Regulation of Vascular Smooth Muscle Cell Proliferation by Nuclear Factor-κB and Its Inhibitor, I-κB*

Proliferation of vascular smooth muscle cells (SMC) is a crucial event in the formation of atherosclerotic tissues and is regulated by nuclear transcriptional factors including nuclear factor-κB (NF-κB). We constructed a reporter gene assay to measure NF-κB-dependent transcriptional activity in SMC. Thrombin receptor-activating peptide (TRAP) and basic fibroblast growth factor (bFGF) stimulated SMC proliferation and rapidly enhanced the NF-κB transcriptional activity in a dose-dependent manner. 4-Cyano-5,5-bis-(methoxyphenyl)4-pentenoic acid (E5510) significantly inhibited SMC proliferation and also suppressed NF-κB transcription stimulated by TRAP and bFGF. In contrast, although tumor necrosis factor (TNF)-α activated NF-κB transcription, E5510 had no effect on TNF-α-induced activation. NF-κB was activated after the stimulation of TRAP, bFGF, and TNF-α in electrophoretic mobility shift assay, and E5510 suppressed the NF-κB activation induced by TRAP and bFGF but not the activation by TNF-α. Western blot analysis of I-κBα and I-κBβ, inhibitors of NF-κB, indicated that I-κBα degradation, rather than I-κBβ degradation, was important in NF-κB activation after the stimulation of TRAP and bFGF. PD98059, an inhibitor of extracellular signal-regulated kinase (ERK) kinase, suppressed NF-κB transcriptional activity and SMC proliferation. The phosphorylation of ERK1/2 was rapidly induced by TRAP and bFGF but not by TNF-α. These results indicate that TRAP and bFGF induced I-κB degradation and NF-κB activation through a distinct pathway from TNF-α and that ERK1/2 may play an important role in NF-κB activation induced by TRAP and bFGF.

SMC proliferation after lumen injury (3,4). In culture, NF-B is activated by growth stimulants and cytokines in SMC (5)(6)(7). Antisense of p65 of NF-B inhibited SMC proliferation and intimal thickening of injured arteries in the rat (8,9). These studies indicate that NF-B is involved in SMC proliferation in vitro and in vivo. Proliferation of SMC plays an important role in the formation of intimal thickening in animals and human with several vascular diseases, such as restenosis after percutaneous transluminal coronary angioplasty and atherosclerosis (10,11). However, intracellular signals leading to NF-B activation in SMC proliferation is still unclear.
I-B is an inhibitory protein of NF-B. The I-B family includes I-B␣, I-B␤, I-B␥, p100, p105, and Bcl-3. After cell stimulation, I-B is phosphorylated and degraded by ubiquitination and cleavage by proteasome enzymes. As a result, NF-B is released as an active form, is localized into nuclei, and transmits signals through binding to DNA (1). I-B degrades immediately after injury in vascular walls (4). Microinjection or liposomal delivery of I-B␣ blocks NF-B activation and inhibits SMC proliferation (12,13). Immunohistochemical study of human atherosclerotic tissues, using the antibody recognizing I-B-binding site of p65 of NF-B, indicated that p65 of NF-B was activated in SMC of atherosclerotic lesions, but not in normal vascular tissues (2). These findings suggest that I-B plays a key role to regulate the activation of NF-B in SMC proliferation on vascular walls.
Proliferation of vascular SMC may be regulated by growth factors and cytokines in the formation of atherosclerotic lesions. A number of factors to regulate SMC proliferation have been identified, such as thrombin, basic fibroblast growth factor (bFGF), and tumor necrosis factor-␣ (TNF-␣). The response of thrombin is mimicked by thrombin receptor-activating peptide (TRAP). These factors have been reported to stimulate NF-B activation in various cells. On the other hand, a previous paper indicated that 4-cyano-5,5-bis-(methoxyphenyl)4pentenoic acid (E5510) suppressed NF-B activation induced by thrombin in SMC (14). In this study, we first focused on NF-B activation and I-B degradation to be regulated by bFGF, TRAP, TNF-␣, and E5510. We have reported in this paper that TRAP and bFGF induced I-B degradation and NF-B activation through an E5510-sensitive pathway and TNF-␣ through an E5510-insensitive pathway. Extracellularregulated kinase (ERK) 1/2 pathway is activated in SMC proliferation in vivo and in vitro (15,16) and plays a key role in NF-B activation in monocytes (17,18). Here we have also reported the role of ERK1/2 in NF-B activation induced by TRAP and bFGF but not by TNF-␣.

MATERIALS AND METHODS
Reagents-TRAP (SFLLRN) was purchased from Peninsula Laboratories; bFGF was from Amersham Pharmacia Biotech, and TNF-␣ was from Genzyme. PD98059 was purchased from Sigma. E5510 (Fig. 1) was prepared in Eisai, according to the method previously reported (19).
Cell Culture-Rat vascular SMC were isolated by the explant method (20). Briefly, aortic explants were obtained from the thoracic aorta of * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
SMC Proliferation Assay-Proliferation activity was determined to measure [ 3 H]thymidine incorporation into SMC (21). Confluent SMC were trypsinized, suspended in DMEM supplemented with 10% fetal bovine serum, and seeded at 2.5 ϫ 10 4 cells per well into 24-well flasks (Corning Glass). After 24 h, the cells were washed with serum-free DMEM and starved with DMEM containing 0.1% bovine serum albumin for 24 h. Then the cells were incubated with growth factors at indicated concentrations for 18 h. In experiments to determine the effect of E5510, the cells were pretreated with E5510 for 2 h. Cells were incubated with [ 3 H]thymidine (Amersham Pharmacia Biotech) for 6 h, and [ 3 H]thymidine incorporation into the cells was counted by a liquid scintillation counter.
To determine the change in cell number, the growth activity was measured by a colorimetric assay using MTT reagent, 3-(4,5-dimethythiazol-2-yl)2,5-diphenyltetrazolium bromide (Sigma) (22). In brief, after SMC were starved for 20 h, growth factors were added to each well. At an indicated time after stimulation, cells were incubated with 0.6 mg/ml MTT reagent for 4 h. The reagent was reduced by living cells to form insoluble blue formazan product. The cells were washed, solubilized with 10% sodium dodecyl sulfate, and quantified as the absorbance at 570 nm.
Assay for NF-B Activity-NF-B dependent transcriptional activity in SMC was measured using placental alkaline phosphatase (PLAP) reporter gene combined with NF-B-dependent human immunodeficiency virus-1-long terminal repeat (HIV-1-LTR) sequence (23,24). SMC were transiently transfected with it using DEAE-dextran (Amersham Pharmacia Biotech). At 12 h after transfection, cells were starved with DMEM containing 0.1% bovine serum albumin for 24 h, and stimulants at indicated concentrations were added to the cells for 24 h. In some experiments, cells were pretreated with E5510 for 2 h before stimulation. Culture supernatant was collected, and alkaline phosphatase activity was measured with a microplate luminometer (EG & G Berthold, Germany).
DNA binding activity of NF-B was studied using electrophoretic mobility shift assay (EMSA). SMC were activated with stimulants for the indicated times, and the cells were collected with a cell scraper. Nuclear extracts were prepared and applied to gel shift assay as described previously (24,25). Briefly, 2 g of nuclear extracts were incubated with a 35-base pair double-stranded 32 P-labeled probe encoding the B consensus sequence (5Ј-GGC TAC AAG GGA CTT TCC GCT GGG GAC TTT CCA GC-3Ј) in the binding buffer containing 10 mM Tris-HCl, 40 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 1% Nonidet P-40, 1% deoxycholate, 3 g/ml polydeoxyinosinic-deoxycytidylic acid at room temperature for 30 min. Then samples were applied to native 5% polyacrylamide gels and analyzed on BAS 2000 (Fuji Photo Film Co., Japan). For competition assay, 20-or 40-fold molar excess unlabeled consensus oligonucleotide was added at 30 min prior to addition of the labeled probe. Components of NF-B proteins were identified by supershift assay using antibodies against p50, p52, p65, c-Rel, and RelB antibodies (Santa Cruz Biotechnology, Inc.). For control, DNA activity of AP-1 was studied using AP-1-specific probes in EMSA and anti-c-Jun and anti-NF-ATc2 antibodies (Santa Cruz Biotechnology, Inc.) in supershift assay (24,25).
Western Blotting-Serum-starved SMC in 100-mm dishes were stimulated with bFGF, TRAP, or TNF-␣ for 15 min and solubilized with ice-cold buffer (pH 7.8) containing 20 mM HEPES, 1.5 mM MgCl 2 , 420 mM NaCl, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM Na 3 VO 4 , 1 mM NaF. The cells were collected with a cell scraper in the ice-cold buffer and then homogenized with 60 strokes of a Dounce homogenizer at 4°C. The homogenates were centrifuged at 4600 ϫ g for 10 min, and 20 g of the cytosolic fraction was subjected to electrophoresis on 10% SDS gels. Protein was transferred to polyvinylidene difluoride membranes and detected with anti-I-B␣ and anti-I-B␤ antibodies (Santa Cruz Biotechnology, Inc.) and horseradish peroxidase-linked donkey anti-rabbit IgG antiserum. Detection was quantitated by a chemiluminescence technique using ECL reagents and ECL hyperfilms (Amersham Pharmacia Biotech).
Assay for ERK1/2 Activity-ERK1/2 activity was measured by MAPK enzyme assay system (Amersham Pharmacia Biotech). Briefly, SMC were starved with DMEM containing 0.1% bovine serum albumin for 24 h. After growth stimulation at the indicated times, cell stimulation was terminated by a rapid aspiration of the medium and addition of 500 l of ice-cold lysis buffer containing 25 mM Tris-HCl (pH 7.4), 25 mM sodium phosphate, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 10 g/ml aprotinin, 1% Triton X-100. Cell lysates were incubated on ice for 20 min and centrifuged for 20 min at 4°C. MAPK activity was assayed by addition of kinase buffer containing 1.0 Ci of [␥-32 P]ATP and a substrate peptide that contains Thr-699 phosphorylation site of epidermal growth factor receptor followed by incubation at 30°C for 30 min. Reaction was terminated by spotting the samples onto binding papers. The papers were immediately washed with distilled water 3 times and placed into scintillation vials, and radioactivity was counted.
Statistical Analysis-Data were analyzed by analysis of variance. When a significant difference was detected, the data were further analyzed by Dunnett's multiple range test. Statistical significance was assumed for p Ͻ 0.05 and p Ͻ 0.01.

RESULTS
Proliferation of vascular SMC may be regulated by growth factors and cytokines in the formation of atherosclerotic lesions. A number of factors to regulate SMC proliferation have been identified, such as thrombin, bFGF, and TNF-␣. The response of thrombin is mimicked by TRAP. Nuclear transcriptional factors such as NF-B regulate SMC proliferation. We first determined the effect of growth factors and cytokine TNF-␣ on DNA synthesis in cultured SMC by [ 3 H]thymidine incorporation assay. TRAP and bFGF induced [ 3 H]thymidine incorporation into SMC in a dose-dependent manner ( Fig. 2A). TRAP at 10 M and bFGF at 1 ng/ml increased the activity up to 3 and 6.5 times, respectively, to the unstimulated activity. TNF-␣ did not affect the activity even at concentrations up to 10 ng/ml. Since previous papers indicated that E5510 inhibited SMC proliferation in intimal thickening in canine vascular tissue (26,27), we determined the effect of E5510 on cultured SMC proliferation. E5510 inhibited [ 3 H]thymidine incorporation induced by TRAP and bFGF (Fig. 2B). E5510 attenuated the activity of TRAP to the basal level at 500 M. E5510 decreased the activity of bFGF in a dose-dependent manner, and the inhibition was significant at 0.5 M and higher concentrations. E5510 had no effect on [ 3 H]thymidine incorporation into untreated and TNF-␣-treated cells.
To determine the effect of E5510 on the increase in cell number, we utilized MTT assay. TRAP and bFGF increased cell number in a time-dependent manner. During 6 days incubation, TRAP increased cell number 3.3 times and bFGF 4.5 times. TNF-␣ induced the slight increase in cell number at 1 day but no significant increase at 3 and 6 days (Fig. 3A). E5510 suppressed the increase in cell number induced by TRAP and bFGF. E5510 diminished the activity of TRAP at 300 M and the activity of bFGF at 100 M to the unstimulated level. E5510 had no effect on cell number of untreated and TNF-␣-treated cells (Fig. 3B). These results show that TRAP and bFGF were potent stimulants for SMC proliferation, whereas TNF-␣ was less potent and that E5510 inhibited DNA synthesis and proliferation of SMC after the stimulation of TRAP and bFGF.
NF-B has been reported to be important in SMC proliferation. Components of NF-B, p65 and p50, were expressed in vascular walls, and their expression was induced during cultured SMC proliferation (3)(4)(5)(6)(7). To measure the NF-B-dependent transcriptional activity, SMC were transfected with HIV-1-LTR PLAP. Tandem NF-B sequence exists in the LTR of HIV-1, and NF-B positively regulates transcription of this gene (23). TRAP, bFGF, and TNF-␣ stimulated the transcriptional activity in a dose-dependent manner (Fig. 4A). TRAP enhanced the activity significantly at 3 M and more. bFGF and TNF-␣ increased the transcriptional activity at 3 and 10 ng/ml. E5510 at 100 M suppressed the activity stimulated by TRAP and bFGF (Fig. 4B). In contrast, E5510 did not affect the TNF-␣-induced transcriptional activity even at 500 M. These results showed that the NF-B-dependent transcriptional activity was induced by TRAP and bFGF through a distinct pathway from that of TNF-␣ in SMC.
Transcriptional activation of genes by NF-B requires its binding to DNA. To determine NF-B activity to bind to DNA after SMC stimulation, the nuclear extracts were analyzed in EMSA using an oligonucleotide containing NF-B consensus sequence (Fig. 5). EMSA showed the two bands corresponding to NF-B, p50/p50 homodimer complex, and p50/p65 heterodimer complex. After the treatment with TRAP, bFGF, and TNF-␣, NF-B activation in binding to DNA was induced, which was in parallel with the NF-B transcriptional activity in the reporter gene assay. When E5510 was added to the culture, the agent suppressed the NF-B activation in the stimulation by TRAP and bFGF. In contrast, E5510 at the same dose had no effect on the activation by TNF-␣. Supershift assay was done using antibodies against p50, p52, p65, c-Rel, and RelB. For control, anti-c-Jun and anti-NF-ATc2 antibodies were used. Anti-p50 and p65 antibodies induced the dramatic shift of the bands, and anti-c-Rel induced the shift of the minor portion of the band. Other antibodies did not affect the migration of the band. 20-or 40-fold molar excess of unlabeled consensus oligonucleotide diminished NF-B band (data not shown). These data showed that NF-B complexes contained p50 and p65 predominantly and c-Rel in minor portion of the band. To study the specificity of NF-B signals, we next determined AP-1 activity in SMC.
We evaluated the same nuclear extract in EMSA using AP-1-specific probes and confirmed that E5510 had no effect on AP-1 activity in the treatment of TRAP, bFGF, and TNF-␣ (data not shown). The results of NF-B activity in EMSA was quantitated in BAS2000 and compared with the results of AP-1 activity (Fig. 6). E5510 suppressed the NF-B activation induced by TRAP by 79.5% and the activation by bFGF by 73.0%. In contrast, E5510 at the same dose had no effect on the Results were shown as average value and S.E. Statistical analysis was done with Dunnett's multiple range test. *, p Ͻ 0.05; **, p Ͻ 0.01 versus control (without stimulants). n ϭ 5 for each point. B, cells were pretreated with E5510 for 2 h and TRAP (10 M), bFGF (10 ng/ml), and TNF-␣ (3 ng/ml) were added for 6 days. *, p Ͻ 0.05; **, p Ͻ 0.01 versus control (without E5510). n ϭ 5 for each point. activation by TNF-␣. E5510 did not decrease the DNA binding activity of AP-1. These results suggest that E5510 suppressed NF-B DNA binding activity induced by TRAP and bFGF but not the activity by TNF-␣ and that the effect was selective to NF-B but not to AP-1.
NF-B activity is regulated by I-B proteins, and the degradation of I-B results in NF-B activation. Cytosolic extracts were isolated from SMC treated with TRAP, bFGF, and TNF-␣, and the degradation of I-B␣ and I-B␤ proteins was determined by Western blot analysis (Fig. 7). I-B␣ was degraded 60 min after stimulation by TRAP and bFGF. After 180 min, the amount of the protein was recovered. In contrast, TNF-␣ did not promote the I-B␣ degradation in SMC. On the other hand, the amount of I-B␤ was once increased 15 and 30 min after stimulation by any of TRAP, bFGF, and TNF-␣ and then degraded after 60 and 180 min. These data suggest that I-B␣ degradation was regulated in a different way from I-B␤ degradation in SMC and that I-B␣ degradation, rather than I-B␤ degradation, might play a role in NF-B activation induced by TRAP and bFGF.
To study the mechanism how E5510 suppressed NF-B activation, the effects of E5510 on the degradation of I-B␣ and I-B␤ proteins were determined by Western blot analysis (Fig.  8). Compared with vehicle-treated cells, E5510-treated cells showed the suppressed degradation of I-B␣ in the activation by TRAP and bFGF. E5510 had no effect on I-B␣ degradation induced by TNF-␣. On the other hand, E5510 suppressed I-B␤ degradation in the activation by bFGF and TNF-␣. But E5510 had no effect on I-B␤ degradation induced by TRAP. These results suggest that E5510 was potent to suppress the degradation of I-B proteins. The degradation of I-B␣, rather than I-B␤, was important in NF-B activation after the stimulation of TRAP and bFGF in SMC.
MAPK is a key component in cell proliferation. Among the MAPK family, ERK1/2 plays a role in stimulating SMC proliferation, and ERK1/2 activity is dramatically induced during SMC proliferation in injured arteries in the rat (16). Therefore, we determined the possibility that ERK1/2 regulated NF-B activity in SMC using an NF-B reporter gene assay and an ERK kinase inhibitor PD98059 (Fig. 9A). PD98059 significantly decreased the NF-B transcriptional activity induced by TRAP and bFGF. At 10 M PD98059, the NF-B transcriptional activity of TRAP and bFGF was reduced to the control level. PD98059 at the same dose had no effect on vehicletreated culture. In addition, the effect of PD98059 on SMC proliferation was determined in MTT assay (Fig. 9B). PD98059 suppressed SMC proliferation induced by TRAP and bFGF. PD98059 decreased the proliferation induced by TRAP and bFGF to 27.5 and 19.8% of the control, respectively. These data indicate that ERK1/2 was important in NF-B activation and SMC proliferation.
Finally, the effect of E5510 on ERK1/2 activity in SMC was determined (Fig. 10). ERK1/2-dependent phosphorylation assay clearly indicated that TRAP and bFGF stimulated ERK1/2 activation. TRAP significantly activated ERK1/2 at 15 and 30 min after the stimulation, and the activity was returned to the level of unstimulated cells at 60 min. bFGF induced the activation at 15 min, and the activity decreased rapidly at 30 and 60 min. In contrast, TNF-␣ had no effect on ERK1/2 activity at concentrations of 3-10 ng/ml, although TNF-␣ at these doses activated NF-B. Then we determined the effect of E5510 on ERK1/2 activity. It was remarkable, however, that E5510 did not affect the ERK1/2 activity stimulated by TRAP and bFGF. Since ERK1/2 was important in NF-B activation and SMC proliferation induced by TRAP and bFGF, these results indicated that E5510 suppressed a signal in the downstream of ERK1/2 activation leading to NF-B activation in SMC proliferation.

DISCUSSION
Proliferation of vascular SMC is a crucial event in the formation of atherosclerotic tissues and is regulated by growth factors. We studied the effects of growth factors on cultured rat SMC proliferation. TRAP and bFGF, but not TNF-␣, stimulated cell proliferation in [ 3 H]thymidine incorporation assay and increased cell number. E5510 dose-dependently inhibited SMC proliferation induced by TRAP and bFGF. In animal experiments, E5510 inhibited SMC proliferation in intimal thickening in canine vein graft and Teflon graft models (26,27). These reports indicate that E5510 is a potent inhibitor of SMC proliferation in culture and in vivo.
TRAP and bFGF activated the NF-B-dependent transcription in SMC. E5510 suppressed NF-B activation of TRAP and bFGF and also blocked TRAP-and bFGF-inducible degradation of I-B␣. These findings indicate that the inhibitory effect of E5510 on NF-B activation was due to the suppression of I-B␣ degradation. We propose the signal cascade of NF-B activation and SMC proliferation, as discussed below (Fig. 11).
The mechanism has not been identified how E5510 inhibited the degradation of I-B␣ and I-B␤. E5510 was originally pre- pared as an anti-platelet agent. In previous studies of ours and others (19,28,29) using human platelets, E5510 had a potent inhibitory effect on platelet aggregation and subsequent release of mitogens. The effect was found to be dependent on the inhibition of cyclooxygenase (COX). Inhibition of COX-1 and -2 activities by E5510 resulted in blocking collagen-and arachidonic acid-induced platelet aggregation, like a COX inhibitor indomethacin. Salicylates, anti-inflammatory agents with an inhibitory activity of COX, inhibit lipopolysaccharide-induced NF-B activation and tissue factor expression through blocking I-B␣ degradation in monocytic THP-1 cells (30). Furthermore, salicylates suppress TNF-␣-induced I-B␣ phosphorylation, degradation, NF-B activation, and adhesion molecules expression (31). However, indomethacin has no effect on the I-B␣ degradation in the same model (31). COXs are induced in SMC and other cells by growth factors and cytokines (32,33). COXs produce prostaglandins and activate cAMP-dependent kinase that suppresses SMC proliferation (15,34). As a consequence, the suppression of COX activity does not inhibit but instead enhances SMC proliferation (32,35). A recent paper has indicated that COX-2 expression is unrelated to NF-B activity in rat SMC (36). Therefore, the inhibitory effect of E5510 on COX activity may not contribute to the suppression of I-B degradation and NF-B activation.
On the other hand, E5510 is a potent inhibitor of phosphodiesterase (PDE) activity, and it may inhibit SMC proliferation through cAMP generation. Inhibition of PDE activities by E5510 results in blocking thrombin-induced platelet aggregation. In the experiments with purified PDE proteins, E5510 suppresses the activity of PDE types II, III, and V and increased cAMP and cGMP levels in platelets (29). The suppression of PDE type III elevates cAMP levels and inhibits SMC proliferation (37). The increase of cAMP and the activation of cAMP-dependent kinase suppress NF-B activation in monocytic THP-1 cells and endothelial cells (38). NF-B-dependent tissue factor gene expression is inhibited by elevated cAMP and by overexpression of catalytic subunit of cAMP-dependent kinase, whereas cAMP does not prevent nuclear translocation of NF-B (38). cAMP-dependent kinase mediated the phosphorylation of cAMP response element-binding protein and the subsequent recruitment of the transcriptional coactivator cAMP response element-binding protein, which inhibits NF-B activity (39). cAMP blocks Raf-1 leading to the activation of ERK kinases (40). Thrombin-induced ERK1/2 activation in airway SMC is suppressed by forskolin which increases cAMP (41). Forskolin also inhibits PDGF-induced ERK activity in aortic SMC (42). But in contrast to cAMP, E5510 did not inhibit ERK activation by TRAP and bFGF. Another possibility should be considered that E5510 blocked NF-B activation in a cAMPindependent manner.
It is important in this study that E5510 suppressed the I-B degradation and NF-B activation by TRAP and bFGF but not the activation by TNF-␣ in SMC. These data indicate that the I-B degradation and NF-B activation by TRAP and bFGF is mediated through a distinct pathway from TNF-␣. So far, a number of agents have been identified to block NF-B activation through the inhibition of I-B degradation, just like E5510. Sanguinarine, a benzophenanthridine alkaloid, inhibited I-B degradation and NF-B activation induced by TNF-␣, interleukin-1, phorbol ester, and okadaic acid but not the activation by hydrogen peroxide and ceramide (43). Tissue factor expression in endothelial cells (EC) is dependent upon NF-B activation, and dilazep, another anti-platelet agent, inhibited the expression of tissue factor mRNA and protein induced by thrombin and phorbol ester but not the expression induced by TNF-␣ (44). The pathway to NF-B activation may be different in the signals by different inducers. Protein kinase C (PKC), MAPK/ERK kinase, and MAPK are important to regulate NF-B activation (45,46). Conventional, novel, and atypical PKC isotypes are involved in ERK1/2 activation during phorbol ester-induced proliferation of Swiss 3T3 cells (47). When SMC are cultured in high glucose condition, NF-B activation is induced through a PKC-dependent pathway (48). Thrombin activates PKC and NF-B in EC (49,50). Enforced expression of MAPK/ERK kinase kinase-1, -2, and -3 induces I-B degradation in HeLa cells (51). Our data that an ERK inhibitor PD98059 attenuated NF-B activation suggest the important role of ERK kinase for NF-B activation in SMC. Since E5510 suppressed the degradation of I-B proteins but not ERK1/2 activity, signals in the downstream of ERK1/2 may be a target that E5510 blocks.
This paper provides the first evidence that the degradation of I-B␣, rather than I-B␤, was important in NF-B activation for SMC proliferation after the stimulation of TRAP and bFGF. E5510-treated cells showed the suppressed degradation of I-B␣ in the activation by TRAP and bFGF, whereas E5510 had no effect on I-B␣ degradation induced by TNF-␣. On the other hand, E5510 suppressed I-B␤ degradation in the activation by bFGF and TNF-␣ without effect on I-B␤ degradation induced by TRAP. There has been accumulating evidence that kinases activate NF-B. Protein tyrosine kinases induce I-B degradation and NF-B activation (52). The serine/threonine phosphatase inhibitors induce the rapid degradation of I-B (53,54). I-B phosphorylates by casein kinase II (55). I-B is phosphorylated and degraded by I-kinase (IKK)-␣ and -␤, and overexpression of NF-B-inducible kinase stimulates the kinase activities of IKK-␣ and -␤, whereas overexpression of MEKK1 preferentially stimulates the kinase activity of IKK-␤ (56). The activation of IKK-␤, but not IKK-␣, is stimulated by the overexpression of PKC (57). Nuclear expression of PKC increases during SMC and EC proliferation (58,59) and thrombin activates PKC , whereas TNF fails to activate it (58,60,61). It is possible that the inhibition of these processes is a target of E5510. However, the mechanism of E5510 to block I-B degradation and NF-B activation needs further studies.
In contrast to TRAP and bFGF, TNF-␣ was a weak stimulant for SMC proliferation in this study, although TNF-␣ was potent to activate NF-B in SMC. The reason is unclear but TNF-␣ may exert bidirectory signals to promote and suppress proliferation. As discussed above, TNF-␣ induces the production of COXs and prostaglandins that suppress SMC proliferation (32). Alternatively, NF-B activation may be important but not sufficient to induce certain biological responses such as cell proliferation. In bovine coronary SMC, the proliferation is induced by thrombin but not by TRAP, although both stimulate NF-B activation (62). In the presence of other mitogens such as serum, TNF-␣ enhances SMC proliferation in culture (63). Matrix metalloproteinase expression is synergistically activated by the combination of TNF-␣ and bFGF through two transcriptional factors NF-B and AP-1. NF-B is induced by TNF-␣ and AP-1 by bFGF (64). Thrombin activates PKC and NF-B, whereas TNF-␣ activated NF-B but not PKC in EC (49,50). Thrombin and TRAP potentiate TNF-␣-induced Eselectin expression in EC (65). Synergistic signal pathways may be necessary for cell proliferation in addition to NF-B activation.
In conclusion, TRAP and bFGF induce I-B degradation and NF-B activation through a distinct pathway from TNF-␣, and ERK1/2 may play an important role in NF-B activation leading to SMC proliferation. Since the suppression of SMC proliferation is the beneficial approach to prevent vascular diseases, E5510 is useful as a therapeutic agent and a good tool to study NF-B-dependent signals for SMC proliferation.