MAP kinase family in endothelial cells (40). Furthermore, it has been shown that TNF{alpha} phosphorylates not only I{kappa}B-{alpha}, but also p65 and p105, to induce DNA-binding activity in HeLa and B cells (41), and a novel serine/threonin kinase that activates NF-{kappa}B by direct phosphorylation of p65 and p50 (NF-{kappa}B kinase) has been identified (42). Thus, it is possible to speculate that cytokines (IL-1 and TNF{alpha}) and SMase/ceramide, depending on cell type, may use multiple signaling pathways other than I{kappa}B kinase-mediated phosphorylation of I{kappa}B-{alpha} to activate NF-{kappa}B (Fig. 9Go).



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  		Figure 9. Hypothetical model for NF-{kappa}B activation pathways by cytokines and SMase in rat VSMCs. A major pathway is indicated by a bold line, and possible minor pathways are indicated by solid lines. TNFR-1, TNF receptor-1; IL-1R, IL-1 receptor, TRAF, TNFR-associated factor; NIK, NF-{kappa}B-inducing kinase; nSMase, neutral sphingomyelinase; aSMase, acidic sphingomyelinase; DAG, diacylglycerol.

 
In conclusion, this study clearly shows an essential role of NF-{kappa}B activation resulting from proteasome-mediated I{kappa}B-{alpha} degradation in IL-1ß- or TNF{alpha}-induced iNOS expression and subsequent NO production in rat VSMCs. Our study also demonstrates the important role of neutral SMase/ceramide in iNOS gene expression via the NF-{kappa}B pathway distinct from the proteasome-mediated I{kappa}B-{alpha} degradation by cytokines, although its exact signaling pathway(s) remains undetermined.


   Note Added in Proof
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
References
 
During submission of our manuscript, Boland and O’Neil have reported that ceramide activates NF-{kappa}ß in HL60 and Jurkat cells by inducing the processing of p105 with a marginal effect on I{kappa}ß{alpha} degradation, whereas TFN-{alpha} stimulated both I{kappa}B{alpha} degradation and p105 processing (43).


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

Received March 3, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 

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