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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3406-3413.)
© 1997 American Heart Association, Inc.

Articles

Inhibition of Tissue Factor Gene Activation in Cultured Endothelial Cells by Curcumin

Suppression of Activation of Transcription Factors Egr-1, AP-1, and NF-B

Usha R. Pendurthi; J. Todd Williams; ; L. Vijaya Mohan Rao

From the Departments of Medical Specialties and Biochemistry, The University of Texas Health Center at Tyler, Texas.

Correspondence to Usha R. Pendurthi, PhD, Department of Medical Specialties, UT Health Center at Tyler, Tyler, TX 75710. E-mail usha{at}uthct.edu

	Abstract

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Abstract Binding of plasma factor VII(a) to tissue factor (TF) initiates the coagulation cascade. In health, TF is not expressed in endothelial cells. However, endothelial cells express TF in response to lipopolysaccharide (LPS), tumor necrosis factor- (TNF), and other biological stimuli. TF expression by endothelial cells is implicated in thrombotic disorders in patients with a variety of clinical disorders. In the present study, we demonstrate that curcumin (diferulolylmethane), a known anticarcinogenic and anti-inflammatory agent, inhibited phorbol 12-myristate 13-acetate (PMA), LPS, TNF, and thrombin-induced TF activity and TF gene transcription in human endothelial cells. The present data show that curcumin prevented the activation of c-Rel/p65, which is essential for TF gene activation in endothelial cells, by impairing the proteolytic degradation inhibitor protein, IB. The data also show that curcumin downregulated AP-1 binding activity. The present studies are the first to demonstrate that PMA, but not LPS, TNF, and thrombin, induced Egr-1 binding to the second serum-responsive region (SRR-2) of TF promoter and that curcumin inhibited the PMA-induced Egr-1 binding to SRR-2. Overall, the data suggest that the anticarcinogenic and anti-inflammatory properties of curcumin may be related to its ability to inhibit cellular gene expression regulated by transcription factors NF-B, AP-1, and Egr-1.

Key Words: tissue factor • transcription factors • endothelium • curcumin

	Introduction

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Tissue factor is a cell surface receptor for coagulation factor VII(a), and the binding of factor VII(a) to TF initiates the coagulation cascade. TF is constitutively expressed in several extravascular cells, such as fibroblasts and pericytes, but not in cells within vasculature, such as endothelial cells and monocytes.¹ In vitro, TF can be induced in these cells by a variety of inflammatory stimuli and immunological mediators, including LPS, TNF, thrombin, and interleukin-1.² Earlier, it was believed that monocytes, but probably not endothelial cells, are stimulated to express TF activity in vivo.³ However, several recent studies document that TF expression is induced in endothelial cells under various pathological conditions. Studies of TF expression in baboons subjected to a lethal dose of E. coli infusion showed the expression of TF in endothelial cells in splenic microvasculature.⁴ Further, expression of TF in vascular endothelial cells had been observed within tumors of patients with breast cancer.⁵ Very recently, Zhang et al⁶ showed that systemic infusion of TNF induced TF expression in capillary endothelial cells of the tumor vascular bed. These studies establish that endothelial cell perturbation does lead to the expression of cell surface TF in vivo but is subject to regulation by other elements in the microenvironment.

Curcumin is a major chemical component of turmeric (Curcuma longa) and is used as a spice to give a specific flavor and yellow color to curry. It is also used as a cosmetic and in some medical preparations.⁷ Curcumin has been shown to display anticarcinogenic properties in animals, as indicated by its ability to inhibit phorbol ester–induced skin tumors in a mouse model system.⁸ Curcumin has also been shown to inhibit TNF and phorbol ester–stimulated human immunodeficiency virus long-terminal-repeat–directed gene expression.⁹ In addition to its anticarcinogenic effects, curcumin is also shown to exhibit anti-inflammatory properties.¹⁰ Curcumin was shown to suppress both phorbol ester–induced c-Jun/AP-1 activation in mouse fibroblast cells¹¹ and TNF-induced NF-B activation in human myelomonoblastic leukemia cells.¹² Whether curcumin inhibits activation of these transcription factors and/or other transcription factors in endothelial cells is not known. Signaling pathways that lead to activation of AP-1 and NF-B differ from one cell type to another type and also among inducers within a cell type.^{13 14 15 16 17 18}

In the present study, we examined the ability of curcumin to inhibit the induction of TF in endothelial cells. The data show that curcumin inhibited the PMA-, LPS-, and TNF-induced TF activity in endothelial cells in a dose-dependent manner. The data establish that curcumin suppresses the binding of Egr-1 and AP-1 to the DNA and inhibits the activation of c-Rel/p65 (NF-B).

	Methods

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Reagents
Curcumin, PMA, LPS (E. coli serotype O111:B4), human recombinant TNF, endothelial cell growth supplement (ECGS), and fibronectin were obtained from Sigma Chemical Co. Stock concentrations of curcumin and PMA were made in DMSO. Culture media F-12 was obtained from GIBCO-BRL, Life Technologies. Fetal bovine serum, trypsin-versene mixture, penicillin-streptomycin, and L-glutamine were from Bio-Whittaker. TRI Reagent was from Molecular Research Center Inc. [³²P]ATP (3000 Ci/mmol) and [³²P]dCTP (3000 Ci/mmol) were from DuPont NEN. Antibodies to various transcription factors (c-fos, c-jun, p50, p65, c-Rel, Sp1, Sp2, Sp3, and Egr-1) were obtained from Santa Cruz Biotechnology. Most of the molecular biology–grade chemicals were obtained from either Boehringer Mannheim or United States Biochemicals.

Cell Culture
In the present study, a human umbilical vein endothelial cell line CRL-1730 was used as a source of endothelial cells. However, primary cultures of HUVECs (passages 2 and 3) were also used in several experiments to confirm the data obtained with the HUVEC cell line. HUVEC cell line was purchased from American Type Culture Collection and maintained at 37°C under 5% CO₂ in T-75 flasks in F-12 medium supplemented with 10% fetal calf serum, 1% penicillin-streptomycin, 1% L-glutamine, 40 µg/mL ECGS, and 15 U/mL heparin. The cells were subcultured by first detaching the cells with trypsin solution and replating them in 24-well culture dishes or in T-75 flasks. Culture flasks and dishes were coated with human fibronectin (0.65 µg/cm²). After the monolayers reached near confluence, ECGS was removed from the media and maintained for 48 hours in media not containing ECGS before the monolayers were used. Primary cultures of HUVECs were obtained from Cell Systems and subcultured essentially as described above.

Cell Survival and Proliferation Assay
Cell survival and proliferation were determined using a tetrazolium-based calorimetric assay.¹⁹ The assay is dependent on the reduction of tetrazolium salt MTT, which results in formation of a blue formazan product, by various dehydrogenase enzymes of viable cells. Briefly, MTT solution (10 µL per 100 µL medium, 5 mg MTT per milliliter in PBS) was added to HUVECs that were cultured in a 96-well culture dish and pretreated with a control medium or media containing varying concentrations of curcumin. The cells were further incubated at 37°C for 4 hours. Acid-isopropanol (100 µL of 0.04N HCl in isopropanol) was added to the wells, mixed thoroughly, and the plates were read on a microplate reader (Molecular Devices Corp) using a test wavelength of 563 nm and a reference wavelength of 650 nm. The plates were normally read within 15 minutes after adding isopropanol.

Coagulant Proteins
Recombinant factor VIIa was a gift from Novo-Nordisk, Gentofte, Denmark. Human plasma factor X²⁰ and factor Xa²¹ were purified as described earlier. Thrombin was purchased from Enzyme Research Laboratories.

Induction of TF
Confluent endothelial cell monolayers were washed three times with F-12 medium. The monolayers were stimulated with PMA (10 ng/mL), LPS (1 µg/mL), TNF (20 ng/mL), or thrombin (5 U/mL). At specific intervals, the medium was removed and the monolayers were washed twice with F-12 medium or buffer A (10 mmol/L HEPES, 0.15 mol/L NaCl, 4 mmol/L KCl, and 11 mmol/L glucose, pH 7.5) and processed further without delay.

Measurement of TF Procoagulant Activity
TF activity was measured as the ability of monolayers or cell lysates to support activation of factor X with the addition of VIIa and CaCl₂. Measurement of cell-surface TF activity was as follows: the monolayers (24-well culture dish) were overlaid with 0.25 mL of buffer B (buffer A containing 5 mg/mL BSA and 5 mmol/L CaCl₂) to which VIIa, 0.5 µg/mL, and factor X, 10 µg/mL, were added. At the end of 30 minutes, 50 µL of subsample was removed from the well, transferred to a microtiter plate, and the amount of factor Xa formed was measured by adding 50 µL of 1.25 mg/mL Chromozym X and recording the initial rate of color development in milli–optical densities per minute at 405 nm with a microplate reader (Molecular Devices). The initial rate was converted to micrograms per milliliter of Xa from a standard curve prepared by adding 50 µL of Chromozym X to 50 µL of serial dilutions of a 1-µg/mL sample of purified human Xa. Cell lysates were prepared by solubilizing the monolayers in 15 mmol/L octyl glucopyranoside and freeze/thawing the cell extracts twice. TF activity in cell lysates was measured essentially as described above (cell lysates obtained from PMA-treated cells were diluted 10 to 20 times before they were used in the assay).

Analysis of TF mRNA
Total RNA was prepared from 2 to 3x10⁶ cells by the acid phenol method using TRI reagent according to the manufacturer's technical bulletin. Ten micrograms of total RNA was size fractionated by gel electrophoresis in 1% agarose/6% formaldehyde gels and transferred onto the nitrocellulose membrane by a capillary blot method. Northern blots were prehybridized at 42°C with a solution containing 50% formamide, 5x SSC, 50 mmol/L Tris-HCl, pH 7.5, 0.1% sodium pyrophosphate, 1% SDS, 1% polyvinylpyrrolidone, 1% Ficoll, 25 mmol/L EDTA, 100 µg/mL denatured salmon sperm DNA, and 1% BSA and hybridized with³² P-labeled TF cDNA probe (10⁶ cpm/mL) as described earlier.²² The filters were exposed to either DuPont NEF or Fuji RX X-ray film.

Preparation of Nuclear and Cytoplasmic Extracts
Nuclear and cytoplasmic extracts were prepared as described,¹² with slight modifications. Briefly, 5 to 6x10⁶ cells (two T-75 flasks) were washed with cold phosphate-buffered saline, and cells were suspended in 0.4 mL of lysis buffer (10 mmol/L HEPES, pH 8.0, 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L DTT, 0.5 mmol/L PMSF, 2.0 µg/mL leupeptin, 2.0 µg/mL aprotinin, and 0.5 mg/mL benzamidine) and incubated on ice for 30 minutes. Then, 12.5 µL of 10% Nonidet P-40 was added and the tube vigorously vortexed for 10 seconds. The nuclei were pelleted by centrifugation for 30 seconds at 4000 rpm in an Eppendorf centrifuge. The supernatant was collected and recentrifuged at 12 000 rpm for 2 minutes at 4°C and used as the cytosolic extract. The nuclear pellet was suspended in 100 µL of ice-cold nuclear extract buffer (20 mmol/L HEPES, pH 8.0, 0.5 mol/L KCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, 1 mmol/L PMSF, 2.0 µg/mL leupeptin, 2.0 µg/mL aprotinin, 0.5 mg/mL benzamidine, and 20% glycerol). Both nuclear and cytosolic extracts were stored at -80°C until used. Protein concentrations in both nuclear and cytoplasmic extracts were determined using a protein assay kit from Bio-Rad.

EMSA
The following oligonucleotides were obtained from The Midland Certified Reagent Company: proximal TF AP-1 site, 5'-CTGGGGTGAGTCATCCCTT-3'; TF B-like site, 5'-GTCCCGGAGTTTCCTACCGGG-3'; a prototypic NF-B site, 5'-CAGAGGGACTTTCCGAGA-3'; TF SRR-2, 5'-GAGCGGCGGGGGCGGGCGCCGG-3' (site-specific sequences are italicized). Double-stranded oligonucleotides were 5' end-labeled with [³²P]ATP. Nuclear extracts (10 µg) were preincubated for 20 minutes on ice in 20 µL of the binding buffer (10 mmol/L HEPES, 100 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L DTT, and 4% glycerol) containing 250 µg/mL poly(dI:dC). After preincubation, 100 000 cpm of the ³² P-labeled oligonucleotide was added and the mixture was incubated at room temperature for 30 minutes. The samples were electrophoresed in a 6% nondenaturing polyacrylamide gel. Electrophoresis was performed in 1x TBE buffer (89 mmol/L Tris-HCl, 89 mmol/L boric acid, and 2 mmol/L EDTA). After electrophoresis, the gel was dried and subjected to autoradiography.

Western Blot Analysis
Cytoplasmic extracts from HUVECs stimulated with TNF (20 ng/mL) for varying times were electrophoresed on 12% SDS-polyacrylamide gels and transferred onto PVDF membrane (Millipore). IB protein was detected according to the ECL protocol (Amersham Corp) using a 1:1000 dilution of anti-IB IgG (Santa Cruz Biotechnology).

	Results

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Curcumin Inhibits the Induction of TF Expression in Endothelial Cells

Endothelial cells were preincubated for 1 hour with different concentrations of curcumin (0 to 30 µmol/L) followed by treatment with PMA (10 ng/mL) for 6 hours. Then, the monolayers were tested for the expression of cell-surface TF activity. The results showed that curcumin inhibited the induction of TF activity in a dose-dependent manner. The suppression of TF expression was evident with as low as 0.5 to 2.5 µmol/L concentration of curcumin, and a concentration of 10 µmol/L curcumin completely blocked the PMA-induced cell-surface TF activity (data not shown).

Next, we investigated the time course for curcumin to inhibit PMA-induced TF activity in HUVECs. For these experiments, curcumin (10 µmol/L) was added to monolayers for varying times, either before or after the addition of PMA to the cells, and the cell-surface TF activity was analyzed 6 hours after the addition of PMA. The data showed that PMA induction of TF activity was completely abolished when the cells were treated with curcumin for between 0 and 60 minutes before the addition of PMA to the monolayers (Fig 1). Curcumin also suppressed the TF induction completely, even if it was added 30 minutes after the addition of PMA. However, if curcumin was added thereafter, there was a time-dependent loss of inhibitory activity. Addition of curcumin 3 hours after the addition of PMA did not suppress the PMA-induced TF activity. The inhibitor activity required the continuous presence of curcumin because its removal permitted the induction of TF activity (data not shown).

View larger version (26K): [in this window] [in a new window]   Figure 1. Effect of addition of curcumin to HUVECs before or after PMA stimulation. The monolayers were incubated with curcumin (10 µmol/L) either before or after the monolayers were stimulated with PMA (arrow represents the addition of PMA). Cell-surface TF activity was measured 6 hours after the addition of PMA. TF activity observed in PMA-stimulated cells in the absence of curcumin treatment was taken as 100% of the activity. The data (mean±SD) from three independent experiments done in duplicate are shown.

In further studies, we investigated the effect of curcumin on LPS- and TNF-induced expression of TF activity in endothelial cells. In initial studies, confluent endothelial cell monolayers were treated with 10 µmol/L curcumin for 60 minutes, and then the monolayers were stimulated with LPS (1 µg/mL) and TNF (20 ng/mL) for 6 hours to induce TF activity. The data of these experiments showed that a 10-µmol/L concentration of curcumin failed to suppress LPS- and TNF-induced expression of TF activity. In further experiments, endothelial cells were treated with varying concentrations of curcumin (10, 20, and 40 µmol/L) before they were stimulated with PMA, LPS or TNF. The data revealed clear differences between doses of curcumin required to inhibit PMA-induced TF activity and LPS- and TNF-induced TF activity. Curcumin, at a 10-µmol/L concentration, markedly inhibited PMA-induced TF activity but had only minimal effect on LPS-induced TF activity and had no effect on TNF-induced TF activity (Fig 2). However, a higher concentration of curcumin (40 µmol/L) completely blocked both LPS- and TNF-induced TF activity.

View larger version (21K): [in this window] [in a new window]   Figure 2. Dose-response inhibition of LPS- and TNF-induced TF activity by curcumin. HUVEC monolayers were incubated with varying concentrations of curcumin, 0 to 40 µmol/L, for 60 minutes and then the cells were stimulated for 6 hours with LPS (1 µg/mL), TNF (20 ng/mL), or PMA (10 ng/mL). TF activity in cell lysates was measured as described in "Methods." TF activity observed with each stimulant in the absence of curcumin was taken as 100% of the activity. The data (mean±SD) from four independent experiments done in duplicate are shown.

Although the concentrations of curcumin used in the above experiments were similar to those employed in previous studies,^{11 12 23} to rule out the possibility that the concentrations of curcumin and the duration of curcumin treatment employed in the present study is toxic to HUVECs, we have evaluated cytotoxicity of curcumin using a sensitive assay (MTT assay). Monolayers of HUVECs were treated for 6 hours with varying concentrations of curcumin (up to 60 µmol/L) and then MTT was added to the monolayers. We found no differences between the control and curcumin-treated cells in their ability to cleave MTT, suggesting that curcumin did not effect the cellular metabolism of HUVECs.

Curcumin Inhibits Induction of TF mRNA
We next examined whether curcumin inhibited induction of TF mRNA. The endothelial cells were preincubated with curcumin (30 µmol/L) for 60 minutes before the cells were stimulated with PMA, LPS, TNF, and thrombin for 2 hours to maximally induce TF mRNA, and accumulation of TF mRNA levels were analyzed by Northern blot analysis. No TF mRNA was detected in unstimulated cells either in the absence or presence of curcumin. Various stimuli induced varying levels of TF mRNA in HUVECs, PMA being a more potent inducer than others. Curcumin completely inhibited the induction of TF mRNA in HUVECs stimulated with all agonists tested (Fig 3).

View larger version (59K): [in this window] [in a new window]   Figure 3. Northern blot analysis of TF mRNA accumulation in HUVECs treated with curcumin. HUVEC monolayers were treated for 60 minutes with curcumin, 30 µmol/L (+), or a control buffer (-). Then, the monolayers were stimulated for 2 hours with PMA (10 ng/mL), LPS (1 µg/mL), TNF (20 ng/mL), or thrombin (Thr; 5 U/mL) to induce TF mRNA. Total RNA was extracted from the cells, and 10 µg of each RNA sample was used for Northern blot analysis. The blots were hybridized with a radiolabeled human TF cDNA probe. The bottom panels show ethidium bromide staining of 28S and 18S ribosomal RNA of the same blots as a measure for RNA loading.

Curcumin Inhibits Transcriptional Activation of TF Gene
To determine whether curcumin suppressed the TF expression by preventing the transcriptional activation of TF gene, we examined the binding of nuclear proteins (of cells treated with or without curcumin) to oligonucleotides containing sequences of the AP-1 site and B-like site of the TF promoter. HUVEC monolayers were pretreated for 60 minutes with or without curcumin (30 µmol/L) before the cells were stimulated for 1 hour with PMA, LPS, TNF, or thrombin. Nuclear extracts were analyzed by EMSA, using radiolabeled oligonucleotides containing an AP-1 (proximal) and B-like site in the TF promoter.

As reported earlier,²⁴ nuclear extracts from both unstimulated and stimulated endothelial cells formed a prominent DNA-protein complex with the AP-1 site. Stimulation of HUVECs with PMA slightly but consistently increased the AP-1 binding activity (Fig 4). In contrast, we have not observed an increased AP-1 binding activity in cells treated with other agonists, such as thrombin (Fig 4), LPS, and TNF (data not shown). Curcumin downmodulated the AP-1 binding activity observed in both unstimulated and stimulated endothelial cells (Fig 4).

View larger version (44K): [in this window] [in a new window]   Figure 4. Inhibition of AP-1 binding activity in HUVECs by curcumin. Nuclear extracts were isolated from HUVECs pretreated with curcumin (30 µmol/L) or a control buffer for 60 minutes before the addition of PMA (10 ng/mL) or thrombin (Thr; 5 U/mL) for 1 hour. Nuclear extracts were incubated with radiolabeled DNA probe containing proximal TF AP-1 site, 5'-CTGGGGTGAGTCATCCCTT-3'. Protein-DNA complexes were analyzed on 6% polyacrylamide gels, and the position of AP-1 complex was indicated by an arrowhead. To demonstrate the specificity of protein binding, a 100-fold molar excess of cold competitor oligonucleotides containing either the TF proximal AP-1 site (lane 5) or Sp1 site (lane 6) were used.

EMSA performed to examine the binding of nuclear proteins to an oligonucleotide containing the TF B-like site showed an inducible complex with nuclear extracts from PMA-, thrombin-, LPS-, and TNF-stimulated cells. The inducible complex migrated as a doublet (Fig 5). (The faster migrating band could be the result of a rapid proteolytic truncation of p65 in nuclear extracts, which retains dimerization and DNA binding properties.²⁵ ) Competition studies performed using unlabeled oligonucleotides containing either the TF B-like site or AP-1 site demonstrated that the inducible complex represents a specific binding to the TF B-like site. Treatment of cells with curcumin for 60 minutes before the addition of stimuli markedly reduced the formation of inducible complex (Fig 5).

View larger version (91K): [in this window] [in a new window]   Figure 5. Inhibition of c-Rel/p65 activation by curcumin. Nuclear extracts were isolated from HUVECs pretreated with curcumin (30 µmol/L) or a control buffer for 60 minutes before the cells were stimulated with PMA (10 ng/mL), thrombin (Thr; 5 U/mL), LPS (1 µg/mL), or TNF (20 ng/mL) for 1 hour. Radiolabeled DNA probe containing TF B-like sequence, 5'-GTCCCGGAGTTT CCTACCGGG-3', was incubated with nuclear extracts, and protein-DNA complexes were analyzed on 6% polyacrylamide gels. The position of the c-Rel/p65 complex is indicated by arrowheads (the faster migrating band could be the result of a rapid proteolytic truncation of p65 in nuclear extracts that retains dimerization and DNA binding properties25 ). To demonstrate the specificity of protein binding, a 100-fold molar excess of cold competitor oligonucleotides containing either TF B-like (lane 5) or TF AP-1 site (lane 6) were used. SC indicates specific competitor; NC, nonspecific competitor; and NS, nonspecific binding.

In additional experiments, we examined the effect of curcumin on the activation of NF-B (p50/p65) by examining the binding of nuclear extracts to a prototypic B site from the mouse light-chain enhancer (Ig). The data showed that treatment of HUVECs with curcumin completely blocked the PMA- and LPS-induced activation of NF-B (Fig 6). Curcumin also suppressed TNF-induced activation of NF-B, but the degree of suppression was less than that observed with PMA-induced activation of NF-B.

View larger version (53K): [in this window] [in a new window]   Figure 6. Inhibition of NF-B activation by curcumin. Nuclear extracts were isolated from HUVECs pretreated with curcumin (30 µmol/L) or a control buffer for 60 minutes before the cells were stimulated with PMA (10 ng/mL), LPS (1 µg/mL), or TNF (20 ng/mL) for 1 hour. Nuclear extracts were incubated with radiolabeled DNA probe containing a prototypic NF-B consensus sequence, 5'-CAGAGGGACTTTCCGAGA-3', and protein-DNA complexes were analyzed on 6% polyacrylamide gels. The position of the p50/p65 complex is indicated by an arrowhead. To demonstrate the specificity of protein binding, a 100-fold molar excess of cold competitor oligonucleotides containing either NF-B sequence (lane 9) or TF AP-1 site (lane 10) were used. SC indicates specific competitor and NC, nonspecific competitor.

The translocation of c-Rel/p65 (and NF-B) to the nucleus is preceded by phosphorylation and degradation of IB in the cytosol.¹⁵ To determine whether curcumin inhibited the activation of c-Rel/p65 by preventing the phosphorylation and the subsequent degradation of IB, the cytoplasmic levels of IB protein was analyzed by immunoblot analysis. In control TNF-treated cells, IB was phosphorylated within 5 minutes of TNF treatment, as indicated by the presence of a slow migrating band (Fig 7). By 15 minutes, most of IB disappeared, suggesting the degradation of phosphorylated IB. The IB level remained low at 30 minutes; however, the newly synthesized IB appeared by 1 hour. Pretreatment of endothelial cells with curcumin before TNF treatment markedly suppressed the phosphorylation of IB, as indicated by the absence of the slow migrating band in cells treated with TNF for 5 minutes. Nonetheless, in many experiments, the IB levels did go down in curcumin-treated cells after 15 minutes' exposure to TNF. The absence of the phosphorylated band but a decrease in IB levels suggests that the rate of degradation exceeds the rate of phosphorylation of IB in curcumin-treated cells. This finding fits with the hypothesis that curcumin suppressed the phosphorylation but not the degradation of IB. In a further experiment, we investigated the translocation of p65 into nuclei in control and curcumin-treated cells by Western blot analysis. The data showed a relatively low level of p65 in nuclear extracts of curcumin-treated cells compared with control cells when the cells were exposed to TNF for 30 minutes (data not shown).

View larger version (59K): [in this window] [in a new window]   Figure 7. Effect of curcumin on TNF-induced phosphorylation and degradation of IB. HUVEC monolayers pretreated for 60 minutes without (A) or with (B) curcumin (30 µmol/L) were incubated for varying times with TNF (20 ng/mL). Cytosolic fractions were subjected to SDS-polyacrylamide gel electrophoresis and analyzed for IB levels by Western blot analysis. The slow migrating band represents phosphorylated IB, whereas the main band represents IB.

Because our present data and the data of an earlier study²⁴ clearly showed that PMA, compared with LPS and TNF, was a more potent agonist to induce TF activity and TF mRNA in endothelial cells and that TF promoter contains serum responsive regions that could act as PMA response elements, we next investigated the effect of curcumin on PMA-induced SRR binding activity. Because our initial experiments showed a clear PMA-inducible binding with SRR-2 but not with other SRRs, we limited our EMSA to a radiolabeled oligonucleotide containing SRR-2. As shown in Fig 8, nuclear extracts of unstimulated cells did not form a complex with SRR-2. PMA stimulation, but not LPS, TNF, or thrombin stimulation, of HUVECs induced a protein complex formation with SRR-2. Pretreatment of HUVECs with curcumin markedly reduced the PMA-induced SRR-2 binding activity (Fig 8). We also observed a single minor complex of SRR-2 in cells treated with curcumin alone. This complex exhibited the same mobility as the PMA-induced complex.

View larger version (69K): [in this window] [in a new window]   Figure 8. Effect of curcumin on the binding activity of SRR-2 of TF promoter. Nuclear extracts were isolated from HUVECs pretreated with curcumin (30 µmol/L) or a control buffer for 60 minutes before the cells were stimulated with PMA (10 ng/mL), LPS (1 µg/mL), or TNF (20 ng/mL) for 1 hour. Nuclear extracts were incubated with radiolabeled DNA probe containing SRR-2 of TF promoter, 5'-GAGCGGCGGGGGCGGGCGCCGG-3', and protein-DNA complexes were analyzed on 6% polyacrylamide gels. The position of the specific band is indicated by an arrowhead. To demonstrate the specificity of protein binding, a 100-fold molar excess of cold competitor oligonucleotides containing either SRR-2 (lane 9) or TF AP-1 site (lane 10) were used. SC indicates specific competitor; NC, nonspecific competitor; and NS, nonspecific binding.

To determine the identity of proteins that bound to SRR-2, monospecific antibodies to various serum-responsive transcription factors (Sp1, Sp2, Sp3, and Egr-1) were used. The data show that the protein-DNA complex formed between SRR-2 and nuclear extracts from PMA-stimulated cells was not inhibited by antibodies against Sp1, Sp2, or Sp3. In contrast, antibodies against Egr-1 completely abolished the complex formation (Fig 9). Overall, these data suggest that the stimulation of HUVECs with PMA, but not LPS, TNF, or thrombin, induces the binding of transcription factor Egr-1 to SRR-2 of TF promoter, and the treatment of HUVECs with curcumin inhibits the PMA-induced Egr-1 activation. Similar experiments performed to identify the protein composition of the minor complex that was observed in nuclear extracts obtained from cells treated with curcumin alone were inconclusive, mainly because the complex was too faint to assess whether a particular antibody reduced the complex in a significant fashion.

View larger version (54K): [in this window] [in a new window]   Figure 9. Identification of the protein in SRR-2–protein complex. Nuclear extracts of PMA-stimulated endothelial cells were incubated without (lane 1) or with monospecific antibodies (100 µg/mL) against Sp1 (lane 2), Sp2 (lane 3), Sp3 (lane 4), and Egr-1 (lane 5) for 60 minutes before the addition of radiolabeled SRR-2 probe. Protein-DNA complex was analyzed on 6% polyacrylamide gels.

	Discussion

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Curcumin is a pharmacologically safe biological compound with known anticarcinogenic and anti-inflammatory properties.^{8 10} However, it is not clear how curcumin carries out these variable functions. Human endothelial cells can be induced to express a variety of genes by stimuli that are elaborated in inflammation and cancer. For example, LPS and cytokines were shown to induce the expression of various adhesion molecules, interleukin-8, TNF, urokinase, and TF.^{26 27} Inducible expression of these genes in endothelial cells is regulated by the NF-B/Rel family of transcription factors.²⁸ In many cases, in addition to the NF-B transcription factor system, a relatively small number of other transcription factors are required to generate unique transcriptional activating complexes.²⁸ Induction of TF in both endothelial cells and monocytes by a diverse group of agents is regulated by functional interactions between transcription factors c-Jun/c-Fos (AP-1) and c-Rel/p65.²⁹

The present study showed that curcumin inhibited PMA-, LPS-, TNF-, and thrombin-induced TF expression in human endothelial cells. Curcumin inhibition of TF activity in HUVECs was due to the inhibition of transcriptional activation of TF gene by curcumin. Curcumin suppressed the constitutive binding of c-Jun/c-Fos to TF AP-1 site, which is essential for the induction of TF gene in HUVECs. These results are in agreement with earlier reports which showed that curcumin not only inhibited the DNA binding activity of c-Jun/AP-1 binding factors^{11 12} but also downmodulated the expression of c-Jun and c-Fos by preventing their transcription.^{11 30}

Our results also show that curcumin blocked the activation of c-Rel/p65 induced by various agents, including PMA, LPS, TNF, and thrombin. Curcumin also blocked the activation of NF-B (p50/p65). The latter observation is in agreement with a recent report which showed that curcumin inhibited TNF-induced activation of NF-B in human myelomonoblastic leukemia cells. Unlike other transcription factors, NF-B/Rel family proteins reside in the cytoplasm by binding to a group of inhibitor proteins, including IB.¹⁵ Proteolytic degradation of IB is required for the activation and nuclear translocation of NF-B and c-Rel/p65 complexes. The present study shows that curcumin suppresses the phosphorylation of IB (Fig 7), thus preventing the degradation of IB and the subsequent activation of c-Rel/p65 and NF-B. In this regard, the inhibitory mechanism of curcumin to suppress TF gene activation in HUVECs is similar to that of salicylates and cyclosporine-mediated inhibition of LPS-induced expression of TF in monocytes^{31 32} but differs from cAMP-mediated inhibition of TF in monocytic cells and endothelial cells.³³ Elevated cAMP was shown not to affect the degradation of IB or the subsequent translocation of c-Rel/p65 into the nucleus.³³

NF-B can be activated by a variety of signals relevant to endothelial cell physiology. Although, there are different early events involved in the activation of NF-B, all of them may converge to phosphorylate IB, which is essential for its degradation and the subsequent translocation of p50/p65 into the nucleus.¹⁵ The present observation that curcumin inhibits the activation of NF-B induced by various agents, including LPS, TNF, and thrombin, suggests that curcumin impairs a step in the signal transduction after all diverse signals converge and before the phosphorylation of IB. Several earlier studies suggest a role for reactive oxygen intermediates and/or protein kinases (protein kinase C and protein tyrosine kinase) as a common and critical denominator in the activation of NF-B.^{34 35 36 37 38} Therefore, it is reasonable to assume that curcumin blocks NF-B activation by either quenching of reactive oxygen intermediate production or by inhibiting a protein kinase. In vitro studies which showed that curcumin inhibited both serine/threonine protein kinase and protein tyrosine kinase activities³⁹ could be viewed as additional support for the above assumption.

The present study and a recent study by Parry and Mackman²⁴ showed that PMA was a much more potent agonist to induce TF activity and TF mRNA in HUVECs than other inducers, such as LPS, TNF, interleukin-1, and thrombin. It is interesting to note that PMA-induced TF activity is suppressed by a lower concentration of curcumin than the concentration of curcumin required for the inhibition of TNF- and LPS-induced TF activity. These observations suggest that other transcription factors in addition to NF-B/Rel and AP-1 family proteins could play an important role in PMA-induced activation of the TF gene and that curcumin effectively blocks the interaction of these factors with TF promoter. Earlier studies suggested the presence of two PMA response elements in the cloned TF promoter: TF enhancer region (-227 to -172 bp, containing two AP-1 sites and a B-like site) and a region (-111 to +121 bp) that contains three overlapping Sp1 and Egr-1 binding sites.²⁴ The present study is the first to demonstrate that PMA, but not other stimulants, induces a specific nuclear protein binding to one of these sites (SRR-2) in endothelial cells. Antibody analysis indicated that the protein bound to SRR-2 is Egr-1. The present data are consistent with a recent observation that Egr-1 sites are required for induction of TF promoter by PMA in human epithelial cells.⁴⁰ PMA was shown to rapidly induce de novo synthesis of Egr-1⁴¹ and also phosphorylation of Egr-1.⁴² The phosphorylated form of Egr-1 is shown to bind to DNA with increased affinity.⁴² Further studies are needed to show whether the suppression of Egr-1 binding to SRR-2 in cells treated with curcumin is the result of inhibition of de novo synthesis of Egr-1 or the diminished phosphorylation of Egr-1.

Egr-1, an immediate early gene, is rapidly and transiently expressed after stimulation with a variety of agents, including serum and growth factors.⁴³ Treatment of quiescent primary human fibroblasts with TNF and interleukin-1 in addition to PMA was shown to induce Egr-1 mRNA and the protein.⁴⁴ However, in the present study, we have not observed TNF-induced Egr-1 binding in human endothelial cells. This raises the possibility that TNF may not induce the synthesis of Egr-1 in endothelial cells. Recent studies showed that Egr-1 expression was markedly induced exclusively at the endothelial wound edge and Egr-1 was shown to interact with a novel element in the proximal platelet-derived growth factor-B promoter and with consensus elements in the promoters of other genes induced by endothelial cell injury.⁴⁵ Since curcumin inhibits Egr-1 activation, it is reasonable to assume that curcumin could inhibit the activation of several other genes, in addition to TF, that are induced in cell injury. In agreement with this hypothesis, we found that curcumin inhibited PMA-induced urokinase-type plasminogen activator receptor and plasminogen activator in endothelial cells (unpublished data).

In fibroblasts, both PMA- and TNF-induced expression of Egr-1 involves the action of protein kinase C,^{41 44} so it is possible that the action of curcumin on a single step, ie, inhibition of protein kinase C activation, could result in inhibition of activation of not only Egr-1 but also NF-B and AP-1. In this regard, it is important to note that curcumin should not be viewed as a nonspecific inhibitor of transcription factors, because curcumin was shown not to inhibit the activity of Sp1 transcription factors.¹²

Overall, our data establish that curcumin affects the function of transcription factors AP-1, NF-B, and Egr-1 in human endothelial cells. This finding could explain many anti-inflammatory, anticarcinogenic, and antiproliferative effects of curcumin. Further, because of its low toxicity and ability to inhibit the induction of the TF gene and probably many other genes that are regulated by the transcription factors of AP-1, NF-B family, and Egr-1, curcumin should be explored for its therapeutic value in bacterial sepsis and other inflammatory diseases and also in suppressing various thrombogenic events in the development of vascular occlusive elements.

Note added in proof. While the present manuscript was under review, Bierhaus et al⁴⁶ reported that curcumin inhibited TNF-induced TF gene expression in bovine aortic endothelial cells. The findings that curcumin reduces endothelial cell TF gene expression by inhibiting the binding of AP-1 to the DNA and the activation of NF-B are similar to the conclusions made in the present study. However, the above study was limited to investigating the effect of curcumin on the activation of the TF gene induced by a single agonist, TNF. Further, the effect of curcumin on Egr-1 binding to the TF promoter was not investigated.

	Selected Abbreviations and Acronyms

 

ECGS = endothelial cell growth supplement EMSA = electrophoretic mobility shift assay HUVEC = human umbilical vein endothelial cell LPS = lipopolysaccharide PMA = phorbol 12-myristate 13-acetate SRR = serum-responsive region TF = tissue factor TNF = tumor necrosis factor

	Acknowledgments

 
This study was supported in part by grants HL-42813 (to Dr Rao) from the National Heart, Lung, and Blood Institute (NHLBI), and a Cancer Research Support Fund No. 30–0428– 9200 (to Dr Pendurthi) from The University of Texas Health Center at Tyler. Dr Pendurthi acknowledges the partial salary support provided by Dr Steven Idell (HL-45018 from NHLBI), The University of Texas Health Center at Tyler. During this investigation, Dr Rao was the recipient of Research Career Development Award (HL-02590) from NHLBI. The recombinant factor VIIa was a gift from Novo-Nordisk, Gentofte, Denmark.

Received March 10, 1997; accepted May 15, 1997.

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