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Cardiovascular Research 2007 76(2):331-339; doi:10.1016/j.cardiores.2007.06.030
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Copyright © 2007, European Society of Cardiology

Role of NAD(P)H:quinone oxidoreductase 1 on tumor necrosis factor-{alpha}-induced migration of human vascular smooth muscle cells

Syng-Ook Leea, Young-Chae Changb, Key Whanga, Cheorl-Ho Kimc,* and In-Seon Leea,*

aDepartment of Food Science and Technology and The Center for Traditional Microorganism Resources (TMR), Keimyung University, Daegu 704-701, Republic of Korea
bDepartment of Pathology, Catholic University of Daegu School of Medicine, Daegu 705-034, Republic of Korea
cMolecular and Cellular Glycobiology Unit, Department of Biological Science, Sungkyunkwan University, Suwon City, Kyunggi 440-746, Republic of Korea

*Corresponding authors. Lee is to be contacted at The Center for Traditional Microorganism Resources (TMR), Keimyung University, 1000 Sindang-Dong, Dalseo-Gu, Daegu 704-701, Republic of Korea. Tel./fax: +82 53 580 5538. Kim, Department of Biological Science, Sungkyunkwan University, Chunchun-Dong 300, Jangan-Gu, Suwon City, Kyunggi-Do 440-746, Republic of Korea. Tel.: +82 31 290 7002; fax: +82 31 290 7015. chkimbio{at}skku.edu inseon{at}kmu.ac.kr

Received 12 December 2006; revised 28 June 2007; accepted 29 June 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objectives In a preliminary study, NAD(P)H:quinone oxidoreductase 1 (NQO1) was found to be highly expressed in cultured human aortic smooth muscle cells (HASMC) and dicumarol, a NQO1 inhibitor and a coumarin-derived natural anticoagulant, suppressed tumor necrosis factor (TNF)-{alpha}-induced HASMC migration. Therefore, it was hypothesized that NQO1 plays an important role in the regulation of vascular smooth muscle cells (VSMC) migration activated by TNF-{alpha}.

Methods and results Gelatin zymography, reporter gene, electrophoretic mobility shift and Western blotting assays showed that dicumarol, but not other coumarin-derived anticoagulants, inhibited TNF-{alpha}-induced HASMC migration and suppressed TNF-{alpha}-induced matrix metalloproteinase (MMP)-9 expression and secretion in a dose-dependent manner. In addition, down-regulation of NQO1 by transfection of its small interfering RNA similarly inhibited TNF-{alpha}-induced MMP-9 secretion, indicating that dicumarol-mediated inhibition of MMP-9 expression is due in large part to inhibition of NQO1. Down-regulation of NQO1 inhibits MMP-9 gene expression by suppressing activation of nuclear factor-kappa B (NF-{kappa}B) and activator protein-1 (AP-1), well-known key elements mediating MMP-9 gene expression in its promoter, via the p38 MAPK and JNK pathways.

Conclusion The results of the present study demonstrate that down-regulation of NQO1 effectively suppresses TNF-{alpha}-induced HASMC migration through inhibition of MMP-9 expression, suggesting that NQO1 may be a potential target for the prevention of vascular disorders related to migration of VSMC.

KEYWORDS Vascular smooth muscle cells; Migration; TNF-{alpha}; NAD(P)H:quinone oxidoreductase 1; Dicumarol; Matrix metalloproteinase-9


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Proliferation and migration of vascular smooth muscle cells (VSMC) play a major role in the development and progression of many cardiovascular diseases, including atherosclerosis and VSMC are the principal cell type in both atherosclerotic and restenotic lesions [1]. During the early stages of atherosclerosis or arterial wall injury, in response to platelet activation, thrombin generation, and the release of various growth factors and cytokines, VSMC migrate into the intimal layer of the arterial wall, causing intimal thickening. In particular, TNF-{alpha} secreted by VSMC in the neointima, as well as by macrophages accumulated in atherosclerotic lesions, induces marked proliferation and migration of VSMC [1,2]. Extracellular matrix (ECM) degradation is an important biological process in the proliferation and migration of VSMC. ECM degradation and remodeling require the action of extracellular proteinases [3]. Recent in vitro and in vivo studies concluded that among the proteinases, MMP-9 is critical for the development of arterial lesions via its regulation of both VSMC migration and proliferation [4,5]. Basal levels of MMP-9 in VSMC usually are low and MMP-9 expression can be induced by treatment with TNF-{alpha} via activation of NF-{kappa}B and AP-1 [3,6,7].

NAD(P)H:quinone oxidoreductase 1 (NQO1, EC 1.6.99.2) is a cytosolic flavoenzyme that catalyzes the obligatory two-electron reduction of a variety of quinone substrates, using both NADH and NADPH as electron donors, to give the corresponding hydroquinone [8]. Dicumarol, a coumarin derivative isolated from sweet clover, is used clinically to inhibit vitamin K (a biological quinone)-dependent blood coagulation. Moreover, the best characterized action of dicumarol on cells is the competitive inhibition of NQO1. A role of NQO1 is thus frequently attributed to biological effects that are inhibited by dicumarol [9,10].

NQO1 inhibition modulates a variety of physiological activities in various cellular systems by altering the function of several signaling pathways, including cell growth and death [9,11,12]. Dicumarol blocks JNK and NF-{kappa}B pathways and potentiates apoptosis induced by TNF-{alpha} in HeLa cells [11]. Dicumarol also inhibits the malignant phenotype of pancreatic cancer cells [9]. In contrast, dicumarol treatment results in degradation of p53 and blocks wild-type p53-mediated apoptosis in {delta}-irradiated normal thymocytes and in M1 myeloid leukemic cells overexpressing wild-type p53 [12]. Dicumarol also stimulates growth by inhibiting the normal blockade in G0/1 in HL-60 cells, which completely lack the p53 gene [10].

Whereas cellular functions of NQO1 in various tumor cells are well known, NQO1 expression and its roles in VSMC remain unknown. In a preliminary study, NQO1 was found to be highly expressed in cultured HASMC and dicumarol, an NQO1-specific inhibitor, suppressed TNF-{alpha}-induced HASMC migration. Therefore, it was hypothesized that NQO1 plays an important role in the regulation of TNF-{alpha}-induced VSMC migration. The present study demonstrates that down-regulation of NQO1 effectively suppresses TNF-{alpha}-induced HASMC migration by inhibiting MMP-9 expression. In addition, this study shows that NQO1 is an important mediator required for TNF-{alpha}-induced NF-{kappa}B and AP-1 activation in HASMC.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1 Cell culture
HASMC were purchased from Bio-Whittaker (California, USA). HASMC were cultured in DMEM (Gibco BRL, Rockville, MD) supplemented with 10% fetal bovine serum (FBS) and 5% CO2 at 37 °C. For all experiments, early passage HASMC were grown to 80–90% confluence and made quiescent by serum starvation (0.1% FBS) for at least 24 h.

2.2 Migration assay
Matrigel migration assay was performed as described previously [13] using Matrigel-coated filter inserts (8 µm pore size; Becton-Dickinson, NJ) that fit into 24-well migration chambers. HASMC to be tested for migration were detached from the tissue culture plates, washed, and resuspended in conditioned medium (2x104 cells/well), then added to the upper compartment of the migration chamber with TNF-{alpha} (100 ng/ml) in the presence or absence of drugs. Conditioned medium (500 µl) was added to the lower compartment of the migration chamber. The chambers were incubated at 37 °C for 24 h in 5% CO2. After incubation, the filter inserts were removed from the wells and the cells on the upper side of the filter were removed using cotton swabs. The filters were fixed, stained, and mounted according to the manufacturer's instructions (Becton-Dickinson). The cells that migrated through the Matrigel and were located on the underside of the filter were counted. Three to five chambers were used per condition.

2.3 Gelatin zymography assay
Conditioned medium was electrophoresed in a polyacrylamide gel containing 0.1% (w/v) gelatin. The gel was then washed at room temperature for 30 min with 2.5% Triton X-100 and subsequently incubated at 37 °C for 24 h in a buffer containing 10 mM CaCl2, 0.01% NaN3, and 50 mM Tris–HCl (pH 7.5). The gel was stained with 0.2% Coomassie brilliant blue and photographed on a light box. Proteolysis was detected as a white zone in a dark blue field.

2.4 Transfection of small interfering RNA (siRNA)
The 19-nucleotide specific sequence for the NQO1 siRNA, which effectively down-regulated the endogenous NQO1 level in HASMC, is as follow: 5'-CUCAACUGACAUAUAGCAU-3'. For transfection of the siRNA duplexes, 1.5x105 cells were plated in 6-well plates and grown overnight. The cells were transfected with 100 pmol of the siRNA duplex for 5 h using Lipofectamine reagent (Invitrogen). After transfection, the cells were washed with PBS and incubated for 48 h in culture medium. The cells were then incubated with drug in serum free medium for additional 24 h, then the conditioned medium was collected for zymography assay and cells were collected for western blot analysis and enzyme assay.

2.5 NQO1 activity assay
NQO1 activity was measured by the method as described [14]. NQO1 activity is described as the dicumarol inhibitable decrease in absorbance at 600 nm with 2,6-dichloroindophenol (DCPIP) as a substrate. The reaction mix contained 25 mM Tris (pH 7.4), 0.07% bovine serum albumin (w/v), 0.01% Tween 20, 5 µM flavin adenine dinucleotide, 0.2 mM NADH, and 40 µM DCPIP, and the activity was measured at 600 nm for 1 min at 25 °C in the presence and absence of 20 µM dicumarol. NQO1 activity is described as the dicumarol inhibitable decrease in absorbance at 600 nm with DCPIP as a substrate. An extinction coefficient of 21/mM/cm was used for the determination of NQO1 specific activity. Protein concentrations were measured using the Bio-Rad protein assay.

2.6 Western blot analysis
HASMC lysates, SDS-PAGE, transfer to an Immobilon-P-membrane (Millipore, USA), and immunoblotting were performed as described previously [13]. To determine the activations of NF-{kappa}B and AP-1, nuclear extracts of cells were isolated by the protocol of electrophoretic mobility shift assay (EMSA). Histone-H1 was used as the internal control for the nuclear extract and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as the internal control for the total cell extract.

2.7 Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted by the RNAzol B reagent (Tel-test, Friendswood, TX, USA) according to the manufacturer's instructions. For RT-PCR, a cDNA was synthesized from 1 µg of total RNA using AMV RNA PCR Kit (Takara, Japan) according to the manufacturer's protocol. The cDNA was amplified by PCR with the following primers: MMP-9 (537 bp), 5'-CGGAGCACGGAGACGGGTAT-3' (sense) and 5'-TGAAGGGGAAGACGCACAGC-3' (antisense); β-actin (247 bp), 5'-CAAGAGATGGCCACGGCTGCT-3' (sense) and 5'-TCCTTCTGCATCCTGTCGGCA-3' (antisense).

2.8 Transient transfection and luciferase reporter assay
MMP-9 wild-type (pGL2-MMP-9WT), AP-1-1 site-mutated MMP-9 (mAP-1-1), AP-1-2 site-mutated MMP-9 (mAP-1-2), and NF-{kappa}B site-mutated MMP-9 luciferase promoter constructs were cloned [7]. Cells were plated onto 6-well plates at a density of 2x105 cells/well and grown overnight. Cells were cotransfected with 1 µg of MMP-9 promoter-luciferase reporter constructs and 0.5 µg of the pCMV-β-galactosidase reporter plasmid for 5 h using Lipofectamine reagent (Invitrogen, San Diego, CA, USA). After transfection, the cells were cultured in 10% FBS medium and incubated with drugs for 24 h. Luciferase and β-galactosidase activities were assayed by using the luciferase and β-galactosidase enzyme assay system (Promega). Luciferase activity was normalized with the β-galactosidase activity in the cell lysate and expressed as an average of three independent experiments.

2.9 Electrophoretic mobility shift assay (EMSA)
The nuclear extract of cells was prepared as described below. Cells were washed with cold PBS and suspended in 0.4 ml of lysis buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 2.0 µg/ml leupeptin, and 2.0 µg/ml aprotinin. The cells were allowed to swell on ice for 15 min, and then 25 µl of 10% Nonidet P-40 was added. The tube was vigorously vortexed for 10 s, and the homogenate centrifuged at 4 °C for 2 min at 13,000 rpm. The nuclear pellet was resuspended in 50 µl of ice-cold nuclear extraction buffer containing 20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 2.0 µg/ml leupeptin, and 2.0 µg/ml aprotinin. The tube was incubated on ice for 15 min with intermittent mixing. The nuclear extract was then centrifuged at 4 °C for 5 min at 13,000 rpm and the supernatant was either used immediately or stored at –70 °C for later use. The protein content was measured using the Bio-Rad protein assay. EMSA was performed using a gel shift assay system kit (Promega, Madison, WI) according to the manufacturer's instructions. Briefly, double-stranded oligonucleotides containing the consensus sequences for AP-1-1 (5'-TGACCCCTGAGTCAGCACTT-3'), and NF-{kappa}B (5'-CCAGTGGAATTCCCCAG-3') were end-labeled with [{delta}-32P] ATP (3000 Ci/mmol) using T4 polynucleotide kinase and used as probes for EMSA. Competition was performed using either the unlabeled AP-1-1 or NF-{kappa}B oligonucleotides. Nuclear extract proteins (2 µg) were preincubated with the gel shift binding buffer (4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris–HCl (pH 7.5), and 0.05 mg/ml poly deoxyinosine–deoxycytosine for 10 min, then incubated with the labeled probe for 20 min at room temperature. Each sample was electrophoresed in a 4% nondenaturing polyacrylamide gel in 0.5xTBE buffer at 250 Volt for 20 min. The gel was dried and exposed to X-ray film overnight.

2.10 Statistical analysis
The results are expressed as means±SE and significance, using one way ANOVA followed by Dunnett's test using SPSS (12.0) statistical software. P-values of 0.05 or less were considered statistically significant. All experiments were performed at least three times.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Down-regulation of NQO1 suppressed TNF-{alpha}-induced HASMC migration through inhibition of MMP-9 expression
As shown in Fig. 1B, the migration of HASMC was increased by treatment with TNF-{alpha} compared with TNF-{alpha}-untreated control cells. Dicumarol inhibited TNF-{alpha}-induced migration of HASMC, reaching the levels of TNF-{alpha}-untreated control cells at a concentration of 25 µM. Neither morphological changes (date not shown) nor growth inhibition (<5%; Fig. 1A) of HASMC was observed at this dicumarol concentration.


Figure 1
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  		Fig. 1 Effects of dicumarol on TNF-{alpha}-induced HASMC migration and MMP-9 expression. Cells were treated with the indicated concentrations of dicumarol for 24 h. Cell viability was determined by an XTT assay (A). A Matrigel migration assay was carried out with 25 µM dicumarol in the presence of TNF-{alpha} (100 ng/ml) after 24 h incubation (B). Cells were treated with the indicated concentrations of dicumarol in the presence of TNF-{alpha} (100 ng/ml) for 24 h. GAPDH and β-actin were used as internal controls for Western blotting, and RT-PCR, respectively (C). Data are mean±SE of 3 independent experiments, expressed relative to the control; *P<0.01 vs control, #P<0.01 vs TNF-{alpha} alone.

 
The induction of MMP-9 contributes to migration of VSMC in vivo and in vitro and TNF-{alpha} stimulates MMP-9 expression in VSMC [4,6,7]. Thus, the role of suppression of MMP-9 expression in TNF-{alpha}-induced HASMC migration was examined. Treatment with TNF-{alpha} (100 ng/ml) induced the secretion of a band with proteolytic MMP-9 activity. The induction of MMP-9 was dramatically inhibited by dicumarol in a dose-dependent manner. Dicumarol also inhibited TNF-{alpha}-induced MMP-9 protein and mRNA expression (Fig. 1C). These results show that dicumarol decreased the migration potential of TNF-{alpha}-induced HASMC in vitro and that the inhibition by dicumarol correlated well with inhibition of MMP-9 expression.

To further evaluate the effects of dicumarol responsible for inhibition of MMP-9 expression, other coumarin-derived compounds, coumarin and warfarin, and NQO1 siRNA, which are used as a RNA-silencing strategy to down-regulate the endogenous NQO1 level, were tested for their capacity to inhibit TNF-{alpha}-induced MMP-9 secretion. Coumarin and warfarin did not inhibit MMP-9 secretion, suggesting that vitamin K metabolism is unrelated to dicumarol inhibition of TNF-{alpha}-induced MMP-9 expression in HASMC (Fig. 2A). In contrast, transfection of a NQO1 siRNA, which did not affect cell viability (data not shown), inhibited TNF-{alpha}-induced MMP-9 secretion and protein expression (Fig. 2D). NQO1 siRNA also dramatically reduced TNF-{alpha}-induced HASMC migration (Fig. 2E). NQO1 protein expression and enzyme activity were concomitantly decreased in cells transfected with NQO1 siRNA compared with cells transfected with negative control siRNA (Fig. 2B,C) by approximately 80 and 60%, respectively. These data suggest that suppression of TNF-{alpha}-induced MMP-9 expression by dicumarol is due in large part to the inhibition of NQO1 in HASMC.


Figure 2
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  		Fig. 2 Suppression of MMP-9 expression by dicumarol was due in large part to inhibition of NQO1. Cells were treated with the indicated concentrations of anticoagulants in the presence of TNF-{alpha} (100 ng/ml) for 24 h (A). Cells transfected with either negative control (NC) siRNA or NQO1 siRNA were treated with TNF-{alpha} (100 ng/ml) for 24 h. Two types of cell lysates were prepared and used to determine NQO1 protein expression (B) and NQO1 activity (C), respectively. Gelatin zymography and Western blotting for MMP-9 were performed. GAPDH was used as the internal control (D). A Matrigel migration assay was carried out with the transfected cells after a 24-h incubation in the presence or absence of TNF-{alpha} (100 ng/ml) (E). Cells were treated with the indicated concentrations of hydroquinone and dicumarol in the presence of TNF-{alpha} for 24 h (F). Cells were treated with the indicated concentrations of hydroquinone in the absence or presence of TNF-{alpha} for 24 h (G). The conditioned medium was prepared and used for gelatin zymography. Data are mean±SE of 3 independent experiments, expressed relative to the NC siRNA control; *P<0.01 vs NC siRNA control.

 
In addition, inhibition of NQO1 by dicumarol is expected to result in a decrease in the levels of reduced quinones in the cell [11]. Therefore, the ability of replacing the reduced quinones to reverse inhibition of MMP-9 secretion was examined. Although dicumarol completely inhibited TNF-{alpha}-induced MMP-9 secretion, hydroquinone reversed inhibition of MMP-9 secretion in a dose-dependent manner (Fig. 2F). In addition, hydroquinone activated TNF-{alpha}-induced MMP-9 secretion (Fig. 2G). These data suggest that the suppression of TNF-{alpha}-induced MMP-9 expression by dicumarol is due in large part to a decrease in reduced quinones, resulting from inhibition of NQO1. This proposed mechanism supports a role for NQO1 in the regulation of intracellular environments that allows induction of MMP-9 expression by TNF-{alpha} in HASMC.

3.2 Dicumarol inhibited TNF-{alpha}-induced promoter activity of MMP-9 via both NF-{kappa}B- and AP-1-dependent pathways
Recently, it has been reported that two AP-1 sites (located at –79 bp and –533 bp) and an NF-{kappa}B site (located at –600 bp) in the MMP-9 promoter are centrally involved in the induction of the MMP-9 gene by TNF-{alpha} in VSMC [7]. To investigate which of these transcription factors are involved in inhibition of MMP-9 transcription by dicumarol in HASMC, cells were transiently transfected with reporter genes, including the wild-type MMP-9 promoter (MMP-9WT) or promoters with mutations in each AP-1 binding site (mAP-1-1 and mAP-1-2) or in the NF-{kappa}B binding site (mNF-{kappa}B). As shown in Fig. 3A, luciferase activity was increased up to 6-fold in cells treated with TNF-{alpha} compared with untreated cells. Dicumarol treatment decreased TNF-{alpha}-stimulated luciferase activity in a dose-dependent manner. Luciferase activity remained unchanged when cells were transfected with the promoterless and enhancerless control vector (pGL2-basic), in the presence or absence of TNF-{alpha} (data not shown). Likewise, luciferase activity was unchanged when cells were transfected with mAP-1-1-, mAP-1-2-, or mNF-{kappa}B-MMP-9 in the absence of TNF-{alpha} (data not shown). As shown in Fig. 3B–D, a mutation in the NF-{kappa}B binding site dramatically decreased, by up to 3-fold, TNF-{alpha}-stimulated MMP-9 promoter activity compared with the promoter activity of MMP-9WT. Mutations in each of the AP-1 binding sites also significantly decreased promoter activity induced by TNF-{alpha}, but not to the extent achieved by a mutation in the NF-{kappa}B binding site (P<0.001, data not shown). The activities of the AP-1 binding site-mutated promoters were about 1.4-fold higher than the activity of the NF-{kappa}B binding site-mutated promoter in the presence of TNF-{alpha}. In addition, TNF-{alpha}-stimulated promoter activity of mAP-1-1-, mAP-1-2-, and mNF-{kappa}B-MMP-9, was completely abolished by treatment with 25 µM dicumarol. These results confirm that both the AP-1 and NF-{kappa}B binding sites in the MMP-9 promoter contribute to promoter activity and show that both sites are primary sites for regulating responses to dicumarol during TNF-{alpha}-induced activation of the MMP-9 promoter.


Figure 3
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  		Fig. 3 Effects of dicumarol on the TNF-{alpha}-induced activation of MMP-9 promoter. pMMP-9WT-(A), pmAP-1-1-(B), pmAP-1-2-(C), and pmNF-{kappa}B-MMP-9-luciferase vector (D) were cotransfected with pCMV-β-galactosidase vector into cells. After 4 h of transfection, the cells were treated with the indicated concentrations of dicumarol in the presence of TNF-{alpha} (100 ng/ml) for 24 h. Luciferase activities were normalized to β-galactosidase activity. Data are mean±SE of 3 independent experiments, expressed relative to the control; *P<0.05 vs TNF-{alpha}, **P<0.01 vs TNF-{alpha}, ***P<0.001 vs TNF-{alpha}.

 
The direct involvement of dicumarol in inhibition of AP-1- and NF-{kappa}B-mediated transcriptional activation of MMP-9 in TNF-{alpha}-induced HASMC was confirmed as follows. The inhibitory effect of dicumarol on binding of AP-1 and NF-{kappa}B is isolated from cells to oligonucleotides that contain the sequence for the AP-1 and NF-{kappa}B binding sites from the MMP-9 promoter using EMSA. HASMC were incubated with different concentrations of dicumarol in the presence of TNF-{alpha} for 4 h, and nuclear extracts were prepared and tested by EMSA. As shown in Fig. 4A, AP-1 and NF-{kappa}B DNA binding activities were induced by TNF-{alpha}, as expected however, dicumarol inhibited activation of AP-1 and NF-{kappa}B in a dose-dependent manner.


Figure 4
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  		Fig. 4 Effect of dicumarol on the TNF-{alpha}-induced AP-1 and NF-{kappa}B activations in HASMC. Cells were treated with the indicated concentrations of dicumarol in the presence of TNF-{alpha} (100 ng/ml) for 4 h. Nuclear extracts were prepared and examined for AP-1 and NF-{kappa}B activation by EMSA (A). Nuclear extracts were used to investigate the phosphorylation level of c-Jun and the expression level of p65. Histone-H1 was used as the internal control for the nuclear extract (B).

 
TNF-{alpha} has been shown to induce the translocation of p65, a major subunit of NF-{kappa}B, to nucleus and phosphorylation of c-Jun, a major subunit of AP-1, which are required for the transcriptional activities [15,16]. In the present study, TNF-{alpha} also induced phosphorylation of c-Jun and nuclear translocation of p65. However, dicumarol inhibited phosphorylation and translocation in a dose-dependent manner (Fig. 4B). These data clearly show that dicumarol regulates the transcriptional activation of MMP-9 by inhibiting TNF-{alpha}-stimulated AP-1 and NF-{kappa}B activities.

3.3 Down-regulation of NQO1 blocked TNF-{alpha}-induced activation of p38 MAPK and JNK in HASMC
In previous studies, we and other research groups found that MMP-9 gene expression can be activated via a number of signal transduction pathways including those involving ERK1/2, p38 MAPK, and JNK, which are the upstream modulators of AP-1 or NF-{kappa}B [6,7,13]. Therefore, it was determined if dicumarol inhibits MMP-9 expression by blocking activation of the ERK, p38 MAPK or JNK pathways (Fig. 5A). TNF-{alpha} induced phosphorylation of all 3 MAPKs, as within 5 min, with maximal phosphorylation at 15 min (data not shown). Dicumarol inhibited phosphorylation of p38 MAPK and JNK pathways in a dose-dependent manner, 15 min after TNF-{alpha} treatment. Transfection of a NQO1 siRNA also inhibited phosphorylation of p38 MAPK and JNK 15 min after TNF-{alpha} treatment (Fig. 5B).


Figure 5
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  		Fig. 5 Effects of dicumarol and NQO1 siRNA on TNF-{alpha}-induced activation of MAPK pathways in HASMC. Cells were treated with TNF-{alpha} for 15 min in the presence or absence of dicumarol, and the phosphorylation levels of ERK1/2, p38 MAPK, and JNK were measured by Western blotting (A). Cells transfected with either negative control siRNA or NQO1 siRNA were treated with TNF-{alpha} (100 ng/ml) for 15 min, and the phosphorylation levels of p38 MAPK and JNK were measured by Western blotting. GAPDH was used as the internal control (B). Cells were treated with the indicated concentrations of dicumarol in the presence of TNF-{alpha} (100 ng/ml) for 24 h. Expression levels of TNFR-1 and -2 were measured by Western blotting and GAPDH was used as the internal control (C).

 
TNF-{alpha} induces a number of biological responses by recognizing two cell surface receptors termed, TNF receptor (TNFR)-1 and TNFR-2 [17]. Therefore, the expression of TNFR-1 and TNFR-2 in HASMC treated with dicumarol in the presence of TNF-{alpha} was examined by Western blot analysis. TNF-{alpha} induced TNFR-1 and -2 expression in HASMC, however, dicumarol did not affect induced activation of the TNFRs (Fig. 5C). These results suggest that dicumarol mediates downstream signaling of TNF-{alpha} after TNF-{alpha} initiates a signal cascade via TNFR binding rather than by directly inhibiting expression of TNFRs.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
Dicumarol exerts a number of physiological effects on cells, which have been widely interpreted to involve NQO1 activity. In the present study, dicumarol and down-regulation of NQO1 by transfection of NQO1 siRNA suppressed TNF-{alpha}-induced HASMC migration to a similar extent through inhibition of MMP-9 expression (Figs. 1,2Go). However, the level of MMP-2 secretion was not altered by TNF-{alpha} and/or dicumarol (data not shown).

As determined by the transient transfection with the promoter construct and EMSA, dicumarol blocked nuclear translocation and DNA binding of AP-1 and NF-{kappa}B, resulted in reduced transcriptional activation of the MMP-9 promoter (Figs. 3 and 4GoA). On the other hand, previous studies showed that the specificity protein-1 (SP-1) site in the MMP-9 promoter also is involved in induction of gene expression in response to TNF-{alpha} in tumor cell lines [18]. In a previous study by our group, however, SP-1 binding activity was not activated by TNF-{alpha} in HASMC. In addition, a reporter vector containing reiterated SP-1 sites was not affected by TNF-{alpha}, with or without dicumarol (data not shown). Dicumarol also inhibited phosphorylation of c-Jun and the nuclear translocation of p65 induced by TNF-{alpha} (Fig. 4B). These findings suggest that dicumarol blocks TNF-{alpha}-induced MMP-9 expression by inhibiting activation of AP-1 and NF-{kappa}B transcription factors, leading to decreased DNA binding activity of the transcription factors.

Several studies have identified signal transduction pathways that are involved in regulation of MMP-9 expression in tumor [19,20] and endothelial cells [21], keratinocytes [22], and in VSMC [6,7]. In our previous study, the effects of various kinase inhibitors on MMP-9 expression were investigated. TNF-{alpha}-induced MMP-9 activation was decreased by ERK1/2, p38 MAPK, and JNK inhibitors but not by PI3K (wortmannin), nor by PKC-{alpha} and -β inhibitor (Gö6976) [13]. Although our observation in this experiment is in general agreement with previous reports showing that TNF-{alpha}-induced MMP-9 activation was inhibited by U0126, SB203580, and SP600125 in VSMC [6,7], results of other investigations indicate that activation of PKC also plays a critical role in the MMP-9 expression induced by other stimulators such as PMA [23], IL-1β [24], or fibroblast growth factor-2 [25] in various cell types. In the present study, signal pathway-mediated regulation of the MMP-9 gene in TNF-{alpha}-induced HASMC in response to down-regulation of NQO1 was identified. The results of this study show that p38 MAPK and JNK are major pathways in NQO1-mediated inhibition of MMP-9 expression in TNF-{alpha}-induced HASMC (Fig. 5). These findings suggest that specific inhibition of the p38 MAPK and JNK signaling pathways, by down-regulation of NQO1, mediates TNF-{alpha}-induced MMP-9 expression in HASMC.

Although it is well known that NQO1 is abundantly expressed in most tumor cells and is linked with various cellular functions such as proliferation, apoptosis, and carcinogenesis [26,27], NQO1 expression and its function in VSMC remain unknown. The results of the present study demonstrate, for the first time, that NQO1 is highly expressed in cultured VSMC (approximately 60% and 40% of the expression levels of HepG2 and MCF-7 cells, respectively, which are tumor cells expressing high levels of NQO1 [28], data not shown) and NQO1 is an important mediator required for TNF-{alpha}-induced migration of VSMC in vitro. Additional studies on the changes in NQO1 expression in VSMC during the atherosclerotic process and in response to arterial wall injury and the possible roles of NQO1 in regulation of VSMC proliferation and migration in vivo are needed to develop a potential therapeutic strategies to inhibit development and progression of both atherosclerotic and restenotic lesions related to the VSMC proliferation and migration.

In conclusion, as illustrated in Fig. 6, dicumarol and down-regulation of NQO1 by transfection with its siRNA suppress TNF-{alpha}-induced MMP-9 expression in HASMC by inhibiting activations of both AP-1 and NF-{kappa}B via the p38 MAPK and JNK pathways. This is the first study showing that NQO1 plays an important role as a signal regulator in TNF-{alpha}-induced VSMC migration, suggesting that NQO1 may be a useful target for the prevention of vascular disorders related to migration of VSMC.


Figure 6
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  		Fig. 6 Schematic model for inhibitory mechanism of dicumarol on TNF-{alpha}-induced MMP-9 expression in HASMC. Down-regulation of NQO1 by dicumarol or siRNA suppresses TNF-{alpha}-induced HASMC migration via inhibition of MMP-9 expression. Down-regulation of NQO1 suppresses TNF-{alpha}-induced MMP-9 expression by inhibiting activation of both AP-1 and NF-{kappa}B transcription factors via the p38 MAPK and JNK pathways. NQO1 also is involved in TNF-{alpha}-mediated activation of MAPK pathways. NQO1 appears to be an important mediator required for TNF-{alpha}-induced migration of HASMC.

 


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by Ministry of Commerce, Industry and Energy (MOCIE) through the Center for Traditional Microorganism Resources (TMR) at Keimyung University, Republic of Korea.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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