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• W2061802019 abstract "G2A is a G protein-coupled receptor that is predominantly expressed in lymphoid tissues and macrophages. G2A can be induced by diverse stimuli to cause cell cycle arrest in the G2/M phase in pro-B and T cells. G2A is also expressed in macrophages within atherosclerotic lesions, suggesting G2A involvement in atherosclerosis. Recently, G2A was discovered to possess proton-sensing ability. In this paper, we report another function of G2A, that is, as a receptor for 9-hydroxyoctadecadienoic acid (9-HODE) and other oxidized free fatty acids. G2A, expressed in CHO-K1 or HEK293 cells, showed 9-HODE-induced intracellular calcium mobilization, inositol phosphate accumulation, inhibition of cAMP accumulation, [35S]guanosine 5′-3-O-(thio)triphosphate binding, and MAP kinase activation. Furthermore, G2A was activated by various oxidized derivatives of linoleic and arachidonic acids, but it was weakly activated by cholesteryl-9-HODE. Oxidized phosphatidylcholine (1-palmitoyl-2-linoleoyl) when hydrolyzed with phospholipase A2 also evoked intracellular calcium mobilization in G2A-expressing cells. These results indicate that G2A is activated by oxidized free fatty acids produced by oxidation and subsequent hydrolysis of phosphatidylcholine or cholesteryl linoleate. Thus, G2A might have a biological role in diverse pathological conditions including atherosclerosis. G2A is a G protein-coupled receptor that is predominantly expressed in lymphoid tissues and macrophages. G2A can be induced by diverse stimuli to cause cell cycle arrest in the G2/M phase in pro-B and T cells. G2A is also expressed in macrophages within atherosclerotic lesions, suggesting G2A involvement in atherosclerosis. Recently, G2A was discovered to possess proton-sensing ability. In this paper, we report another function of G2A, that is, as a receptor for 9-hydroxyoctadecadienoic acid (9-HODE) and other oxidized free fatty acids. G2A, expressed in CHO-K1 or HEK293 cells, showed 9-HODE-induced intracellular calcium mobilization, inositol phosphate accumulation, inhibition of cAMP accumulation, [35S]guanosine 5′-3-O-(thio)triphosphate binding, and MAP kinase activation. Furthermore, G2A was activated by various oxidized derivatives of linoleic and arachidonic acids, but it was weakly activated by cholesteryl-9-HODE. Oxidized phosphatidylcholine (1-palmitoyl-2-linoleoyl) when hydrolyzed with phospholipase A2 also evoked intracellular calcium mobilization in G2A-expressing cells. These results indicate that G2A is activated by oxidized free fatty acids produced by oxidation and subsequent hydrolysis of phosphatidylcholine or cholesteryl linoleate. Thus, G2A might have a biological role in diverse pathological conditions including atherosclerosis. G2A (derived from G2 accumulation) was first identified as a stress inducible G protein-coupled receptor (GPCR) 2The abbreviations used are: GPCRG protein-coupled receptorLPClysophosphatidylcholineSPCsphingosylphosphorylcholineMAP kinasemitogen-activated protein kinaseGPR4G protein-coupled receptor 4TDAG8T cell death-associated gene 8OGR1ovarian cancer G protein-coupled receptor 1IPinositol phosphateHODEhydroxyoctadecadienoic acidLDLlow density lipoproteinPCphosphatidylcholineHPODEhydroperoxyoctadecadienoic acidHETEhydroxyeicosatetraenoic acidPTXpertussis toxinCHO-K1Chinese hamster ovary K1HEK293human embryonic kidney 293BSAbovine serum albuminERKextracellular signal-regulated kinaseJNKc-Jun NH2-terminal kinaseAMVN2,2′-azobis(2,4-dimethylvaleronitrile)PLA2phospholipase A2sPLA2secretory type of phospholipase A2sPLA2-Xgroup X secretory type of phospholipase A2GTPγSguanosine 5′-3-O-(thio)triphosphateEPPSN′-(2-hydroxyethyl)piperazine-N′-3-propanesulfonic acidOxPCoxidized phosphatidylcholineMES2-(N-morpholino)ethanesulfonic acid. (1Weng Z. Fluckiger A.C. Nisitani S. Wahl M.I. Le L.Q. Hunter C.A. Fernal A.A. Le Beau M.M. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12334-12339Crossref PubMed Scopus (127) Google Scholar). G2A causes cell cycle arrest in the G2/M-phase in pro-B and T cells when these cells are treated with various DNA-damaging stimuli (1Weng Z. Fluckiger A.C. Nisitani S. Wahl M.I. Le L.Q. Hunter C.A. Fernal A.A. Le Beau M.M. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12334-12339Crossref PubMed Scopus (127) Google Scholar). Mice lacking G2A were shown to develop a late-onset autoimmune syndrome similar to systemic lupus erythematosus (2Le L.Q. Kabarowski J.H. Weng Z. Satterthwaite A.B. Harvill E.T. Jensen E.R. Miller J.F. Witte O.N. Immunity. 2001; 14: 561-571Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), suggesting that G2A plays a critical role in controlling peripheral lymphocyte homeostasis. Involvement of G2A in the pathogenesis of atherosclerosis was also suggested, as G2A is expressed in macrophages in murine, rabbit, and human atherosclerotic plaques (3Rikitake Y. Hirata K. Yamashita T. Iwai K. Kobayashi S. Itoh H. Ozaki M. Ejiri J. Shiomi M. Inoue N. Kawashima S. Yokoyama M. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 2049-2053Crossref PubMed Scopus (62) Google Scholar). G protein-coupled receptor lysophosphatidylcholine sphingosylphosphorylcholine mitogen-activated protein kinase G protein-coupled receptor 4 T cell death-associated gene 8 ovarian cancer G protein-coupled receptor 1 inositol phosphate hydroxyoctadecadienoic acid low density lipoprotein phosphatidylcholine hydroperoxyoctadecadienoic acid hydroxyeicosatetraenoic acid pertussis toxin Chinese hamster ovary K1 human embryonic kidney 293 bovine serum albumin extracellular signal-regulated kinase c-Jun NH2-terminal kinase 2,2′-azobis(2,4-dimethylvaleronitrile) phospholipase A2 secretory type of phospholipase A2 group X secretory type of phospholipase A2 guanosine 5′-3-O-(thio)triphosphate N′-(2-hydroxyethyl)piperazine-N′-3-propanesulfonic acid oxidized phosphatidylcholine 2-(N-morpholino)ethanesulfonic acid. Although G2A was initially identified as an orphan receptor, Kabarowski et al. (4Kabarowski J.H. Zhu K. Le L.Q. Witte O.N. Xu Y. Science. 2001; 293: 702-705Crossref PubMed Scopus (276) Google Scholar) once reported that lysophosphatidylcholine (LPC) and sphingosylphosphorylcholine (SPC) were potent ligands for G2A. By using G2A-expressing cells, they demonstrated that radiolabeled LPC and SPC specifically bound to cell membranes, and that LPC and SPC induced intracellular signaling such as a transient [Ca2+]i increase and mitogen-activated protein kinase (MAP kinase) activation. Furthermore, in their subsequent papers, they showed T cell (5Radu C.G. Yang L.V. Riedinger M. Au M. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 245-250Crossref PubMed Scopus (161) Google Scholar) and macrophage (6Yang L.V. Radu C.G. Wang L. Riedinger M. Witte O.N. Blood. 2005; 105: 1127-1134Crossref PubMed Scopus (138) Google Scholar) chemotaxis to LPC via G2A. However, they recently retracted the first paper (4Kabarowski J.H. Zhu K. Le L.Q. Witte O.N. Xu Y. Science. 2001; 293: 702-705Crossref PubMed Scopus (276) Google Scholar) because they were unable to reproduce the specific binding of LPC to G2A-expressing cells (7Witte O.N. Kabarowski J.H. Xu Y. Le L.Q. Zhu K. Science. 2005; 307: 206Crossref PubMed Google Scholar). Although the possibility of a direct interaction between LPC and G2A cannot be ruled out, the indirect action of LPC on G2A via an as yet identified mechanism is more likely. G2A forms a GPCR subfamily defined by amino acid sequence homology along with three other GPCRs, namely, G protein-coupled receptor 4 (GPR4) (8Heiber M. Docherty J.M. Shah G. Nguyen T. Cheng R. Heng H.H. Marchese A. Tsui L.C. Shi X. George S.R. DNA Cell Biol. 1995; 14: 25-35Crossref PubMed Scopus (84) Google Scholar, 9Mahadevan M.S. Baird S. Bailly J.E. Shutler G.G. Sabourin L.A. Tsilfidis C. Neville C.E. Narang M. Korneluk R.G. Genomics. 1995; 30: 84-88Crossref PubMed Scopus (31) Google Scholar), T cell death-associated gene 8 (TDAG8, also known as GPR65) (10Choi J.W. Lee S.Y. Choi Y. Cell. Immunol. 1996; 168: 78-84Crossref PubMed Scopus (90) Google Scholar), and ovarian cancer G protein-coupled receptor 1 (OGR1, also known as GPR68) (11Xu Y. Casey G. Genomics. 1996; 35: 397-402Crossref PubMed Scopus (78) Google Scholar). A series of recent studies have shown that these four receptors function as proton-sensing receptors (12Ludwig 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, 13Murakami N. Yokomizo T. Okuno T. Shimizu T. J. Biol. Chem. 2004; 279: 42484-42491Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 14Wang 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, 15Ishii S. Kihara Y. Shimizu T. J. Biol. Chem. 2005; 280: 9083-9087Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Murakami et al. (13Murakami N. Yokomizo T. Okuno T. Shimizu T. J. Biol. Chem. 2004; 279: 42484-42491Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) demonstrated that G2A mediates acidic pH-sensitive accumulation of inositol phosphates (IP) and activation of zif 268 promoter, and that LPC inhibits these pH-dependent activations of G2A in a dose-dependent manner. However, Radu et al. (16Radu C.G. Nijagal A. McLaughlin J. Wang L. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1632-1637Crossref PubMed Scopus (162) Google Scholar) reported more recently that G2A is less sensitive to pH fluctuations than the other three receptors in terms of IP and cAMP accumulation. By using receptor-deficient mice, they showed that TDAG8, but not G2A, is required for acidic pH-induced cAMP accumulation in splenocytes, although both the receptors are coexpressed in the cells (16Radu C.G. Nijagal A. McLaughlin J. Wang L. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1632-1637Crossref PubMed Scopus (162) Google Scholar). Thus, G2A might have another function in immune cells that has not yet been identified. During the course of a de-orphaning project of some orphan GPCRs, we found that G2A expressed in mammalian cells responded to some oxidized free fatty acids, such as 9-hydroxyoctadecadienoic acid (HODE). 9-HODE is an oxidized derivative of linoleic acid and one of the major lipid components of oxidized low-density lipoproteins (LDLs). In this report, we demonstrate that 9-HODE causes G2A activation resulting in intracellular calcium mobilization, [35S]GTPγS binding, inhibition of cAMP formation, and MAP kinase activation. 9-HODE in its free fatty acid form, but not in the form incorporated in phosphatidylcholine (PC) or cholesteryl ester, is effective in activating G2A. Based on the expression of G2A in lymphocytes, macrophages, and atherosclerotic plaques, our results suggest the newly identified aspects of G2A that might be involved in atherosclerosis and other oxidative pathological conditions. Materials—Various HODE, hydroperoxyoctadecadienoic acid (HPODE), and hydroxyeicosatetraenoic acid (HETE) species, and cholesteryl-9-HODE were purchased from Cayman Chemical. Linoleic acid, arachidonic acid, LPC (1-palmitoyl), and PC (1-palmitoyl-2-linoleoyl) were from Sigma. Pertussis toxin (PTX) was from Calbiochem. [35S]GTPγS was from PerkinElmer Life Sciences. Synthesis of 9(S)-HODE—Potato 5-lipoxygenase that functions as 9-lipoxygenase of linoleic acid was purified from potato tubers according to the method described by Shimizu et al. (17Shimizu T. Radmark O. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 689-693Crossref PubMed Scopus (263) Google Scholar). Linoleic acid was incubated with potato 5-lipoxygenase in a reaction buffer (20 mm Tris-HCl, pH 7.4, 0.1 mm diethylenetriaminepentaacetic acid), followed by reduction with NaBH4. The reaction products were separated from nonreacted substrates by silica column chromatography and analyzed by liquid chromatography-mass spectrometry. The purified reaction products mainly consisted of 9-HODE (purity >95%) and trace amounts of 13-HODE (data not shown). Plasmid Constructions—An additional sequence of the FLAG epitope tag was inserted between the amino-terminal initiator methionine and the second amino acid of human G2A (NCBI accession number AF083955) by PCR using Pyrobest (Takara) and oligonucleotides (sense primer containing BamHI and FLAG tag sequences, 5′-CGGGATCCACCATGGATTACAAGGACGACGATGACAAGTGCCCAATGCTACTGAAAAAC-3′; antisense primer containing EcoRI sequence, 5′-GGAATTCTCAGCAGGACTCCTCAATC-3′), and then subcloned into mammalian expression vector pCXN2.1, which is a slightly modified version of pCXN2 (18Niwa H. Yamamura K. Miyazaki J. Gene (Amst.). 1991; 108: 193-200Crossref PubMed Scopus (4597) Google Scholar) with multiple cloning sites. This construct was designated as pCXN2.1-G2A. A DNA sequence containing the entire open reading frame of Gαqi was subcloned into a pcDNA3.1/Zeo vector (Invitrogen). Gαqi protein is a chimeric protein in which 9 carboxyl-terminal peptides of murine Gαq protein (NCBI accession number M55412) were replaced with corresponding residues of murine Gαi protein (NCBI accession number M13963). The sequence of each construct was confirmed by DNA sequencing using a LIC-4200L DNA sequencing system (Aloka). Cell Culture, Transfection, and Flow Cytometry—Chinese hamster ovary K1 (CHO-K1) and human embryonic kidney 293 (HEK293) cells were maintained in Ham's F-12 medium (Sigma) and Dulbecco's modified Eagle's medium (Sigma), respectively, containing 10% fetal bovine serum at 37 °C in a humidified incubator with 5% CO2. Cells were transfected with plasmid DNAs using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. To observe the expression of FLAG-tagged G2A proteins on cell surfaces, cells were incubated with 10 μg/ml M5 anti-FLAG antibodies (Sigma) for 1 h at room temperature in phosphate-buffered saline containing 1% bovine serum albumin (BSA) without cell permeabilization, followed by staining with fluorescein isothiocyanate-conjugated anti-mouse IgG for 30 min at room temperature and analysis using an EPICS XL flow cytometer system (Beckman Coulter). Stable Expression of G2A and/or Gαqi Protein in CHO-K1 Cells— CHO-K1 cells were transfected with pCXN2.1-G2A using Lipofectamine 2000 reagent. Stably transfected clones resistant to 1 mg/ml Geneticin (Invitrogen) were selected, and the expression levels of G2A were confirmed by reverse transcriptase-PCR and flow cytometric analysis. Some clones that highly expressed G2A (designated as CHO-G2A cells) were further transfected with pcDNA3.1/Zeo-Gqi, and the stably transfected clones were selected in the presence of 1 mg/ml Zeocin (Invitrogen) and reverse transcriptase-PCR analysis (CHO-G2A-Gqi cells). CHO-K1 cells stably expressing Gαqi protein were also cloned (CHO-Gqi cells). Measurement of Intracellular Calcium Concentration—CHO cells were loaded with 5 μm Fura-2 AM (Dojindo) in HEPES/Tyrode's/BSA buffer (25 mm HEPES-NaOH, pH 7.4, 140 mm NaCl, 2.7 mm KCl, 1.0 mm CaCl2, 12 mm NaHCO3, 5.6 mm d-glucose, 0.37 mm NaH2PO4, 0.49 mm MgCl2, and 0.01% fatty acid-free BSA) containing 1.25 mm probenecid and 0.02% Pluronic F127 for 1 h at 37°C. The cells were washed with HEPES/Tyrode's/BSA buffer, and changes in intracellular calcium concentrations upon ligand stimulation were monitored with a FLEX-station scanning fluorometer system (Molecular Devices) in 96-well microtiter plates or with a RF5300PC spectrofluorometer (Shimadzu) in glass tubes. Measurement of Intracellular cAMP Concentration—CHO cells seeded onto the 96-well plates were washed with HEPES/Tyrode's/BSA buffer and pretreated with 1 mm 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor) for 10 min at 37 °C. The cells were costimulated with various concentrations of 9(S)-HODE and 3 μm forskolin for 30 min at 37 °C, and the accumulated cAMP concentrations were measured using a cAMP-Screen System (Applied Biosystems) according to the manufacturer's instructions. In some cases, the cells were pretreated with 50 ng/ml PTX for 16 h before stimulation. GTPγS Binding Assay—CHO-G2A or HEK293 cells transfected with pCXN2.1-G2A were disrupted by sonication in a homogenizing buffer (20 mm Tris-HCl, pH 7.4, 0.25 m sucrose, 10 mm MgCl2, 1 mm EDTA, and Complete protease inhibitor mixture (Roche)). The homogenates were centrifuged for 10 min at 12,000 × g, and the resulting supernatants were further centrifuged for 60 min at 100,000 × g. The precipitates (membrane fractions) were resuspended in the homogenizing buffer, and the protein concentrations were determined by BCA protein assay reagent (Pierce) using BSA as a standard. The membrane fractions (20 μg of proteins) were incubated with 0.5 nm [35S]GTPγS and various concentrations of 9(S)-HODE in 200 μl of binding buffer (20 mm Tris-HCl, pH 7.5, 5 mm MgCl2, 100 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol, 5 μm GDP, and 0.1% fatty acid-free BSA) for 30 min at 30 °C in the presence or absence of unlabeled 20 μm GTPγS. The reactions were terminated by rapid filtration through GF/C glass fiber filters (Whatman). The filters were intensively washed with phosphate-buffered saline, dried at 50 °C, and then immersed in the Aquasol II scintillation mixture (Packard). The radioactivity of the filters was measured with a LS6500 scintillation system (Beckman). Analysis of MAP Kinase Activation—CHO cells were disrupted by sonication after various stimulations, and the cell debris was removed by centrifugation at 10,000 × g for 5 min. An aliquot of protein (15 μg) was separated on 10% SDS-polyacrylamide gel electrophoresis, transferred to a Hybond ECL nitrocellulose membrane (Amersham Biosciences), blocked in Tris-buffered saline containing 20% Applie Duo blocking solution (Seikagaku Corp.) at 4 °C overnight, and then incubated with primary antibodies in Tris-buffered saline containing 2% Applie Duo blocking solution for 1 h at room temperature. The primary antibodies to detect total and phosphorylated extracellular signal-regulated kinase (ERK) 1/2, p38, and c-Jun NH2-terminal kinase (JNK) were supplied as a part of the MAP Kinase Activation Sampler Kit (BD Bioscience), and used according to the manufacturer's instructions. After incubation with horseradish peroxidase-conjugated secondary antibodies, the signals were visualized using an ECL plus Western blotting Detection System (Amersham Biosciences). In Vitro Peroxidation and PLA2 Treatment of PC—In vitro peroxidation of 1-palmitoyl-2-linoleoyl PC by a radical initiator, 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN), was performed according to the method described by Yoshida et al. (19Yoshida Y. Ito N. Shimakawa S. Niki E. Biochem. Biophys. Res. Commun. 2003; 305: 747-753Crossref PubMed Scopus (30) Google Scholar). In brief, PC (2 mg) was dissolved in acetonitrile/tert-butyl alcohol (4:1 in volume) at a final concentration of 5 mm, and peroxidation was induced by 2 mm AMVN for 3 h at 37 °C with mild agitation. The reaction was terminated by adding excess ethanol, and the solvent was evaporated under nitrogen gas. For the hydrolysis by phospholipase A2 (PLA2), the lipids were redissolved in diethyl ether, and mixed with the same volume of 100 mm Tris-HCl (pH 7.4), and CaCl2 was added at a final concentration of 2.5 mm. Hydrolysis was induced by adding 20 units of PLA2 from Naja mossambica mossambica (Sigma) followed by incubation for 2 h at room temperature. The reaction was terminated by adding EDTA (2.5 mm), and the lipids were extracted by the Bligh and Dyer method (20Bligh E.G. Dyer W.J. Can J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42865) Google Scholar). After evaporation of the solvent under nitrogen gas, the lipids were dissolved in HEPES/Tyrode's/BSA buffer for the FLEXstation assay. Measurement of Proton-sensing Activity—As described by Ludwig et al. (12Ludwig 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), Ham's F-12 medium containing 0.1% fatty acid-free BSA and HEPES/EPPS/MES (7.5 mm each) was prepared to achieve a wide range of pH (referred as F12-HEM). EPPS was from Wako. The pH of the buffer was adjusted under the experimental conditions using a carefully calibrated pH meter (Beckman). IP accumulation assay was performed as described by Murakami et al. (13Murakami N. Yokomizo T. Okuno T. Shimizu T. J. Biol. Chem. 2004; 279: 42484-42491Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). In brief, CHO cells seeded onto 12-well plates were labeled with 1 μCi/ml myo-[3H]inositol (Amersham Biosciences) for 18 h in Ham's F-12 medium containing 10% fetal bovine serum. Cells were washed twice and exposed to F12-HEM containing 20 mm LiCl with or without 10 μm 9(S)-HODE for 45 min at 37 °C. Accumulated IPs were isolated by anion-exchange chromatography (AG 1-X8 resin, Bio-Rad), and the radioactivity was measured with a LS6500 scintillation system. Intracellular Calcium Mobilization Evoked by 9(S)-HODE in CHO Cells Expressing G2A—While determining ligands for orphan GPCRs by a calcium mobilization assay using a FLEXstation system, we found that G2A, which was stably expressed in CHO-K1 cells (CHO-G2A), responded to 9(S)-HODE. As shown in Fig. 1A, 9(S)-HODE evoked intracellular calcium mobilization in CHO-G2A cells in a dose-dependent manner. The concentration of 9(S)-HODE required to induce half-maximal activation was ∼2 μm. On the other hand, parental CHO-K1 cells did not respond to 9(S)-HODE at all. To facilitate the detection of calcium signals, we utilized Gαqi chimeric protein in which 9 COOH-terminal peptides of Gαq protein were replaced with the corresponding residues of the Gαi protein. The COOH-terminal region of the Gα protein plays an important role in specifying receptor interactions, and this chimeric protein can mediate stimulation of phospholipase C by receptors otherwise coupled exclusively to Gαi (21Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Nature. 1993; 363: 274-276Crossref PubMed Scopus (609) Google Scholar). As illustrated in Fig. 1A, cells that stably expressed both G2A and Gαqi (CHO-G2A-Gqi) showed largely enhanced responses to 9(S)-HODE. In these cells, the concentration of 9(S)-HODE required to induce half-maximal activation was reduced to submicromolar levels. On the contrary, CHO cells that stably expressed only Gαqi (CHO-Gqi) did not respond to 9(S)-HODE. To eliminate the possibility of specific stable clones responding to 9(S)-HODE because of some unexpected changes during the selection processes, we performed the calcium mobilization assay in multiple stable clones and obtained essentially the same results (data not shown). We also analyzed the calcium response of G2A to 9(S)-HODE in a transient expression system. CHO-Gqi cells were transfected with pCXN2.1-G2A or the empty vector (pCXN2.1), and the calcium response to 9(S)-HODE was examined after 24 h. Only after the pCXN2.1-G2A was transfected, did the CHO-Gqi cells respond to 9(S)-HODE (Fig. 1B). These results show that G2A responds to 9(S)-HODE and mediates intracellular calcium mobilization, which is greatly enhanced by coexpression of Gαqi protein. Ligand Specificity of G2A—To elucidate the ligand specificity of G2A, various oxidized derivatives of linoleic and arachidonic acid, 1 μm each, were assessed in the calcium mobilization assay. We chose a series of oxidized free fatty acids, as they could be produced in vivo by the enzymatic reactions of lipoxygenases and cytochrome P450 enzymes (22Funk C.D. Science. 2001; 294: 1871-1875Crossref PubMed Scopus (3048) Google Scholar, 23Capdevila J.H. Falck J.R. Harris R.C. J. Lipid Res. 2000; 41: 163-181Abstract Full Text Full Text PDF PubMed Google Scholar, 24Wang H. Zhao Y. Bradbury J.A. Graves J.P. Foley J. Blaisdell J.A. Goldstein J.A. Zeldin D.C. Mol. Pharmacol. 2004; 65: 1148-1158Crossref PubMed Scopus (59) Google Scholar), or by nonenzymatic radical reactions (19Yoshida Y. Ito N. Shimakawa S. Niki E. Biochem. Biophys. Res. Commun. 2003; 305: 747-753Crossref PubMed Scopus (30) Google Scholar). Among them, 9(S)-HODE and 11-HETE showed the strongest ability to mobilize intracellular calcium in CHO-G2A-Gqi cells (Fig. 2A; the molecular structures of these two lipids are shown in the inset). In this respect, the ability of 9(S)-HPODE was comparable with that of 9(S)-HODE, but that of 13(S)-HODE and 13(S)-HPODE were much weaker. 9-HODE in its cholesteryl ester form showed little activity. We also examined the effects of LPC (1-palmitoyl) and SPC in the range from 10 nm to 10 μm on calcium mobilization, but neither showed any significant response under these assay conditions. The results with 10 μm LPC are shown in Fig. 2A. Next, we investigated the chiral specificity of the ligands. The concentrations of 9(S)- and 9(R)-HODE (Cayman Chemical) were adjusted by quantification using a spectrophotometer just prior to use. As shown in Fig. 2B, 9(S)-HODE showed little but significantly higher ability to mobilize intracellular calcium in CHO-G2A-Gqi cells than 9(R)-HODE in concentrations above 0.3 μm. However, the chiral selectivity of G2A was not highly specific. These results indicate that the common structure from the hydroxy or hydroperoxy group to the ω end (2Z,4E-decadien-1-hydro(pero)xide) of 9-H(P)ODE and 11-HETE is important for ligand recognition of G2A. 9(S)-HODE-induced GTPγS Binding—Next, we examined whether 9(S)-HODE stimulated the GDP/GTP exchange on Gα protein using membrane preparations from G2A-expressing cells. As shown in Fig. 3A, the membrane of CHO-G2A cells exhibited 9(S)-HODE-evoked [35S]GTPγS binding in a concentration-dependent manner, whereas the membrane of CHO-K1 cells did not evoke such a binding. To confirm the involvement of G2A in 9(S)-HODE-evoked [35S]GTPγS binding, we performed another analysis in the transient expression system using HEK293 cells. Membranes were prepared from HEK293 cells 24 h after transfection of plasmid DNAs. Although 9(S)-HODE-evoked [35S]GTPγS binding was not observed in the membranes transfected with G2A alone (data not shown), the specific binding became apparent in the membranes after cotransfection of Gαi with G2A (Fig. 3B). On the other hand, transfection of Gαi with the empty vector did not show the 9(S)-HODE-evoked [35S]GTPγS binding. These results indicate that 9(S)-HODE can stimulate the activation of Gα protein via G2A. Involvement of Gαi Protein in G2A-mediated Signal—As the intracellular calcium mobilization evoked by 9(S)-HODE was largely potentiated by Gαqi (Fig. 1A), and the GTPγS binding was observed by coexpression of G2A with Gαi in HEK293 cells (Fig. 3B), it was inferred that Gαi protein is involved in G2A-mediated signaling. To examine this possibility, CHO-G2A cells were pretreated with 50 ng/ml PTX for 16 h, and analyzed in a calcium mobilization assay. PTX pretreatment partially suppressed intracellular calcium mobilization evoked by 9(S)-HODE, although PTX did not affect the calcium mobilization induced by 100 μm ATP (Fig. 4A). This result indicates that G2A induces intracellular calcium mobilization through both Gαi and Gαq pathways. Next, we examined the effects of 9(S)-HODE on adenylyl cyclase activity by measuring cAMP accumulation. When CHO-K1 or CHO-G2A cells were stimulated with 9(S)-HODE, no increase in cAMP concentration was observed (data not shown). On the other hand, in the presence of 3 μm forskolin (adenylyl cyclase activator), 9(S)-HODE inhibited cAMP accumulation in a dose-dependent manner, and this inhibition was abolished almost completely by PTX pretreatment (Fig. 4B). These results indicate that G2A couples with Gαi and inhibits adenylyl cyclase activity. JNK Activation by 9(S)-HODE—The family of serine/threonine kinases referred to as MAP kinases is activated after cell stimulation by various stimuli and plays pivotal roles in multiple signal transduction pathways. Three important kinases, namely, ERK1/2, p38, and JNK, belong to this family. We examined whether 9(S)-HODE activated these MAP kinases. CHO-K1 and CHO-G2A cells were treated with 9(S)-HODE for 0-30 min, and whole cell lysates were prepared by sonication. Activation of MAP kinases was assessed by Western blotting using phospho-specific antibodies. As shown in Fig. 5A, JNK was activated by 9(S)-HODE in CHO-G2A cells, but not in CHO-K1 cells. The activation of JNK was maximal at ∼10 min with 1 μm 9(S)-HODE stimulation, and it was diminished after 30 min. As shown in Fig. 5B, 9(S)-HODE activated JNK in a dose-dependent manner in CHO-G2A cells. On the other hand, activation of neither ERK1/2 nor p38 was observed with 9(S)-HODE both in CHO-K1 and CHO-G2A cells (Fig. 5A). Oxidation and Hydrolysis of Phosphatidylcholine—Linoleic acid esters are the most abundant polyunsaturated fatty acid esters both in human plasma and membrane lipids, and are continuously exposed to oxidative stresses to yield hydroxy and hydroperoxy esters. To mimic the production of oxidized free fatty acids in vitro, 1-palmitoyl-2-linoleoyl PC was oxidized by AMVN and then followed by hydrolysis of the ester bond of phospholipids at the sn-2 position by PLA2. AMVN, used as a radical initiator, was reported to hydroperoxidize 1-palmitoyl-2-linoleoyl PC to generate 9- and 13-HPODE esters (19Yoshida Y. Ito N. Shimakawa S. Niki E. Biochem. Biophys. Res. Commun. 2003; 305: 747-753Crossref PubMed Scopus (30) Google Scholar). PLA2 from N. mossambica mossambica hydrolyzed almost all PC to produce free fatty acids and LPC in our assay conditions, as judged from thin layer chromatography analysis (data not shown). As shown in Fig. 6, neith" @default.
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• W2061802019 cites W1748919512 @default.
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• W2061802019 cites W1991069383 @default.
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• W2061802019 cites W2007462786 @default.
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• W2061802019 cites W2024694801 @default.
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• W2061802019 cites W2046828784 @default.
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