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• W2005055445 abstract "Engagement of the IgE receptor (FcϵRI) on mast cells leads to the release of preformed and newly formed mediators as well as of cytokines. The signaling pathways responsible for these responses involve tyrosine phosphorylation of multiple proteins. We previously reported the phosphorylation on tyrosine of phospholipid scramblase 1 (PLSCR1) after FcϵRI aggregation. Here, PLSCR1 expression was knocked down in the RBL-2H3 mast cell line using short hairpin RNA. Knocking down PLSCR1 expression resulted in significantly impaired degranulation responses after FcϵRI aggregation and release of vascular endothelial growth factor, whereas release of MCP-1 was minimally affected. The release of neither leukotriene C4 nor prostaglandin D2 was altered by knocking down of PLSCR1. Analysis of FcϵRI-dependent signaling pathways revealed that whereas tyrosine phosphorylation of ERK and Akt was unaffected, tyrosine phosphorylation of LAT was significantly reduced in PLSCR1 knocked down cells. Tyrosine phosphorylation of phospholipase Cγ1 and consequently the mobilization of calcium were also significantly reduced in these cells. In nonactivated mast cells, PLSCR1 was found in part in lipid rafts where it was further recruited after cell activation and was constitutively associated with Lyn and Syk but not with LAT or Fyn. Altogether, these data identify PLSCR1 as a novel amplifier of FcϵRI signaling that acts selectively on the Lyn-initiated LAT/phospholipase Cγ1/calcium axis, resulting in potentiation of a selected set of mast cell responses. Engagement of the IgE receptor (FcϵRI) on mast cells leads to the release of preformed and newly formed mediators as well as of cytokines. The signaling pathways responsible for these responses involve tyrosine phosphorylation of multiple proteins. We previously reported the phosphorylation on tyrosine of phospholipid scramblase 1 (PLSCR1) after FcϵRI aggregation. Here, PLSCR1 expression was knocked down in the RBL-2H3 mast cell line using short hairpin RNA. Knocking down PLSCR1 expression resulted in significantly impaired degranulation responses after FcϵRI aggregation and release of vascular endothelial growth factor, whereas release of MCP-1 was minimally affected. The release of neither leukotriene C4 nor prostaglandin D2 was altered by knocking down of PLSCR1. Analysis of FcϵRI-dependent signaling pathways revealed that whereas tyrosine phosphorylation of ERK and Akt was unaffected, tyrosine phosphorylation of LAT was significantly reduced in PLSCR1 knocked down cells. Tyrosine phosphorylation of phospholipase Cγ1 and consequently the mobilization of calcium were also significantly reduced in these cells. In nonactivated mast cells, PLSCR1 was found in part in lipid rafts where it was further recruited after cell activation and was constitutively associated with Lyn and Syk but not with LAT or Fyn. Altogether, these data identify PLSCR1 as a novel amplifier of FcϵRI signaling that acts selectively on the Lyn-initiated LAT/phospholipase Cγ1/calcium axis, resulting in potentiation of a selected set of mast cell responses. Mast cells actively participate in the defense against bacterial and parasitic infections (1Malaviya R. Abraham S.N. Immunol. Rev. 2001; 179: 16-24Crossref PubMed Scopus (128) Google Scholar, 2Pennock J.L. Grencis R.K. Chem. Immunol. Allergy. 2006; 90: 128-140PubMed Google Scholar) and are key players in the allergic reaction. They express receptors that bind IgE with a high affinity (FcϵRI) and that are aggregated by the cognate allergen when they are occupied by allergen-specific IgE (3Metzger H. Clin. Exp. Allergy. 1991; 21: 269-279Crossref PubMed Scopus (71) Google Scholar). This results in a number of cellular responses, such as immediate release of granule contents (e.g. histamine and β-hexosaminidase), synthesis of the lipid mediators of inflammation leukotrienes (LT) 6The abbreviations used are: LTleukotrieneBSAbovine serum albuminDNP-HSAdinitrophenyl coupled to human serum albuminFcϵRIhigh affinity IgE receptorIP3inositol 1,4,5-trisphosphatePGprostaglandinPLCphospholipase CPLSCR1phospholipid scramblase 1VEGFvascular endothelial growth factorERKextracellular signal-regulated kinasePI3Kphosphatidylinositol 3-kinasesiRNAsmall interfering RNAshRNAsmall hairpin RNAELISAenzyme-linked immunosorbent assayPBSphosphate-buffered salineFACSfluorescence-activated cell sortermAbmonoclonal antibodyIP3R1IP3 receptor 1. 6The abbreviations used are: LTleukotrieneBSAbovine serum albuminDNP-HSAdinitrophenyl coupled to human serum albuminFcϵRIhigh affinity IgE receptorIP3inositol 1,4,5-trisphosphatePGprostaglandinPLCphospholipase CPLSCR1phospholipid scramblase 1VEGFvascular endothelial growth factorERKextracellular signal-regulated kinasePI3Kphosphatidylinositol 3-kinasesiRNAsmall interfering RNAshRNAsmall hairpin RNAELISAenzyme-linked immunosorbent assayPBSphosphate-buffered salineFACSfluorescence-activated cell sortermAbmonoclonal antibodyIP3R1IP3 receptor 1. and prostaglandins (PG), and delayed production and release of cytokines and chemokines (4Kraft S. Kinet J.P. Nat. Rev. Immunol. 2007; 7: 365-378Crossref PubMed Scopus (432) Google Scholar). leukotriene bovine serum albumin dinitrophenyl coupled to human serum albumin high affinity IgE receptor inositol 1,4,5-trisphosphate prostaglandin phospholipase C phospholipid scramblase 1 vascular endothelial growth factor extracellular signal-regulated kinase phosphatidylinositol 3-kinase small interfering RNA small hairpin RNA enzyme-linked immunosorbent assay phosphate-buffered saline fluorescence-activated cell sorter monoclonal antibody IP3 receptor 1. leukotriene bovine serum albumin dinitrophenyl coupled to human serum albumin high affinity IgE receptor inositol 1,4,5-trisphosphate prostaglandin phospholipase C phospholipid scramblase 1 vascular endothelial growth factor extracellular signal-regulated kinase phosphatidylinositol 3-kinase small interfering RNA small hairpin RNA enzyme-linked immunosorbent assay phosphate-buffered saline fluorescence-activated cell sorter monoclonal antibody IP3 receptor 1. The pathways leading from FcϵRI aggregation to cellular responses have been extensively studied and depend on tyrosine phosphorylations (5Benhamou M. Gutkind J.S. Robbins K.C. Siraganian R.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5327-5330Crossref PubMed Scopus (199) Google Scholar, 6Benhamou M. Stephan V. Robbins K.C. Siraganian R.P. J. Biol. Chem. 1992; 267: 7310-7314Abstract Full Text PDF PubMed Google Scholar). The two main pathways involve tyrosine kinases belonging to the Src family, Lyn and Fyn (4Kraft S. Kinet J.P. Nat. Rev. Immunol. 2007; 7: 365-378Crossref PubMed Scopus (432) Google Scholar). Lyn is constitutively associated with FcϵRI β chain (7Eiseman E. Bolen J.B. Nature. 1992; 355: 78-80Crossref PubMed Scopus (413) Google Scholar). Upon aggregation of the receptor it phosphorylates both the β and the γ chains of FcϵRI on tyrosine residues within the immunoreceptor tyrosine-based activation motif that is found in their intracellular domains (8Paolini R. Jouvin M.H. Kinet J.P. Nature. 1991; 353: 855-858Crossref PubMed Scopus (219) Google Scholar). This results in the recruitment of additional Lyn molecules and of the tyrosine kinase Syk to the phosphorylated receptors and in the activation of Syk (9Benhamou M. Ryba N.J. Kihara H. Nishikata H. Siraganian R.P. J. Biol. Chem. 1993; 268: 23318-23324Abstract Full Text PDF PubMed Google Scholar, 10Yamashita T. Mao S.Y. Metzger H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11251-11255Crossref PubMed Scopus (157) Google Scholar, 11Minoguchi K. Benhamou M. Swaim W.D. Kawakami Y. Kawakami T. Siraganian R.P. J. Biol. Chem. 1994; 269: 16902-16908Abstract Full Text PDF PubMed Google Scholar). Lyn and Syk then phosphorylate a number of substrates including the scaffold proteins LAT (12Saitoh S. Arudchandran R. Manetz T.S. Zhang W. Sommers C.L. Love P.E. Rivera J. Samelson L.E. Immunity. 2000; 12: 525-535Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar) and LAT2 (13Brdicka T. Imrich M. Angelisova P. Brdickova N. Horvath O. Spicka J. Hilgert I. Luskova P. Draber P. Novak P. Engels N. Wienands J. Simeoni L. Osterreicher J. Aguado E. Malissen M. Schraven B. Horejsi V. J. Exp. Med. 2002; 196: 1617-1626Crossref PubMed Scopus (177) Google Scholar, 14Zhu M. Liu Y. Koonpaew S. Granillo O. Zhang W. J. Exp. Med. 2004; 200: 991-1000Crossref PubMed Scopus (105) Google Scholar, 15Volna P. Lebduska P. Draberova L. Simova S. Heneberg P. Boubelik M. Bugajev V. Malissen B. Wilson B.S. Horejsi V. Malissen M. Draber P. J. Exp. Med. 2004; 200: 1001-1013Crossref PubMed Scopus (114) Google Scholar). Activation of PLCγ1 and PLCγ2 recruited to phosphorylated LAT leads to the release in the cytosol of inositol 1,4,5-trisphosphate (IP3) and of diacylglycerols. IP3 releases calcium from the intracellular stores with a subsequent influx of calcium from the extracellular milieu, thus participating in the calcium signal that is required for mast cell activation and, together with diacylglycerol, activates various protein kinase C. Fyn is also constitutively associated with FcϵRI and is activated after receptor aggregation (16Parravicini V. Gadina M. Kovarova M. Odom S. Gonzalez-Espinosa C. Furumoto Y. Saitoh S. Samelson L.E. O'Shea J.J. Rivera J. Nat. Immunol. 2002; 3: 741-748Crossref PubMed Scopus (396) Google Scholar, 17Gomez G. Gonzalez-Espinosa C. Odom S. Baez G. Cid M.E. Ryan J.J. Rivera J. J. Immunol. 2005; 175: 7602-7610Crossref PubMed Scopus (93) Google Scholar). It then phosphorylates the adaptor protein Gab2 leading to activation of PI3K and of its downstream effectors PDK1 and Akt (16Parravicini V. Gadina M. Kovarova M. Odom S. Gonzalez-Espinosa C. Furumoto Y. Saitoh S. Samelson L.E. O'Shea J.J. Rivera J. Nat. Immunol. 2002; 3: 741-748Crossref PubMed Scopus (396) Google Scholar, 18Ali K. Bilancio A. Thomas M. Pearce W. Gilfillan A.M. Tkaczyk C. Kuehn N. Gray A. Giddings J. Peskett E. Fox R. Bruce I. Walker C. Sawyer C. Okkenhaug K. Finan P. Vanhaesebroeck B. Nature. 2004; 431: 1007-1011Crossref PubMed Scopus (353) Google Scholar). It also results in activation of sphingosine kinases that phosphorylate sphingosine into sphingosine 1 phosphate, thus modulating calcium influx (19Choi O.H. Kim J.H. Kinet J.P. Nature. 1996; 380: 634-636Crossref PubMed Scopus (383) Google Scholar, 20Olivera A. Urtz N. Mizugishi K. Yamashita Y. Gilfillan A.M. Furumoto Y. Gu H. Proia R.L. Baumruker T. Rivera J. J. Biol. Chem. 2006; 281: 2515-2525Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 21Olivera A. Mizugishi K. Tikhonova A. Ciaccia L. Odom S. Proia R.L. Rivera J. Immunity. 2007; 26: 287-297Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Therefore, both the Lyn and the Fyn pathways participate in regulating the calcium signal in FcϵRI-dependent mast cell activation. Production of LT and PG depends on the production of their common precursor arachidonic acid following cleavage of membrane phospholipids by phospholipase A2. Cytosolic phospholipase A2 is activated downstream of the ERK/mitogen-activated protein kinase (MAPK) cascade (22Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1995; 154: 5391-5402PubMed Google Scholar) that depends on both the Lyn-initiated and the Fyn-initiated pathways (4Kraft S. Kinet J.P. Nat. Rev. Immunol. 2007; 7: 365-378Crossref PubMed Scopus (432) Google Scholar). Similarly, both pathways participate in the activation of transcription factors leading to the synthesis of cytokines. Recently, we reported the phosphorylation on tyrosine of phospholipid scramblase 1 (PLSCR1) after FcϵRI aggregation (23Pastorelli C. Veiga J. Charles N. Voignier E. Moussu H. Monteiro R.C. Benhamou M. J. Biol. Chem. 2001; 276: 20407-20412Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). PLSCR1 has been initially proposed as the protein responsible for the rapid redistribution of phospholipids between the two leaflets of the plasma membrane after cell activation or apoptosis, leading to the disruption of their asymetric distribution (24Zhou Q. Zhao J. Stout J.G. Luhm R.A. Wiedmer T. Sims P.J. J. Biol. Chem. 1997; 272: 18240-18244Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). To date however, contradictory results have been reported, and this issue is still unresolved (25Zhou Q. Zhao J. Wiedmer T. Sims P.J. Blood. 2002; 99: 4030-4038Crossref PubMed Scopus (192) Google Scholar, 26Wiedmer T. Zhao J. Li L. Zhou Q. Hevener A. Olefsky J.M. Curtiss L.K. Sims P.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13296-13301Crossref PubMed Scopus (40) Google Scholar). Additional roles have been described for PLSCR1. Although PLSCR1 contains a consensus transmembrane domain, it requires palmitoylation to be stabilized at the plasma membrane (27Wiedmer T. Zhao J. Nanjundan M. Sims P.J. Biochemistry. 2003; 42: 1227-1233Crossref PubMed Scopus (92) Google Scholar). In the absence of palmitoylation, it is found in the nucleus where it can bind DNA and activate the transcription of the IP3 receptor 1 gene (28Ben-Efraim I. Zhou Q. Wiedmer T. Gerace L. Sims P.J. Biochemistry. 2004; 43: 3518-3526Crossref PubMed Scopus (56) Google Scholar, 29Zhou Q. Ben-Efraim I. Bigcas J.L. Junqueira D. Wiedmer T. Sims P.J. J. Biol. Chem. 2005; 280: 35062-35068Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). When palmitoylated it participates in the epidermal growth factor receptor signaling (30Sun J. Nanjundan M. Pike L.J. Wiedmer T. Sims P.J. Biochemistry. 2002; 41: 6338-6345Crossref PubMed Scopus (75) Google Scholar) by amplifying the activation of the tyrosine kinase Src (31Nanjundan M. Sun J. Zhao J. Zhou Q. Sims P.J. Wiedmer T. J. Biol. Chem. 2003; 278: 37413-37418Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The tyrosine phosphorylation of PLSCR1 following FcϵRI aggregation strongly suggested the involvement of this protein in FcϵRI-dependent mast cell activation. Yet this role was still elusive (32Smrz D. Lebduska P. Dráberová L. Korb J. Dráber P. J. Biol. Chem. 2008; 283: 10904-10918Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). To address this question, we used an RNA silencing approach. We show that a mast cell line with a knocked down PLSCR1 expression had significantly impaired degranulation and VEGF release after FcϵRI engagement that was preceded by a decreased tyrosine phosphorylation of LAT and PLCγ1 and a decreased calcium mobilization. Antibodies and Reagents—Monoclonal anti-rat PLSCR1 antibodies 129.2 and 17.3 and anti-FcϵRIβ monoclonal antibody 30.9 have been already described (23Pastorelli C. Veiga J. Charles N. Voignier E. Moussu H. Monteiro R.C. Benhamou M. J. Biol. Chem. 2001; 276: 20407-20412Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 33Charles N. Monteiro R.C. Benhamou M. J. Biol. Chem. 2004; 279: 12312-12318Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Antibody 17.3 recognizes also a p28 in addition to PLSCR1 (33Charles N. Monteiro R.C. Benhamou M. J. Biol. Chem. 2004; 279: 12312-12318Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Rabbit anti-Syk polyclonal antibody has been described (34Launay P. Lehuen A. Kawakami T. Blank U. Monteiro R.C. J. Leukocyte Biol. 1998; 63: 636-642Crossref PubMed Scopus (48) Google Scholar). Anti-phosphotyrosine monoclonal antibody 4G10 was used as a culture supernatant of the 4G10 hybridoma or as horseradish peroxidase-coupled antibody (Calbiochem, La Jolla, CA). The anti-DNP monoclonal IgE DNP48 was a kind gift from Dr. R. P. Siraganian (NIDCR, National Institutes of Health, Bethesda, MD). Mouse anti-LAT monoclonal IgG was a generous gift from Dr. P. Dráber (Laboratory of Signal Transduction, Institute of Molecular Genetics, Prague, Czech Republic). Anti-Fyn polyclonal IgG is a kind gift from Dr. S. Latour (INSERM U768, Necker Hospital, Paris, France). Other reagents were from the following sources: anti-Lyn polyclonal antibody (Santa Cruz Biotech, Santa Cruz, CA); Alexa Fluor 647 streptavidin, Lipofectamine 2000 (Invitrogen); anti-IP3R1 polyclonal antibody (Chemicon International, Temecula, CA); anti-phospho-ERK (Tyr202/Tyr204), -Akt (Ser473), -PLCγ1 (Tyr783), and -LAT (Tyr191 and Tyr132), and anti-total-ERK, -Akt, -PLCγ1 polyclonal antibodies (Cell Signaling Tech, Danvers, MA); anti-PLCγ1 monoclonal antibody (Upstate Biotech Inc, Lake Placid, NY); horseradish peroxidase-labeled secondary antibodies, Fura 2-AM, Triton X-100, SDS, bovine serum albumin (BSA), DNP-human serum albumin (DNP-HSA), proteases and phosphatases inhibitors (leupeptin, aprotinin, NaF, and Na3VO4), and polybrene (Sigma-Aldrich); Dulbecco's modified Eagle's medium, fetal calf serum, and other culture reagents as well as goat serum (Invitrogen); puromycin (Invivogen, San Diego, CA); and Vectashield mounting medium (Vector Labs, Burlingame, CA). Cells and Cell Lines—The rat mast cell line RBL-2H3 and human embryonic kidney cells HEK 293 were cultured in Dulbecco's modified Eagle's medium with Glutamax supplemented with 10% heat-inactivated fetal calf serum and with penicillin and streptomycin. siRNA, shRNA Design, and Stable Expression—Three siRNA were selected that were specific for rat PLSCR1. These corresponded to the following sequences: siRNA1, 5′-GUG-GCU-UUC-CUG-UCC-AAC-A-3′; siRNA2, 5′-GGC-AGG-ACG-UUC-UAA-AGG-U-3′; and siRNA3, 5′-CCU-UGA-GGA-UCC-UGG-AUA-A-3′; control siRNA (scrambled siRNA3) was 5′-UGC-UAG-UGA-CCU-GCC-AUA-A-3′;. The siRNA duplexes were transfected into RBL-2H3 cells by electroporation. Two days later, the cells were lysed. The presence of PLSCR1 in the lysates was examined by immunoblotting with the monoclonal antibody 17.3 that recognizes both PLSCR1 and a 28-kDa protein (p28). This protein served as a control both for loading and for silencing specificity during the screening test. The sequence of the most efficient siRNA (and of the control siRNA) were then selected to design a shRNA. The mammalian expression vector pSuper.retro.puro (Oligo-Engine, Seattle, WA) was used for expression of these shRNA in RBL-2H3 cells. The sequences were inserted between the HindIII and BglII sites of the vector, and after amplification the cloned sequences were confirmed. To generate retrovirus-containing supernatants, the packaging cell line HEK 293 was plated at 3 × 106 cells/10-cm plate 48 h prior to transfection of the recombinant vectors with Lipofectamine. A green fluorescent protein expression system was used to control for transfection efficiency by flow cytometry. The culture medium was changed 24 h after transfection, and 48 h later the retrovirus-containing supernatant was collected, supplemented with polybrene (8 μg/ml), and added to RBL-2H3 cell cultures. The infected cells were resuspended 48 h later in fresh medium containing puromycin at 1 μg/ml for selection. Silencing of PLSCR1 expression was confirmed by Western blotting and confocal microscopy. For functional analyses, puromycin was omitted at least 72 h before experiments without detectable alleviation of PLSCR1 silencing. Cell Stimulation and Measurement of β-Hexosaminidase Release—One or 1.5 × 106 RBL-2H3 cells were incubated overnight in the presence of a 1:100 dilution DNP48 anti-DNP IgE ascitic fluid. After two washes in Tyrode's solution (10 mm Hepes, pH 7.3, 135 mm NaCl, 5 mm KCl, 5.6 mm glucose, 1.8 mm CaCl2, 1 mm MgCl2, 0.5 mg/ml BSA), the cells were stimulated at 37 °C with DNP-HSA. Release of the granule marker β-hexosaminidase was measured in the supernatants as described (36Benhamou M. Ninio E. Salem P. Hieblot C. Bessou G. Pitton C. Liu F.T. Mencia-Huerta J.M. J. Immunol. 1986; 136: 1385-1392PubMed Google Scholar). Prostaglandin and Leukotriene Measurements—Supernatants from cells stimulated in Tyrode's buffer containing fatty acid-free BSA were centrifuged at 1400 rpm for 5 min and used for the determination of LTC4 and PGD2 using an enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's recommendations. VEGF and MCP-1 Release—Supernatants of cells stimulated in culture medium were centrifuged to eliminate cell debris. Secreted VEGF was measured using ELISA DuoSet kits (R & D Systems, Minneapolis, MN). MCP-1 was measured with the rat MCP-1 ELISA development kit from Peprotech (Paris, France). Calcium Imaging—5 × 105 IgE-sensitized RBL-2H3 cells were plated on coverslips and loaded with 1 μl of pluronic acid F-127 (InVitrogen) and 1 μm Fura 2-AM at 37 °C for 30 min. Fluorescence measurements were performed with a combination of a scanning monochromator and a CCD camera operated by TILLvisION software (Till Imago, TILL Photonics, Gräfelfing, Germany). The camera was mounted to an inverted microscope (Axiovert 100; Carl Zeiss, Jena, Germany) with a 40× oil objective (Nikon). The cells were exposed to alternating excitation light pulses of 340- and 380-nm wavelengths of 120 μs each. Fluorescence values of the collected emission images were corrected for background fluorescence before starting data collection. The results are compiled from three experiments, and per experiment up to 5–6 cells were analyzed simultaneously. The fluorescence intensity ratio was calculated by the ratio of the fluorescence values at 340- and 380-nm excitation. Fluorescence signals were plotted as (F/F0) - 1 with F as fluorescence during the experimentation and F0 as the initial level of fluorescence (37Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). The areas under the curve were calculated as arbitrary units. Confocal Microscopy—2.5 × 105 RBL-2H3 cells were plated on coverslips inside 24-well plates. After each step, the cells were washed three times with PBS. The cells were fixed in PBS, 3% paraformaldehyde for 15 min at 4 °C and incubated with 50 nm of NH4Cl in PBS (to reduce aldehyde groups) at room temperature. After a 1-h saturation in A1 buffer (0.25% BSA, 0.025% saponin, and 10% of goat serum in PBS), the cells were incubated with biotinylated 129.2 antibody (20 μg/ml) in A1 buffer for 1 h. The cells were then incubated with streptavidin coupled to AlexaFluor 647 (1:400 dilution) for 45 min in 0.25% BSA, 0.00125% saponin, 10% goat serum in PBS. Slides were mounted using Vectashield mounting medium and analyzed by confocal microscopy with a Zeiss LSM-5 confocal scanning laser microscope, equipped with argon and helium-neon lasers, using a plan-aprochromat 63× objective (numerical aperture, 1.40; oil immersion) and a crop ×2, at room temperature. Red fluorescence was observed with a 560-nm long pass emission filter under 543-nm laser illumination. Images were collected every 0.4 μm along the z-axis. Imaging was obtained using LSM imaging software. FACS Analysis—The cells were washed twice in PBS containing 0.5% BSA and 0.01% NaN3 (PBA) at 4 °C. After a 30-min incubation with 100 μg/ml rabbit IgG in PBA, 5 × 105 cells were further incubated with or without 10 μg/ml IgE at 4 °C for 1 h. After two washes in PBA, the cells were incubated for 1 h at 4 °C with goat anti-mouse IgE antibody coupled to fluorescein isothiocyanate. After two final washes in PBA, the cell-associated fluorescence was analyzed with a FACSCalibur (Becton Dickinson, Franklin Lakes, NJ) flow cytometer. Metabolic Labeling with [3H]Palmitate—One million RBL-2H3 cells were incubated overnight with 200 μCi/ml of [3H]palmitate (45 Ci/mmol; MP Biomedicals, Irvine, CA) in a fatty-acid free medium containing fetal calf serum dialyzed against PBS. The cells were then washed two times in PBS prior to lysis as described below. Cell Lysis and Immunoprecipitation—One or 10 × 106 cells were lysed in 200 μl or 1 ml of lysis buffer (50 mm NaCl, 50 mm NaF, 1 mm sodium orthovanadate, 0.5% Triton X-100, 50 mm Hepes, and anti-proteases: 10 μg/ml leupeptin, 10 μg/ml aprotinin) for 10 min at 4 °C and centrifuged to obtain the soluble fraction. Protein concentration was determined by the Bradford protein assay from Bio-Rad. For immunoprecipitation, rabbit polyclonal or mouse monoclonal anti-PLCγ1 or anti-FcϵRIβ chain monoclonal antibody 30.9 was incubated with Protein A/G-agarose beads (VWR, Paris, France) and with 200 μl of cell lysates on a rotating wheel for 2–3 h at 4 °C. The beads were washed six times with ice-cold lysis buffer, and the bound material was eluted by boiling for 5 min in 50 μl of Laemmli sample buffer. For coimmunoprecipitation, Sepharose 4B beads coupled with anti-PLSCR1 mAb 129.2 or with a control antibody were used. Western Blotting—Proteins resolved by SDS-PAGE were transferred onto polyvinylidene difluoride membranes that were then blocked by incubation in Tris-buffered saline containing 0.1% Tween 20 (TBST) and 4% BSA. The blots were incubated for 1 h at room temperature or overnight at 4 °C with primary antibodies. After several washes in TBST, horseradish peroxidase-labeled anti-rabbit or anti-mouse IgGs were used for a 1-h incubation at room temperature. The blots were washed, and labeling was visualized using the super signal West Pico chemoluminescence kit from Pierce followed by exposure to X-Omat Kodak films. For immunoprecipitated PLCγ1 samples, after first probing for tyrosine phosphorylation, the membranes were stripped by two 10-min incubations in 2% SDS and two 10-min incubations in 0.2 n NaOH followed by three washes in TTBS. The membranes were then reprobed with antibodies directed against PLCγ1 for loading control. For quantifications, control and PLSCR1 knocked down samples of individual experiments were resolved in the same gel and blotted on the same membrane. The bands were analyzed with the National Institutes of Health ImageJ software and were quantified relative to the PLCγ1 or to the p28 band analyzed in the corresponding lane. For analysis of individual experiments, normalization was done by setting arbitrarily the value thus obtained for the unstimulated control as “1.” For statistical analysis, a different procedure was followed. Each individual value obtained for control cells in a given experiment was arbitrarily set as “100,” and the value obtained for the corresponding point in knocked down cells in the same experiment was normalized according to it, i.e. as the percentage of the value obtained with control cells for the corresponding experimental point. This procedure allowed elimination of the intrinsic variations in “absolute” values between experiments resulting from the ImageJ analysis procedure and to analyze the paired differences between control and PLSCR1 knocked down cells. Student's t test was then used with the values thus calculated. Isolation of Lipid Raft Microdomains by Sucrose Gradient Centrifugation—Lipid rafts were isolated from freshly harvested cells. The procedure was adapted from that of Pombo et al. (38Pombo I. Rivera J. Blank U. FEBS Lett. 2003; 550: 144-148Crossref PubMed Scopus (42) Google Scholar). Eight million RBL-2H3 cells were plated on 15-cm dishes, and 24 h later the cells were sensitized with a 1:102 dilution of the DNP-48 monoclonal IgE ascitic fluid for an overnight incubation. The cells were stimulated with 1 μg/ml DNP-HSA in Tyrode's buffer for 30 min and washed two times in ice-cold PBS followed by two washes in ice-cold TNV buffer (10 mm Tris-HCl, pH 7.5, 150 mm NaCl). The cells were solubilized in 1 ml of ice-cold TNV containing 0.1% Triton X-100, 1 mm sodium orthovanadate, 1000 units/ml aprotinin, and 20 μg/ml leupeptin and harvested by scraping. The lysate was transferred to a Dounce homogenizer and subjected to 15 strokes. The homogenate was mixed in an Ultra-clear TM centrifuge tube (Beckman) with an equal volume of 85% sucrose. This mixture was successively overlaid with 5 ml of 30% sucrose and 2 ml of 5% sucrose, neither of which contained Triton X-100. The tubes were then centrifuged at 200,000 × g without break at 4 °C, in a Beckman SW40Ti rotor for 16 h. Sequential pooled fractions 1–3 (1713 μl), 4–6 (2499 μl), 7–9 (2499 μl), and 10–12 (3000 μl) were harvested from the top of the gradient. An opaque band at the interface between the 5 and 30% layers was routinely harvested in fractions 4–6 and contained the lipid rafts. The samples were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with the indicated antibodies. Knocking Down of PLSCR1 Expression—The function of PLSCR1 in mast cell activation through the FcϵRI remained unclear, although its involvement was strongly suggested by its phosphorylation on tyrosine residues after FcϵRI aggregation (23Pastorelli C. Veiga J. Charles N. Voignier E. Moussu H. Monteiro R.C. Benhamou M. J. Biol. Chem. 2001; 276: 20407-20412Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). To address this issue, we chose a siRNA approach. Three siRNA were tested for their ability to decrease PLSCR1 expression. For this screening we chose an immunoblotting procedure to follow protein expression. The siRNA2 was the most potent for silencing PLSCR1 expression (supplemental Fig. S1), whereas the other siRNA were somewhat less efficient. The sequences corresponding to siRNA2 and siRNA3 were selected to design shRNA. These sequences and a control shRNA corresponding to the scrambled sequence of siRNA3 were inserted into an expression vector that was transduced into cells with a retroviral system to allow for a stable and selectable decrease in PLSCR1 expression. By immunoblotting (Fig. 1A) the decrease in PLSCR1 expression reached over 99% in PLSCR1 shRNA-infected cells as compared with control shRNA-infected cells or to noninfected cells. This was confirmed by confocal analysis (Fig. 1B). By contrast, expression of FcϵRI was unaffected in these cells (Fig. 1C). PLSCR1 Amplifies Degranulation and Release of VEGF—Cell responses of mast cells in this virtual absence of PLSCR1 were the" @default.
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• W2005055445 date "2008-09-01" @default.
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• W2005055445 title "Phospholipid Scramblase 1 Modulates a Selected Set of IgE Receptor-mediated Mast Cell Responses through LAT-dependent Pathway" @default.
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• W2005055445 doi "https://doi.org/10.1074/jbc.m705320200" @default.
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