Eur J Biochem 271, 3470–3480 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04242.x Phospholipase C, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, and redox state are involved in epigallocatechin gallate-induced phospholipase D activation in human astroglioma cells Shi Yeon Kim1, Bong-Hyun Ahn1, Joonmo Kim1, Yoe-Sik Bae2, Jong-Young Kwak2, Gyesik Min3, Taeg Kyu Kwon4, Jong-Soo Chang5, Young Han Lee6, Shin-Hee Yoon1 and Do Sik Min1 Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul, Korea; 2Medical Research Center for Cancer Molecular Therapy and Department of Biochemistry, College of Medicine, Dong-A University, Busan, Korea; 3Department of Microbiological Engineering, Jinju National University, Korea; 4Department of Immunology, School of Medicine, Keimyung University, Daegu, Korea; 5Department of Life Science, Daejin University, Kyeongggido, Korea; 6Division of Molecular and Life Science, College of Science and Technology, Hanyang University, Ansan, Korea We show that epigallocatechin-3 gallate (EGCG), a major component of green tea, stimulates phospholipase D (PLD) activity in U87 human astroglioma cells EGCG-induced PLD activation was abolished by the phospholipase C (PLC) inhibitor and a lipase inactive PLC-c1 mutant, which is dependent on intracellular or extracellular Ca2+, with the possible involvement of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) EGCG induced translocation of PLC-c1 from the cytosol to the membrane and PLC-c1 interaction with PLD1 EGCG regulates the activity of PLD by modulating the redox state of the cells, and antioxidants reverse this effect Moreover, EGCG-induced PLD Phospholipase D (PLD) catalyzes the hydrolysis of the most abundant membrane phospholipid, phosphatidylcholine, to generate phosphatidic acid and choline and is assumed to have an important function in cell regulation [1] Signaldependent activation of PLD has been demonstrated in numerous cell types stimulated by various hormones, growth factors, cytokines, neurotransmitters, adhesion molecules, drugs, and physical stimuli [2] Pathways leading to PLD activation include protein serine/threonine kinases, e.g protein kinase C (PKC), small GTPases, e.g ADPribosylation factor, RhoA and Ral, phosphatidylinositol 4,5-bisphosphate, and tyrosine kinases [2–4] To date, two Correspondence to D S Min, Department of Molecular Biology, College of Natural Science, Pusan National University, Geumjeong-gu, Busan 609-735, Korea Fax: +82 51 513 9258, Tel.: +82 51 510 1775 (from September 2004) Abbreviations: CaM kinase II, Ca2+/calmodulin-dependent protein kinase II; DCFH, 2¢,7¢-dichlorofluorescein diacetate; DCF, 2¢,7¢dichlorofluorescein; DMEM, Dulbecco’s modified Eagle’s medium; EC, epicatechin; ECG, epicatechin-3-gallate; EGC, epigallocatechin; EGCG, epigallocatechin-3-gallate; PKC, protein kinase C: PLC, phospholipase C; PLD, phospholipase D; PtdBut, phosphatidylbutanol; ROS, reactive oxygen species (Received 29 March 2004, revised 25 May 2004, accepted June 2004) activation was reduced by PKC inhibitors or down-regulation of PKC Taken together, these results show that, in human astroglioma cells, EGCG regulates PLD activity via a signaling pathway involving changes in the redox state that stimulates a PLC-c1 [Ins(1,4,5)P3-Ca2+]–CaM kinase II–PLD pathway and a PLC-c1 (diacylglycerol)–PKC–PLD pathway Keywords: Ca2+/calmodulin-dependent protein kinase II; epigallocatechin-3 gallate; phospholipase C-c1; phospholipase D; reactive oxygen species distinct isoforms of mammalian PLD have been cloned, PLD1 and PLD2 These isoforms share about 50% amino acid similarity, but exhibit quite different regulatory properties [5,6] Both proteins appear to be complexly regulated, usually in an agonist-specific and cell-specific manner, and the molecular mechanisms underlying their functions have not been fully elucidated Green tea (Camellia sinensis) is a popular beverage world wide, and its possible health benefits have received a great deal of attention Documented beneficial effects of green tea and its active components include cancer chemoprevention, inhibition of the growth, invasion and metastasis of tumor cells, as well as antiviral and anti-inflammatory activities [7] Green tea contains the characteristic polyphenolic compounds epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin-3-gallate (ECG) and epicatechin (EC) EGCG is considered to be the constituent primarily responsible for the green tea effects [8,9] Although the activity of EGCG in some biological events has been investigated, its effect on the signal transduction cascade is not yet fully defined Recently, it has been reported that EGCG produces reactive oxygen species (ROS) including H2O2 [10] Oxidant-induced PLD activation and redox regulation of PLD have been reported in a variety of cells such as Swiss 3T3 fibroblasts [11], PC12 cells [12,13], and endothelial cells [14] ROS such as H2O2 and superoxide have been shown to be generated in a variety of cells stimulated with cytokines, growth factors, and agonists of Ó FEBS 2004 Regulation of phospholipase D by EGCG (Eur J Biochem 271) 3471 G protein-linked receptors, and it has been suggested that they may act as second messengers [15] However, no information is available on how EGCG affects PLDmediated signaling pathways Therefore, we investigated PLD regulation by EGCG We show that EGCG significantly stimulates PLD activity and that EGCG-induced PLD activation is mediated via a signaling pathway involving redox-dependent changes in the cell, which stimulate the PLC-c1 [Ins(1,4,5)P3–Ca2+]–Ca2+/calmodulin-dependent protein kinase II (CaM kinase II)–PLD pathway and the PLC-c1 (diacylglycerol)–PKC–PLD pathway Experimental procedures PLD assay PLD activity was assessed by measuring the formation of [3H]PtdBut, the product of PLD-mediated transphosphatidylation, in the presence of butan-1-ol Cells were subcultured in six-well plates at · 105 cells per well and serum-starved in the presence of lCiỈmL)1 [3H]myristic acid After overnight starvation, the cells were washed three times with mL NaCl/Pi and pre-equilibrated in serum-free DMEM for h For the final 10 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 TLC were performed as described previously [16] Materials Subcellular fractionation Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum and LipofectAMINE were purchased from Invitrogen EGCG, EGC, ECG and EC were obtained from Sigma Protein A–Sepharose was from Amersham Biosciences Biotech Antibody to PLC-c1 was from Upstate Biotechnology PD98059, U-73122, U-73343, Ro-31-8220, and calphostin C were purchased from Biomol Research Laboratories (Plymouth Meeting, PA, USA) KN-92, KN-93, sphingosine 1-phosphate, and pertussis toxin were obtained from Calbiochem Other chemicals were purchased from Sigma Rabbit polyclonal antibody that recognizes both PLD1 and PLD2 was generated as described previously [16] Authentic phosphatidylbutanol (PtdBut) standard was from Avanti Polar Lipid myo-[2-3H]Inositol and [9,10-3H]myristate were purchased from Perkin-Elmer Life Sciences AG 1-X8 anion-exchange resin was bought from Bio-Rad Silica gel 60A TLC plates were from Whatman Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG were from Kirkegaard and Perry Laboratory (Gaithersburg, MD, USA) The ECL Western blotting detection kit was from Amersham Biosciences Biotech Serum-starved cells were treated with 500 lM EGCG for 10 min, and washed with NaCl/Pi and harvested by microcentrifugation The cells were then resuspended in lysis buffer (20 mM Hepes, pH 7.4, 10% glycerol, mM EDTA, mM EGTA, mM dithiothreitol, mM phenylmethanesulfonyl fluoride and 10 lgỈmL)1 leupeptin) and lysed by 20 passages through a 25-gauge needle Trypan blue staining of the lysate indicated > 95% disruption of the cells The lysates were then spun at 100 000 g for h at °C to separate the cytosolic and membrane fractions Membrane fractions were washed twice with the buffer to remove cytosolic proteins Cell culture and transfection U87 human astroglioma were maintained in DMEM supplemented with 10% (v/v) fetal bovine serum under 5% CO2 Cells were transiently transfected for 40 h with plasmids encoding empty vector, PLD1, PLD2, or a lipase inactive mutant PLC-c1 (H335Q) expression vectors using LipofectAMINE according to the manufacturer’s instructions Digital calcium imaging Intracellular calcium was measured as described previously [17] Cells were plated on to glass coverslips and loaded with lM fura-2 acetoxymetyl ester (Molecular Probes) for 45 at 37°C The coverglass was then mounted in a flowthrough chamber The chamber containing the fura-2labeled cells was mounted and alternately excited at 340 or 380 nm Digital fluorescence images were collected with a cooled CCD camera [Ca2+]i was calculated from the ratio of the two background-subtracted digital images Ratios were converted into free [Ca2+]i by the equation ẵCa2 ỵ i ẳ Kb R Rmin Þ=ðRmax À RÞ in which R is the 340/380-nm fluorescence emission ratio and K ¼ 224 nM, the dissociation constant for fura-2 [18] Immunoprecipitation Measurement of phosphoinositide hydrolysis by PLC The cells were labeled with myo-[2-3H]inositol (2 lCiỈmL)1) in inositol-free DMEM for 20 h Subsequently, the labeled cells were pretreated with 20 mM LiCl for 15 After stimulation with EGCG, the reaction was terminated by the addition of ice-cold 5% HClO4 The extracts were applied to a Bio-Rad Dowex AG 1-X8 anion-exchange column The column was then washed with 10 mL distilled water followed by 10 mL 60 mM ammonium formate containing mM sodium tetraborate Total inositol phosphates were eluted with a solution containing M ammonium formate and 0.1 M formic acid U87 cells were harvested and lysed with lysis buffer (20 mM Hepes, pH 7.2, 1% Triton X-100, 1% sodium deoxycholate, 0.2% SDS, 150 mM NaCl, mM Na3VO4, mM NaF, 10% glycerol, 10 lgỈmL)1 leupeptin, 10 lgỈmL)1 aprotinin, mM phenymethanesulfonyl fluoride) The cells were then centrifuged at 10 000 g for h, and the resulting supernatant was incubated with antibody to PLD or PLC-c1 and Protein A–Sepharose for h at °C with rocking Protein concentrations were determined using the Bio-Rad Protein Assay with BSA as standard The immune complexes were collected by centrifugation and washed five times with buffer (20 mM Tris/HCl, pH 7.5, mM EDTA, mM Ó FEBS 2004 3472 S Y Kim et al (Eur J Biochem 271) EGTA, 150 mM NaCl, mM Na3VO4, 10% glycerol and 1% Nonidet P40) and resuspended in sample buffer The final pellet was loaded on to a polyacrylamide gel for immunoblot analysis reaction was mediated by 30% NaOH which specifically transformed the substituted product (thioether) obtained with GSH into a chromophoric thione Results Immunoblot analysis Proteins were denatured by boiling for at 95 °C in Laemmli sample buffer [19], separated by SDS/PAGE, and transferred to nitrocellulose membranes After being blocked in Tris/Tween-buffered saline containing 5% skimmed milk powder, the membranes were incubated with individual monoclonal or polyclonal antibodies and then further incubated with anti-mouse or anti-rabbit IgG coupled to horseradish peroxidase Blots were detected using the enhanced chemiluminescence kit according to the manufacturer’s instructions Confocal immunofluorescence microscopy U87 cells grown on poly(L-lysine)-coated glass coverslips were serum-starved for 24 h After stimulation with EGCG, the cells were fixed in 3.7% (w/v) formaldehyde for 15 and quenched using 50 mM NH4Cl for 10 After permeabilization using 1% Triton X-100 for min, the cells were incubated with blocking buffer (1% goat serum in NaCl/Pi) at room temperature for h, and then with primary antibody overnight at °C, and then with subclass-specific secondary antibodies [fluorescein isothiocyanate-conjugated donkey anti-(mouse IgG) (Jackson ImmunoResearch, West Grove, PA, USA) or Texas Red-conjugated goat anti-(rabbit IgG) (Jackson ImmunoResearch)] for h After being washed, the coverslips were mounted on to slides in Prolong (Molecular Probes) Images in the Figures were acquired using a Zeiss MRC 1024 microscope (Bio-Rad) Detection of intracellular ROS generation Intracellular ROS production was monitored using 2¢,7¢dichlorofluorescein diacetate (DCFH) (Sigma-Aldrich), which is oxidized to the fluorescent product 2¢7¢-dichlorofluorescein (DCF) by ROS [20] Briefly, U87 cells grown on coverslips were loaded with ROS-sensitive dye (10 lM) After 15 at room temperature, the cells were washed three times with serum-free medium, and treated with vehicle alone or EGCG ROS produced were monitored using an excitation wavelength of 490 nm and emission fluorescence at 520 nm with a confocal Microscope (Zeiss) Determination of glutathione concentration Cells treated with EGCG were washed in NaCl/Pi and then scraped into 5% metaphosphoric acid Reduced glutathione (GSH) was quantified using a commercially available GSH determination kit (Calbiochem) Briefly, the method was based on a chemical reaction which proceeded in two steps The first step led to the formation of substitution products (thioethers) between 4-chloro-1-methyl-7-trifluromethylquinolinum methylsulfate and all mercaptans which were present in the sample The second step included a b-elimination reaction under alkaline conditions This EGCG stimulates PLD activity in U87 human astroglioma cells We investigated whether green tea polyphenols activate PLD in U87 human astroglioma cells Cells were treated for 30 with EGCG, ECG, EGC or EC The data presented in Fig 1A show that these polyphenolic compounds significantly stimulated PLD activity, with EGCG being the most potent activator EGCG-induced [3H]PtdBut formation increased in a time- and concentrationdependent manner (Fig 1B,C) Activation of PLD by EGCG continued up to 50 and then remained constant up to 100 min; maximum activation was observed at mM EGCG Using PLD antibodies, we detected PLD1, but not PLD2, in U87 cells However, transient transfection of cells with PLD1 and PLD2 expression vectors revealed that EGCG activates both PLD1 and PLD2 (Fig 2) Role of PLC in EGCG-induced PLD activation Numerous studies have implicated PLC in the activation of PLD [21,22]; however, the results of other studies have suggested that PLC is not involved [23,24] To determine whether PLC activity or G-protein-mediated signaling was involved in EGCG-induced PLD activation in U87 cells, we examined the effects of pertussis toxin and the phosphoinositide-specific PLC inhibitor, U-73122 Pretreatment with pertussis toxin (100 ngỈmL)1 for 24 h) inhibited sphingosine 1-phosphate-induced PLD activation, suggesting that this activation reaction is dependent on the Gi protein-mediated signaling response in these cells However, pertussis toxin had no effect on EGCG-induced PLD activation (Fig 3A) EGCG-induced PLD activation was significantly attenuated by the PLC-specific inhibitor U73122, in a dose-dependent manner, but not by its inactive analog U-73343 (Fig 3B) These data suggest that phosphoinositide-specific PLC activation via a pertussis toxininsensitive pathway plays a critical role in EGCG-induced PLD activity in these cells We also investigated whether EGCG induces PLC activity in U87 cells The data presented in Fig 3C show that EGCG treatment stimulates PLC activity, as measured by formation of [3H]inositol phosphates, which peaked after 10 and was sustained for at least 50 In a control experiment, the PLC inhibitor U73122 actually inhibited PLC activity in cells stimulated by EGCG (Fig 3C) We found that PLC-c1 was the predominantly expressed PLC in U87 cells, indicating that the PLC activity shown in these cells may be due mainly to PLC-c1 We found that ectopic expression of the lipase inactive mutant PLC-c1 (His335 fi Gln) [25] attenuated endogenous PLC activity by EGCG, suggesting surprising effectiveness of the catalytically inactive PLC-c1 mutant expression plasmid on the suppression of EGCG-stimulated PLC activity Therefore, we examined the involvement of PLC-c1 in the PLD activation by EGCG in U87 cells Ó FEBS 2004 Regulation of phospholipase D by EGCG (Eur J Biochem 271) 3473 Fig EGCG activates both PLD1 and PLD2 U87 cells were transiently transfected for 40 h with plasmids encoding empty vector, PLD1, or PLD2 expression vectors using LipofectAMINE according to the manufacturer’s instructions, labeled with [3H]myristic acid, and treated with EGCG (500 lM) for 30 PLD activity was measured as described in Experimental procedures Results are means ± SD from three independent experiments AM Figure shows simultaneous measurement of [Ca2+]i increases in different cells, using digital calcium imaging One trace represents [Ca2+]i increase in one cell, and the different traces represent each [Ca2+]i increase pattern in the different cells The rise in [Ca2+]i after EGCG stimulation peaked within and then decreased (Fig 4A) An EGCG-stimulated increase in [Ca2+]i may result from an influx of extracellular calcium To test this possibility, we treated cells with EGCG in the presence of Ca2+-free buffer The level of [Ca2+]i after EGCG treatment was visualized by loading the cells with Fura-2/AM For cells in Ca2+-free buffer, EGCG caused only a very small increase in [Ca2+]i (Fig 4B) These results clearly show that treatment of U87 cells with EGCG results in an increase in cytosolic calcium Furthermore, the results suggest that an influx of calcium from the extracellular medium is mainly responsible for this rise Fig Green tea polyphenols stimulate PLD activity in U87 human astroglioma cells Cells were cultured in six-well plates, labeled with [3H]myristate, and treated for 30 without or with 500 lM EC, ECG, EGC, or EGCG in the presence of 0.3% butanol (A) [3H]Myristate-labeled cells were treated with 500 lM EGCG for the indicated time (B) or with the indicated concentration of EGCG for 50 (C) The radioactivity incorporated into PtdBut was measured as described in Experimental procedures Results are means ± SD from three independent experiments Interestingly, expression of the lipase inactive mutant PLCc1 significantly attenuated EGCG-induced PLD activation (Fig 3D), suggesting that PLC-c1 is involved in this process EGCG induces a rise in [Ca2+]i in U87 cells As EGCG stimulates PLC activity, it might induce an increase in [Ca2+]i in U87 cells [Ca2+]i after EGCG treatment was visualized by loading the cells with Fura-2/ EGCG induces translocation of PLC-c1 and its interaction with PLD1 After growth factor stimulation, PLC-c1 is translocated from the cytosol to the membrane, where its substrate molecules reside [26] We examined whether EGCG induced PLC-c1 translocation Incubation with EGCG for 10 significantly increased the amount of PLC-c1 associated with the membrane fraction in U87 cells (Fig 5A) Using confocal immunofluorescence microscopy, we confirmed that PLC-c1 translocation to membrane regions increased after EGCG treatment Furthermore, colocalization of PLD1 and PLC-c1 increased in the membraneous region after EGCG stimulation (Fig 5B) We sought to confirm this apparent interaction between PLD1 and PLC-c1 in EGCG-stimulated U87 cells We found that PLD1 showed a mild interaction with PLC-c1 in unstimulated cells, and this association increased after treatment of EGCG for 10 (Fig 5C) These data suggest that PLD1 associates with PLC-c1 during EGCGinduced PLD activation 3474 S Y Kim et al (Eur J Biochem 271) Ó FEBS 2004 Fig PLC is involved in EGCG-induced PLD activation (A) Quiescent U87 cells were pretreated with 200 ngỈmL)1 pertussis toxin for 24 h, labeled with [3H]myristate, and stimulated with lM sphingosine 1-phosphate or 500 lM EGCG for 30 (B) [3H]Myristate-labeled cells were pretreated with the indicated concentrations of U-73122 or U-73343, and stimulated with EGCG for 30 (C) Cells transfected with or without a catalytically inactive mutant of PLC-c1 (H335Q) were labeled with lCiỈmL)1 myo-[2-3H]inositol, pretreated with or without U-73122 (20 lM), and stimulated with EGCG for the indicated time PLC activity was measured as described in Experimental procedures (D) U87 cells were transiently transfected with a catalytically inactive mutant of PLC-c1 (H335Q), labeled with [3H]myristic acid, and treated with EGCG for 30 *P < 0.05 compared with cells transfected with vector and treated with EGCG The radioactivity incorporated into PtdBut was measured as described in Experimental procedures Results are means ± SD from three independent experiments Pretreatment with antioxidants abolishes activation of PLC and PLD induced by EGCG It has been demonstrated that PLC-c1 is activated in response to oxidant exposure [27,28] In addition, oxidative stress stimulates PLD activity in a various cells [11–14] Therefore, we examined the effect of antioxidants on the PLC and PLD activation induced by EGCG Pretreatment with N-acetylcysteine, a glutathione precursor and scavenger of ROS, decreased EGCG-induced PLC activation in a dose-dependent manner (Fig 6A) Moreover, pretreatment with the antioxidants, catalase and N-acetylcysteine, abolished EGCG-induced PLD activation in a dose-dependent manner (Fig 6B,C) These results suggest that EGCG may increase ROS production and induce activation of PLC and PLD Furthermore, we found that incubation of the astrocytoma cells with H2O2 led to PLD activation (Fig 6D) These results demonstrate the role of ROS such as H2O2 in the EGCG effect on the activation of PLC and PLD EGCG has pro-oxidant activity in U87 astrocytoma cells It is possible that pro-oxidative activity of EGCG in astrocytoma cells could explain the activation of PLD U87 cells were incubated with DCFH to test whether EGCG increases ROS production ROS produced in cells causes oxidation of DCFH, yielding the fluorescent product DCF [20] The cells were treated in the presence or absence of EGCG, and DCF fluorescence was measured (Fig 7) EGCG significantly increased fluorescence This suggests that EGCG has pro-oxidant activity in astrocytoma cells The EGCG-mediated increase in DCF fluorescence was abolished by pretreating the cells with N-acetylcysteine, a glutathione precursor and scavenger of ROS (Fig 7) These results suggest that EGCG increases ROS production in U87 cells We next measured the glutathione (GSH) content in the cells treated with EGCG in the presence or absence of N-acetylcysteine to support the redox state of the cells EGCG treatment decreased the GSH concentration, and the decrease in GSH content by EGCG in cells pretreated with N-acetylcysteine was recovered, suggesting that treatment of cells with EGCG decreases GSH EGCG-induced PLD activation is dependent on intracellular or extracellular Ca2+ and mediated by CaM kinase II Several examples of the participation of Ca2+ in the regulation of PLD activity have been reported, although the effector molecules involved have not been fully characterized [29,30] We found that 1,2-bis-(2-aminophenoxy)ethane-N,N,N¢,N¢,-tetra-acetic acid acetoxymethyl ester (BAPTA/AM), an intracellular chelator of Ca2+, Ó FEBS 2004 Regulation of phospholipase D by EGCG (Eur J Biochem 271) 3475 specific CaM kinase II inhibitor, inhibited EGCG-induced PLD activation, but not KN-92, a negative control of KN-93 As a result for the specificity of KN-92, we found that, at 20 lM, KN-92 did not affect PKC activity (data not shown) These data suggest that EGCG-induced PLD Fig EGCG stimulates [Ca2+]i increases in U87 cells (A) Serumstarved cells were treated with EGCG (500 lM) for min, and [Ca2+]i was measured (B) After the removal of extracellular Ca2+ (0 Ca2+), the quiescent cells were treated with EGCG for min, and then [Ca2+]i was measured Measurements of [Ca2+]i were derived from fura-2-based digital images as described in Experimental procedures Data are representative of three experiments significantly reduced EGCG-induced PLD activity (Fig 8A), indicating a role for [Ca2+]i in this process We also measured EGCG-stimulated PtdBut accumulation in a mM EGTA/Ca2+-free buffer system We found that EGCG-stimulated PtdBut accumulation was completely abolished when cells were incubated in this Ca2+-free buffer (Fig 8B), suggesting that extracellular Ca2+ influx is also required for EGCG-induced PLD activation in U87 cells The possible mechanisms by which [Ca2+]i regulates EGCG-stimulated PLD activity were investigated We examined whether CaM kinase II mediates PLD activation in response to EGCG As shown in Fig 8C, KN-93, a Fig EGCG induces translocation of PLC-c1 and its interaction with PLD1 in U87 cells Serum-starved cells were treated with 500 lM EGCG for 10 (A) Lysates were separated into cytosol and membrane fractions which were immunoblotted using antibodies to PLC-c1 or PLD (B) U87 cells were cultured on coverslips and starved for 24 h, after which they were stimulated with EGCG for 10 Coverslips were fixed and stained with the indicated antibody and incubated with fluorescein isothiocyanate-conjugated or Texas Redconjugated IgG Immunoreactive cells were visualized by confocal microscopy Superimposed images display colocalization of PLD1labeled and PLC-c1-labeled cells The results shown are representative of three separate experiments (C) Serum-starved cells were stimulated with EGCG for 10 min, after which cell lysates were prepared and immunoprecipitated with antibodies to PLD or PLC-c1 and then immunoblotted using PLC-c1 or PLD antibodies, respectively Data are representative of three experiments Ó FEBS 2004 3476 S Y Kim et al (Eur J Biochem 271) Fig Effect of antioxidants on EGCG-induced PLC and PLD activation (A) Quiescent cells were labeled with lCiỈmL)1 myo-[2-3H]inositol, pretreated with the indicated concentrations of N-acetylcysteine (NAC) for 40 and stimulated with EGCG (500 lM) for 30 PLC activity was measured as described in Experimental procedures [3H]Myristate-labeled cells were pretreated with the indicated concentrations of N-acetylcysteine (B) or catalase (C) for 40 and stimulated with EGCG for 30 (D) [3H]Myristate-labeled cells were treated with 500 lM H2O2 for 30 in the presence of 0.3% butanol The radioactivity incorporated into phosphatidylbutanol was measured as described in Experimental Procedures Results are means ± SD from three independent experiments activation is dependent on Ca2+ and possibly involves the Ca2+-activated protein kinase, CaM kinase II (Fig 9D) These data suggest that EGCG activates PKC-a in U87 cells Involvement of PKC in EGCG-induced PLD activation Discussion Phosphoinositide-specific PLC activation by EGCG leads to the production of two second messengers, Ins(1,4,5)P3 and diacylglycerol, which induce the release of Ca2+ from intracellular stores and PKC activation, respectively To investigate the possible role of PKC in EGCG-stimulated PLD activity, we applied two approaches, namely the use of PKC inhibitors and depletion of enzyme by prolonged exposure of cells to 4b-phorbol 12-myristate 13-acetate Using immunoblotting with PKC isozyme-specific antibodies, we first investigated which PKC isozymes were expressed in U87 cells We found that PKC-a (a conventional PKC) and PKC-e (a novel PKC) were predominantly expressed, and that PKC-b, -d, and -f were present at low levels (data not shown) The potent and selective PKC inhibitors and down-regulation of PKC were shown to decrease EGCG-stimulated PLD activity (Fig 9A,B), suggesting that PKC is involved in EGCG-stimulated PLD activation in U87 cells Activation of PKC, which is a consequence of PLC activity, should in turn stimulate PLD Therefore, we examined whether EGCG induces PKC activation EGCG treatment stimulated PKC-a translocation to the plasma membrane, and it appears that all of the enzyme associates with the membrane on stimulation with EGCG (Fig 9C) This translocation event was also confirmed using confocal immunofluorescence microscopy Many studies have provided evidence of the highly complex regulation of PLD by extracellular ligands In this study, we show that EGCG, a natural substance isolated from green tea, stimulates PLD activity via a network of signaling molecules in U87 human astroglioma cells PLD plays an important role in controling many biological functions, including exocytosis, phagocytosis, and secretion PLD in mammalian cells can be activated by a range of extracellular signals [4] The mechanisms underlying PLD activation are highly dependent on the model system used, and are still under investigation in numerous laboratories The recent cloning of the two mammalian PLD isozymes has led to an explosion of research in the field, principally driven by the availability of molecular tools Despite a great deal of research on the biological properties of EGCG, until now nothing has been reported on its effects on PLD-mediated signal transduction In this study, we show that EGCG, a major component of green tea, significantly stimulated PLD activity, and induced inositol phosphate production and [Ca2+]i in astroglioma cells EGCG-induced PLD activation was suppressed by the phosphoinositide-specific PLC inhibitor PLC-c1 was the predominantly expressed PLC in U87 cells, indicating that the PLC activity demonstrated in these cells Ó FEBS 2004 Regulation of phospholipase D by EGCG (Eur J Biochem 271) 3477 Fig Effect of EGCG on ROS production and the cellular GSH content (A) DCFH-loaded U87 cells were stimulated with vehicle alone or EGCG (500 lM) for 20 U87 cells were preincubated with N-acetylcysteine (10 mM) for h before stimulation with EGCG for 20 ROS produced were measured as described in Experimental procedures (B) The cellular GSH contents were determined in U87 cells pretreated with or without 10 mM N-acetylcysteine for h, and then stimulated with EGCG (500 lM) for 30 Results are means ± SD from three independent experiments is due mainly to PLC-c1 This led us to assume that PLC-c1 may be involved in EGCG-induced PLD activation A transfection experiment using a lipase-inactive PLC-c1 mutant revealed significant attenuation of EGCG-induced PLD activation, suggesting that PLD lies downstream of PLC-c1 in the signaling pathway Expression of the inactive mutant of PLC-c1 also attenuated EGCG-induced PLC activation In resting cells, PLC-c1 is located predominantly in the cytosol, and translocates to the membrane fraction upon activation [25]; hence translocation is a widely accepted measure of PLC-c1 activation We observed that EGCG induced translocation of PLC-c1 from the cytosol to the membrane, where its substrate molecules reside Furthermore, EGCG induced the interaction of PLC-c1 with PLD1, as well as colocalization of these two molecules in membrane In this study, we report on two signaling phospholipase complexes composed of PLC-c1 and PLD1 Recently, it was reported that, on stimulation with epidermal growth factor, PLC-c1 interacts directly with PLD2 [31] Moreover, EGCG induced tyrosine phosphorylation of PLC-c1 which was inhibited by pretreatment of antioxidant (data not shown) The effects of EGCG on the activation of PLC and PLD are reversed by N-acetylcysteine and catalase, suggesting a role for ROS in this process Recently, it has been reported that EGCG displays two opposing activities: antioxidant and pro-oxidant [32] Some studies have implicated inhibition of growth and induction of apoptosis in human cancer cells by EGCG [7–9]; however, the results of other studies suggest that EGCG Fig EGCG-induced PLD activation is dependent on intracellular or extracellular Ca2+ and is mediated by CaM kinase II (A) U87 cells were labeled with [3H]myristate, preincubated with the indicated concentrations of BAPTA/AM, and then stimulated with EGCG (500 lM) for 30 (B) [3H]Myristate-labeled cells were preincubated with or without extracellular Ca2+-free buffer and then stimulated with EGCG for 30 (C) [3H]Myristate-labeled cells were pretreated with the indicated concentration of KN-92 or KN-93, and then stimulated with EGCG for 30 PLD activity was measured as described in Experimental procedures Results are means ± SD from three independent experiments 3478 S Y Kim et al (Eur J Biochem 271) Ó FEBS 2004 Fig Role of PKC in EGCG-induced PLD activation U87 cells were pretreated with various PKC inhibitors (5 lM) for 30 and stimulated with 500 lM EGCG for 30 R, Ro-31-8220; C, calphostin C (A) For down-regulation of PKC, cells were pretreated with 500 nM 4b-phorbol 12-myristate 13-acetate for 24 h, and then stimulated with 500 lM EGCG for 30 (B) Radioactivity incorporated into PtdBut was measured as described in Experimental procedures Results are means ± SD from three independent experiments Serum-starved U87 cells were treated with EGCG for 10 Lysates were fractionated into cytosolic and membrane fractions Each fraction was immunoblotted using antibodies specific for PKC-a The intensity of PKC-a immunoreactive bands quantified by densitometry of the immunoblot was expressed as relative intensity of the bands (C) U87 cells were cultured on coverslips, starved for 24 h, and then stimulated with EGCG for 10 Coverslips were fixed and stained with PKC-a antibody, and then incubated with Texas Red-conjugated IgG Immunoreactive cells were visualized by confocal microscopy (D) Data are representative of three experiments has protective effects against Ab-induced neurotoxicity in human SY5Y neuroblastoma cells [33], and prevent neuronal cell death via PKC activation and the modulation of the expression of several cell survial/cell cycle genes [34] Furthermore, it was suggested that the antioxidant activity might be a driving force to inhibit carcinogenesis or apoptosis [32], whereas the pro-oxidant activity might generate cytotoxicity However, at present, the mechanism by which EGCG converts the antioxidant activity from prooxidant activity and vice versa is unclear In U87 astrocytoma cells, EGCG is a pro-oxidant This is not completely unexpected because other compounds, such as ascorbate, can act as either an antioxidant or pro-oxidant, depending on the cellular environment [32] Curcumin, the phytochemical responsible for the color of tumeric, has antioxidant activity in many different cell types but displays pro-oxidant qualities in the presence of transition metals, such as copper, which exist in the kidney and liver at relatively high concentrations [35] The data presented here suggest that EGCG regulates PLD activity by modulating the redox state of the cell We also found that EGCG induced translocation and activation of PKC-a (a calciumdependent PKC), and PKC was involved in EGCG-induced PLD activation Furthermore, we found that treatment of the astrocytoma cells with H2O2 led to PLD activation In this respect, oxidant-induced PLD activation is comparable to PLD activation via ROS induced by EGCG, suggesting the specificity of the ROS cascade induced by EGCG EGCG increased [Ca2+]i in U87 cells, and chelation of [Ca2+]i by BAPTA/AM abolished EGCG-induced PLD activation It is therefore assumed that the increase in [Ca2+]i may be due to EGCG-induced PLC activation and subsequent Ins(1,4,5)P3 production Indeed, as CaM kinase II is activated via the PLC pathway in many cell types [36], and the inhibitor attenuated EGCG-induced increases in PLD activity, PLC probably regulates PLD through stimulation of this kinase Interestingly, the increase in PLD activity caused by EGCG is dependent on extracellular Ca2+, with removal of extracellular Ca2+ from the medium abolishing EGCG-induced PLD activation and the Ó FEBS 2004 Regulation of phospholipase D by EGCG (Eur J Biochem 271) 3479 increase in [Ca2+]i The EGCG-evoked increase in [Ca2+]i was inhibited by the nonspecific Ca2+ channel inhibitor lanthanum, and the PLC inhibitor U73122, but not by pretreatment with the L-type Ca2+ channel blocker, nifedipine (data not shown) These results suggest that, in U87 cells, EGCG-induced increases in [Ca2+]i result from mobilization of Ins(1,4,5)P3-sensitive [Ca2+]i stores EGCG has been shown, in an animal study, to pass the blood–brain barrier and reach brain parenchyma, and detection of EGCG in rat brain suggests polyphenols can modulate neuronal activity [37] We also observed that EGCG induced PLD activity in NG108-15 neuronal cells (data not shown) The observation that tea drinking affects mood suggests possible neuronal effects Recently, it was reported that green tea polyphenols modulate ionic currents and stimulus–secretion coupling in neuroendocrine cells [38] PLD is an important component of the exocytotic machinery in neuroendocrine cells and plays a major role in neurotransmission, most likely by controlling the number of functional release sites at nerve terminals [39,40] Therefore, it is possible that modulation of synaptic transmission via PLD signaling may explain part of the effect of tea drinking on mood change However, the role of PLD activated by EGCG in glial cells is not known at present In summary, we show that EGCG regulates PLD activity by modulating the redox state of the glial cells, the major cell population in the central nervous system, which stimulates the PLC-c1 [Ins(1,4,5)P3–Ca2+]–CaM kinase II–PLD and PLC-c1 (diacylglycerol)–PKC–PLD pathways This study identifies PLD as a new target for EGCG in human astroglioma cells Although the physiological role of PLD and overall signal-transduction pathways associated with EGCG-induced PLD activation in glial cells remain to be determined, these effects of EGCG provide insight into the mechanisms of action of polyphenols on PLD-mediated signaling pathways Acknowledgements We thank Dr Pann-Ghill Suh (POSTECH, Pohang, Korea) for providing cDNA encoding a lipase inactive mutant PLC-c1 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Y.H (2000) Phospholipase C, protein kinase C, Ca2 +/calmodulin-dependent protein kinase II, and tyrosine phosphorylation are involved in carbachol-induced phospholipase D activation in Chinese hamster... signaling pathway involving redox- dependent changes in the cell, which stimulate the PLC-c1 [Ins(1,4,5)P3? ?Ca2 +]? ?Ca2 +/calmodulin-dependent protein kinase II (CaM kinase II)–PLD pathway and the... EGCG-induced PLD activation is dependent on intracellular or extracellular Ca2 + and is mediated by CaM kinase II (A) U87 cells were labeled with [3H]myristate, preincubated with the indicated concentrations