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Cytosolic Phospholipase A2 Is Required for Cytokine-induced Expression of Type IIA Secretory Phospholipase A2 That Mediates Optimal Cyclooxygenase-2-dependent Delayed Prostaglandin E2 Generation in Rat 3Y1 Fibroblasts

1998; Elsevier BV; Volume: 273; Issue: 3 Linguagem: Inglês

10.1074/jbc.273.3.1733

ISSN

1083-351X

Autores

Hiroshi Kuwata, Yoshihito Nakatani, Makoto Murakami, Ichiro Kudo,

Tópico(s)

NF-κB Signaling Pathways

Resumo

Activation of rat fibroblastic 3Y1 cells with interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) induced delayed prostaglandin (PG) E2 generation over 6–48 h, which occurred in parallel with de novo induction of type IIA secretory phospholipase A2 (sPLA2) and cyclooxygenase (COX)-2, without accompanied by changes in the constitutive expression of type IV cytosolic PLA2(cPLA2) and COX-1. Types V and IIC sPLA2s were barely detectable in these cells. Studies using an anti-type IIA sPLA2 antibody, sPLA2 inhibitors, and a type IIA sPLA2-specific antisense oligonucleotide revealed that IL-1β/TNFα-induced delayed PGE2 generation by these cells was largely dependent on inducible type IIA sPLA2, which was functionally linked to inducible COX-2. Delayed PGE2 generation was also suppressed markedly by the cPLA2 inhibitor arachidonoyl trifluoromethyl ketone (AACOCF3), which attenuated induction of type IIA sPLA2, but not COX-2, expression. AACOCF3inhibited the initial phase of cytokine-stimulated arachidonic acid release, and supplementing AACOCF3-treated cells with exogenous arachidonic acid partially restored type IIA sPLA2 expression. These results suggest that certain metabolites produced by the cPLA2-dependent pathway are crucial for the subsequent induction of type IIA sPLA2 expression and attendant delayed PGE2generation. Some lipoxygenase-derived products might be involved in this event, since IL-1β/TNFα-induced type IIA sPLA2induction and PGE2 generation were reduced markedly by lipoxygenase, but not COX, inhibitors. In contrast, Ca2+ionophore-stimulated immediate PGE2 generation was regulated predominantly by the constitutive enzymes cPLA2and COX-1, even when type IIA sPLA2 and COX-2 were maximally induced after IL-1β/TNFα treatment, revealing functional segregation of the constitutive and inducible PG biosynthetic enzymes. Activation of rat fibroblastic 3Y1 cells with interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) induced delayed prostaglandin (PG) E2 generation over 6–48 h, which occurred in parallel with de novo induction of type IIA secretory phospholipase A2 (sPLA2) and cyclooxygenase (COX)-2, without accompanied by changes in the constitutive expression of type IV cytosolic PLA2(cPLA2) and COX-1. Types V and IIC sPLA2s were barely detectable in these cells. Studies using an anti-type IIA sPLA2 antibody, sPLA2 inhibitors, and a type IIA sPLA2-specific antisense oligonucleotide revealed that IL-1β/TNFα-induced delayed PGE2 generation by these cells was largely dependent on inducible type IIA sPLA2, which was functionally linked to inducible COX-2. Delayed PGE2 generation was also suppressed markedly by the cPLA2 inhibitor arachidonoyl trifluoromethyl ketone (AACOCF3), which attenuated induction of type IIA sPLA2, but not COX-2, expression. AACOCF3inhibited the initial phase of cytokine-stimulated arachidonic acid release, and supplementing AACOCF3-treated cells with exogenous arachidonic acid partially restored type IIA sPLA2 expression. These results suggest that certain metabolites produced by the cPLA2-dependent pathway are crucial for the subsequent induction of type IIA sPLA2 expression and attendant delayed PGE2generation. Some lipoxygenase-derived products might be involved in this event, since IL-1β/TNFα-induced type IIA sPLA2induction and PGE2 generation were reduced markedly by lipoxygenase, but not COX, inhibitors. In contrast, Ca2+ionophore-stimulated immediate PGE2 generation was regulated predominantly by the constitutive enzymes cPLA2and COX-1, even when type IIA sPLA2 and COX-2 were maximally induced after IL-1β/TNFα treatment, revealing functional segregation of the constitutive and inducible PG biosynthetic enzymes. Two kinetically different prostaglandin (PG) 1The abbreviations used are: PG, prostaglandin; PLA2, phospholipase A2; sPLA2, secretory PLA2; cPLA2, cytosolic PLA2; COX, cyclooxygenase; LOX, lipoxygenase; IL, interleukin; TNFα, tumor necrosis factor α; TBS-T, Tris-buffered saline containing 0.05% Tween 20; FCS, fetal calf serum; DMEM, Dulbecco's modified Eagle medium, RT-PCR, reverse transcriptase-polymerase chain reaction; PCR, polymerase chain reaction; AACOCF3, arachidonoyl trifluoromethyl ketone; NDGA, nordihydroguaiaretic acid. -generating pathways from endogenous arachidonic acid, the immediate and delayed phases, imply the recruitment of different sets of biosynthetic enzymes, expression and activation of which are tightly regulated by distinct transmembrane signalings (1Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Curr. Rev. Immunol. 1997; 17: 225-284Crossref PubMed Google Scholar). The immediate phase of PG biosynthesis, occurring within several minutes of stimulation, is elicited by agonists that mobilize intracellular Ca2+ and characterized by a burst release of arachidonic acid initiated by phospholipase A2 (PLA2) and subsequent conversion to bioactive PGs by the sequential actions of cyclooxygenase (COX) and terminal PG synthases. The delayed phase of PG biosynthesis is accompanied by the continuous supply of arachidonic acid and its conversion to PGs, often PGE2, over long culture periods following growth or proinflammatory stimuli. Segregated utilization of the two COX isoforms in these two distinct phases has been demonstrated in several systems (2Murakami M. Matsumoto R. Austen K.F. Arm J.P. J. Biol. Chem. 1994; 269: 22269-22275Abstract Full Text PDF PubMed Google Scholar, 3Reddy S.T. Herschman H.R. J. Biol. Chem. 1994; 269: 15473-15480Abstract Full Text PDF PubMed Google Scholar, 4Murakami M. Bingham III, C.O. Matsumoto R. Austen K.F. Arm J.P. J. Immunol. 1995; 155: 4445-4453PubMed Google Scholar). COX-1 is constitutively expressed in most cells and tissues and is generally thought to serve certain physiologic housekeeping functions, whereas COX-2 is dramatically induced in response to a wide variety of stimuli, and is thought to contribute to the generation of PGs at certain stages of cell proliferation and differentiation and at sites of inflammation (5Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1852) Google Scholar). The absolute requirement for COX-2 in the delayed PG generation, irrespective of the constitutive presence of COX-1, has been shown by studies using COX-2-selective inhibitors, antisense oligonucleotides, and knockout mice (2Murakami M. Matsumoto R. Austen K.F. Arm J.P. J. Biol. Chem. 1994; 269: 22269-22275Abstract Full Text PDF PubMed Google Scholar, 3Reddy S.T. Herschman H.R. J. Biol. Chem. 1994; 269: 15473-15480Abstract Full Text PDF PubMed Google Scholar, 4Murakami M. Bingham III, C.O. Matsumoto R. Austen K.F. Arm J.P. J. Immunol. 1995; 155: 4445-4453PubMed Google Scholar, 6Morham 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 (1027) Google Scholar). COX-1 is involved in immediate PG generation, such as thromboxane A2 generation by activated platelets (7Langenbach 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 (1044) Google Scholar) and the IgE-dependent immediate phase of PGD2 generation by mast cells (2Murakami M. Matsumoto R. Austen K.F. Arm J.P. J. Biol. Chem. 1994; 269: 22269-22275Abstract Full Text PDF PubMed Google Scholar, 4Murakami M. Bingham III, C.O. Matsumoto R. Austen K.F. Arm J.P. J. Immunol. 1995; 155: 4445-4453PubMed Google Scholar). In contrast, interleukin (IL)-1α-primed, platelet-derived growth factor-initiated immediate PGE2 generation by mouse calvaria cells depends on IL-1α-induced COX-2 in preference to constitutive COX-1 (8Chen Q.-R. Miyaura C. Higashi S. Murakami M. Kudo I. Saito S. Hiraide T. Shibasaki Y. Suda T. J. Biol. Chem. 1997; 272: 5952-5958Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Several explanations of the functional segregation of the two COX isoforms have been proposed, such as their different subcellular localizations (9Morita I. Schindler M. Regier M.K. Otto J.C. Hori T. DeWitt D.L. Smith W.L. J. Biol. Chem. 1995; 270: 10902-10908Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar) and their different substrate concentration requirements (10Kulmacz R.J. Wang L.-H. J. Biol. Chem. 1995; 270: 24019-24023Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). In fact, the finding that the utilization of arachidonic acid by COX-2 is favored over COX-1 at low peroxide concentrations (10Kulmacz R.J. Wang L.-H. J. Biol. Chem. 1995; 270: 24019-24023Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar) appears consistent with the involvement of COX-1 in the burst of events that follow immediate activation and of COX-2 in the delayed phase, in which only a limited amount of arachidonic acid would be supplied continuously. Moreover, preferential coupling between particular PLA2s, COXs, and terminal PG synthases have been suggested by several recent studies (11Bingham 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, 12Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 13Matsumoto H. Naraba H. Murakami M. Kudo I. Yamaki K. Ueno A. Oh-ishi S. Biochem. Biophys. Res. Commun. 1997; 230: 110-114Crossref PubMed Scopus (145) Google Scholar). The enzymatic properties of type IV 85-kDa cytosolic PLA2(cPLA2), i.e. arachidonate selectivity, Ca2+-dependent translocation, and phosphorylation-dependent activation via kinases belonging to the mitogen-activated protein kinase family (14Clark 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 (1462) Google Scholar, 15Lin 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 (1657) Google Scholar), are in agreement with its role in immediate PG biosynthesis, which is usually accompanied by rapid and transient cytoplasmic Ca2+mobilization. Under such conditions, cPLA2 translocates to the perinuclear envelope and endoplasmic reticular membranes (16Glover S.M.S. Bayburt T. Jonas M. Chi E. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15367Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 17Schievella A.R. Regier M.K. Smith W.L. Lin L.-L. J. Biol. Chem. 1995; 270: 30749-30754Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar), where downstream COXs, 5-lipoxygenase (LOX), 5-LOX-activating protein, and some terminal PG and leukotriene synthases colocalize (9Morita I. Schindler M. Regier M.K. Otto J.C. Hori T. DeWitt D.L. Smith W.L. J. Biol. Chem. 1995; 270: 10902-10908Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar, 18Woods J.W. Evans J.F. Ethier D. Scott S. Vickers P.J. Hearn L. Heibein J.A. Charleson S. Singer I.I. J. Exp. Med. 1993; 178: 1935-1946Crossref PubMed Scopus (361) Google Scholar, 19Ohashi K. Ruan K.-H. Kulmacz R.J. Wu K.K. Wang L.-H. J. Biol. Chem. 1992; 267: 789-793Abstract Full Text PDF PubMed Google Scholar, 20Penrose J.F. Spector J. Baldassaro M. Xu K. Boyce J. Arm J.P. Austen K.F. Lam B.K. J. Biol. Chem. 1996; 271: 11356-11361Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The functional association of cPLA2 with constitutive COX-1 resulting in immediate PG biosynthesis has been demonstrated in several studies (21Riendeau D. Guay J. Weech P.K. Laliberte F. Yergey J. Li C. Desmarais S. Perrier H. Liu S. Nicoll-Griffith D. Street I.P. J. Biol. Chem. 1994; 269: 15619-15624Abstract Full Text PDF PubMed Google Scholar, 22Huang Z. Payette P. Abdullah K. Cromlish W.A. Kennedy B.P. Biochemistry. 1996; 35: 3712-3721Crossref PubMed Scopus (86) Google Scholar). cPLA2 has been also implicated in the inducible COX-2-dependent delayed PG generation lasting for hours (12Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 23Roshak A. Sathe G. Marshall L.A. J. Biol. Chem. 1994; 269: 25999-26005Abstract Full Text PDF PubMed Google Scholar, 24Murakami 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) and COX-2-dependent immediate PGE2 generation (8Chen Q.-R. Miyaura C. Higashi S. Murakami M. Kudo I. Saito S. Hiraide T. Shibasaki Y. Suda T. J. Biol. Chem. 1997; 272: 5952-5958Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) that are initiated and primed, respectively, by inflammatory stimuli. Secretory PLA2 (sPLA2) enzymes comprise a growing family of distinct enzymes with similar molecular masses of about 14 kDa. To date, five mammalian sPLA2s, designated types I, IIA, IIC, V, and X, have been identified (1Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Curr. Rev. Immunol. 1997; 17: 225-284Crossref PubMed Google Scholar, 25Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (758) Google Scholar, 26Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 27Cupillard L. Koumanov K. Matte M.-G. Ladzdunski M. Lambeau G. J. Biol. Chem. 1997; 272: 15145-15152Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Type I sPLA2 is abundantly expressed in the pancreas, where it functions as a digestive enzyme for dietary phospholipids. It is also expressed in several nondigestive organs, where it may act as a regulator of cellular functions via specific sPLA2 receptor (28Lambeau G. Ancian P. Barhanin J. Lazdunski M. J. Biol. Chem. 1994; 269: 1575-1578Abstract Full Text PDF PubMed Google Scholar, 29Tohkin M. Kishino J. Ishizaki J. Arita H. J. Biol. Chem. 1993; 268: 2865-2871Abstract Full Text PDF PubMed Google Scholar). Type IIA sPLA2 was originally isolated from inflamed sites and inflammatory cells, is induced by proinflammatory stimuli, and is thought to play some roles in cellular inflammatory responses (1Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Curr. Rev. Immunol. 1997; 17: 225-284Crossref PubMed Google Scholar, 30Kudo I. Murakami M. Hara S. Inoue K. Biochim. Biophys. Acta. 1993; 1170: 217-231Crossref PubMed Scopus (371) Google Scholar). Type IIA sPLA2 has been shown to augment the delayed phase of PG generation by several cells in response to proinflammatory cytokines (31Pfeilschifter J. Schalkwijk C. Briner V.A. van den Bosch H. J. Clin. Invest. 1993; 92: 2516-2523Crossref PubMed Scopus (210) Google Scholar, 32Murakami M. Kudo I. Inoue K. J. Biol. Chem. 1993; 268: 839-844Abstract Full Text PDF PubMed Google Scholar, 33Suga H. Murakami M. Kudo I. Inoue K. Eur. J. Biochem. 1993; 218: 807-813Crossref PubMed Scopus (70) Google Scholar, 34Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), and further support for these biological actions has been supplied by several animal studies (35Tanaka K. Kato T. Matsumoto K. Yoshida T. Inflammation. 1993; 17: 107-119Crossref PubMed Scopus (31) Google Scholar, 36Murakami M. Kudo I. Nakamura H. Yokoyama Y. Mori H. Inoue K. FEBS Lett. 1990; 268: 113-116Crossref PubMed Scopus (59) Google Scholar, 37Chapdelaine J.M. Ciofalo V.B. Grass D.S. Felkner R.H. Wallace R.E. Swanson M.E. Arthritis Rheum. 1995; 38: S293Google Scholar). Recently, two groups have demonstrated that type V sPLA2, the transcript of which was originally found to be expressed in the heart and lung (38Chen J. Engle S.J. Seilhamer J.J. Tischfield J.A. J. Biol. Chem. 1994; 269: 2365-2368Abstract Full Text PDF PubMed Google Scholar), is crucial for stimulus-initiated PG biosynthesis by mouse macrophages (39Balboa 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) and mast cells (40Reddy 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). Type IIC sPLA2 is abundantly expressed in rodent testes, whereas it is a nonfunctional pseudogene in human (41Chen J. Engle S.J. Seilhamer J.J. Tischfield J.A. J. Biol. Chem. 1994; 269: 23018-23024Abstract Full Text PDF PubMed Google Scholar). The overall structures of types IIA, IIC, and V sPLA2s, the genes of which are tightly linked on human chromosome 1, are more closely related to one another than to that of type I sPLA2, the gene of which maps to human chromosome 12 (26Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). More recently, a novel group X sPLA2 has been cloned; it is distributed in immune tissues, exhibits some features characteristic of both types I and IIA sPLA2s, and maps to human chromosome 16 (27Cupillard L. Koumanov K. Matte M.-G. Ladzdunski M. Lambeau G. J. Biol. Chem. 1997; 272: 15145-15152Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Thus, the sPLA2 family is growing rapidly; therefore, it has been proposed that some of the previously described functions of sPLA2s, particularly type IIA sPLA2, need to be reevaluated, since studies based upon enzyme activities and using inhibitors or antibodies against type IIA sPLA2 may not discriminate these sPLA2s (26Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Here, we report the regulation of PGE2 biosynthesis in rat fibroblastic 3Y1 cells, which have the capacity to express high levels of type IIA sPLA2, cPLA2 and the two COXs and to exhibit both immediate and delayed PGE2 generation in response to appropriate stimuli. Pharmacologic, immunochemical, and genetic studies have provided evidence that both cPLA2 and type IIA sPLA2 are required for COX-2-dependent delayed, whereas cPLA2, but not type IIA sPLA2, is utilized for COX-1-dependent immediate, PGE2biosynthesis by 3Y1 cells. Of particular interest is that functional cPLA2 may be crucial for the subsequent type IIA sPLA2 induction and attendant COX-2-dependent delayed PGE2 generation. Thus, this study (i) reconfirmed the involvement of type IIA sPLA2 in biological responses, (ii) demonstrated significant cross-talk between the two Ca2+-dependent PLA2s where one enzyme is required for the induction of the other, and (iii) revealed segregated coupling of discrete PLA2 and COX enzymes in the different phases of PG biosynthesis. Mouse IL-1β was purchased from Genzyme. Human TNFα was provided by Dr. H. Ishimaru (Asahi Chemical Industry). Rabbit antiserum to human cPLA2 was provided by J. D. Clark (Genetics Institute), mouse cPLA2 cDNA by M. Tsujimoto (RIKEN Institute), rabbit antiserum to mouse COX-1 by W. L. Smith (Michigan State University), mouse COX-2 cDNA and the COX-2 inhibitor NS-398 (42Futaki N. Yoshikawa K. Hamasaka Y. Arai I. Higuchi S. Iizuka H. Otomo S. Gen. Pharmacol. 1993; 24: 105-110Crossref PubMed Scopus (361) Google Scholar) by J. Trzaskos (Merck DuPont), rat type V sPLA2 cDNA by J. A. Tischfield (Indiana University School of Medicine), and mouse β-actin cDNA by J. P. Arm (Harvard Medical School). Mouse COX-1 cDNA, rabbit antiserum to COX-2, arachidonic acid, and the PGE2 enzyme immunoassay kit were purchased from Cayman Chemical. The LOX inhibitors, including nordihydroguaiaretic acid (NDGA; general LOX inhibitor), AA-861 (5-LOX inhibitor), cinnamyl-3,4-dihydroxy-α-cyanocinnamate (12-LOX inhibitor) and 5,8,11,14-eicosatetraynoic acid (15-LOX inhibitor) (43Rao G.N. Glasgow W.C. Eling T.E. Runge M.S. J. Biol. Chem. 1996; 271: 27760-27764Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), were purchased from BIOMOL Research Laboratories. Rat type IIA sPLA2 cDNA (44Komada M. Kudo I. Mizushima H. Kitamura N. Inoue K. J. Biochem. (Tokyo). 1989; 106: 545-547Crossref PubMed Scopus (65) Google Scholar) and rabbit polyclonal antibody against rat type IIA sPLA2 (45Murakami M. Kudo I. Natori Y. Inoue K. Biochim. Biophys. Acta. 1989; 1043: 34-42Crossref Scopus (50) Google Scholar) were prepared as described previously. The cDNA probes for rat cPLA2 and rat type IIC sPLA2 were obtained by the reverse transcriptase-polymerase chain reaction (RT-PCR) using RNA extracted from 3Y1 cells (34Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and rat testis (41Chen J. Engle S.J. Seilhamer J.J. Tischfield J.A. J. Biol. Chem. 1994; 269: 23018-23024Abstract Full Text PDF PubMed Google Scholar), respectively, as described previously. The antisense and sense oligonucleotides and RT-PCR primers were obtained from Greiner Japan. The type IIA sPLA2inhibitor thielocin A1 (35Tanaka K. Kato T. Matsumoto K. Yoshida T. Inflammation. 1993; 17: 107-119Crossref PubMed Scopus (31) Google Scholar, 36Murakami M. Kudo I. Nakamura H. Yokoyama Y. Mori H. Inoue K. FEBS Lett. 1990; 268: 113-116Crossref PubMed Scopus (59) Google Scholar) was donated by Dr. T. Yoshida (Shionogi Pharmaceutical). A23187 and the cPLA2 inhibitor arachidonoyl trifluoromethyl ketone (AACOCF3) (47Street I.P. Lin H.-K. Laliberte F. Ghomashchi F. Wang Z. Perrier H. Tremblay N.M. Huang Z. Weech P.K. Gelb M.H. Biochemistry. 1993; 32: 5935-5940Crossref PubMed Scopus (419) Google Scholar) were purchased from Calbiochem. Aspirin, dexamethasone, and heparin were purchased from Sigma. CellFectin reagent, Opti-MEM medium, and TRIzol reagent were from Life Technologies, Inc. The IL-3-dependent mouse bone marrow-derived mast cell line MC-MKM, which originated from BALB/cJ mice (48Murakami M. Kudo I. Inoue K. Biochim. Biophys. Acta. 1992; 1124: 17-22Crossref PubMed Scopus (11) Google Scholar), was cultured in enriched medium supplemented with 50% (v/v) WEHI-3B-conditioned medium as a source of IL-3 and 100 ng/ml recombinant mouse c-kitligand, which had been expressed using a baculovirus system (2Murakami M. Matsumoto R. Austen K.F. Arm J.P. J. Biol. Chem. 1994; 269: 22269-22275Abstract Full Text PDF PubMed Google Scholar). The 3Y1 cells were a gift from Dr. Y. Uehara (National Institute of Infectious Disease, Tokyo, Japan) and maintained in culture medium composed of Dulbecco's modified Eagle's medium (DMEM; Nissui Pharmaceutical) supplemented with 10% (v/v) fetal calf serum (FCS), penicillin/streptomycin (100 units/ml and 100 μg/ml, respectively) (Flow Laboratories) and 2 mmglutamine (Life Technologies, Inc.). The media of 3Y1 cells that had attained 60–80% confluence in six-well plates (Iwaki) were replaced with 2 ml of DMEM supplemented with 2% FCS. After culture for 24 h, 1 ng/ml IL-1β, 100 units/ml human TNFα, or both were added to the cultures to assess the delayed response. Replicated cells were activated with 1 μm A23187 for 10 min in DMEM supplemented with 2% FCS to assess the immediate response. The supernatants were taken for PGE2 enzyme immunoassay. For immunoblot analysis and the PLA2 assay (see below), the cells were trypsinized, washed once with 10 mm phosphate buffer, pH 7.4, containing 150 mm NaCl (phosphate-buffered saline), resuspended in 10 mm Tris-HCl (pH 7.4) containing 1 mm EDTA and 250 mmsucrose at 1 × 107 cells/ml, and disrupted by 1-min pulse sonication (50% work cycle, setting 4) with a Branson Sonifier (Branson Sonic Power Co.). As for the RNA blot analysis, TRIzol was directly added to the cell monolayer. All the procedures were performed as described elsewhere (34Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Briefly, equal amounts (10 μg) of total RNA, purified using TRIzol reagent, 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 sequentially probed with cPLA2, type IIA sPLA2, COX-2, and β-actin cDNA probes that had been labeled with [32P]dCTP (Amersham Life Science) by random priming (Takara Biomedicals). All hybridizations were carried out at 42 °C overnight in a solution comprising 50% (v/v) formamide, 0.75m NaCl, 75 mm sodium citrate, 0.1% (w/v) SDS, 1 mm EDTA, 10 mm sodium phosphate, pH 6.8, 5 × Denhardt's solution (Sigma), 10% (w/v) dextran sulfate (Sigma), and 100 μg/ml salmon sperm DNA (Sigma). The membranes were washed three times at room temperature with 150 mm NaCl, 15 mm sodium citrate, 1 mm EDTA, 0.1% SDS, and 10 mm sodium phosphate, pH 6.8, for 5 min each, followed by two washes at 55 °C with 30 mm NaCl, 3 mmsodium citrate, 1 mm EDTA, 0.1% SDS, and 10 mmsodium phosphate, pH 6.8, for 15 min each. The blots were visualized by autoradiography with Kodak X-Omat AR films and double intensifying screens at −80 °C. The relative amount of each transcript was estimated by quantitating the associated radioactivity using a BAS-III bioimaging analyzer (Fuji Film). The -fold increase in steady-state mRNA was calculated as the ratio of radioactivity associated with a specific transcript in treated cells compared with that in untreated cells and was corrected for changes in steady-state levels of β-actin transcript to adjust for differences in loading between lanes. Specific primers for the PCR, based on the sequences of sPLA2s reported previously (38Chen J. Engle S.J. Seilhamer J.J. Tischfield J.A. J. Biol. Chem. 1994; 269: 2365-2368Abstract Full Text PDF PubMed Google Scholar, 41Chen J. Engle S.J. Seilhamer J.J. Tischfield J.A. J. Biol. Chem. 1994; 269: 23018-23024Abstract Full Text PDF PubMed Google Scholar, 45Murakami M. Kudo I. Natori Y. Inoue K. Biochim. Biophys. Acta. 1989; 1043: 34-42Crossref Scopus (50) Google Scholar), were synthesized. The type IIA sPLA2 primers used were 5′-ATG AAG GTC CTC CTC CTG CTA G-3′ and 5′-TCA GCA TTT GGG CTT CTT CC-3′ (45Murakami M. Kudo I. Natori Y. Inoue K. Biochim. Biophys. Acta. 1989; 1043: 34-42Crossref Scopus (50) Google Scholar), type IIC sPLA2 primers were 5′-ATG GAC CTC CTG GTC TCC TCA GG-3′ and 5′-CTA GCA ATG AGT TTG TCC CTG C-3′ (41Chen J. Engle S.J. Seilhamer J.J. Tischfield J.A. J. Biol. Chem. 1994; 269: 23018-23024Abstract Full Text PDF PubMed Google Scholar), and type V sPLA2 primers were 5′-CAG GGG GCT TGC TAG AAC TCA A-3′ and 5′-AAG AGG GTT GTA AGT CCA GAG G-3′ (38Chen J. Engle S.J. Seilhamer J.J. Tischfield J.A. J. Biol. Chem. 1994; 269: 2365-2368Abstract Full Text PDF PubMed Google Scholar). The RT-PCR was carried out using a RNA PCR kit (avian myeloblastosis virus) version-2 (Takara Biomedicals), according to the manufacturer's instructions, using 1 μg of total RNA from IL-1β/TNFα-treated 3Y1 or mast cells as a template. Equal amounts of each RT product were amplified by PCR withex Taq polymerase (Takara Biomedicals) for 30 cycles consisting of 30 s each at 94 °C, 55 °C and 72 °C. The amplified cDNA fragments were resolved electrophoretically on 1.5% (w/v) agarose gels and visualized by ethidium bromide. The fragments were then transferred onto Immobilon-N membranes and probed with [32P]dCTP-labeled cDNAs for types IIA, IIC, and V sPLA2s. Hybridization and subsequent washing were carried out as described above for RNA blot analysis. All the procedures were performed as described previously (34Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Briefly, cell lysates were subjected to SDS-polyacrylamide gel electrophoresis under nonreducing (for type IIA sPLA2) and reducing (for cPLA2, COX-1, and COX-2) 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 washed once with 10 mmTris-HCl (pH 7.2) containing 150 mm NaCl and 0.1% (v/v) Tween 20 (TBS-T) and then blocked for 1 h in TBS-T containing 3% (w/v) skim milk. After washing the membranes with TBS-T, antibodies against cPLA2, type IIA sPLA2, COX-1, and COX-2 were added at a dilution of 1:5,000 in TBS-T and incubated for 2 h. Following three washes with TBS-T, the membranes were treated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymed Laboratories Inc.; diluted 1:7,000) in TBS-T. After six washes with TBS-T, the protein bands were visualized using an ECL Western blot analysis system (Amersham Life Science). The PLA2 activities in the resulting lysates were measured after incubation with 100 mm Tris-HCl, pH 9.0, 4 mm CaCl2, and 2 μm1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphoethanolamine (Amersham Life Science) or 1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphoethanolamine (NEN Life Science Products), as the substrates, for 30 min at 37 °C (34Murakami M. Nakatani Y. Kudo I. J. Biol. Chem. 1996; 271: 30041-30051Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The antisense (5′-TAG CAA CAG GAG GAC CTT CAT-3′) and sense (5′-ATG AAG GTC CTC CTG TTG CTA-3′) oligonucleotides for rat type IIA sPLA2, corresponding to the translation initiation site, 100 nm each, were incubated individually with CellFectin reagent (Life Technologies, Inc.) in 200 μl of Opti-MEM medium (Life Technologies, Inc.) for 15 min at room temperature and then added to cells that had attained 60–80% confluence in 6-well plates and been supplemented with 800 μl of Opti-MEM. After incubation for 6 h at 37 °C, the medium was replaced with 2 ml of DMEM supplemented with 2% FCS in the continued presence of 100 nmoligonucleotide.

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