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• W2029372505 abstract "We previously demonstrated that secretory phospholipase A2 (sPLA2) and lysophosphatidylcholine (LPC) exhibit neurotrophin-like neuritogenic activity in the rat pheochromocytoma cell line PC12. In this study, we further analyzed the mechanism whereby sPLA2 displays neurite-inducing activity. Exogenously added mammalian group X sPLA2 (sPLA2-X), but not group IB and IIA sPLA2s, induced neuritogenesis, which correlated with the ability of sPLA2-X to liberate LPC into the culture media. In accordance, blocking the effect of LPC by supplementation of bovine serum albumin or phospholipase B attenuated neuritogenesis by sPLA2 or LPC. Overproduction or suppression of G2A, a G-protein-coupled receptor involved in LPC signaling, resulted in the enhancement or reduction of neuritogenesis induced by sPLA2 treatment. These results indicate that the neuritogenic effect of sPLA2 is mediated by generation of LPC and subsequent activation of G2A. We previously demonstrated that secretory phospholipase A2 (sPLA2) and lysophosphatidylcholine (LPC) exhibit neurotrophin-like neuritogenic activity in the rat pheochromocytoma cell line PC12. In this study, we further analyzed the mechanism whereby sPLA2 displays neurite-inducing activity. Exogenously added mammalian group X sPLA2 (sPLA2-X), but not group IB and IIA sPLA2s, induced neuritogenesis, which correlated with the ability of sPLA2-X to liberate LPC into the culture media. In accordance, blocking the effect of LPC by supplementation of bovine serum albumin or phospholipase B attenuated neuritogenesis by sPLA2 or LPC. Overproduction or suppression of G2A, a G-protein-coupled receptor involved in LPC signaling, resulted in the enhancement or reduction of neuritogenesis induced by sPLA2 treatment. These results indicate that the neuritogenic effect of sPLA2 is mediated by generation of LPC and subsequent activation of G2A. Phospholipase A2 (PLA2) 1The abbreviations used are: PLA2, phospholipase A2; sPLA2, secretory PLA2; BSA, bovine serum albumin; FCS, fetal calf serum; GPCR, G-protein-coupled receptor; LPC, lysophosphatidylcholine; NGF, nerve growth factor; PC, phosphatidylcholine; PLB, phospholipase B; shRNA, short hairpin RNA; SPC, sphingosylphosphorylcholine; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; EGFP, enhanced GFP; RT, reverse transcription. is an enzyme that cleaves sn-2 ester linkage of glycerophospholipids thereby releasing fatty acids and 2-lysophospholipids (1Murakami M. Kudo I. Adv. Immunol. 2001; 77: 163-194Crossref PubMed Scopus (135) Google Scholar, 2Kudo I. Murakami M. Prostaglandins Other Lipid Mediat. 2002; 68-69: 3-58Crossref PubMed Scopus (659) Google Scholar, 3Murakami M. Kudo I. Biol. Pharm. Bull. 2004; 27: 1158-1164Crossref PubMed Scopus (110) Google Scholar). The secreted type of PLA2, sPLA2, is a small (13-20 kDa), Ca2+-dependent, disulfide-rich protein composed of extremely diverse members present in venoms, digestive exudates, inflammation sites, various mammalian tissues, and in microorganisms. In mammals, 11 genes encoding distinct sPLA2 isozymes that display overlapping yet distinct tissue distributions have been identified through the extensive genomic search, but the precise roles for each individual isozyme largely remain to be specified. Group IB (sPLA2-IB) and group IIA (sPLA2-IIA) sPLA2s are the two well characterized sPLA2s, known as pancreatic and non-pancreatic/inflammatory sPLA2s, respectively. sPLA2-IB has been thought to be involved in the digestion of dietary phospholipids in the gastrointestinal tract (4Richmond B.L. Boileau A.C. Zheng S. Huggins K.W. Granholm N.A. Tso P. Hui D.Y. Gastroenterology. 2001; 120: 1193-1202Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), whereas sPLA2-IIA is present in high levels in rheumatoid synovial fluid, and its expression is induced or repressed by pro- or anti-inflammatory stimuli, respectively (5Kramer R.M. Hession C. Johansen B. Hayes G. McGray P. Chow E.P. Tizard R. Pepinsky R.B. J. Biol. Chem. 1989; 264: 5768-5775Abstract Full Text PDF PubMed Google Scholar, 6Oka S. Arita H. J. Biol. Chem. 1991; 266: 9956-9960Abstract Full Text PDF PubMed Google Scholar, 7Nakano T. Ohara O. Teraoka H. Arita H. J. Biol. Chem. 1990; 265: 12745-12748Abstract Full Text PDF PubMed Google Scholar). sPLA2-IIA is also enriched in human tears and is the principal bactericidal factor against Gram-positive bacteria (8Qu X.D. Lehrer R.I. Infect. Immun. 1998; 66: 2791-2797Crossref PubMed Google Scholar, 9Foreman-Wykert A.K. Weiss J. Elsbach P. Infect. Immun. 2000; 68: 1259-1264Crossref PubMed Scopus (30) Google Scholar). Recently, these sPLA2 isoforms have been implicated in neuronal apoptosis both in vitro and in vivo through the generation of reactive oxygen species generated in the course of arachidonic acid metabolism (10Yagami T. Ueda K. Asakura K. Hata S. Kuroda T. Sakaeda T. Takasu N. Tanaka K. Gemba T. Hori Y. Mol. Pharmacol. 2002; 61: 114-126Crossref PubMed Scopus (120) Google Scholar, 11Yagami T. Ueda K. Asakura K. Hayasaki Kajiwara Y. Nakazato H. Sakaeda T. Hata S. Kuroda T. Takasu N. Hori Y. J. Neurochem. 2002; 81: 449-461Crossref PubMed Scopus (62) Google Scholar, 12Yagami T. Ueda K. Asakura K. Sakaeda T. Hata S. Kuroda T. Sakaguchi G. Itoh N. Hashimoto Y. Hori Y. Brain Res. 2003; 960: 71-80Crossref PubMed Scopus (30) Google Scholar, 13Yagami T. Ueda K. Asakura K. Nakazato H. Hata S. Kuroda T. Sakaeda T. Sakaguchi G. Itoh N. Hashimoto Y. Hori Y. J. Neurochem. 2003; 85: 749-758Crossref PubMed Scopus (57) Google Scholar). Furthermore, in addition to these biological functions, which are dependent on their enzymatic activity, receptor-mediated actions of sPLA2-IB and sPLA2-IIA have also been proposed (14Lambeau G. Lazdunski M. Trends Pharmacol. Sci. 1999; 20: 162-170Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar, 15Hanasaki K. Arita H. Prostaglandins Other Lipid Mediat. 2002; 68-69: 71-82Crossref PubMed Scopus (116) Google Scholar, 16Hanasaki K. Biol. Pharm. Bull. 2004; 27: 1165-1167Crossref PubMed Scopus (49) Google Scholar). Groups X sPLA2 (sPLA2-X) is unique in that it has the prominent ability to liberate free fatty acids, including arachidonic acid, when added exogenously to the culture media of adherent mammalian cells, whereas other groups of sPLA2 do not, except for group V sPLA2 (17Bezzine S. Koduri R.S. Valentin E. Murakami M. Kudo I. Ghomashchi F. Sadilek M. Lambeau G. Gelb M.H. J. Biol. Chem. 2000; 275: 3179-3191Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 18Singer A.G. Ghomashchi F. Le Calvez C. Bollinger J. Bezzine S. Rouault M. Sadilek M. Nguyen E. Lazdunski M. Lambeau G. Gelb M.H. J. Biol. Chem. 2002; 277: 48535-48549Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). This difference apparently results from the distinct interfacial binding affinity of sPLA2s toward phosphatidylcholine (PC)-rich outer leaflet of mammalian plasma membrane, because sPLA2-X binds efficiently to vesicles rich in PC, and sPLA2-IIA exhibits very poor binding affinity for charge-neutral PC-rich vesicles in marked contrast to anionic vesicles and thus displays virtually no enzymatic activity to PC-enriched vesicles (18Singer A.G. Ghomashchi F. Le Calvez C. Bollinger J. Bezzine S. Rouault M. Sadilek M. Nguyen E. Lazdunski M. Lambeau G. Gelb M.H. J. Biol. Chem. 2002; 277: 48535-48549Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 19Bezzine S. Bollinger J.G. Singer A.G. Veatch S.L. Keller S.L. Gelb M.H. J. Biol. Chem. 2002; 277: 48523-48534Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The distinct tissue distribution of sPLA2 enzymes, together with the variability in the substrate specificity, further argues for the existence of different physiological functions for each sPLA2 enzyme. The OGR1 subfamily of G-protein-coupled receptors (GPCRs), OGR1, G2A, GPR4, and TDAG8, displays unique ligand specificity in that they were initially proposed to recognize lysolipid molecules as ligands. OGR1 was shown to bind with high affinity to sphingosylphosphorylcholine (SPC) (20Xu Y. Zhu K. Hong G. Wu W. Baudhuin L.M. Xiao Y. Damron D.S. Nat. Cell Biol. 2000; 2: 261-267Crossref PubMed Scopus (176) Google Scholar), and G2A and GPR4 were later reported to bind to SPC and lysophosphatidylcholine (LPC) with distinct affinities (21Kabarowski J.H. Zhu K. Le L.Q. Witte O.N. Xu Y. Science. 2001; 293: 702-705Crossref PubMed Scopus (276) Google Scholar, 22Zhu K. Baudhuin L.M. Hong G. Williams F.S. Cristina K.L. Kabarowski J.H. Witte O.N. Xu Y. J. Biol. Chem. 2001; 276: 41325-41335Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar). Galactosylsphingosine (psychosine) was identified as a ligand for TDAG8 (23Im D.S. Heise C.E. Nguyen T. O'Dowd B.F. Lynch K.R. J. Cell Biol. 2001; 153: 429-434Crossref PubMed Scopus (154) Google Scholar). Upon binding to the respective receptors, these lysolipid ligands activate various second messenger pathways, including inositol phosphate accumulation, intracellular Ca2+ mobilization, and increase or decrease of cAMP content. More recently, however, these receptors have been shown to respond to changes in extracellular pH; Ludwig et al. (24Ludwig M.G. Vanek M. Guerini D. Gasser J.A. Jones C.E. Junker U. Hofstetter H. Wolf R.M. Seuwen K. Nature. 2003; 425: 93-98Crossref PubMed Scopus (527) Google Scholar) reported that OGR1 and GPR4 are proton-sensing receptors that accumulate inositol phosphate and cAMP, respectively, in response to acidic pH of the extracellular milieu. Furthermore, they described that SPC and LPC do not exert any effects on the generation of second messengers. Subsequently, pH-dependent activation of G2A (25Murakami N. Yokomizo T. Okuno T. Shimizu T. J. Biol. Chem. 2004; 279: 42484-42491Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) and TDAG8 (26Wang J.Q. Kon J. Mogi C. Tobo M. Damirin A. Sato K. Komachi M. Malchinkhuu E. Murata N. Kimura T. Kuwabara A. Wakamatsu K. Koizumi H. Uede T. Tsujimoto G. Kurose H. Sato T. Harada A. Misawa N. Tomura H. Okajima F. J. Biol. Chem. 2004; 279: 45626-45633Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 27Ishii S. Kihara Y. Shimizu T. J. Biol. Chem. 2005; 280: 9083-9087Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) was reported; Murakami et al. (25Murakami N. Yokomizo T. Okuno T. Shimizu T. J. Biol. Chem. 2004; 279: 42484-42491Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) showed that previously proposed ligand of G2A, LPC, inhibited pH-dependent accumulation of inositol phosphate in cells expressing G2A, whereas Wang et al. (26Wang J.Q. Kon J. Mogi C. Tobo M. Damirin A. Sato K. Komachi M. Malchinkhuu E. Murata N. Kimura T. Kuwabara A. Wakamatsu K. Koizumi H. Uede T. Tsujimoto G. Kurose H. Sato T. Harada A. Misawa N. Tomura H. Okajima F. J. Biol. Chem. 2004; 279: 45626-45633Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) described that cAMP generation in TDAG8-expressing cells was inhibited by psychosine. The latter authors also demonstrated that psychosine acts antagonistically to the pH-dependent generation of second messengers in GPR4- or OGR1-expressing cells. Thus, although the controversial results remain to be reconciled, they raise the possibility that OGR1 subfamily receptors can respond to proton in addition to lysolipid ligands, which act either agonistically or antagonistically depending on the experimental systems employed. Our previous work with fungal, bacterial, and bee venom sPLA2s has demonstrated that sPLA2s display neurotrophin-like neurite-inducing activity in PC12 cells (28Wakatsuki S. Arioka M. Dohmae N. Takio K. Yamasaki M. Kitamoto K. J. Biochem. 1999; 126: 1151-1160Crossref PubMed Scopus (20) Google Scholar, 29Wakatsuki S. Yokoyama T. Nakashima S. Nishimura A. Arioka M. Kitamoto K. Biochim. Biophys. Acta. 2001; 1522: 74-81Crossref PubMed Scopus (14) Google Scholar, 30Nakashima S. Wakatsuki S. Yokoyama T. Arioka M. Kitamoto K. Biosci. Biotechnol. Biochem. 2003; 67: 77-82Crossref PubMed Scopus (4) Google Scholar, 31Nakashima S. Kitamoto K. Arioka M. Brain Res. 2004; 1015: 207-211Crossref PubMed Scopus (24) Google Scholar). Unlike nerve growth factor (NGF), neuritogenesis of PC12 cells by sPLA2s was insensitive to tyrosine kinase inhibitor K-252a. Conversely, inhibition of L-type Ca2+ channel or depletion of extracellular Ca2+, which were ineffective in blocking NGF-induced neuritogenesis, inhibited sPLA2-induced neurite outgrowth. Our subsequent work demonstrated that sPLA2-induced neuritogenesis requires the activation of src and ras proteins and is accompanied by the activation of mitogen-activated protein kinase cascade (28Wakatsuki S. Arioka M. Dohmae N. Takio K. Yamasaki M. Kitamoto K. J. Biochem. 1999; 126: 1151-1160Crossref PubMed Scopus (20) Google Scholar). The enzymatic activity of sPLA2s was required for induction of neurites, although direct addition of arachidonic acid (as well as oleic acid) failed to induce neurites. Also, inhibitors of cyclooxygenase and lipoxygenase did not attenuate the neuritogenic activity of sPLA2. These results suggest that arachidonic acid release and subsequent conversion to eicosanoids is not involved in the neuritogenesis of PC12 cells. In contrast, we found that lysophosphatidylcholine (LPC), but not other lysophospholipids, induced neurite formation, suggesting that sPLA2 induces neurites via LPC generation (32Nakashima S. Ikeno Y. Yokoyama T. Kuwana M. Bolchi A. Ottonello S. Kitamoto K. Arioka M. Biochem. J. 2003; 376: 655-666Crossref PubMed Scopus (37) Google Scholar). In this study, we first compared the neurite-inducing activity of a subset of sPLA2s and found that sPLA2-X exhibits potent neuritogenic activity as did the sPLA2s from other species. We then demonstrated that LPC was actually generated by sPLA2 treatment. To elucidate the mode of action of LPC, we modified the expression of G2A, a GPCR involved in LPC signaling, and found that the expression level of G2A correlates with the sensitivity of PC12 cells toward sPLA2 or LPC treatments. Collectively, these results strongly suggest that neuritogenic action of sPLA2 is mediated by LPC generation and subsequent activation of G2A. Materials—1-Palmitoyl-sn-glycero-3-phosphocholine (Sigma, L5254) was used as LPC throughout this study unless specified otherwise. 1-Myristoyl-sn-glycero-3-phosphocholine (C14:0; L6629), 1-stearoyl-sn-glycero-3-phosphocholine (C18:0; P1418), lysophosphatidylinositol (L7635), lysophosphatidylethanolamine (L4754), lysophosphatidylserine (L3401), and 1-oleyl-sn-glycero-3-phosphate (L7260), sphingosylphosphorylcholine (SPC; S4257), phospholipase B from Vibrio sp. (PLB; P8914), and nicardipine (N7510) were purchased from Sigma. 1-Lauroyl-sn-glycero-3-phosphocholine (C12:0) was from Avanti Polar Lipids (855475P). Methylcarbamyl platelet activating factor C-16 was from Cayman (catalog no. 60908). Fatty acid-free bovine serum albumin (BSA) was from Wako (017-15416). [3H]Oleic acid (9,10-3H-labeled, 15 Ci/mmol) was from PerkinElmer Life Sciences (NET-289). [methyl-14C]Choline chloride (50 mCi/mmol) was from Amersham Biosciences (CFA424). Silica 60 TLC plates (8 × 8 cm) were from Merck. Production of carboxyl-terminally hemagglutinin- and hexahistidine-tagged mouse sPLA2-IB and sPLA2-IIA (32Nakashima S. Ikeno Y. Yokoyama T. Kuwana M. Bolchi A. Ottonello S. Kitamoto K. Arioka M. Biochem. J. 2003; 376: 655-666Crossref PubMed Scopus (37) Google Scholar) was conducted by using the baculovirus expression system. Proteins were purified with the nickel-nitrilotriacetic acid-agarose chromatography. Recombinant human group X sPLA2 was a generous gift from Dr. M. Gelb at the University of Washington (17Bezzine S. Koduri R.S. Valentin E. Murakami M. Kudo I. Ghomashchi F. Sadilek M. Lambeau G. Gelb M.H. J. Biol. Chem. 2000; 275: 3179-3191Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Preparation of recombinant p15 and phospholipase activity assay were described previously (32Nakashima S. Ikeno Y. Yokoyama T. Kuwana M. Bolchi A. Ottonello S. Kitamoto K. Arioka M. Biochem. J. 2003; 376: 655-666Crossref PubMed Scopus (37) Google Scholar). PC12 Cell Culture and Neurite Outgrowth Assay—Rat pheochromocytoma PC12 cells were maintained in DMEM (Dulbecco's modified Eagle's medium, high glucose type, Invitrogen) supplemented with 5% horse serum and 5% fetal calf serum. Cells were passaged every 3-4 days and maintained at 37 °C in 10% CO2 in humidified air. In a typical neurite-induction experiment, PC12 cells were seeded in the growth medium at 4.5 × 103 cells/cm2 in collagen type I-coated 24-well culture plates (BD Biosciences), allowed to grow for 24 h, and then supplemented with each of the various protein and/or non-protein additives specified in the text. When neuritogenesis in G2A-EGFP stable transfectants were examined, DMEM containing 1% fetal calf serum (FCS) and indicated amount of LPC were used. After 24 h, neurite outgrowth was quantified by taking four random photographs/well; cells bearing processes longer than the cell diameter were judged as positive. The data are mean ± S.D. of at least two independent experiments. [3H]Oleic acid release from live PC12 cells was determined as described (32Nakashima S. Ikeno Y. Yokoyama T. Kuwana M. Bolchi A. Ottonello S. Kitamoto K. Arioka M. Biochem. J. 2003; 376: 655-666Crossref PubMed Scopus (37) Google Scholar). The percent oleic acid release was calculated by dividing the total counts present in the medium by the sum of the counts measured in the medium and in the corresponding cell lysate; background radioactivity, measured in phospholipase-unsupplemented, control incubations was subtracted from each data point. Assay of Phospholipase-mediated LPC Release from Cells—PC12 cells grown at 1.0 × 105 cells/cm2 in 24-well culture plates were incubated for 24 h in the presence of [14C]choline chloride (0.75 μCi/ml) in 400 μl of DMEM containing sera. After washing three times with pre-warmed phosphate-buffered saline, cells were treated with sPLA2 and PLB for 4 h at 37°C. When the culture supernatants of COS1 cells transfected with each sPLA2 construct were examined, they were adjusted to 5% fetal calf serum and 5% horse serum by 2-fold dilution with fresh DMEM containing 10% horse serum, and were applied to the PC12 cell culture. Culture media were then recovered, and cells were washed and suspended in 400 μl of phosphate-buffered saline. Lipids were extracted with 600 μl of chloroform/methanol (1:2, v/v), and the organic phase was separated by silica TLC, using chloroform/methanol/acetic acid/water (60:30:8:5, v/v). Radioactive lipids were visualized and quantified using a fluorescence image analyzer (FLA3000, Fuji). RT-PCR—The expression of GPCR mRNA in the mouse brains and PC12 cells were examined by RT-PCR. Total RNA (15 μg) extracted from the mouse brains at various stages of development or PC12 cells 24 h after seeding was reverse-transcribed with oligo(dT16-30) primer and PowerScript™ reverse transcriptase (Clontech, catalog no. 8460-1). An aliquot of cDNA corresponding to 1 μg of total RNA was used as a template for the PCR reaction (94 °C for 3 min; 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min; then 72 °C for 3 min) using the specific primer sets corresponding to GenBank™ sequence of mouse G2A (G2As1: 5′-GGTGACTGCTTACATCTTCTTCTGC-3′ and G2Aas1: 5′-CTGTGTGGATTCTGGACACTTCTTG-3′), OGR1 (5′-TCTGGCCCAAAGATGGGGAACATCA-3′ and 5′-AGCCCACGCTGATGTAAATGTTCTC-3′), GPR4 (5′-ATATCAGCATCGCCTTCCTGTGCTG-3′ and 5′-CAGCCACACAATTGAGGCTGGTGAA-3′), TDAG8 (5′-TGGACTTTCTCTCCCACCTTGTGCA-3′ and 5′-AGTACAGAATGGGATCGGCAACACA-3′), β-actin (5′-GTGGGCCGCTCTAGGCACCAA-3′ and 5′-CTCTTTGATGTCACGCACGATTTC-3′), and glyceraldehyde-3-phosphate dehydrogenase (5′-GACCACAGTCCATGCCATCACT-3′ and 5′-TCCACCACCCTGTTGCTGTAG-3′). cDNA Cloning and Isolation of Stable Transfectants—Rat G2A, mouse GPR4, and TDAG8 cDNAs were amplified by PCR using Pfx DNA polymerase and cDNA libraries from PC12 cells (G2A) and mouse fetal brain (GPR4 and TDAG8) as templates. The following oligonucleotides were utilized as amplification primers: 5′-tcgcaagcttATGAGATCAGAACCTACCAA-3′ and 5′-tatgaattcGGCAGAGCTCGTCAGGCAGTC-3′ for G2A; 5′-atgtaagcttATGGACAACAGCACGGGCAC-3′ and 5′-tatgaattcGCTGTGCCGGGGGCAGCAGCA-3′ for GPR4; 5′-cgctaagcttATGGCGATGAACAGCATGTG-3′ and 5′-tatgaattcCGTCTATAATCTCTAATTCTA-3′ for TDAG8. Capital letters correspond to the coding regions, and lowercase underlined sequences indicate the HindIII and EcoRI sites used for cDNA cloning. Amplified cDNA fragments were digested with HindIII and EcoRI, and ligated to the pEGFP-N1 (Clontech), generating pEGFP-G2A, pEGFP-GPR4, and pEGFP-TDAG8. For localization studies, we used Neuro2A cells because the internalization of EGFP-tagged GPCRs were more easily detected in this cell line than in PC12 cells. Neuro2A cells were transfected with these plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction. After 24 h, cells were starved for additional 24 h in serum-free medium and were subjected to various treatments in the final 2 h of incubation. They were then fixed in 4% paraformaldehyde/phosphate-buffered saline and observed by fluorescence microscopy (BX52, Olympus, Tokyo, Japan). To generate stable PC12 cell transfectants, pEGFP-N1, pEGFP-G2A, pEGFP-GPR4, and pEGFP-TDAG8 were transfected into PC12 cells using Lipofectamine 2000. At 24 h after transfection, cells were detached and re-plated at a 1:50 dilution into medium containing 1000 μg/ml G418 (Invitrogen). After 12 days, G418-resistant clones were isolated, amplified, and tested for G2A-EGFP expression and for neurite outgrowth assay. Immunoblotting—PC12 cells were scraped and suspended in SDS sample buffer (125 mm Tris-HCl, pH 6.9, 10% glycerol, 5% mercaptoethanol, 2% SDS, 0.05% bromphenol blue). An equal volume of cell lysates was subjected to SDS-PAGE immediately. SDS-PAGE was performed on 12.5% acrylamide gels under reducing conditions. Western blotting with anti-GFP antibody (1:5,000 dilution, Invitrogen, catalog no. R970-01) was performed according to the standard procedure. RNA Interference Experiment—The oligonucleotides used for expression of short hairpin (sh) RNA were (i) r1 sense, 5′-tttGTCCTACAAAGGAACGTGCcttcctgtcaGCACGTTTCTCTGTAGGACtttttggatc-3′; r1 antisense, 5′-ctaggatccaaaaaGTCCTACAGAGAAACGTGCtgacaggaagGCACGTTCCTTTGTAGGA-3′; (ii) r2 sense, 5′-tttGTGACAGCCTGCATCTTCTcttcctgtcaAGAAGATGTAAGCTGTCACtttttggatc-3′; r2 antisense, 5′-ctaggatccaaaaaGTGACAGCTTACATCTTCTtgacaggaagAGAAGATGCAGGCTGTCA-3′; (iii) r3 sense, 5′-tttGCTCAGTAATAGTCTGAGCcttcctgtcaGCTCAGGCTATCACTGAGCtttttggatc-3′; r3 antisense, 5′-ctaggatccaaaaaGCTCAGTGATAGCCTGAGCtgacaggaagGCTCAGACTATTACTGAG-3′; and (iv) m1 sense, 5′-tttGAGTAGTTCTGATGGTAGTgtgtgctgtccACCACCACCAGGACCACTCtttttggatc-3′; m1 antisense, 5′-ctaggatccaaaaaGAGTGGTCCTGGTGGTGGTggacagcacacACTACCATCAGAACTACT-3′. The central small letters indicate the 10- to 11-bp loop sequences. Underlined sequences are substitutions introduced in the sense strand of shRNA to stabilize the double strand RNA. Italicized nucleotides in m1 indicate mismatches at the single position to the rat G2A mRNA sequence. These oligonucleotides were dissolved in TE (10 mm Tris, 1 mm EDTA, pH 8.0) at 250 μm, and 20 μl each of sense and antisense DNA was mixed with 5 μl each of distilled water and 10× annealing buffer (1 m NaCl, 100 mm Tris, pH 7.4). The mixture was heated in the boiling water for 5 min and spontaneously annealed by cooling down to the room temperature. The annealed double-stranded DNA was diluted by 4,000-fold in TE and ligated to pmU6pro vector (kindly provided by Dr. Dave Turner, University of Michigan) digested with BbsI and XbaI. The resultant plasmids were verified by DNA sequencing. These plasmids or the control plasmid pMT (which does not contain EGFP and shRNA sequences (32Nakashima S. Ikeno Y. Yokoyama T. Kuwana M. Bolchi A. Ottonello S. Kitamoto K. Arioka M. Biochem. J. 2003; 376: 655-666Crossref PubMed Scopus (37) Google Scholar), 2 μg each) were co-transfected with the equal amount of pEGFP-G2A into Neuro2A cells, which were subjected to RT-PCR and flow cytometry analyses 24 h after transfection. Quantitative RT-PCR was performed using the LightCycler-FastStart DNA Master SYBR Green I (Roche Applied Science, catalog no. 3003230). The copy number of G2A transcript was quantified using the calibration curve drawn using the standard solution containing the fixed amount of the plasmid carrying G2A cDNA. Normalization was done against the amount of glyceraldehyde-3-phosphate dehydrogenase transcript. For the expression of shRNA sequences by adenovirus vector-mediated method, a HindIII-NotI fragment containing the U6 promoter, shRNA sequence, and SV40 poly(A) sequence was blunt-ended and ligated to the cloning site (SwaI site) of the adenovirus cosmid vector, pAxCAwtit (Riken DNA bank, catalog no. 3121). The resultant cosmid DNA was processed as described below. The recombinant adenoviruses thus prepared were infected to 1.0 × 106 PC12 cells grown in a 100-mm dish for 1 h at a multiplicity of infection of 100, and 24 h after the infection, cells were processed for RT-PCR and neurite outgrowth experiments. In a single neurite outgrowth assay, more than 100 cells were counted for individual treatment. The data are mean ± S.D. of three independent experiments. Flow Cytometry—24 h after transfection, Neuro2A cells were washed twice with phosphate-buffered saline, and cells were detached from the culture dishes by trypsin. After fixation in 4% paraformaldehyde, cells were washed twice with phosphate-buffered saline, triturated by pipetting, and filtrated through the nylon membrane to prepare dissociated cell suspension. They were then subjected to flow cytometry analysis (BD LSR, BD Biosciences) in which ∼10,000 cells were counted. The areas counted for EGFP-positive and -negative cells were determined by control experiments conducted with cells transfected with pEGFP-N1 (positive control) and pMT lacking EGFP sequence (negative control), respectively. Adenovirus Vector Construction and Infection—A PCR-based mutagenesis protocol (33Imai Y. Matsushima Y. Sugimura T. Terada M. Nucleic Acids Res. 1991; 19: 2785Crossref PubMed Scopus (314) Google Scholar) was used to generate a silent mutation at Csp45I site in G2A, which is necessary for linearization of adenoviral vector. Using the pEGFP-G2A plasmid as a template, mutated DNA fragments were amplified by PCR using the following pairs of oligonucleotides as primers: G2A antisense (used for cDNA cloning)/mutant sense (5′-GTGTGACTTCGAGAACAGGCTGTAC-3′) and G2A sense/mutant antisense (5′-GTACAGCCTGTTCTCGAAGTCACAC-3′). Underlined nucleotides correspond to the silent mutation. The two resulting PCR fragments were mixed, followed by a second amplification, carried out with the G2A sense and antisense primers. The amplified DNA fragment thus obtained was then digested with HindIII and EcoRI and ligated to the pEGFP-N1 vector. The mutated G2A-EGFP fragment was blunt-ended and ligated to the SwaI site of pAxCAwtit. The resultant cosmid DNA (1 μg) was packaged in vitro using 6 μl of LAMBDA INN (Nippon Gene, catalog no. 317-01741), infected to Escherichia coli DH5α, and amplified. Cosmid DNA (10 μg) was then purified, linearized by digestion with Csp45I, and transfected to HEK293 cells grown in a 60-mm dish using 30 μl of TransFast (Promega, catalog no. E2431). Cells were recovered 24 h later in phosphate-buffered saline, transferred to a 100-mm dish, and grown in 10 ml of 5% FCS/DMEM. Five days after, DMEM containing 10% FCS (5 ml) was added. Every 5 days, 5 ml of culture medium was removed, and the same volume of fresh 10% FCS/DMEM was added until most of the cells became detached (typically 7-30 days). Then the cells and culture medium were harvested together, freeze-thawed, and centrifuged to obtain the adenovirus-enriched supernatants. Aliquots of the supernatants were added to fresh HEK293 cells, and the recombinant adenovirus was amplified by another 3 times. The resultant adenovirus-containing media were used as virus stocks, and the titers were determined by the 50% tissue culture infectious doses method, using the plaque forming assay with 293A cells. Typically, 109 plaque forming units/ml viral stocks were obtained. 24 h after plating, PC12 cells were infected with either adenoviral-EGFP or adenoviral-G2A-EGFP, each at a multiplicity of infection of 50. After infection, cells were incubated for another 24 h and were subjected to the neurite outgrowth assay as described above. Statistical Analysis—The results shown were from one experiment representative of at least two independent experiments, each done in triplicate. Data are presented as means (±S.D.). Differences were analyzed by Student's t test, and the values of p < 0.05 were taken as significant. The experiments with TLC plates, gels, and blots were carried out at least twice with duplicates, and one representative result is shown. Group X sPLA2 Induces Neurite Outgrowth in PC12 Cells via L-type Ca2+ Channel Activity—We previously reported that exogenously added fungal sPLA2, p15, induces neurite outgrowth in PC12 cells via L-type Ca2+ channel activity (32Nakashima S. Ikeno Y. Yokoyama T. Kuwana M. Bolchi A. Ottonello S. Kitamoto K. Arioka M. Biochem. J. 2003; 376: 655-666Crossref PubMed Scopus (37) Google Scholar). Interestingly, when neuritogenesis by supernatants of COS1 cells transfected with mouse sPLA2s was examined, only the supernatant containing sPLA2-X, but not sPLA2-IB nor sPLA2-IIA, elicited neurites. To unequivocally show the neuritogenic response of PC12 cells by mammalian sPLA2s, we prepared purified, recombinant mouse sPLA2-IB and sPLA2-IIA produced by the bacu" @default.
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• W2029372505 title "Secretory Phospholipases A2 Induce Neurite Outgrowth in PC12 Cells through Lysophosphatidylcholine Generation and Activation of G2A Receptor" @default.
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