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A Novel Role of Group VIB Calcium-independent Phospholipase A2 (iPLA2γ) in the Inducible Expression of Group IIA Secretory PLA2 in Rat Fibroblastic Cells*

  • Hiroshi Kuwata
    Affiliations
    Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
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  • Chikako Fujimoto
    Affiliations
    Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
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  • Emiko Yoda
    Affiliations
    Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
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  • Satoko Shimbara
    Affiliations
    Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
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  • Yoshihito Nakatani
    Affiliations
    Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
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  • Shuntaro Hara
    Affiliations
    Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
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  • Author Footnotes
    1 Supported by Precursory Research for Embryonic Science and Technology from the Japan Science and Technology Agency.
    Makoto Murakami
    Footnotes
    1 Supported by Precursory Research for Embryonic Science and Technology from the Japan Science and Technology Agency.
    Affiliations
    Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan

    Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyou-ku, Tokyo 113-8613, Japan

    Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan
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  • Ichiro Kudo
    Correspondence
    To whom correspondence should be addressed. Tel.: 81-3-3784-8196; Fax: 81-3-3784-8245
    Affiliations
    Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
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  • Author Footnotes
    * This work was supported in part by a Showa University special grant-in-aid for Innovative Collaborative Research Projects, and grants-in-aid for Scientific Research from the Ministry of Education, Science, Culture, Sports, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3 and Table S1.
    1 Supported by Precursory Research for Embryonic Science and Technology from the Japan Science and Technology Agency.
Open AccessPublished:May 01, 2007DOI:https://doi.org/10.1074/jbc.M611883200
      Group IIA secretory phospholipase A2 (sPLA2-IIA) is a prototypic sPLA2 enzyme that may play roles in modification of eicosanoid biosynthesis as well as antibacterial defense. In several cell types, inducible expression of sPLA2 by pro-inflammatory stimuli is attenuated by group IVA cytosolic PLA2 (cPLA2α) inhibitors such as arachidonyl trifluoromethyl ketone, leading to the proposal that prior activation of cPLA2α is required for de novo induction of sPLA2. However, because of the broad specificity of several cPLA2α inhibitors used so far, a more comprehensive approach is needed to evaluate the relevance of this ambiguous pathway. Here, we provide evidence that the induction of sPLA2-IIA by pro-inflammatory stimuli requires group VIB calcium-independent PLA2 (iPLA2γ), rather than cPLA2α, in rat fibroblastic 3Y1 cells. Results with small interfering RNA unexpectedly showed that the cytokine induction of sPLA2-IIA in cPLA2α knockdown cells, in which cPLA2α protein was undetectable, was similar to that in replicate control cells. By contrast, knockdown of iPLA2γ, another arachidonyl trifluoromethyl ketone-sensitive intracellular PLA2, markedly reduced the cytokine-induced expression of sPLA2-IIA. Supporting this finding, the R-enantiomer of bromoenol lactone, an iPLA2γ inhibitor, suppressed the cytokine-induced sPLA2-IIA expression, whereas (S)-bromoenol lactone, an iPLA2β inhibitor, failed to do so. Moreover, lipopolysaccharide-stimulated sPLA2-IIA expression was also abolished by knockdown of iPLA2γ. These findings open new insight into a novel regulatory role of iPLA2γ in stimulus-coupled sPLA2-IIA expression.
      Phospholipases A2 (PLA2)
      The abbreviations used are: PLA2, phospholipase A2; cPLA2, cytosolic PLA2; iPLA2, calcium-independent PLA2; COX, cyclooxygenase; LOX, lipoxygenase; sPLA2, secretory PLA2; IL-1β, interleukin-1β; TNFα, tumor necrosis factor-α; LPS, lipopolysaccharide; PGE2, prostaglandin E2; AACOCF3, arachidonyl trifluoromethyl ketone; Rac-BEL, racemic bromoenol lactone; cPGES, cytosolic PGE synthase; KD, knockdown; siRNA, small interference RNA.
      3The abbreviations used are: PLA2, phospholipase A2; cPLA2, cytosolic PLA2; iPLA2, calcium-independent PLA2; COX, cyclooxygenase; LOX, lipoxygenase; sPLA2, secretory PLA2; IL-1β, interleukin-1β; TNFα, tumor necrosis factor-α; LPS, lipopolysaccharide; PGE2, prostaglandin E2; AACOCF3, arachidonyl trifluoromethyl ketone; Rac-BEL, racemic bromoenol lactone; cPGES, cytosolic PGE synthase; KD, knockdown; siRNA, small interference RNA.
      are a family of enzymes that catalyze the hydrolysis of the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids. These products are metabolized to various bioactive lipid mediators, such as prostaglandins (PG), leukotrienes, and platelet-activating factor. Currently, a number of mammalian PLA2s have been identified and classified into several families, including secretory PLA2 (sPLA2), cytosolic PLA2 (cPLA2), calcium-independent PLA2 (iPLA2), platelet-activating factor acetylhydrolases, and lysosomal PLA2s (
      • Kudo I.
      • Murakami M.
      ,
      • Murakami M.
      • Kudo I.
      ,
      • Six D.A.
      • Dennis E.A.
      ,
      • Schaloske R.H.
      • Dennis E.A.
      ). Although the existence of multiple PLA2 enzymes suggests their diverse functions in the modulation of various pathophysiological conditions, the roles of most PLA2 enzymes are not fully understood until date.
      Among PLA2 enzymes, group IV (cPLA2) and group VI (iPLA2) families represent intracellular enzymes with a catalytic serine in their lipase consensus motif. Various studies, including gene targeting, have revealed that group IVA cPLA2 (cPLA2α), which is regulated by Ca2+-dependent membrane translocation and mitogen-activated protein kinase-dependent phosphorylation, plays a central role in stimulus-dependent eicosanoid biosynthesis (
      • Uozumi N.
      • Kume K.
      • Nagase T.
      • Nakatani N.
      • Ishii S.
      • Tashiro F.
      • Komagata Y.
      • Maki K.
      • Ikuta K.
      • Ouchi Y.
      • Miyazaki J.
      • Shimizu T.
      ,
      • Bonventre J.V.
      • Huang Z.
      • Taheri M.R.
      • O'Leary E.
      • Li E.
      • Moskowitz M.A.
      • Sapirstein A.
      ). Of the iPLA2 family, group VIA iPLA2 (iPLA2β) and group VIB iPLA2 (iPLA2γ) mainly exhibit PLA2 activity, whereas iPLA2δ shows lysophospholipase activity (
      • Quistad G.B.
      • Barlow C.
      • Winrow C.J.
      • Sparks S.E.
      • Casida J.E.
      ) and iPLA2∊ (adiponutrin), iPLA2ζ (adipose triglyceride lipase), and iPLA2η possess triglyceride lipase and transacylase activities in marked preference to PLA2 activity (
      • Jenkins C.M.
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      ). Group VIA iPLA2β, the most extensively studied iPLA2 isozyme, has been implicated in various cellular events such as phospholipid remodeling (
      • Balsinde J.
      • Balboa M.A.
      • Dennis E.A.
      ), eicosanoid formation (
      • Tay H.K.
      • Melendez A.J.
      ), cell proliferation (
      • Herbert S.P.
      • Walker J.H.
      ), apoptosis (
      • Atsumi G.
      • Tajima M.
      • Hadano A.
      • Nakatani Y.
      • Murakami M.
      • Kudo I.
      ), and activation of store-operated channels and capacitative Ca2+ influx (
      • Smani T.
      • Zakharov S.I.
      • Csutora P.
      • Leno E.
      • Trepakova E.S.
      • Bolotina V.M.
      ). Disruption of the iPLA2β gene causes impaired sperm motility (
      • Bao S.
      • Miller D.J.
      • Ma Z.
      • Wohltmann M.
      • Eng G.
      • Ramanadham S.
      • Moley K.
      • Turk J.
      ), mitigated insulin secretion (
      • Bao S.
      • Song H.
      • Wohltmann M.
      • Ramanadham S.
      • Jin W.
      • Bohrer A.
      • Turk J.
      ), and neuronal disorders (
      • Morgan N.V.
      • Westaway S.K.
      • Morton J.E.
      • Gregory A.
      • Gissen P.
      • Sonek S.
      • Cangul H.
      • Coryell J.
      • Canham N.
      • Nardocci N.
      • Zorzi G.
      • Pasha S.
      • Rodriguez D.
      • Desguerre I.
      • Mubaidin A.
      • Bertini E.
      • Trembath R.C.
      • Simonati A.
      • Schanen C.
      • Johnson C.A.
      • Levinson B.
      • Woods C.G.
      • Wilmot B.
      • Kramer P.
      • Gitschier J.
      • Maher E.R.
      • Hayflick S.J.
      ). Group VIB iPLA2γ is a membrane-bound iPLA2 enzyme with some unique features such as utilization of distinct translation initiation sites producing different sizes of enzymes with distinct subcellular localizations (
      • Mancuso D.J.
      • Jenkins C.M.
      • Gross R.W.
      ,
      • Tanaka H.
      • Takeya R.
      • Sumimoto H.
      ,
      • Yang J.
      • Han X.
      • Gross R.W.
      ,
      • Murakami M.
      • Masuda S.
      • Ueda-Semmyo K.
      • Yoda E.
      • Kuwata H.
      • Takanezawa Y.
      • Aoki J.
      • Arai H.
      • Sumimoto H.
      • Ishikawa Y.
      • Ishii T.
      • Nakatani Y.
      • Kudo I.
      ,
      • Kinsey G.R.
      • McHowat J.
      • Beckett C.S.
      • Schnellmann R.G.
      ,
      • Mancuso D.J.
      • Jenkins C.M.
      • Sims H.F.
      • Cohen J.M.
      • Yang J.
      • Gross R.W.
      ) and phospholipid selectivity in terms of sn-1/sn-2 positional specificity that differs among substrates (
      • Yan W.
      • Jenkins C.M.
      • Han X.
      • Mancuso D.J.
      • Sims H.F.
      • Yang K.
      • Gross R.W.
      ). Although an overexpression study has shown the unique functional coupling of iPLA2γ with cyclooxygenase (COX)-1 for prostanoid production (
      • Murakami M.
      • Masuda S.
      • Ueda-Semmyo K.
      • Yoda E.
      • Kuwata H.
      • Takanezawa Y.
      • Aoki J.
      • Arai H.
      • Sumimoto H.
      • Ishikawa Y.
      • Ishii T.
      • Nakatani Y.
      • Kudo I.
      ), the true cellular functions of iPLA2γ yet remain unknown.
      The sPLA2 family represents an evolutionally conserved group of low molecular weight PLA2 enzymes that have a catalytic histidine residue adjacent to a Ca2+-binding loop and are typically disulfide-rich. Group IIA sPLA2 (sPLA2-IIA) is a prototypic inducible sPLA2 isozyme that is often highly elevated in the circulation and locally in the tissues associated with various pathological situations such as rheumatoid arthritis, sepsis, and atherosclerosis (
      • Seilhamer J.J.
      • Pruzanski W.
      • Vadas P.
      • Plant S.
      • Miller J.A.
      • Kloss J.
      • Johnson L.K.
      ,
      • Menschikowski M.
      • Kasper M.
      • Lattke P.
      • Schiering A.
      • Schiefer S.
      • Stockinger H.
      • Jaross W.
      ,
      • Guidet B.
      • Piot O.
      • Masliah J.
      • Barakett V.
      • Maury E.
      • Bereziat G.
      • Offenstadt G.
      ,
      • Kramer R.M.
      • Hession C.
      • Johansen B.
      • Hayes G.
      • McGray P.
      • Chow E.P.
      • Tizard R.
      • Pepinsky R.B.
      ). Consequently, it is thought that sPLA2-IIA participates in the progression of various pro-inflammatory reactions by modifying eicosanoid biosynthesis. In addition, recent evidence has also shown that sPLA2-IIA is abundantly present in extracellular fluids such as tears and seminal plasma and has potent antibacterial activity in vitro and in vivo, thereby contributing to the first line of antibacterial defense of the host independent to eicosanoid production (
      • Weiss J.
      • Inada M.
      • Elsbach P.
      • Crowl R.M.
      ,
      • Piris-Gimenez A.
      • Paya M.
      • Lambeau G.
      • Chignard M.
      • Mock M.
      • Touqui L.
      • Goossens P.L.
      ,
      • Koduri R.S.
      • Gronroos J.O.
      • Laine V.J.
      • Le Calvez C.
      • Lambeau G.
      • Nevalainen T.J.
      • Gelb M.H.
      ,
      • Qu X.D.
      • Lehrer R.I.
      ).
      Although the expression of sPLA2-IIA is up-regulated in a wide variety of cells and tissues following pro-inflammatory stimuli, such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), and lipopolysaccharide (LPS) (
      • Oka S.
      • Arita H.
      ,
      • Crowl R.M.
      • Stoller T.J.
      • Conroy R.R.
      • Stoner C.R.
      ,
      • Akiba S.
      • Hatazawa R.
      • Ono K.
      • Kitatani K.
      • Hayama M.
      • Sato T.
      ,
      • Pfeilschifter J.
      • Schalkwijk C.
      • Briner V.A.
      • van den Bosch H.
      ), the mechanisms leading to sPLA2-IIA induction by particular stimuli appear to be diverse (
      • Menschikowski M.
      • Hagelgans A.
      • Siegert G.
      ). In some cases, attenuation of stimulus-dependent expression of sPLA2-IIA (or related isozymes such as sPLA2-V) by cPLA2α inhibitors, such as arachidonyl trifluoromethyl ketone (AACOCF3), has been found in several cell types, leading to the proposal that prior activation of cPLA2α is required for optimal induction of sPLA2 (
      • Kuwata H.
      • Nakatani Y.
      • Murakami M.
      • Kudo I.
      ,
      • Couturier C.
      • Brouillet A.
      • Couriaud C.
      • Koumanov K.
      • Bereziat G.
      • Andreani M.
      ,
      • Beck S.
      • Lambeau G.
      • Scholz-Pedretti K.
      • Gelb M.H.
      • Janssen M.J.
      • Edwards S.H.
      • Wilton D.C.
      • Pfeilschifter J.
      • Kaszkin M.
      ,
      • Ni Z.
      • Okeley N.M.
      • Smart B.P.
      • Gelb M.H.
      ,
      • Shinohara H.
      • Balboa M.A.
      • Johnson C.A.
      • Balsinde J.
      • Dennis E.A.
      ). However, because it is now clear that AACOCF3 and related compounds, which have been used frequently to analyze the cellular functions of cPLA2α, can also inhibit several other PLA2 enzymes with a catalytic serine (
      • Ackermann E.J.
      • Conde-Frieboes K.
      • Dennis E.A.
      ,
      • van Tienhoven M.
      • Atkins J.
      • Li Y.
      • Glynn P.
      ), we cannot rule out the possibility that some functions of cPLA2α previously described on the basis of inhibition by cPLA2α inhibitors might be attributable to other PLA2 enzymes.
      In an effort to address this question, we examined whether knockdown of particular intracellular PLA2s by respective small interfering RNAs (siRNAs) would affect stimulus-dependent sPLA2-IIA expression in rat fibroblastic 3Y1 cells, in which we have previously shown that the sPLA2-IIA induction is suppressed by AACOCF3 (
      • Kuwata H.
      • Nakatani Y.
      • Murakami M.
      • Kudo I.
      ,
      • Kuwata H.
      • Yamamoto S.
      • Takekura A.
      • Murakami M.
      • Kudo I.
      ,
      • Kuwata H.
      • Yamamoto S.
      • Miyazaki Y.
      • Shimbara S.
      • Nakatani Y.
      • Suzuki H.
      • Ueda N.
      • Yamamoto S.
      • Murakami M.
      • Kudo I.
      ). Our results indicate that iPLA2γ, rather than cPLA2α, participates in the regulation of sPLA2-IIA induction in IL-1β/TNFα- and LPS-treated 3Y1 cells, and that cPLA2α is involved in delayed PGE2 biosynthesis through a mechanism independent of sPLA2-IIA regulation. Thus, these findings not only argue against the proposed contribution of cPLA2α to sPLA2-IIA induction but also reveal an unexplored signaling function of iPLA2γ.

      EXPERIMENTAL PROCEDURES

      Materials—Mouse IL-1β and human TNFα were purchased from R & D Systems. AACOCF3; racemic, (S)- and (R)-bromoenol lactone (BEL); and the enzyme immunoassay kit for PGE2 were obtained from Cayman Chemicals. A23187 was purchased from Calbiochem. LPS (Escherichia coli 0111:B4) was purchased from Sigma. Mouse monoclonal antibody against human cPLA2α and goat polyclonal antibody against human COX-2 and human iPLA2β (T-14) were purchased from Santa Cruz Biotechnology. The cDNA and rabbit polyclonal antibody for rat sPLA2-IIA were prepared as described previously (
      • Komada M.
      • Kudo I.
      • Mizushima H.
      • Kitamura N.
      • Inoue K.
      ,
      • Murakami M.
      • Kudo I.
      • Natori Y.
      • Inoue K.
      ). Rabbit polyclonal antibody against rat cytosolic PGE synthase (cPGES; p23) was prepared as described previously (
      • Tanioka T.
      • Nakatani Y.
      • Semmyo N.
      • Murakami M.
      • Kudo I.
      ). For preparing the antibody for iPLA2γ, the N-terminal half (amino acid residues 1–400) of human iPLA2γ was expressed in competent E. coli (BL21(DE3), Invitrogen) as a His6-tagged protein, purified by an Ni2+-chelating column (Qiagen), and then immunized into a rabbit with Freund's complete and then incomplete adjuvants. The specificity of the resulting anti-iPLA2γ antibody was verified by immunoblotting of iPLA2γ protein in several cell lines and mouse tissues as well as by neutralization of the enzymatic activity of the Sf9-expressed iPLA2γ.
      E. Yoda, Y. Nakatani, and I. Kudo, unpublished observation.
      The pRNA-U6.1/Hygro vector was purchased from GenScript Corp. Lipofectamine 2000, CellFECTIN, Opti-MEM medium, TRIzol reagent, hygromycin, and monoclonal antibody against V5-epitope were obtained from Invitrogen. Polymyxin B was purchased from InvivoGen. 3Y1 cells were generously given by Dr. Y. Uehara (National Institute of Infectious Disease, Tokyo). These were maintained in culture medium composed of Dulbecco's modified Eagle's medium (Nissui Pharmaceutical) supplemented with 10% (v/v) fetal calf serum, penicillin/streptomycin (100 units/ml and 100 μg/ml, respectively), and 2 mm glutamine (Invitrogen) at 37 °C in a CO2 incubator flushed with 5% CO2 in humidified air.
      Activation of 3Y1 Cells—The media of 3Y1 cells or its transfectants that had attained 80% confluence in 6-well plates (Iwaki Glass) were replaced with 2 ml of Dulbecco's modified Eagle's medium supplemented with 2% fetal calf serum. After culture for 24 h, 1 ng/ml mouse IL-1β and 2 ng/ml human TNFα or 100 ng/ml LPS were added to the cultures to assess the sPLA2-IIA induction. For RNA blotting (see below), TRIzol reagent was added directly to the cell monolayer. For calcium ionophore stimulation, 1 μm A23187 was added to the cells for 10 min. The culture supernatants were taken for enzyme immunoassay for PGE2, as required.
      RNA Blot Analysis—All blotting procedures were performed as described previously (
      • Kuwata H.
      • Nakatani Y.
      • Murakami M.
      • Kudo I.
      ). Briefly, equal amounts (5 μg) of total RNA were applied to each lane of 1.2% (w/v) formaldehyde-agarose gels, electrophoresed, and then transferred to Immobilon-N membranes (Millipore). The resulting blots were then probed with cDNA inserts and labeled with [32P]dCTP (PerkinElmer Life Sciences) by random priming (Takara Biomedicals). Hybridization was conducted at 42 °C overnight in a solution comprising 50% (v/v) formamide, 0.75 m NaCl, 75 mm sodium citrate, 0.1% (w/v) SDS, 1 mm EDTA, 10 mm sodium phosphate, pH 6.8, 5× Denhardt's solution (Nakarai Tesque), 10% (w/v) dextran sulfate (Sigma), and 100 μg/ml salmon sperm DNA (Wako). 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 mm sodium citrate, 1 mm EDTA, 1% SDS, and 10 mm sodium 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.
      Cloning and Recombinant Expression of iPLA2β—RNA obtained from 3Y1 cells was subjected to reverse transcription using SuperScript III reverse transcriptase (Invitrogen). The cDNA encoding rat iPLA2β was prepared as follows: sense primer (5′-ATG CAG TTC TTT GGA CGC CTC-3′) and antisense primer (5′-GGG AGA TAG CAG CAG CTG GAC-3′) were paired to amplify a 2421-bp cDNA product from the 3Y1 cell-derived cDNA by PCR using high fidelity KOD plus DNA polymerase (Toyobo, 35 cycles at 94 °C for 10 s and 68 °C for 2.5 m). The cDNA insert was further subjected to PCR with ExTaq polymerase (Takara Biomedicals) for 10 min at 68 °C to add an adenosine base to its 3′-ends on both the strands, followed by subcloning into pcDNA3.1/V5/His-TOPO mammalian expression vector (Invitrogen). The plasmid was digested with BamHI/PmeI, and the resulting insert was ligated into pFASTBac1 vector (Invitrogen) at the BamHI/StuI sites. The plasmid was used for bacmid preparation using the Bac-to-Bac Baculovirus Expression System (Invitrogen) according to the manufacturer's instruction, and the purified bacmid was then transfected into Sf9 cells (Invitrogen) with CellFECTIN to produce baculovirus bearing the C-terminally V5-tagged iPLA2β. Amplification of the virus was performed by adding an aliquot of the initial virus pool to fresh, sub-confluent Sf9 cells in 150-mm dishes (Iwaki). After incubation for 48 h at 27 °C, all the infected cells were pelleted by centrifugation, washed with ice-cold saline, resuspended in 500 μl of SET buffer (250 mm sucrose, 10 mm Tris-HCl, pH 7.4, and 1 mm EDTA), sonicated (3 × 10 s bursts) using a BLANSON sonicator at 30% output, and centrifuged at 2300 × g for 5 min. The supernatant was used as an enzyme source for PLA2 enzyme assay (see below). The expression of C-terminally V5-tagged iPLA2β protein in Sf9 cells was assessed by immunoblotting with anti-V5 antibody (supplemental Fig. S1).
      Cloning and Recombinant Expression of iPLA2γ—Total RNA from mouse brain was subjected to a reverse transcription reaction using an RNA PCR kit (avian myeloblastosis virus, Takara Biomedicals). The obtained cDNA was subjected to PCR using mouse iPLA2γ sense (5′-TGG AAT GAG AGA ATG TCA CAT AG-3′) and antisense (5′-TCA CAA TTT TGA AAA AAA TGG AAG GCC-3′) primers (35 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 m). The PCR product was subcloned into pCR3.1 mammalian expression vector (Invitrogen). The insert was digested out and subcloned into pFASTBac1 vector at KpnI/NotI sites. Baculovirus expressing recombinant iPLA2γ and the homogenate of the virus-infected Sf9 cells were prepared as described above. The expression of iPLA2γ protein was assessed by immunoblotting with anti-iPLA2γ antibody described above (supplemental Fig. S1).
      Establishment of Cell Lines Expressing cPLA2α- or iPLA2γ-directed siRNA—Synthesized oligonucleotides (BEX) with the target sequences for cPLA2α and iPLA2γ siRNAs were each subcloned into pRNA-U6.1/Hygro (GenScript Corp.) at the HindIII/BamHI sites. The sequences of the oligonucleotides used were as follows: 5′-GAT CCC GCA TCA ACT TCA GAA AGT ACT TCA AGA GAG TAC TTT CTG AAG TTG ATG TTT TTT GGA AA-3′ and 5′-AGC TTT TCC AAA AAA CAT CAA CTT CAG AAA GTA CTC TCT TGA AGT ACT TTC TGA AGT TGA TGC GG-3′ for cPLA2α; and 5′-GAT CCC GAT AGA CAG CTT CAG GAC TTC AAG AGA GTC CTG AAG CTG TCT ATC TTT TTT GGA AA-3′ and 5′-AGC TTT TCC AAA AAA GAT AGA CAG CTT CAG GAC TCT CTT GAA GTC CTG AAG CTG TCT ATC GG-3′ for iPLA2γ. The plasmids were transfected into 3Y1 cells with Lipofectamine 2000 according to the manufacturer's instructions. Three days after transfection, the cells were seeded into 96-well plates in the presence of 100 μg/ml hygromycin to establish stable transfectants. The expression levels of cPLA2α and iPLA2γ in the obtained transfectants in comparison with those in mock-transfected control cells were assessed by RNA blotting and immunoblotting.
      Immunoblot Analysis—Cell lysates (105 cells equivalent) were subjected to SDS-PAGE using 10% (w/v) gels under reducing conditions (for cPLA2α, iPLA2γ, and COX-2) or 15% (w/v) gels under non-reducing conditions (for sPLA2-IIA and cPGES). The separated proteins were electroblotted onto nitrocellulose membranes (Schleicher & Schuell) with a semidry blotter (Bio-Rad) according to the manufacturer's instructions. After blocking for 2 h with 3% (w/v) skimmed milk in 10 mm Tris-HCl, pH 7.4, containing 150 mm NaCl and 0.05% Tween 20, the membranes were probed for 2 h with the respective antibodies (1:5,000 for sPLA2-IIA, iPLA2γ, COX-2, and cPGES and 1:2,000 for cPLA2α), followed by incubation with horseradish peroxidase-conjugated anti-mouse (1:2,000 for cPLA2α), anti-rabbit (1:5,000 for sPLA2-IIA, cPGES, and iPLA2γ), or anti-goat (1:10,000 for COX-2) IgG. After being washed, the membranes were visualized with Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences), as described previously (
      • Murakami M.
      • Nakatani Y.
      • Kudo I.
      ).
      Measurement of iPLA2 Activity—Appropriate amounts of the enzyme preparations were preincubated with various concentrations of PLA2 inhibitors on ice for 20 min, and then incubated with 2 μm 1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphoethanolamine (Amersham Biosciences) in 100 mm Tris-HCl, pH 8.0, containing 1 mm EGTA for 10 min at 37 °C. The released [14C]linoleic acid was extracted by Dole's reagent, and radioactivity was counted, as described previously (
      • Hamaguchi K.
      • Kuwata H.
      • Yoshihara K.
      • Masuda S.
      • Shimbara S.
      • Ohishi S.
      • Murakami M.
      • Kudo I.
      ).

      RESULTS

      Analyses with Group IVA cPLA2α Knockdown 3Y1 Cells—Our previous studies have shown that, in 3Y1 cells, IL-1β/TNFα-stimulated induction of sPLA2-IIA is markedly reduced by AACOCF3, a cPLA2α inhibitor (
      • Kuwata H.
      • Nakatani Y.
      • Murakami M.
      • Kudo I.
      ,
      • Kuwata H.
      • Yamamoto S.
      • Takekura A.
      • Murakami M.
      • Kudo I.
      ,
      • Kuwata H.
      • Yamamoto S.
      • Miyazaki Y.
      • Shimbara S.
      • Nakatani Y.
      • Suzuki H.
      • Ueda N.
      • Yamamoto S.
      • Murakami M.
      • Kudo I.
      ). To address whether cPLA2α is involved in the cytokine-dependent sPLA2-IIA induction or the previous observations merely reflect the nonspecific effect of AACOCF3, we first established cPLA2α-knockdown (cPLA2α-KD) 3Y1 cells by using an siRNA strategy. Fig. 1A depicts the expression levels of several constitutively expressed arachidonate-metabolizing enzymes, including cPLA2α, COX-1, and cPGES, in cells stably transfected with cPLA2α-specific siRNA or with mock vector, as assessed by immunoblotting. The expression of cPLA2α protein disappeared to an undetectable level in cPLA2α-KD cells, whereas the expression levels of COX-1 and cPGES were similar in both the cells (Fig. 1A). When these cells were incubated for 10 min with the calcium ionophore A23187, the immediate PGE2 biosynthesis was nearly absent in cPLA2α-KD cells as compared with those in mock-transfectants in which robust PGE2 synthesis was evident (Fig. 1B), thus substantiating our previous studies employing the cPLA2α inhibitor (
      • Kuwata H.
      • Nakatani Y.
      • Murakami M.
      • Kudo I.
      ,
      • Kuwata H.
      • Nonaka T.
      • Murakami M.
      • Kudo I.
      ). We also found that IL-1β/TNFα stimulated delayed PGE2 biosynthesis, which we had proposed to be mediated by synergistic action of cPLA2α and sPLA2-IIA on the basis of the inhibitory effects of AACOCF3 and sPLA2-IIA-directed antisense oligonucleotide, respectively (
      • Kuwata H.
      • Nakatani Y.
      • Murakami M.
      • Kudo I.
      ), was decreased by ∼60% at 24 h and ∼40% at 48 h in cPLA2α-KD cells as compared with that in the control cells (Fig. 1C). These results support the notion that cPLA2α contributes almost entirely to the A23187-elicited immediate and partially to the cytokine-induced delayed PGE2-synthetic responses. Of note, relative contribution of cPLA2α to the cytokine-induced response was greater during the first 24 h than after 24–48 h (Fig. 1C), suggesting that the later phase of the delayed response depends more on the cPLA2α-independent, probably sPLA2-IIA-mediated (as sPLA2-IIA induction reaches a maxima over this period (
      • Kuwata H.
      • Nakatani Y.
      • Murakami M.
      • Kudo I.
      )), PGE2 synthesis in this setting.
      Figure thumbnail gr1
      FIGURE 1PGE2 production and sPLA2-IIA expression in cPLA2α knockdown 3Y1 cells. A, expression of cPLA2α, COX-1, and cPGES proteins in cPLA2α-siRNA transfectants (cPLA2α-KD, right lane) and control cells (mock, left lane) was assessed by immunoblotting. B, effect of cPLA2α knockdown on immediate PGE2 biosynthesis. Mock or cPLA2α-KD cells were stimulated with 1 μm A23187 for 10 min, and the supernatants were taken for PGE2 enzyme immunoassay. Values are means ± S.E. of four independent experiments (*, p < 0.01). C, effect of cPLA2α knockdown on delayed PGE2 biosynthesis. Mock or cPLA2α-KD cells were stimulated with or without IL-1β/TNFα for indicated periods, and the supernatants were taken for PGE2 enzyme immunoassay. Values are means ± S.E. of four independent experiments (*, p < 0.05 versus IL-1β/TNFα-stimulated control cells).D, effect of cPLA2α knockdown on IL-1β/TNFα-induced expression of sPLA2-IIA mRNA. Mock or cPLA2-KD cells were cultured for 24 h with (+) or without (–) IL-1β/TNFα in the presence (+) or absence (–) of 10 μm AACOCF3. Equal loading of samples in each lane was verified by staining of rRNA with ethidium bromide in agarose gels. Result is a representative of three independent experiments.
      Unexpectedly, however, when cPLA2α-KD cells were cultured for 24 h with IL-1β/TNFα, the inducible expression of sPLA2-IIA mRNA was comparable to that in replicate mocktransfectants (Fig. 1D, lanes 1, 2, 4, and 5). In addition, we found that the sPLA2-IIA mRNA induction in both the cPLA2α-KD and control cells was attenuated equally by the addition of the cPLA2α inhibitor AACOCF3 (Fig. 1D, lanes 3 and 6). Upon incubation with various doses of AACOCF3, the suppression of cytokine-dependent sPLA2-IIA induction at each dose was comparable between these cells (data not shown). These results indicate that AACOCF3 does not exert a suppressive effect on sPLA2-IIA induction by inhibiting cPLA2α but, rather, raise the possibility that certain AACOCF3-sensitive PLA2 enzyme(s) other than cPLA2α may participate in the regulation of this process in IL-1β/TNFα-treated 3Y1 cells.
      Pharmacological Inhibition of iPLA2γ Attenuates Cytokine-dependent sPLA2-IIA Induction—Because AACOCF3 is known to inhibit several enzymes, including group VIA calcium-independent PLA2β (iPLA2β) (
      • Ackermann E.J.
      • Conde-Frieboes K.
      • Dennis E.A.
      ,
      • van Tienhoven M.
      • Atkins J.
      • Li Y.
      • Glynn P.
      ), we suspected that the enzymes in the iPLA2 family could be involved in the regulation of cytokine-dependent sPLA2-IIA expression. To clarify this hypothesis, we next examined the effects of racemic BEL (Rac-BEL), a catalytic site-directed inhibitor of iPLA2 activity (
      • Ackermann E.J.
      • Conde-Frieboes K.
      • Dennis E.A.
      ,
      • Hazen S.L.
      • Zupan L.A.
      • Weiss R.H.
      • Getman D.P.
      • Gross R.W.
      ), on A23187-induced immediate and IL-1β/TNFα-induced delayed PGE2 generation as well as on the cytokine-mediated sPLA2-IIA induction. As shown in Fig. 2A, when 3Y1 cells were stimulated with A23187 for 10 min in the presence or absence of 10 μm Rac-BEL, the immediate, cPLA2α-mediated (Fig. 1B) PGE2 synthesis by Rac-BEL-treated cells did not differ significantly from that by the replicate cells without the inhibitor. A23187-induced immediate PGE2 release was markedly attenuated by AACOCF3 (Fig. 2A and Ref.
      • Kuwata H.
      • Nakatani Y.
      • Murakami M.
      • Kudo I.
      ). These results suggest that Rac-BEL failed to inhibit cPLA2α at the concentration used and further support our conclusion that the A23187-elicited response depends entirely on cPLA2α but not on iPLA2s.
      Figure thumbnail gr2
      FIGURE 2Effects of intracellular PLA2 inhibitors on immediate (A) and delayed (B) PGE2 biosynthesis and on sPLA2-IIA expression (C) in 3Y1 cells. A and B, 3Y1 cells were stimulated for 10 min with A23187 (A) or for 24 h with IL-1β/TNFα (B) in the presence or absence of 10 μm AACOCF3 or 10 μm Rac-BEL. The supernatants were taken for PGE2 enzyme immunoassay. C, 3Y1 cells were stimulated for 24 h with (+) or without (–) IL-1β/TNFα in the presence or absence of the indicated concentration of Rac-BEL. The expression of sPLA2-IIA, COX-2, and cPGES proteins was assessed by immunoblotting. Equal loading of samples in each lane was verified by immunoblotting with cPGES-specific antibody. Values are means ± S.E. of three independent experiments in A and B.A representative result of three independent experiments is shown in C.
      On the other hand, IL-1β/TNFα-dependent delayed PGE2 biosynthesis was inhibited not only by AACOCF3 but also by Rac-BEL (Fig. 2B). When 3Y1 cells were cultured with IL-1β/TNFα for 24 h in the presence or absence of various concentrations of Rac-BEL, the sPLA2-IIA induction was dose-dependently suppressed by Rac-BEL, whereas the expression of COX-2 was slightly increased (rather than inhibited) in the presence of the inhibitor, implying that some iPLA2 enzymes might be involved in sPLA2-IIA expression (Fig. 2C). Because attenuation of sPLA2-IIA expression by its specific antisense oligonucleotide led to a concomitant reduction of the delayed PGE2 biosynthesis (
      • Kuwata H.
      • Nakatani Y.
      • Murakami M.
      • Kudo I.
      ), we reasoned that the suppression of the PGE2 biosynthesis by Rac-BEL might result mainly from the reduction of sPLA2-IIA expression.
      The above observations that AACOCF3 and Rac-BEL have similar abilities to inhibit sPLA2-IIA expression prompted us to investigate whether iPLA2 enzymes, which are sensitive to both inhibitors, would regulate sPLA2-IIA expression. To clarify which iPLA2 isozymes are endogenously expressed in 3Y1 cells, reverse transcription-PCR was conducted using primers specific for six isoforms of iPLA2 (β, γ, δ, ∊, ζ, and η). After 35 cycles of amplification with primers listed under “Experimental Procedures” and supplemental Table S1, clear signals for iPLA2β, iPLA2γ, iPLA2δ, iPLA2ζ, and iPLA2η, as well as a weak but obvious signal for iPLA2∊, were detected in unstimulated 3Y1 cells (supplemental Fig. S2). None of the expression of these enzymes increased after cytokine stimulation (data not shown). Given the reported substrate specificity for each iPLA2 member (
      • Quistad G.B.
      • Barlow C.
      • Winrow C.J.
      • Sparks S.E.
      • Casida J.E.
      ,
      • Jenkins C.M.
      • Mancuso D.J.
      • Yan W.
      • Sims H.F.
      • Gibson B.
      • Gross R.W.
      ), iPLA2β and iPLA2γ are the dominant iPLA2 enzymes with a main “PLA2” activity in 3Y1 cells. As shown in Fig. 3A, expression of iPLA2β and iPLA2γ mRNAs was readily detected in 3Y1 cells by RNA blotting.
      Figure thumbnail gr3
      FIGURE 3Detection of iPLA2 isozymes in 3Y1 cells (A) and effect of AACOCF3 on recombinant iPLA2 activities (B). A, expression of iPLA2β (left lane) and iPLA2γ (right lane) in 3Y1 cells was assessed by RNA blotting. A representative result of two independent experiments is shown. B, recombinant iPLA2s expressed in Sf9 cells were preincubated for 20 min with the indicated concentrations of AACOCF3, and then their enzymatic activities were assessed, as described under “Experimental Procedures.” Residual PLA2 activities (percent relative to the activity without AACOCF3) are shown. Values are means ± S.E. (*, p < 0.05 versus iPLA2β activity) of three independent experiments.
      We then examined the inhibitory effects of AACOCF3 on the enzymatic activities of recombinant iPLA2β and iPLA2γ, which were expressed in Sf9 cells by the baculovirus expression system (supplemental Fig. S1). As shown in Fig. 3B, both the enzymes were potently inhibited by AACOCF3 in a dose-dependent manner, with iPLA2γ showing a slightly higher sensitivity than iPLA2β (IC50 of ∼0.4 and 1.2 μm, respectively).
      A previous inhibition experiment with enantiomers of BEL showed that (R)- and (S)-BEL are potent inhibitors of iPLA2γ and iPLA2β, respectively (
      • Jenkins C.M.
      • Han X.
      • Mancuso D.J.
      • Gross R.W.
      ). We confirmed the selectivity of these stereo-specific inhibitors on Sf-9-expressed iPLA2β and iPLA2γ:IC50 values of (S)-BEL against iPLA2β and iPLA2γ were ∼0.2 and 1 μm, respectively, and those of (R)-BEL were ∼1 and 0.1 μm, respectively (Fig. 4A). We then examined the effects of (R)- and (S)-BEL on IL-1β/TNFα-dependent sPLA2-IIA induction. As shown in Fig. 4B, the induction of sPLA2-IIA, but not COX-2, was dose-dependently suppressed in (R)-BEL-treated cells (Fig. 4B, lanes 5 and 6). By contrast, treatment with (S)-BEL had little effect on sPLA2-IIA expression, but rather tended to increase COX-2 expression (Fig. 4B, lanes 3 and 4). Thus, the inhibitory effect of (R)-BEL on sPLA2-IIA expression (Fig. 4B) was similar to that of Rac-BEL (Fig. 2C).
      Figure thumbnail gr4
      FIGURE 4Effect of (S)- or (R)-BEL on IL-1β/TNFα-induced sPLA2-IIA expression in 3Y1 cells. A, recombinant iPLA2s expressed in Sf9 cells were preincubated for 20 min with the indicated concentrations of (S)- or (R)-BEL, and then their enzymatic activities were assessed as described under “Experimental Procedures.” Residual PLA2 activities (percent relative to the activity without inhibitors) are shown. B, 3Y1 cells were cultured for 24 h with or without IL-1β/TNFα in the presence or absence of the indicated concentrations of (S)-BEL or (R)-BEL. The expression of sPLA2-IIA, COX-2, and cPGES was assessed by immunoblotting. Equal loading of samples in each lane was verified by immunoblotting with cPGES-specific antibody. Representative results of two reproducible experiments are shown.
      siRNA-mediated Knockdown of iPLA2γ Attenuates Cytokine-dependent sPLA2-IIA Induction—Although the above data suggest a vital role of iPLA2γ in the regulation of IL-1β/TNFα-dependent sPLA2-IIA expression, BEL is also known to inhibit several enzymes other than iPLA2 enzymes (
      • Balsinde J.
      • Dennis E.A.
      ), and, therefore, the observed inhibitory effects of the (R)-enantiomer of BEL on sPLA2-IIA induction (Fig. 4B) might merely reflect an event that occurred independently of iPLA2 inhibition. To exclude this possibility and corroborate the pharmacological studies shown above, we aimed to knockdown the expression of iPLA2γ with an iPLA2γ-targeted siRNA. The siRNA treatment resulted in >70% reduction of iPLA2γ expression at both mRNA and protein levels in comparison with those in the control (mock) cells (Fig. 5A). This iPLA2γ siRNA effect was specific, because it did not affect the expression of cPLA2α and iPLA2β (Fig. 5A, right panel). Concomitantly, the knockdown of iPLA2γ reduced the IL-1β/TNFα-induced expression of sPLA2-IIA mRNA and protein by >70% relative to the replicate control cells (Fig. 5B), consistent with the inhibitor studies (Figs. 2, 3, 4). Similar results were observed in the transient transfection study of iPLA2γ siRNA (supplemental Fig. S3). Thus, we conclude that iPLA2γ is a prerequisite signaling component for IL-1β/TNFα-dependent sPLA2-IIA induction in 3Y1 cells.
      Figure thumbnail gr5
      FIGURE 5Knockdown of iPLA2γ attenuates IL-1β/TNFα-induced sPLA2-IIA expression. A, expression of iPLA2γ transcripts (left panel) and iPLA2γ, cPLA2α, and iPLA2β proteins (right panel) in iPLA2γ siRNA transfectants (iPLA2γ-KD, right lane) and control cells (mock, left lane) were assessed by RNA blotting and immunoblotting, respectively. B, mock or iPLA2γ-KD cells were cultured for 24 h with (+) or without (–) IL-1β/TNFα, and the expression of sPLA2-IIA mRNA (left panel) or protein (right panel) was assessed. Equal loading of samples in each lane was verified by staining of rRNA with ethidium bromide in agarose gels (A and B, left panels) or immunoblotting with anti-cPLA2α (A, right panel) and anti-cPGES (B, right panel) antibodies. Representative results of three independent experiments are shown.
      Involvement of iPLA2γ in LPS-induced sPLA2-IIA Expression—To better understand the general role of iPLA2γ in inducible sPLA2-IIA expression, we next examined whether siRNA-directed down-regulation of iPLA2γ would affect LPS-stimulated sPLA2-IIA expression. The LPS receptor, Toll-like receptor 4, was detected in 3Y1 cells by reverse transcription-PCR (data not shown). Treatment of 3Y1 cells with LPS markedly up-regulated sPLA2-IIA expression, and this level was comparable to that elicited by IL-1β/TNFα (Fig. 6A, lanes 2 and 5). The sPLA2-IIA expression induced by LPS was blocked completely by treatment with polymyxin B, an LPS inhibitor (Fig. 6A, lanes 3 and 6), revealing LPS specificity. The LPS-dependent induction of sPLA2-IIA expression was suppressed by addition of AACOCF3 (Fig. 6B, lanes 1–3). Furthermore, LPS-induced sPLA2-IIA expression was markedly decreased in iPLA2γ-KD cells as compared with that in the control cells (Fig. 6, lanes 4–6). The residual expression of sPLA2-IIA in the KD cells was sensitive to AACOCF3 (Fig. 6, lanes 5 and 6), likely because a substantial level of iPLA2γ remained in the KD cells (Fig. 5A). These results suggest that the activation of iPLA2γ following pro-inflammatory signaling commonly evoked by LPS and cytokines is crucial for subsequent sPLA2-IIA induction in 3Y1 cells.
      Figure thumbnail gr6
      FIGURE 6Knockdown of iPLA2γ attenuates LPS-induced sPLA2-IIA expression. A, 3Y1 cells were cultured for 24 h with or without IL-1β/TNFα (lanes 1–3) or 100 ng/ml LPS (lanes 4–6) in the presence or absence of the 10 μg/ml polymyxin B. B, mock- or iPLA2γ-KD cells were cultured for 24 h with (+) or without (–) LPS in the presence or absence of 10 μm AACOCF3, and the expression of sPLA2-IIA and cPGES was assessed by immunoblotting. Representative results of three independent experiments are shown.

      DISCUSSION

      Numerous experimental studies, both in vitro and in vivo, have revealed that cPLA2α, an intracellular Ca2+-dependent enzyme that exhibits arachidonic acid preference, is the most important PLA2 enzyme involved in stimulus-coupled production of bioactive lipid mediators (
      • Uozumi N.
      • Kume K.
      • Nagase T.
      • Nakatani N.
      • Ishii S.
      • Tashiro F.
      • Komagata Y.
      • Maki K.
      • Ikuta K.
      • Ouchi Y.
      • Miyazaki J.
      • Shimizu T.
      ,
      • Bonventre J.V.
      • Huang Z.
      • Taheri M.R.
      • O'Leary E.
      • Li E.
      • Moskowitz M.A.
      • Sapirstein A.
      ,
      • Uozumi N.
      • Shimizu T.
      ). Not only does cPLA2α directly supply arachidonic acid to COX and lipoxygenase (LOX) enzymes, but also it has been proposed to modulate eicosanoid production in cooperation with sPLA2s (
      • Mounier C.M.
      • Ghomashchi F.
      • Lindsay M.R.
      • James S.
      • Singer A.G.
      • Parton R.G.
      • Gelb M.H.
      ,
      • Kim Y.J.
      • Kim K.P.
      • Han S.K.
      • Munoz N.M.
      • Zhu X.
      • Sano H.
      • Leff A.R.
      • Cho W.
      ). The concept that the inducible expression of sPLA2s is often regulated by cPLA2α has relied mostly on the observations that several cPLA2α inhibitors so far available could considerably block the expression of sPLA2s (
      • Kuwata H.
      • Nakatani Y.
      • Murakami M.
      • Kudo I.
      ,
      • Couturier C.
      • Brouillet A.
      • Couriaud C.
      • Koumanov K.
      • Bereziat G.
      • Andreani M.
      ,
      • Beck S.
      • Lambeau G.
      • Scholz-Pedretti K.
      • Gelb M.H.
      • Janssen M.J.
      • Edwards S.H.
      • Wilton D.C.
      • Pfeilschifter J.
      • Kaszkin M.
      ,
      • Ni Z.
      • Okeley N.M.
      • Smart B.P.
      • Gelb M.H.
      ,
      • Shinohara H.
      • Balboa M.A.
      • Johnson C.A.
      • Balsinde J.
      • Dennis E.A.
      ). However, studies using chemical inhibitors should cautiously interpret results because of their uncertain specificity toward various cellular targets. Although LPS-stimulated induction of sPLA2-V in P388D1 cells was reported to be reversed by methylarachidonyl fluorophosphonate, a cPLA2α inhibitor often used in several studies (
      • Shinohara H.
      • Balboa M.A.
      • Johnson C.A.
      • Balsinde J.
      • Dennis E.A.
      ), this scenario was subsequently retracted, because pyrroridine-2, a more specific cPLA2α inhibitor, did not affect the sPLA2-V expression (
      • Kessen U.A.
      • Schaloske R.H.
      • Stephens D.L.
      • Killermann Lucas K.
      • Dennis E.A.
      ). In rat gastric cells, pyrroridine-2 abolished the cytokine-elicited induction of sPLA2-IIA, yet it appeared that marked reduction of cPLA2α expression by its siRNA was accompanied by only a modest reduction of sPLA2-IIA expression (
      • Ni Z.
      • Okeley N.M.
      • Smart B.P.
      • Gelb M.H.
      ), raising the question whether the target of pyrroridine-2 is only cPLA2α or some other unknown targets for this agent exist.
      In the case of rat fibroblastic 3Y1 cells, which have been currently studied, the well known cPLA2α inhibitor AACOCF3 potently inhibits both A23187-induced immediate and cytokine-initiated delayed PGE2-synthetic responses and, more strikingly, it markedly suppresses the cytokine-induced expression of sPLA2-IIA (
      • Kuwata H.
      • Nakatani Y.
      • Murakami M.
      • Kudo I.
      ). These observations, together with our previous finding that the antisense inhibition of sPLA2-IIA expression dramatically reduces the late phase of PGE2 production in these cells (
      • Kuwata H.
      • Nakatani Y.
      • Murakami M.
      • Kudo I.
      ), suggest that cPLA2α, a key regulator of the stimulus-dependent eicosanoid synthesis, is a prerequisite for the cytokine induction of sPLA2-IIA. Moreover, these two enzymes act cooperatively in supplying arachidonic acid for PGE2 generation in the delayed response (
      • Kuwata H.
      • Nakatani Y.
      • Murakami M.
      • Kudo I.
      ). As mentioned above, however, these inhibitor studies alone cannot define whether the activation of cPLA2α is indeed functionally coupled with sPLA2-IIA-directed delayed PG biosynthesis, because AACOCF3 also inhibits several high molecular weight PLA2s, such as other members of the cPLA2 family and iPLA2s, in addition to cPLA2α.
      In an effort to re-evaluate the roles of cPLA2α in stimulus-dependent PGE2 synthesis and sPLA2-IIA induction in 3Y1 cells, using siRNA technique, we found that iPLA2γ, rather than cPLA2α, is profoundly involved in the regulation of sPLA2-IIA induction, and that cPLA2α regulates delayed PGE2 synthesis in a manner independent of sPLA2-IIA induction. Five lines of evidence support this conclusion. First, treatment of cPLA2α-KD 3Y1 cells with IL-1β/TNFα resulted in increased sPLA2-IIA expression that was comparable to that occurred in the replicated control cells. This result clearly indicates that cPLA2α is dispensable for sPLA2-IIA expression. Second, treatment of the cells with (R)-BEL, an iPLA2γ-selective inhibitor, but not (S)-BEL, an iPLA2β-selective one, markedly attenuated IL-1β/TNFα-dependent sPLA2-IIA induction. Third, AACOCF3 dose-dependently suppressed the enzymatic activity of recombinant iPLA2γ (as well as that of iPLA2β, as reported (
      • Ackermann E.J.
      • Conde-Frieboes K.
      • Dennis E.A.
      )) in vitro. Fourth, suppression of endogenous iPLA2γ expression by iPLA2γ-directed siRNA markedly decreased cytokine-induced sPLA2-IIA expression. This again supports that iPLA2γ participates in the regulation of cytokine-dependent sPLA2-IIA expression. Finally, knockdown of cPLA2α by siRNA significantly, even if not entirely, reduced IL-1β/TNFα-induced delayed PGE2 biosynthesis without influencing sPLA2-IIA expression in 3Y1 cells, indicating that cPLA2α promotes delayed PGE2 biosynthesis irrespective of the expression levels of sPLA2-IIA, most likely by supplying arachidonic acid directly to downstream COX-2. Supporting this, double knockdown of cPLA2α and iPLA2γ (which suppresses the sPLA2-IIA induction) markedly attenuated delayed PGE2 biosynthesis (supplemental Fig. S3). Thus, it is likely that the suppression of delayed PGE2 generation by AACOCF3 results from both direct inhibition of cPLA2α-directed PGE2 biosynthesis and indirect abrogation of sPLA2-IIA-mediated PGE2 biosynthesis due to the blockage of iPLA2γ-dependent sPLA2-IIA induction. On the other hand, the role of iPLA2β, an enzyme that has recently been implicated in various biological processes (
      • Balsinde J.
      • Balboa M.A.
      • Dennis E.A.
      ,
      • Tay H.K.
      • Melendez A.J.
      ,
      • Herbert S.P.
      • Walker J.H.
      ,
      • Atsumi G.
      • Tajima M.
      • Hadano A.
      • Nakatani Y.
      • Murakami M.
      • Kudo I.
      ,
      • Smani T.
      • Zakharov S.I.
      • Csutora P.
      • Leno E.
      • Trepakova E.S.
      • Bolotina V.M.
      ), in this process seems minimal, because the iPLA2β-selective inhibitor (S)-BEL failed to decrease sPLA2-IIA expression and because overexpression of iPLA2β did not increase (or even tended to reduce) sPLA2-IIA expression in 3Y1 cells in our preliminary experiment.
      H. Kuwata, C. Fujimoto, M. Murakami, and I. Kudo, unpublished observation.
      iPLA2γ is a membrane-associated iPLA2 isoform that was originally identified from a search of nucleic acid data base as a molecule with a region homology to the catalytic domain of iPLA2β (
      • Mancuso D.J.
      • Jenkins C.M.
      • Gross R.W.
      ,
      • Tanaka H.
      • Takeya R.
      • Sumimoto H.
      ). iPLA2γ has four potential translation initiation sites (
      • Mancuso D.J.
      • Jenkins C.M.
      • Gross R.W.
      ,
      • Mancuso D.J.
      • Jenkins C.M.
      • Sims H.F.
      • Cohen J.M.
      • Yang J.
      • Gross R.W.
      ), which produce distinct sizes of its protein, and the expression patterns of the individual iPLA2γ translation products were unique between cell types (
      • Murakami M.
      • Masuda S.
      • Ueda-Semmyo K.
      • Yoda E.
      • Kuwata H.
      • Takanezawa Y.
      • Aoki J.
      • Arai H.
      • Sumimoto H.
      • Ishikawa Y.
      • Ishii T.
      • Nakatani Y.
      • Kudo I.
      ). Remarkably, iPLA2γ has a mitochondrial localization signal in the N-terminal region and a peroxisomal localization signal near the C terminus, and the 88-kDa full-length and the 63-kDa translation products of iPLA2γ are preferentially distributed in the mitochondria and peroxisome, respectively (
      • Murakami M.
      • Masuda S.
      • Ueda-Semmyo K.
      • Yoda E.
      • Kuwata H.
      • Takanezawa Y.
      • Aoki J.
      • Arai H.
      • Sumimoto H.
      • Ishikawa Y.
      • Ishii T.
      • Nakatani Y.
      • Kudo I.
      ,
      • Kinsey G.R.
      • McHowat J.
      • Beckett C.S.
      • Schnellmann R.G.
      ,
      • Mancuso D.J.
      • Jenkins C.M.
      • Sims H.F.
      • Cohen J.M.
      • Yang J.
      • Gross R.W.
      ). Because our immunoblot analysis showed that 3Y1 cells dominantly express the full-size iPLA2γ protein (
      • Murakami M.
      • Masuda S.
      • Ueda-Semmyo K.
      • Yoda E.
      • Kuwata H.
      • Takanezawa Y.
      • Aoki J.
      • Arai H.
      • Sumimoto H.
      • Ishikawa Y.
      • Ishii T.
      • Nakatani Y.
      • Kudo I.
      ), we speculate that the full-size form may be mainly localized and functions in the mitochondria, where it regulates the stimulus-coupled sPLA2-IIA expression through an unidentified manner, in 3Y1 fibroblasts.
      In addition to the contribution of iPLA2γ to the delayed response as demonstrated in this study, we previously reported that 12/15-LOX, which generates 12(S)- or 15(S)-hydroperoxyeicosatetraenoic acid from free arachidonic acid (
      • Kuhn H.
      • Walther M.
      • Kuban R.J.
      ,
      • Yamamoto S.
      • Suzuki H.
      • Ueda N.
      ) and is capable of oxidizing polyunsaturated fatty acyl chains of phospholipids (
      • Schewe T.
      • Halangk W.
      • Hiebsch C.
      • Rapoport S.M.
      ), also contributes to the regulation of cytokine-dependent sPLA2-IIA expression in 3Y1 cells (
      • Kuwata H.
      • Yamamoto S.
      • Miyazaki Y.
      • Shimbara S.
      • Nakatani Y.
      • Suzuki H.
      • Ueda N.
      • Yamamoto S.
      • Murakami M.
      • Kudo I.
      ). Thus, it appears that the stimulus-dependent production of certain bioactive lipid mediator(s) via the iPLA2γ-12/15-LOX pathway may precede the control of sPLA2-IIA expression. Furthermore, considering that iPLA2γ exhibits either PLA2 or PLA1 activity, depending upon the substrates (
      • Yan W.
      • Jenkins C.M.
      • Han X.
      • Mancuso D.J.
      • Sims H.F.
      • Yang K.
      • Gross R.W.
      ), it could be speculated that certain oxidized fatty acid or lysolipid derivative(s) may act as a second messenger for switching on the transcriptional activation of the sPLA2-IIA gene.
      Studies with knockdown of iPLA2γ revealed that sPLA2-IIA expression following multiple pro-inflammatory stimuli (cytokines and LPS) is regulated by iPLA2γ, suggesting that the signaling pathway involving MyD88, a common adapter molecule for the Toll/IL-1 receptor family, is functionally linked to iPLA2γ activation and thereby sPLA2-IIA expression. The updated signaling module leading to sPLA2-IIA expression, as revealed by our current studies, is illustrated in Fig. 7. Nonetheless, because regulation of the inducible expression of sPLA2-IIA or related isozymes appears to be cell type-specific, efforts to identify specific cell types that exhibit iPLA2γ-dependent regulation of sPLA2s, particularly in vivo, would be important to shed new light on the as yet unclarified pathophysiological roles of sPLA2 family members as well as iPLA2γ enzyme.
      Figure thumbnail gr7
      FIGURE 7The signaling pathway leading to the sPLA2-IIA gene expression in cytokine- and endotoxin-treated rat fibroblastic 3Y1 cells. Proinflammatory cytokines or endotoxins generate certain lipid metabolite(s) via the iPLA2γ-12/15-LOX pathway, which in turn activates the sPLA2-IIA gene expression. Whether iPLA2γ-mediated lipolysis precedes 12/15-LOX or 12/15-LOX-catalyzed phospholipid oxygenation lies upstream of iPLA2γ remains unknown. See text for details.
      The finding that the siRNA-based knockdown of cPLA2α ablated A23187-induced generation of PGE2 implies that cPLA2α is indeed essential for the immediate PGE2 biosynthesis in 3Y1 cells and is in agreement with the fact that rapid production of prostanoids, leukotrienes, and platelet-activating factor is blunted in cPLA2α-null mice in many (even if not all) cases (
      • Uozumi N.
      • Kume K.
      • Nagase T.
      • Nakatani N.
      • Ishii S.
      • Tashiro F.
      • Komagata Y.
      • Maki K.
      • Ikuta K.
      • Ouchi Y.
      • Miyazaki J.
      • Shimizu T.
      ). By contrast, it is notable that the suppressive effect of cPLA2α siRNA on the cytokine-induced delayed PGE2 response (40–60% reduction over 24–48 h) was less effective than that on the A23187-elicited immediate response (>90% reduction). This difference could be explained by the contribution of the iPLA2γ-sPLA2-IIA axis to the delayed, but not to the immediate, PGE2-biosynthetic response reported in this study. Nonetheless, even though the occurrence of cPLA2α-regulated sPLA2-IIA induction is now unlikely in 3Y1 cells, our finding does not rule out the possibility that the coordinate membrane hydrolysis by sPLA2-IIA and cPLA2α could occur in some other cell systems. For instance, exogenous sPLA2-V leads to cPLA2α-dependent and -independent leukotriene production in human neutrophils (
      • Kim Y.J.
      • Kim K.P.
      • Han S.K.
      • Munoz N.M.
      • Zhu X.
      • Sano H.
      • Leff A.R.
      • Cho W.
      ,
      • Munoz N.M.
      • Kim Y.J.
      • Meliton A.Y.
      • Kim K.P.
      • Han S.K.
      • Boetticher E.
      • O'Leary E.
      • Myou S.
      • Zhu X.
      • Bonventre J.V.
      • Leff A.R.
      • Cho W.
      ). sPLA2-initiated production of lipid mediators, such as eicosanoids or lysophospholipids, or binding of sPLA2 to the sPLA2 receptor can trigger subsequent cPLA2α activation (
      • Hernandez M.
      • Burillo S.L.
      • Crespo M.S.
      • Nieto M.L.
      ). Furthermore, macrophages derived from cPLA2α knock-out mice produce eicosanoids only minimally (
      • Uozumi N.
      • Kume K.
      • Nagase T.
      • Nakatani N.
      • Ishii S.
      • Tashiro F.
      • Komagata Y.
      • Maki K.
      • Ikuta K.
      • Ouchi Y.
      • Miyazaki J.
      • Shimizu T.
      ), whereas there is also ∼50% reduction of eicosanoid production in macrophages derived from sPLA2-V-deficient mice (
      • Satake Y.
      • Diaz B.L.
      • Balestrieri B.
      • Lam B.K.
      • Kanaoka Y.
      • Grusby M.J.
      • Arm J.P.
      ). Thus, in such situations, sPLA2s may work together with cPLA2α leading to maximal eicosanoid production according to the types of cell and stimulus.
      In summary, we have shown a novel functional aspect of iPLA2γ through which it can participate in the regulation of pro-inflammatory cytokine- and endotoxin-dependent expression of sPLA2-IIA, a pro-inflammatory sPLA2. This suggests that the iPLA2γ-dependent signaling may be linked to the development and progression of inflammatory diseases under particular conditions in which inducible sPLA2s would be involved. To fully understand the roles of iPLA2γ in various cellular events, particularly in its interplay with sPLA2-IIA, the next challenges will include the identification of the particular bioactive lipid products that are produced by iPLA2γ, search of target genes other than sPLA2-IIA that are regulated by these bioactive lipids, and clarification of the regulatory mechanisms leading to iPLA2γ activation following the particular transmembrane and/or intracellular signals.

      Supplementary Material

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