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, Ca
2+
/calmodulin-dependent
protein kinase II, and redox state are involved in epigallocatechin
gallate-induced phospholipase D activation in human astroglioma cells
Shi Yeon Kim
1
, Bong-Hyun Ahn
1
, Joonmo Kim
1
, Yoe-Sik Bae
2
, Jong-Young Kwak
2
, Gyesik Min
3
,
Taeg Kyu Kwon
4
, Jong-Soo Chang
5
, Young Han Lee
6
, Shin-Hee Yoon
1
and Do Sik Min
1
1
Department of Physiology, College of Medicine, The Catholic University of Korea, Seoul, Korea;
2

Medical Research Center for
Cancer Molecular Therapy and Department of Biochemistry, College of Medicine, Dong-A University, Busan, Korea;
3
Department of
Microbiological Engineering, Jinju National University, Korea;
4
Department 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 w as abolished b y t he phospholipase C
(PLC) inhibitor a nd a lipase inactive PLC-c1mutant,which
is dependent on intracellular o r extracellular Ca
2+
,withthe
possible involvement of Ca
2+
/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 p athway involving c hanges in the redox state
that stimulates a PLC-c1 [Ins(1,4,5)P
3
-Ca
2+
]–CaM kinase
II–PLD pathway and a PLC-c1 (diacylglycerol)–PKC–PLD
pathway.
Keywords:Ca
2+
/calmodulin-dependent protein kinase II;
epigallocatechin-3 gallate; phospholipase C-c1; phospho-
lipase D; reactive oxygen s pecies.
Phospholipase D (PLD) c atalyzes the hydrolysis of the most
abundant membrane phospholipid, phosphatidylcholine, to
generate phosphatidic acid a nd 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 s hare 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 e ffects 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), e pigallocate-
chin (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
H
2
O
2
[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 H
2
O
2

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 B iology,
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 Sept ember 2004).
Abbreviations: CaM ki nase II, Ca
2+
/calmodulin-dependent protein
kinase II; DCFH, 2 ¢,7¢-dichlorofluorescein diacetate; DCF, 2¢,7¢-
dichlorofluorescein; DMEM, Dulbecco’s modified Eagle’s m edium;
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 M arch 2004, revised 25 May 2004,
accepted 3 June 2004)
Eur. J. Biochem. 271, 3470–3480 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04242.x
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 EGC G.
We show that EGCG significantly stimulates PLD
activity and that EGCG-in duced 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)P
3

–Ca
2+
]–Ca
2+
/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 (DM EM), fetal bovine
serum and LipofectAMINE were purchased from Invitro-
gen. EGCG, EGC, ECG and E C were obtained f rom S igma.
Protein A–Sepharose was from Amersham Biosciences
Biotech. Antibody to PLC-c1 was fr om Upstate B iotechno-
logy. PD98059, U-73122, U-73343, Ro-31-8220, and
calphostin C were purchased from Bio mol Research
Laboratories (Plymouth Meeting, PA, U SA). 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 a nd PLD2 was generated as described previously
[16]. Authentic phosphatidylb utanol (PtdBut) standard
was from Avanti Polar Lipid. myo-[2-
3
H]Inositol and
[9,10-
3
H]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 I gG 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% CO
2
. 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-
3
H]inositol (2 lCiÆmL
)1
)
in inositol-free DMEM for 20 h. Subsequently, the labeled
cells were pretreated with 20 m
M
LiCl for 15 min. After
stimulation with EGCG, the r eaction w as terminate d by the
addition of ice-cold 5% HClO
4
. The extracts were applied
toaBio-RadDowexAG1-X8anion-exchangecolumn.
The column was then washed with 10 mL distilled water

followedby10mL60m
M
ammonium formate containing
5m
M
sodium tetraborate. Total inositol phosphates were
eluted with a solution containing 1
M
ammonium formate
and 0.1
M
formic acid.
PLD assay
PLD activity was assessed by measuring the formation of
[
3
H]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 · 10
5
cells per well and
serum-starved in the presence of 1 lCiÆmL
)1
[
3
H]myristic
acid. After overnight starvation, the cells were washed three
times with 5 mL NaCl/P
i
and p re-equilibrated i n s erum-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 l
M
EGCG for
10 min, and w ashed with N aCl/P
i
and harvested by
microcentrifugation. The cells were then resuspended in
lysis buffer (20 m
M
Hepes, pH 7.4, 10% glycerol, 1 m
M
EDTA, 1 m
M
EGTA, 1 m
M
dithiothreitol, 1 m
M
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 t hen spun at 100 000 g for 1 h at

4 °C 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]. C ells were plated on to glass coverslips and loaded with
2 l
M
fura-2 acetoxymetyl ester (Molecular Probes) for
45 min at 37°C. The coverglass was then mounted in a flow-
through chamber. The chamber containing the fura-2-
labeled cells was mounted and alternately excited a t 340 or
380 nm. Digital fluorescence images were collected with a
cooled CCD camera. [Ca
2+
]
i
was calculated from the ratio
of the two background-subtracted digital images. Ratios
were converted into free [Ca
2+
]
i
by the equation
½Ca
2 þ

i
¼ K
b

ðR À R
min
Þ=ðR
max
À RÞ
in which R is the 340/380-nm fluorescence emission ratio
and K ¼ 224 n
M
, the dissociation constant for f ura-2 [18].
Immunoprecipitation
U87 cells were harvested and lysed with lysis buffer ( 20 m
M
Hepes, pH 7.2, 1% Triton X-100, 1% sodium deoxycho-
late, 0.2% SDS, 150 m
M
NaCl, 1 m
M
Na
3
VO
4
,1m
M
NaF,
10% glycerol, 10 lgÆmL
)1
leupeptin, 10 lgÆmL
)1
aprotinin,
1m

M
phenymethanesulfonyl 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-c1and
Protein A–Sepharose for 4 h at 4 °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 m
M
Tris/HCl, pH 7.5, 1 m
M
EDTA, 1 m
M
Ó FEBS 2004 Regulation of phospholipase D by EGCG (Eur. J. Biochem. 271) 3471
EGTA, 150 m
M
NaCl, 2 m
M
Na
3
VO
4
, 10% glycerol and
1% Nonidet P40) and resuspended in sample buffer. The
final pellet w as loaded on to a polyacrylamide gel for
immunoblot analysis.
Immunoblot analysis
Proteins were denatured by boiling for 5 min at 95 °Cin
Laemmli sample buffer [19], separated by SDS/PAGE,

and transferred to nitrocellulose membranes. After being
blocked in Tris/Tween-buffered s aline containing 5%
skimmed milk powder, the m embranes 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-st arved for 24 h. After stimulation with EGCG,
the cells were fixed in 3.7% (w/v) formaldehyde for 15 min
and quenched using 50 m
M
NH
4
Cl for 10 min. After
permeabilization using 1% Triton X-100 for 5 min, t he ce lls
were incubated with blocking buffer (1% goat serum in
NaCl/P
i
) at room temperature for 1 h, and then with
primary antibody overnight at 4 °C,andthenwith
subclass-specific secondary antibodies [fluorescein isothio-
cyanate-conjugated donkey anti-(mouse I gG) (Jackson
ImmunoResearch, West G rove, 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 w ith ROS-sensitive dye (10 l
M
).
After 15 min a t room temperature, the cells were washed
three times with serum-fre e 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/P
i
and then
scraped i nto 5% metaphosphoric acid. Reduced glutathione
(GSH) was quantified using a commercially available GSH
determination kit (Calbiochem). Briefly, the method was
basedonachemicalreactionwhichproceededintwosteps.
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. T he second step included a
b-elimination reaction under alkaline c onditions. 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 t o 5 0 min and then remained
constant up to 100 min; m aximum activation was
observed at 1 m
M
EGCG. Using PLD antibodies, we
detected PLD1, but not PLD2, in U87 cells. H owever,
transient transfection o f 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 P LC is not involved [23,24]. To determine
whether PLC activity or G-protein-mediated signaling was
involved in EGCG-induced PLD activation in U 87 ce lls, 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
G
i
protein-mediated signaling response i n 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 [
3
H]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-c1was
the predominantly expressed PLC in U87 cells, indicating
that the P LC 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-c1mutant
expression plasmid on the suppression of EGCG-stimulated
PLC a ctivity. Therefore, we examine d the involvement of
PLC-c1 in the PLD activation by EGCG in U87 cells.
3472 S. Y. Kim et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Interestingly, expression of the lipase inactive mutant PLC-
c1 s ignificantly attenuated EGCG-induced PLD activation
(Fig. 3D), suggesting that PLC-c1 is involved in this
process.
EGCG induces a rise in [Ca
2+
]
i
in U87 cells
As EGCG stimulates PLC activity, it might induce an
increase in [Ca
2+
]
i
in U87 cells. [Ca
2+
]
i
after EGCG
treatment was visualized by loading the cells with Fura-2/
AM. Figure 4 shows simultaneous measurement of [Ca
2+
]

i
increases in different cells, using digital calcium imaging.
One trace represents [Ca
2+
]
i
increase in one cell, and the
different traces represent each [ Ca
2+
]
i
increase pattern in the
different cells. The rise in [Ca
2+
]
i
after EGCG stimulation
peaked within 3 min and then decreased (Fig. 4A). An
EGCG-stimulated increase in [Ca
2+
]
i
may result fro m an
influx of extracellular calcium. To test this possibility, we
treated cells with EGCG in the presence of Ca
2+
-free
buffer. The level of [Ca
2+
]

i
after EGCG treatment was
visualized by loading the cells with Fura-2/AM. For cells in
Ca
2+
-free buffer, EGCG caused only a very small increase
in [Ca
2+
]
i
(Fig. 4B). T hese results clearly show that
treatment of U87 cells with EGCG results i n an increase
in cytosolic calcium. Furthermore, the results sugge st 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-c1istranslocated
from the cytosol to the membrane, where its su bstrate
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 i n U87 ce lls
(Fig. 5A). U sing confocal immunofluorescence microscopy,
we confirmed that PLC-c1 translocation to membrane
regions increased after EGCG treatment. Furthermore,
colocalization o f PLD1 a nd PLC-c1increasedinthe
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-c1in
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 m anufacturer’s instructions, labeled with [
3
H]myristic acid, and
treated with E GCG (500 l
M
) for 30 min. PLD activity was measured
as described in Exp erimental 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
[
3
H]myristate, and t reated for 30 min without or w ith 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 c oncentratio n o f EGCG for

50 min (C). The radioactivity incorporated into PtdBut was me asured
as described in Experimental procedures. Results are means ± SD
from three independent experiments.
Ó FEBS 2004 Regulation of phospholipase D by EGCG (Eur. J. Biochem. 271) 3473
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 PL C 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 H
2
O
2
led to PLD act ivation
(Fig. 6D). These results demonstrate the role of ROS such
as H
2
O
2
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 R OS (Fig. 7). These
results suggest that EGCG increases ROS production in
U87 cells. We n ext m easured t he glutathione (GSH) c ontent
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 Ca
2+
and mediated
by CaM kinase II
Several examples of the participation of Ca
2+
in the

regulation of PLD activity have been reported, although the
effector molecules i nvolved have not been fully character-
ized [29,30]. We found that 1,2-bis-(2-amin ophen-
oxy)ethane-N,N,N¢,N¢,-tetra-acetic acid acetoxymethyl
ester (BAPTA/AM), an intracellular chelator of Ca
2+
,
Fig. 3. PLC is involved in EGCG-induced PLD activation. (A) Quiescent U87 cells were pretreated with 200 ngÆmL
)1
pertussis toxin for 24 h,
labeled with [
3
H]myristate,andstimulatedwith1l
M
sphingosine 1-phosphate or 500 l
M
EGCG f or 3 0 min. (B ) [
3
H]Myristate-labeled cells were
pretreated with the indicated concentrations of U-73122 or U-73343, and stimulated with E GCG for 30 min. (C) Cells transfected with or without a
catalytically inactive mutant of PLC-c1 (H335Q) were labeled with 1 lCiÆmL
)1
myo-[2-
3
H]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 [
3

H]myristic acid, a nd treated with EGCG for 30 min.
*P<0 .05 compared with cells transfected with vecto r a nd t reat ed with EGCG. The radioact ivity i ncorp orated i nto PtdBut was measured as
described in Experimental pro cedure s. Results are means ± SD from three independent exp eriments.
3474 S. Y. Kim et al.(Eur. J. Biochem. 271) Ó FEBS 2004
significantly reduced EGCG-induced PLD activity
(Fig. 8A), indicating a role for [Ca
2+
]
i
in this process. We
also measured EGCG-stimula ted PtdBut a ccumulation in a
3m
M
EGTA/Ca
2+
-free buffer system. We found that
EGCG-stimulated PtdBut accumulation was completely
abolished when cells were incubated in t his Ca
2+
-free buffer
(Fig. 8B), suggesting that extracellular Ca
2+
influx is also
required for EGCG-induced PLD activation in U87 cells.
The possible mechanisms by which [Ca
2+
]
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 2 0 l
M
, KN-92 did n ot 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
)for3min,and[Ca
2+
]
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
[Ca
2+
]
i

was measured. Measurements of [Ca
2+
]
i
were derived from
fura-2-based d igital images as described in Experimental procedures.
Data are representative of t hree experiments.
Fig. 5. EGCG induces tr anslocation of PLC-c1 a nd its interaction with
PLD1inU87cells.Serum-starvedcellsweretreatedwith500l
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 m in.
Coverslips w ere fixed and stained with the indicated antibody and
incubated with fluorescein isothiocyanate-conjugated or Texas Red-
conjugated IgG. Immunoreactive ce lls were visualized by confo cal
microscopy. Superimposed images display colocalization of PLD1-
labeled and PLC-c1-labeled cells. T he results s hown are representative
of thre e separate e xperiments. (C) Serum-starved c ells were stimulated
with EGCG for 10 min, after which cell lysates were prepared and
immunoprecipitated with antibodies to PLD or PLC-c1andthen
immunoblotted using PLC-c1 or PLD antibodies, respectively. Da ta
are representative of three experiments.
Ó FEBS 2004 Regulation of phospholipase D by EGCG (Eur. J. Biochem. 271) 3475
activation is dependent on Ca
2+
and possibly involves the
Ca
2+

-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)P
3
and diacylglycerol, which induce the release of Ca
2+
from
intracellular stores and PKC activation, respectively. To
investigate the possible role of PKC in EGCG-stimulated
PLD activity, we applied t wo approaches, namely the use of
PKC inhibitors and d epletion of enzyme by prolonged
exposure of cells to 4b-phorbol 12-myristate 13-acetate.
Using immunoblotting with PKC isozyme-specific anti-
bodies, w e fi rst investigated which PKC isozymes were
expressed i n U87 cells. We f ound that PKC-a (a conven-
tional P KC) 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 a nd 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 P KC, which is a
consequence of P LC 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 p lays an important role in controling many
biological functions, including exocytosis, phagocytosis,
and secretion. PLD in mammalian cells can be activated by
a r ange 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 fi eld, 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 transductio n.
In this study, we show that EGCG, a major component
of green tea, significantly stimulated PLD activity, and
induced inositol phosphate production and [Ca
2+
]
i
in
astroglioma cells. EGCG-induced PLD activation was
suppressed by the phosphoinositide-specific PLC inhibitor.

PLC-c1 was the predominantly expressed PLC in U 87 cells,
indicating that the PLC activity demonstrated in these cells
Fig. 6. Effect of antioxidants on EGCG-induced PLC and PLD a ctivation. (A) Quiescent cells were labeled with 1 lCiÆmL
)1
myo-[2-
3
H]inositol,
pretreated with the indicat ed concentrations of N-acetylcysteine ( NAC) for 40 min and stimulated w ith EGCG (500 l
M
) f or 30 min. PLC a ctivity
was measured as described in Experimental procedures. [
3
H]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 radioact ivity incorporated into phosphatidylbu tanolwasmeasuredasdescribedin
Experimental Procedures. Results are means ± SD from three independent experiments.
3476 S. Y. Kim et al.(Eur. J. Biochem. 271) Ó FEBS 2004
is due mainly to PLC-c1. This led us t o assume that PLC-c1
may be involved in EGCG-indu ced 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 o f
PLC-c1 in t he signaling p athway. Expression of the inactive

mutant of PLC-c1 also attenuated EGCG-induced PLC
activation. In resting cells, PLC-c1 i s located predominantly
in the cytosol, a nd 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-c1with
PLD1, as well as colocalization of these two m olecules in
membrane. In this study, we report on two signaling
phospholipase complexes composed of PLC-c1andPLD1.
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 P LC and PLD are reversed by N-acetylcysteine
and catalase, suggesting a r ole for ROS in t his 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 Ca
2+
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
)for30min.(B)[
3
H]Myristate-labeled c ells were preincubated
with or without extracellular Ca
2+
-free buffer and th en stimulated
with EGCG for 30 min. (C) [
3
H]Myristate-labeled cells were pre-
treated with the indicated concentration of K N-92 or KN -93, and then
stimulated with EG CG for 30 min. PLD activity was measured as
described i n Experimental p rocedures. 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 c ells were stimulated with vehicle
alone o r EGCG (500 l
M
) for 20 min. U 87 cells were pre incubated with
N-acetylcysteine (10 m
M
) for 1 h before stimulation with EGCG for
20 m in. ROS produced were me asured as described i n E xperimental
procedures. (B) The cellular GSH contents were determined in U87
cells p retreated with or without 10 m
M
N-acetylcysteine for 1 h, and
then stimulated with EGCG (500 l

M
)for30min.Resultsare
means ± SD from three indep enden t experiments.
Ó FEBS 2004 Regulation of phospholipase D by EGCG (Eur. J. Biochem. 271) 3477
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 H
2

O
2
ledtoPLDactivation.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 [ Ca
2+
]
i
in U87 cells, and chelation of
[Ca
2+
]
i
by BAPTA/AM abolished EGCG-induced PLD
activation. It is therefore assumed that the increase in
[Ca
2+
]
i
may be due to EGCG-induced PLC activation and
subsequent Ins(1,4,5)P
3
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 a ctivity, PLC probably r egulates PLD through
stimulation of this kinase. Interestingly, the increase in
PLD activity caused by EGCG i s dependent on extracel-

lular Ca
2+
, with removal of extracellular Ca
2+
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-phorb ol
12-myristate 13-acetate for 24 h, and then stimulated with 500 l
M
EGCG for 30 m in. (B) Radioactivity incorporated into PtdBu t was measured as
described in E xperimental proc edures. Results a re means ± SD from three i ndepend ent e xperiments. S erum-s tarved U87 c ells were treated with
EGCG for 10 min. L ysates were fractionated into c ytosolic and membrane fractions. E ach fraction was immunoblotted using antibodies specific for
PKC-a. The intensity of PKC-a immunoreactive bands quantified by densitometry of the immunoblo t was expressed as re lative intensity of the
bands. (C) U87 cells were cultured on coverslips, starved f or 24 h, and thenstimulatedwithEGCGfor10min.Coverslipswerefixedandstained
with PKC-a antibody, and th en incubated with Texas Red-conjugated IgG. Immunoreactive c ells we re visualized by c onfocal microscopy (D). Data
are representative of three experiments.
3478 S. Y. Kim et al.(Eur. J. Biochem. 271) Ó FEBS 2004
increase in [Ca
2+
]
i
. The EGCG-evoked increase in [Ca
2+

]
i
was inhibited by the nonspecific Ca
2+
channel inhibitor
lanthanum, and the PLC inhibitor U73122, but not by
pretreatment with the
L
-type C a
2+
channel blocker, n ifedi-
pine (data not shown). These results s uggest that, in U87
cells, EGCG-induced increases i n [Ca
2+
]
i
result from
mobilization of Ins(1,4,5)P
3
-sensitive [Ca
2+
]
i
stores.
EGCG has been shown, in an animal study, to pass the
blood–brain barrier and reach brain parenchyma, and
detectionofEGCGinratbrain suggests polyph enols 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 ne rve
terminals [39,40]. Therefore, it is poss ible that m odulation of
synaptic transmission via PLD signaling may explain part of
the e ffect o f tea d rinking 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 o f the glial cells, the major cell
population in the central nervous system, which stimulates
the PLC-c1 [Ins(1,4,5)P
3
–Ca
2+
]–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 t he
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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