The Confluence-dependent Interaction of Cytosolic Phospholipase A2-α with Annexin A1 Regulates Endothelial Cell Prostaglandin E2 Generation
2007; Elsevier BV; Volume: 282; Issue: 47 Linguagem: Inglês
10.1074/jbc.m701541200
ISSN1083-351X
AutoresShane P. Herbert, Adam F. Odell, Sreenivasan Ponnambalam, John H. Walker,
Tópico(s)Vitamin K Research Studies
ResumoThe 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-α (cPLA2α) 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 cPLA2α with the Golgi apparatus of confluent endothelial cells correlates with decreased prostaglandin synthesis. Golgi association blocks arachidonic acid release and prevents functional coupling between cPLA2α and COX-mediated prostaglandin synthesis. When inactivated at the Golgi apparatus of confluent endothelial cells, cPLA2α 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 cPLA2α and annexin A1 at the Golgi acts as a novel molecular switch controlling cPLA2α activity and endothelial cell prostaglandin generation. 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-α (cPLA2α) 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 cPLA2α with the Golgi apparatus of confluent endothelial cells correlates with decreased prostaglandin synthesis. Golgi association blocks arachidonic acid release and prevents functional coupling between cPLA2α and COX-mediated prostaglandin synthesis. When inactivated at the Golgi apparatus of confluent endothelial cells, cPLA2α 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 cPLA2α and annexin A1 at the Golgi acts as a novel molecular switch controlling cPLA2α activity and endothelial cell prostaglandin generation. Cytosolic phospholipase A2-α (cPLA2α) 2The abbreviations used are: cPLA2α, cytosolic phospholipase A2-α; 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. is an 85-kDa, Ca2+-sensitive member of the phospholipase A2 (PLA2) family of enzymes (1Clark J.D. Schievella A.R. Nalefski E.A. Lin L.L. J. Lipid Mediat. Cell Signal. 1995; 12: 83-117Crossref PubMed Scopus (425) Google Scholar, 2Hirabayashi T. Murayama T. Shimizu T. Biol. Pharm. Bull. 2004; 27: 1168-1173Crossref PubMed Scopus (192) Google Scholar) which includes the Ca2+-independent (iPLA2) and secretory phospholipases A2 (3Akiba S. Sato T. Biol. Pharm. Bull. 2004; 27: 1174-1178Crossref PubMed Scopus (110) Google Scholar). The PLA2 enzymes hydrolyze the sn-2 fatty acyl bond of phospholipids to simultaneously generate free fatty acid and lysophospholipids (4Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (762) Google Scholar). Upon agonist stimulation and cytosolic Ca2+ elevation, cPLA2α translocates to intracellular membranes utilizing an N-terminal Ca2+-dependent lipid binding (CalB) domain (5Nalefski E.A. Sultzman L.A. Martin D.M. Kriz R.W. Towler P.S. Knopf J.L. Clark J.D. J. Biol. Chem. 1994; 269: 18239-18249Abstract Full Text PDF PubMed Google Scholar, 6Channon J.Y. Leslie C.C. J. Biol. Chem. 1990; 265: 5409-5413Abstract Full Text PDF PubMed Google Scholar, 7Evans J.H. Spencer D.M. Zweifach A. Leslie C.C. J. Biol. Chem. 2001; 276: 30150-30160Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Upon membrane binding, cPLA2α preferentially cleaves phospholipids containing arachidonic acid (AA) at the sn-2 position to liberate free AA (4Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (762) Google Scholar). Consequently, cPLA2α is seen as the rate-limiting enzyme in receptor-mediated AA release (8Kramer R.M. Sharp J.D. FEBS Lett. 1997; 410: 49-53Crossref PubMed Scopus (237) Google Scholar). Ca2+ elevation can induce relocation of cPLA2α to the specific intracellular membranes in which the downstream AA-metabolizing cyclooxygenase (COX) enzymes are also located (2Hirabayashi T. Murayama T. Shimizu T. Biol. Pharm. Bull. 2004; 27: 1168-1173Crossref PubMed Scopus (192) Google Scholar). There are two isoforms of COX that have been extensively characterized (COX-1 and -2) (9Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1868) Google Scholar), and more recently an alternative COX-1 splice variant, COX-3, has also been cloned (10Chandrasekharan N.V. Dai H. Roos K.L. Evanson N.K. Tomsik J. Elton T.S. Simmons D.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13926-13931Crossref PubMed Scopus (1702) Google Scholar). The spatiotemporal co-localization of cPLA2α with COX can couple these enzymes to facilitate efficient conversion of AA into prostaglandins (2Hirabayashi T. Murayama T. Shimizu T. Biol. Pharm. Bull. 2004; 27: 1168-1173Crossref PubMed Scopus (192) Google Scholar, 11Murakami M. Das S. Kim Y. Cho W. Kudo I. FEBS Lett. 2003; 546: 251-256Crossref PubMed Scopus (20) Google Scholar). Chimeric cPLA2α mutants specifically targeted to intracellular membranes in which COX does not reside do not couple with COX and drastically reduce prostaglandin production (11Murakami M. Das S. Kim Y. Cho W. Kudo I. FEBS Lett. 2003; 546: 251-256Crossref PubMed Scopus (20) Google Scholar). Thus, the subcellular targeting of cPLA2α to specific intracellular membranes is essential for the regulation of both AA and prostaglandin production. Despite this, the subcellular targeting of cPLA2α 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 (12Vane J.R. Anggard E.E. Botting R.M. N. Engl. J. Med. 1990; 323: 27-36Crossref PubMed Scopus (1802) Google Scholar, 13Simmons D.L. Botting R.M. Hla T. Pharmacol. Rev. 2004; 56: 387-437Crossref PubMed Scopus (1388) Google Scholar). 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 (14Evans C.E. Billington D. McEvoy F.A. Prostaglandins Leukot. Med. 1984; 14: 255-266Abstract Full Text PDF PubMed Scopus (22) Google Scholar, 15Whatley R.E. Satoh K. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Clin. Invest. 1994; 94: 1889-1900Crossref PubMed Scopus (41) Google Scholar, 16Herbert S.P. Ponnambalam S. Walker J.H. Mol. Biol. Cell. 2005; 16: 3800-3809Crossref PubMed Scopus (41) Google Scholar), which has been attributed to elevated cPLA2α activity. Despite this, the actual mechanism of this differential regulation of cPLA2α activity has not been defined. Inhibition of cPLA2α 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 (17Flower R.J. Rothwell N.J. Trends Pharmacol. Sci. 1994; 15: 71-76Abstract Full Text PDF PubMed Scopus (334) Google Scholar, 18Lim L.H. Pervaiz S. FASEB J. 2007; 21: 968-975Crossref PubMed Scopus (333) Google Scholar, 19Parente L. Solito E. Inflamm. Res. 2004; 53: 125-132Crossref PubMed Scopus (246) Google Scholar). Annexin A1 is known to inhibit cPLA2α activity upon interaction with the CalB domain of cPLA2α in vitro (20Kim S. Rhee H.J. Ko J. Kim H.G. Yang J.M. Choi E.C. Na D.S. J. Biol. Chem. 2001; 276: 15712-15719Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 21Kim S. Ko J. Kim J.H. Choi E.C. Na D.S. FEBS Lett. 2001; 489: 243-248Crossref PubMed Scopus (45) Google Scholar); 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 cPLA2α at the Golgi apparatus of confluent cells. Cell Culture and Materials—Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords as previously described (22Jaffe E.A. Biology of Endothelial Cells. Martinus Nijhoff Publishers, Boston, MA1984: 1-13Google Scholar, 23Howell G.J. Herbert S.P. Smith J.M. Mittar S. Ewan L.C. Mohammed M. Hunter A.R. Simpson N. Turner A.J. Zachary I. Walker J.H. Ponnambalam S. Mol. Membr. Biol. 2004; 21: 413-421Crossref PubMed Scopus (40) Google Scholar). 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-cPLA2α (C20; Santa Cruz Biotechnology), anti-cPLA2α (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 cPLA2α 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 (16Herbert S.P. Ponnambalam S. Walker J.H. Mol. Biol. Cell. 2005; 16: 3800-3809Crossref PubMed Scopus (41) Google Scholar). 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 (24Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212178) Google Scholar). For immunoblotting, protein was transferred onto nitrocellulose membranes for 3 h at 300 mA (25Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (46130) Google Scholar). 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). cPLA2α was immunoprecipitated from cell lysates with anti-cPLA2α 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. 26Dominguez M. Fazel A. Dahan S. Lovell J. Hermo L. Claude A. Melancon P. Bergeron J.J. J. Cell Biol. 1999; 145: 673-688Crossref PubMed Scopus (22) Google Scholar. Interfaces were removed, diluted in homogenization media, and collected by centrifugation at 100,000 × g for 60 min. Isolated fractions were analyzed by SDS-PAGE and immunoblotting. Iodixanol gradients were performed essentially as described by Yang et al. (27Yang M. Ellenberg J. Bonifacino J.S. Weissman A.M. J. Biol. Chem. 1997; 272: 1970-1975Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), except a 10-30% gradient was utilized. Differential centrifugation enrichment of membrane fractions was performed as described previously by Lamour et al. (28Lamour N.F. Stahelin R.V. Wijesinghe D.S. Maceyka M. Wang E. Allegood J.C. Merrill Jr., A.H. Cho W. Chalfant C.E. J. Lipid Res. 2007; 48: 1293-1304Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Immunofluorescence—Immunofluorescence microscopy was performed as previously described (16Herbert S.P. Ponnambalam S. Walker J.H. Mol. Biol. Cell. 2005; 16: 3800-3809Crossref PubMed Scopus (41) Google Scholar). 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 as previously (29Grewal S. Ponnambalam S. Walker J.H. J. Cell Sci. 2003; 116: 2303-2310Crossref PubMed Scopus (34) Google Scholar, 30Grewal S. Smith J. Ponnambalam S. Walker J. Eur. J. Biochem. 2004; 271: 69-77Crossref PubMed Scopus (7) Google Scholar). 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 × 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 (16Herbert S.P. Ponnambalam S. Walker J.H. Mol. Biol. Cell. 2005; 16: 3800-3809Crossref PubMed Scopus (41) Google Scholar). 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 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 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). 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. Cytosolic Ca2+ Elevation Targets cPLA2α to Intracellular Membranes in Subconfluent Endothelial Cells—cPLA2α activity is greater in subconfluent endothelial cells than in quiescent, confluent endothelial cells (15Whatley R.E. Satoh K. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Clin. Invest. 1994; 94: 1889-1900Crossref PubMed Scopus (41) Google Scholar, 16Herbert S.P. Ponnambalam S. Walker J.H. Mol. Biol. Cell. 2005; 16: 3800-3809Crossref PubMed Scopus (41) Google Scholar). We have previously shown that subconfluent and confluent endothelial cells (see supplemental Fig. 1) express equal amounts of cPLA2α (16Herbert S.P. Ponnambalam S. Walker J.H. Mol. Biol. Cell. 2005; 16: 3800-3809Crossref PubMed Scopus (41) Google Scholar), indicating that mechanisms other than control of cPLA2α expression are responsible for confluence-dependent changes in its activity. In response to elevated cytosolic Ca2+, cPLA2α is activated by relocation to intracellular membranes. Recruitment to specific membranes is required for the regulation of cPLA2α activity (2Hirabayashi T. Murayama T. Shimizu T. Biol. Pharm. Bull. 2004; 27: 1168-1173Crossref PubMed Scopus (192) Google Scholar, 11Murakami M. Das S. Kim Y. Cho W. Kudo I. FEBS Lett. 2003; 546: 251-256Crossref PubMed Scopus (20) Google Scholar); however, the precise membranes to which cPLA2α relocates in primary endothelial cells have not been defined. Therefore, we investigated the Ca2+-induced relocation of cPLA2α. In HUVECs, cPLA2α was detectable as a 110 kDa band by Western blotting using a well characterized affinity-purified antibody specific to the C-terminal region of cPLA2α (16Herbert S.P. Ponnambalam S. Walker J.H. Mol. Biol. Cell. 2005; 16: 3800-3809Crossref PubMed Scopus (41) Google Scholar, 31Grewal S. Herbert S.P. Ponnambalam S. Walker J.H. FEBS J. 2005; 272: 1278-1290Crossref PubMed Scopus (27) Google Scholar). Additionally, the immunoreactivity was removed by preabsorption of the antibody with the antigenic peptide (supplemental Fig. 2A). By immunofluorescence microscopy, cPLA2α 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 (16Herbert S.P. Ponnambalam S. Walker J.H. Mol. Biol. Cell. 2005; 16: 3800-3809Crossref PubMed Scopus (41) Google Scholar, 32Bunt G. de Wit J. van den Bosch H. Verkleij A.J. Boonstra J. J. Cell Sci. 1997; 110: 2449-2459PubMed Google Scholar, 33Grewal S. Morrison E.E. Ponnambalam S. Walker J.H. J. Cell Sci. 2002; 115: 4533-4543Crossref PubMed Scopus (24) Google Scholar). To study the relocation of cPLA2α in response to cytosolic Ca2+ elevation, we used the Ca2+ ionophore, A23187. This agent is ideal for studying confluence-dependent changes in cPLA2α relocation, since A23187 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 (34Grazia Lampugnani M. Zanetti A. Corada M. Takahashi T. Balconi G. Breviario F. Orsenigo F. Cattelino A. Kemler R. Daniel T.O. Dejana E. J. Cell Biol. 2003; 161: 793-804Crossref PubMed Scopus (351) Google Scholar, 35Sorby M. Ostman A. J. Biol. Chem. 1996; 271: 10963-10966Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In subconfluent HUVECs, upon elevation of cytosolic Ca2+, cPLA2α relocated to the nuclear periphery and less disperse cytoplasmic structures (see supplemental Fig. 2). Relocation occurred rapidly (<1 min) and cPLA2α 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) (36Opas M. Dziak E. Fliegel L. Michalak M. J. Cell. Physiol. 1991; 149: 160-171Crossref PubMed Scopus (132) Google Scholar, 37Schweizer A. Fransen J.A. Bachi T. Ginsel L. Hauri H.P. J. Cell Biol. 1988; 107: 1643-1653Crossref PubMed Scopus (396) Google Scholar, 38Schweizer A. Fransen J.A. Matter K. Kreis T.E. Ginsel L. Hauri H.P. Eur. J. Cell Biol. 1990; 53: 185-196PubMed Google Scholar). Quantitation of this co-localization revealed a 2.5-fold increase in overlap between cPLA2α and calreticulin and a 2.3-fold increase in overlap between cPLA2α and ERGIC-53 upon A23187 treatment (Fig. 1A). A23187-induced relocation of cPLA2α 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). cPLA2α 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 cPLA2α 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 (2Hirabayashi T. Murayama T. Shimizu T. Biol. Pharm. Bull. 2004; 27: 1168-1173Crossref PubMed Scopus (192) Google Scholar, 11Murakami M. Das S. Kim Y. Cho W. Kudo I. FEBS Lett. 2003; 546: 251-256Crossref PubMed Scopus (20) Google Scholar). 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 cPLA2α 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 (12Vane J.R. Anggard E.E. Botting R.M. N. Engl. J. Med. 1990; 323: 27-36Crossref PubMed Scopus (1802) Google Scholar, 13Simmons D.L. Botting R.M. Hla T. Pharmacol. Rev. 2004; 56: 387-437Crossref PubMed Scopus (1388) Google Scholar), this functional coupling between AA release and prostaglandin synthesis has never been investigated in endothelial cells. We predicted that recruitment of cPLA2α to the ER of subconfluent HUVECs would promote functional coupling, since both COX-1 and -2 are located at the ER of endothelial cells (39Morita I. Schindler M. Regier M.K. Otto J.C. Hori T. DeWitt D.L. Smith W.L. J. Biol. Chem. 1995; 273: 9886-9893Google Scholar). Subconfluent cells were stimulated to elevate intracellular Ca2+, and the co-distribution of cPLA2α 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 cPLA2α and COX-1 and a 2.3-fold increase between cPLA2α 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 (40Kamiyama M. Pozzi A. Yang L. DeBusk L.M. Breyer R.M. Lin P.C. Oncogene. 2006; 25: 7019-7028Crossref PubMed Scopus (104) Google Scholar, 41Tang L. Loutzenhiser K. Loutzenhiser R. Circ. Res. 2000; 86: 663-670Crossref PubMed Scopus (88) Google Scholar). Prior to A23187 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 (42Ackermann E.J. Conde-Frieboes K. Dennis E.A. J. Biol. Chem. 1995; 270: 445-450Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar), 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 cPLA2α with AACOCF3 (43Street I. Lin H.K. Laliberte F. Ghomashchi F. Wang Z. Perrier H. Tremblay N.M. Huang Z. Weeck P.K. Gelb M.H. Biochemistry. 1993; 32: 5935-5940Crossref PubMed Scopus (422) Google Scholar) inhibited PGE2 generation by 88% (Fig. 2D). Thus, Ca2+-induced PGE2 generation was almost entirely dependent on cPLA2α 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 cPLA2α 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 cPLA2α is the rate-limiting step in PGE2 production in endothelial cells. Sequestration of cPLA2α at the Golgi Apparatus of Quiescent Endothelial Cells Blocks Its Translocation to Other Membranes—By immunofluorescence microscopy, in confluent endothelial cells, cPLA2α 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 cPLA2α (N-terminal and C-terminal linked constructs; Fig. 2B) and with a separate antibody targeted to the C terminus of cPLA2α (supplemental Fig. 3). In other cell types, association of cPLA2α with the Golgi apparatus promotes AA release (44Pettus B.J. Bielawska A. Subramanian P. Wijesinghe D.S. Maceyka M. Leslie C.C. Evans J.H. Freiberg J. Roddy P. Hannun Y.A. Chalfant C.E. J. Biol. Chem. 2004; 279: 11320-11326Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 45Grimmer S. Ying M. Walchli S. van Deurs B. Sandvig K. Traffic. 2005; 6: 144-156Crossref PubMed Scopus (53) Google Scholar), whereas in confluent endothelial cells, cPLA2α activity is inhibited (14Evans C.E. Billington D. McEvoy F.A. Prostaglandins Leukot. Med. 1984; 14: 255-266Abstract Full Text PDF PubMed Scopus (22) Google Scholar, 15Whatley R.E. Satoh K. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Clin. Invest. 1994; 94: 1889-1900Crossref PubMed Scopus (41) Google Scholar, 16Herbert S.P. Ponnambalam S. Walker J.H. Mol. Biol. Cell. 2005; 16: 3800-3809Crossref PubMed Scopus (41) Google Scholar). Furthermore, interaction with the Golgi blocked targeting of cPLA2α to the ER and ERGIC upon Ca2+ elevation (Fig. 2, C-E). In confluent HUVECs, the co-distribution of cPLA2α with calreticulin (Fig. 2C) and ERGIC-53 (Fig. 2D) positive structures was not enhanced upon cytosolic Ca2+ elevation. Quantitation revealed that overlap between cPLA2α and calreticulin was only 4% that of subconfluent cells upon cytosolic Ca2+ elevation (Fig. 2E). Overlap between cPLA2α and ERGIC-53 was also reduced by 47% relative to A23187-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 cPLA2α with the ER and ERGIC was not enhanced upon cytosolic Ca2+ elevation (Fig. 2E). Thus, association with the Golgi apparatus sequesters cPLA2α away from its intracellular substrate, accounting for the reduced AA release seen at endothelial cell confluence (15Whatley R.E. Satoh K. Zimmerman G.A. McIntyre T.M. Prescott S.M. J. Clin. Invest. 1994; 94: 1889-1900Crossref PubMed Scopus (41) Google Scholar, 16Herbert S.P. Ponnambalam S. Walker J.H. Mol. Biol. Cell. 2005; 16: 3800-3809Crossref PubMed Scopus (41) Google Scholar). Sequestration of cPLA2α at the Golgi Apparatus Inhibits Its Functional Coupling with the COX Enzymes—Confluent endothelial cells generate lower levels of prostaglandins than subconfluent cells (14Evans C.E. Billington D. McEvoy F.A. Prostaglandins Leukot. Med. 1984; 14: 255-266Abstract Full Text PDF PubMed Scopus (22) Google Scholar
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