4-O-methylgallic acid down-regulates endothelial adhesion molecule expression by inhibiting NF-κB-DNA-binding activity

European Journal of Pharmacology 551 (2006) 143 – 151 www.elsevier.com/locate/ejphar

4-O-methylgallic acid down-regulates endothelial adhesion molecule expression by inhibiting NF-κB-DNA-binding activity Gwangsoo Lee a,c , Hee-Jun Na a,b , Seung Namkoong a , Ho Jeong Kwon d , Sanghwa Han c , Kwon-Soo Ha a,b , Young-Guen Kwon e , Hansoo Lee a , Young-Myeong Kim a,b,⁎ b

a Vascular System Research Center, School of Medicine, Kangwon National University, Chunchon, 200-701, Republic of Korea Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chunchon, 200-701, Republic of Korea c Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chunchon, 200-701, Republic of Korea d Department of Biotechnology, Yonsei University, Seoul, 120-749, Republic of Korea e Department of Biochemistry, Yonsei University, Seoul, 120-749, Republic of Korea

Received 5 April 2006; received in revised form 17 August 2006; accepted 28 August 2006 Available online 8 September 2006

Abstract We here investigated the functional effect of 4-O-methylgallic acid (4-OMGA), a major metabolite of gallic acid abundant in red wine, on vascular inflammation and its action mechanism. 4-OMGA inhibited the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) stimulated with tumor necrosis factor-α (TNF-α), resulting in the suppression of leukocyte adhesion to HUVECs. In addition, 4-OMGA inhibited the promoter activities of ICAM-1 and VCAM-1 and the activity of nuclear factor-κB (NF-κB) without affecting cytosolic IκB kinase (IKK) activation, inhibitor of κB (IκB) phosphorylation and degradation, and nuclear translocation of NF-κB. This compound did not alter nitric oxide (NO) generation, but inhibited reactive oxygen species (ROS) production in TNF-α-stimulated HUVECs, suggesting that NO and ROS are not involved in 4-OMGA-mediated inhibition of NF-κB activity. Moreover, 4-OMGA directly blocked the binding activity of NF-κB to its consensus DNA oligonucleotide, when pre-incubated with the nuclear extract from TNF-α-stimulated HUVECs, but not with the oligonucleotide alone. This inhibition was completely abolished by the addition of dithiothreitol. 4-OMGA exhibits an anti-inflammatory property by interfering with the formation of the NF-κB-DNA complex in the nuclei through direct and redox-sensitive interactions and may play an important role in the prevention of inflammatory responses such as the atherosclerotic process. © 2006 Elsevier B.V. All rights reserved. Keywords: 4-O-methylgallic acid; ICAM-1; VCAM-1; NF-κB; Endothelial cell

1. Introduction Vascular inflammation is a primary event in the pathogenesis of many human diseases, including atherosclerosis, hypertension, restenosis, and septic shock (Ross, 1999; Schiffrin, 2002; Shah, 2003). The vascular inflammatory reaction is mediated by complex interactions between circulating leukocytes and the endothelium. In healthy blood vessels, the endothelial cell ⁎ Corresponding author. Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chunchon, 200-701, Republic of Korea. Tel.: +82 33 250 8831; fax: +82 33 244 3286. E-mail address: [email protected] (Y.-M. Kim). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.08.061

surface of the lumen is a comparatively nonadhesive and nonthrombogenic conduit for the cellular and macromolecular constituents of blood. In certain disease states, some adhesive interactions between the endothelial cells and constituents of blood or the extracellular matrix are changed by the expression of adhesion molecules and their shedding onto the surfaces of endothelial cells and leukocytes (Ross, 1999). The activation of endothelial cells by pro-inflammatory molecules including TNF-α increases adhesion molecule expression and leukocyte adhesion to the vascular endothelium, which are critical initiating steps in atherosclerosis (Kutuk and Basaga, 2003). Furthermore, there is in vivo evidence of increased expression of the endothelial adhesion molecules ICAM-1 and VCAM-1 in

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inflammatory animal models and in human atherosclerotic plaques (Cybulsky and Gimbrone, 1991). TNF-α provides cell signals which result in the activation of NF-κB, which plays an important role in the development of inflammatory responses by up-regulating the expression of many inflammatory mediators (Ghosh et al., 1998; Rahman and MacNee, 1998). In most resting cells, NF-κB is sequestered in the cytoplasm in an inactive form associated with IκB. Upon stimulation of endothelial cells by inflammatory cytokines such as TNF-α, IκB becomes phosphorylated and proteolytically degraded, permitting NF-κB to translocate into the nucleus where NF-κB binds to κB enhancer elements of inflammatory target genes, including ICAM-1 and VCAM-1, to induce their transcription (Beg et al., 1993; Chen et al., 1995). These evidences suggest that selective suppression of the NF-κB signaling pathway prevents various inflammatory diseases including atherosclerosis. A variety of natural substances such as hematein and gallates have been reported to possess anti-atherogenic properties (Choi et al., 2003; Hong et al., 2001; Murase et al., 1999). These compounds have been shown to inhibit cytokine-induced expression of ICAM-1 and VCAM-1 in endothelial cells, probably by inhibiting NF-κB activation. 4-OMGA is a major metabolite of gallic acid which is abundant in red wine, tea, and legumes (Shahrzad et al., 2001). We here investigated the antiinflammatory effect and molecular action mechanism of 4OMGA in TNF-α-stimulated HUVECs. We show that 4OMGA prominently reduces the expression of ICAM-1 and VCAM-1 and also the adhesion of monocytes to TNF-α-treated HUVECs by inhibiting the redox-sensitive DNA-binding activity of NF-κB. These results indicate that 4-OMGA has potential for the treatment of human vascular inflammatory diseases including atherosclerosis. 2. Materials and methods 2.1. Materials Medium 199 (M199), RPMI-1640, TRIzol reagent kit, LipofectAMINE reagents, and penicillin/streptomycin were purchased from Invitrogen Corp (Grand Island, NY). Fetal bovine serum and basic fibroblast growth factor were obtained from Hyclone (Logan, Utah) and Upstate Biotechnology (Lake Placid, NY), respectively. Luciferase assay system was purchased from Promega Corp (Madison, WI). Antibodies procured from Santa Cruz Biotechnology (Santa Cruz, CA) were as follows: β-Actin, ICAM-1, IκB-α, phospho-IκB-α, NF-κB p65, VCAM-1, and anti-goat IgG-rhodamine. Antiphospho-IKK-α/β antibody was procured from Cell Signaling Technology, Inc (Beverly, MA). Anti-PARP-1 antibody was purchased from Oncogene Research Products (Cambridge, MA). Fluorescein isothiocyanate (FITC)-conjugated mouse anti-human ICAM-1 and VCAM-1 antibodies were obtained from Serotec Co (Oxford, UK). DAF-FM diacetate and 2′,7′Dichlorofluorescin diacetate (DCFH2-DA) were purchased from Molecular Probes, Inc (Eugene, OR). 5,5-Dimethyl-1pyrroline-1-oxide (DMPO) was obtained from Sigma-Aldrich,

Inc. N G -monomethyl-L-arginine (NMA) was purchased from Calbiochem (La Jolla, CA). 4-OMGA was prepared as formerly stated (Jeon et al., 2005). 2.2. Cell culture HUVECs from human umbilical cord veins were isolated as previously described (Jaffe et al., 1973), and used for experiments in passages 3 to 7. HUVECs were grown in M199 medium supplemented with 20% of fetal bovine serum, 3 ng/ml of basic fibroblast growth factor, 1% of penicillin/ streptomycin, and 5 units/ml of heparin at 37°C under a humidified 95% to 5% (v/v) mixture of air and CO2. Prior to drug treatment, HUVECs were incubated in fresh M199 medium containing 5% FBS for 5 h. U937 cells were grown in RPMI-1640 supplemented with 10% of fetal bovine serum and 1% of penicillin/streptomycin. 2.3. Western blot analysis Cells were harvested, washed twice with ice-cold phosphatebuffered saline (PBS), and resuspended in PBS containing 0.1 mM phenylmethylsulfonyl fluoride. The suspension was lysed by three cycles of freezing and thawing. The cytosolic fractions were obtained from the supernatant after centrifugation at 12,000 ×g at 4 °C for 20 min. For NF-κB translocation experiment, the nuclear and cytosolic fractions were prepared according to the same method as previously described (Kim et al., 1997). The protein concentrations were determined by the BCA method (Pierce, Rockford, IL). Samples (30–40 μg of protein) were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane as previously described (Min et al., 2005). After blocking, the membrane was blotted with the described primary antibody and subsequently incubated with the corresponding horseradish peroxidase-conjugated secondary antibody. The immunoreactive bands were detected by enhanced chemiluminescent (ECL) reagents. 2.4. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis TOTAL cellular mRNA was isolated from HUVECs by using the TRIzol reagent kit. One microgram of the mRNA was converted to cDNA by treatment with 200 U of reverse transcriptase and 500 ng of oligo(dT) primer in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, and 1 mM dNTPs at 42 °C for 1 h. The reaction was stopped by heating at 70 °C for 15 min. One microliter of the cDNA mixture was then used for subsequent PCR amplification performed with the GeneAmp PCR System 9700 thermal cycler (Applied Biosystems; CA, USA) under the following conditions: denaturation at 94 °C for 5 min for the first cycle and for 30 s starting from the second cycle, annealing at 55 °C (GAPDH) and 60 °C (ICAM-1 and VCAM-1) for 30 s, and extension at 72 °C for 30 s for 25 cycles. Final extension was at 72 °C for 5 min. The final concentration of the reaction components was as follows: 50 mM

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KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.2 mM dNTPs, 2.5 U of Taq DNA polymerase, and 0.4 μM each of primers. The primers used were as follows: 5′-CAGTGACCATCTACAGCT TTCCGG-3′ (sense) and 5′-GCTGCTACCACAGTGATGAT-GA CAA-3′ (antisense) for ICAM-1, 5′-GATACAACCGTCTTGGT CAGCCC-3′ (sense) and 5′-CGCATCCTTCAACTGGCCTT-3′ (antisense) for VCAM-1, and 5′-CGCCACAGTTTCCCGGAG GG-3′ (sense) and 5′-CCCTCCAAAATCAAGTGGGG-3′ (antisense) for GAPDH. 2.5. Reporter gene assay The ICAM-1 and VCAM-1 luciferase plasmids containing regions spanning −1350 to +45 bp (full length) and −485 to +45 (truncated form) of the human ICAM-1 promoter and regions spanning −1716 to +119 bp (full length) and −213 to +119 (truncated form) of the human VCAM-1 promoter were used as previously reported (Kim et al., 2001). HUVECs were transiently transfected with 1 μg of the plasmids and 1 μg of the control pCMV-β-gal plasmid using LipofectAMINE reagents. After 12 h recovery, cells were incubated in M199 medium containing 5% FBS for 5 h and treated with 10 ng/ml TNF-α for 8 h following pretreatment with 10 μg/ml 4-OMGA for 30 min. After cell extracts were prepared, the protein concentrations were determined by the BCA method and adjusted to equal concentrations in all samples. Luciferase activities were assayed using the Luciferase Assay System, and fitted in parallel to β-galactosidase activities to correct differences in transfection efficiencies. 2.6. Electrophoretic mobility shift assay (EMSA) The nuclear protein extracts were prepared from HUVECs as formerly described (Kim et al., 1997). The nuclear extracts (10 μg of protein) were incubated with ∼ 40,000 cpm (∼ 0.5 ng) of [32P]-labeled NF-κB oligonucleotide (probe) for 30 min at room temperature as previously described (Kim et al., 1997). For analyzing the direct effect of 4-OMGA on NF-κB-DNA-binding

Fig. 2. 4-OMGA suppresses TNF-α-induced expression of ICAM-1 and VCAM-1 in HUVECs. HUVECs were treated with TNF-α in the presence or absence of 10 μg/ml with the indicated concentrations of 4-OMGA for 6 h and the indicated time periods. (A and B) Total protein levels of ICAM-1 and VCAM-1 were determined by Western blot analysis. Cell surface levels of ICAM-1 (C) and VCAM-1 (D) were determined by flow cytometric analysis. Data shown are the mean channel fluorescence (MCF) ± S.D. (n = 3). ⁎P b 0.05 vs. TNF-α alone.

activity, nuclear proteins were isolated using dithiothreitol-free extraction buffers, and the nuclear extracts or probes were directly incubated with 4-OMGA, followed by binding reactions with counter pairs, viz. probes or nuclear extracts. DNA-NF-κB complexes were resolved by electrophoresis in a 5% native gel. The gel was then dried and subjected to autoradiography. The specific interaction between DNA and NF-κB was demonstrated by a competitive inhibition assay with a 100-fold excess of unlabelled oligonucleotide (cold probe) and super-shift assay of the DNA-NF-κB complex using a p65 antibody. 2.7. Cell adhesion assay HUVECs were plated on 0.2% gelatin-coated 96-well plates at a density of 1 × 104 cells/well. 12 h afterward, cells were incubated in M199 containing 5% FBS for 5 h and pretreated with 4-OMGA for 30 min. After treatment of TNF-α (10 ng/ml) for 6 h, HUVECs were washed 3 times with PBS, replenished with M199 containing 5% FBS, and incubated with human U937 cells (5 × 104 cells/ml) for 30 min. After washing 3 times with PBS, the attached cells were fixed, stained with Diff-Quick Solutions (Baxter Healthcare Corp), and then counted in 5 randomly selected microscopic fields in each well. 2.8. Immunocytochemical localization of NF-κB p65 subunit

Fig. 1. 4-OMGA reduces TNF-α-induced adhesion of leukocytes to HUVECs. (A) HUVECs were stimulated with TNF-α (10 ng/ml) for 6 h after pretreatment with 4-OMGA for 30 min. Adhesion to U937 human monocytes was then measured, as described in Section 2 of Methods. a: control; b: TNF-α alone; c: TNF-α + 5 μg/ml 4-OMGA; d: TNF-α + 10 μg/ml 4-OMGA. (B) Data are the means ± S.D. (n = 3). ⁎⁎P b 0.01 vs. TNF-α alone.

Succinctly, treated cells were fixed with 2% paraformaldehyde and permeabilized with ice-cold 0.1% saponin. After washing with PBS, the fixed cells were blocked with 3% bovine serum albumin for 1 h and incubated with goat polyclonal anti-p65/RelA antibody (1:100). After 2 h incubation, the cells were washed and incubated

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with anti-goat IgG-rhodamine (1:100) for 1 h. Following mounting the cells on slide glasses with mounting medium, p65/RelA localization was observed by confocal laser scanning microscopy.

meter conditions were as follows: microwave frequency=9.42 GHz, microwave power=8.02 mW, modulation frequency=100 kHz, and field center=337.25 mT.

2.9. Measurements of NO and ROS

2.10. Flow cytometric analysis

After incubation in fresh M199 medium containing 5% FBS for 5 h, HUVECs were pretreated with 4-OMGA (10 μg/ml) for 30 min, followed by TNF-α (10 ng/ml) treatment for 6 h. The intracellular level of NO was measured by confocal microscopy using DAF-FM diacetate (10 μM) as described earlier (Kojima et al., 1999). To quantify NO production, the fluorescence images were processed for densitometric determination at the single-cell level. For measuring intracellular ROS level, HUVECs were treated with 4-OMGA (10 μg/ml) in M199 medium containing 5% FBS for 30 min, followed by TNF-α (10 ng/ml) treatment for 1 h. After incubating for 10 more minutes following the addition of DCFH2-DA (10 μM), cells were washed 3 times with PBS. The intracellular level of ROS was measured by confocal microscopy. In vitro hydroxyl radical generation was measured in the reaction mixture of 100 μM H2O2, 200 μM Fe2SO4, 100 mM DMPO and 10 μg/ml 4-OMGA using electron paramagnetic resonance (EPR) spectrometer. The reaction was initiated by the addition of H2O2, and after 3 min DMPO-OH adduct was recorded. EPR spectro-

After incubation in fresh M199 medium containing 5% FBS for 5 h, subconfluent HUVECs were pretreated with 4-OMGA (10 μg/ml) for 30 min, followed by TNF-α (10 ng/ml) treatment for 6 h. After washing twice with PBS, HUVECs were detached gently by treating with PBS containing 10 mM EDTA, and then washed two times with PBS. Subsequently, the cells were incubated with FITC-conjugated mouse anti-human VCAM-1 and ICAM-1 antibodies for 30 min in ice-cold PBS containing 2% bovine serum albumin, fixed with 2% paraformaldehyde, and analyzed by flow cytometry in a fluorescence-activated cell sorter (Becton Dickinson). 2.11. Statistical analysis The data are presented as the mean ± S.D., and statistical comparisons between groups were performed using one-way analysis of variance (ANOVA) followed by the Student's t test.

Fig. 3. 4-OMGA inhibits TNF-α-induced transcription of ICAM-1 and VCAM-1 and their promoter activities. (A) HUVECs were treated with TNF-α for 3 h in the presence or absence of 4-OMGA. ICAM-1 and VCAM-1 mRNA levels were determined by RT-PCR. (B and C) HUVECs were transiently transfected with luciferase plasmids containing the full length and truncated ICAM-1 and VCAM-1 promoter regions. Cells were treated with TNF-α for 8 h in the presence or absence of 4-OMGA (10 μg/ml) or PDTC (100 μM). Luciferase activity was assayed in the cell lysates. Data are the mean ± S.D. (n = 4). ⁎P b 0.05 and ⁎⁎P b 0.01 vs. TNF-α alone.

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3. Results 3.1. 4-OMGA reduces TNF-α-induced adhesion of leukocytes to HUVECs It is well known that the adhesion of leukocytes from circulating blood to vascular endothelial cells is the earliest and most essential process in vascular inflammatory responses as well as in the initiation of atherosclerosis (Ross, 1999). We first examined whether 4-OMGA regulates leukocyte adhesion to TNF-α-stimulated HUVECs. Confluent HUVECs were treated with TNF-α (10 ng/ml) for 6 h following pretreatment with or without 4-OMGA for 30 min and co-cultured with human monocytic U937 cells. Adhesion of U937 cells to TNF-αstimulated HUVECs was increased about 2.5-fold compared to control, and this adhesion was markedly decreased in a dosedependent manner by treatment with 4-OMGA (Fig. 1A and B).

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3.2. 4-OMGA suppresses TNF-α-induced expression of ICAM-1 and VCAM-1 in HUVECs To investigate whether 4-OMGA-induced inhibition of U937 cell adhesion to TNF-α-stimulated HUVECs depends on the reduced expression of adhesion molecules including ICAM-1, VCAM-1, E-selectin, and P-selectin, we examined the effects of 4-OMGA on the total expression levels of adhesion molecules in TNF-α-stimulated endothelial cells. Treatment of HUVECs with TNF-α resulted in a significant increase in the protein levels of ICAM-1 and VCAM-1, with peak protein production at 6 to 8 h, and this increase was effectively suppressed by pretreatment with 4-OMGA (Fig. 2A). This inhibitory effect was also enhanced in a dose-dependent manner (Fig. 2B). We further verified the inhibitory effect of 4-OMGA on the levels of endothelial cell surface adhesion molecules using flow cytometric analysis. 4-OMGA treatment significantly blocked TNF-

Fig. 4. 4-OMGA blocks NF-κB-DNA-binding activity without affecting NF-κB activation in HUVECs stimulated by TNF-α. (A) HUVECs were treated with TNF-α for the indicated time periods in the presence or absence of 4-OMGA (10 μg/ml). Phosphorylation of IκB-α and IKK were determined by Western blot analysis. (B) Cells were treated with TNF-α for 1 h in the presence or absence of 4-OMGA. The NF-κB p65 subunit level of cytosolic fractions and nuclear extracts was visualized by Western blot analysis. (C) Cells were treated with TNF-α for 1 h in the presence or absence of 4-OMGA. The localization of the NF-κB p65 subunit was visualized by immunocytochemical analysis. a: control; b: TNF-α alone; c: 4-OMGA alone; d: TNF-α + 4-OMGA. (D) Nuclear extracts were prepared from HUVECs treated with TNF-α for 1 h in the presence or absence of 4-OMGA using dithiothreitol-free extraction buffers. Nuclear NF-κB activity was analyzed by EMSA in the presence or absence of an excess amount of cold probe or NF-κB p65 antibody. (E) Nuclear extracts isolated as described in (D) were incubated with or without 1 mM dithiothreitol for 10 min at room temperature. NF-κB-DNA-binding activity was analyzed by EMSA

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α-induced increases in the endothelial cell surface expression of the adhesion molecules ICAM-1 and VCAM-1 (Fig. 2C and D). However, 4-OMGA did not alter TNF-α-induced increases in E-selectin and P-selectin expression on the surface of endothelial cells as determined by Western blot and flow cytometric analyses (data not shown). 3.3. 4-OMGA inhibits TNF-α-induced transcription of ICAM-1 and VCAM-1 and their promoter activities To determine whether the reduced protein expression of ICAM-1 and VCAM-1 by 4-OMGA is due to the transcriptional suppression of these genes, their mRNA levels were determined by RT-PCR analysis. Treatment of HUVECs with TNF-α increased the mRNA levels of both ICAM-1 and VCAM-1, and these increases were markedly suppressed in a dose-dependent manner by treatment with 4-OMGA (Fig. 3A). This transcriptional down-regulation was further verified by assaying transcriptional activities of human ICAM-1 and VCAM-1 promoters. The promoter regions of both adhesion molecules possess several transcription factor binding sites, such as NFκB, AP-1, and STAT for the 1.2 kb ICAM-1 promoter (Roebuck and Finnegan, 1999) and NF-κB, TRE, and GATA for the 1.8 kb VCAM-1 promoter (Minami and Aird, 2001), as depicted in Fig. 3B. HUVECs were transiently transfected with these promoter constructs and stimulated with TNF-α in the presence or absence of 4-OMGA. TNF-α treatment resulted in significant increases in these promoter activities, and these increased activities were suppressed by pretreatment with 4-OMGA (Fig. 3B). Although a number of cis-acting elements in the distal and proximal promoter regions contribute to ICAM-1 and VCAM-1 expression, the proximal NF-κB-binding sites located at ∼200 bp (ICAM-1) and both 65 and 75 bp (VCAM-1) upstream of the transcription start site have been shown to be particularly important (Minami and Aird, 2001; Roebuck and Finnegan, 1999), as depicted in Fig. 3C. The enhancement of transcriptional activity of these truncated ICAM-1 and VCAM1 promoters by TNF-α was blocked by treatment with 4OMGA and the NF-κB inhibitor PDTC. These results indicate that 4-OMGA controls TNF-α-mediated expression of ICAM-1 and VCAM-1 at the transcriptional level by inhibiting the NFκB pathway. 3.4. 4-OMGA blocks NF-κB-DNA-binding activity without affecting NF-κB activation in HUVECs stimulated by TNF-α NF-κB activation requires a sequential cascade such as IκB kinase (IKK-α/β)-dependent IκB phosphorylation, ubiquitination and degradation, translocation of cytosolic NF-κB to the nucleus, and binding to its consensus sequence in several gene promoters (Ghosh et al., 1998). Treatment of HUVECs with TNF-α rapidly led to marked increases in the phosphorylation of IKK-α/β and IκB-α, and then to IκB-α degradation, but these processes were not affected by 4-OMGA pretreatment (Fig. 4A). We next determined the effect of 4-OMGA on the nuclear translocation and DNA-binding ability of NF-κB. TNFα significantly increased the nuclear translocation of the p65

subunit of NF-κB as determined by both Western blot and immunocytochemical analyses, but this translocation was not altered by 4-OMGA (Fig. 4B and C). However, 4-OMGA distinctly inhibited NF-κB-DNA-binding activity in the nuclear extracts from HUVECs treated with TNF-α (Fig. 4D), and this inhibition was clearly abolished by the addition of dithiothreitol, a potent reducing agent, into the nuclear extract (Fig. 4E). This result indicates that 4-OMGA may directly block the DNA-binding activity of NF-κB in the nucleus without affecting the degradation of IκB and nuclear translocation of NF-κB. 3.5. 4-OMGA-mediated inhibition of NF-κB-DNA-binding is not associated with NO and ROS production NO can interact with redox-sensitive protein thiols and inhibit their biological activity. NO has been shown to inhibit the DNA-binding activity of NF-κB by redox-sensitive modification (Marshall and Stamler, 2001). Hence, we measured the intracellular levels of NO production in HUVECs

Fig. 5. 4-OMGA-mediated inhibition of NF-κB-DNA-binding is not associated with NO and ROS production. (A) HUVECs were treated with TNF-α for 6 h in the presence or absence of 10 μg/ml 4-OMGA or 4 mM NMA. The NO levels were determined using confocal microscopy. Data shown are the mean ± S.D. (n = 3). (B) Cells were treated with TNF-α for 6 h in the presence or absence of 10 μg/ml 4-OMGA or 4 mM NMA. The levels of ICAM-1 and VCAM-1 were measured by Western blot analysis. (C) Cells were treated with 4-OMGA (10 μg/ml) for 30 min, followed by TNF-α (10 ng/ml) treatment for 1 h. Cells were incubated with DCFH2-DA (10 μM) for another 10 min and washed 3 times with PBS. The intracellular level of ROS was measured by confocal microscopy, and the relative ROS levels were quantified. Data shown are the mean ± S.D. (n = 3). ⁎P b 0.05. (D) Hydroxyl radical production was measured by EPR spectroscopy in the reaction mixture of 100 μM H2O2, 200 μM Fe2SO4, and the spin trapping agent DMPO (100 mM), with or without 10 μg/ml 4-OMGA. a: H2O2; b: H2O2 + Fe2SO4; c: H2O2 + 4-OMGA; d: H2O2 + Fe2SO4 + 4-OMGA.

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treated with TNF-α in the presence and absence of 4-OMGA or NMA, an inhibitor of NO synthase (NOS) (Fig. 5A). Treatment with TNF-α slightly increased NO production compared with control, and this NO level was not altered by the addition of 4-OMGA, whereas the addition of NMA significantly inhibited intracellular NO production. The addition of NMA did not noticeably affect the inhibitory effect of 4-OMGA and the inducing effect of TNF-α on the expression of ICAM-1 and VCAM-1 (Fig. 5B). High levels of ROS can inhibit the DNAbinding activity of NF-κB (Meyer et al., 1993), by oxidative modification of NF-κB subunits (Stevens et al., 2006). We tested whether 4-OMGA regulates intracellular ROS generation in TNF-α-stimulated HUVECs. 4-OMGA treatment strongly inhibited the TNF-α-mediated increase in intracellular ROS production in cultured HUVECs (Fig. 5C) and the production of hydroxyl radicals by the in vitro reaction of H2O2 with ferrous iron (Fig. 5D). These data indicate that both NO and ROS are not directly involved in the inhibitory effects of 4-OMGA on the binding activity of NF-κB to DNA and the expression of ICAM-1 and VCAM-1 in HUVECs treated with TNF-α. 3.6. 4-OMGA directly suppresses the DNA-binding activity of NF-κB We further examined whether 4-OMGA directly blocks the binding activity of NF-κB to DNA. The nuclear extract isolated from TNF-α-stimulated HUVECs was incubated with 4OMGA for 10 min, followed by additional incubation with [32P]-labeled NF-κB oligonucleotide. 4-OMGA inhibited the binding activity of nuclear NF-κB to DNA, and this inhibition

Fig. 6. 4-OMGA directly suppresses the DNA-binding activity of NF-κB. (A) HUVECs were treated with TNF-α for 1 h and nuclear extracts were prepared using dithiothreitol-free extraction buffer. The nuclear proteins were incubated with 4-OMGA (10 μg/ml) for 10 min in the presence or absence of 1 mM dithiothreitol and further incubated with the labeled probes for 30 min. (B) The labeled probes were incubated with 4-OMGA (10 μg/ml) in the presence or absence of 1 mM dithiothreitol for the indicated time periods and further incubated for 30 min with nuclear extracts from TNF-α-stimulated HUVECs.

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was completely abolished by the addition of dithiothreitol to the in vitro incubation mixture of nuclear extract and 4-OMGA (Fig. 6A). To examine whether 4-OMGA modifies DNA and then modulates its binding affinity to NF-κB, the probe DNA was first incubated with 4-OMGA for 10 and 30 min and subsequently mixed with the nuclear extracts from HUVECs treated with TNF-α, and then DNA-binding activity of NF-κB was analyzed. Pre-incubation of the probe DNA with 4-OMGA did not alter the binding affinity of NF-κB to DNA compared with the non-incubated control (Fig. 6B). These results suggest that 4-OMGA directly inhibits the binding of NF-κB to DNA by modifying the DNA-binding domain of NF-κB. 4. Discussion The naturally occurring phenolic compounds derived from red wine, such as resveratrol and gallic acid, have received considerable attention during the last decade because they are likely to reduce the risk of coronary artery disease (the French paradox) (Appeldoorn et al., 2005; Renaud and de Lorgeril, 1992). The atheroprotective effect of phenolic compounds has been tentatively attributed to their capacity to inhibit lowdensity lipoprotein (LDL) oxidation (Frankel et al., 1993) and to inhibit the expression of endothelial–leukocyte adhesion molecules on the surface of the vascular endothelium covering atherosclerotic and inflammatory lesions (Murase et al., 1999). These adhesion molecules include ICAM-1 and VCAM-1, whose expression can be transcriptionally induced by inflammatory cytokines including TNF-α. 4-OMGA has been found to be a major metabolite of gallic acid in vivo, and its level in the plasma and urine was rapidly increased after oral administration of gallic acid (Shahrzad et al., 2001). Furthermore, 4-OMGA regulated the pathophysiological function of endothelial cells stimulated with basic fibroblast growth factor (Jeon et al., 2005). Results from these studies suggest that 4-OMGA can regulate inflammatory gene expression in endothelial cells exposed to immune stimulants. Many studies have shown that TNF-α up-regulates a number of vascular inflammation-associated genes such as ICAM-1 and VCAM-1, which depend on the function of NF-κB (Beg et al., 1993; Collins et al., 1995; Ghosh et al., 1998; Kutuk and Basaga, 2003). The up-regulation of ICAM-1 and VCAM-1 by NF-κB activation elevates the adhesiveness of circulating leukocytes to endothelial cells, which has been linked to a wide range of pathological processes of vascular inflammatory diseases including atherosclerosis (Cybulsky and Gimbrone, 1991; Ross, 1999). Although leukocyte adhesion to the endothelium is a complex process, earlier studies have shown that ICAM-1 and VCAM-1 are the main adhesion molecules induced in the activated endothelium. It has been also shown that the expression levels of ICAM-1 and VCAM-1 were elevated in atherosclerotic lesions of rabbits fed a cholesterol diet and in human atherosclerotic plaques (Davies et al., 1993; Oh et al., 2001). These evidences indicate that the adhesion of circulating inflammatory cells to activated endothelial cells is one of the early important steps in atherogenesis (Kutuk and Basaga, 2003; Ross, 1999; Schiffrin, 2002). The suppression of

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leukocyte adhesion to endothelial cells by modulating ICAM-1 and VCAM-1 expression prevented against the pathogenic process of atherosclerosis (Choi et al., 2003; Nageh et al., 1997; Oh et al., 2001). We found that treatment with 4-OMGA significantly attenuated leukocyte adhesion to TNF-α-stimulated HUVECs by inhibiting ICAM-1 and VCAM-1 expression (Figs. 1 and 2), indicating that this compound can prevent the early pathogenesis of atherosclerosis by modulating vascular inflammation. Activation of NF-κB appears to play a central role in the regulation of transcriptional expression of a variety of genes, including ICAM-1 and VCAM-1, relevant to atherogenesis as well as inflammatory responses. It suggests that the modulation of the NF-κB pathway is an extremely attractive target for the control of atherogenesis and vascular inflammation. There is a high correlation between NF-κB activation and the levels of adhesion molecules in atherosclerotic lesions in vivo (Oh et al., 2001). It has been reported that inhibitors of NF-κB ameliorate atherosclerotic lesions by suppressing the expression of these genes (Oh et al., 2001; Zheng et al., 2005). 4-OMGA suppressed TNF-α-mediated increases in the promoter activities of ICAM-1 and VCAM-1 and the binding activity of nuclear NF-κB to DNA without affecting the degradation of IκB-α and nuclear translocation of NF-κB (Figs. 3 and 4). These results imply that 4-OMGA may block the interaction between the DNA-binding domain of NF-κB and its consensus DNA sequence. NO, produced by endothelial nitric oxide synthase or NO donors, possesses many anti-atherogenic properties including its ability to inhibit vascular smooth muscle cell proliferation, to reduce platelet aggregation, and to prevent monocyte chemotaxis and adhesion (Napoli and Ignarro, 2001). In addition, NO can attenuate endothelium–leukocyte interactions and limit the extent of atherosclerotic lesions (Cooke et al., 1992; Kubes et al., 1991), probably by blocking the DNA-binding activity of NF-κB through S-nitrosylation of cysteine 62 of its p50 subunit (Marshall and Stamler, 2001), which is a critical thiol group in the DNA-interacting p50 subunit. We showed that treatment of HUVECs with TNF-α resulted in a slight increase in NO production, which was not noticeably changed by the addition of 4-OMGA. Although the addition of NMA, a NOS inhibitor, significantly inhibited intracellular NO production, this inhibitor did not alter the TNF-α-induced increase in the expression of ICAM-1 and VCAM-1 as well as the suppressive effect of 4-OMGA on TNF-α-mediated elevation of ICAM-1 and VCAM-1 expression. These results indicate that NO is not involved in 4-OMGA-mediated suppression of NF-κB-DNAbinding activity and the expression of ICAM-1 and VCAM-1. ROS have been regarded as dual-functional molecules that lead to either NF-κB activation or the inhibition of DNAbinding activity of NF-κB (Han et al., 2001; Meyer et al., 1993). Low or physiological levels of ROS activate NF-κB mainly through the IKK-dependent phosphorylation of IκB-α on Ser32 and Ser36, which is subsequently ubiquitinated and degraded via the proteasome pathway (Gloire et al., 2006). In contrast, high levels of ROS interfere with the direct interaction between activated NF-κB and DNA, probably by oxidative modification

of the redox-sensitive thiols on NF-κB (Stevens et al., 2006). Thus, it may be possible that 4-OMGA elevates intracellular ROS generation, thus resulting in the inhibition of NF-κBDNA-binding via oxidative modification of redox-sensitive cysteine 62 of the p50 subunit. Our results, however, showed that 4-OMGA potently inhibits the TNF-α-induced increase in ROS generation in HUVECs and hydroxyl radical production in the in vitro system of H2O2 plus ferrous iron (Fig. 5C and D), indicating that the elevation of intracellular ROS is not important for TNF-α-mediated cytosolic NF-κB activation via the phosphorylation and degradation of IκB in HUVECs (Fig. 4). These data suggest that the inhibition of NF-κB-DNA-binding activity by 4-OMGA is not due to intracellular ROS generation. Some NF-κB inhibitors, such as CAPE (Natarajan et al., 1996) and herbimycin A (Mahon and O'Neill, 1995), have been shown to exhibit anti-inflammatory activity by blocking the binding activity of NF-κB to target DNA sites without affecting the degradation of IκB, and their inhibitory effects were abolished by the addition of reducing agents dithiothreitol and 2-mercaptoethanol. It has been also shown that CAPE and herbimycin A can directly modify a critical sulfhydryl group at cysteine 62 of the p50 subunit. Similarly, we showed that the inhibition of NF-κB-DNA-binding activity by 4-OMGA was completely abolished by the addition of dithiothreitol to the nuclear extract from HUVECs co-treated with TNF-α and 4-OMGA (Fig. 4E). This inhibition was ascribed to direct interaction of 4-OMGA with NF-κB, but not with DNA (Fig. 6A and B). In addition, a molecular modeling study demonstrated that gallic acid can form five hydrogen bonds with amino acid residues, one of which is cysteine 62, in the DNA-binding region of the p50 subunit, thereby inhibiting its DNA-binding activity (Sharma et al., 2005). 4-OMGA is very similar to gallic acid in its structure in which a hydrogen atom is substituted with methyl. Therefore, it is highly probable that 4-OMGA can form multiple hydrogen bonds with the DNAbinding region of the p50 subunit and subsequently modify its redox-sensitive cysteine 62, resulting in the inhibition of its binding to DNA. Taken together, these data suggest that 4-OMGA may play an important role in the prevention of atherosclerosis and inflammatory diseases. Further studies related to in vivo antiinflammatory function of 4-OMGA may provide new insights into the development of therapeutic drugs for atherosclerosis. Acknowledgments This work was supported by Vascular System Research Center grant from KOSEF. References Appeldoorn, C.C., Bonnefoy, A., Lutters, B.C., Daenens, K., van Berkel, T.J., Hoylaerts, M.F., Biessen, E.A., 2005. Gallic acid antagonizes P-selectinmediated platelet–leukocyte interactions: implications for the French paradox. Circulation 111, 106–112. Beg, A.A., Finco, T.S., Nantermet, P.V., Baldwin Jr., A.S., 1993. Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of IκB-α: a mechanism for NF-κB activation. Mol. Cell. Biol. 13, 3301–3310.

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