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Green Tea Inhibits Human Inducible Nitric-Oxide Synthase Expression by Down-Regulating Signal Transducer and Activator of Transcription-1 Activation

Biochemistry Section, Department of Neuroscience and Vision, University of Verona, Verona, Italy (E.T., M.M., H.S.); and Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany (Y.Y., U.F., H.K.)

Received May 6, 2003; accepted September 24, 2003

	Abstract

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Green tea has been reported to show anti-inflammatory properties because of its inhibitory effects on the expression of several pro-inflammatory genes. Because the inducible nitricoxide synthase (iNOS) plays an important role in chronic inflammatory diseases, we have focused our attention on the regulation of iNOS expression by green tea in two different human epithelial cell lines, alveolar A549/8 and colon DLD-1 cells. With the use of electrophoretic mobility shift assays, we found a green tea-mediated down-regulation of the DNA binding activity of the transcription factor signal transducer and activator of transcription-1 (STAT-1), but not of nuclear factor-B. This down-regulation of the STAT-1 DNA binding was shown to result from reduced tyrosine phosphorylation of the STAT-1 protein and not from antioxidative effects of the green tea extract. Green tea extract inhibited human iNOS expression in a concentration-dependent manner, quantified in terms of iNOS mRNA, iNOS protein, and nitric oxide production in both cell lines. This inhibitory effect of green tea resulted from transcriptional inhibition as shown in reporter gene experiments. These data suggest that green tea extracts may be promising at least as an auxiliary anti-inflammatory principle in chronic inflammatory diseases.

The regulation of iNOS expression is cell- and speciesspecific, and a variety of signal transduction pathways are involved (Kleinert et al., 2000). Regulation of the transcription of the iNOS gene is believed to be the most important control mechanism for iNOS expression. iNOS induction in human cells seems to be partially dependent on activation of the NF-B signal pathway (Kleinert et al., 1998). In addition to NF-B activation, the Janus kinase/signal transducer and activator of transcription (JAK-STAT) pathway has been shown to be essential for human and murine iNOS expression (Gao et al., 1997; Kleinert et al., 1998; Ganster et al., 2001; Menegazzi et al., 2001; Yao et al., 2003). Transient transfection experiments with human A549/8 or DLD-1 cells have demonstrated that maximal induction of the human iNOS promoter depends on transcription factor binding sites upstream of position -5 kb (de Vera et al., 1996; Ganster et al., 2001; and our unpublished results). The published sequence of the human iNOS promoter exhibits homologies to numerous transcription factor binding sites (GenBank accession no. AC005697 [GenBank] ). Studies using the human iNOS promoter showed important regulation of the iNOS promoter activity by the transcription factors NF-B, activator protein 1, and STAT-1 (de Vera et al., 1996; Kleinert et al., 1998; Kleinert et al., 2000; Ganster et al., 2001). Ganster et al. (2001) described the dependence of human iNOS promoter activity on a composite GAS/NF-B element located at position -5.8 kb in the human iNOS promoter (Ganster et al., 2001). In human A549/8 or DLD-1 cells, the cytokine-induced activity of a stably transfected 16-kb human iNOS promoter fragment was inhibited by tyrphostin AG490, a specific inhibitor of the JAK2-tyrosine kinase (Kleinert et al., 1999), or by fungal metabolites blocking the JAK/STAT pathway (Yao et al., 2003). Therefore, the IFN-–JAK–STAT pathway seems to be a good target for the inhibition of human iNOS promoter activation and hence iNOS expression in pathophysiologic situations.

Experimental and epidemiologic studies have demonstrated that green tea can inhibit tumor formation and tumor growth (Fujiki et al., 2002; Lin, 2002). Green tea has an inhibitory effect toward inflammatory diseases such as collagen-induced arthritis (Haqqi et al., 1999), inflammatory bowel disease (Varilek et al., 2001), and the carrageenan-induced paw edema in mice (Das et al., 2002). Only recently have some hints on the possible molecular mechanism of the antitumor or anti-inflammatory effect of green tea emerged. Epigallocatechin gallate (EGCG), the main polyphenol present in green tea (and theaflavin-3,3'-digallate, a polyphenol isolated from black tea) have been shown to inhibit LPS/cytokine-induced iNOS expression in human chondrocytes (Singh et al., 2002) and murine macrophages (Lin and Lin, 1997) by blocking LPS/cytokine-induced NF-B activation. In addition, ECGC was recently reported to be a specific and potent inhibitor of IFN-–elicited STAT-1 activation in a number of human carcinoma cell lines (Menegazzi et al., 2001).

In the current study, we analyzed the effect of green tea extract on the cytokine-induced activation of NF-B and STAT-1 and on the expression of iNOS in two human epithelial carcinoma cell lines, A549/8 and DLD-1. Our data show that green tea extract exerts an efficient inhibitory action on STAT-1 activation (but not NF-B activation) and on iNOS expression. This leads to a drastic reduction in NO production in these cells.

	Materials and Methods

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Reagents
Trypsin, glutamine, and pyruvate solutions, agarose, tRNA, N-acetyl-cysteine (NAC), ascorbate (ASC), and bovine serum albumin were purchased from Sigma (Deisenhofen, Germany). Isotopes were obtained from ICN Biomedicals (Eschwege, Germany). T3 and T7 RNA polymerase, RNase A, RNase T1, the cell proliferation reagent WST-1, and DNase I were obtained from Roche Diagnostics (Mannheim, Germany). Human interferon- (IFN-), interleukin (IL) 1 and tumor necrosis factor- were from Strathmann, Hannover, Germany. Fetal calf serum (FCS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from PAN-Systems, Nürnberg, Germany. The Dual-Luciferase Reporter Assay System and Passive Lysis buffer were purchased from Promega, Heidelberg, Germany. Restriction enzymes, Klenow DNA polymerase, dNTPs, NTPs and the PhosphoPlus® STAT-1 (Tyr701) Antibody Kit were from New England Biolabs, Frankfurt a. M., Germany. The anti-STAT3 (C20) was purchased from Santa Cruz Biotechnology, Heidelberg, Germany. The polyclonal anti-iNOS and anti-actin antibodies were obtained from Transduction Laboratories, Lexington, KY. The green tea extract was obtained from Indena, Milan, Italy.

Cell Culture, Cytokine Treatment, RNA Isolation and Nitrite Measurement
The human alveolar epithelial A549/8 cells, human colon carcinoma DLD-1 cells and human ECV304 cells were grown in DMEM with 5 to 10% fetal bovine serum, 2 mM L-glutamine, penicillin and streptomycin. For RNA isolation, they were plated onto 10 cm-diameter (58 cm²/well) dishes, whereas those experiments involving luciferase activity determinations or NO production studies were performed with cells plated onto 6-well plates (9.6 cm²/well) or 24-well plates (1.75 cm²/well). Eighteen hours beforecytokine induction, cells were washed with PBS solution and incubated with DMEM containing 2 mM L-glutamine in the absence of serum and phenol red. After this preincubation time cells were pretreated with different concentrations of green tea extract for 1 h. A549/8 and DLD-1 cells were induced with a cytokine mixture (CM) composed of IFN- (100 U/ml), IL-1 (50 U/ml) and tumor necrosis factor- (10 ng/ml) for the time periods indicated in the figure legends. Then the supernatant of the cells (300 µl) was used to measure by the Griess reaction, and cells were processed for RNA isolation by guanidinium thiocyanate/phenol/chloroform extraction as described previously (Chomczynski and Sacchi, 1987; Kleinert et al., 1998).

RNase Protection Analysis
For the generation of radiolabeled human iNOS-, human -actin, and human GAPDH-antisense probes for RNase protection assays, 0.5 µg of the linearized plasmids pCR_iNOS_human (Kleinert et al., 1996), pCR_-actin_human (Kleinert et al., 1996), or pXcm_GAPDH-_human (Witteck et al., 2003) were in vitro-transcribed using T3 or T7 RNA polymerase and [-³²P]UTP. To quantify human iNOS mRNA levels, RNase protection experiments were performed as described previously (Kleinert et al., 1998). In all experiments, GAPDH or -actin mRNA expression was determined for normalization purposes. Densitometric analyses were performed using a Molecular Imager FX Pro (Bio-Rad, Munich, Germany). The protected fragments of human iNOS, -actin, and human GAPDH-mRNA were 386 nt or 195 nt, 108 nt, and 105 nt, respectively.

Analysis of the Human iNOS Promoter Activity and Human eNOS Promoter Activity
To investigate the effect of green tea extract or antioxidants, such as NAC or ASC, on cytokine-induced iNOS promoter activity or GAS-dependent promoter activity, pools of stably transfected A549/8 or DLD-1 cells [containing a 16-kb fragment of the human iNOS promoter cloned in front of a luciferase reporter gene or pGAS-Luc (Yao et al., 2003)] were incubated for 18 h with DMEM without FCS and without phenol red. Before cytokine induction, the cells were pretreated with green tea extract, NAC, or ASC in the concentrations indicated. After CM incubation for 4.5 h in the presence or absence of green tea extract, NAC or ASC cells were lysed in 1x passive lysis buffer.

To investigate the effect of green tea extract on the constitutive human eNOS promoter activity, pools of stably transfected ECV 304 cells [containing a 3.5-kb fragment of the human eNOS promoter cloned in front of a luciferase reporter gene; (Yao et al., 2003)] were incubated for 24 h with DMEM without FCS and without phenol red. Then the cells were incubated with or without different concentrations of green tea extract for 4 to 5 h, before being lysed in 1x passive lysis buffer.

Firefly luciferase activity was determined using the dual-luciferase assay kit. Protein concentrations of the extracts were determined by Bradford reagent using bovine serum albumin as standard. Protein content of the extracts was used for normalization of the luciferase activity.

Western Blot Experiments
Detection of STAT-1 Phosphorylation. A549/8 and DLD-1 cells incubated with or without CM in the presence or absence of green tea extract for 0.5 h were lysed on ice with 20 mM HEPES, pH 7.4, 420 mM NaCl, 1% Nonidet P40, 1 mM EGTA, and 1 mM EDTA for 15 min. After centrifugation for 15 min at 12,000 rpm, proteins (50 µg/lane) were fractionated by SDS-polyacrylamide gel electrophoresis in a 7.5% gel, electroblotted onto polyvinylidene difluoride membrane (Millipore S.p.A., Rome, Italy), and reacted with anti-STAT-1 phosphotyrosine701 diluted 1:700 (New England Biolabs, Hetchin, England) and after stripping with an anti-STAT-1 or an anti-actin antibody diluted 1:1000 according to standard procedures. Immune complexes were detected by using anti-rabbit horseradish peroxidase-conjugated immunoglobulin for detection of the primary antibody. The immunoreactive proteins on the blot were detected by the enhanced chemiluminescence detection system (ECL; Amersham Biosciences, Little Chalfont, Buckinghamshire, UK).

Detection of iNOS Protein. DLD-1 cells incubated with or without CM in the presence or absence of green tea extract for 24 h were lysed on ice with radioimmunoprecipitation assay buffer (20 mM HEPES, pH 7.4, 420 mM NaCl, 1% Nonidet P40, 1 mM EGTA, and 1 mM EDTA) for 20 min. After centrifugation for 20 min at 12,000 rpm, proteins (50 µg/lane) were fractionated by SDS-polyacrylamide gel electrophoresis in a 7.5% gel, electroblotted onto polyvinylidene difluoride membrane (Millipore S.p.A.), and reacted with a polyclonal anti-iNOS antibody (BD Biosciences Transduction Laboratories, Lexington, KY) diluted 1:200 and after stripping with anti-STAT-1 or anti-STAT3 antibody diluted 1:1000 according to standard procedures. Immune complexes were detected by using anti-rabbit horseradish peroxidase-conjugated immunoglobulin for detection of the primary antibody. The immunoreactive proteins on the blot were detected by ECL detection system (Amersham Biosciences).

Electrophoretic Mobility Shift Assay. Nuclear extracts were prepared from cells treated with or without cytokines in the presence or absence of green tea extract, NAC, or ASC according to Osborne et al. (1997) The protease inhibitors leupeptin (10 µg/ml), antipain (5 µg/ml), and pepstatin (5 µg/ml) and phenylmethylsulfonyl fluoride (1 mM) were present. Protein concentration in the nuclear extracts was determined by using the method of Bradford (1976). Nuclear extract (10 µg) were incubated at room temperature for 20 min with a ³²P-labeled double-stranded oligonucleotide containing the STAT-1 binding site (sis-inducible factor-binding recognition element, SIE/m67) from the c-fos promoter (5'-gtcgaCATTTCCCGTAAATCg-3'; lower case letters represent additional sequences not present in these promoters) (Wagner et al., 1990), or the NF-B binding sequence from the IL-6 promoter (5'-gatcCAGAGGGGACTTTCCGAGt-3') (Promega, Milan, Italy), in a 15-µl reaction mixture containing 20 mM HEPES, pH 7.9, 50 mM KCl, 10% glycerol, 0.5 mM dithiothreitol, 0.1 mM EDTA, 2 µg of poly(dI-dC), and 1 µg of salmon sperm DNA. Products were fractioned on a nondenaturing 5% polyacrylamide gel in Tris-borate/EDTA buffer (1.08% Tris, pH 8.3, 0.55% boric acid, and 20 mM EDTA).

Determination of Cellular Viability
The effect of green tea extract on cell viability was measured by a colorimetric assay based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases (cell proliferation reagent WST-1; Roche).

Cells were seeded at the concentration of 10³ cells per well in flat-bottomed, 96-well plates in 0.5 ml of medium. Cells were preincubated in medium containing 2% FCS for 18 h. Then cells were treated with or without a CM in the presence or absence of green tea extract. After 24 h of incubation, 10 µl of the WST-1 solution was added, and the cells were incubated for additional 4 h. Afterward, the optical density at 450 nm was determined.

Calculations
All data are presented as mean ± S.E.M. Differences between means were tested for statistical significance using factorial analysis of variance followed by Fisher's protected least significant difference test as the post hoc test (StatView software; SAS Institute, Cary, NC). Concentrations of compounds producing half-maximal inhibition were determined using Prism 3 software (GraphPad Software Inc., San Diego, CA).

	Results

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Green Tea Extract Inhibited STAT-1 DNA Binding, but Did Not Affect NF-B DNA Binding. To study the effect of green tea extract on the DNA binding activity of inflammatory transcription factors in A549/8 and DLD-1 cells, we performed electrophoretic mobility shift assays for STAT-1 and NF-B, transcription factors known to play a critical role in human iNOS expression. As shown in Figs. 1 and 2, in both cell lines, cytokine treatment rapidly induced both STAT-1 and NF-B DNA binding activity. However, only STAT-1 DNA binding was reduced in a concentration-dependent manner by coincubation of cytokine-treated cells with green tea extract (Fig. 1); activation of NF-B DNA binding remained unchanged (Fig. 2).

View larger version (77K): [in this window] [in a new window]   Fig. 1. Effect on green tea extracts on cytokine-induced STAT-1 DNA binding activity n human A549/8 and DLD-1 cells. Gel shift experiments using a radiolabeled oligonucleotide containing a consensus STAT-1 binding site and nuclear extracts from untreated A549/8 and DLD-1 cells, or A549/8 and DLD-1 cells stimulated with a CM, consisting of 100 U/ml IFN-, 50 U/ml IL-1 and 10 ng/ml TNF-, in the presence or absence of GX (2–40 µg/ml). The position of the DNA protein complex (STAT) and the free oligonucleotide is indicated. The gel is representative of four gels showing similar results.

View larger version (84K): [in this window] [in a new window]   Fig. 2. Effect on green tea extracts on cytokine-induced NF-B DNA binding activity in human A549/8 and DLD-1 cells. Gel shift experiments using a radiolabeled oligonucleotide containing a consensus NF-B binding site and nuclear extracts from untreated A549/8 and DLD-1 cells or A549/8 and DLD-1 cells stimulated with a CM in the presence or absence of GX (2–40 µg/ml). The position of the DNA protein complex (NF-B) and the free oligonucleotide is indicated. The gel is representative of three gels showing similar results.

Incubation of A549/8 or DLD-1 Cells with the Potent Antioxidants NAC and ASC Did Not Change Cytokine-Induced STAT-1 DNA Binding. Because green tea extract is known to contain different compounds with antioxidative properties, we analyzed the effect of the potent antioxidative compounds NAC (0.01–10 mM) and ASC (0.01–10 mM) on cytokine-induced activation of STAT-1 DNA binding activity. As shown in Fig. 3, neither NAC nor ASC incubation changed the STAT-1 DNA binding activity induced by cytokine treatment in A549/8 (Fig. 3A) or DLD-1 cells (Fig. 3B). NAC and ASC also did not modify cytokine-induced activity of an INF-–dependent promoter containing five -activated sites (GAS, binding site of STAT-1) stably transfected in A549/8 cells or human iNOS promoter activity (data not shown).

View larger version (57K): [in this window] [in a new window]   Fig. 3. Effect of antioxidants on cytokine-induced STAT-1 DNA binding activity in human A549/8 and DLD-1 cells. Gel shift experiments using a radiolabeled oligonucleotide containing a consensus STAT-1 binding site and nuclear extracts from untreated A549/8 (A) and DLD-1 cells (B), or A549/8 and DLD-1 cells stimulated with a CM in the presence or absence of ASC (0.1–10 mM), NAC (0.1–10 mM), or GX (40 µg/ml). The position of the DNA protein complex (STAT) and the free oligonucleotide is indicated. Each gel is representative of three gels showing similar results.

Green Tea Extract Inhibited the Activation of STAT-1 in A549/8 Cells and DLD-1 Cells. To further confirm the mechanism of action of the green tea extract on DNA binding activity in DLD-1 and A548/9 cells, we studied STAT-1 activation by immunoblot analyses using an antibody that specifically detects the tyrosine (Tyr701)-phosphorylated form of STAT-1 (Darnell et al., 1994; Ihle et al., 1994; Ihle, 1995). This phosphorylation is critical for the activation of the STAT-1 protein and enables it to dimerize, to migrate into the nucleus, and to bind to specific STAT-1 binding sites on the DNA (the GAS elements). Incubation of A549/8 and DLD-1 cells for 30 min with a CM consisting of 100 U/ml interferon-, 50 U/ml IL-1, and 10 ng/ml tumor necrosis factor-, resulted in a marked enhancement of STAT-1 tyrosine phosphorylation (Fig. 4). Coincubation of CM-treated A549/8 and DLD-1 cells with green tea extract (40 µg/ml) markedly inhibits the Tyr701 phosphorylation, in line with the inhibition of DNA binding activity seen upon treatment with green tea (Fig. 1).

View larger version (53K): [in this window] [in a new window]   Fig. 4. Effect of green tea extract on cytokine-induced activation of STAT-1 in human A549/8 and DLD-1 cells. A, A549/8 cells were preincubated for 18 h with DMEM (without FCS and phenol red). Then, GX (40 µg/ml) was added for 1 h. Subsequently, the cells were incubated for an additional 30 min with or without a CM in the presence or absence of green tea extract. Nuclear extracts were prepared and 50 µg of nuclear proteins were analyzed for STAT-1 tyrosine-(Tyr701) phosphorylation by Western blotting using the PhosphoPlus STAT-1 (Tyr701) antibody kit as described under Materials and Methods. The blots shown are representative for four experiments showing the same results. Top, a Western blot using a STAT-1-tyrosine-(Tyr701)-phosphate-specific antibody. Middle, the same blot (after stripping) using a STAT-1 antibody, which detects phosphorylated and nonphosphorylated STAT-1. Bottom, the same blot (after stripping) using an actin antibody. B, experiments similar to those in A. Results are generated with nuclear extracts obtained from DLD-1 cells.

Green Tea Extract Inhibited Human iNOS mRNA- and Protein Expression and Nitrite Production in A549/8 and DLD-1 Cells. As shown in Fig. 5, green tea extract (GX) inhibited CM-elicited iNOS mRNA expression in A549/8 and DLD-1 cells in a concentration-dependent manner, as measured by RNase protection assay. In addition, the cytokine-induced expression of iNOS protein was inhibited by green tea extract (Fig. 6). Accordingly, inhibition of cytokine-induced NO production was also observed (Fig. 7) by measuring nitrite in cell supernatants using the Griess assay.

View larger version (61K): [in this window] [in a new window]   Fig. 5. Effect of green tea extract on cytokine-induced human iNOS mRNA expression in epithelial A549/8 and DLD-1 cells A, representative RNase protection assay performed with total RNA from human A549/8 cells incubated for 8 h in serum-free medium alone or in serum-free medium containing a CM in the presence or absence of GX (2–200 µg/ml). Cells were preincubated for 18 h in serum-free medium and for an additional 1 h with the different concentrations of GX. Experiments were performed using antisense RNA probes for human iNOS and GAPDH, which was used for normalization. The positions of the protected iNOS- and GAPDH fragments are indicated. M, molecular weight standard; X174-restricted with HinFI; I, iNOS antisense probe; G, GAPDH antisense probe. B, densitometric analyses of six different gels. Columns and symbols (means ± S.E.M.) represent relative iNOS mRNA levels at the different concentrations of green tea extract (***, p < 0.001; ns, not significant versus CM). Half-maximal inhibition was achieved with 15.1 µg/ml. C, representative RNase protection assay performed with total RNA from human DLD-1 cells treated as described above for A549/8 cells. Experiments were performed using antisense RNA probes for human iNOS and -actin, which was used for normalization. The positions of the protected iNOS- and -actin fragments are indicated. T, tRNA control; M, molecular weight standard, X174-restricted with HinFI; I, iNOS antisense probe; A, -actin antisense probe. D, data obtained using RNA isolated from human DLD-1 cells. Columns and symbols (means ± S.E.M., n = 5) represent relative iNOS mRNA levels at the different concentrations of green tea extract (***, p < 0.001; ns, not significant versus CM). Half-maximal inhibition was achieved with 2.2 µg/ml.

View larger version (55K): [in this window] [in a new window]   Fig. 6. Effect of green tea extract cytokine-induced iNOS protein expression in human DLD-1 cells. DLD-1 cells were preincubated for 18 h with DMEM (without FCS and phenol red). Then cells were incubated for 24 h with or without a CM in the presence or absence of GX (40 µg/ml). Total cell extracts (80 µg of total proteins) were analyzed for iNOS- and (for normalization) for STAT-1 and STAT3 protein expression by Western blotting using a polyclonal anti-iNOS antibody, a monoclonal STAT-1 antibody, and a polyclonal STAT3 antibody, respectively, as described under Materials and Methods. The blot shown is representative for three experiments showing the same results.

View larger version (26K): [in this window] [in a new window]   Fig. 7. Effect of green tea extract on cytokine-induced nitrite production in human epithelial A549/8 and DLD-1cells. A, statistical analysis of 9 different Griess assays performed with supernatants from untreated A549 cells or cells stimulated for 24 h with a CM in the presence or absence of GX (2–200 µg/ml). Cells were preincubated for 18 h in serum-free medium and additionally for 1 h with or without GX. Columns and symbols (means ± S.E.M.) represent the relative nitrite levels determined under the conditions indicated (100% corresponds to 400 pmol/ml/24 h; ***, p < 0.001 versus CM). Half-maximal inhibition was achieved with 1.5 µg/ml. B, data generated with DLD-1 cells. Columns and symbols (means ± S.E.M.) represent the relative nitrite levels determined under the conditions indicated (100% corresponds to 400 pmol/ml/24 h; **, p < 0.01; ***, p < 0.001 versus CM). Half-maximal inhibition was achieved with 4.7 µg/ml.

Green Tea Extract Inhibited Human iNOS Promoter Activity in Stably Transfected A549/8 and DLD-1 Cells. To analyze the effect of green tea extract on human iNOS promoter activity, we incubated A549/8 and DLD-1 cells stably transfected with pNOSII(16)Luc (de Vera et al., 1996; Hausding et al., 2000), with different concentrations of green tea extract. The plasmid pNOSII(16)Luc contains a 16-kb fragment of the human iNOS promoter, cloned in front of a luciferase reporter gene. In line with previous reports (Hausding et al., 2000; Witteck et al., 2003; Yao et al., 2003), CM incubation of these pools of stably transfected A549/8 and DLD-1 cells (Fig. 8) resulted in a 5-fold enhancement of luciferase expression. Green tea extract concentration-dependently inhibited the CM-induced iNOS promoter activity in these cells (Fig. 8).

View larger version (33K): [in this window] [in a new window]   Fig. 8. Effect of green tea extract cytokine-induced human iNOS promoter activity in stably transfected A549/8 and DLD-1 cells A549/8 and DLD-1 cells, stably transfected with pNOSII(16)Luc (de Vera et al., 1996), a construct containing a 16-kb fragment of the human iNOS promoter in front of a luciferase reporter gene, were preincubated in serum-free medium for 18 h and then with GX (0.4–200 µg/ml) for an additional 1 h. Then, cells were incubated with a cytokine mixture (CM) for another 4 h in the presence or absence of the green tea extract. Luciferase activity and protein content in cell extracts were determined. A, columns and symbols (means ± S.E.M.; n = 18) represent the relative luciferase activities (percentage of the CM effect) in extracts of stably transfected A549/8 cells, incubated with GX (***, p < 0.001; **, p < 0.01; ns, not significant versus CM). Half-maximal inhibition was achieved with 1.5 µg/ml. B, columns and symbols (means ± S.E.M.; n = 16) represent the relative luciferase activities (percentage of the CM effect) in extracts of stably transfected DLD-1 cells, incubated with GX (***, p < 0.001; ns, not significant versus CM). Half-maximal inhibition was achieved with 1.3 µg/ml.

Green Tea Extract Has No Effect on the Viability of Human A549/8 and DLD-1 Cells. To determine whether the inhibition of iNOS expression was a result of a cytotoxic action of the green tea extract, the effect of green tea extract on the cellular viability of A549/8 and DLD-1 cells was analyzed using the WST-1 cell viability assay (Roche). Incubation of A549/8 and DLD-1 cells with different concentrations of green tea extract resulted in no reduction of cell viability (see Fig. 9).

View larger version (38K): [in this window] [in a new window]   Fig. 9. Effect of green tea extract on the cell viability of A549/8 and DLD-1 cells. A549/8 and DLD-1 cells were preincubated in medium containing 2% FCS for 18 h and then with or without a CM in the presence or absence of GX (5–50 µg/ml) for an additional 24 h. Then, 10 µl of the WST-1 solution (Roche) was added and the optical density at 450 nm was analyzed after an additional 4 h. Columns and symbols (means ± S.E.M.; n = 24) represent relative optical densities at 450 nm (percentage of CM-treated) in the presence of the different concentrations of the green tea extract (*, p < 0.05; ns, not significant versus CM; nsx, not significant versus control).

Green Tea Extract Had Only a Modest Effect on the Activity of the Human 3.5-kb eNOS Promoter. To determine whether the inhibition of iNOS expression was a result of the inhibition of general transcription factors, the effect of green tea extract on a constitutively active promoter was analyzed. For this purpose ECV-pNOS III-Hu-3500-Luc-neo cells (Yao et al., 2003) were used. These cells derived from human ECV304 cells, which had been transfected with the plasmid pNOS III-Hu-3500-Luc-neo, containing a 3.5-kb fragment of the human eNOS promoter, cloned in front of a luciferase reporter gene. This promoter displayed a significant constitutive activity in the stably transfected cells (see Fig. 10). Incubation of ECV-pNOS III-Hu-3500-Luc-neo cells with the highest concentrations of green tea extract resulted in only a moderate reduction in eNOS promoter activity at high concentrations of green tea extract (control cells, 100%; green tea extract, 200 µg/ml, 58.3 ± 6.5%, n = 12).

View larger version (22K): [in this window] [in a new window]   Fig. 10. Effect of green tea extract on constitutive human eNOS promoter activity in stably transfected ECV-pNOS III-Hu-3500-Luc-neo cells ECV-pNOS III-Hu-3500-Luc-neo cells (Yao et al., 2003) stably transfected with pNOS III-Hu-3500-Luc-neo, a construct containing a 3.5-kb human eNOS promoter fragment in front of a luciferase reporter gene were preincubated with serum-free medium for 18 h. Then, cells were incubated with or without GX (4–200 µg/ml) for an additional 6 h in serum-free medium. Cell extracts were prepared and luciferase activity and protein content of the extracts were determined. Columns and symbols (means ± S.E.M.; n = 12) represent relative luciferase activities (percentage of control cells) in the presence of the different concentrations of the green tea extract (***, p < 0.001; ns, not significant versus control). With 200 µg/ml green tea extract, the human eNOS promoter activity was reduced to 60% of control.

	Discussion

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An aberrant expression of iNOS leading to inappropriate NO production has been implicated in human autoimmune and pro-inflammatory diseases, such as rheumatoid arthritis, asthma, short-term and chronic lung diseases, neurodegenerative diseases (such as Alzheimer's and Parkinson's diseases), as well as inflammatory bowel diseases, tumor development, and septic shock (Kröncke et al., 1998). One possible way to inhibit excessive NO production by iNOS is specific enzyme inhibition. However, the use of inhibitors of the iNOS enzyme activity is reported to provoke secondary undesired effects (Husain, 2002; Kang et al., 2002). An alternative approach to block iNOS-dependent NO production is the suppression of iNOS induction (Yao et al., 2003). In contrast to eNOS and neuronal nitric-oxide synthase, which are constitutively expressed, expression of iNOS has to be induced (Kleinert et al., 2000). The transcription factors involved in the expression of the three nitric-oxide synthase isoforms differ markedly (Kleinert et al., 2000). Therefore, it seems likely that specific inhibitors of iNOS expression can be generated. In recent years, several authors described the dependence of human iNOS induction on the IFN-–JAK–STAT pathway (Kleinert et al., 1998, 2000; Dell'Albani et al., 2001; Ganster et al., 2001; Ohmori and Hamilton, 2001). Therefore, the IFN-–JAK–STAT pathway seems to be a reasonable target for the development of inhibitors of iNOS expression.

EGCG, the main component of green tea, has been shown to inhibit the activation of the STAT-1 transcription factor by suppressing Tyr701 phosphorylation of the STAT-1 protein (Menegazzi et al., 2001). Therefore, we focused our attention on green tea extract, a natural product whose main component is EGCG, to identify a new principle to inhibit iNOS expression. Green tea extract is the most used ingredient for all the food and cosmetic green tea-flavored products on the market. The maximum green tea extract concentrations investigated are in the range of the plasma concentrations found in humans drinking 6 to 10 cups of green tea per day (Yang et al., 1997).

As a cellular model, we choose two epithelium-derived human cell lines: DLD-1 colon carcinoma and A548/9 alveolar carcinoma cells. As reported already, there are many diseases in the colon and in the lung that seem to be correlated to an aberrant expression of the iNOS enzyme (Barnes and Liew, 1995; Guslandi, 1998; Kröncke et al., 1998; Cavicchi and Whittle, 1999; van Straaten et al., 1999; Kleinert et al., 2000; Bogdan, 2001).

Data presented in the current work clearly show that, in line with previous reports on EGCG, green tea extract also exerts a concentration-dependent, specific inhibitory action toward IFN-–elicited STAT-1 activation in epithelial cell lines (Figs. 1 and 4). Because the potent antioxidative compounds N-acetyl-cysteine and ascorbate did not influence cytokine-induced STAT-1 DNA binding, this green tea-mediated inhibition of STAT-1 activity is unlikely to result from a general antioxidative effect of the extract. The mechanism of the green tea extract-mediated inhibition of STAT-1 activation seems to operate mainly via inhibition of cytokine induced STAT-1 tyrosine phosphorylation (see Fig. 4). In addition, however, a green tea extract-related reduction of STAT-1 expression may partly explain the effect. Furthermore, green tea-mediated inhibition of cytokine-induced iNOS promoter activity (Fig. 8) leads to the inhibition of iNOS synthesis (mRNA and protein; Figs. 5 and 6) and NO production (Fig. 7) in these cell lines. In all cases, the concentrations of green tea extract required for half-maximal inhibition displayed no cytotoxicity in A549/8 and DLD-1 cells (Fig. 9) and were below the concentrations showing a significant effect on the activity of the constitutively active eNOS promoter (Fig. 10). Only at very high concentrations of green tea extract was a relevant effect on eNOS promoter activity seen (Fig. 10). These results show that the inhibitory effect of green tea extract on iNOS expression is unlikely to result from inhibition of general transcription or cell viability. Inhibition of STAT-1 activation and thereby iNOS expression by the green tea seems to be a rather specific effect. Inhibition of the INF-–JAK–STAT-1 pathway by green tea extract may be an efficient way to reduce massive iNOS-derived NO production. In view of the fact that drugs currently used in the treatment of inflammatory bowel disease, such as steroids, do not substantially reduce intestinal NO synthesis (Leonard et al., 1998), the above effect of green tea may be of therapeutic relevance. Furthermore, in chronic inflammatory lung diseases, especially in neonates, the use of steroids has a number of undesired effects (Shanley et al., 2002). Therefore, in inflammatory diseases, green tea may be a suitable adjuvant to reduce the doses of steroids needed.

In contrast to other reports (Lin and Lin, 1997; Singh et al., 2002), we observed no inhibitory effect of green tea extract on the LPS/cytokine-induced NF-B DNA binding activity. Therefore, in human epithelial A549/8 or DLD-1 cells, inhibition of NF-B activity is unlikely to be involved in the inhibition of iNOS expression by green tea extract as described for human chondrocytes or murine macrophages. In addition, as shown in our previous reports, induction of iNOS expression depends only partially on NF-B activation in A549/8 or DLD-1 cells (Kleinert et al., 1998). Therefore, the effect of green tea extract (or ECGC) on iNOS expression seems to be cell-specific.

In conclusion, green tea extract may be as effective as EGCG in inhibiting iNOS expression by suppressing IFN-–elicited STAT-1 activation. As a possible anti-inflammatory drug, green tea extract may be more suitable than EGCG because green tea extract is far more economical than EGCG and, more important, is more stable because of the presence of different antioxidants in the extract.

	Acknowledgements

 
We gratefully acknowledge the expert technical assistance of I. Ihrig-Biedert and K. Masch.

	Footnotes

 
This work was supported by grant 8312-38 62 61/322a, b from the Innovation Foundation of the State of Rhineland-Palatinate, grant Kl 1020/4-1 from the Deutsche Forschungsgemeinschaft (to H.K.), by the Collaborative Research Center SFB 553 (Project A7 to H.K.) and Italian Ministry for University and Scientific Research grants 2000–2002 (to H.S.). E.T. is supported by a doctoral fellowship in Biochemical Science of University of Verona.

This article contains data from the thesis work of E.T.

ABBREVIATIONS: iNOS, inducible nitric-oxide synthase; JAK, Janus kinase; STAT, signal transducer and activator of transcription; kb, kilobase(s); NF-B, nuclear factor B; GAS, -activated site; tyrphostin AG490, N-benzyl-3,4-dihydroxy-benzylidenecyanoacetamide; ECGC, epigallocatechin-3-gallate; LPS, lipopolysaccharide; IFN, interferon; IL, interleukin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NAC, N-acetyl-cysteine; ASC, ascorbate; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; CM, cytokine mixture; eNOS, endothelial nitric-oxide synthase; GX, green tea extract; TNF, tumor necrosis factor.

Address correspondence to: Dr. Hartmut Kleinert, Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Str. 67, 55101 Mainz, Germany. E-mail: kleinert{at}mail.uni-mainz.de

	References

Top Abstract Materials and Methods Results Discussion References

 
Barnes PJ and Liew FY (1995) Nitric oxide and asthmatic inflammation. Immunol Today 16: 128-130.[CrossRef][Medline]

Bogdan C (2001) Nitric oxide and the immune response. Nat Immunol 2: 907-916.[CrossRef][Medline]

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.[CrossRef][Medline]

Cavicchi M and Whittle BJ (1999) Regulation of induction of nitric oxide synthase and the inhibitory actions of dexamethasone in the human intestinal epithelial cell line, Caco-2: influence of cell differentiation. Br J Pharmacol 128: 705-715.[CrossRef][Medline]

Chomczynski P and Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159.[Medline]

Darnell JE Jr, Kerr IM, and Stark GR (1994) Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science (Wash DC) 264: 1415-1421.[Abstract/Free Full Text]

Das M, Sur P, Gomes A, Vedasiromoni JR and Ganguly DK (2002) Inhibition of tumour growth and inflammation by consumption of tea. Phytother Res 16: S40-S44.

de Vera ME, Shapiro RA, Nüssler AK, Mudgett JS, Simmons RL, Morris SM, Billiar TR, and Geller DA (1996) Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter. Proc Natl Acad Sci USA 93: 1054-1059.[Abstract/Free Full Text]

Dell'Albani P, Santangelo R, Torrisi L, Nicoletti VG, de Vellis J, and Giuffrida Stella AM (2001) JAK/STAT signaling pathway mediates cytokine-induced iNOS expression in primary astroglial cell cultures. J Neurosci Res 65: 417-424.[CrossRef][Medline]

Fujiki H, Suganuma M, Imai K, and Nakachi K (2002) Green tea: cancer preventive beverage and/or drug. Cancer Lett 188: 9-13.[CrossRef][Medline]

Ganster RW, Taylor BS, Shao L, and Geller DA (2001) Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-kappa B. Proc Natl Acad Sci USA 98: 8638-8643.[Abstract/Free Full Text]

Gao J, Morrison DC, Parmely TJ, Russell SW, and Murphy WJ (1997) An interferon--activated site (GAS) is necessary for full expression of the mouse iNOS gene in response to interferon-g and lipopolysaccharide. J Biol Chem 272: 1226-1230.[Abstract/Free Full Text]

Guslandi M (1998) Nitric oxide and inflammatory bowel diseases. Eur J Clin Investig 28: 904-907.[CrossRef][Medline]

Haqqi TM, Anthony DD, Gupta S, Ahmad N, Lee MS, Kumar GK, and Mukhtar H (1999) Prevention of collagen-induced arthritis in mice by a polyphenolic fraction from green tea. Proc Natl Acad Sci USA 96: 4524-4529.[Abstract/Free Full Text]

Hausding M, Witteck A, Rodriguez-Pascual F, von Eichel-Streiber M, Förstermann U, and Kleinert H (2000) Inhibition of small G proteins of the Rho family enhances cytokine-mediated induction of NO synthase II. Br J Pharmacol 131: 553-561.[CrossRef][Medline]

Husain K (2002) Exercise conditioning attenuates the hypertensive effects of nitric oxide synthase inhibitor in rat. Mol Cell Biochem 231: 129-137.[CrossRef][Medline]

Ihle JN (1995) Cytokine receptor signalling. Nature (Lond) 377: 591-594.[CrossRef][Medline]

Ihle JN, Witthuhn BA, Quelle FW, Yamamoto K, Thierfelder WE, Kreider B, and Silvennoinen O (1994) Signaling by the cytokine receptor superfamily: JAKs and STATs. Trends Biochem Sci 19: 222-227.[CrossRef][Medline]

Jaiswal M, LaRusso NF and Gores GJ (2001) Nitric oxide in gastrointestinal epithelial cell carcinogenesis: linking inflammation to oncogenesis. Am J Physiol 281: G626-G634.

Kang DG, Hur TY, Lee GM, Oh H, Kwon TO, Sohn EJ, and Lee HS (2002) Effects of Cudrania tricuspidata water extract on blood pressure and renal functions in NO-dependent hypertension. Life Sci 70: 2599-2609.[CrossRef][Medline]

Kleinert H, Boissel JP, Schwarz PM, and Förstermann U (2000) Regulation of the expression of nitric oxide synthase isoforms, in Nitric Oxide: Biology and Pathobiology (Ignarro LJ ed) pp 105-128, Academic Press, New York.

Kleinert H, Euchenhofer C, Ihrig-Biedert I, and Förstermann U (1996) Glucocorticoids inhibit the induction of nitric oxide synthase II by down-regulating cytokine-induced activity of transcription factor nuclear factor-B. Mol Pharmacol 49: 15-21.[Abstract]

Kleinert H, Geller DA, and Förstermann U (1999) Analysis of the molecular mechanism regulating the induction of the human NO synthase II promoter in human A549/8 alveolar carcinoma cells. Naunyn-Schmiedeberg's Arch Pharmacol 359: R37.

Kleinert H, Wallerath T, Fritz G, Ihrig-Biedert I, Rodriguez-Pascual F, Geller DA, and Förstermann U (1998) Cytokine induction of NO Synthase II in human DLD-1 cells: roles of the JAK-STAT, AP-1 and NF-kB-signaling pathways. Br J Pharmacol 125: 193-201.[CrossRef][Medline]

Kröncke KD, Fehsel K, and Kolb-Bachofen V (1998) Inducible nitric oxide synthase in human diseases. Clin Exp Immunol 113: 147-156.[CrossRef][Medline]

Leonard N, Bishop AE, Polak JM, and Talbot IC (1998) Expression of nitric oxide synthase in inflammatory bowel disease is not affected by corticosteroid treatment. J Clin Pathol 51: 750-753.[Abstract]

Lin JK (2002) Cancer chemoprevention by tea polyphenols through modulating signal transduction pathways. Arch Pharm Res (NY) 25: 561-571.

Lin YL and Lin JK (1997) (-)-Epigallocatechin-3-gallate blocks the induction of nitric oxide synthase by down-regulating lipopolysaccharide-induced activity of transcription factor nuclear factor-B. Mol Pharmacol 52: 465-472.[Abstract/Free Full Text]

MacMicking J, Xie QW, and Nathan C (1997) Nitric oxide and macrophage function. Annu Rev Immunol 15: 323-350.[CrossRef][Medline]

Menegazzi M, Tedeschi E, Dussin D, De Prati AC, Cavalieri E, Mariotto S, and Suzuki H (2001) Anti-interferon gamma action of epigallocatechin-3-gallate mediated by specific inhibition of STAT1 activation. FASEB J 15: 1309-1311.[Free Full Text]

Ohmori Y and Hamilton TA (2001) Requirement for STAT1 in LPS-induced gene expression in macrophages. J Leukoc Biol 69: 598-604.[Abstract/Free Full Text]

Osborne KA, Robichon A, Burgess E, Butland S, Shaw RA, Coulthard A, Pereira HS, Greenspan RJ, and Sokolowski MB (1997) Natural behavior polymorphism due to a cGMP-dependent protein kinase of Drosophila. Science (Wash DC) 277: 834-836.[Abstract/Free Full Text]

Shanley TP, Zhao B, Macariola DR, Denenberg A, Salzman AL, and Ward PA (2002) Role of nitric oxide in acute lung inflammation: lessons learned from the inducible nitric oxide synthase knockout mouse. Crit Care Med 30: 1960-1968.[CrossRef][Medline]

Singh R, Ahmed S, Islam N, Goldberg VM, and Haqqi TM (2002) Epigallocatechin-3-gallate inhibits interleukin-1beta-induced expression of nitric oxide synthase and production of nitric oxide in human chondrocytes: suppression of nuclear factor kappaB activation by degradation of the inhibitor of nuclear factor kappaB. Arthritis Rheum 46: 2079-2086.[CrossRef][Medline]

van Straaten EA, Koster-Kamphuis L, Bovee-Oudenhoven IM, van der Meer R, and Forget PP (1999) Increased urinary nitric oxide oxidation products in children with active coeliac disease. Acta Paediatr 88: 528-531.[CrossRef][Medline]

Varilek GW, Yang F, Lee EY, deVilliers WJ, Zhong J, Oz HS, Westberry KF, and McClain CJ (2001) Green tea polyphenol extract attenuates inflammation in interleukin-2-deficient mice, a model of autoimmunity. J Nutr 131: 2034-2039.[Abstract/Free Full Text]

Wagner BJ, Hayes TE, Hoban CJ, and Cochran BH (1990) The SIF binding element confers sis/PDGF inducibility onto the c-fos promoter. EMBO (Eur Mol Biol Organ) J 9: 4477-4484.[Medline]

Witteck A, Yao Y, Fechir M, Förstermann U, and Kleinert H (2003) Human iNOS gene expression is regulated by Rho protein-mediated changes in the structure of the actin cytoskeleton. Exp Cell Res 287: 106-115.[CrossRef][Medline]

Yang CS, Lee MJ, Chen L, and Yang GY (1997) Polyphenols As Inhibitors of Carcinogenesis. Environ Health Perspect 105 (Suppl 4): 971-976.

Yao Y, Hausding M, Erkel G, Anke T, Forstermann U, and Kleinert H (2003) Sporogen, S14–95 and S-curvularin, three inhibitors of human inducible nitricoxide synthase expression isolated from fungi. Mol Pharmacol 63: 383-391.[Abstract/Free Full Text]

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