Báo cáo khóa học: Phospholipase C, protein kinase C, Ca 2+ /calmodulin-dependent protein kinase II, and redox state are involved in epigallocatechin gallate-induced phospholipase D activation in human astroglioma cells ppt

Phospholipase C, protein kinase C, Ca2+/calmodulin-dependentprotein kinase II, and redox state are involved in epigallocatechin gallate-induced phospholipase D activation in human astrog

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 Yoon1and Do Sik Min1

1

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; 5 Department of Life Science, Daejin University, Kyeongggido, Korea; 6 Division 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

pro-tein 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

antioxi-dants reverse this effect Moreover, EGCG-induced PLD

activation was reduced by PKC inhibitors or down-regula-tion 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; phospho-lipase D; reactive oxygen species

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]

Signal-dependent 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

ADP-ribosylation factor, RhoA and Ral, phosphatidylinositol

4,5-bisphosphate, and tyrosine kinases [2–4] To date, two

distinct isoforms of mammalian PLD have been cloned, PLD1 and PLD2 These isoforms share about 50% amino acid similarity, but exhibit quite different regulatory prop-erties [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 com-pounds epigallocatechin-3-gallate (EGCG), epigallocate-chin (EGC), epicateepigallocate-chin-3-gallate (ECG) and epicateepigallocate-chin (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

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 1 September 2004).

Abbreviations: CaM kinase II, Ca2+/calmodulin-dependent protein

kinase II; DCFH, 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 3 June 2004)

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

PLD-mediated signaling pathways Therefore, we investigated

PLD regulation by EGCG

We show that EGCG significantly stimulates PLD

activity and that EGCG-induced PLD activation is

medi-ated 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

Materials

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine

serum and LipofectAMINE were purchased from

Invitro-gen EGCG, EGC, ECG and EC were obtained from Sigma

Protein A–Sepharose was from Amersham Biosciences

Biotech Antibody to PLC-c1 was from Upstate

Biotechno-logy 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

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

instruc-tions

Measurement of phosphoinositide hydrolysis by PLC

The cells were labeled with myo-[2-3H]inositol (2 lCiÆmL)1)

in inositol-free DMEM for 20 h Subsequently, the labeled

cells were pretreated with 20 mM LiCl for 15 min 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 mMammonium formate containing

5 mM sodium tetraborate Total inositol phosphates were

eluted with a solution containing 1Mammonium formate

and 0.1 formic acid

PLD assay PLD activity was assessed by measuring the formation of [3H]PtdBut, the product of PLD-mediated transphosphati-dylation, in the presence of butan-1-ol Cells were sub-cultured in six-well plates at 2· 105cells per well and serum-starved in the presence of 1 lCiÆmL)1[3H]myristic acid After overnight starvation, the cells were washed three times with 5 mL NaCl/Piand pre-equilibrated in serum-free DMEM for 1 h For the final 10 min of preincubation, 0.3% butan-1-ol was included At the end of the preincu-bation, cells were treated with agonists for the indicated times The extraction and characterization of lipids by TLC were performed as described previously [16]

Subcellular fractionation 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, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenyl-methanesulfonyl fluoride and 10 lgÆmL)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 1 h at

4C to separate the cytosolic and membrane fractions Membrane fractions were washed twice with the buffer to remove cytosolic proteins

Digital calcium imaging Intracellular calcium was measured as described previously [17] Cells were plated on to glass coverslips and loaded with

2 lM fura-2 acetoxymetyl ester (Molecular Probes) for

45 min at 37C The coverglass was then mounted in a flow-through chamber The chamber containing the fura-2-labeled cells was mounted and alternately excited at 340 or

380 nm Digital fluorescence images were collected with a cooled CCD camera [Ca2+]iwas calculated from the ratio

of the two background-subtracted digital images Ratios were converted into free [Ca2+]iby the equation

½Ca2þ
i ¼ KbðR  RminÞ=ðRmax  RÞ

in which R is the 340/380-nm fluorescence emission ratio and K¼ 224 nM, the dissociation constant for fura-2 [18] Immunoprecipitation

U87 cells were harvested and lysed with lysis buffer (20 mM Hepes, pH 7.2, 1% Triton X-100, 1% sodium deoxycho-late, 0.2% SDS, 150 mMNaCl, 1 mMNa3VO4, 1 mMNaF, 10% glycerol, 10 lgÆmL)1leupeptin, 10 lgÆmL)1aprotinin,

1 mMphenymethanesulfonyl fluoride) The cells were then centrifuged at 10 000 g for 1 h, and the resulting superna-tant was incubated with antibody to PLD or PLC-c1 and Protein A–Sepharose for 4 h at 4C 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, 1 mM EDTA, 1 mM

EGTA, 150 mMNaCl, 2 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

Immunoblot analysis

Proteins were denatured by boiling for 5 min at 95C 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 min

and quenched using 50 mM NH4Cl for 10 min After

permeabilization using 1% Triton X-100 for 5 min, the cells

were incubated with blocking buffer (1% goat serum in

NaCl/Pi) at room temperature for 1 h, and then with

primary antibody overnight at 4C, and then with

subclass-specific secondary antibodies [fluorescein

isothio-cyanate-conjugated donkey anti-(mouse IgG) (Jackson

ImmunoResearch, West Grove, PA, USA) or Texas

Red-conjugated goat anti-(rabbit IgG) (Jackson

ImmunoResearch)] for 1 h After being washed, the

cover-slips 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¢-dichloro-fluorescein (DCF) by ROS [20] Briefly, U87 cells grown on

coverslips were loaded with ROS-sensitive dye (10 lM)

After 15 min 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/Piand 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-trifluromethyl-quinolinum methylsulfate and all mercaptans which were

present in the sample The second step included a

b-elimination reaction under alkaline conditions This

reaction was mediated by 30% NaOH which specifically transformed the substituted product (thioether) obtained with GSH into a chromophoric thione

Results 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 min with EGCG, ECG, EGC or EC The data presented in Fig 1A show that these polyphenolic com-pounds significantly stimulated PLD activity, with EGCG being the most potent activator EGCG-induced [3 H]Ptd-But formation increased in a time- and concentration-dependent manner (Fig 1B,C) Activation of PLD by EGCG continued up to 50 min and then remained constant up to 100 min; maximum activation was observed at 1 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 phospho-inositide-specific PLC inhibitor, U-73122 Pretreatment with pertussis toxin (100 ngÆmL)1 for 24 h) inhibited sphingosine 1-phosphate-induced PLD activation, suggest-ing that this activation reaction is dependent on the

Giprotein-mediated signaling response in these cells How-ever, pertussis toxin had no effect on EGCG-induced PLD activation (Fig 3A) EGCG-induced PLD activation was significantly attenuated by the PLC-specific inhibitor

U-73122, in a dose-dependent manner, but not by its inactive analog U-73343 (Fig 3B) These data suggest that phos-phoinositide-specific PLC activation via a pertussis toxin-insensitive 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 min and was sustained for at least 50 min 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 (His335fi 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

Interestingly, expression of the lipase inactive mutant

PLC-c1 significantly attenuated EGCG-induced PLD activation

(Fig 3D), suggesting that PLC-c1 is involved in this

process

EGCG induces a rise in [Ca2+]iin 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/

AM Figure 4 shows simultaneous measurement of [Ca2+]i increases in different cells, using digital calcium imaging One trace represents [Ca2+]iincrease in one cell, and the different traces represent each [Ca2+]iincrease pattern in the different cells The rise in [Ca2+]iafter EGCG stimulation peaked within 3 min and then decreased (Fig 4A) An EGCG-stimulated increase in [Ca2+]imay 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

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 min 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 min (Fig 5C) These data suggest that PLD1 associates with PLC-c1 during EGCG-induced PLD activation

Fig 2 EGCG activates both PLD1 and PLD2 U87 cells were tran-siently transfected for 40 h with plasmids encoding empty vector, PLD1, or PLD2 expression vectors using LipofectAMINE according

to the manufacturer’s instructions, labeled with [ 3 H]myristic acid, and treated with EGCG (500 l M ) for 30 min PLD activity was measured

as described in Experimental procedures Results are means ± SD from three independent experiments.

Fig 1 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 min without or with 500 l M EC,

ECG, EGC, or EGCG in the presence of 0.3% butanol (A).

[ 3 H]Myristate-labeled cells were treated with 500 l M EGCG for the

indicated time (B) or with the indicated concentration of EGCG for

50 min (C) 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

scaven-ger of ROS, decreased EGCG-induced PLC activation in a

dose-dependent manner (Fig 6A) Moreover, pretreatment

with the antioxidants, catalase and N-acetylcysteine,

abol-ished 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 H2O2in 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 treat-ment 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 character-ized [29,30] We found that 1,2-bis-(2-aminophen-oxy)ethane-N,N,N¢,N¢,-tetra-acetic acid acetoxymethyl ester (BAPTA/AM), an intracellular chelator of Ca2+,

Fig 3 PLC is involved in EGCG-induced PLD activation (A) Quiescent U87 cells were pretreated with 200 ngÆmL)1pertussis toxin for 24 h, labeled with [ 3 H]myristate, and stimulated with 1 l M sphingosine 1-phosphate or 500 l M EGCG for 30 min (B) [ 3 H]Myristate-labeled cells were pretreated with the indicated concentrations of U-73122 or U-73343, and stimulated with EGCG for 30 min (C) Cells transfected with or without a catalytically inactive mutant of PLC-c1 (H335Q) were labeled with 1 lCiÆmL)1myo-[2-3H]inositol, pretreated with or without U-73122 (20 l M ), 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 min.

*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.

significantly reduced EGCG-induced PLD activity

(Fig 8A), indicating a role for [Ca2+]iin this process We

also measured EGCG-stimulated PtdBut accumulation in a

3 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

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 4 EGCG stimulates [Ca 2+ ] i increases in U87 cells (A)

Serum-starved cells were treated with EGCG (500 l M ) for 3 min, and [Ca2+] i

was measured (B) After the removal of extracellular Ca 2+ (0 Ca 2+ ),

the quiescent cells were treated with EGCG for 3 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.

Fig 5 EGCG induces translocation of PLC-c1 and its interaction with

PLD1 in U87 cells Serum-starved cells were treated with 500 l M

EGCG for 10 min (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 min.

Coverslips were fixed and stained with the indicated antibody and

incubated with fluorescein isothiocyanate-conjugated or Texas

Red-conjugated IgG Immunoreactive cells were visualized by confocal

microscopy Superimposed images display colocalization of

PLD1-labeled and PLC-c1-PLD1-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.

activation is dependent on Ca2+and possibly involves the

Ca2+-activated protein kinase, CaM kinase II

Involvement of PKC in EGCG-induced PLD activation

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

anti-bodies, we first investigated which PKC isozymes were

expressed in U87 cells We found that PKC-a (a

conven-tional 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),

sug-gesting 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

transloca-tion 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

(Fig 9D) These data suggest that EGCG activates PKC-a

in U87 cells

Discussion 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

Fig 6 Effect of antioxidants on EGCG-induced PLC and PLD activation (A) Quiescent cells were labeled with 1 lCiÆmL)1myo-[2- 3 H]inositol, pretreated with the indicated concentrations of N-acetylcysteine (NAC) for 40 min and stimulated with EGCG (500 l M ) for 30 min 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 min and stimulated with EGCG for 30 min (D) [ 3 H]Myristate-labeled cells were treated with 500 l M

H 2 O 2 for 30 min 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.

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

Fur-thermore, 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

epider-mal growth factor, PLC-c1 interacts directly with PLD2

[31] Moreover, EGCG induced tyrosine phosphorylation

of PLC-c1 which was inhibited by pretreatment of

anti-oxidant (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 8 EGCG-induced PLD activation is dependent on intracellular or extracellular Ca2+and is mediated by CaM kinase II (A) U87 cells were labeled with [ 3 H]myristate, preincubated with the indicated con-centrations of BAPTA/AM, and then stimulated with EGCG (500 l M ) for 30 min (B) [ 3 H]Myristate-labeled cells were preincubated with or without extracellular Ca 2+ -free buffer and then stimulated with EGCG for 30 min (C) [3H]Myristate-labeled cells were pre-treated with the indicated concentration of KN-92 or KN-93, and then stimulated with EGCG for 30 min PLD activity was measured as described in Experimental procedures Results are means ± SD from three independent experiments.

Fig 7 Effect of EGCG on ROS production and the cellular GSH

content (A) DCFH-loaded U87 cells were stimulated with vehicle

alone or EGCG (500 l M ) for 20 min U87 cells were preincubated with

N-acetylcysteine (10 m M ) for 1 h before stimulation with EGCG for

20 min ROS produced were measured as described in Experimental

procedures (B) The cellular GSH contents were determined in U87

cells pretreated with or without 10 m M N-acetylcysteine for 1 h, and

then stimulated with EGCG (500 l M ) for 30 min Results are

means ± SD from three independent experiments.

has protective effects against Ab-induced neurotoxicity in

human SY5Y neuroblastoma cells [33], and prevent

neur-onal 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

pro-oxidant activity and vice versa is unclear In U87

astrocy-toma 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 calcium-dependent PKC), and PKC was involved in EGCG-induced PLD activation Furthermore, we found that treatment of the astrocytoma cells with H2O2led 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+]iin U87 cells, and chelation of [Ca2+]i by BAPTA/AM abolished EGCG-induced PLD activation It is therefore assumed that the increase in [Ca2+]imay be due to EGCG-induced PLC activation and subsequent Ins(1,4,5)P3production 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 extracel-lular Ca2+, with removal of extracellular Ca2+from the medium abolishing EGCG-induced PLD activation and the

Fig 9 Role of PKC in EGCG-induced PLD activation U87 cells were pretreated with various PKC inhibitors (5 l M ) for 30 min and stimulated with 500 l M EGCG for 30 min R, Ro-31-8220; C, calphostin C (A) For down-regulation of PKC, cells were pretreated with 500 n M 4b-phorbol 12-myristate 13-acetate for 24 h, and then stimulated with 500 l M EGCG for 30 min (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 min 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 min 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.

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 theL-type Ca2+channel blocker,

nifedi-pine (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+]istores

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 (H335Q).

This study was supported by a grant from the Korea Health 21 R and

D Project, Ministry of Health and Welfare, Republic of Korea

(02-PJ1-PG10-20706-0001).

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