Functional Coupling Between Various Phospholipase A2s and Cyclooxygenases in Immediate and Delayed Prostanoid Biosynthetic Pathways
1999; Elsevier BV; Volume: 274; Issue: 5 Linguagem: Inglês
10.1074/jbc.274.5.3103
ISSN1083-351X
AutoresMakoto Murakami, Terumi Kambe, Satoko Shimbara, Ichiro Kudo,
Tópico(s)Vitamin K Research Studies
ResumoSeveral distinct phospholipase A2s (PLA2s) and two cyclooxygenases (COXs) were transfected, alone or in combination, into human embryonic kidney 293 cells, and their functional coupling during immediate and delayed prostaglandin (PG)-biosynthetic responses was reconstituted. Signaling PLA2s, i.e. cytosolic PLA2 (cPLA2) (type IV) and two secretory PLA2s (sPLA2), types IIA (sPLA2-IIA) and V (sPLA2-V), promoted arachidonic acid (AA) release from their respective transfectants after stimulation with calcium ionophore or, when bradykinin receptor was cotransfected, with bradykinin, which evoked the immediate response, and interleukin-1 plus serum, which induced the delayed response. Experiments on cells transfected with either COX alone revealed subtle differences between the PG-biosynthetic properties of the two isozymes in that COX-1 and COX-2 were favored over the other in the presence of high and low exogenous AA concentrations, respectively. Moreover, COX-2, but not COX-1, could turn on endogenous AA release, which was inhibited by a cPLA2 inhibitor. When PLA2 and COX were coexpressed, AA released by cPLA2, sPLA2-IIA and sPLA2-V was converted to PGE2 by both COX-1 and COX-2 during the immediate response and predominantly by COX-2 during the delayed response. Ca2+-independent PLA2 (iPLA2) (type VI), which plays a crucial role in phospholipid remodeling, failed to couple with COX-2 during the delayed response, whereas it was linked to ionophore-induced immediate PGE2 generation via COX-1 in marked preference to COX-2. Finally, coculture of PLA2 and COX transfectants revealed that extracellular sPLA2s-IIA and -V, but neither intracellular cPLA2 nor iPLA2, augmented PGE2 generation by neighboring COX-expressing cells, implying that the heparin-binding sPLA2s play a particular role as paracrine amplifiers of the PG-biosynthetic response signal from one cell to another. Several distinct phospholipase A2s (PLA2s) and two cyclooxygenases (COXs) were transfected, alone or in combination, into human embryonic kidney 293 cells, and their functional coupling during immediate and delayed prostaglandin (PG)-biosynthetic responses was reconstituted. Signaling PLA2s, i.e. cytosolic PLA2 (cPLA2) (type IV) and two secretory PLA2s (sPLA2), types IIA (sPLA2-IIA) and V (sPLA2-V), promoted arachidonic acid (AA) release from their respective transfectants after stimulation with calcium ionophore or, when bradykinin receptor was cotransfected, with bradykinin, which evoked the immediate response, and interleukin-1 plus serum, which induced the delayed response. Experiments on cells transfected with either COX alone revealed subtle differences between the PG-biosynthetic properties of the two isozymes in that COX-1 and COX-2 were favored over the other in the presence of high and low exogenous AA concentrations, respectively. Moreover, COX-2, but not COX-1, could turn on endogenous AA release, which was inhibited by a cPLA2 inhibitor. When PLA2 and COX were coexpressed, AA released by cPLA2, sPLA2-IIA and sPLA2-V was converted to PGE2 by both COX-1 and COX-2 during the immediate response and predominantly by COX-2 during the delayed response. Ca2+-independent PLA2 (iPLA2) (type VI), which plays a crucial role in phospholipid remodeling, failed to couple with COX-2 during the delayed response, whereas it was linked to ionophore-induced immediate PGE2 generation via COX-1 in marked preference to COX-2. Finally, coculture of PLA2 and COX transfectants revealed that extracellular sPLA2s-IIA and -V, but neither intracellular cPLA2 nor iPLA2, augmented PGE2 generation by neighboring COX-expressing cells, implying that the heparin-binding sPLA2s play a particular role as paracrine amplifiers of the PG-biosynthetic response signal from one cell to another. Phospholipase A2(PLA2), 1The abbreviations used are: PLA2, phospholipase A2; sPLA2, secretory PLA2; cPLA2, cytosolic PLA2; iPLA2, Ca2+-independent PLA2; AA, arachidonic acid; PG, prostaglandin; COX, cyclooxygenase; IL-1, interleukin-1; FCS, fetal calf serum; MAFP, methyl arachidonylfluorophosphate; CHO, Chinese hamster ovary; BK, bradykinin; BKR, bradykinin receptor. 1The abbreviations used are: PLA2, phospholipase A2; sPLA2, secretory PLA2; cPLA2, cytosolic PLA2; iPLA2, Ca2+-independent PLA2; AA, arachidonic acid; PG, prostaglandin; COX, cyclooxygenase; IL-1, interleukin-1; FCS, fetal calf serum; MAFP, methyl arachidonylfluorophosphate; CHO, Chinese hamster ovary; BK, bradykinin; BKR, bradykinin receptor.which regulates the release of arachidonic acid (AA) from membrane phospholipids, and cyclooxygenase (COX), which converts AA to the intermediate prostaglandin (PG) precursor PGH2, represent the two crucial rate-limiting steps for the PG-biosynthetic pathway. To date, more than 10 PLA2 (1Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (756) Google Scholar, 2Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar) and 2 COX (3Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1838) Google Scholar) isozymes have been identified in mammals. The existence of two kinetically distinct PG-biosynthetic responses, the immediate and delayed phases, implies the recruitment of different sets of biosynthetic enzymes to this pathway. Although several works have suggested that preferential coupling between these biosynthetic enzymes accounts for the differential regulation of the immediate and delayed responses, conflicting evidence has been yielded by different experimental systems (4Murakami M. Matsumoto R. Austen K.F. Arm J.P. J. Biol. Chem. 1994; 269: 22269-22275Abstract Full Text PDF PubMed Google Scholar, 5Bingham III, C.O. Murakami M. Fujishima H. Hunt J.E. Austen K.F. Arm J.P. J. Biol. Chem. 1996; 271: 25936-25944Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 6Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 7Marshall L.A. Bolognese B. Winkler J.D. Roshak A. J. Biol. Chem. 1997; 272: 759-765Crossref PubMed Scopus (59) Google Scholar, 8Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar, 9Murakami M. Kuwata H. Amakasu Y. Shimbara S. Nakatani Y. Atsumi G. Kudo I. J. Biol. Chem. 1997; 272: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 10Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 11Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar). A rapidly expanding body of evidence suggests that the two COXs, the constitutive COX-1 and inducible COX-2, play distinct roles in regulating AA metabolism (3Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1838) Google Scholar). Several investigators have proposed that COX-1 and COX-2 respectively metabolize exogenous and endogenous arachidonic acid (AA) to PGs (12Reddy S.T. Herschman H.R. J. Biol. Chem. 1994; 269: 15473-15480Abstract Full Text PDF PubMed Google Scholar, 13Shitashige M. Morita I. Murota S. Biochim. Biophys. Acta. 1998; 1389: 57-66Crossref PubMed Scopus (70) Google Scholar). This statement, however, is an oversimplification, because in certain situations, COX-1 metabolizes endogenous AA to PGs, e.g. thromboxane generation by platelets (14Langenbach R. Morham S.G. Tiano H.F. Loftin C.D. Ghanayem B.I. Chulada P.C. Mahler J.F. Lee C.A. Goulding E.H. Kluckman K.D. Kim H.S. Smithies O. Cell. 1995; 83: 483-492Abstract Full Text PDF PubMed Scopus (1037) Google Scholar) and macrophages (11Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar) and PGD2 generation by mast cells (4Murakami M. Matsumoto R. Austen K.F. Arm J.P. J. Biol. Chem. 1994; 269: 22269-22275Abstract Full Text PDF PubMed Google Scholar, 5Bingham III, C.O. Murakami M. Fujishima H. Hunt J.E. Austen K.F. Arm J.P. J. Biol. Chem. 1996; 271: 25936-25944Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 6Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), and COX-2 also utilizes exogenous AA (11Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar). More generally, utilization of COX-1 is observed during the early phase of PG biosynthesis occurring within several minutes of stimulation, whereas COX-2-dependent PG generation proceeds over several hours in parallel with the induction of COX-2 expression (4Murakami M. Matsumoto R. Austen K.F. Arm J.P. J. Biol. Chem. 1994; 269: 22269-22275Abstract Full Text PDF PubMed Google Scholar, 5Bingham III, C.O. Murakami M. Fujishima H. Hunt J.E. Austen K.F. Arm J.P. J. Biol. Chem. 1996; 271: 25936-25944Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 6Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 10Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 11Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar,15Morham S.G. Langenbach R. Loftin C.D. Tiano H.F. Vouloumanos N. Jennette J.C. Mahler J.F. Kluckman K.D. Ledford A. Lee C.A. Smithies O. Cell. 1995; 83: 473-482Abstract Full Text PDF PubMed Scopus (1023) Google Scholar). Although subtle differences in the subcellular distributions of these two isozymes may be responsible for their separate functions, a recent electron microscopic analysis showed that their locations in the perinuclear and endoplasmic reticular membranes were indistinguishable (16Spencer A.G. Woods J.W. Arakawa T. Singler I.I. Smith W.L. J. Biol. Chem. 1998; 273: 9886-9893Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Whether distinct PLA2s are utilized selectively in the different PG-biosynthetic phases and couple specifically with each COX isozyme is controversial. PLA2s are subdivided into several classes, among which cytosolic PLA2 (cPLA2; recently called cPLA2α after the recent discovery of two related isozymes cPLA2 β and γ (17Underwood K.W. Song C. Kriz R.W. Chang X.J. Knopf J.L. Lin L.-L. J. Biol. Chem. 1998; 273: 21926-21932Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar)), a family of secretory PLA2s (sPLA2s), and Ca2+-independent PLA2 (iPLA2) have been paid considerable attention. The increased cytoplasmic Ca2+ concentration and activation of mitogen-activated protein kinases, which are prerequisite for cPLA2activation, occur rapidly and are often transient, implying that cPLA2 plays a crucial role in immediate AA release (18Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar, 19Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1455) Google Scholar, 20Lin L.-L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1649) Google Scholar). Evidence is accumulating that cPLA2 is also involved in the delayed response (9Murakami M. Kuwata H. Amakasu Y. Shimbara S. Nakatani Y. Atsumi G. Kudo I. J. Biol. Chem. 1997; 272: 19891-19897Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 21Lin L.-L. Lin A.Y. DeWitt D.L. J. Biol. Chem. 1992; 267: 23451-23454Abstract Full Text PDF PubMed Google Scholar, 22Roshak A. Sathe G. Marshall L.A. J. Biol. Chem. 1994; 269: 25999-26005Abstract Full Text PDF PubMed Google Scholar), although the mechanism whereby cPLA2 is activated under such a Ca2+-free condition is poorly understood. Some studies showed that sPLA2 couples selectively with COX-1 during the immediate phase (6Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar), whereas others demonstrated that sPLA2, particularly type IIA (sPLA2-IIA), functions during the delayed phase, as its expression is often induced markedly in response to proinflammatory stimuli and correlates with ongoing PG biosynthesis (10Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 23Nakazato Y. Simonson M.S. Herman W.H. Konieczkowski M. Sedor J.R. J. Biol. Chem. 1991; 266: 14119-14127Abstract Full Text PDF PubMed Google Scholar, 24Murakami M. Kudo I. Inoue K. J. Biol. Chem. 1993; 268: 839-844Abstract Full Text PDF PubMed Google Scholar, 25Pfeilschifter J. Schalkwijk C. Briner V.A. van den Bosch H. J. Clin. Invest. 1993; 92: 2516-2523Crossref PubMed Scopus (209) Google Scholar). Several types of cell express type V sPLA2 (sPLA2-V), which appears to mediate certain phases of PG biosynthesis (26Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 27Reddy S.T. Winstead M.V. Tischfield J.A. Herschman H.R. J. Biol. Chem. 1997; 272: 13591-13596Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Supportive (28Wolf M.J. Gross R.W. J. Biol. Chem. 1996; 271: 20989-20992Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 29Wolf M.J. Wang J. Turk J. Gross R.W. J. Biol. Chem. 1997; 272: 1522-1526Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) and contradictory (30Balboa M.A. Balsinde J. Jones S.S. Dennis E.A. J. Biol. Chem. 1997; 272: 8576-8580Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 31Balsinde J. Dennis E.A. J. Biol. Chem. 1997; 272: 16069-16072Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar) evidence for the involvement of iPLA2 in stimulus-dependent AA release has been reported. The conflicting observations reported so far may be due to the limitations of studies using chemical inhibitors, antibodies, and even antisense oligonucleotides, which often cannot gain access to certain cellular compartments and may cause cross-inhibition or undesirable side effects, thereby leading to misinterpretation. Recently, in an attempt to elucidate the general functions of each PLA2 in AA release, we analyzed the functional effects of transfecting various PLA2 isozymes into mammalian cell lines, such as human embryonic kidney 293 cells and Chinese hamster ovary (CHO) cells. This approach enabled us to assess the overlapping and different functions of five distinct PLA2s, namely cPLA2, sPLA2s (IIA, IIC, and V), and iPLA2 (32Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). We demonstrated that cPLA2 and the two heparin-binding sPLA2s, sPLA2-IIA and sPLA2-V, act as "signaling" PLA2s that promote stimulus-dependent AA release during both the immediate and delayed responses, whereas iPLA2 mediates spontaneous fatty acid release during culture, consistent with its proposed role in "phospholipid remodeling" rather than signaling (30Balboa M.A. Balsinde J. Jones S.S. Dennis E.A. J. Biol. Chem. 1997; 272: 8576-8580Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 31Balsinde J. Dennis E.A. J. Biol. Chem. 1997; 272: 16069-16072Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). In this study, we extended our previous study in order to investigate particular functional cooperation between PLA2 and COX enzymes during different phases of PG biosynthesis by cotransfecting each PLA2 and COX into 293 cells. Reconstitution of the immediate and delayed PGE2 biosynthetic responses of these transfectants confirmed that distinct functional coupling between different PLA2 and COX enzymes occurs during each phase. The cDNAs for mouse cPLA2, mouse sPLA2-IIA, and its mutants (G30S, H48E, and KE4), rat sPLA2-V, rat sPLA2-IIC, and hamster iPLA2 were described previously (32Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). Human COX-1 and COX-2 cDNAs were provided by Dr. S. Nagata (Osaka University) and subcloned into pcDNA3.1 (Invitrogen). Rat bradykinin receptor (BKR) B2 cDNA (33McEachern A.E. Shelton E.R. Bhakta S. Obernolte R. Bach C. Zuppan P. Fujisaki J. Aldrich R.W. Jarnagin K. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7724-7728Crossref PubMed Scopus (403) Google Scholar), obtained from a rat genomic library (CLONTECH) by performing the reverse transcription-polymerase chain reaction using a RNA polymerase chain reaction kit (AMV) version-2 (Takara Shuzo), was subcloned into pCR3.1 (Invitrogen). Human embryonic kidney 293 cells were obtained from Riken Cell Bank, and CHO-K1 cells stably expressing human COX-1 and COX-2 were provided by Dr. M. Sugimoto (Chugai Pharmaceutical Co. Ltd.). The rabbit anti-human cPLA2 antibody and sPLA2inhibitor LY311727 (34Schevitz R.W. Bach N.J. Carlson D.G. Chirgadze N.Y. Clawson D.K. Dillard R.D. Draheim S.E. Hartley L.W. Jones N.D. Mihelich E.D. Olkowski J.L. Snyder D.W. Sommers C. Wery J.-P. Nature Struct. Biol. 1995; 2: 458-465Crossref PubMed Scopus (235) Google Scholar) were provided by Dr. R. M. Kramer (Lilly Research). The rabbit anti-rat sPLA2-IIA antibody was prepared as described previously (35Murakami M. Kudo I. Natori Y. Inoue K. Biochim. Biophys. Acta. 1989; 1043: 34-42Crossref Scopus (50) Google Scholar). The goat anti-human COX-2 antibody was purchased from Santa Cruz. The rabbit anti-human COX-1 antibody and COX-1 inhibitor valeryl salicylate (36Bhattacharyya D.K. Lecomte M. Dunn J. Morgans D.J. Smith W.L. Arch. Biochem. Biophys. 1995; 317: 19-24Crossref PubMed Scopus (99) Google Scholar) were provided by Dr. W. L. Smith (Michigan State University). The COX-2 inhibitor NS-398 (37Futaki N. Yoshikawa K. Hamasaka Y. Arai I. Higuchi S. Iizuka H. Otomo S. Gen. Pharmacol. 1993; 24: 105-110Crossref PubMed Scopus (361) Google Scholar) was provided by Dr. J. Trzaskos (DuPont Merck Pharmaceutical Co.). The cPLA2 inhibitor methyl arachidonylfluorophosphate (MAFP) (38Huang Z. Liu S. Street I. Laliberte F. Abdullah K. Desmarais S. Wang Z. Kennedy B. Payette P. Riendeau D. Weech P. Gresser M. Mediat. Inflamm. 1994; 3: 307-308Google Scholar), the iPLA2 inhibitor bromoenol lactone (39Ackermann E.J. Conde-Frieboes K. Dennis E.A. J. Biol. Chem. 1995; 270: 445-450Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar), AA, and the PGE2 enzyme immunoassay kit were purchased from Cayman Chemical. A23187 was purchased from Calbiochem. BK was purchased from Sigma. Human and mouse interleukin (IL)-1βs were purchased from Genzyme. LipofectAMINE PLUS reagent, Opti-MEM medium, and Trizol reagent were obtained from Life Technologies. RPMI 1640 medium was purchased from Nissui Pharmaceutical. Transformants of 293 cells that stably expressed cPLA2, sPLA2-IIA, sPLA2-V, sPLA2-IIC, and iPLA2 were established as described previously (32Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). COX-1 and COX-2 cDNAs were transfected into 293 cells using LipofectAMINE PLUS, according to the manufacturer's instructions. Briefly, 1 μg of plasmid was mixed with 5 μl of LipofectAMINE PLUS in 200 μl of Opti-MEM medium for 30 min and then added to cells that had attained 40–60% confluence in 6-well plates (Iwaki) containing 1 ml of Opti-MEM. After incubation for 6 h, the medium was replaced with 2 ml of fresh culture medium comprising RPMI 1640 containing 10% (v/v) fetal calf serum (FCS). After overnight culture, the medium was replaced again with 2 ml of fresh medium, and culture was continued at 37 °C in a CO2 incubator flushed with 5% CO2 in humidified air. For transient expression analysis, the cells were harvested 3 days after transfection and used immediately. In order to establish stable transfectants, cells transfected with each cDNA were cloned by limiting dilution in 96-well plates in culture medium supplemented with 800 μg/ml G418 (Life Technologies). After culture for 2–4 weeks, wells containing a single colony were chosen, and the expression of each COX was assessed by immunoblotting. The established clones were expanded and used for the experiments as described below. In order to establish PLA2/COX double transformants, 293 transformants expressing each COX were subjected to a second transfection with each PLA2 cDNA, which had been subcloned into pcDNA3.1/Zeo (+) (Invitrogen). Three days after transfection, the cells were used for the experiments or seeded into 96-well plates to be cloned by culture in the presence of 50 μg/ml zeocin (Invitrogen) in order to establish stable transformants overexpressing both PLA2 and COX. The expression of each PLA2 was examined by RNA blotting, and in the case of sPLA2s, by measuring PLA2 activities released into the supernatants. A similar strategy was employed to produce PLA2/BKR double transformants: after the second transfection with cPLA2 or sPLA2-IIA cDNA in pcDNA3.1/Zeo (+), G418-resistant 293 cells stably expressing rat B2-type BKR were selected with zeocin. Cells in 1 ml of culture medium were seeded, at a density of 5 × 104cells/ml, into each well of 24-well plates. After culture for 4 days, the supernatants were collected, and the cells were incubated for a further 15 min at 37 °C with 1 ml of culture medium containing 1m NaCl. This allowed cell surface-associated sPLA2s to be recovered quantitatively from the medium, as described previously (32Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). The PLA2 activity was assayed by measuring the amounts of free radiolabeled fatty acids released from the substrates 1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphoethanolamine (NEN Life Science Products) and 1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphoethanolamine (Amersham Pharmacia Biotech) for sPLA2-V and sPLA2-IIA, respectively. Each reaction mixture consisted of an aliquot of the required sample, 100 mm Tris-HCl, pH 6.0 (for sPLA2-V) or 7.4 (for sPLA2-IIA), 4 mm CaCl2, and 2 μm substrate. After incubation for 10–30 min at 37 °C, the [14C]fatty acids released were extracted by the method of Dole and Meinertz (40Dole V.P. Meinertz H. J. Biol. Chem. 1960; 235: 2595-2599Abstract Full Text PDF PubMed Google Scholar), and the radioactivity was counted. Approximately equal amounts (∼10 μg) of the total RNAs obtained from transfected cells were applied to each lane of 1.2% (w/v) formaldehyde-agarose gels, electrophoresed, and transferred to Immobilon-N membranes (Millipore). The resulting blots were then probed with the respective cDNA probes that had been labeled with [32P]dCTP (Amersham Pharmacia Biotech) by random priming (Takara Shuzo). All hybridizations were carried out as described previously (10Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Cell lysates (105 cell equivalents) were subjected to SDS-polyacrylamide gel electrophoresis (7.5% (w/v) for cPLA2 and 10% for COX-1 and COX-2) under reducing conditions. In order to analyze sPLA2-IIA, the cells were treated for 15 min with culture medium containing 1 m NaCl, and aliquots of the resulting supernatants were subjected to 15% SDS-polyacrylamide gel electrophoresis under nonreducing conditions. The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher & Schuell) using a semidry blotter (MilliBlot-SDE system; Millipore), according to the manufacturer's instructions. The membranes were probed with the respective antibodies and visualized using the ECL Western blot analysis system (Amersham Pharmacia Biotech), as described previously (10Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Cells (5 × 104 in 1 ml of culture medium) were seeded into each well of 24-well plates. In order to assess AA release, 0.1 μCi/ml [3H]AA (Amersham Pharmacia Biotech) was added to the cells in each well on day 3, when they had nearly reached confluence, and culture was continued for another day. After three washes with fresh medium, 250 μl of RPMI 1640 medium with or without 10 μm A23187, 10 μm BK, or 1 ng/ml IL-1β and/or 10% FCS was added to each well and the amount of free [3H]AA released into each supernatant during culture (up to 30 min after the addition of A23187 and BK and up to 8 h after IL-1β) was measured. The percentage release of AA was calculated using the formula (S/(S + P)) × 100, where Sand P are the radioactivities measured in equal portions of the supernatant and cell pellet, respectively. The supernatants from replicate cells cultured without added radiolabeled fatty acids were subjected to the PGE2 enzyme immunoassay. Conversion of exogenous AA to PGE2 was determined by treating cells with AA for 30 min and immunoassaying the PGE2 produced. Human COX-1 and COX-2 cDNAs, subcloned into a mammalian expression vector with a neomycin-resistant marker, were individually transfected into 293 and CHO cells to establish stable transformants. Expression of COX-1 and COX-2 proteins was barely detectable in parental 293 cells but was detected clearly in the respective transformants (Fig. 1 A). In order to assess immediate and delayed PGE2 generation from endogenous AA by each COX, we stimulated the transformants with 10 μm A23187 for 30 min and 1 ng/ml IL-1 in the presence of 10% FCS for 4 h, respectively (Fig. 1 B). Whereas treatment of parental 293 cells with A23187 resulted in minimal PGE2 generation, consistent with undetectable expression levels of both COX isozymes (Fig. 1 A), replicate cells expressing COX-1 and COX-2 produced approximately 15 and 35 times more PGE2 in response to A23187 than the control cells (Fig.1 B, top). When COX-2-expressing cells were stimulated with IL-1/FCS, delayed PGE2 generation increased markedly, reaching approximately 14 times the amount generated by the control cells (Fig. 1 B, bottom). In contrast, COX-1-expressing cells produced only minimal amounts of PGE2 that did not differ significantly from the amounts produced by the control cells. To investigate the functional differences between COX-1 and COX-2 further, we measured the COX activities by supplying the transfectants with exogenous AA (Fig. 1 C). In our assay system, COX-1 transfectants produced PGE2 when the exogenous AA concentration exceeded 10 μm, reaching nearly 100 ng of PGE2 in the presence of 50 μm AA, whereas conversion to PGE2 was undetectable when the AA concentration was below 10 μm. COX-2-expressing cells produced substantial amounts of PGE2 even when the AA concentration was below 10 μm. Approximately 20 ng of PGE2 was produced by COX-2-expressing cells treated with 50 μm AA, only 15 the amount produced by COX-1 transfectants. No appreciable conversion of AA to PGE2 by the control cells was observed. Conversion of exogenous AA to PGE2 by cells expressing COX-1 and COX-2 was inhibited by the COX-1-specific inhibitor valeryl salicylate and the COX-2-specific inhibitor NS-398, respectively, but not vice versa (Fig.1 D). Collectively, these data suggest that COX-1 is favored over COX-2 when excess AA is supplied, whereas COX-2 is more active than COX-1 when the supply of AA is limited. We observed that some PGE2 was produced by cells expressing COX-2, but not COX-1, even in the absence of exogenous AA or any extracellular stimulus (Fig. 1, C and E), indicating that COX-2 could utilize endogenous AA in this situation. Interestingly, COX-2-expressing cells not only spontaneously produced more PGE2 but also released more AA than the control cells (Fig. 1 E). This increased AA release induced by COX-2 transfection was significantly suppressed by the cPLA2inhibitor MAFP but not by the iPLA2 inhibitor bromoenol lactone, the COX-2 inhibitor NS-398 (Fig. 1 E), or the sPLA2 inhibitor LY311727 (data not shown), whereas PGE2 generation by COX-2 transfectants was suppressed by MAFP and NS-398 but not by bromoenol lactone. Immunoblot analysis revealed a trace amount of cPLA2 (Fig. 1 F) but none of other PLA2s examined in this study (data not shown) in parental 293 cells. These observations suggest that COX-2 can activate AA release mediated by the MAFP-sensitive cPLA2through a mechanism independent of COX activity. In order to verify that the observations described above was also applicable to other cells, we carried out similar experiments using CHO cells transfected with COX-1 and COX-2 (Fig. 1, F andG). CHO cells expressing COX-1 and COX-2 produced 16 and 25 times more PGE2, respectively, than the parental cells during the immediate response to A23187 (Fig. 1 G, left). Significant and comparable increases in PGE2 generation by parental and COX-1-expressing cells after stimulation with IL-1 plus FCS was observed (Fig. 1 G, middle). This increase was suppressed almost completely by NS-398 (data not shown) (32Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstra
Referência(s)