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 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 Seema Grewal*, Shane P Herbert, Sreenivasan Ponnambalam and John H Walker School of Biochemistry and Microbiology, University of Leeds, UK 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 colocalization 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 patterns 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-resembling 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 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-terminal calcium-binding lipid domain (CaLB, or C2 domain), where it then acts upon its phospholipid substrate In addition, cPLA2-a can be regulated by phosphorylation 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 membrane 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 Abbreviations Con A, concanavalin A; COX, cyclooxygenase; cPLA2-a, cytosolic phospholipase A2-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 S Grewal et al tone and prevention of thrombosis formation [5] Following this, cPLA2-a has become an attractive target for the development of novel pharmacological therapeutics against various pathological conditions [6,7] To date, however, very little is known about the exact mechanisms involved in the regulation of this important enzyme Cyclooxygenase (COX) enzymes function downstream of cPLA2-a and convert arachidonic acid into prostaglandin H2 [8,9] To date, two distinct COX isoforms, 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 demonstrated 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, antibodies 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, studies 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 membrane (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 Previous experiments with human umbilical vein endothelial cells (HUVECs) and rabbit coronary endothelial 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 Following this, Tashiro et al have recently confirmed the FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS cPLA2-a localization in endothelial cells 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 resolution 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 compartments 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 compared 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 HUVECs In contrast, A549 cells, with which the HUVECs 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 concerning 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 concentration in the nucleus Following A23187 stimulation, 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 1279 cPLA2-a localization in endothelial cells A S Grewal et al C B D after 30 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 nuclear membrane In addition, a highly punctate staining pattern throughout the cytosol and nucleus of a resting cell was observed HeLa cells showed similar redistribution patterns to the EA.hy.926 and HUVECs, however, a high proportion of intranuclear staining remained following stimulation Similar relocation patterns were observed following stimulation of EA.hy.926 cells with the natural agonists, histamine and thrombin (Fig 1D) This relocation was also seen to be dependent on an influx of extracellular calcium, consistent with previous work on HUVECs [32], which demonstrated that agonistevoked prostacyclin production is dependent on extracellular free [Ca2+] In conclusion, it was observed 1280 Fig cPLA2-a expression levels and subcellular 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-cPLA2-a serum was used to detect cPLA2-a (as described in Experimental procedures) (B) Quantification of the amount of cPLA2-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 fluorescence microscope Scale bar, 20 mm (D) EA.hy.926 cells were stimulated with 10 mM histamine or mL)1 thrombin for in the presence or absence of mM extracellular calcium Cells were then fixed and analysed by microscopy as described above Scale bar, 20 mm 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 relocation 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 further characterize the precise site of membrane relocation 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 FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS S Grewal et al cPLA2-a localization in endothelial cells Fig Double-labelling of cells with ER, Golgi apparatus and plasma membrane markers Cells were stimulated with A23187 (5 lM in the presence of mM extracellular calcium for min), fixed and permeabilized (as described in Experimental procedures) Cells were then incubated with goat polyclonal anti-cPLA2-a followed by FITC-conjugated anti-goat IgG and either rhodamineconjugated Con A or 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, lm indicated 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, complete overlap was not seen, and only patches of colocalization, particularly at the nuclear envelope, were evident Finally, in order to selectively label only the ER, cells were counterstained with a rabbit polyclonal anticalreticulin 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 membrane, cells were permeabilized with digitonin When used at low concentrations, this nonionic detergent selectively permeabilizes membranes with high cholesterol content, such as the plasma membrane Under FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS these conditions the nuclear membrane is not permeabilized 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 digitonin-permeabilized cells was lower than that seen in Triton 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 present 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 presence 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 1281 cPLA2-a localization in endothelial cells A S Grewal et al B C Fig 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 lM A23187 for in the presence of mM 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 polyclonal 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 membrane 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 previously for GFP–cPLA2-a [34], cPLA2-a was present in the cytosol and at a higher concentration in the nucleus of resting cells (Fig 4A) Following stimulation with A23187, relocation 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 demonstrated 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 1282 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 Annexin V, for example, has been shown to exhibit similar membrane relocation kinetics to cPLA2-a [35] and several reports have demonstrated that annexin V FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS cPLA2-a localization in endothelial cells S Grewal et al A B Fig 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 lg of GFP–cPLA2-a plasmid DNA or GFP–annexin V plasmid DNA Forty-eight hours after transfection, the cells were fixed, permeabilized and counterstained with DAPI (1 mgỈmL)1 for 2min) For A23187 stimulations, cells were incubated with lM A23187 for in the presence of 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 is 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 interacts with the head domain of the intermediate filament protein vimentin These studies demonstrated that this interaction was necessary for cPLA2-a-mediated arachidonic acid release, and suggested that vimentin may function as an adapter protein for FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS 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 collagen-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 structurally distinct sites of esterified arachidonic acid and thus represent a nonmembrane site of eicosanoid generation COX isoforms have also been found in similar 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 caveo1283 cPLA2-a localization in endothelial cells A B S Grewal et al C Colocalization with cyclooxygenase isoforms In order to investigate whether cPLA2-a colocalizes with COX proteins, EA.hy.926 cells were stained with antibodies 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 surfaces 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 cPLA2a colocalizes specifically with the COX-1 isoform in A549 epithelial cells thus to determine if similar interactions 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 demonstrated 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 following A23187 stimulation only the COX-2 isoform colocalized 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 nonstimulated cells Fig Colocalization of cPLA2-a Cells were grown on coverslips overnight and stimulated with lM A23187 for in the presence of mM extracellular calcium Cells were then fixed and permeabilized and incubated with goat polyclonal anti-cPLA2-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 antibodies 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 lin in EA.hy.926 cells, and determined whether any of these candidate cPLA2-a-interacting proteins colocalized 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 1284 Discussion The subcellular location of cPLA2-a has been a controversial issue in recent years, and the exact cellular membrane or structure to which this enzyme translocates has not been confirmed In endothelial cells, this protein has been reported to translocate following stimulation 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 previously to be a sufficient model for endothelial cells with regards to its morphology, expression of endothelial-specific markers and response to physiological agonists [30,47], expressed levels of cPLA2-a that were almost identical to those expressed by the parental HUVECs Also, in terms of prostacyclin production, it has been shown previously that the EA.hy.926 cell line is capable of sustaining basal and stimulated FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS cPLA2-a localization in endothelial cells S Grewal et al A Fig Colocalization with cyclooxygenase isoforms Cells were grown on coverslips overnight and were either fixed directly (A) or stimulated with lM A23187 for in the presence of mM 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 TRITCconjugated anti-mouse secondary sera Fluorescence was viewed using a Leica TCS NT confocal fluorescence microscope Scale bar, 20 lm B levels 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 stimulation 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 cPLA2-a relocation has arisen due to a variety of antibodies and cell types being studied Here, comparative 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 FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS 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 expression, 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 membrane 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 metabolism 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 1285 cPLA2-a localization in endothelial cells 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 colocalization 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 production, such as COX-1, COX-2 and prostacyclin synthase, which have all been shown to localize to this subcellular region In addition, any free arachidonic acid released from the membrane must be rapidly converted to arachidonyl-CoA and re-esterified into phospholipids to avoid over synthesis of eicosanoids Because the enzyme responsible for this, arachidonylCoA-1-acyl-lysophosphatide acyltransferase, is present in ER-like structures, compartmentalization of eicosanoid biosynthesis at this site would be beneficial to the cell Consistent with this, we show a partial colocalization 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 whether the location of cytoplasmic COX-2 changes upon stimulation COX-2 staining patterns observed before stimulation appear similar to patterns seen after stimulation 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 systems 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 lipopolysaccharideinduced 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 investi1286 S Grewal et al 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 different localization patterns and thus differential colocalization 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 membrane markers Interestingly, however, we show that cPLA2-a colocalizes specifically with the COX-2 isoform but not COX-1 This suggests a novel compartmentalization of cPLA2-a, which could possibly aid the process of eicosanoid generation by placing this enzyme in an appropriate position for catalysis, perhaps in close proximity to lipid-rich bodies or microdomains, and other proteins involved in eicosanoid biosynthesis Experimental procedures Reagents Tissue culture media, enzymes and antibiotics were purchased from Invitrogen (Paisley, UK) The N-terminal GFP–cPLA2-a plasmid construct was a gift from Dr R Williams (MRC LMB, Cambridge, UK) The pEGFP-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 Biotechnology (Santa Cruz, CA, USA) Mouse monoclonal antivimentin serum was obtained from Sigma (Poole, UK) Anti-caveolin sera were a gift from N M Hooper (University 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 Citifluor 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 FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS S Grewal et al (human lung epithelial carcinoma) cells were from ATCC EA.hy.926 cells were cultured at 37 °C in a humid atmosphere containing 5% CO2 in air Cells were grown in Dulbecco’s modified Eagles’ medium (DMEM) supplemented with 10% foetal bovine serum, penicillin (100 mL)1), streptomycin (100 lgỈmL)1) and HAT (100 lm hypoxanthine, 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, penicillin (100 mL)1) and streptomycin (100 lgỈmL)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 ⁄ Pi and fixed in prewarmed 10% formalin in neutral-buffered saline ( 4% formaldehyde, Sigma) for For A23187 stimulations, cells were washed with NaCl ⁄ Pi and incubated with lm A23187 in Hepes ⁄ Tyrode’s buffer containing mm calcium for 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 ⁄ Pi for and fixed once again for The cells were then washed three times with NaCl ⁄ Pi and incubated in sodium borohydride solution (1 mgỈmL)1 in NaCl ⁄ Pi) for or in ammonium chloride (50 mm) for 10 to reduce autofluorescence Following three further NaCl ⁄ Pi wash steps, the cells were blocked in 5% rabbit serum in NaCl ⁄ Pi for h The cells were then incubated with primary antibodies (diluted : 100 into NaCl ⁄ Pi ⁄ 5% serum) overnight Cells were washed eight times with NaCl ⁄ Pi then incubated with AlexaFluor 488 and 594-conjugated secondary antibodies, or rhodamine-conjugated WGA or Con A (10 lgỈmL)1) for 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 represent 0.485 lm sections taken through the centre of the nucleus To capture double-labelled samples, sequential scanning of each fluorescence channel was performed (according to the manufacturer’s guidelines) to avoid cross-contamination of fluorescence signals For p11- and annexin II- FEBS Journal 272 (2005) 1278–1290 ª 2005 FEBS cPLA2-a localization in endothelial cells 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 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 lg plasmid DNA per coverslip Six hours post-transfection, the DNA–calcium phosphate 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, mm EDTA, mm EGTA containing lgỈmL)1 pepstatin, 10 lgỈmL)1 leupeptin, 10 lgỈmL)1 aprotinin and mm phenylmethanesulfonyl fluoride) Protein concentrations were determined using the BCA assay (Sigma) according to the manufacturer’s instructions Standard curves were constructed using known concentrations of bovine serum albumin Proteins (20 lg per well) were separated on SDS–polyacrylamide gels using a discontinuous buffer system [56] For Western blot analysis, proteins were transferred to nitrocellulose [57] Subsequently, the nitrocellulose blots were blocked in 5% nonfat milk in NaCl ⁄ Pi ⁄ 0.1% Triton X-100 for h Primary antibody incubations (1 : 1000) were carried out overnight at room temperature, followed by h incubations with the appropriate horseradish peroxidase-conjugated secondary antibody For antigenic adsorption, the antibody was incubated with its corresponding blocking peptide (1 : ratio of lg antibody to lg antigen) for 30 at room temperature prior to being incubated with the nitrocellulose blot Immunoreactive bands were visualized using an ECL detection kit (Pierce, Rockford, IL, USA) according to the manufacturers 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 Foundation and the BBSRC We thank Dr C J Edgell for the gift of the 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(1996) ? ?Cytosolic? ?? phospholipase A2 is in the nucleus of subconfluent endothelial cells but confined to the cytoplasm of confluent endothelial cells and redistributes to the nuclear envelope and cell... vein endothelial cells (HUVECs) and rabbit coronary endothelial cells have reported a redistribution of cPLA2-a to the nuclear envelope [26,27] The localization of cPLA2-a in human endothelial cells. .. 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,