- Email: [email protected]
Involvement of ERK and Protein Tyrosine Phosphatase Signaling Pathways in EGCG-Induced Cyclooxygenase-2 Expression in Raw 264.7 Cells
Biochemical and Biophysical Research Communications 286, 721–725 (2001) doi:10.1006/bbrc.2001.5415, available online at http://www.idealibrary.com on
Involvement of ERK and Protein Tyrosine Phosphatase Signaling Pathways in EGCG-Induced Cyclooxygenase-2 Expression in Raw 264.7 Cells Jong-Wook Park,* Yoon Jung Choi,* Seong-IL Suh,† and Taeg Kyu Kwon* ,1 *Department of Immunology and †Department of Microbiology, School of Medicine, Keimyung University, 194 DongSan-Dong Jung-Gu, Taegu, 700-712, South Korea
Received July 5, 2001
Prostaglandins play regulatory roles in a variety of physiological and pathological processes in immune response and inflammation. Epigallocatechin-3-gallate (EGCG) is known to potent antitumor agent with antioxidant property. We first investigated the effect of EGCG on the production of prostaglandin E 2 (PGE 2) and the expression of cyclooxygenase-2 (COX-2), the ratelimiting enzyme in the synthesis of PGE 2, using macrophage cell line, Raw264.7. Our results showed that COX-2 expression and PGE 2 production are upregulated by EGCG treatment and that this induction of COX-2 is regulated in part at the transcriptional level. In addition, we demonstrated the signal transduction pathway of mitogen-activated protein kinase (MAP kinase) in EGCG-mediated COX-2 expression. The MEK inhibitor (PD098059) prevented EGCG-induced COX-2 expression, whereas sodium orthovanadate (protein-tyrosine phosphatase inhibitor) significantly enhanced COX-2 expression and PGE 2 production. These results suggest that EGCG mediated COX-2 expression and PGE 2 production is associated with the activation of both the ERK and protein-tyrosine phosphatase signaling pathways. © 2001 Academic Press
Key Words: EGCG; COX-2; signal transduction; prostaglandin, COX-2 promoter.
Tea is one of the most popular beverages in the world, and the possible beneficial health effects have received a great deal of attention. Polyphenols are the most significant group of tea components, especially the catechin group of the flavonols. The major tea catechins are epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG), epicatechin (EG), (⫹)-gallocathechin, and (⫹)-catechin. EGCG has been considered to be a major constituent of tea (1–3). These polyphenol components are known to 1
To whom correspondence should be addressed. Fax: 82-53-2551398. E-mail: [email protected].
have antioxidative activities due to their radical scavenging and metal chelating functions as well as antimutagenic activities (4 – 6). Recently, Lin et al. reported that EGCG could prevent the binding of NFB to the inducible nitric oxide synthase (iNOS) promoter, thereby inhibiting the induction of iNOS transcription. It has been suggested that EGCG may play role in preventing carcinogenesis and anti-inflammation (7). The cyclooxygenase (COX) catalyze the rate-limiting step in prostaglandin synthesis. At least two isoforms of the enzyme are expressed in mammalian tissues, COX-1 and COX-2. COX-1 is constitutively expressed in tissues and is thought to be involved in homeostatic prostanoid biosynthesis (8 –10). COX-2 is thought to be the predominant isoform involved in the inflammatory response (8 –10). Prostaglandins have a central role in many normal and pathophysiological responses (11). Prostaglandin synthesis can be stimulated by various physical and chemical agents in mammalian cells (12). However, the effects of EGCG on prostaglandin E 2 (PGE 2) production still remain uncertain. Therefore, we determined whether EGCG influences COX-2 expression and PGE 2 production in macrophage cells, since these cells play a central role in immune regulation and inflammation. Here we first reported that EGCG upregulates COX-2 expression and PGE 2 production. This induction of COX-2 is regulated in part at the transcriptional level. In addition, we demonstrate the importance of the activation of both the ERK and protein-tyrosine phosphatase signaling pathways in COX-2 induction. MATERIALS AND METHODS Cell culture and reagents. The macrophage cell line Raw264.7 was obtained from ATCC (Rockville, MD). The cells were cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and 10% FCS. The cells were grown at 37°C, 5% CO 2 in fully humidified air and subcultured twice weekly. Cells were seeded on 6-well plates at 1 ⫻ 10 6 cells/well. The cells were stimulated for various lengths of time ranging from 1 to
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Vol. 286, No. 4, 2001
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24 h in presence of EGCG with or without inhibitors. Anti-COX-2 and anti-Hsp70 antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-phospho-ERK and anti-phosphoJNK were purchased from New England Biolabs Inc. (Beverly, MA). RO 31-8220, SB 203580, PD 098059, PDTC (pyrrolidinedithiocarbamate) and LY 294002 were purchased from Biomol (Biomol Research Laboratories, Inc., PA). Other chemicals were purchased from Sigma. Western blot analysis. Cellular lysates were prepared by suspending 1 ⫻ 10 6 cells in 100 l of lysis buffer (137 mM NaCl, 15 mM EGTA, 1 mM sodium orthovanadate, 15 mM MgCl 2, 0.1% Triton X-100, 25 mM Mops, 2 g/ml proteinase inhibitor E64, adjusted to pH 7.2). The cells were disrupted by sonication and extracted at 4°C for 30 min. Fifty micrograms of cell lysate were electrophoresed on 10% SDS–polyacrylamide gels. The proteins were electro-transferred to Immobilon-P membranes (Millipore Corporation, Bedford, MA). Detection of the specific proteins was carried out with an ECL kit following the manufacturer’s instructions. RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA was isolated according to Chomczymski and Sacchi (13). Single-strand cDNA was synthesized from 2 g of total RNA using M-MLV reverse transcriptase (Gibco-BRL, Gaithersburg, MD). The cDNA for COX-2 and actin were amplified by PCR with specific primers. The sequences of the sense and antisense primers for COX-2 were 5⬘-CCGTGGTGAATGTATGAGCA-3⬘ and 5⬘CCTCGCTTCTGATCTGTCTT-3⬘, respectively. Conditions for PCR were 1⫻ (94°C, 3 min); 30⫻ (94°C, 45 s; 58°C, 45 s; and 72°C, 1 min); and 1⫻ (72°C, 10 min). PCR products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide. DNA transfection and luciferase assay. COX-2 promoter constructs were generously provided by Dr. H. Inoue (National Cardiovascular Center Research Institute, Japan). Briefly, the region from ⫺1432 bp to ⫹59 bp of COX-2 promoter was cloned into pGL2. COX-2 promoter plasmid was transfected into human embryonic kidney (HEK) 293 cells using the Lipofectamine reagent (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions. To assess the COX-2 promoter luciferase, cells were collected and disrupted by sonication in lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM EDTA, 1% Triton X-100, and 10% glycerol). After centrifugation, aliquots of the supernatants were tested for luciferase activity using the luciferase assay system (Promega, Madison, WI) according to the manufacturer’s instructions. PGE 2 production assay. Cells were plated in 6-well plates at 10 6 cell per well and allowed to culture for 24 h. Twenty-four hours later, the culture medium was replaced with fresh serum-free medium with or without EGCG for various time points. PGE 2 secretion into the culture medium was measured by a PGE 2 enzyme immunoassay (EIA) kit (Cayman Chemicals, Ann Arbor, MI). All experiments were performed in duplicate.
RESULTS EGCG Induces COX-2 Enzyme Activity Lipopolysaccharide (LPS) by itself activates mouse macrophages to express COX-2 and produce PGE 2. To determine whether EGCG, major compound of green tea, was involved in signal pathway transduction pathway leading to PGE 2 releasing and COX-2 expression caused by LPS, we monitored PGE 2 concentrations in the culture media of cells stimulated with EGCG or LPS or EGCG plus LPS. As shown in Fig. 1, LPS significantly induced COX-2 in Raw264.7 cells. Cotreatment and pretreatment with EGCG did not alter
FIG. 1. Effects of EGCG on COX-2 expression and PGE 2 production caused by LPS in Raw 264.7 cells. (A) Cellular lysate protein (50 g/lane) was loaded onto a 10% SDS–polyacrylamide gel. Immunoblot was probed with antibody specific for COX-2. Lysates were form Raw 264.7 cell treated with vehicle (lane 1), 25 M EGCG (lane 2), 1 g/ml LPS (lane 3), EGCG pretreated for 2 h and treated with LPS (lane 4), EGCG plus LPS (lane 5), and 10% FBS serum (lane 6). The equal loading in each lane was demonstrated by the similar intensities of Hsp 70. (B) Cells were incubated with vehicle, 25 M EGCG, 1 mg/ml LPS, and 25 M EGCG plus 1 mg/ml LPS for 10 h. The medium was removed, and the production of PGE 2 was measured by PGE 2 enzyme immunoassay kit. Results are expressed as means and standard deviation of three independent experiments performed in duplicate.
amounts of COX-2 and PGE 2 production. Interestingly, 25 M EGCG upregulated COX-2 expression level in Raw264.7 cells. Characterization of COX-2 Expression Induced by EGCG in Raw264.7 Cells To investigate whether EGCG could induce PGE 2 production in the Raw264.7 cells, we monitored PGE 2 concentrations in the culture media of cells stimulated with EGCG. Treatment with EGCG (25–100 M, for 12 h) caused a concentration-dependent increase in the release of PGE 2 and the expression of a 70 kDa COX-2 protein in Raw264.7 cells (Fig. 2A). There was a correlation between the release of PGE 2 and the expression level of COX-2 protein. To further elucidate the mechanism responsible for the changes in amounts of COX-2 protein, we determined levels of COX-2 mRNA by RTPCR. Treatment with EGCG resulted in marked increases COX-2 mRNA levels, an effect that was induced by EGCG in a concentration-dependent manner (Fig. 2B). In further studies of the relationship between COX-2 protein and COX-2 mRNA in Raw264.7 cells, we carried out time kinetic studies of EGCG treated Raw264.7 cells. Incubation with EGCG caused a time-
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FIG. 2. Effects of EGCG on PGE 2 production and COX-2 expression. (A) Concentration-dependent increases in the PGE 2 production and COX-2 expression. Cells were incubated with various concentrations of EGCG for 12 h and then harvested in lysis buffer. Equal amounts of soluble lysates (50 g) were subjected to electrophoresis. The blots were analyzed using a specific antibody against COX-2 and Hsp70. The medium was removed, and the production of PGE 2 was measured by PGE 2 enzyme immunoassay kit. Results are expressed as means and standard deviation of three independent experiments performed in duplicate. (B) COX-2 mRNA induction by EGCG treatment. Cells were incubated for 12 h with various concentrations of EGCG and RT-PCR analysis was performed. The expression of COX-2 mRNA increased in a concentration-dependent manner. (C) Time courses of COX-2 protein expression and COX-2 mRNA induced by EGCG (75 M). Cells were incubated at 37°C for the periods indicated in the presence or absence of EGCG. Levels of COX-2 protein and mRNA were detected by Western blot analysis and RT-PCR, respectively. The results were confirmed by repeating two independent experiments. (D) Induction of COX-2 promoter activity by EGCG treatment. Human COX-2 promoter construct (⫺1432/⫹59) was transfected into HEK 293 cells and incubated with or without EGCG (75 M) for 12 h. Cells were harvested and assayed for luciferase. Data are mean values obtained from three independent experiments and bars represent standard deviations.
dependent increase in COX-2 protein expression. At the highest peak of COX-2 expression, observed 12 h and then sustained up to 24 h after treatment. EGCG caused an induction of COX-2 mRNA in Raw264.7 cells, which peaked at 8 h and then declined up to 24 h after the treatment (Fig. 2C). To investigate whether or not EGCG-induced COX-2 induction is due to promoter activity, transient transfection of COX-2 reporter gene construct was performed. Used COX-2 promoters contain several binding sites of the potential transcription factors within 1432 bases upstream of the COX-2 transcription start site (14 –16). EGCG treatment significantly increased the promoter activity compared with no treatment (Fig. 2D). These results suggest that EGCG treatment could stimulate the COX-2 promoter region.
MAPK Signal Pathway after EGCG Treatment It is well established that mitogen-activated protein kinase (MAPK) signaling pathways mediate COX-2 induction in a number of cell types (17–19). To examine the role of the cell signaling pathway in the induction of COX-2 by EGCG, we measured phosphorylation level of MAPK in EGCG treated Raw264.7 cells by Western blotting using a phosphorylation specific MAPKs antibodies (Fig. 3A). By treatment with EGCG (75 M), phosphorylation of ERK was induced 10 min after incubation and then declined. Preincubation with PD 098059 (50 M) in culture medium before the addition of EGCG significantly decreased ERK phosphorylation. EGCG-induced phosphorylation of ERK at 30 min was not suppressed by SB 203580 (p38 MAPK inhibitor) at 10 M. Phosphorylation of c-Jun
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The Effect of Sodium Orthovanadate on COX-2 Expression Sodium orthovanadate is generally regarded as a protein tyrosine phosphatases inhibitor (20). To examine whether protein tyrosine phosphatase was involved in the signal transduction pathway leading to PGE 2 release and COX-2 expression caused by EGCG, Raw264.7 cells were treated with EGCG in the presence or absence of sodium orthovanadate, and COX-2 expression was measured (Fig. 4). When Raw264.7 cells were cotreated with EGCG and sodium orthovanadate, further potentiations of the COX-2 expression were observed after 8 h of treatment. DISCUSSION Polyphenolic compounds in green tea have been associated with lower risk of some diseases, including cancer (21, 22). Based on many studies, it is believed that the biological responses of green tea are mediated by its major polyphenolic constituent EGCG that has been shown to possess exceptional antioxidant potential. In additional, EGCG has anticancer effects and is effective for reducing free radical-mediated injury (23– 25). These findings suggest that EGCG has anti-
FIG. 3. Effects of SB 203580, PD 98059, and RO 31-8220 on EGCG-induced phosphorylation of MAP kinase and COX-2 expression. (A) Raw 264.7 cells were incubated for indicated time to determine phosphorylation of ERK and JNK in the presence or absence of EGCG and indicated inhibitor. Equal amounts of soluble lysates (50 g) were subjected to electrophoresis. The blots were analyzed using a specific antibody against phosphorylated ERK and phosphorylated JNK. The lower panel shows the same blot stripped and reprobed with ERK and JNK antibodies as an internal control of the protein contents per lane. (B) Cells were pretreated with various inhibitors (50 M PD 98059, 10 M SB 203580, 2 M RO 31-8220, 25 M LY 294002, and 100 M PDTC) for 90 min and then washed. Cells were incubated with EGCG (75 M) for 8 h. Equal amounts of soluble lysates (50 g) were subjected to electrophoresis. The blots were analyzed using a specific antibody against COX-2. The equal loading in each lane was demonstrated by the similar intensities of Hsp 70.
N-terminal kinase (JNK) was also observed at 10 min and sustained until 30 min. These results suggest that EGCG induce activation of ERK and JNK. To determine whether or not the MAPK signaling pathways, is involved in EGCG-induced COX-2 upregulation, Raw264.7 cells were pretreated with or without MAPK specific inhibitors in the presence of EGCG (Fig. 3B). PD 098059 significantly inhibited the induction of COX-2 protein expression, suggesting that EGCGinduced COX-2 expression was mediated, at least in part, through the ERK signaling pathway.
FIG. 4. Effects of sodium orthovanadate on EGCG-induced COX-2 expression and PGE 2 production. Cells were incubated with vehicle, 60 M EGCG, 10 M sodium orthovanadate, 50 M sodium orthovanadate, 60 M EGCG plus 10 mM sodium orthovandate, and 60 M EGCG plus 10 M sodium orthovandate for 8 h. Cells were harvested in lysis buffer. Equal amounts of soluble lysates (50 g) were subjected to electrophoresis. The blots were analyzed using a specific antibody against COX-2 and Hsp70. The medium was removed, and the production of PGE 2 was measured by PGE 2 enzyme immunoassay kit. Results are expressed as means and standard deviation of three independent experiments performed in duplicate.
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inflammatory effects. However, the present study demonstrated that EGCG upregulates COX-2 expression and PGE 2 production in Raw264.7 macrophage cells, suggesting that EGCG enhances inflammatory processes. In the present study, COX-2 protein induction coincided with the secretion of PGE2 into the culture medium. Previous studies have demonstrated that COX-2 expression can be regulated by transcriptional as well as posttranscriptional mechanism (26 –27). Our results obtained by RT-PCT assay and transient transfection assay using report constructs, in which the report is under the control of various lengths of the COX-2 regulatory region, support the conclusion that EGCG regulates COX-2 gene expression at least in part at the transcriptional level. The region from ⫺840 bp to ⫹123 bp includes the binding sites for many transcriptional factors such as NFB, C/EBP, CRE/ATF, E-box, STAT3, AP2 and NF-IL6 (14 – 16). Recently, it was reported that COX-2 expression can be regulated through different MAP kinase signaling pathways and that the particular signaling pathway involved is dependent on the type of stimuli (17–19). The induction of COX-2 by PDGF has been demonstrated to require activation of the ERK signaling pathway (17), while constitutively active MEKK1 has been shown to induce COX-2 expression by activating the SEK1/MKK4p38 kinase pathway (18). Our data suggested that EGCG induces PGE2 production via activation of PKC and partially by activation of ERK. The suppression of EGCGinduced COX-2 expression by MEK inhibitor PD 98059 is in agreement with the concept that ERK signaling pathway is important in the regulation of COX-2 by EGCG. It has been reported that ERK can phosphorylate and activate CRE/ATF, E-box and NF-IL6 (28 –30). These finding suggest that EGCG treatment may activate these transcriptional factors through ERK signaling pathway, leading to COX-2 expression and subsequent PGE2 production. In addition, a role for protein-tyrosine kinases (PTKs) in control of COX-2 expression was also reported for interleukin-1␣ treated human endothelial cells (31). Synergistic enhancement of expression of the COX-2 expression was observed on stimulation of Raw264.7 with vanadate plus EGCG. In conclusion, the present study is first report showing that COX-2 expression and PGE 2 production are regulated by EGCG. We provide evidence showing that activation of ERK MAP kinase and protein-tyrosine kinase signaling pathways an important role in the control of COX-2 expression. The EGCG may regulate inflammatory processes by modulating PGE 2 as well as other inflammatory mediators. ACKNOWLEDGMENT This work was supported by a Korea Research Foundation Grant (KRF-2000-041-F00103).
REFERENCES 1. Yang, C. S., and Wang, Z., Y. (1993) J. Natl. Cancer Inst. 58, 1038 –1049. 2. Stoner, G. D., and Mukhtar, H. (1995) J. Cell Biochem. 22, 169 –180. 3. Katiyar, S. K., and Mukhtar, H. (1996) Int. J. Oncol. 8, 221–238. 4. Lin, Y. L., Juan, I. M., Chen, Y. L., Liang, Y. C., and Lin, J. K. (1996) J. Agric. Food Chem. 44, 1387–1394. 5. Katiyar, S. K., Agarwal, R., Zaim, M. T., and Mukhtar, H. (1993) Carcinogenesis 14, 849 – 855. 6. Shiraki, M., Hara, Y., Osawa, T., Kumon, H., Nakauama, T., and Kawakishi, S. (1994) Mutat. Res. 323, 29 –34. 7. Lin, Y. L., and Lin, J, K. (1997) Mol. Pharmacol. 52, 465– 472. 8. Smith, W. L., and DeWitt, D. L. (1996) Adv. Immunol. 62, 167– 215. 9. Griswold, D. E., and Adams, J. L. (1996) Med. Res. Rev. 16, 181–206. 10. Herschman, H. R. (1996) Biochim. Biophys. Acta 1299, 125–140. 11. DeWitt, D. L. (1991) Biochim. Biophys. Acta 1083, 121–134. 12. Weksler, B. B., Eldor, A., Falcone, D., Levin, R. I., Jaffe, E. A., and Minick, C. R. (1982) Cardiovascular Pharmacology of the Prostaglandins (Herman, A. G., Vanhoutte, P. M., Denolin, H., and Goossens, A., Eds.), pp. 137–148, Raven Press, New York. 13. Chomczymski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156 –159. 14. Inoue, H., Yokoyama, C., Hara, S., Tone, Y., and Tanabe, T. (1995) J. Biol. Chem. 270, 24965–24971. 15. Lukiw, W. J., Pelaez, R. P., Martinez, J., and Bazan, N. G. (1998) Proc. Natl. Acad. Sci. USA 95, 3914 –3919. 16. Kim, Y., and Fischer, S. M. (1998) J. Biol. Chem. 273, 27686 – 27694. 17. Xie, W., and Herschman, H. R. (1996) J. Biol. Chem. 271, 31742– 31748. 18. Guan, Z., Buckman, S. Y., Pentland, A. P., Templeton, D. J., and Morrison, A. R. (1998) J. Biol. Chem. 273, 12901–12908. 19. Adderley, S. R., and Fitzgerald, D. J. (1999) J. Biol. Chem. 273, 27686 –27694. 20. Li, L., Eisen, A. Z., Sturman, E., and Seltzer, J. L. (1998) Biochim. Biophys. Acta 1405, 110 –120. 21. Bushman, J. L. (1998) Nutr. Cancer 31, 151–159. 22. Ahmad, N., and Mukhtar, H. (1999) Nutr. Rev. 57, 78 – 83. 23. Garbisa, S., Biggin, S., Cavallarin, N., Sartor, L., Benelli, R., and Albini, A. (1999) Nat. Med. 5, 1216. 24. Garbisa, S., Sartor, L., Biggin, S., Salvato, B., Benelli, R., and Albini, A. (2001) Cancer 91, 822– 832. 25. Ahmad, N., Gupta, S., and Mukhtar, H. (2000) Arch. Biochem. Biophys. 376, 338 –346. 26. Mestre, J. R., Subbaramaiah, K., Sacks, P. G., Schantz, S. P., Tanabe, T., Inoue, H., and Dannenberg, A. J. (1997) Cancer Res. 57, 1081–1085. 27. Daen, J. L., Brook, M., Clark, A. R., and Saklatvala, J. (1999) J. Biol. Chem. 274, 264 –269. 28. Nakajima, T., Kinoshita, S., Sasagawa, T., Sasaki, K., Naruto, M., Kishimoto, T., and Akira, S. (1993) Proc. Natl. Acad. Sci. USA 90, 2207–2211. 29. Prasad, K. S., and Brandt, S. J. (1997) J. Biol. Chem. 272, 11457–11462. 30. Davis, S., Vanhoutte, P., Pages, C., Caboche, J., and Laroche, S. (2000) J. Neurosci. 20, 4563– 4572. 31. Hirai, K., Takayama, H., Tomo, K., and Okuma, M. (1997) Biochem. J. 322, 373–377.
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Involvement of ERK and Protein Tyrosine Phosphatase Signaling Pathways in EGCG-Induced Cyclooxygenase-2 Expression in Raw 264.7 Cells Jong-Wook Park,* Yoon Jung Choi,* Seong-IL Suh,† and Taeg Kyu Kwon* ,1 *Department of Immunology and †Department of Microbiology, School of Medicine, Keimyung University, 194 DongSan-Dong Jung-Gu, Taegu, 700-712, South Korea
Received July 5, 2001
Prostaglandins play regulatory roles in a variety of physiological and pathological processes in immune response and inflammation. Epigallocatechin-3-gallate (EGCG) is known to potent antitumor agent with antioxidant property. We first investigated the effect of EGCG on the production of prostaglandin E 2 (PGE 2) and the expression of cyclooxygenase-2 (COX-2), the ratelimiting enzyme in the synthesis of PGE 2, using macrophage cell line, Raw264.7. Our results showed that COX-2 expression and PGE 2 production are upregulated by EGCG treatment and that this induction of COX-2 is regulated in part at the transcriptional level. In addition, we demonstrated the signal transduction pathway of mitogen-activated protein kinase (MAP kinase) in EGCG-mediated COX-2 expression. The MEK inhibitor (PD098059) prevented EGCG-induced COX-2 expression, whereas sodium orthovanadate (protein-tyrosine phosphatase inhibitor) significantly enhanced COX-2 expression and PGE 2 production. These results suggest that EGCG mediated COX-2 expression and PGE 2 production is associated with the activation of both the ERK and protein-tyrosine phosphatase signaling pathways. © 2001 Academic Press
Key Words: EGCG; COX-2; signal transduction; prostaglandin, COX-2 promoter.
Tea is one of the most popular beverages in the world, and the possible beneficial health effects have received a great deal of attention. Polyphenols are the most significant group of tea components, especially the catechin group of the flavonols. The major tea catechins are epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG), epicatechin (EG), (⫹)-gallocathechin, and (⫹)-catechin. EGCG has been considered to be a major constituent of tea (1–3). These polyphenol components are known to 1
To whom correspondence should be addressed. Fax: 82-53-2551398. E-mail: [email protected].
have antioxidative activities due to their radical scavenging and metal chelating functions as well as antimutagenic activities (4 – 6). Recently, Lin et al. reported that EGCG could prevent the binding of NFB to the inducible nitric oxide synthase (iNOS) promoter, thereby inhibiting the induction of iNOS transcription. It has been suggested that EGCG may play role in preventing carcinogenesis and anti-inflammation (7). The cyclooxygenase (COX) catalyze the rate-limiting step in prostaglandin synthesis. At least two isoforms of the enzyme are expressed in mammalian tissues, COX-1 and COX-2. COX-1 is constitutively expressed in tissues and is thought to be involved in homeostatic prostanoid biosynthesis (8 –10). COX-2 is thought to be the predominant isoform involved in the inflammatory response (8 –10). Prostaglandins have a central role in many normal and pathophysiological responses (11). Prostaglandin synthesis can be stimulated by various physical and chemical agents in mammalian cells (12). However, the effects of EGCG on prostaglandin E 2 (PGE 2) production still remain uncertain. Therefore, we determined whether EGCG influences COX-2 expression and PGE 2 production in macrophage cells, since these cells play a central role in immune regulation and inflammation. Here we first reported that EGCG upregulates COX-2 expression and PGE 2 production. This induction of COX-2 is regulated in part at the transcriptional level. In addition, we demonstrate the importance of the activation of both the ERK and protein-tyrosine phosphatase signaling pathways in COX-2 induction. MATERIALS AND METHODS Cell culture and reagents. The macrophage cell line Raw264.7 was obtained from ATCC (Rockville, MD). The cells were cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and 10% FCS. The cells were grown at 37°C, 5% CO 2 in fully humidified air and subcultured twice weekly. Cells were seeded on 6-well plates at 1 ⫻ 10 6 cells/well. The cells were stimulated for various lengths of time ranging from 1 to
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Vol. 286, No. 4, 2001
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24 h in presence of EGCG with or without inhibitors. Anti-COX-2 and anti-Hsp70 antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-phospho-ERK and anti-phosphoJNK were purchased from New England Biolabs Inc. (Beverly, MA). RO 31-8220, SB 203580, PD 098059, PDTC (pyrrolidinedithiocarbamate) and LY 294002 were purchased from Biomol (Biomol Research Laboratories, Inc., PA). Other chemicals were purchased from Sigma. Western blot analysis. Cellular lysates were prepared by suspending 1 ⫻ 10 6 cells in 100 l of lysis buffer (137 mM NaCl, 15 mM EGTA, 1 mM sodium orthovanadate, 15 mM MgCl 2, 0.1% Triton X-100, 25 mM Mops, 2 g/ml proteinase inhibitor E64, adjusted to pH 7.2). The cells were disrupted by sonication and extracted at 4°C for 30 min. Fifty micrograms of cell lysate were electrophoresed on 10% SDS–polyacrylamide gels. The proteins were electro-transferred to Immobilon-P membranes (Millipore Corporation, Bedford, MA). Detection of the specific proteins was carried out with an ECL kit following the manufacturer’s instructions. RNA isolation and reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA was isolated according to Chomczymski and Sacchi (13). Single-strand cDNA was synthesized from 2 g of total RNA using M-MLV reverse transcriptase (Gibco-BRL, Gaithersburg, MD). The cDNA for COX-2 and actin were amplified by PCR with specific primers. The sequences of the sense and antisense primers for COX-2 were 5⬘-CCGTGGTGAATGTATGAGCA-3⬘ and 5⬘CCTCGCTTCTGATCTGTCTT-3⬘, respectively. Conditions for PCR were 1⫻ (94°C, 3 min); 30⫻ (94°C, 45 s; 58°C, 45 s; and 72°C, 1 min); and 1⫻ (72°C, 10 min). PCR products were analyzed by agarose gel electrophoresis and visualized by ethidium bromide. DNA transfection and luciferase assay. COX-2 promoter constructs were generously provided by Dr. H. Inoue (National Cardiovascular Center Research Institute, Japan). Briefly, the region from ⫺1432 bp to ⫹59 bp of COX-2 promoter was cloned into pGL2. COX-2 promoter plasmid was transfected into human embryonic kidney (HEK) 293 cells using the Lipofectamine reagent (Life Technologies, Grand Island, NY) according to the manufacturer’s instructions. To assess the COX-2 promoter luciferase, cells were collected and disrupted by sonication in lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM EDTA, 1% Triton X-100, and 10% glycerol). After centrifugation, aliquots of the supernatants were tested for luciferase activity using the luciferase assay system (Promega, Madison, WI) according to the manufacturer’s instructions. PGE 2 production assay. Cells were plated in 6-well plates at 10 6 cell per well and allowed to culture for 24 h. Twenty-four hours later, the culture medium was replaced with fresh serum-free medium with or without EGCG for various time points. PGE 2 secretion into the culture medium was measured by a PGE 2 enzyme immunoassay (EIA) kit (Cayman Chemicals, Ann Arbor, MI). All experiments were performed in duplicate.
RESULTS EGCG Induces COX-2 Enzyme Activity Lipopolysaccharide (LPS) by itself activates mouse macrophages to express COX-2 and produce PGE 2. To determine whether EGCG, major compound of green tea, was involved in signal pathway transduction pathway leading to PGE 2 releasing and COX-2 expression caused by LPS, we monitored PGE 2 concentrations in the culture media of cells stimulated with EGCG or LPS or EGCG plus LPS. As shown in Fig. 1, LPS significantly induced COX-2 in Raw264.7 cells. Cotreatment and pretreatment with EGCG did not alter
FIG. 1. Effects of EGCG on COX-2 expression and PGE 2 production caused by LPS in Raw 264.7 cells. (A) Cellular lysate protein (50 g/lane) was loaded onto a 10% SDS–polyacrylamide gel. Immunoblot was probed with antibody specific for COX-2. Lysates were form Raw 264.7 cell treated with vehicle (lane 1), 25 M EGCG (lane 2), 1 g/ml LPS (lane 3), EGCG pretreated for 2 h and treated with LPS (lane 4), EGCG plus LPS (lane 5), and 10% FBS serum (lane 6). The equal loading in each lane was demonstrated by the similar intensities of Hsp 70. (B) Cells were incubated with vehicle, 25 M EGCG, 1 mg/ml LPS, and 25 M EGCG plus 1 mg/ml LPS for 10 h. The medium was removed, and the production of PGE 2 was measured by PGE 2 enzyme immunoassay kit. Results are expressed as means and standard deviation of three independent experiments performed in duplicate.
amounts of COX-2 and PGE 2 production. Interestingly, 25 M EGCG upregulated COX-2 expression level in Raw264.7 cells. Characterization of COX-2 Expression Induced by EGCG in Raw264.7 Cells To investigate whether EGCG could induce PGE 2 production in the Raw264.7 cells, we monitored PGE 2 concentrations in the culture media of cells stimulated with EGCG. Treatment with EGCG (25–100 M, for 12 h) caused a concentration-dependent increase in the release of PGE 2 and the expression of a 70 kDa COX-2 protein in Raw264.7 cells (Fig. 2A). There was a correlation between the release of PGE 2 and the expression level of COX-2 protein. To further elucidate the mechanism responsible for the changes in amounts of COX-2 protein, we determined levels of COX-2 mRNA by RTPCR. Treatment with EGCG resulted in marked increases COX-2 mRNA levels, an effect that was induced by EGCG in a concentration-dependent manner (Fig. 2B). In further studies of the relationship between COX-2 protein and COX-2 mRNA in Raw264.7 cells, we carried out time kinetic studies of EGCG treated Raw264.7 cells. Incubation with EGCG caused a time-
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FIG. 2. Effects of EGCG on PGE 2 production and COX-2 expression. (A) Concentration-dependent increases in the PGE 2 production and COX-2 expression. Cells were incubated with various concentrations of EGCG for 12 h and then harvested in lysis buffer. Equal amounts of soluble lysates (50 g) were subjected to electrophoresis. The blots were analyzed using a specific antibody against COX-2 and Hsp70. The medium was removed, and the production of PGE 2 was measured by PGE 2 enzyme immunoassay kit. Results are expressed as means and standard deviation of three independent experiments performed in duplicate. (B) COX-2 mRNA induction by EGCG treatment. Cells were incubated for 12 h with various concentrations of EGCG and RT-PCR analysis was performed. The expression of COX-2 mRNA increased in a concentration-dependent manner. (C) Time courses of COX-2 protein expression and COX-2 mRNA induced by EGCG (75 M). Cells were incubated at 37°C for the periods indicated in the presence or absence of EGCG. Levels of COX-2 protein and mRNA were detected by Western blot analysis and RT-PCR, respectively. The results were confirmed by repeating two independent experiments. (D) Induction of COX-2 promoter activity by EGCG treatment. Human COX-2 promoter construct (⫺1432/⫹59) was transfected into HEK 293 cells and incubated with or without EGCG (75 M) for 12 h. Cells were harvested and assayed for luciferase. Data are mean values obtained from three independent experiments and bars represent standard deviations.
dependent increase in COX-2 protein expression. At the highest peak of COX-2 expression, observed 12 h and then sustained up to 24 h after treatment. EGCG caused an induction of COX-2 mRNA in Raw264.7 cells, which peaked at 8 h and then declined up to 24 h after the treatment (Fig. 2C). To investigate whether or not EGCG-induced COX-2 induction is due to promoter activity, transient transfection of COX-2 reporter gene construct was performed. Used COX-2 promoters contain several binding sites of the potential transcription factors within 1432 bases upstream of the COX-2 transcription start site (14 –16). EGCG treatment significantly increased the promoter activity compared with no treatment (Fig. 2D). These results suggest that EGCG treatment could stimulate the COX-2 promoter region.
MAPK Signal Pathway after EGCG Treatment It is well established that mitogen-activated protein kinase (MAPK) signaling pathways mediate COX-2 induction in a number of cell types (17–19). To examine the role of the cell signaling pathway in the induction of COX-2 by EGCG, we measured phosphorylation level of MAPK in EGCG treated Raw264.7 cells by Western blotting using a phosphorylation specific MAPKs antibodies (Fig. 3A). By treatment with EGCG (75 M), phosphorylation of ERK was induced 10 min after incubation and then declined. Preincubation with PD 098059 (50 M) in culture medium before the addition of EGCG significantly decreased ERK phosphorylation. EGCG-induced phosphorylation of ERK at 30 min was not suppressed by SB 203580 (p38 MAPK inhibitor) at 10 M. Phosphorylation of c-Jun
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The Effect of Sodium Orthovanadate on COX-2 Expression Sodium orthovanadate is generally regarded as a protein tyrosine phosphatases inhibitor (20). To examine whether protein tyrosine phosphatase was involved in the signal transduction pathway leading to PGE 2 release and COX-2 expression caused by EGCG, Raw264.7 cells were treated with EGCG in the presence or absence of sodium orthovanadate, and COX-2 expression was measured (Fig. 4). When Raw264.7 cells were cotreated with EGCG and sodium orthovanadate, further potentiations of the COX-2 expression were observed after 8 h of treatment. DISCUSSION Polyphenolic compounds in green tea have been associated with lower risk of some diseases, including cancer (21, 22). Based on many studies, it is believed that the biological responses of green tea are mediated by its major polyphenolic constituent EGCG that has been shown to possess exceptional antioxidant potential. In additional, EGCG has anticancer effects and is effective for reducing free radical-mediated injury (23– 25). These findings suggest that EGCG has anti-
FIG. 3. Effects of SB 203580, PD 98059, and RO 31-8220 on EGCG-induced phosphorylation of MAP kinase and COX-2 expression. (A) Raw 264.7 cells were incubated for indicated time to determine phosphorylation of ERK and JNK in the presence or absence of EGCG and indicated inhibitor. Equal amounts of soluble lysates (50 g) were subjected to electrophoresis. The blots were analyzed using a specific antibody against phosphorylated ERK and phosphorylated JNK. The lower panel shows the same blot stripped and reprobed with ERK and JNK antibodies as an internal control of the protein contents per lane. (B) Cells were pretreated with various inhibitors (50 M PD 98059, 10 M SB 203580, 2 M RO 31-8220, 25 M LY 294002, and 100 M PDTC) for 90 min and then washed. Cells were incubated with EGCG (75 M) for 8 h. Equal amounts of soluble lysates (50 g) were subjected to electrophoresis. The blots were analyzed using a specific antibody against COX-2. The equal loading in each lane was demonstrated by the similar intensities of Hsp 70.
N-terminal kinase (JNK) was also observed at 10 min and sustained until 30 min. These results suggest that EGCG induce activation of ERK and JNK. To determine whether or not the MAPK signaling pathways, is involved in EGCG-induced COX-2 upregulation, Raw264.7 cells were pretreated with or without MAPK specific inhibitors in the presence of EGCG (Fig. 3B). PD 098059 significantly inhibited the induction of COX-2 protein expression, suggesting that EGCGinduced COX-2 expression was mediated, at least in part, through the ERK signaling pathway.
FIG. 4. Effects of sodium orthovanadate on EGCG-induced COX-2 expression and PGE 2 production. Cells were incubated with vehicle, 60 M EGCG, 10 M sodium orthovanadate, 50 M sodium orthovanadate, 60 M EGCG plus 10 mM sodium orthovandate, and 60 M EGCG plus 10 M sodium orthovandate for 8 h. Cells were harvested in lysis buffer. Equal amounts of soluble lysates (50 g) were subjected to electrophoresis. The blots were analyzed using a specific antibody against COX-2 and Hsp70. The medium was removed, and the production of PGE 2 was measured by PGE 2 enzyme immunoassay kit. Results are expressed as means and standard deviation of three independent experiments performed in duplicate.
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inflammatory effects. However, the present study demonstrated that EGCG upregulates COX-2 expression and PGE 2 production in Raw264.7 macrophage cells, suggesting that EGCG enhances inflammatory processes. In the present study, COX-2 protein induction coincided with the secretion of PGE2 into the culture medium. Previous studies have demonstrated that COX-2 expression can be regulated by transcriptional as well as posttranscriptional mechanism (26 –27). Our results obtained by RT-PCT assay and transient transfection assay using report constructs, in which the report is under the control of various lengths of the COX-2 regulatory region, support the conclusion that EGCG regulates COX-2 gene expression at least in part at the transcriptional level. The region from ⫺840 bp to ⫹123 bp includes the binding sites for many transcriptional factors such as NFB, C/EBP, CRE/ATF, E-box, STAT3, AP2 and NF-IL6 (14 – 16). Recently, it was reported that COX-2 expression can be regulated through different MAP kinase signaling pathways and that the particular signaling pathway involved is dependent on the type of stimuli (17–19). The induction of COX-2 by PDGF has been demonstrated to require activation of the ERK signaling pathway (17), while constitutively active MEKK1 has been shown to induce COX-2 expression by activating the SEK1/MKK4p38 kinase pathway (18). Our data suggested that EGCG induces PGE2 production via activation of PKC and partially by activation of ERK. The suppression of EGCGinduced COX-2 expression by MEK inhibitor PD 98059 is in agreement with the concept that ERK signaling pathway is important in the regulation of COX-2 by EGCG. It has been reported that ERK can phosphorylate and activate CRE/ATF, E-box and NF-IL6 (28 –30). These finding suggest that EGCG treatment may activate these transcriptional factors through ERK signaling pathway, leading to COX-2 expression and subsequent PGE2 production. In addition, a role for protein-tyrosine kinases (PTKs) in control of COX-2 expression was also reported for interleukin-1␣ treated human endothelial cells (31). Synergistic enhancement of expression of the COX-2 expression was observed on stimulation of Raw264.7 with vanadate plus EGCG. In conclusion, the present study is first report showing that COX-2 expression and PGE 2 production are regulated by EGCG. We provide evidence showing that activation of ERK MAP kinase and protein-tyrosine kinase signaling pathways an important role in the control of COX-2 expression. The EGCG may regulate inflammatory processes by modulating PGE 2 as well as other inflammatory mediators. ACKNOWLEDGMENT This work was supported by a Korea Research Foundation Grant (KRF-2000-041-F00103).
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