Phospholipase D Isozymes Mediate Epigallocatechin Gallate-induced Cyclooxygenase-2 Expression in Astrocyte Cells

Little is known about the effect of epigallocatechin-3 gallate (EGCG), a major constituent of green tea, on the expression of cyclooxygenase (COX)-2. Here, we studied the role of phospholipase D (PLD) isozymes in EGCG-induced COX-2 expression. Stimulation of human astrocytoma cells (U87) with EGCG induced formation of phosphatidylbutanol, a specific product of PLD activity, and synthesis of COX-2 protein and its product, prostaglandin E2 (PGE2). Pretreatment of cells with 1-butanol, but not 3-butanol, suppressed EGCG-induced COX-2 expression and PGE synthesis. Furthermore, evidence that PLD was involved in EGCG-induced COX-2 expression was provided by the observations that COX-2 expression was stimulated by overexpression of PLD1 or PLD2 isozymes and treatment with phosphatidic acid (PA), and that prevention of PA dephosphorylation by 1-propranolol significantly potentiated COX-2 expression induced by EGCG. EGCG induced activation of p38 mitogen-activated protein kinase (p38 MAPK), and specific inhibition of p38 MAPK dramatically abolished EGCG-induced PLD activation, COX-2 expression, and PGE2 formation. Moreover, protein kinase C (PKC) inhibition suppressed EGCG-induced p38 MAPK activation, COX-2 expression, and PGE2 accumulation. The same pathways as those obtained 2in the astrocytoma cells were active in primary rat astrocytes, suggesting the relevance of the findings. Collectively, our results demonstrate for the first time that PLD isozymes mediate EGCG-induced COX-2 expression through PKC and p38 in immortalized astroglial line and normal astrocyte cells. Little is known about the effect of epigallocatechin-3 gallate (EGCG), a major constituent of green tea, on the expression of cyclooxygenase (COX)-2. Here, we studied the role of phospholipase D (PLD) isozymes in EGCG-induced COX-2 expression. Stimulation of human astrocytoma cells (U87) with EGCG induced formation of phosphatidylbutanol, a specific product of PLD activity, and synthesis of COX-2 protein and its product, prostaglandin E2 (PGE2). Pretreatment of cells with 1-butanol, but not 3-butanol, suppressed EGCG-induced COX-2 expression and PGE synthesis. Furthermore, evidence that PLD was involved in EGCG-induced COX-2 expression was provided by the observations that COX-2 expression was stimulated by overexpression of PLD1 or PLD2 isozymes and treatment with phosphatidic acid (PA), and that prevention of PA dephosphorylation by 1-propranolol significantly potentiated COX-2 expression induced by EGCG. EGCG induced activation of p38 mitogen-activated protein kinase (p38 MAPK), and specific inhibition of p38 MAPK dramatically abolished EGCG-induced PLD activation, COX-2 expression, and PGE2 formation. Moreover, protein kinase C (PKC) inhibition suppressed EGCG-induced p38 MAPK activation, COX-2 expression, and PGE2 accumulation. The same pathways as those obtained 2in the astrocytoma cells were active in primary rat astrocytes, suggesting the relevance of the findings. Collectively, our results demonstrate for the first time that PLD isozymes mediate EGCG-induced COX-2 expression through PKC and p38 in immortalized astroglial line and normal astrocyte cells. Cyclooxygenase (COX) 1The abbreviations used are: COX, cyclooxygenase; PLD, phospholipase D; PGE2, prostaglandin E2; PKC, protein kinase C; DMEM, Dulbecco's modified Eagle's medium; EGCG, epigallocatechin-3 gallate; MAPK, mitogen-activated protein kinase. is the key enzyme in the metabolic pathway leading to prostaglandin (PG) and thromboxane A2 formation from arachidonic acid (1DuBois R.N. Abramson S.B. Crofford L. Gupta R.A. Simon L.S. Van De Putte L.B. Lipsky P.E. FASEB J. 1998; 12: 1063-1073Crossref PubMed Scopus (2231) Google Scholar). Two isoforms have been identified, COX-1 and COX-2 (2Smith W.L. Garavito M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1858) Google Scholar). COX-1 is constitutively expressed in nearly all normal mammalian tissues and mediates the synthesis of PGs required for physiological tissue homeostasis. In contrast, COX-2 expression is rapidly induced in response to various stimuli, including inflammatory signals, mitogens, cytokines, and growth factors in a wide variety of cells such as macrophages, microglia, and astrocytes (3Bauer M.K. Lieb K. Schulze-Osthoff K. Berger M. Gebicke-Haerter P.J. Bauer J. Fiebich B.L. Eur. J. Biochem. 1997; 243: 726-731Crossref PubMed Scopus (232) Google Scholar, 4O'Banion M.K. Miller J.C. Chang J.W. Kaplan M.D. Coleman P.D. J. Neurochem. 1996; 66: 2352-2540Google Scholar, 5van Ryn J. Pairet M. Inflamm. Res. 1999; 48: 247-254Crossref PubMed Scopus (39) Google Scholar). COX-2 has also been in normal brain, in discrete populations of neurons (6Yamagata K. Andreasson K.I. Kaufmann W.E. Barnes C.A. Worley P.F. Neuron. 1993; 11: 371-386Abstract Full Text PDF PubMed Scopus (1093) Google Scholar, 7Chen J. Marsh T. Zhang J.S. Graham S.H. Neuroreport. 1995; 6: 245-248Crossref PubMed Scopus (110) Google Scholar). The function of basal prostanoid production in brain is unclear. As COX-2 expression appears to be up-regulated by physiological synaptic activity, it has been suggested that normal COX-2 expression in neurons may be related to regulation of the sleep/wake cycle, hormone release, and neuronal signaling (6Yamagata K. Andreasson K.I. Kaufmann W.E. Barnes C.A. Worley P.F. Neuron. 1993; 11: 371-386Abstract Full Text PDF PubMed Scopus (1093) Google Scholar, 8Hayaishi O. FASEB J. 1991; 5: 2575-2581Crossref PubMed Scopus (237) Google Scholar, 9Pairet M. Engelhardt G. Fundam. Clin. Pharmacol. 1996; 10: 1-17Crossref PubMed Scopus (194) Google Scholar). The expression of COX-1 and COX-2 has been reported to be associated with complex changes observed during a variety of diseases of the brain. Prostanoids are considered important mediators for various brain functions and have recently been implicated in the pathogenesis of cerebral ischemia, seizures, and other neural injuries (6Yamagata K. Andreasson K.I. Kaufmann W.E. Barnes C.A. Worley P.F. Neuron. 1993; 11: 371-386Abstract Full Text PDF PubMed Scopus (1093) Google Scholar, 10Kijubu D.A. Fletcher B.S. Varnum B.C. Lim R.W. Herschman H.R. J. Biol. Chem. 1991; 266: 12866-12872Abstract Full Text PDF PubMed Google Scholar, 11DuBois R.N. Awad J. Morrow J. Roberts L.J. Bishop P.R. J. Clin. Investig. 1994; 93: 493-498Crossref PubMed Scopus (379) Google Scholar, 12Nogawa S. Zhang F. Ross M.E. Iadecola C. J. Neurosci. 1997; 17: 2746-2755Crossref PubMed Google Scholar, 13Ellis E.F. Chao J. Heizer M.L. J. Neurosurg. 1989; 71: 437-442Crossref PubMed Scopus (56) Google Scholar). The regulation and function of COX-2 and prostaglandin synthesis in the central nervous system are not completely understood. Polyphenolic compounds in green tea have recently received increasing attention as preventive agents against hippocampal neuronal damage following transient global ischemia, cardiovascular disease and cancer (14Lee S.R Suh S.I Kim S.P. Lett. 2000; 287: 191-194Google Scholar, 15Dufresne C.J. Farnworth E.R. J. Nutr. Biochem. 2001; 12: 404-421Crossref PubMed Scopus (423) Google Scholar, 16Yang C.S. Chung J.Y. Yang G. Chhabra S.K. Lee M.J. J. Nutr. 2000; 130: 472S-478SCrossref PubMed Google Scholar, 17Stoner G.D. Mukhtar H. J. Cell. Biochem. 1995; 22: 169-180Crossref Scopus (586) Google Scholar). Green tea polyphenols, which comprise 30% of the dry weight of green tea leaves, include epigallocatechin-3-gallate (EGCG), epigallocatechin, epicatechin-3-gallate, and epicatechin. EGCG is the most abundant of these catechins and it has been attributed many healthful benefits. However, a recent study demonstrates that EGCG up-regulates COX-2 expression and prostaglandin E2 (PGE2) production in Raw 264.7 macrophage cells (18Park J.-W. Choi Y.J. Suh S.-I. Kwon T.K. Biochem. Biophys. Res. Commun. 2001; 286: 721-725Crossref PubMed Scopus (63) Google Scholar), suggesting that EGCG may enhance inflammatory processes. However, the downstream effectors linking EGCG stimulation with COX-2 expression and PG production remains unidentified, although the effects of EGCG on COX-2 expression still remain uncertain. Recently, several studies have implicated a PLD-derived signaling pathway in the generation of prostaglandin in many cell types (19Steed P.M. Chow A.H.M. Curr. Pharm. Biotech. 2001; 2: 241-256Crossref PubMed Scopus (46) Google Scholar). PLD catalyzes the hydrolysis of phosphatidylcholine to generate a lipid mediator, phosphatidic acid (PA) (20Frohman M.A. Morris A.J. Chem. Phys. Lipids. 1999; 98: 127-140Crossref PubMed Scopus (100) Google Scholar). PA mediates the biological and physiological functions of PLD either directly or indirectly by its metabolism to lysophosphatidic acid (LPA) or diacylglycerol (DAG). In mammals, two isoforms of PLD, PLD1 and PLD2, have been cloned and are being characterized for regulation and cellular function (20Frohman M.A. Morris A.J. Chem. Phys. Lipids. 1999; 98: 127-140Crossref PubMed Scopus (100) Google Scholar). However, segregated roles of the two PLD isoforms in cellular responses are still poorly understood. Activation of PLD occurs through interactions of the ARF and Rho families as well as with protein kinase C (PKC) (20Frohman M.A. Morris A.J. Chem. Phys. Lipids. 1999; 98: 127-140Crossref PubMed Scopus (100) Google Scholar). The relative contribution of these factors to PLD activation is highly dependent on the cell type and signaling model examined. Several lines of evidence have suggested a functional role for PLD in COX-2 regulation during cell activation (21Sciorra V.A. Daniel L.W. J. Biol. Chem. 1996; 271: 14226-14232Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 22Angel J. Audubert F. Bismuth G. Fournier C. J. Immunol. 1994; 152: 5032-5040PubMed Google Scholar). However, the role of PLD isozymes in EGCG-induced COX-2 expression has not been studied in any biological system. We therefore investigated a role of PLD in the regulation of COX-2 expression in glial cells treated with EGCG. We selected the normal astrocyte cell and immortalized astroglial cell line (U87) for several reasons. 1) Astrocytes cells are the major cell population in the central nervous system (23O'Banion M.K. Miller J.C. Chang J.W. Kaplan M.D. Coleman P.D. J. Neurochem. 1996; 66: 2532-2540Crossref PubMed Scopus (196) Google Scholar), and reports using astrocytes cells are lacking. 2Smith W.L. Garavito M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1858) Google Scholar) PG and lipid metabolites formed by astrocytes may contribute to central nervous system physiology and pathology (24Pistritto G. Mancuso C. Tringali G. Perretti M. Preziosi P. Navarra P. Neurosci. Lett. 1998; 246: 45-48Crossref PubMed Scopus (31) Google Scholar, 25Amruthesh S.C. Boerschel M.F. McKinney J.S. Willoughby K.A. Ellis E.F. J. Neurochem. 1993; 61: 150-159Crossref PubMed Scopus (109) Google Scholar). In this study, we demonstrate that EGCG activates PLD through an upstream protein kinase C to elicit p38 activation and finally induce COX-2 expression in normal rat astrocyte cells and glioma cells. This is the first study to show the involvement of PLD isozymes in mediating EGCG-induced COX-2 expression in any biological system. Materials—Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, and LipofectAMINE were purchased from Invitrogen. Phospho-ERK1/2, ERK, phospho-p38, and p38 antibodies were from Cell Signaling. Rabbit polyclonal COX-2 antibody and goat anti-actin antibody were from Santa Cruz Biotechnology. A polyclonal antibody that recognizes both PLD1 and PLD2 was generated as previously described (26Min D.S. Ahn B.-H. Rhie D.-J. –H. S. Yoon S.J. Hahn M.-S. Kim Jo Y.H. Neuropharmacology. 2001; 41: 384-391Crossref PubMed Scopus (50) Google Scholar). Phosphatidylbutanol (PtdBut) standard was from Avanti Polar Lipid. 1-propranolol, 1-butanol, dioctanoyl PA, and anti-β-tubulin antibody were from Sigma, and PD98059, SB203580, and Gö6976 were from Biomol (Plymouth Meeting, PA). [9, 10-13H]Myristate was purchased from PerkinElmer Life Sciences. Silica gel 60 A thin layer chromatography plates were from Whatman. Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG were from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Enhanced chemiluminescence (ECL) reagents and the PGE2 enzyme immunoassay kit were from Amersham Biosciences. Cell Culture and Transfection—U87 MG human astroglioma was obtained from the American Type Culture Collection. Cells were maintained in DMEM supplemented with 10% (v/v) fetal bovine serum under 5% CO2. U87 cells were transiently transfected for 40 h with expression plasmid encoding empty vector or a catalytically inactive mutant of p38 MAPK (T180A, Y182F), using LipofectAMINE according to the manufacturer's instructions. U87 cells stably overexpressing PLD isozyme were obtained by transfection, using LipofectAMINE. Transfected cells were selected with G418 (700 μg/ml) for 21 days at 37 °C. At that time antibiotic-resistant colonies were pooled and expanded for further analysis under selective conditions. Preparation of Primary Astrocytes and Microglia—Primary astrocytes were cultured from 1–3-day-old Sprague-Dawley rats as previously described (27Giulian D. Baker T.J. J. Neurosci. 1986; 6: 2163-2178Crossref PubMed Google Scholar, 28Pyo H.K. Ilo J. Chung S.Y. Hong S.M. Joe E.H. NeuroReport. 1998; 9: 871Crossref PubMed Scopus (198) Google Scholar). The cortices were triturated into single cells in minimal essential medium (Invitrogen) containing 10% fetal bovine serum (Hyclone, Logan, Utah), and then plated into 75 cm2 T-flasks (0.5 hemisphere/flask) for 10–14 days. To prepare pure astrocytes, microglia were removed from the T-flasks by mild shaking. The cells remaining in the flasks after removal of the microglia were harvested with 0.1% trypsin and plated into dishes or plates. One hour later, the cells were washed to remove unattached cells before being used in experiments. In Vivo PLD Activity—In vivo PLD activity was determined as described previously (29Ahn B.H. Kim S.Y. Kim E.H. Choi K.S. Kwon T.K. Lee Y.H. Chang J.S. Kim M.S. Jo Y.H. Min D.S. Mol. Cell. Biol. 2003; 23: 3103-3115Crossref PubMed Scopus (87) Google Scholar). PLD activity was assessed by measuring the formation of [3H]phosphatidylbutanol (PtdBut), the product of PLD-mediated transphosphatidylation, in the presence of 1-butanol. Cells in 6-well plates were serum-starved in the presence of 2 μCi/ml [3H]myristic acid. After overnight starvation, the cells were washed three times with 5 ml of phosphate-buffered saline and pre-equilibrated in serumfree DMEM for 1 h. For the final 10 min of preincubation, 0.3% butan-1-ol was included. At the end of the preincubation, cells were treated with agonists for the indicated times. The extraction and characterization of lipids by thin-layer chromatography were performed as previously described (29Ahn B.H. Kim S.Y. Kim E.H. Choi K.S. Kwon T.K. Lee Y.H. Chang J.S. Kim M.S. Jo Y.H. Min D.S. Mol. Cell. Biol. 2003; 23: 3103-3115Crossref PubMed Scopus (87) Google Scholar). Radioactivity incorporated into total phospholipids was measured, and the results were presented as percentage of total lipid cpm incorporated into phosphatidylbutanol to normalize the results. Western Blot—Cells were washed twice with ice-cold phosphate-buffered saline and then lysed in the extraction buffer (20 mm Hepes, pH 7.2, 1% Triton X-100, 1% sodium deoxycholate, 0.2% SDS, 200 mm NaCl, 1 mm Na3VO4, 1 mm NaF, 10% glycerol, 10 μg/ml leupeptin, 10μg/ml aprotinin, 1 mm phenymethylsulfonyl fluoride). The resulting cell lysates were spun at 15,000 × g in an Eppendorf microcentrifuge for 10 min at 4 °C to pellet the unbroken cells. Protein concentrations were determined using Bradford method with bovine serum albumin as a standard (4O'Banion M.K. Miller J.C. Chang J.W. Kaplan M.D. Coleman P.D. J. Neurochem. 1996; 66: 2352-2540Google Scholar). Protein samples were analyzed by SDS-polyacrylamide gel electrophoresis on 8% gels and were transferred to a nitrocellulose membrane. The blots were then blocked with 5% nonfat milk in Tris-buffered saline-Tween 20 (25 mm Tris-HCl, 150 mm NaCl, and 0.05% Tween 20) and incubated with appropriate primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibody. Immunoreactive bands were detected using enhanced chemiluminescence, resuspended in sample buffer. PGE2 Production Assay—PGE2 levels were determined using an enzyme immunoassay kit according to the manufacturer's instruction. Briefly, 50 μl of standard or sample was pipetted into the wells of a 96-well plate. Aliquots of mouse polyclonal PGE2 antibody and PGE2 conjugated to alkaline phosphatase were then added to each well, and the plate was incubated at room temperature for 1 h. After incubation, the wells were washed six times with 200 μl of PBS containing 0.05% Tween 20, and the TMB substrate was added. Wells were read at 670 nm with an enzyme-linked immunosorbent assay reader 30 min after adding substrate. Reverse Transcription-Polymerase Chain Reaction—Total RNA was isolated using RNAzol B (TEL-TEST, Inc. Friendwood, TX), and cDNA was prepared using reverse transcriptase that originated from Avian Myeloblastosis Virus (Takara, Japan) according to the manufacturer's instructions. PCR was performed with 30 cycles of sequential reactions as follows: 94 °C for 30 s, and 55 °C for 30 s, and 72 °C for 30 s. Oligonucleotide primers were purchased from Bioneer (Seoul, Korea). The sequence of PCR primers are as follows: The PCR primers for the COX-2 gene were 5′-ACACTCTATCACTGGCATCC-3′ (sense primer) and 5′-GAAGGGACACCCTTTCACAT (antisense primer). 5′-AGATCCACAACGGATACATT-3′ and (forward) 5′-TCCCTCAAGATTGTCAGCAA-3′ for glyceraldehyde-3-phosphate dehydrogenase. PCR products were separated by electrophoresis in a 1.5% agarose gel and detected under UV light. EGCG Induces COX-2 Expression and PGE2 Production—We investigated whether EGCG, a major compound of green tea, is involved in the signal transduction pathways leading to COX-2 expression and PGE2 production in U87 MG human astroglioma cells. Treatment of the cells with EGCG resulted in significantly increased levels of COX-2 expression in a dose- and time-dependent manner (Fig. 1A). The increase in COX-2 expression was apparent 12 h after 50 μm EGCG treatment, reaching a maximal level at 100 μm EGCG. Furthermore, the induction of COX-2 appeared in a time-dependent manner, and 100 μm EGCG treatment showed an increase in COX-2 protein within 6 h, which peaked at 18 h and then sustained up to 24 h after treatment. Because COX-2 catalyzes biosynthesis of PGs, we examined whether this enzyme was responsible for EGCG-induced PGE2 production in the culture media of cells stimulated with EGCG. As shown in Fig. 1B, COX-2 protein expression induced by EGCG was accompanied by an increase in PGE2 accumulation in a dose-dependent manner. The results indicate that EGCG can lead to COX-2 protein expression and subsequently PGE2 biosynthesis in U87 MG human astroglioma cells. PLD Mediates EGCG-induced COX-2 Expression—We next investigated how EGCG-induced COX-2 protein expression is regulated during glioma cell activation. There is also evidence that a PLD-derived signaling pathway is involved in the generation of PGs in several cell types (21Sciorra V.A. Daniel L.W. J. Biol. Chem. 1996; 271: 14226-14232Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 22Angel J. Audubert F. Bismuth G. Fournier C. J. Immunol. 1994; 152: 5032-5040PubMed Google Scholar). Therefore, to address the possible involvement of PLD activation in EGCG-induced COX-2 expression and PGE2 biosynthesis, cells prelabeled with [3H]myristate were stimulated with 100 μm EGCG for various times, and PLD activity was measured by the formation of [3H]PtdBut from 1-butanol, a product specific to PLD activity. As shown in Fig. 2, EGCG induced PtdBut formation, in a time-dependent manner. Although the experiments showed that PLD was activated in EGCG-treated human glioma cells, they provided no direct evidence that PLD was involved in the induction of COX-2 expression. A role for PLD in the pathway leading to COX-2 expression received further support when 1-butanol was used to block PA production by PLD, by virtue of the formation of phosphatidylbutanol through the transphosphatidylation reaction. U87 MG cells were stimulated with EGCG in the presence of 1% 1-butanol or 3-butanol. As shown in Fig. 3A, 1-butanol, but not 3-butanol, inhibited EGCG-induced COX-2 expression and PGE2 biosynthesis. To confirm that the effect of 1-butanol on COX-2 expression and PGE2 production is caused by the inhibition of PA formation by PLD, we examined COX-2 expression by EGCG in cells pretreated with propranolol, well established PA phosphohydrolase (PAP) inhibitor. Fig. 3B shows that propranolol potentiated EGCG-induced COX-2 expression in a dose-dependent manner. To further establish that PA was involved in the induction of COX-2 expression, cells were treated with various concentrations of dioctanoyl PA. Fig. 4 shows that PA significantly induced COX-2 expression in a dose- and time-dependent manner. These results demonstrate that PLD activity and the intracellular accumulation of PA are importantly involved in EGCG-induced COX-2 expression in U87 MG human astroglioma cells.Fig. 3Effect of 1-butanol and 1-propranolol on EGCG-induced COX-2 expression and PGE2 production. A, U87 cells were stimulated with 100 μm EGCG in the presence of 1% 1-butanol or 3-butanol for 6 h. B, cells were treated with EGCG in the presence of indicated concentrations of 1-propranolol for 6 h. The release of PGE2 was measured from supernatants, and the extracted proteins were immunodetected with anti-COX-2 or anti-β-tubulin antibody. The values for PGE2 production are shown as the mean ± S.E., and the COX-2 protein levels are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Phosphatidic acid induces COX-2 expression. A, cells were stimulated for 6 h with the indicated concentrations of PA. B, U87 cells were treated with or without PA (100 μm) for the indicated times. The data shown are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PKC Is Involved in the COX-2 Protein Expression and PGE2 Formation Induced by EGCG—It has been suggested that PKC is an important regulator of PLD. Moreover, PKC is known to regulate COX-2 expression and PGE2 production by various agonists (19Steed P.M. Chow A.H.M. Curr. Pharm. Biotech. 2001; 2: 241-256Crossref PubMed Scopus (46) Google Scholar, 30Molina-Holgado E. Ortiz S. Molina-Holgado F. Guaza C. Br. J. Pharmacol. 2000; 131: 152-159Crossref PubMed Scopus (175) Google Scholar). Using Western blot analysis and confocal immunofluorescence microscopy, we have found that EGCG induces translocation of PKC-α from the cytosol to the membrane (data not shown), which is well documented as a measure of PKC activation. Therefore, we investigated whether EGCG-induced PLD activation and COX-2 expression is regulated by PKC. As shown in Fig. 5, Ca2+-dependent PKC-specific inhibitor, Gö6976, significantly inhibited not only EGCG-induced PLD activation but also EGCG-induced COX-2 expression, and subsequently PGE2 biosynthesis, in a dose-dependent manner, suggesting that PKC might be involved in EGCG-induced PLD activation, COX-2 expression, and PGE2 formation. p38 Mediates EGCG-induced PLD Activation, COX-2 Expression, and PGE2 Synthesis—It has been reported that COX-2 expression can be regulated by p38 MAPK or p42/44 MAPK (31Fiebich B.L. Schleicher S. Spleiss O. Czygan M. Hull M. J. Neurochem. 2001; 79: 950-958Crossref PubMed Scopus (114) Google Scholar, 32Beyaert R. Cuenda A. Vanden B.W. Plaisance S. Lee J.C. Haegeman G. Cohen P. Fiers W. EMBO J. 1996; 15: 1914-1923Crossref PubMed Scopus (603) Google Scholar). Therefore, we evaluated the ability of p38 and p42/44 inhibition to block COX-2 expression and PGE2 production in astroglioma cells. The p38 MAPK-specific inhibitor, SB203580, but not the ERK upstream inhibitor PD98059, dramatically suppressed EGCG-induced COX-2 expression and PGE2 biosynthesis (Fig. 6, A and B), suggesting that the p38 kinase signaling pathway may be involved in the EGCG-induced COX-2 expression and PGE2 production. In addition, we found that SB203580, an inhibitor specific for p38, caused a dose-dependent decrease in EGCG-stimulated PLD activity (Fig. 7A). Moreover, expression of a catalytically inactive mutant of p38 MAPK (T180A/Y182F) significantly attenuated EGCG-induced PLD activation in U87 MG cells (Fig. 7B), suggesting that p38 is involved in EGCG-induced PLD activation.Fig. 7Role of p38 in EGCG-induced PLD activation. A, U87 cells were labeled with 2 μCi/ml [3H]myristic acid, treated with the indicated concentrations of SB203580 for 30 min and stimulated with 500 μm EGCG for 30 min. B, U87 cells were transiently transfected for 40 h with expression plasmid encoding empty vector or a catalytically inactive mutant of p38 (T180A, Y182F), using LipofectAMINE (Invitrogen) according to the manufacturer's instructions and labeled with [3H]myristic acid. Cells were left untreated or treated with EGCG (500 μm) for 30 min in the presence of 0.3% 1-butanol. The amount of [3H]PtdBut formed was quantified as described in “Experimental Procedures.” Results are depicted as means ± S.E. of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) EGCG Induces Activation of p38 MAPK via PKC—We next determined whether EGCG activates ERK and p38 MAPK in human astroglioma cells. As shown in Fig. 6C, the phosphorylation of ERK by EGCG peaked at 10 min and decreased thereafter. EGCG stimulation significantly induced activation of p38 MAPK. The phosphorylation of p38 MAPK induced by EGCG peaked at 30 min and then declined. Reprobing the immunoblot with anti-ERK or anti-p38 MAPK antibodies showed the equal loading of proteins in each lane. To examine whether p38-mediated COX-2 expression is exerted via PKC, we stimulated the cells with EGCG in the presence of PKC inhibitors and determined the change of phosphorylation of p38. As shown in Fig. 8, pretreatment with the PKC inhibitor Gö6976 suppressed EGCG-induced p38 phosphorylation, suggesting that EGCG induces activation of p38 via PKC. Both PLD1 and PLD2 Mediate EGCG-induced COX-2 Expression via PKC and p38 MAPK Pathway—To investigate which isozyme of PLD is involved in EGCG-induced COX-2 expression, we generated U87 MG cells stably overexpressing vector, PLD1, or PLD2 (Fig. 9A). In unstimulated cells, overexpression of PLD1 or PLD2 led to higher basal expression of COX-2 protein (Fig. 9B). In EGCG-stimulated cells, overexpression of PLD 1 or 2 dramatically increased COX-2 expression, compared with that of control cells (Fig. 9B), suggesting that overexpression of PLD1 or PLD2 significantly enhances EGCG-induced COX-2 expression. In addition, inhibitors of PKC and p38 MAPK abolished EGCG-induced COX-2 expression in cells overexpressing PLD1 and PLD2 (Fig. 9C). COX-2 expression in PLD1-expressing cells (U87-PLD1) was more sensitive to PKC inhibition than that in PLD2-expressing cells (U87-PLD2). COX-2 expression in both PLD1- and PLD2-expressing cells showed similar response to inhibitor of p38 MAPK. These results suggest that both PLD1 and PLD2 isozymes mediate EGCG-induced COX-2 expression via same pathway, PKC and p38. PLD Also Mediates EGCG-induced COX-2 Expression through PKC and p38 in Primary Astrocyte Cells—U87 cells are transformed glial cells. The relevance of the findings are questionable in that experiments were done in an immortalized astroglial line that may or may not mimic the in vivo situation. Therefore, we tried to ascertain whether treatment with EGCG stimulates COX-2 expression and the same pathways as those obtained in the glioma cells are active in primary astroglial cell cultures. Rat primary astrocytes and microglial cells were stimulated with EGCG or PA for the indicated times, and total RNA was extracted for reverse transcriptase-PCR analysis. As shown in Fig. 10, A and B, addition of EGCG rapidly increased the mRNA levels of COX-2 in both primary astrocytes and microglia. Moreover, treatment with PA significantly induced transcription of the COX-2 gene in these cells. Treatment of the primary rat astrocytes with EGCG significantly resulted in increased levels of COX-2 expression in a dose- and time-dependent manner (Fig. 10C). These findings demonstrate that EGCG induces the COX-2 in normal glial cells. We next investigated how EGCG-induced COX-2 protein expression is regulated during normal astroglial cell activation. Pretreatment of normal glial cells with 1% 1-butanol caused great inhibition of EGCG-induced COX-2 expression, but the same concentration of 3-butanol, an inactive analogue for PLD-mediated PA formation, had no significant effect (Fig. 11A). Pretreatment propranolol, an inhibitor of PA phosphatase, also stimulated EGCG-induced COX-2 expression (Fig. 11B). These data indicate that EGCG induces COX-2 expression by PLD-mediated pathway in primary astroglial cells. In addition, EGCG stimulated PLD activity and pre