Báo cáo khoa học: Cytosolic phospholipase A2-a and cyclooxygenase-2 localize to intracellular membranes of EA.hy.926 endothelial cells that are distinct from the endoplasmic reticulum and the Golgi apparatus pdf

Cytosolic phospholipase A
2
-a and cyclooxygenase-2
localize to intracellular membranes of EA.hy.926
endothelial cells that are distinct from the endoplasmic
reticulum and the Golgi apparatus
Seema Grewal*, Shane P. Herbert, Sreenivasan Ponnambalam and John H. Walker
School of Biochemistry and Microbiology, University of Leeds, UK
Cytosolic phospholipase A
2
-a (cPLA
2
-a) is an 85 kDa
protein that specifically cleaves the sn-2 fatty acyl of
phospholipid to liberate free fatty acids [1,2]. In
response to a variety of extracellular stimuli, cPLA
2
-a
translocates to intracellular membranes, via its N-ter-
minal calcium-binding lipid domain (CaLB, or C2
domain), where it then acts upon its phospholipid sub-
strate. In addition, cPLA
2
-a can be regulated by phos-
phorylation by a number of protein kinases, including
the p38 and ERK mitogen-activated protein kinases
(MAPKs) [3,4].
Within the endothelium, cPLA
2
-a plays a pivotal
role in the generation of arachidonic acid from mem-

brane phospholipids. As this arachidonic acid is the
precursor for prostacyclin, a member of the eicosanoid
family of inflammatory mediators, it can be seen that
cPLA
2
-a plays a crucial role in endothelium functions
such as haemostasis, angiogenesis, control of vascular
Keywords
arachidonic acid; calcium; cPLA
2
-a;
cyclooxygenase; endothelium
Correspondence
J. H. Walker, School of Biochemistry and
Microbiology, University of Leeds, Leeds
LS2 9JT, UK
Fax: +44 113 3433167
Tel. +44 113 3433119
E-mail:
*Present address
Department of Developmental and Cell
Biology, University of California, USA
(Received 29 October 2004, revised 21
December 2004, accepted 11 January 2005)
doi:10.1111/j.1742-4658.2005.04565.x
Cytosolic phospholipase A
2
-a (cPLA
2
-a) is a calcium-activated enzyme that

plays an important role in agonist-induced arachidonic acid release. In
endothelial cells, free arachidonic acid can be converted subsequently into
prostacyclin, a potent vasodilator and inhibitor of platelet activation,
through the action of cyclooxygenase (COX) enzymes. Here we study the
relocation of cPLA
2
-a in human EA.hy.926 endothelial cells following
stimulation with the calcium-mobilizing agonist, A23187. Relocation of
cPLA
2
-a was seen to be highly cell specific, and in EA.hy.926 cells occurred
primarily to intracellular structures resembling the endoplasmic reticulum
(ER) and Golgi. In addition, relocation to both the inner and outer
surfaces of the nuclear membrane was observed. Colocalization studies
with markers for these subcellular organelles, however, showed colocaliza-
tion of cPLA
2
-a with nuclear membrane markers but not with ER or Golgi
markers, suggesting that the relocation of cPLA
2
-a occurs to sites that are
separate from these organelles. Colocalization with annexin V was also
observed at the nuclear envelope, however, little overlap with staining pat-
terns for the potential cPLA
2
-a interacting proteins, annexin I, vimentin,
p11 or actin, was seen in this cell type. In contrast, cPLA
2
-a was seen to
partially colocalize specifically with the COX-2 isoform at the ER-resem-

bling structures, but not with COX-1. These studies suggest that cPLA
2
-a
and COX-2 may function together at a distinct and novel compartment for
eicosanoid signalling.
Abbreviations
Con A, concanavalin A; COX, cyclooxygenase; cPLA
2
-a, cytosolic phospholipase A
2
-a; DMEM, Dulbecco’s modified Eagle’s medium; ER,
endoplasmic reticulum; FITC, fluoroscein isothiocyanate; GFP, green fluorescent protein; HUVEC, human umbilical vein endothelial cell;
MAPK, mitogen-activated protein kinase; WGA, wheatgerm agglutinin.
1278 FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS
tone and prevention of thrombosis formation [5]. Fol-
lowing this, cPLA
2
-a has become an attractive target
for the development of novel pharmacological thera-
peutics against various pathological conditions [6,7].
To date, however, very little is known about the exact
mechanisms involved in the regulation of this import-
ant enzyme.
Cyclooxygenase (COX) enzymes function down-
stream of cPLA
2
-a and convert arachidonic acid into
prostaglandin H
2
[8,9]. To date, two distinct COX iso-

forms, COX-1 and COX-2, have been identified and
characterized, and an alternative splice variant of
COX-1, termed COX-3 has been cloned recently [10].
COX-1 is generally considered the housekeeping
enzyme that is constitutively expressed and involved in
the maintenance of haemostasis. COX-2, by contrast,
is the inducible isoform that is expressed primarily in
disease states and in response to inflammatory and
mitogenic stimuli [11,12]. Both isoforms have been
shown to localize at the nuclear envelope and in the
endoplasmic reticulum (ER) of endothelial cells [13].
In addition, immunoelectron microscopy studies dem-
onstrated that both were constitutively present on the
lumenal surfaces of the ER and on the inner and outer
membranes of the nuclear envelope [14].
Within the last decade, many groups have studied
the relocation of cPLA
2
-a following an increase in
[Ca
2+
]
i
. However, because a variety of cell types, anti-
bodies and methods have been used, the exact site of
cPLA
2
-a relocation remains unknown, and appears to
be cell specific. In the majority of studies, cPLA
2

-a has
been reported to translocate to the nuclear periphery
and structures within the cytosol, sites presumed to be
the nuclear envelope, ER and Golgi apparatus. These
include studies on rat mast cells [15], Chinese hamster
ovary cells [16,17], rat alveolar epithelial cells [18] and
rat glomerular epithelial cells [19]. More recently, stud-
ies using over-expression of green fluorescent protein
(GFP)-tagged cPLA
2
-a have also shown relocation to
Golgi and ER membranes [20–22]. In contrast, some
reports have shown relocation to the plasma mem-
brane (Chinese hamster ovary cells [23] and glomerular
epithelial cells [19]). Interestingly, studies on fibroblasts
[24,25] show that cPLA
2
-a is localized within cytosolic
clusters in stimulated as well as unstimulated cells. Pre-
vious experiments with human umbilical vein endothel-
ial cells (HUVECs) and rabbit coronary endothelial
cells have reported a redistribution of cPLA
2
-a to the
nuclear envelope [26,27]. The localization of cPLA
2
-a
in human endothelial cells has also been shown to be
dependent on cell density [26,28], with proliferating
cells showing a higher level of nuclear cPLA

2
-a. Fol-
lowing this, Tashiro et al. have recently confirmed the
presence of nuclear localization and export signals
within the cPLA
2
-a protein [29]. Few studies on
cPLA
2
-a in endothelial cells, however, have attempted
to characterize its localization at high spatial resolu-
tion. Here we study the calcium-induced relocation of
cPLA
2
-a in EA.hy.926 endothelial cells. We show that
cPLA
2
-a relocates to intracellular membrane compart-
ments that are distinct from the ER and the Golgi
apparatus. More importantly, we demonstrate that, at
this specific site, cPLA
2
-a colocalizes with COX-2 but
not COX-1.
Results
Expression of cPLA
2
-a varies according
to cell type
EA.hy.926 cells are a hybrid derived from HUVEC

and A549 cells [30]. In order to establish the validity
of the EA.hy.926 cells as a model for studies on
cPLA
2
-a in endothelial cells, expression levels of
cPLA
2
-a in EA.hy.926 cells and HUVECs were com-
pared. Equivalent amounts of protein were separated
by SDS ⁄ PAGE and immunoblotted for cPLA
2
-a.
Lysates from the parental A549 cell line and HeLa
cells were also analysed. The results (Fig. 1A,B) show
that the EA.hy.926 cells express cPLA
2
-a at levels that
are similar to those exhibited by their parent HU-
VECs. In contrast, A549 cells, with which the HU-
VECs were fused to generate the EA.hy.926 cells,
showed much higher levels of cPLA
2
-a expression. In
addition, HeLa cells were seen to express very high
levels of cPLA
2
-a.
The precise membrane to which cPLA
2
-a

relocates is dependent on cell type
The location and relocation of cPLA
2
-a in EA.hy.926
cells was compared with that seen in their parent
HUVEC and A549 cells. In addition, HeLa cells,
which were shown to express high levels of cPLA
2
-a,
were studied. In order to avoid any complications con-
cerning cell-specific responses to physiological stimuli,
cells were stimulated with the calcium ionophore,
A23187. cPLA
2
-a was then detected using a specific
goat polyclonal anti-cPLA
2
-a serum (Fig. 1C). As
reported previously [26,28,31], cPLA
2
-a in all cell types
was located in the cytosol and at a higher concentra-
tion in the nucleus. Following A23187 stimulation,
EA.hy.926 cells showed a loss in nuclear staining and
relocation of cPLA
2
-a to the nuclear envelope and
nuclear periphery. Relocation occurred as soon as 30 s
following stimulation and staining patterns observed
S. Grewal et al. cPLA

2
-a localization in endothelial cells
FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS 1279
after 30 min stimulation were similar to those obtained
after shorter stimulations (data not shown). Similar
redistribution of cPLA
2
-a was seen in HUVECs. In
contrast, and in agreement with previous studies [31], a
confocal section through an A23187-stimulated A549
cell showed relocation to structures resembling the
Golgi, with very little staining in the region of the nuc-
lear membrane. In addition, a highly punctate staining
pattern throughout the cytosol and nucleus of a resting
cell was observed. HeLa cells showed similar redistri-
bution patterns to the EA.hy.926 and HUVECs, how-
ever, a high proportion of intranuclear staining
remained following stimulation.
Similar relocation patterns were observed following
stimulation of EA.hy.926 cells with the natural agon-
ists, histamine and thrombin (Fig. 1D). This reloca-
tion was also seen to be dependent on an influx of
extracellular calcium, consistent with previous work
on HUVECs [32], which demonstrated that agonist-
evoked prostacyclin production is dependent on extra-
cellular free [Ca
2+
]. In conclusion, it was observed
that the hybrid EA.hy.926 cell line closely resembles
its HUVEC parental line with regards to both levels

of cPLA
2
-a expression and site of cPLA
2
-a reloca-
tion.
Comparison of the location of cPLA
2
-a in
EA.hy.926 endothelial cells with markers for
specific cellular organelles
The relocation of cPLA
2
-a in endothelial cells has been
reported to occur to sites referred to as the ER, Golgi
and nuclear membrane [26,27]. However, in very few
of these cases have direct double-labelling studies been
performed to confirm these locations. Hence, to fur-
ther characterize the precise site of membrane reloca-
tion of cPLA
2
-a, endothelial cells were counterstained
with markers for intracellular membranes.
First, fluoroscein isothiocyanate (FITC)-conjugated
Concanavalin A (Con A), which binds selectively to
a-d-mannosyl and a -d-glucosyl residues, was used to
label the ER and nuclear envelope (Fig. 2). The results
A
B
D

C
Fig. 1. cPLA
2
-a expression levels and sub-
cellular distribution in EA.hy.926, HUVEC,
A549 and HeLa cells. (A) Total cell lysates
from EA.hy.926, HUVEC, A549 and HeLa
cells were prepared and 20 lg protein of
each lysate was separated by SDS ⁄ PAGE.
Following Western blotting, goat polyclonal
anti-cPLA
2
-a serum was used to detect
cPLA
2
-a (as described in Experimental pro-
cedures). (B) Quantification of the amount
of cPLA
2
-a (in arbitrary units) in the cell
lysates (expressed as an average from three
independent sets of cell lysates ± SEM). (C)
Cells were grown on coverslips, stimulated,
fixed, permeabilized and incubated with goat
polyclonal anti-cPLA
2
-a serum followed by
FITC-conjugated anti-goat serum. Cells were
viewed using a Leica TCS NT confocal fluor-
escence microscope. Scale bar, 20 mm.

(D) EA.hy.926 cells were stimulated with
10 m
M histamine or 1 UÆmL
)1
thrombin for
1 min in the presence or absence of 1 m
M
extracellular calcium. Cells were then fixed
and analysed by microscopy as described
above. Scale bar, 20 mm.
cPLA
2
-a localization in endothelial cells S. Grewal et al.
1280 FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS
indicated that, although the cPLA
2
-a (green) and
Con A (red) staining patterns appeared similar, a
direct overlay of high-resolution 0.485 lm sections
through the cell revealed very few regions of overlap
(yellow), indicating that cPLA
2
-a relocates to only a
subdomain of these regions.
Wheatgerm agglutinin (WGA), which selectively
labels N-acetyl-b-d-glucosaminyl residues in the plasma
membrane, Golgi apparatus and nuclear envelope, was
also used as a counter stain (Fig. 2). Once again, com-
plete overlap was not seen, and only patches of colo-
calization, particularly at the nuclear envelope, were

evident.
Finally, in order to selectively label only the ER,
cells were counterstained with a rabbit polyclonal anti-
calreticulin serum followed by a Texas Red-conjugated
secondary antibody (Fig. 2). When overlain on the
cPLA
2
-a image, the characteristic ER-like calreticulin
staining pattern did not show areas of colocalization.
Secondary-only controls and controls in which goat
polyclonal antibody was followed by anti-rabbit and
vice versa were blank.
Relocation of cPLA
2
-a occurs to both the inner
and the outer surfaces of the nuclear membrane
In order to assess whether cPLA
2
-a was relocating to
the inner, outer or both surfaces of the nuclear mem-
brane, cells were permeabilized with digitonin. When
used at low concentrations, this nonionic detergent
selectively permeabilizes membranes with high choles-
terol content, such as the plasma membrane. Under
these conditions the nuclear membrane is not permea-
bilized hence the interior of the nucleus is inaccessible
to antibodies.
When cells were permeabilized with 0.05% digitonin,
very little nuclear staining was evident in resting cells
(Fig. 3A). Following stimulation with A23187, the

intensity of staining of the nuclear membrane in digito-
nin-permeabilized cells was lower than that seen in Tri-
ton X-100-permeabilized cells (Fig. 3B). Quantification
of the intensity of nuclear membrane staining showed
a significant decrease (from 88.7 ± 3.28 to 53.0 ±
1.46 arbitrary units) in the level of staining when using
digitonin instead of Triton X-100 (Fig. 3C). Such a
decrease suggests that some relocation is occurring to
the inner surface of the nuclear membrane, where the
cPLA
2
-a is not accessible to antibodies. However,
because some staining of the nuclear membrane
remained evident in digitonin-permeabilized cells and
not all nuclear membrane staining was abolished, it is
clear that relocation must be occurring to the outer
surface of the nuclear membrane, too. It was also
observed that several intranuclear speckles were pre-
sent in the digitonin-permeabilized cells, suggesting an
association of cPLA
2
-a with intranuclear membranous
invaginations of the nuclear membrane [33].
To further investigate the nuclear localization and
nuclear membrane relocation, GFP–cPLA
2
-a was
expressed and its distribution in the absence and pres-
ence of stimuli was followed. In addition, cells were
counterstained with DAPI, a fluorescent DNA-binding

dye. Consistent with the results obtained from indirect
immunofluorescence studies above and with those seen
Fig. 2. Double-labelling of cells with ER,
Golgi apparatus and plasma membrane mar-
kers. Cells were stimulated with A23187
(5 l
M in the presence of 1 mM extracellular
calcium for 1 min), fixed and permeabilized
(as described in Experimental procedures).
Cells were then incubated with goat
polyclonal anti-cPLA
2
-a followed by FITC-co-
njugated anti-goat IgG and either rhodamine-
conjugated Con A or rhodamine-conjugated
WGA. For calreticulin staining, cells were
blocked in donkey serum and incubated
with goat polyclonal anti-cPLA
2
-a and rabbit
polyclonal anti-calreticulin sera, followed by
donkey FITC-conjugated anti-sheep and
donkey Texas Red-conjugated anti-rabbit
sera. Cells were visualized using a Leica
TCS SP confocal microscope. Scale bar,
20 mm. Also shown is an enlarged section
(outlined in box) of the overlay. Scale bar,
5 lm.
S. Grewal et al. cPLA
2

-a localization in endothelial cells
FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS 1281
previously for GFP–cPLA
2
-a [34], cPLA
2
-a was pre-
sent in the cytosol and at a higher concentration in the
nucleus of resting cells (Fig. 4A). Following stimula-
tion with A23187, relocation to the nuclear envelope
and cytosolic structures was observed. An overlay of
the GFP–cPLA
2
-a and DAPI staining patterns
revealed a small region of overlap on the inner surface
of the nuclear envelope. A densitometrical plot
(Fig. 4B) of staining across the cell further demonstra-
ted the overlap between the FITC (green) and DAPI
(red) channels, suggesting that cPLA
2
-a was present
on both the inner and outer surfaces of the nuclear
envelope. In contrast, GFP–annexin V, which showed
similar relocation to the nuclear envelope, exhibited a
complete overlap with the DAPI staining, indicating
that it was relocating to primarily the inner surface of
the nuclear membrane.
Colocalization with other proteins
Previous studies on cPLA
2

-a have suggested that
accessory or binding proteins may play a role in the
regulation of cPLA
2
-a activity and localization. Ann-
exin V, for example, has been shown to exhibit sim-
ilar membrane relocation kinetics to cPLA
2
-a [35]
and several reports have demonstrated that annexin V
A
B
C
Fig. 3. The location and relocation of cPLA
2
-a in Triton X-100- and digitonin-permeabilized cells. (A) Cells were grown on coverslips overnight
and either directly fixed or stimulated with 5 l
M A23187 for 1 min in the presence of 1 mM extracellular calcium prior to fixation. Cells were
then fixed and permeabilized with either 0.1% Triton X-100 or 0.05% digitonin. cPLA
2
-a was then detected using affinity-purified goat poly-
clonal anti-cPLA
2
. In the case of the digitonin-permeabilized cells, all incubations and washes included 0.05% digitonin. Cells were viewed
using a Leica TCS SP confocal fluorescence microscope. Scale bar,10 lm. (B) The intensity of nuclear membrane staining (in arbitrary units)
was measured using the
TCS NT software. Densitometric plots were constructed and the maximum levels (corresponding to nuclear mem-
brane staining) were recorded. (C) Plot of the average intensity of nuclear membrane staining (± SEM, n ¼ 90 sections) in Triton X-100 and
digitonin-permeabilized cells.
cPLA

2
-a localization in endothelial cells S. Grewal et al.
1282 FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS
is able to interact with and inhibit cPLA
2
-a activity
[36–38]. Studies also revealed that a specific peptide
derived from the N-terminus of annexin I was able to
inhibit phosphorylation and activation of cPLA
2
-a
[39]. Furthermore, studies using recombinant annexin
proteins showed that annexin I–cPLA
2
-a complexes
could be coimmunoprecipitated, suggesting that these
two proteins can interact directly [38]. p11, a member
of the S100 family of calcium-binding proteins that
forms a heterotetramer with annexin II, has also been
shown to interact with and inhibit the activity of
cPLA
2
-a [40]. Previous studies by Nakatani and
coworkers [41] have also shown that cPLA
2
-a inter-
acts with the head domain of the intermediate fila-
ment protein vimentin. These studies demonstrated
that this interaction was necessary for cPLA
2

-a-medi-
ated arachidonic acid release, and suggested that
vimentin may function as an adapter protein for
cPLA
2
-a at its site of localization. Several studies
have also implied that cPLA
2
-a may be regulated by
cytoskeletal interactions. Cytochalasin B, an inhibitor
of actin polymerization, was shown to reduce colla-
gen-induced arachidonic acid production in platelets
[42]. Furthermore, PLA
2
activity was found in the
Triton X-100-insoluble cytoskeletal fraction isolated
from thrombin-stimulated platelets [43]. Finally,
cPLA
2
-a has been shown to be present in punctate
cytosolic structures [44]. These structures were shown
to correspond to lipid-rich inclusions, which are struc-
turally distinct sites of esterified arachidonic acid and
thus represent a nonmembrane site of eicosanoid gen-
eration. COX isoforms have also been found in sim-
ilar cytosolic vesicles, where they were shown to
interact with caveolin-1A [45].
Following this, we examined the localization of
annexin V, annexin I, p11, vimentin, actin and caveo-
A

B
Fig. 4. GFP–cPLA
2
-a transfected cells double-labelled with DAPI: a comparison with relocation of GFP–annexin V. (A) EA.hy.926 cells were
seeded onto coverslips and transfected with 5 lg of GFP–cPLA
2
-a plasmid DNA or GFP–annexin V plasmid DNA. Forty-eight hours after
transfection, the cells were fixed, permeabilized and counterstained with DAPI (1 mgÆmL
)1
for 2min). For A23187 stimulations, cells were
incubated with 5 l
M A23187 for 1 min in the presence of 1 mM extracellular calcium prior to fixation. Fluorescence was viewed using a
Leica TCS NT confocal fluorescence microscope. Scale bar, 20 lm. (B) Plots comparing the distribution of fluorescence emitted by each
channel across the section.
S. Grewal et al. cPLA
2
-a localization in endothelial cells
FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS 1283
lin in EA.hy.926 cells, and determined whether any of
these candidate cPLA
2
-a-interacting proteins colocal-
ized with cPLA
2
-a following A23187 stimulation. The
results of these studies (Fig. 5) demonstrate that,
although a substantial pool of annexin V appeared to
colocalize with cPLA
2
-a at the nuclear membrane,

very little colocalization with any of the proteins
examined occurred at the ER-like structures and
vesicles.
Colocalization with cyclooxygenase isoforms
In order to investigate whether cPLA
2
-a colocalizes with
COX proteins, EA.hy.926 cells were stained with anti-
bodies raised specifically against COX-1 and COX-2
proteins. Both COX isoforms have been shown to be
constitutively localized on both the inner and outer sur-
faces of the nuclear membrane [14], and recently,
COX-1 and prostacyclin have been shown to colocalize
at the ER-like structures and at the nuclear membrane
of endothelial cells [46], thus it is possible that cPLA
2
-a
may associate with these enzymes to form a functional
complex. We have previously demonstrated that cPLA
2
-
a colocalizes specifically with the COX-1 isoform in
A549 epithelial cells thus to determine if similar inter-
actions were occurring in EA.hy.926 cells, we costained
cells with antibodies raised against COX-1 and COX-2.
Consistent with previous reports, our studies demon-
strated that both COX-1 and COX-2 were present in
cytosolic structures resembling the ER and also around
the periphery of the nuclear membrane in both resting
(Fig. 6A) and stimulated (Fig. 6B) cells. In addition,

COX-1 showed high levels of nuclear localization.
Most importantly, however, we observed that follow-
ing A23187 stimulation only the COX-2 isoform colo-
calized with cPLA
2
-a. This partial overlap occurred at
distinct cytosolic structures around the periphery of
the nucleus (indicated by arrows in Fig. 6B) and at
structures on the nuclear envelope. No colocalization
with either COX isoform was observed in nonstimu-
lated cells.
Discussion
The subcellular location of cPLA
2
-a has been a con-
troversial issue in recent years, and the exact cellular
membrane or structure to which this enzyme translo-
cates has not been confirmed. In endothelial cells, this
protein has been reported to translocate following sti-
mulation to sites at the nuclear envelope, ER and
plasma membrane [26,27]. Here, using high-resolution
confocal sectioning and the EA.hy.926 cell line as a
model for endothelial cells, the relocation of cPLA
2
-a
was studied.
The EA.hy.926 cell line, which has been shown pre-
viously to be a sufficient model for endothelial
cells with regards to its morphology, expression of
endothelial-specific markers and response to physiolo-

gical agonists [30,47], expressed levels of cPLA
2
-a that
were almost identical to those expressed by the paren-
tal HUVECs. Also, in terms of prostacyclin produc-
tion, it has been shown previously that the EA.hy.926
cell line is capable of sustaining basal and stimulated
A
BC
Fig. 5. Colocalization of cPLA
2
-a. Cells were grown on coverslips
overnight and stimulated with 5 l
M A23187 for 1 min in the pres-
ence of 1 m
M extracellular calcium. Cells were then fixed and per-
meabilized and incubated with goat polyclonal anti-cPLA
2
-a serum
and mouse monoclonal antibodies against annexin V, annexin I,
p11, caveolin, actin or vimentin. Cells were then incubated with
FITC-conjugated goat and TRITC-conjugated mouse secondary anti-
bodies. Fluorescence was viewed using a Leica TCS NT confocal
fluorescence microscope. The figure shows staining in the FITC
(green; A) and TRITC (red; B) channels along with a merged image
of the two (C). Scale bar, 20 lm.
cPLA
2
-a localization in endothelial cells S. Grewal et al.
1284 FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS

levels of prostacyclin, similar to those exhibited by
HUVECs [48]. Finally, using immunofluorescence
microscopy, the location and relocation of cPLA
2
-a in
EA.hy.926 was shown to be analogous to that seen in
both HUVECs and other endothelial cells [26,27].
Therefore, it is apparent that this hybrid endothelial
cell line behaves more like HUVECs than A549 cells,
further supporting its use as a model for endothelial
cells.
In EA.hy.926 cells, cPLA
2
-a was seen to relocate to
structures resembling the ER and the inner and outer
surfaces of the nuclear membrane following stimula-
tion with A23187. Identical relocation patterns were
also seen following stimulation with histamine and
thrombin. In addition, and consistent with previous
data [49], this relocation was shown to increase with
time (data not shown) and to be dependent on an
influx of extracellular calcium.
Much of the controversy over the exact site of
cPLA
2
-a relocation has arisen due to a variety of
antibodies and cell types being studied. Here, compar-
ative studies using one specific antibody on several cell
types confirmed that the relocation of cPLA
2

-a is
dependent on cell type. In EA.hy.926 cells and
HUVECs, relocation to the nuclear membrane and ER
was evident. In contrast, relocation primarily to the
Golgi was seen in A549 cells. Furthermore, in HeLa
cells, which displayed high levels of cPLA
2
-a expres-
sion, only a small proportion of the total protein was
seen to relocate. This relocation occurred mainly to
the nuclear membrane, and very little Golgi or ER
staining was evident. These distinct contrasts in mem-
brane relocation could be dependent on the specific
role played by each of these cell types. The fate of the
arachidonic acid released by the action of cPLA
2
-a is
entirely dependent on cell type, hence the expression of
the downstream enzymes involved in arachidonic meta-
bolism also varies considerably from cell to cell. The
subcellular locations of these proteins also vary, with
some being present in the ER, others in the nuclear
membrane and some present at both these locations.
Thus the relocation of cPLA
2
-a may be dependent on
an association or complex formation with downstream
enzymes in eicosanoid biosynthesis, in which case the
relocation of this enzyme would be expected to vary.
Many of the reports on cPLA

2
-a describe relocation
to the ER and nuclear membrane, however, to date
few detailed direct double-labelling or colocalization
A
B
Fig. 6. Colocalization with cyclooxygenase
isoforms. Cells were grown on coverslips
overnight and were either fixed directly (A)
or stimulated with 5 l
M A23187 for 1 min in
the presence of 1 m
M extracellular calcium
prior to fixation (B). Cells were then
permeabilized and incubated with goat
polyclonal anti-cPLA
2
-a antibody and mouse
monoclonal antibodies against either COX-1
or COX-2. Cells were then incubated with
FITC-conjugated anti-goat and TRITC-
conjugated anti-mouse secondary sera.
Fluorescence was viewed using a Leica TCS
NT confocal fluorescence microscope. Scale
bar, 20 lm.
S. Grewal et al. cPLA
2
-a localization in endothelial cells
FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS 1285
experiments have been published. The high-resolution

confocal microscopy studies presented here suggest
that, although the staining pattern closely resembles
typical ER staining, thin 0.485 lm sections through
the cell demonstrate that there is very little colocaliza-
tion of cPLA
2
-a with ER, nuclear membrane and
Golgi markers such as Con A, WGA and calreticulin.
Thus it appears that cPLA
2
-a relocates to ER-like
structures or microdomains of the ER and nuclear
membrane. It may be possible that such a domain
contains other proteins involved in eicosanoid produc-
tion, such as COX-1, COX-2 and prostacyclin syn-
thase, which have all been shown to localize to this
subcellular region. In addition, any free arachidonic
acid released from the membrane must be rapidly con-
verted to arachidonyl-CoA and re-esterified into
phospholipids to avoid over synthesis of eicosanoids.
Because the enzyme responsible for this, arachidonyl-
CoA-1-acyl-lysophosphatide acyltransferase, is present
in ER-like structures, compartmentalization of eicosa-
noid biosynthesis at this site would be beneficial to
the cell. Consistent with this, we show a partial colo-
calization of cPLA
2
-a with COX-2, but not COX-1, at
these intracellular cytosolic structures and sites on the
nuclear envelope. A recent study [45] has suggested

that the COX-2 isoform colocalizes and interacts with
caveolin-1 in caveolae of human fibroblasts, however,
in the EA.hy.926 cells we observed little colocalization
of cPLA
2
-a with caveolin. It is also not known whe-
ther the location of cytoplasmic COX-2 changes upon
stimulation. COX-2 staining patterns observed before
stimulation appear similar to patterns seen after stimu-
lation and show no overlap with cPLA
2
-a staining,
but it is possible that increases in cytosolic calcium
concentrations also cause local relocation of COX-2
to these cytosolic structures.
Differential functional coupling between COX and
cPLA
2
-a has been reported previously in several sys-
tems. In rat peritoneal macrophages, for example, the
A23187-induced immediate response, which resulted
primarily in thromboxane B
2
, production was shown
to be dependent on coupling between cPLA
2
-a and
COX-1. In contrast, cPLA
2
-a and COX-2 were

functionally coupled to produce the lipopolysaccharide-
induced delayed release of prostaglandin E
2
[50]. In
COS-1 epithelial cells, however, cPLA
2
-a was found to
be coupled to both COX-1 and COX-2 to produce
prostaglandin E
2
[51]. The physical colocalization of
COX and cPLA
2
-a in these systems, however, has not
been studied, and this is one of few studies that shows
distinct colocalization of cPLA
2
-a with a COX
isoform. Functional coupling of cPLA
2
-a and COX
isoforms in endothelial cells has not yet been investi-
gated, however, it is possible that the preferential
A23187-induced colocalization of cPLA
2
-a with
COX-2 and not COX-1 observed here may be reflected
in preferential functional coupling. Moreover, it may
also be possible that stimulation with other agonists
(e.g. histamine, thrombin) may result in subtly differ-

ent localization patterns and thus differential colocali-
zation with COX isoforms.
In conclusion, cPLA
2
-a relocates to structures that
resemble the ER and nuclear membrane, however, it
does not colocalize directly with ER and nuclear mem-
brane markers. Interestingly, however, we show that
cPLA
2
-a colocalizes specifically with the COX-2 iso-
form but not COX-1. This suggests a novel compart-
mentalization of cPLA
2
-a, which could possibly aid
the process of eicosanoid generation by placing this
enzyme in an appropriate position for catalysis, per-
haps in close proximity to lipid-rich bodies or micro-
domains, and other proteins involved in eicosanoid
biosynthesis.
Experimental procedures
Reagents
Tissue culture media, enzymes and antibiotics were pur-
chased from Invitrogen (Paisley, UK). The N-terminal
GFP–cPLA
2
-a plasmid construct was a gift from
Dr R. Williams (MRC LMB, Cambridge, UK). The pEG-
FP-C1–annexin V plasmid was generously provided by
R. Sainson (University of Leeds, UK). Goat polyclonal

antibodies to cPLA
2
-a and mouse monoclonal antibodies
to annexin V were obtained from Santa Cruz Biotechno-
logy (Santa Cruz, CA, USA). Mouse monoclonal anti-
vimentin serum was obtained from Sigma (Poole, UK).
Anti-caveolin sera were a gift from N. M. Hooper (Univer-
sity of Leeds, UK). Mouse monoclonal C4 actin antibody
was obtained from ICN Biomedicals (Irvine, CA, USA)
and Chemicon (Temecula, CA, USA). Rabbit polyclonal
anti-(annexin I) was a gift from E. Solito (Paris, France).
Mouse monoclonal anti-p11 S100 serum was acquired from
Swant (Bellinzona, Switzerland). Secondary FITC- and
rhodamine-conjugated antibodies were from Sigma. Citi-
fluor mounting medium was obtained from Agar Scientific
(Stansted, UK). All other standard reagents and chemicals
were from Sigma or BDH (Poole, UK).
Cell culture
The EA.hy.926 cells were a generous gift from Dr C. J.
Edgell (University of North Carolina, USA). HUVECs
(passaged three times since their initial isolation) were
obtained from Dr S. M. Parkin (University of Bradford,
UK). HeLa (human epithelial carcinoma) and A549
cPLA
2
-a localization in endothelial cells S. Grewal et al.
1286 FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS
(human lung epithelial carcinoma) cells were from ATCC.
EA.hy.926 cells were cultured at 37 °C in a humid atmo-
sphere containing 5% CO

2
in air. Cells were grown in Dul-
becco’s modified Eagles’ medium (DMEM) supplemented
with 10% foetal bovine serum, penicillin (100 UÆmL
)1
),
streptomycin (100 lgÆmL
)1
) and HAT (100 lm hypoxan-
thine, 0.4 lm aminopterin, 16 lm thymidine). HUVECs
were cultured on gelatin-coated surfaces [52] in the above
medium. HeLa, A549 and HEK293 cells were cultured in
DMEM supplemented with 10% foetal bovine serum, peni-
cillin (100 UÆmL
)1
) and streptomycin (100 lgÆmL
)1
).
Immunofluorescence microscopy
The method for immunofluorescence microscopy was
adapted from Barwise & Walker [53] and Heggeness
et al. [54]. Cells were grown on glass coverslips in six-well
dishes overnight. Media was removed and the cells were
washed three times with prewarmed (37 °C) NaCl ⁄ P
i
and
fixed in prewarmed 10% formalin in neutral-buffered sal-
ine ( 4% formaldehyde, Sigma) for 5 min. For A23187
stimulations, cells were washed with NaCl ⁄ P
i

and incuba-
ted with 5 lm A23187 in Hepes ⁄ Tyrode’s buffer contain-
ing 1 mm calcium for 1 min prior to fixation. All
subsequent steps were performed at room temperature
(20 °C). After fixation, the cells were permeabilized with
0.1% Triton X-100 in NaCl ⁄ P
i
for 5 min and fixed once
again for 5 min. The cells were then washed three times
with NaCl ⁄ P
i
and incubated in sodium borohydride solu-
tion (1 mgÆmL
)1
in NaCl ⁄ P
i
) for 5 min or in ammonium
chloride (50 mm) for 10 min to reduce autofluorescence.
Following three further NaCl ⁄ P
i
wash steps, the cells
were blocked in 5% rabbit serum in NaCl ⁄ P
i
for 3 h.
The cells were then incubated with primary antibodies
(diluted 1 : 100 into NaCl ⁄ P
i
⁄ 5% serum) overnight. Cells
were washed eight times with NaCl ⁄ P
i

then incubated
with AlexaFluor 488 and 594-conjugated secondary anti-
bodies, or rhodamine-conjugated WGA or Con A
(10 lgÆmL
)1
) for 3 h. The cells were then washed eight
times with NaCl ⁄ P
i
and mounted onto slides in Citifluor
mounting medium (Agar Scientific).
Confocal imaging
Confocal fluorescence microscopy was performed using a
Leica TCS NT spectral confocal imaging system coupled to a
Leica DM IRBE inverted microscope. Each confocal section
was the average of four scans to obtain optimal resolution.
The system was used to generate individual sections that
were 0.485 lm thick. All figures shown in this study repre-
sent 0.485 lm sections taken through the centre of the
nucleus. To capture double-labelled samples, sequential scan-
ning of each fluorescence channel was performed (according
to the manufacturer’s guidelines) to avoid cross-contamin-
ation of fluorescence signals. For p11- and annexin II-
labelled samples, confocal images were taken using a Zeiss
LSM 510 Meta system through a Zeiss Axioplan confocal
microscope. Each section was the average of four scans and
the system generated sections that were 1 lm thick.
Transfection of EA.hy.926 cells
Cells were transfected using the calcium phosphate–DNA
coprecipitation method as described in Jordan et al. [55].
For transient transfections, cells (2 · 10

5
) were seeded onto
coverslips in six-well dishes and grown overnight. One hour
prior to transfection, the cells were fed with fresh medium.
Cells were transfected with 5 lg plasmid DNA per cover-
slip. Six hours post-transfection, the DNA–calcium phos-
phate mixture was removed and cells were rinsed three
times with NaCl ⁄ P
i
. The cells were then grown for a further
42 h before being analysed by fluorescence microscopy.
SDS/PAGE and Western blotting
Cells were grown in flasks to confluency and lysates were
prepared by scraping the cells into ice-cold lysis solution
(2% SDS, 1 mm EDTA, 1 mm EGTA containing
1 lgÆmL
)1
pepstatin, 10 lgÆmL
)1
leupeptin, 10 lgÆmL
)1
aprotinin and 1 mm phenylmethanesulfonyl fluoride). Pro-
tein concentrations were determined using the BCA assay
(Sigma) according to the manufacturer’s instructions.
Standard curves were constructed using known concentra-
tions of bovine serum albumin. Proteins (20 lg per well)
were separated on SDS–polyacrylamide gels using a discon-
tinuous buffer system [56]. For Western blot analysis, pro-
teins were transferred to nitrocellulose [57]. Subsequently,
the nitrocellulose blots were blocked in 5% nonfat milk in

NaCl ⁄ P
i
⁄ 0.1% Triton X-100 for 1 h. Primary antibody
incubations (1 : 1000) were carried out overnight at room
temperature, followed by 1 h incubations with the appro-
priate horseradish peroxidase-conjugated secondary anti-
body. For antigenic adsorption, the antibody was incubated
with its corresponding blocking peptide (1 : 5 ratio of lg
antibody to lg antigen) for 30 min at room temperature
prior to being incubated with the nitrocellulose blot. Immu-
noreactive bands were visualized using an ECL detection
kit (Pierce, Rockford, IL, USA) according to the manufac-
turers instructions. Developed films were photographed and
captured using the FujiFilm Intelligent dark Box II with
the Image Reader Las-1000 package.
Acknowledgements
This work was funded by the British Heart Founda-
tion and the BBSRC. We thank Dr C. J Edgell for the
gift of the EA.hy.926 cells and Dr R. Williams for
the cPLA
2
-a–GFP plasmid. We are also grateful to
Dr R. C. A. Sainson for providing the annexin V–GFP
S. Grewal et al. cPLA
2
-a localization in endothelial cells
FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS 1287
plasmid and to Dr E. E. Morrison for assistance with
confocal imaging.
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