Annexin A6-induced Inhibition of Cytoplasmic Phospholipase A2 Is Linked to Caveolin-1 Export from the Golgi
2008; Elsevier BV; Volume: 283; Issue: 15 Linguagem: Inglês
10.1074/jbc.m706618200
ISSN1083-351X
AutoresLaia Cubells, Sandra Vilà de Muga, Francesc Tebar, Joseph V. Bonventre, Jesús Balsinde, Albert Pol, Thomas Grewal, Carlos Enrich,
Tópico(s)Ion channel regulation and function
ResumoThe molecular mechanisms regulating the exit of caveolin from the Golgi complex are not fully understood. Cholesterol and sphingolipid availability affects Golgi vesiculation events and involves the activity of cytoplasmic phospholipase A2 (cPLA2). We recently demonstrated that high expression levels of annexin A6 (AnxA6) perturb the intracellular distribution of cellular cholesterol, thereby inhibiting caveolin export from the Golgi complex. In the present study we show that in Chinese hamster ovary cells overexpressing AnxA6, sequestration of cholesterol in late endosomes, leading to reduced amounts of cholesterol in the Golgi, inhibits cPLA2 activity and its association with the Golgi complex. This correlates with the blockage of caveolin export from the Golgi in cells treated with methyl arachidonyl fluorophosphonate, a Ca2+-dependent cPLA2 inhibitor. AnxA6-mediated down-regulation of cPLA2 activity was overcome upon the addition of exogenous cholesterol or transfection with small interfering RNA targeting AnxA6. These findings indicate that AnxA6 interferes with caveolin transport through the inhibition of cPLA2. The molecular mechanisms regulating the exit of caveolin from the Golgi complex are not fully understood. Cholesterol and sphingolipid availability affects Golgi vesiculation events and involves the activity of cytoplasmic phospholipase A2 (cPLA2). We recently demonstrated that high expression levels of annexin A6 (AnxA6) perturb the intracellular distribution of cellular cholesterol, thereby inhibiting caveolin export from the Golgi complex. In the present study we show that in Chinese hamster ovary cells overexpressing AnxA6, sequestration of cholesterol in late endosomes, leading to reduced amounts of cholesterol in the Golgi, inhibits cPLA2 activity and its association with the Golgi complex. This correlates with the blockage of caveolin export from the Golgi in cells treated with methyl arachidonyl fluorophosphonate, a Ca2+-dependent cPLA2 inhibitor. AnxA6-mediated down-regulation of cPLA2 activity was overcome upon the addition of exogenous cholesterol or transfection with small interfering RNA targeting AnxA6. These findings indicate that AnxA6 interferes with caveolin transport through the inhibition of cPLA2. Annexins are a family of Ca2+ and membrane-binding proteins involved in membrane trafficking and various other processes including signaling, proliferation, differentiation, and inflammation (1Raynal P. Pollard H.B. Biochim. Biophys. Acta. 1994; 1197: 63-93Crossref PubMed Scopus (1022) Google Scholar, 2Gerke V. Creutz C.E. Moss S.E. Nat. Rev. Mol. Cell. Biol. 2005; 6: 449-461Crossref PubMed Scopus (1109) Google Scholar, 3Grewal T. Enrich C. BioEssays. 2006; 28: 1211-1220Crossref PubMed Scopus (48) Google Scholar). Each annexin consists of a unique N-terminal tail and a common, well conserved C-terminal core domain containing 4 (or 8 for AnxA6 3The abbreviations used are:AnxA6annexin A6AAarachidonic acidAnxA1annexin A1cavcaveolinCHOChinese hamster ovarycPLA2cytoplasmic phospholipase A2EGFRepidermal growth factor receptorp120GAPp120 GTPase-activating proteinGFPgreen fluorescent proteinHELSSE-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (haloenol lactone suicide substrate)iFRAPinverse fluorescence recovery after photobleachingMAFPmethyl arachidonyl fluorophosphonatewtwild typePTRFpolymerase I and transcript release factorDMEMDulbecco's modified Eagle's mediumRNAiRNA-mediated interferenceGSTglutathione S-transferase.3The abbreviations used are:AnxA6annexin A6AAarachidonic acidAnxA1annexin A1cavcaveolinCHOChinese hamster ovarycPLA2cytoplasmic phospholipase A2EGFRepidermal growth factor receptorp120GAPp120 GTPase-activating proteinGFPgreen fluorescent proteinHELSSE-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (haloenol lactone suicide substrate)iFRAPinverse fluorescence recovery after photobleachingMAFPmethyl arachidonyl fluorophosphonatewtwild typePTRFpolymerase I and transcript release factorDMEMDulbecco's modified Eagle's mediumRNAiRNA-mediated interferenceGSTglutathione S-transferase.) repeats of a highly homologous 70-amino acid sequence which facilitates their Ca2+ and phospholipid binding.We and others showed that AnxA6 is located at the plasma membrane, in the endocytic compartment, and in caveolae. AnxA6 has been implicated in endo- and exocytic membrane trafficking pathways and regulates low density lipoprotein (LDL) receptor-mediated endocytosis (4Grewal T. Heeren J. Mewawala D. Schnitgerhans T. Wendt D. Salomon G. Enrich C. Beisiegel U. Jackle S. J. Biol. Chem. 2000; 275: 33806-33813Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 5Kamal A. Ying Y. Anderson R.G. J. Cell Biol. 1998; 142: 937-947Crossref PubMed Scopus (85) Google Scholar), is crucial for LDL degradation and its transport from late endosomes/pre-lysosomes to lysosomes (6Pons M. Grewal T. Rius E. Schnitgerhans T. Jackle S. Enrich C. Exp. Cell Res. 2001; 269: 13-22Crossref PubMed Scopus (42) Google Scholar), and is recruited to cholesterol-laden late endosomes (7de Diego I. Schwartz F. Siegfried H. Dauterstedt P. Heeren J. Beisiegel U. Enrich C. Grewal T. J. Biol. Chem. 2002; 277: 32187-32194Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In addition, AnxA6 stimulates the membrane recruitment of the GTPase-activating protein p120GAP and protein kinase C to modulate the Ras signaling pathway (8Rentero C. Evans R. Wood P. Tebar F. Vila de Muga S. Cubells L. de Diego I. Hayes T.E. Hughes W.E. Pol A. Rye K.A. Enrich C. Grewal T. Cell. Signal. 2006; 18: 1006-1016Crossref PubMed Scopus (35) Google Scholar, 9Grewal T. Evans R. Rentero C. Tebar F. Cubells L. de Diego I. Kirchhoff M.F. Hughes W.E. Heeren J. Rye K.A. Rinninger F. Daly R.J. Pol A. Enrich C. Oncogene. 2005; 24: 5809-5820Crossref PubMed Scopus (82) Google Scholar). These multifunctional features of AnxA6 are most probably a consequence of (a) its dynamic spatiotemporal behavior in a Ca2+ and/or cholesterol-dependent manner but also (b) the promiscuity to interact with a large set of other molecules (10Gerke V. Moss S.E. Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1611) Google Scholar), in particular through the flexible linker region (amino acids 320-378) which connects the N- and C-terminal AnxA6 repeats (4 repeats each).Expression of AnxA6 and other annexins varies in cells and tissues as well as in different pathophysiological situations (1Raynal P. Pollard H.B. Biochim. Biophys. Acta. 1994; 1197: 63-93Crossref PubMed Scopus (1022) Google Scholar, 10Gerke V. Moss S.E. Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1611) Google Scholar). We recently compared wild type Chinese hamster ovary cells (CHOwt, contain very low AnxA6 levels) and AnxA6-deficient human epithelial carcinoma A431 cells with stable cell lines expressing high amounts of AnxA6 (CHOanx6, A431anx6). In these studies we showed that high AnxA6 levels result in an accumulation of cholesterol in the late endocytic compartment, leading to reduced amounts of cholesterol in the Golgi and the plasma membrane. This overall imbalance of cellular cholesterol is accompanied by an inhibition of caveolin export from the Golgi complex. Consequently, a significant diminution in the number of caveolae at the cell surface and a strong reduction of cholesterol efflux was observed (11Cubells L. Vila de Muga S. Tebar F. Wood P. Evans R. Ingelmo-Torres M. Calvo M. Gaus K. Pol A. Grewal T. Enrich C. Traffic. 2007; 8: 1568-1589Crossref PubMed Scopus (80) Google Scholar). However, the molecular mechanism(s) regulating the exit of caveolin from the Golgi complex is not fully understood, and recently the participation of syntaxin 6 (12Choudhury A. Marks D.L. Proctor K.M. Gould G.W. Pagano R.E. Nat. Cell Biol. 2006; 8: 317-328Crossref PubMed Scopus (74) Google Scholar) or PTRF-cavin (13Hill M.M. Bastiani M. Luetterforst R. Kirkham M. Kirkham A. Nixon S.J. Walser P. Abankwa D. Oorschot V.M. Martin S. Hancock J.F. Parton R.G. Cell. 2008; 132: 113-124Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar, 14Liu L. Pilch P.F. J. Biol. Chem. 2007; 283: 4314-4322Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) in this process has also been demonstrated.Changes in the availability of cellular cholesterol can be directly related to the vesiculation of the Golgi apparatus. Vesiculation and tubulation events regulating membrane traffic and cargo export from the Golgi require the activity of cholesterol-dependent cPLA2 (15Grimmer S. Ying M. Walchli S. van Deurs B. Sandvig K. Traffic. 2005; 6: 144-156Crossref PubMed Scopus (53) Google Scholar). Therefore, we addressed the possibility of AnxA6 interfering with caveolin export from Golgi membranes through the inhibition of cPLA2. In the present study we show that overexpression of AnxA6 indirectly inhibits cPLA2 activity and its association with the Golgi complex through the reduction of cholesterol availability. In support of cPLA2 being involved in caveolin transport, CHOwt treated with the Ca2+-dependent cPLA2 inhibitor, methyl arachidonyl fluorophosphonate (MAFP), but not the Ca2+-independent cPLA2 inhibitor haloenol lactone suicide substrate (HELSS), mimics the phenotype of AnxA6 overexpression and leads to an accumulation of caveolin in the Golgi complex.EXPERIMENTAL PROCEDURESReagents and Antibodies—Nutrient Mixture Ham's F-12, DMEM, cycloheximide, water-soluble cholesterol, and saponin were from Sigma. [3H]Arachidonic acid ([3H]AA) was from Amersham Biosciences. cPLA2 inhibitors E-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (HELSS), MAFP, and U18666A were from BIOMOL. Paraformaldehyde was from Electron Microscopy Sciences, and Mowiol from Calbiochem. The cloning of the green fluorescent protein (GFP)-tagged caveolin-3 (GFP-cav3) expression vector has been described previously (16Pol A. Martin S. Fernandez M.A. Ingelmo-Torres M. Ferguson C. Enrich C. Parton R.G. Mol. Biol. Cell. 2005; 16: 2091-2105Crossref PubMed Scopus (169) Google Scholar). GFP-tagged T-lymphocyte maturation-associated protein (MAL; GFP-MAL) and untagged AnxA1 expression vectors were kindly provided by Dr. M. A. Alonso (Centro de Biologia Molecular "Severo Ochoa," Universidad Autónoma de Madrid, Spain) and V. Gerke (Institute of Medical Biochemistry, University of Münster, Germany), respectively. The construction of the epidermal growth factor receptor (EGFR)-GFP fusion construct (EGFR-GFP) has been described previously (17Llado A. Tebar F. Calvo M. Moreto J. Sorkin A. Enrich C. Mol. Biol. Cell. 2004; 15: 4877-4891Crossref PubMed Google Scholar). The construction of full-length cytosolic group IVA phospholipase A2α-GFP (GFP-cPLA2) was described previously (18Casas J. Gijon M.A. Vigo A.G. Crespo M.S. Balsinde J. Balboa M.A. J. Biol. Chem. 2006; 281: 6106-6116Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). RNAi targeting human AnxA6, GFP, and the mouse monoclonal anti-cPLA2 (4-4B-3C; sc-454) were from Santa Cruz Biotechnology. Glutathione S-transferase (GST) and GST-AnxA6 fusion proteins were expressed in the Escherichia coli strain BL21 pLysE and purified by glutathione-Sepharose chromatography as described (6Pons M. Grewal T. Rius E. Schnitgerhans T. Jackle S. Enrich C. Exp. Cell Res. 2001; 269: 13-22Crossref PubMed Scopus (42) Google Scholar). Polyclonal anti-caveolin (C13630), anti-p120GAP, and mouse anti-GM130 were from BD Transduction Laboratories. Polyclonal anti-actin was from MP Biomedicals. Polyclonal anti-α-mannosidase II was kindly provided by Dr. A. Velasco (Universidad de Sevilla). Horseradish peroxidase-conjugated secondary antibodies were from Zymed Laboratories Inc.. Alexa Fluor-conjugated secondary antibodies were from Molecular Probes.Cell Culture—CHO cells were grown in Ham's F-12, and HeLa and COS-1 cells were grown together in DMEM with 10% fetal calf serum, l-glutamine (2 mm), penicillin (100 units/ml), and streptomycin (100 μg/ml) at 37 °C, 5% CO2. The generation of the stable AnxA6-overexpressing CHO cell line (CHOanx6) was described in detail (4Grewal T. Heeren J. Mewawala D. Schnitgerhans T. Wendt D. Salomon G. Enrich C. Beisiegel U. Jackle S. J. Biol. Chem. 2000; 275: 33806-33813Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 6Pons M. Grewal T. Rius E. Schnitgerhans T. Jackle S. Enrich C. Exp. Cell Res. 2001; 269: 13-22Crossref PubMed Scopus (42) Google Scholar). For the intracellular accumulation of cholesterol, cells were treated for 24 h with U18666A (2 μg/ml) as described (7de Diego I. Schwartz F. Siegfried H. Dauterstedt P. Heeren J. Beisiegel U. Enrich C. Grewal T. J. Biol. Chem. 2002; 277: 32187-32194Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). For the inhibition of Ca2+-independent and -dependent cPLA2 activity, cells were incubated as described with either 5 μm HELSS for 30 min or 15 μm MAFP for 60 min, respectively (15Grimmer S. Ying M. Walchli S. van Deurs B. Sandvig K. Traffic. 2005; 6: 144-156Crossref PubMed Scopus (53) Google Scholar). For transient transfections, cells (40-70% confluence) were transfected with 1 μg of DNA/ml using Effectene (Qiagen) following the manufacturer's instructions.Cycloheximide/Cholesterol Treatments—To inhibit protein synthesis in some experiments, 10 μg/ml cycloheximide (in 100 mm Hepes, pH 7.5) was added to the media for 90 min. For cholesterol addition, cells were incubated with 30 μg/ml cholesterol (premixed for 30 min in DMEM by gentle agitation) for 90 min.RNAi-mediated Inhibition of AnxA6—To specifically knock down gene expression of human AnxA6, 1-2 × 106 HeLa cells were transfected in 2 ml of medium with 10 nm AnxA6 small interfering RNA (Santa Cruz, sc-29688) and 6 μl of Lipofectamine 2000 reagent (Invitrogen) according to the instructions of the manufacturer. Experiments were conducted 72 h after transfection when the depletion of AnxA6 was more significant (see Western blot in Fig. 1C). GFP small interfering RNA was used as a negative control.Release of [3H]AA—Cells were incubated overnight in 12 wells with 0.25 μCi/ml [3H]AA in DMEM, 0.5% fetal calf serum. Cells were washed and incubated with DMEM, 0.2% BSA for 10 min. Then the media and cells were harvested and measured by scintillation counting (15Grimmer S. Ying M. Walchli S. van Deurs B. Sandvig K. Traffic. 2005; 6: 144-156Crossref PubMed Scopus (53) Google Scholar).Pulldown Assays with Purified GST-AnxA6—Cells were incubated with and without 30 μg/ml cholesterol, placed in cold lysis buffer (50 mm Tris, 100 mm NaCl, 1% Triton X-100, 0.1 mm CaCl2 plus protease inhibitors), scraped, and centrifuged. Then 75 μg of GST-AnxA6 and 21 μg of GST were incubated for 90 min with glutathione-Sepharose at 4 °C in phosphate-buffered saline. After washing with lysis buffer, Sepharose-bound GST-AnxA6 (and GST as control) was then incubated with 600 μg of cell extract for 2 h at 4 °C, and proteins bound to the column were collected by centrifugation and analyzed by Western blotting.Immunoprecipitation—CHO and CHOanx6 cells grown on 100-mm dishes were transfected with pEGFP-cPLA2. After washing in phosphate-buffered saline and solubilizing by scraping with a rubber policeman in TGH buffer (1% Triton X-100, 10% glycerol, 50 mm NaCl, 50 mm HEPES, pH 7.3, 1 mm sodium orthovanadate, 10 mm sodium fluoride, 1 mm phenylmethylsulfonyl fluoride, 10 mg/ml leupeptin, 10 mg/ml aprotinin) followed by gentle rotation for 10 min at 4 °C, lysates were then centrifuged at 14,000 × g for 10 min at 4 °C. Supernatants of transfected or not transfected cells were incubated with rabbit polyclonal anti-GFP or polyclonal anti-annexin A6 antibodies for 2 h at 4 °C and then for 60 min after the addition of protein A-Sepharose. Immunoprecipitates were washed twice in TGH supplemented with 150 mm NaCl and then once without NaCl. 10% SDS-polyacrylamide gels were used to separate proteins. Proteins were then transferred to Immobilon-P and immunoblotted using anti-annexin A6 or anti-GFP followed by the appropriate peroxidase-conjugated secondary antibody and ECL detection.Immunofluorescence Microscopy—Cells were grown on coverslips and fixed with 4% paraformaldehyde, washed, permeabilized with 0.1% saponin, and incubated with primary and secondary antibodies as described elsewhere (19Pons M. Ihrke G. Koch S. Biermer M. Pol A. Grewal T. Jackle S. Enrich C. Exp. Cell Res. 2000; 257: 33-47Crossref PubMed Scopus (35) Google Scholar). For anti-cPLA2 labeling, cells were fixed with cold methanol for 2 min. For double labeling with anti-cPLA2 and anti-α-mannosidase II, cells were first fixed with cold methanol followed by 4% paraformaldehyde for 12 min. Finally, samples were mounted in the anti-fading media Mowiol (Calbiochem), and cells were observed using an oil immersion Plan-Apo63x/1.4 objective in an Axio-plan Zeiss microscope. Images were captured with an AxioCam HRc camera and were digitally treated with Axio-Vision 3.1 software. In some experiments a Leica TCS SL laser-scanning confocal spectral microscope (Leica Microsystems) with argon and HeNe lasers attached to a Leica DMIRE2 inverted microscope was used. Image analysis was performed with Adobe Photoshop 7.0 software. To quantify the fluorescence intensity staining, images were captured using identical contrast and exposure times. Using NIH image software (Image J), the area to be quantified (the plasma membrane) was selected, the pixel intensity was determined, and the average staining intensity (20 cells from 3 experiments) was calculated. Colocalization of cPLA2 and α-mannosidase II was quantified using the Image J program (National Institutes of Health) and the "Highlighting Colocalization" plugin (Pierre Bourdoncle, Institut Jacques Monod, Service Imagerie, Paris), which highlights the colocalized points of two images. Two points were considered to colocalize if their intensity was higher than 50%, and an image of colocalized points was generated. Then in each cell the Golgi was defined as the region of interest, and the co-localization (%) therein was calculated. In each experiment at least 50 cells per cell line were analyzed. In some experiments the colocalization of mannosidase II with transiently transfected GFP-cPLA2 was analyzed. The transfection procedures have been described elsewhere (11Cubells L. Vila de Muga S. Tebar F. Wood P. Evans R. Ingelmo-Torres M. Calvo M. Gaus K. Pol A. Grewal T. Enrich C. Traffic. 2007; 8: 1568-1589Crossref PubMed Scopus (80) Google Scholar).Photobleaching Experiments and Time-lapse Confocal Microscopy—Fluorescence recovery after photobleaching (iFRAP) experiments were carried out in COS-1 cells using a Leica TCS SL laser-scanning confocal spectral microscope (Leica Microsystems) with argon and HeNe lasers attached to a Leica DMIRE2 inverted microscope equipped with an incubation system with temperature and CO2 control (16Pol A. Martin S. Fernandez M.A. Ingelmo-Torres M. Ferguson C. Enrich C. Parton R.G. Mol. Biol. Cell. 2005; 16: 2091-2105Crossref PubMed Scopus (169) Google Scholar). For visualization of GFP, images were acquired using 63× oil immersion objective lens (NA 1.32; 488 nm laser line; excitation beam splitter RSP 500, emission range detection 500-600 nm). The confocal pinhole was set at 2-3 Airy units to minimize changes in fluorescence due to GFP-tagged proteins moving away from the plane of focus. The whole cytoplasm, except the Golgi of a GFP fusion protein transfected cell, was photobleached using 50-80 scans with the 488-nm laser line at full power. Pre- and post-bleach images were monitored at 30-s intervals for 40 min. The excitation intensity was attenuated down to ∼5% of the half laser power to avoid significant photobleaching. The relative loss of fluorescence intensity in the unbleached region of interest and overall photobleaching in the whole cell during the time series were quantified using Image Processing Leica Confocal Software. Background fluorescence was measured in a random field outside of the cells. Fluorescence correction and normalization of GFP-tagged proteins were calculated according to Rabut and Ellenberg (20Rabut G. Ellenberg J. Goldman R.D. Spector D.L. Live Imaging: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York2005: 101-126Google Scholar).Isolation of Golgi Membranes—A modified method of Balch (21Balch W.E. Dunphy W.G. Braell W.A. Rothman J.E. Cell. 1984; 39: 405-416Abstract Full Text PDF PubMed Scopus (479) Google Scholar) described in detail by Brügger et al. (22Brügger B. Sandhoff R. Wegehingel S. Gorgas K. Malsam J. Helms J.B. Lehmann W.D. Nickel W. Wieland F.T. J. Cell Biol. 2000; 151: 507-518Crossref PubMed Scopus (184) Google Scholar) was used to isolate Golgi membrane fractions. All procedures were carried out at 4 °C. In brief, 4-5 × 109 cells were harvested, washed twice with phosphate-buffered saline, twice with BB (breaking buffer; 0.25 m sucrose in 10 mm Tris-HCl, pH 7.4), and finally resuspended in four volumes of BB. Cells were homogenized by passing 25 times through a ball-bearing homogenizer (Balch homogenizer), which disrupts early and late endosomes, but not Golgi vesicles, and brought to a sucrose concentration of 37% (w/w) by the addition of 62% (w/w) sucrose. 14 ml of sample was overlaid with 15 ml of 35% (w/w) sucrose and 9 ml of 29% (w/w) sucrose (in 10 mm Tris-HCl, pH 7.4) and centrifuged for 2.5 h at 25,000 rpm. Typically, 2 ml of a Golgi-enriched membrane fraction was recovered at the 35-29% interphase. In some experiments, before homogenization cells were preincubated with and without cholesterol for 90 min (30 μg/ml). The purity of the isolated Golgi membranes was confirmed by Western blot analysis with markers for Golgi (TGN38, GM130, GMAP210), early and late endosomes (Rab5, Rab7), plasma membrane (Na+K+ ATPase), and endoplasmic reticulum (KDEL; data not shown).Western Blot Analysis—CHOwt, CHOanx6, and HeLa cell lysates, samples from isolated Golgi membranes, and GST-AnxA6 pulldown assays were separated by 10% SDS-PAGE and transferred to Immobilon-P (Millipore) followed by incubation with primary antibodies and the appropriate peroxidase-conjugated secondary antibodies and ECL detection (Amersham Biosciences). Protein content was measured by the method of Lowry et al. (23Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar).RESULTSAnxA6 Expression Levels Affect Cholesterol-dependent cPLA2 Activity—Regulation of membrane traffic and cargo export from the Golgi complex involves cPLA2 activity (24de Figueiredo P. Drecktrah D. Polizotto R.S. Cole N.B. Lippincott-Schwartz J. Brown W.J. Traffic. 2000; 1: 504-511Crossref PubMed Scopus (56) Google Scholar, 25de Figueiredo P. Drecktrah D. Katzenellenbogen J.A. Strang M. Brown W.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8642-8647Crossref PubMed Scopus (123) Google Scholar). To address whether the AnxA6-dependent reduction of cholesterol export from late endosomes (11Cubells L. Vila de Muga S. Tebar F. Wood P. Evans R. Ingelmo-Torres M. Calvo M. Gaus K. Pol A. Grewal T. Enrich C. Traffic. 2007; 8: 1568-1589Crossref PubMed Scopus (80) Google Scholar) could be linked to cholesterol- and cPLA2-dependent secretory pathways from the Golgi to the plasma membrane, we first compared the activity of cPLA2 in CHOwt and CHOanx6 and measured the cellular release of radiolabeled arachidonic acid ([3H]AA) as described by Grimmer et al. (15Grimmer S. Ying M. Walchli S. van Deurs B. Sandvig K. Traffic. 2005; 6: 144-156Crossref PubMed Scopus (53) Google Scholar). CHOwt express very low amounts of AnxA6 (4Grewal T. Heeren J. Mewawala D. Schnitgerhans T. Wendt D. Salomon G. Enrich C. Beisiegel U. Jackle S. J. Biol. Chem. 2000; 275: 33806-33813Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 6Pons M. Grewal T. Rius E. Schnitgerhans T. Jackle S. Enrich C. Exp. Cell Res. 2001; 269: 13-22Crossref PubMed Scopus (42) Google Scholar, 8Rentero C. Evans R. Wood P. Tebar F. Vila de Muga S. Cubells L. de Diego I. Hayes T.E. Hughes W.E. Pol A. Rye K.A. Enrich C. Grewal T. Cell. Signal. 2006; 18: 1006-1016Crossref PubMed Scopus (35) Google Scholar, 9Grewal T. Evans R. Rentero C. Tebar F. Cubells L. de Diego I. Kirchhoff M.F. Hughes W.E. Heeren J. Rye K.A. Rinninger F. Daly R.J. Pol A. Enrich C. Oncogene. 2005; 24: 5809-5820Crossref PubMed Scopus (82) Google Scholar), whereas AnxA6 levels in CHOanx6 are rather similar to AnxA6 expression levels in other commonly used cell lines, such as HeLa (11Cubells L. Vila de Muga S. Tebar F. Wood P. Evans R. Ingelmo-Torres M. Calvo M. Gaus K. Pol A. Grewal T. Enrich C. Traffic. 2007; 8: 1568-1589Crossref PubMed Scopus (80) Google Scholar). As shown in Fig. 1A, both cell lines express comparable levels of cPLA2 (Fig. 1B). Importantly, these experiments clearly showed a 1.5-2.0-fold reduction of [3H]AA release in CHOanx6 cells (p < 0.01) (Fig. 1A).To find out whether AnxA6 down-regulation leads to increased cPLA2 activity, HeLa cells, which express high levels of AnxA6 (11Cubells L. Vila de Muga S. Tebar F. Wood P. Evans R. Ingelmo-Torres M. Calvo M. Gaus K. Pol A. Grewal T. Enrich C. Traffic. 2007; 8: 1568-1589Crossref PubMed Scopus (80) Google Scholar) (Fig. 1C), were transfected with RNAi targeting AnxA6 (RNAi-AnxA6), and the release of AA was measured. Upon AnxA6 knockdown, the cellular release of AA increased significantly (2.0-fold, Fig. 1C). RNAi-GFP-transfected cells were used as a control and did not show any changes in the release of AA. Then, to address if reduced cPLA2 activity could be overcome by the addition of exogenous cholesterol in AnxA6-expressing cells, [3H]AA-labeled CHOwt and CHOanx6 cells were incubated with and without cholesterol, and the amount of released radioactivity was determined as described above (Fig. 1D). Similar to the results described in Fig. 1A, release of [3H]AA is reduced in CHOanx6 cells compared with the controls (100 ± 6 and 65 ± 7% for CHOwt and CHOanx6, respectively). In agreement with previous data (15Grimmer S. Ying M. Walchli S. van Deurs B. Sandvig K. Traffic. 2005; 6: 144-156Crossref PubMed Scopus (53) Google Scholar), exogenous cholesterol increased cPLA2 activity 1.54 ± 0.10- and 2.12 ± 0.37-fold in both cell lines, respectively (n = 3). Thus, the addition of cholesterol compensates for the inhibitory effect of AnxA6 on cPLA2 activity in CHOanx6 cells to result in a release of [3H]AA, which is comparable with that of the controls. It is tempting to speculate that the retention of cholesterol in late endosomes of CHOanx6 cells (11Cubells L. Vila de Muga S. Tebar F. Wood P. Evans R. Ingelmo-Torres M. Calvo M. Gaus K. Pol A. Grewal T. Enrich C. Traffic. 2007; 8: 1568-1589Crossref PubMed Scopus (80) Google Scholar) leads to an impaired supply of cholesterol for cPLA2, thereby interfering with Golgi vesiculation.Impaired Translocation of cPLA2 to the Golgi Complex in AnxA6-expressing Cells—Then, to address if elevated AnxA6 levels perturb cPLA2 translocation to the Golgi, we isolated Golgi membranes from CHOwt and CHOanx6 cells and compared the amount of cPLA2. Western blot analysis (Fig. 2A) revealed reduced cPLA2 levels in the Golgi-enriched membrane fraction of CHOanx6 cells. Because CHOanx6 are characterized by an accumulation of cholesterol in late endosomes leading to reduced cholesterol levels in the Golgi (11Cubells L. Vila de Muga S. Tebar F. Wood P. Evans R. Ingelmo-Torres M. Calvo M. Gaus K. Pol A. Grewal T. Enrich C. Traffic. 2007; 8: 1568-1589Crossref PubMed Scopus (80) Google Scholar), we concluded that reduced cPLA2 levels in the Golgi of CHOanx6 cells could be due to an ineffective delivery of cholesterol from late endosomes to the Golgi complex. In support of this hypothesis, treatment of CHOwt cells with U18666A, a pharmacological agent to accumulate cholesterol in late endosomes, also resulted in a clear reduction of cPLA2 in the Golgi fractions without affecting total amounts of cPLA2 (Fig. 2B, compare the second and fourth lanes). To confirm these findings by means of immunofluorescence microscopy, the staining pattern of cPLA2 and a Golgi marker (α-mannosidase II) in CHOwt and CHOanx6 was compared (Fig. 3A). In both cell lines punctuate and in part diffuse staining of cPLA2 was observed throughout the cell and was more intense in the perinuclear region, in particular in CHOwt cells. In the CHOanx6 cell line, cPLA2 appeared cytosolic with minor cPLA2 staining detected within the Golgi complex as judged by colocalization with anti-α-mannosidase II and anti-cPLA2 (see Fig. 3A, arrows in panel a-c). Quantification of fluorescence intensity confirmed the increased co-localization (∼3-fold) of cPLA2 with α-mannosidase II in CHOwt compared with CHOanx6 cells (Fig. 3B). In addition, when Golgi membranes were isolated from CHOwt or CHOanx6 cells in the presence of cholesterol (90 min) and analyzed by Western blotting, a significant increase of cPLA2 levels was observed (Fig. 3C). Thus, in CHOanx6 cells the reduced localization of cPLA2 in the Golgi correlates with a decreased enzymatic activity of cPLA2 (Fig. 1A).FIGURE 2Cholesterol impaired translocation of cPLA2 into Golgi membranes. A, reduced cPLA2 levels in Golgi fractions isolated from CHOanx6 cells (anx6) compared with controls (wt) are shown. AnxA6 expression does not affect cPLA2 levels in whole cell lysates (WCL, A and B). B, cPLA2 levels in isolated Golgi fractions from CHOwt cells with and without U18666A were analyzed. cPLA2 is not detectable in the Golgi fraction upon U18666A treatment, indicating that cPLA2 translocation to the Golgi requires late endosomal cholesterol.View Large Image Figure ViewerDownload Hi-res
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