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Originally published In Press as doi:10.1074/jbc.M701541200 on September 16, 2007

J. Biol. Chem., Vol. 282, Issue 47, 34468-34478, November 23, 2007
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The Confluence-dependent Interaction of Cytosolic Phospholipase A2-{alpha} with Annexin A1 Regulates Endothelial Cell Prostaglandin E2 Generation*Formula

Shane P. Herbert, Adam F. Odell, Sreenivasan Ponnambalam, and John H. Walker1

From the Faculty of Biological Sciences, Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom

Received for publication, February 21, 2007 , and in revised form, September 13, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The regulated generation of prostaglandins from endothelial cells is critical to vascular function. Here we identify a novel mechanism for the regulation of endothelial cell prostaglandin generation. Cytosolic phospholipase A2-{alpha} (cPLA2{alpha}) cleaves phospholipids in a Ca2+-dependent manner to yield free arachidonic acid and lysophospholipid. Arachidonic acid is then converted into prostaglandins by the action of cyclooxygenase enzymes and downstream synthases. By previously undefined mechanisms, nonconfluent endothelial cells generate greater levels of prostaglandins than confluent cells. Here we demonstrate that Ca2+-independent association of cPLA2{alpha} with the Golgi apparatus of confluent endothelial cells correlates with decreased prostaglandin synthesis. Golgi association blocks arachidonic acid release and prevents functional coupling between cPLA2{alpha} and COX-mediated prostaglandin synthesis. When inactivated at the Golgi apparatus of confluent endothelial cells, cPLA2{alpha} is associated with the phospholipid-binding protein annexin A1. Furthermore, the siRNA-mediated knockdown of endogenous annexin A1 significantly reverses the inhibitory effect of confluence on endothelial cell prostaglandin generation. Thus the confluence-dependent interaction of cPLA2{alpha} and annexin A1 at the Golgi acts as a novel molecular switch controlling cPLA2{alpha} activity and endothelial cell prostaglandin generation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytosolic phospholipase A2-{alpha} (cPLA2{alpha})2 is an 85-kDa, Ca2+-sensitive member of the phospholipase A2 (PLA2) family of enzymes (1, 2) which includes the Ca2+-independent (iPLA2) and secretory phospholipases A2 (3). The PLA2 enzymes hydrolyze the sn-2 fatty acyl bond of phospholipids to simultaneously generate free fatty acid and lysophospholipids (4). Upon agonist stimulation and cytosolic Ca2+ elevation, cPLA2{alpha} translocates to intracellular membranes utilizing an N-terminal Ca2+-dependent lipid binding (CalB) domain (5-7). Upon membrane binding, cPLA2{alpha} preferentially cleaves phospholipids containing arachidonic acid (AA) at the sn-2 position to liberate free AA (4). Consequently, cPLA2{alpha} is seen as the rate-limiting enzyme in receptor-mediated AA release (8).

Ca2+ elevation can induce relocation of cPLA2{alpha} to the specific intracellular membranes in which the downstream AA-metabolizing cyclooxygenase (COX) enzymes are also located (2). There are two isoforms of COX that have been extensively characterized (COX-1 and -2) (9), and more recently an alternative COX-1 splice variant, COX-3, has also been cloned (10). The spatiotemporal co-localization of cPLA2{alpha} with COX can couple these enzymes to facilitate efficient conversion of AA into prostaglandins (2, 11). Chimeric cPLA2{alpha} mutants specifically targeted to intracellular membranes in which COX does not reside do not couple with COX and drastically reduce prostaglandin production (11). Thus, the subcellular targeting of cPLA2{alpha} to specific intracellular membranes is essential for the regulation of both AA and prostaglandin production. Despite this, the subcellular targeting of cPLA2{alpha} in endothelial cells and its functional coupling with downstream COX enzymes has received little attention.

Release of prostaglandins by endothelial cells, the cells lining the luminal surface of all blood vessels, is essential to the control of vascular tone and thrombus formation (12, 13). Therefore, regulation of endothelial prostaglandin generation is critical to the maintenance of normal vascular function. Nonconfluent endothelial cells generate much greater levels of AA and prostaglandin than confluent cells (14-16), which has been attributed to elevated cPLA2{alpha} activity. Despite this, the actual mechanism of this differential regulation of cPLA2{alpha} activity has not been defined.

Inhibition of cPLA2{alpha} activity by the phospholipid-binding protein, annexin A1, and the resulting block in AA metabolite release is a mechanism by which glucocorticoids exert their anti-inflammatory action (17-19). Annexin A1 is known to inhibit cPLA2{alpha} activity upon interaction with the CalB domain of cPLA2{alpha} in vitro (20, 21); however, the relevance of this interaction to processes other than inflammation remains unclear. Here we demonstrate that annexin A1 acts as a novel regulator of endothelial cell AA and prostaglandin generation upon interaction with cPLA2{alpha} at the Golgi apparatus of confluent cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Materials—Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords as previously described (22, 23). Human dermal micro-vascular endothelial cells were purchased from PromoCell. Cells were cultured in endothelial cell basal medium supplemented with endothelial cell growth factor kit 2 (PromoCell). All cells were grown on 0.1% (w/v) gelatin-coated cultureware and were not used in excess of four passages. The following antibodies were purchased: anti-annexin A1 (ANEX 5E4/1; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-cPLA2{alpha} (C20; Santa Cruz Biotechnology), anti-cPLA2{alpha} (ab9014; Abcam), anti-mannosidase II (Serotec Ltd.), anti-calreticulin (Stressgen), anti-vimentin (Sigma), anti-p11 (SWant), anti-COX-2 and anti-COX-2 (Cayman Chemical), horseradish peroxidase-conjugated secondary antibodies (Pierce), and Alexa-fluor-conjugated secondary antibodies (Molecular Probes). Anti-ERGIC-53 and rabbit anti-annexin A1 antibodies were provided by H. P. Hauri (Basel, Switzerland) and E. Solito (Imperial College, London), respectively. Anti-TGN46 antibodies were supplied by S. Ponnambalam (University of Leeds, UK). N-terminal and C-terminal GFP-tagged cPLA2{alpha} constructs were kind gifts from R. Williams (Medical Research Council Laboratory of Molecular Biology, Cambridge, UK) and T. Hirabayashi (University of Tokyo), respectively. Arachidonyl trifluoromethylketone (AACOCF3) and bromoenol lactone (BEL) were purchased from BioMol. All other reagents were obtained from Sigma or Invitrogen unless otherwise stated.

Biochemistry—Lysate preparation and Western analysis were performed as described previously (16). Briefly, samples (20 µg of protein) were resolved for 60 min at 30 mA/gel on 10% SDS-polyacrylamide minigels using a discontinuous buffer system (24). For immunoblotting, protein was transferred onto nitrocellulose membranes for 3 h at 300 mA (25). Membranes were blocked in 5% (w/v) nonfat milk in phosphate-buffered saline for 30 min and then incubated overnight with primary antibody (1:500) at room temperature. After incubation with horseradish peroxidase-conjugated anti-goat IgG (1:3000) for 1 h, immunoreactive bands were visualized using a West Pico enhanced chemiluminescence detection kit (Pierce). Images were captured on a Fuji Film Intelligent dark box II image reader. Band intensities were determined densitometrically using Aida (Advanced Image Data Analyzer) 2.11 software. For immunoprecipitations, confluent HUVECs were lysed in lysis buffer (25 mM Tris-HCl, pH 7.4, 0.5% Nonidet P-40, 150 mM NaCl, 2 mM EGTA, 2 mM EDTA, 1:250 protease inhibitor mixture) for 30 min on ice. The annexin A1 immunoprecipitations were performed in 0.5% CHAPS lysis buffer (10 mM Tris-HCl, pH 7.4, 0.5% CHAPS, 140 mM NaCl, 0.5 mM CaCl2, 0.5 mM MgCl2). cPLA2{alpha} was immunoprecipitated from cell lysates with anti-cPLA2{alpha} antibodies overnight at 4 °C. Supernatants were then incubated with protein G-agarose for 3 h at 4 °C. Bead complexes were washed with ice-cold lysis buffer and boiled with SDS-PAGE sample buffer prior to immunoblotting. For subcellular fractionation, confluent endothelial cells were mechanically disrupted in homogenization buffer (0.25 M sucrose, 5 mM Tris-HCl, pH 7.4, 25 mM KCl, 5 mM MgCl2, 4.5 mM CaCl2) in the presence or absence of 1 mM glutaraldehyde and fractionated on a discontinuous sucrose gradient according to Ref. 26. Interfaces were removed, diluted in homogenization media, and collected by centrifugation at 100,000 x g for 60 min. Isolated fractions were analyzed by SDS-PAGE and immunoblotting. Iodixanol gradients were performed essentially as described by Yang et al. (27), except a 10-30% gradient was utilized. Differential centrifugation enrichment of membrane fractions was performed as described previously by Lamour et al. (28).

Immunofluorescence—Immunofluorescence microscopy was performed as previously described (16). Briefly, cells were grown to the required level of confluence on 0.1% (w/v) gelatin-coated coverslips. Cells were then fixed in 10% (v/v) formalin in neutral buffered saline (HT50-1-128; Sigma) for 5 min at 37 °C. Prior to fixation, some cells were stimulated for 1 min with 5 µM A23187 [GenBank] as previously (29, 30). All ensuing steps were performed at 25 °C. After permeabilization with 0.1% (v/v) Triton X-100 for 5 min, cells were refixed (5 min), washed with phosphate-buffered saline, and then incubated in 50 mM ammonium chloride for 10 min. Following phosphate-buffered saline washes, nonspecific binding sites were blocked with 5% (v/v) donkey serum for 3 h. Cells were incubated overnight with primary antibody followed by the appropriate secondary antibodies. Finally, coverslips were mounted on microscope slides in Fluoromount-G mounting medium (Southern Biotech).

Microscopy and Quantitation—Deconvolution fluorescence microscopy was performed using an Olympus IX-70 inverted fluorescence microscope (63 x 1.5 oil immersion lens) and DeltaVision deconvolution system (Applied Precision Inc.). Individual optical sections of 0.2 µm were generated from 15 iterative cycles of deconvolution. Quantification of co-localization was determined using the IMARIS software suite (Bitplane AG). Gray scale values below 10% of the maximum pixel intensity were eliminated as background. Co-localized pixels were expressed as percentages of the total pixels selected. Some images were captured using an inverted Zeiss LSM 510 META Axiovert 200M confocal microscope.

Determination of Cytosolic Ca2+ Concentration—HUVECs cultured on glass bottom dishes were washed with HEPES/Tyrode's buffer and incubated with 2.5 µM Fluo3-AM (Molecular Probes) for 30 min. Subsequently, cells were washed and then incubated with HEPES/Tyrode's buffer (plus 1 mM Ca2+) for 20 min. Cells were placed in a heated chamber (37 °C) above an Olympus IX-70 inverted fluorescence microscope. Fluctuations in cytosolic Ca2+ were monitored by acquisition of fluorescence images.

AA Release—This technique was performed as previously (16). Briefly, HUVECs were labeled for 24 h with 1 µCi/ml [3H]AA, washed with phosphate-buffered saline, and then incubated with 10 µM BEL for 30 min to inhibit background iPLA2 activity. Cells were then stimulated with 5 µM A23187 [GenBank] in serum-free media (plus 0.3% (w/v) fatty acid-free bovine serum albumin). Aliquots of media and cell lysate were counted by liquid scintillation for radioactivity.

Prostaglandin E2 Generation—HUVECs were cultured to the required cell density in 6-well culture dishes. Cells were washed and then in some cases incubated with 50 µM AACOCF3 and/or 10 µM BEL for 30 min prior to treatment with 5 µM A23187 [GenBank] in HEPES/Tyrode's buffer with 1 mM CaCl2 for 15 min. Aliquots of media were assayed for prostaglandin E2 (PGE2) content using a high sensitivity ELISA (Assay Designs).


Figure 1
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  		FIGURE 1.
cPLA2{alpha} targets to the ER/ERGIC and functionally couples with the COX enzymes in nonconfluent endothelial cells. A, subconfluent HUVECs were directly fixed or stimulated with 5 µM A23187 for 1 min prior to fixation. cPLA2{alpha} and either calreticulin or ERGIC-53 were detected by immunofluorescence microscopy. Quantification of cPLA2{alpha} co-distribution with calreticulin and ERGIC-53 was performed, and the percentage of co-localization was calculated using the IMARIS computer package as described under "Experimental Procedures" (n = 13, ±S.E.). B, subconfluent cells loaded with 1 µCi/ml [3H]AA were stimulated with 5 µM A23187 for 15 min, and released AA was determined by scintillation counting. Results are expressed as a percentage of the total [3H]AA incorporated (n = 3, ±S.E.). *, p < 0.001 versus unstimulated cells. C, subconfluent HUVECs were directly fixed or stimulated with 5 µM A23187 for 1 min prior to fixation. cPLA2{alpha} and either COX-1 or -2 were detected by immunofluorescence microscopy, and the co-distribution of cPLA2{alpha} with COX-1 and -2 was quantified. Percentage co-localization was calculated using the IMARIS computer package as described under "Experimental Procedures" (n = 15, ±S.E.). D, generation of PGE2 by subconfluent HUVECs in response to A23187 stimulation. Cells were treated with combinations of AACOCF3 and/or BEL for 30 min prior to stimulation with A23187 for 15 min in the presence or absence of 10 µM AA. PGE2 generation was quantified using a high sensitivity ELISA (n = 3, ±S.E.). *, p < 0.001 versus unstimulated cells. **, p < 0.001 versus A23187-stimulated cells. All results are representative of three separate experiments.

 
RNA Interference—HUVECs were transfected with either no siRNA (control), 50 nM nontargeting control siRNA (mock; D-001210-01; Dharmacon), or 50 nM annealed annexin A1 siRNA (siRNA; 593139; Ambion) for 4 h using the Lipo-fectamine2000 transfection reagent (Invitrogen). Cells were recovered for 48 h prior to lysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cytosolic Ca2+ Elevation Targets cPLA2{alpha} to Intracellular Membranes in Subconfluent Endothelial Cells—cPLA2{alpha} activity is greater in subconfluent endothelial cells than in quiescent, confluent endothelial cells (15, 16). We have previously shown that subconfluent and confluent endothelial cells (see supplemental Fig. 1) express equal amounts of cPLA2{alpha} (16), indicating that mechanisms other than control of cPLA2{alpha} expression are responsible for confluence-dependent changes in its activity. In response to elevated cytosolic Ca2+, cPLA2{alpha} is activated by relocation to intracellular membranes. Recruitment to specific membranes is required for the regulation of cPLA2{alpha} activity (2, 11); however, the precise membranes to which cPLA2{alpha} relocates in primary endothelial cells have not been defined. Therefore, we investigated the Ca2+-induced relocation of cPLA2{alpha}.

In HUVECs, cPLA2{alpha} was detectable as a 110 kDa band by Western blotting using a well characterized affinity-purified antibody specific to the C-terminal region of cPLA2{alpha} (16, 31). Additionally, the immunoreactivity was removed by preabsorption of the antibody with the antigenic peptide (supplemental Fig. 2A). By immunofluorescence microscopy, cPLA2{alpha} was present as both diffuse and structured pools throughout the cytoplasm and nucleus of subconfluent HUVECs (supplemental Fig. 2B), similar to previous observations with endothelial cells and fibroblasts (16, 32, 33). To study the relocation of cPLA2{alpha} in response to cytosolic Ca2+ elevation, we used the Ca2+ ionophore, A23187. [GenBank] This agent is ideal for studying confluence-dependent changes in cPLA2{alpha} relocation, since A23187 [GenBank] raises cytosolic Ca2+ to similar levels in both subconfluent and confluent endothelial cells (supplemental Fig. 2C). Signaling mediated by other agonists that elevate cytosolic Ca2+ varies with endothelial cell density (34, 35). In subconfluent HUVECs, upon elevation of cytosolic Ca2+, cPLA2{alpha} relocated to the nuclear periphery and less disperse cytoplasmic structures (see supplemental Fig. 2). Relocation occurred rapidly (<1 min) and cPLA2{alpha} immunoreactivity co-distributed extensively with calreticulin and ERGIC-53 (Fig. 1A and supplemental Fig. 2, D-E), consistent with translocation to the endoplasmic reticulum (ER) and ER-Golgi intermediate compartments (ERGIC) (36-38). Quantitation of this co-localization revealed a 2.5-fold increase in overlap between cPLA2{alpha} and calreticulin and a 2.3-fold increase in overlap between cPLA2{alpha} and ERGIC-53 upon A23187 [GenBank] treatment (Fig. 1A). A23187 [GenBank] -induced relocation of cPLA2{alpha} to the ER and ERGIC promoted its interaction with membrane substrate, resulting in a 12-fold increase in AA release from subconfluent cells (Fig. 1B).

cPLA2{alpha} Is Coupled to COX-1 and -2 in Subconfluent Endothelial Cells—AA may be converted into prostaglandin H2 by the action of the COX enzymes. Targeting of cPLA2{alpha} to the specific intracellular membranes in which COX enzymes are located can lead to the coupling of these enzymes to facilitate efficient conversion of AA into prostaglandins (2, 11). Functional coupling does not require the direct interaction of AA synthesizing and metabolizing enzymes but relies on both enzymes being in close apposition. Consequently, AA released by cPLA2{alpha} is statistically more likely to encounter the required downstream enzyme than if synthesized at a distant site. Despite the central importance of prostaglandins to numerous vascular processes, including angiogenesis (12, 13), this functional coupling between AA release and prostaglandin synthesis has never been investigated in endothelial cells. We predicted that recruitment of cPLA2{alpha} to the ER of subconfluent HUVECs would promote functional coupling, since both COX-1 and -2 are located at the ER of endothelial cells (39).


Figure 2
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  		FIGURE 2.
Sequestration of cPLA2{alpha} at the Golgi apparatus inhibits membrane recruitment. A, the localization of cPLA2{alpha} insubconfluent (sub-confl.) and confluent (confl.) HUVECs was determined by immunofluorescence microscopy. B, HUVECs were transfected with either recombinant GFP alone or recombinant cPLA2{alpha} tagged with GFP to the N terminus (GFP-cPLA2{alpha}) or C terminus (cPLA2{alpha}-GFP). After 48 h, transfected HUVECs were directly fixed, and both GFP and TGN46 were detected by immunofluorescence microscopy. C and D, confluent HUVECs were directly fixed or stimulated with 5 µM A23187 for 1 min prior to fixation. cPLA2{alpha} and either calreticulin (C) or ERGIC-53 (D) were detected by immunofluorescence microscopy. E, quantification of cPLA2{alpha} co-distribution with calreticulin and ERGIC-53. Percentage co-localization was calculated using the IMARIS computer package as described under "Experimental Procedures" (n = 13, ±S.E.). *, p < 0.001 versus subconfluent cells. All results are representative of three separate experiments. All images represent 0.2-µm sections through cell nuclei. *, EC nuclei. Scale bar, 25 µm.

 
Subconfluent cells were stimulated to elevate intracellular Ca2+, and the co-distribution of cPLA2{alpha} with COX-1 and -2 was assessed (Fig. 1C and supplemental Fig. 2, F and G). Quantitation revealed a 3.2-fold increase in overlap between cPLA2{alpha} and COX-1 and a 2.3-fold increase between cPLA2{alpha} and COX-2 upon cytosolic Ca2+ elevation (Fig. 1C). We then assessed the ability of subconfluent HUVECs to generate PGE2, a major downstream product of both COX-1 and -2 activity (Fig. 1D). PGE2 plays a key role in a variety of key vascular processes, such as angiogenesis and the regulation of vascular tone (40, 41). Prior to A23187 [GenBank] treatment, PGE2 generation was minimal (0.35 pg/1000 cells) but rose 24-fold upon cytosolic Ca2+ elevation. Pretreatment of HUVECs with BEL, an inhibitor of iPLA2 activity (42), had no effect on Ca2+-induced PGE2 generation. This suggests that iPLA2-mediated AA release is not involved in PGE2 production in endothelial cells. Inhibition of cPLA2{alpha} with AACOCF3 (43) inhibited PGE2 generation by 88% (Fig. 2D). Thus, Ca2+-induced PGE2 generation was almost entirely dependent on cPLA2{alpha} activity. The maximal capacity of the cells to produce PGE2 was assessed by incubating cells with excess exogenous AA. Exogenous AA reversed the inhibitory effect of AACOCF3 but did not elevate PGE2 generation any higher than that liberated by cytosolic Ca2+ elevation. Thus, specific targeting of cPLA2{alpha} to the ER/ERGIC and co-localization with the COX enzymes appears to result in maximal conversion of AA into PGE2. This demonstrates coupling between these enzymes in endothelial cells and indicates that release of AA by cPLA2{alpha} is the rate-limiting step in PGE2 production in endothelial cells.


Figure 3
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  		FIGURE 3.
Sequestration of cPLA2{alpha} at the Golgi apparatus inhibits functional coupling. A and B, confluent HUVECs were directly fixed or stimulated with 5 µM A23187 for 1 min prior to fixation. cPLA2{alpha} and either COX-1 (A) or -2 (B) were detected by immunofluorescence microscopy. C, quantification of cPLA2{alpha} co-distribution with COX-1 and -2. Percentage co-localization was calculated using the IMARIS computer package as described under "Experimental Procedures" (n = 15, ±S.E.). D, generation of PGE2 from HUVECs in response to A23187 stimulation. Subconfluent and confluent cells were treated with combinations of AACOCF3 and/or BEL for 30 min prior to stimulation with A23187. PGE2 release was quantified using a high sensitivity ELISA (n = 3, ±S.E.). E, assessment of endogenous COX activity. Subconfluent and confluent HUVECs were pretreated with AACOCF3 and BEL for 30 min to inhibit endogenous cPLA2{alpha} and iPLA2 activity. Cells were then incubated with 5 µM A23187 for 15 min in the presence or absence of 10 µM AA. PGE2 generation was assessed using a high sensitivity ELISA (n = 3, ± S.E.). *, p < 0.001 versus subconfluent cells. All results are representative of three separate experiments. All images represent 0.2-µm sections through cell nuclei. *, EC nuclei. Scale bar, 25 µm.

 
Sequestration of cPLA2{alpha} at the Golgi Apparatus of Quiescent Endothelial Cells Blocks Its Translocation to Other Membranes—By immunofluorescence microscopy, in confluent endothelial cells, cPLA2{alpha} was seen to become associated with a reticular juxtanuclear region (Fig. 2A) corresponding to the Golgi apparatus. Similar results were obtained with recombinant GFP-tagged cPLA2{alpha} (N-terminal and C-terminal linked constructs; Fig. 2B) and with a separate antibody targeted to the C terminus of cPLA2{alpha} (supplemental Fig. 3). In other cell types, association of cPLA2{alpha} with the Golgi apparatus promotes AA release (44, 45), whereas in confluent endothelial cells, cPLA2{alpha} activity is inhibited (14-16). Furthermore, interaction with the Golgi blocked targeting of cPLA2{alpha} to the ER and ERGIC upon Ca2+ elevation (Fig. 2, C-E). In confluent HUVECs, the co-distribution of cPLA2{alpha} with calreticulin (Fig. 2C) and ERGIC-53 (Fig. 2D) positive structures was not enhanced upon cytosolic Ca2+ elevation. Quantitation revealed that overlap between cPLA2{alpha} and calreticulin was only 4% that of subconfluent cells upon cytosolic Ca2+ elevation (Fig. 2E). Overlap between cPLA2{alpha} and ERGIC-53 was also reduced by 47% relative to A23187 [GenBank] -treated subconfluent cells (Fig. 2E). The reduction in overlap with ERGIC-53 was not to the extent seen with calreticulin but is probably due to background overlap with ERGIC-53-positive vesicles that have fused with the Golgi apparatus. Most importantly, when quantified, overlap of cPLA2{alpha} with the ER and ERGIC was not enhanced upon cytosolic Ca2+ elevation (Fig. 2E). Thus, association with the Golgi apparatus sequesters cPLA2{alpha} away from its intracellular substrate, accounting for the reduced AA release seen at endothelial cell confluence (15, 16).


Figure 4
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  		FIGURE 4.
Immobilization of cPLA2{alpha} at the Golgi apparatus of confluent endothelial cells is Ca2+-independent. A, confluent HUVECs were directly fixed or stimulated with 5 µM A23187 for 1 min prior to fixation. cPLA2{alpha} and mannosidase II were then detected by immunofluorescence microscopy. B, quantification of cPLA2{alpha} co-distribution with mannosidase II. Percentage co-localization was calculated using the IMARIS computer package as described under "Experimental Procedures" (n = 13, ±S.E.). C, confluent HUVECs were either directly fixed or incubated with 25 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid acetoxymethyl ester (BAPTA-AM) or 3 mM EGTA for 1 h prior to fixation. cPLA2{alpha} was then detected by immunofluorescence microscopy. The percentage of the total cells displaying Golgi-localized cPLA2{alpha} was then quantified. All results are representative of three separate experiments. All images represent 0.2-µm sections through cell nuclei. *, EC nuclei. Scale bar, 25 µm. *, p < 0.01 versus nonconfluent cells.

 
Sequestration of cPLA2{alpha} at the Golgi Apparatus Inhibits Its Functional Coupling with the COX Enzymes—Confluent endothelial cells generate lower levels of prostaglandins than subconfluent cells (14, 15). We predicted that the sequestration of cPLA2{alpha} at confluence would be important to the control of prostaglandin generation. In confluent HUVECs, no significant overlap between cPLA2{alpha} and COX-1 or -2 was observed prior to A23187 [GenBank] stimulation (Fig. 3, A and B). Furthermore, sequestration at the Golgi apparatus blocks the Ca2+-induced co-localization of cPLA2{alpha} with COX-1 and -2 (Fig. 3, A and B). Quantitation revealed that overlap of cPLA2{alpha} with COX-1 and -2 was only 6 and 22% that of subconfluent cells upon cytosolic Ca2+ elevation (Fig. 3C). As a result, in confluent HUVECs, Ca2+-induced PGE2 generation was inhibited by 95.6% relative to subconfluent cells (Fig. 3D). Furthermore, cytosolic Ca2+ elevation did not induce any greater PGE2 generation than unstimulated controls (Fig. 3D). The low levels of PGE2 generated from confluent HUVECs were not due to iPLA2- or cPLA2{alpha}-mediated AA release, since preincubation with BEL and AACOCF3 had no effect (Fig. 3D). These results were not due to variations in endogenous COX activity, since both subconfluent and confluent HUVECs generate similar levels of PGE2 when supplied with exogenous AA (Fig. 3E). Thus, sequestration of cPLA2{alpha} at the Golgi apparatus of endothelial cells represents a novel mechanism for the regulation of prostaglandin generation by blocking its targeting to the specific intracellular membranes at which COX-1 and -2 reside.

The Interaction of cPLA2{alpha} with the Golgi Apparatus is Ca2+-independent—Treatment of subconfluent HUVECs with A23187 [GenBank] does not enhance the co-distribution of cPLA2{alpha} with the Golgi-resident protein mannosidase II (46) (Fig. 4A, ManII). This is despite other reports documenting the Ca2+-dependent association of cPLA2{alpha} with the Golgi apparatus of other cell types (29, 47). In quiescent, confluent HUVECs, cPLA2{alpha} co-distributed extensively with ManII both before and after A23187 [GenBank] treatment (Fig. 4A). Quantitation revealed that the overlap between cPLA2{alpha} and ManII was not influenced by intracellular Ca2+ elevation (Fig. 4B). Thus, in confluent HUVECs, cPLA2{alpha} is immobilized at the Golgi apparatus and is insensitive to Ca2+ elevation. The interaction of cPLA2{alpha} with the Golgi is entirely Ca2+-independent, since treatment of confluent monolayers with the intracellular Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, did not affect the Golgi association of cPLA2{alpha} (Fig. 4C). Furthermore, the Golgi association of cPLA2{alpha} was not affected by incubation with the extracellular Ca2+ chelator, EGTA (Fig. 4C), which is known to decrease intracellular Ca2+ levels (7).


Figure 5
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  		FIGURE 5.
cPLA2{alpha} interacts with annexin A1 at the Golgi apparatus of confluent endothelial cells. A, confluent HUVECs were directly fixed, and then cPLA2{alpha} and either vimentin, p11, or annexin A1 were detected by immunofluorescence microscopy. B, confluent and subconfluent HUVECs were directly fixed, and then annexin A1 and TGN46 were detected by confocal immunofluorescence microscopy. C, cPLA2{alpha} was immunoprecipitated (I.P.) from equal volumes of a confluent HUVEC lysate with either anti-cPLA2{alpha} antibody or anti-cPLA2{alpha} antibody preadsorbed with antigenic peptide. Immunoprecipitated proteins and resulting supernatant were subjected to SDS-PAGE and immunoblotted (W.B.) for cPLA2{alpha} and annexin A1. D, equal quantities of either confluent or subconfluent HUVEC lysates were immunoprecipitated with either anti-annexin A1 antibody or control mouse IgG (mse), and bound proteins were subjected to SDS-PAGE and immunoblotted with the indicated antibodies. All results are representative of three separate experiments. All images represent 0.2-µm sections through cell nuclei. *, EC nuclei. Scale bar, 25 µm.

 
Since this interaction has no requirement for Ca2+, it cannot be mediated by the Ca2+-dependent lipid binding property of the CalB domain of cPLA2{alpha}. Thus, we hypothesized that association with the Golgi apparatus may occur via a novel mechanism.

cPLA2{alpha} Interacts with Annexin A1 at the Golgi Apparatus of Confluent Endothelial Cells—There is previous evidence to suggest that cPLA2{alpha} activity can be modulated by its interaction with a number of binding partners. The CalB domain of cPLA2{alpha} binds the head domain of vimentin in far Western and co-immunoprecipitation experiments (48). In addition, overexpression of the head domain of vimentin inhibits AA and prostanoid production in rat fibroblasts (48). However, when confluent HUVECs were co-stained for cPLA2{alpha} and vimentin, no evidence for a co-association was apparent at the Golgi apparatus (Fig. 5A). From yeast two-hybrid and immunoprecipitation experiments, the catalytic domain of cPLA2{alpha} has also been found to bind p11, a member of the S100 family of calcium-binding proteins (49). In vitro, p11 inhibits cPLA2{alpha}, and in vivo knockdown of p11 elevates AA release in human bronchial epithelial cell lines. However, from immunofluorescence co-staining experiments, no evidence for an association of p11 with cPLA2{alpha} was found at the Golgi apparatus of confluent HUVECs (Fig. 5A).

The phospholipid-binding protein, annexin A1, is known to interact with the CalB domain of cPLA2{alpha} in vitro (20, 21). In addition, annexin A1 has been implicated in the regulation of cellular cPLA2{alpha} activity in a number of studies (17, 19). We therefore compared the locations of cPLA2{alpha} and annexin A1 in confluent endothelial cells (Fig. 5A). Results showed that a pool of annexin A1 was located at the Golgi apparatus of confluent endothelial cells and that this pool extensively co-distributed with cPLA2{alpha} and the Golgi marker protein, TGN46 (Fig. 5, A and B). In subconfluent endothelial cells, annexin A1 is not enriched at the Golgi apparatus to the same extent as confluent cells (Fig. 5B). Cellular levels of annexin A1 protein do not vary between confluent and subconfluent endothelial cells (supplemental Fig. 4); therefore, an enrichment of annexin A1 at the Golgi apparatus of confluent HUVECs must be a consequence of the cell density-dependent redistribution of annexin A1. Evidence for a physical association between annexin A1 and cPLA2{alpha} was further obtained upon co-immunoprecipitation of annexin A1 with cPLA2{alpha} from confluent HUVEC lysates (Fig. 5C). To confirm the specificity of the interaction, immunoprecipitations were also performed after preincubation of anti-cPLA2{alpha} antibodies with antigenic peptide. Under these conditions, neither cPLA2{alpha} nor annexin A1 were immunoprecipitated (Fig. 5C). To further assess the confluence dependence of the association between annexin A1 and cPLA2{alpha}, immunoprecipitations of annexin A1 were performed from confluent and subconfluent HUVEC lysates and immunoblotted for the presence of cPLA2{alpha}. Consistent with the immunofluorescence data, only annexin A1 from confluent endothelial cells was associated with cPLA2{alpha} (Fig. 5D).

Interaction of cPLA2{alpha} with Annexin A1 Inhibits Prostaglandin Synthesis in Confluent Endothelial Cells—To test the hypothesis that interaction with annexin A1 at the Golgi apparatus is responsible for inhibition of cPLA2{alpha} activity in confluent endothelial cells, the siRNA knockdown of annexin A1 was performed. In agreement with the hypothesis, knockdown of endogenous annexin A1 resulted in a significant increase in AA-dependent PGE2 generation relative to HUVECs transfected with nontargeting siRNA (Fig. 6, A and B, mock). However, the knockdown of endogenous annexin A1 was not sufficient to elevate PGE2 generation from HUVECs stimulated in the presence of free AA (Fig. 6C). Thus, the increased ability of siRNA-treated HUVECs to generate PGE2 was a consequence of an increased ability to release free AA and not due to an effect on downstream aspects of PGE2 generation. Furthermore, this effect was not a consequence of increased cPLA2{alpha}, COX-1, or COX-2 expression, since knockdown of endogenous annexin A1 had no effect on their protein levels (Fig. 6A). To determine whether the interaction of cPLA2{alpha} with annexin A1 is responsible for sequestering cPLA2{alpha} at the Golgi apparatus of confluent HUVECs, the subcellular localization of cPLA2{alpha} after annexin A1 knockdown was investigated (Fig. 6D). Upon knockdown, endogenous annexin A1 cPLA2{alpha} becomes distributed throughout the cell in a manner similar to subconfluent cells. Furthermore, in annexin A1 siRNA-treated cells, cPLA2{alpha} does not appear to co-distribute as extensively with the Golgi marker protein, TGN46, relative to control siRNA-treated HUVECs (Fig. 6D). Quantification of this overlap confirmed that significantly less cPLA2{alpha} co-distributed with the Golgi apparatus of annexin A1 siRNA-treated cells versus control cells (Fig. 6E). Thus, the association of cPLA2{alpha} with annexin A1 at the Golgi apparatus represents a novel mechanism for the control of endothelial cell AA release and prostaglandin production.


Figure 6
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  		FIGURE 6.
Inhibition of cPLA2{alpha} activity at confluence occurs upon interaction with annexin A1. A, confluent HUVECs were transfected with either no siRNA (control), nontargeted control siRNA (mock), or annexin A1-targeted siRNA (siRNA). 48 h after transfection, cells were lysed, and annexin A1 levels were analyzed by Western blotting. Membranes were reprobed for cPLA2{alpha}, COX1, and COX2 to show equal protein loading. Relative amounts of annexin A1, cPLA2{alpha}, COX1, and COX2 immunoreactivity were determined using Aida densitometry software and then plotted (n = 3, ±S.E.). B, transfected cells were incubated with 5 µM A23187 for 15 min, and the ability of cells to generate PGE2 was then assessed using an ELISA (n = 3, ±S.E.). C, transfected cells were incubated with 5 µM A23187 and 10 µM arachidonic acid for 15 min, and the ability of cells to generate PGE2 was then assessed using an ELISA (n = 5, ±S.E.). D, confluent transfected cells were directly fixed, and then cPLA2{alpha}, annexin A1, and TGN46 were detected by confocal immunofluorescence microscopy. E, quantification of the co-distribution of cPLA2{alpha} with TGN46 upon the siRNA-mediated knockdown of annexin A1 performed using LSM510Meta software (Zeiss). Results represent the ratio of cPLA2{alpha}/TGN46 fluorescence intensity at TGN46-positive Golgi structures (n = 40, ±S.E.). *, p < 0.05 versus mock.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here we have defined a novel mechanism for the regulation of prostaglandin biosynthesis in endothelial cells. Generation of prostaglandins by the endothelium regulates vascular homeostasis. For example, PGE2 acts as a potent vasodilator/vasoconstrictor and proangiogenic stimuli (40, 41). Here we find that targeting of cPLA2{alpha} to the ER/ERGIC is required for its functional coupling to the COX enzymes and maximal PGE2 generation. Furthermore, sequestration of cPLA2{alpha} at the Golgi apparatus is sufficient to abolish Ca2+-induced prostaglandin generation in confluent endothelial cells (Fig. 7). This represents a novel molecular switch for the control of endothelial prostaglandin generation and accounts for earlier observations that confluent endothelial cells generate lower levels of prostaglandins compared with nonconfluent endothelial cells (14, 15).


Figure 7
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  		FIGURE 7.
A model for the regulation of cPLA2{alpha} activity in endothelial cells. In subconfluent cells, cPLA2{alpha} is free to associate with the ER and ERGIC upon cytosolic Ca2+ elevation. The resulting spatiotemporal colocalization with the COX enzymes then facilitates the conversion of AA into prostaglandins. Upon cell confluence, cPLA2{alpha} becomes associated with the Golgi apparatus and interacts with the cPLA2{alpha}-inhibitory protein, annexin A1. Consequently, the blocking of membrane targeting and inhibition of cPLA2{alpha} activity abolishes cPLA2{alpha}-mediated signaling.

 
In this study, we describe the Ca2+-induced association of cPLA2{alpha} with the ER/ERGIC of nonconfluent HUVECs. Previously, we demonstrated that intracellular Ca2+ elevation targets cPLA2{alpha} to membranes distinct from ER/Golgi apparatus of EA.hy.926 cells (31). The EA.hy.926 cell line is a continuous hybrid line formed from the fusion of primary HUVECs and the human lung carcinoma cell line, A549. Consequently, any difference in the localization of cPLA2{alpha} in EA.hy.926 cells versus their HUVEC donor line must represent characteristics associated with the A549 fusion partner. These differences emphasize the highly cell type-specific nature of the subcellular localization of cPLA2{alpha} and highlight the potential deficiencies of hybrid cell lines as model cell systems. Sequestration at the Golgi apparatus and inactivation of cPLA2{alpha} appears to be unique to endothelial cells and is not displayed by any other cell type tested (HeLa, Madin-Darby canine kidney, A549, saphenous vein smooth muscle, and EA.hy.926 cells; data not shown). Furthermore, this mechanism appears to be common to all endothelial cells, since we find that Golgi-localized cPLA2{alpha} is seen in confluent cell cultures of HUVECs, human dermal microvascular endothelial cells, and HCAEC (not shown). Golgi-localized cPLA2{alpha} is also seen in endothelial cells in isolated rat mesenteric arteries (data not shown).

It has been suggested previously that confluence-dependent fluctuations in endothelial cell PGE2 generation are a consequence of differential COX activity (50). However, the previous study was conducted on serum-starved, unstimulated endothelial cells in the absence of intracellular Ca2+ elevation. In their model system, AA must be derived from cPLA2{alpha}-independent sources, and the resultant PGE2 production is minimal (<1.4 pg/1000 cells). In addition, we find that total COX activity remains constant independent of endothelial cell density, consistent with previous observations (15). Thus, regulation of prostaglandin generation from endothelial cells is critically dependent on the ability of cPLA2{alpha} to be targeted to specific intracellular membranes.

Here we establish that inhibition of cPLA2{alpha} at the Golgi apparatus of confluent endothelial cells occurs upon interaction with annexin A1. A number of cPLA2{alpha}-binding proteins that negatively regulate cPLA2{alpha} activity have been previously identified and include the intermediate filament protein vimentin (48, 51), the calcium-binding protein p11/S100A10 (49), and the phospholipid-binding protein annexin A1 (20, 21). Using co-immunoprecipitation assays, we found no evidence for binding of cPLA2{alpha} to vimentin or p11/S100A10 in confluent cells (data not shown); however, cPLA2{alpha} was seen to specifically associate with annexin A1 only at the Golgi apparatus of confluent unstimulated endothelial cells. This interaction was responsible for inhibition of cPLA2{alpha} activity, since siRNA-mediated knockdown of annexin A1 released cPLA2{alpha} from the Golgi and significantly elevated AA-dependent PGE2 production. Thus, interaction of annexin A1 with cPLA2{alpha} at the Golgi apparatus represents a novel regulatory mechanism for the control of endothelial cell AA release and prostaglandin production. This is consistent with previous studies documenting the importance of annexin A1-mediated inhibition of cPLA2{alpha} to the anti-inflammatory action of glucocorticoids (17-19).

In this study, we also describe the cell density-dependent relocation of annexin A1 to the Golgi apparatus of confluent endothelial cells. Consequently, the accumulation of annexin A1 at the Golgi apparatus inactivates cPLA2{alpha} upon its interaction with annexin A1 and the sequestration of cPLA2{alpha} away from its intracellular substrates. When endothelial cells reach confluence, they characteristically undergo the contact inhibition of cellular proliferation and exit the cell cycle to form quiescent monolayers. The molecular mechanisms regulating the contact inhibition are not clearly defined but are known to involve the inhibition of growth factor receptor signaling, activation of protein phosphatases, and inactivation of Rac upon the formation of cell-cell contacts (34, 52, 53). Similar mechanisms may promote the confluence-dependent redistribution of annexin A1 to the Golgi apparatus and subsequent inactivation of cPLA2{alpha}. However, the distinct mechanisms involved in the redistribution of annexin A1 remain to be elucidated.

In subconfluent endothelial cells, cPLA2{alpha} is distributed throughout the cytoplasm and nucleus of resting cells and remains largely inactive. Upon Ca2+ elevation, cPLA2{alpha} can then interact with intracellular membrane substrates via its Ca2+-dependent lipid-binding domain (C2 domain). In confluent cells, the Ca2+-dependent ability of cPLA2{alpha} to interact with membranes is blocked upon its sequestration at the Golgi apparatus. Sequestration of signaling proteins away from their endogenous substrates is an emerging concept in the regulation of a variety of enzymes. The Golgi apparatus is known to possess an "exoskeleton" of structural proteins, which includes the molecular scaffold Sef. Interaction with Sef sequesters MEK/ERK at the Golgi apparatus and excludes it from nuclear targets (54). Similarly, annexin A1 is also known to form membrane scaffolds (55). However, isolation of endothelial cell Golgi membranes using differential centrifugation, sucrose density gradients, or iodixanol (OptiPrep) gradients does not produce annexin A1- or cPLA2{alpha}-enriched Golgi fractions when isolated from confluent cells lysed in the absence of high Ca2+ concentrations (>1mM) (supplemental Figs. 5 and 6, A and B). It is only upon the addition of Ca2+ and the absence of chelators that cPLA2{alpha} and annexin A1 become associated with membrane-containing fractions. Thus, in confluent endothelial cells, cPLA2{alpha} may not directly interact with components of the Golgi membrane but may interact with an associated structure or scaffold that is disrupted upon mechanical lysis and subcellular fractionation. The Golgi-localized pool of annexin A1 may represent such a labile scaffold to which cPLA2{alpha} is immobilized in confluent endothelial cells. An analogous situation has been reported for phospholipase D, which is blocked from accessing its phospholipid substrate by binding to the cytoskeletal, spectrin-related protein, fodrin (56). The next challenge will be to elucidate the molecular details of annexin A1-mediated inhibition of cPLA2{alpha} in the endothelial cell.


   FOOTNOTES
 
* This work was funded by a Biotechnology and Biological Sciences Research Council Ph.D. studentship (to S. P. H.), a Yorkshire Cancer Research pump priming grant (to S. P. H. and J. H. W.), a British Heart Foundation project grant (to S. P. and J. H. W.), and a Wellcome Trust project grant (to J. H. W. and S. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-6. Back

1 To whom correspondence should be addressed: Faculty of Biological Sciences, Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom. Tel.: 44-113-3433119; Fax: 44-113-3433167; E-mail: j.h.walker{at}leeds.ac.uk.

2 The abbreviations used are: cPLA2{alpha}, cytosolic phospholipase A2-{alpha}; PLA2, phospholipase A2; iPLA2, calcium-independent phospholipase A2; AA, arachidonic acid; AACOCF3, arachidonyl trifluoromethylketone; BEL, bromoenol lactone; COX, cyclooxygenase; ERGIC, endoplasmic reticulum-Golgi intermediate compartment; HUVEC, human umbilical vein endothelial cell; ManII, mannosidase II; PGE2, prostaglandin E2; CalB, Ca2+-dependent lipid binding; GFP, green fluorescent protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ELISA, enzyme-linked immunosorbent assay; siRNA, small interfering RNA; ER, endoplasmic reticulum. Back


   ACKNOWLEDGMENTS
 
We thank G. J. Howell for assistance with all aspects of bioimaging.



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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
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