Colocalization studies with markers for these subcellular organelles, however, showed colocaliza-tion of cPLA2-a with nuclear membrane markers but not with ER or Golgi markers, suggestin
Trang 1localize 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 A2-a (cPLA2-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, cPLA2-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, cPLA2-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, cPLA2-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 cPLA2-a plays a crucial role in endothelium functions such as haemostasis, angiogenesis, control of vascular
Keywords
arachidonic acid; calcium; cPLA2-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: J.H.Walker@bmb.leeds.ac.uk
*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 A2-a (cPLA2-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 cPLA2-a in human EA.hy.926 endothelial cells following stimulation with the calcium-mobilizing agonist, A23187 Relocation of cPLA2-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 cPLA2-a with nuclear membrane markers but not with ER or Golgi markers, suggesting that the relocation of cPLA2-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 cPLA2-a interacting proteins, annexin I, vimentin, p11 or actin, was seen in this cell type In contrast, cPLA2-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 cPLA2-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.
Trang 2tone and prevention of thrombosis formation [5]
Fol-lowing this, cPLA2-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 cPLA2-a and convert arachidonic acid into
prostaglandin H2 [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 cPLA2-a following an increase in
[Ca2+]i However, because a variety of cell types,
anti-bodies and methods have been used, the exact site of
cPLA2-a relocation remains unknown, and appears to
be cell specific In the majority of studies, cPLA2-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 cPLA2-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 cPLA2-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 endothelendothel-ial
cells have reported a redistribution of cPLA2-a to the
nuclear envelope [26,27] The localization of cPLA2-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 cPLA2-a
Fol-lowing this, Tashiro et al have recently confirmed the
presence of nuclear localization and export signals within the cPLA2-a protein [29] Few studies on cPLA2-a in endothelial cells, however, have attempted
to characterize its localization at high spatial resolu-tion Here we study the calcium-induced relocation of cPLA2-a in EA.hy.926 endothelial cells We show that cPLA2-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, cPLA2-a colocalizes with COX-2 but not COX-1
Results
Expression of cPLA2-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 cPLA2-a in endothelial cells, expression levels of cPLA2-a in EA.hy.926 cells and HUVECs were com-pared Equivalent amounts of protein were separated
by SDS⁄ PAGE and immunoblotted for cPLA2-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 cPLA2-a at levels that are similar to those exhibited by their parent VECs In contrast, A549 cells, with which the HU-VECs were fused to generate the EA.hy.926 cells, showed much higher levels of cPLA2-a expression In addition, HeLa cells were seen to express very high levels of cPLA2-a
The precise membrane to which cPLA2-a relocates is dependent on cell type The location and relocation of cPLA2-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 cPLA2-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 cPLA2-a was then detected using a specific goat polyclonal anti-cPLA2-a serum (Fig 1C) As reported previously [26,28,31], cPLA2-a in all cell types was located in the cytosol and at a higher concentra-tion in the nucleus Following A23187 stimulaconcentra-tion, EA.hy.926 cells showed a loss in nuclear staining and relocation of cPLA2-a to the nuclear envelope and nuclear periphery Relocation occurred as soon as 30 s following stimulation and staining patterns observed
Trang 3after 30 min stimulation were similar to those obtained
after shorter stimulations (data not shown) Similar
redistribution of cPLA2-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 [Ca2+] 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 cPLA2-a expression and site of cPLA2-a reloca-tion
Comparison of the location of cPLA2-a in EA.hy.926 endothelial cells with markers for specific cellular organelles
The relocation of cPLA2-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 cPLA2-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 cPLA2-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-cPLA2-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)1thrombin 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.
Trang 4indicated that, although the cPLA2-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 cPLA2-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
cPLA2-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 cPLA2-a occurs to both the inner
and the outer surfaces of the nuclear membrane
In order to assess whether cPLA2-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 cPLA2-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 cPLA2-a with intranuclear membranous invaginations of the nuclear membrane [33]
To further investigate the nuclear localization and nuclear membrane relocation, GFP–cPLA2-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 m M 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-rhodamine-conjugated
WGA For calreticulin staining, cells were
blocked in donkey serum and incubated
with goat polyclonal anti-cPLA2-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.
Trang 5previously for GFP–cPLA2-a [34], cPLA2-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, relocastimula-tion to the nuclear envelope
and cytosolic structures was observed An overlay of
the GFP–cPLA2-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 cPLA2-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 cPLA2-a have suggested that accessory or binding proteins may play a role in the regulation of cPLA2-a activity and localization Ann-exin V, for example, has been shown to exhibit sim-ilar membrane relocation kinetics to cPLA2-a [35] and several reports have demonstrated that annexin V
C
Fig 3 The location and relocation of cPLA2-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 m M extracellular calcium prior to fixation Cells were then fixed and permeabilized with either 0.1% Triton X-100 or 0.05% digitonin cPLA2-a was then detected using affinity-purified goat poly-clonal anti-cPLA2 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 memmem-brane staining (± SEM, n ¼ 90 sections) in Triton X-100 and digitonin-permeabilized cells.
Trang 6is able to interact with and inhibit cPLA2-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 cPLA2-a
[39] Furthermore, studies using recombinant annexin
proteins showed that annexin I–cPLA2-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
cPLA2-a [40] Previous studies by Nakatani and
coworkers [41] have also shown that cPLA2-a
inter-acts with the head domain of the intermediate
fila-ment protein vifila-mentin These studies demonstrated
that this interaction was necessary for cPLA2
-a-medi-ated arachidonic acid release, and suggested that
vimentin may function as an adapter protein for
cPLA2-a at its site of localization Several studies have also implied that cPLA2-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, PLA2 activity was found in the Triton X-100-insoluble cytoskeletal fraction isolated from thrombin-stimulated platelets [43] Finally, cPLA2-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–cPLA2-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)1for 2min) For A23187 stimulations, cells were incubated with 5 l M A23187 for 1 min in the presence of 1 m M 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.
Trang 7lin in EA.hy.926 cells, and determined whether any of
these candidate cPLA2-a-interacting proteins
colocal-ized with cPLA2-a following A23187 stimulation The
results of these studies (Fig 5) demonstrate that,
although a substantial pool of annexin V appeared to
colocalize with cPLA2-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 cPLA2-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 cPLA2-a may associate with these enzymes to form a functional complex We have previously demonstrated that cPLA2
-a coloc-alizes specific-ally 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 cPLA2-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 cPLA2-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 cPLA2-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 cPLA2-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
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.
Trang 8levels of prostacyclin, similar to those exhibited by
HUVECs [48] Finally, using immunofluorescence
microscopy, the location and relocation of cPLA2-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, cPLA2-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 relocastimula-tion 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
cPLA2-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 cPLA2-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 cPLA2-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 cPLA2-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 cPLA2-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 cPLA2-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-cPLA2-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.
Trang 9experiments 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 cPLA2-a with ER, nuclear membrane and
Golgi markers such as Con A, WGA and calreticulin
Thus it appears that cPLA2-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 cPLA2-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 cPLA2-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 cPLA2-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
cPLA2-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 B2, production was shown
to be dependent on coupling between cPLA2-a and
COX-1 In contrast, cPLA2-a and COX-2 were
functionally coupled to produce the
lipopolysaccharide-induced delayed release of prostaglandin E2 [50] In
COS-1 epithelial cells, however, cPLA2-a was found to
be coupled to both COX-1 and COX-2 to produce
prostaglandin E2 [51] The physical colocalization of
COX and cPLA2-a in these systems, however, has not
been studied, and this is one of few studies that shows
distinct colocalization of cPLA2-a with a COX
isoform Functional coupling of cPLA2-a and COX
isoforms in endothelial cells has not yet been
investi-gated, however, it is possible that the preferential A23187-induced colocalization of cPLA2-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 differdiffer-ential colocali-zation with COX isoforms
In conclusion, cPLA2-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 cPLA2-a colocalizes specifically with the COX-2 iso-form but not COX-1 This suggests a novel compart-mentalization of cPLA2-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–cPLA2-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 cPLA2-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
Trang 10(human lung epithelial carcinoma) cells were from ATCC.
EA.hy.926 cells were cultured at 37C in a humid
atmo-sphere containing 5% CO2in 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 (37C) NaCl ⁄ Pi 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⁄ Pi 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
(20C) After fixation, the cells were permeabilized with
0.1% Triton X-100 in NaCl⁄ Pi for 5 min and fixed once
again for 5 min The cells were then washed three times
with NaCl⁄ Pi and incubated in sodium borohydride
solu-tion (1 mgÆmL)1 in NaCl⁄ Pi) for 5 min or in ammonium
chloride (50 mm) for 10 min to reduce autofluorescence
Following three further NaCl⁄ Pi wash steps, the cells
were blocked in 5% rabbit serum in NaCl⁄ Pi for 3 h
The cells were then incubated with primary antibodies
(diluted 1 : 100 into NaCl⁄ Pi⁄ 5% serum) overnight Cells
were washed eight times with NaCl⁄ Pi 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⁄ Pi 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· 105) 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⁄ Pi 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⁄ Pi⁄ 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 cPLA2-a–GFP plasmid We are also grateful to
Dr R C A Sainson for providing the annexin V–GFP