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Wortmannin Inhibits Mitogen-activated Protein Kinase Activation by Platelet-activating Factor through a Mechanism Independent of p85/p110-type Phosphatidylinositol 3-Kinase (∗)

  • Ingvar M. Ferby
    Affiliations
    Department of Biochemistry, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan
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  • Iwao Waga
    Affiliations
    Department of Biochemistry, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan
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  • Mitsunobu Hoshino
    Affiliations
    Department of Biochemistry, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan
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  • Kazuhiko Kume
    Affiliations
    Department of Biochemistry, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan
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  • Takao Shimizu
    Correspondence
    To whom correspondences should be addressed. Tel.: 81-3-5802-2925; Fax: 81-3-3813-8732
    Affiliations
    Department of Biochemistry, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan
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  • Author Footnotes
    ∗ The work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan and by grants from Yamanouchi Foundation of Metabolic Disorders and the Human Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:May 17, 1996DOI:https://doi.org/10.1074/jbc.271.20.11684
      We have shown previously that wortmannin partially inhibits mitogen-activated protein kinase (MAPK) activated by platelet-activating factor (PAF) in guinea pig neutrophils (Ferby, M. I., Waga, I., Sakanaka, C., Kume, K., and Shimizu, T.(1994) J. Biol. Chem. 269, 30485-30488). To identify whether p85-dependent phosphatidylinositol 3-kinase is a target molecule of wortmannin in this inhibitory process, we established a murine macrophage cell line (P388D1), inducibly expressing a dominant-negative p85, Δp85. Upon induction of Δp85 by isopropyl-β-D-thiogalactopyranoside, PAF still induced unaltered activation of MAPK, which was inhibited completely by wortmannin and 1,2-bis-(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester in an additive manner. Thus, PAF activates MAPK in P388D1 cells via two distinct pathways, one calcium-dependent and another calcium-independent, but wortmannin-sensitive. The inhibition of calcium-independent activation of MAPK by wortmannin does not involve p85-dependent phosphatidylinositol 3-kinase.

      INTRODUCTION

      The phospholipid mediator platelet-activating factor (PAF)
      The abbreviations used are: PAF
      platelet-activating factor
      MAPK
      mitogen-activated protein kinase
      PI 3-K
      phosphatidylinositol 3-kinase
      MLCK
      myosin light chain kinase
      BAPTA/AM
      1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester
      GM-CSF
      granulocyte-macrophage colony-stimulating factor
      CHO cells
      Chinese hamster ovary cells
      PIP
      phosphatidylinositol 3-phosphate
      PTX
      pertussis toxin
      HBSS
      Hanks' balanced salt solution
      IPTG
      isopropyl-β-D-thiogalactopyranoside
      BSA
      bovine serum albumin
      ELISA
      enzyme-linked immunosorbent assay
      TBS
      Tris-buffered saline.
      acts through a heterotrimeric G-protein-coupled receptor to mediate divergent biological activities in a wide variety of blood cells such as platelets, neutrophils, macrophages, eosinophils, and lymphocytes(
      • Valone F.H.
      • Coles E.
      • Reinold V.R.
      • Goetzl E.J.
      ,
      • Dhar A.
      • Shukla S.D.
      ,
      • Ingraham L.M.
      • Coates T.D.
      • Allen J.M.
      • Higgins C.P.
      • Baehner R.L.
      • Boxer L.A.
      ,
      • Hayashi H.
      • Kudo I.
      • Nojima S.
      • Inoue K.
      ,
      • Asmis R.
      • Randriamampita C.
      • Tsien R.Y.
      • Dennis E.A.
      ,
      • Shute J.K.
      • Rimmer S.J.
      • Akerman C.L.
      • Church M.K.
      • Holgate S.T.
      ,
      • Schulam P.G.
      • Kuruvilla A.
      • Putcha G.
      • Mangus L.
      • Franklin-Johnson J.
      • Shearer T.W.
      ), thus making it a remarkably versatile mediator of inflammation and immune responses(
      • Hanahan D.J.
      ,
      • Prescott S.J.
      • Zimmerman G.A.
      • McIntyre T.M.
      ). PAF triggers various early signaling events, including activation of phospholipases C, D, and A2(
      • Shukla S.D.
      ,
      • Reinhold S.L.
      • Prescott S.M.
      • Zimmermann G.A.
      • McIntyre T.M.
      ,
      • Chao W.
      • Olson M.S.
      ), as well as phosphatidylinositol 3-kinase (PI 3-K)(
      • Stephens L.
      • Jackson T.
      • Hawkins P.T.
      ,
      • Stephens L.
      • Jackson T.
      • Hawkins P.T.
      ). Furthermore, PAF has been shown to activate mitogen-activated protein kinase (MAPK) in human platelets(
      • Samei M.
      • Sanghara J.S.
      • Pelesh S.L.
      ), human B cell lines(
      • Franklin R.A.
      • Mazar B.
      • Sawani H.
      • Mills G.B.
      • Terada N.
      • Lucas J.J.
      • Gelfand E.W.
      ), guinea pig neutrophils(
      • Ferby I.M.
      • Waga I.
      • Sakanaka C.
      • Kume K.
      • Shimizu T.
      ), and CHO cells expressing the cloned PAF receptor (
      • Honda Z.
      • Takano T.
      • Gotoh Y.
      • Nishida E.
      • Ito K.
      • Shimizu T.
      ). MAPK is a widely distributed serine-threonine kinase considered to be a key mediator of mainly proliferative and mitogenic responses through regulating activities of several transcription factors (
      • Alvarez E.
      • Northwood I.C.
      • Gonzalez F.A.
      • Latour D.A.
      • Seth A.
      • Abate C.
      • Curran T.
      • Davis R.J.
      ) and cytosolic PLA2 etc.(
      • Lin L.-L.
      • Wartmann M.
      • Lin A.Y.
      • Knopf J.L.
      • Seth A.
      • Davis R.J.
      ,
      • Nemenoff R.A.
      • Winitz S.
      • Qian N.X.
      • Van Putten V.
      • Johnson G.L.
      • Heasley L.E.
      ).
      PAF has been shown recently to activate PI 3-K in neutrophils (
      • Stephens L.
      • Jackson T.
      • Hawkins P.T.
      ) and human B cells(
      • Kuruvilla A.
      • Pielop C.
      • Shearer W.T.
      ). PI 3-K is a phospholipid kinase that has received much attention lately since its main physiological product, phosphatidylinositol(
      • Ingraham L.M.
      • Coates T.D.
      • Allen J.M.
      • Higgins C.P.
      • Baehner R.L.
      • Boxer L.A.
      ,
      • Hayashi H.
      • Kudo I.
      • Nojima S.
      • Inoue K.
      ,
      • Asmis R.
      • Randriamampita C.
      • Tsien R.Y.
      • Dennis E.A.
      ) -trisphosphate, appears to be a second messenger involved in membrane ruffling, superoxide generation in neutrophils, glucose transport control in adipocytes, and neurite outgrowth on PC12 cells(
      • Stephens L.
      • Jackson T.
      • Hawkins P.T.
      ,
      • Gardner A.M.
      • Vaillancourt R.R.
      • Johnson G.L.
      ,
      • Pelech S.L.
      • Sanghera J.S.
      ). Growth factor receptors, cytokine receptors, and G-protein-coupled receptors are capable of stimulating PI 3-K activity(
      • Okuda K.
      • Sanghera J.S.
      • Pelech S.L.
      • Kanakura Y.
      • Hallek M.
      • Griffin J.D.
      • Druker B.J.
      ,
      • Crews C.M.
      • Erikson L.
      ,
      • Blenis J.
      ). The PI 3-K pathway has been fairly well described in growth factor signaling, much thanks to the molecular cloning of PI 3-K activated by platelet-derived growth factor(
      • Escobedo J.A.
      • Navankasattusas S.
      • Kavanaugh W.M.
      • Milfay D.
      • Fried V.A.
      • Williams L.T.
      ,
      • Skolnik E.Y.
      • Margolis B.
      • Mohammadi M.
      • Lowenstein E.
      • Ficher R.
      • Drepps A.
      • Ullrich A.
      • Schlessinger J.
      ). This enzyme consists of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit. Enzyme activity requires association of the regulatory and catalytic subunits and by disrupting the binding site for p110 on the p85 subunit, PI 3-K activity is lost(
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.
      • Jacsorn T.R.
      • Hawkins R.T.
      • Dharn R.
      • Clark A.E.
      • Holman G.D.
      • Waterfield M.D.
      • Kasuga M.
      ). The characterization of G-protein-mediated PI 3-K activity is currently unclear and has been proposed to involve both heterodimeric p85-dependent PI 3-K (
      • Thomason P.A.
      • James S.R.
      • Casey P.J.
      • Downes C.P.
      ) and a distinct form of PI 3-K regulated by the βγ-subunit of heterotrimeric G-proteins(
      • Stephens L.
      • Smrcka A.
      • Cooke F.T.
      • Jackson T.R.
      • Sternweis P.C.
      • Hawkins P.T.
      ,
      • Stoyanov B.
      • Volinia S.
      • Hanck T.
      • Rubio I.
      • Loubtchenkov M.
      • Malek D.
      • Stoyanova S.
      • Vanhaesebroeck B.
      • Dhand R.
      • Nurnberg B.
      • Waterfield M.D.
      • Wetzker R.
      ).
      Wortmannin is a fungal metabolite that blocks many functional responses in neutrophils and has been characterized as an inhibitor of PI 3-K at nanomolar order (
      • Okada T.
      • Sakuma L.
      • Fukui Y.
      • Hazeki O.
      • Ui M.
      ,
      • Ninomiya N.
      • Hazeki K.
      • Fukui Y.
      • Seya T.
      • Okada T.
      • Hazeki O.
      • Ui M.
      ,
      • Woscholski R.
      • Kodaki T.
      • McKinnon M.
      • Waterfield M.D.
      • Parker P.J.
      ) and myosin light chain kinase (MLCK) at micromolar order(
      • Nakanishi S.
      • Kakita S.
      • Takahashi I.
      • Kawahara K.
      • Tsukuda E.
      • Sano T.
      • Yamada K.
      • Yoshida M.
      • Kase H.
      • Matsuda Y.
      • Hashimoto Y.
      • Nonomura Y.
      ). Recent studies in our laboratory and others have demonstrated that wortmannin partially inhibits MAPK activation caused by PAF in guinea pig neutrophils (
      • Ferby I.M.
      • Waga I.
      • Sakanaka C.
      • Kume K.
      • Shimizu T.
      ) and CHO cells2 or vasopressin (V1)-induced MAPK activation in rat 3Y1 cells(
      • Nishioka N.
      • Hirai S.
      • Mizuno K.
      • Osada S.
      • Suzuki A.
      • Kosaka K.
      • Ohno S.
      ). In these cells, wortmannin in combination with the use of calcium chelator or down-regulation of calcium-dependent protein kinase C completely inhibited the ligand-induced MAPK activation in an additive manner. Therefore, it was speculated that wortmannin targets on a molecule(s) involved in a calcium-independent and protein kinase C-independent pathway. The present study was undertaken to identify whether a p85-dependent PI 3-K is the target of wortmannin involved in PAF-induced activation of MAPK. By inducibly expressing dominant-negative p85, Δp85, in the macrophage cell line, P388D1, we here demonstrate that wortmannin inhibits MAPK activation by PAF through a mechanism independent of the conventional p85/p110 heterodimeric PI 3-K.

      EXPERIMENTAL PROCEDURES

      Materials

      P388D1 cells were obtained from ATCC (Rockville, MD). Vectors, p3′SS and pOPRSVICAT were purchased in form of a LacSwitch(™) Inducible Mammalian Expression System from Stratagene (La Jolla, CA). The Δp85 gene was kindly provided by Dr. M. Kasuga (Kobe University, Japan)(
      • Kotani K.
      • Yonezawa K.
      • Hara K.
      • Ueda H.
      • Kitamura Y.
      • Sakaue H.
      • Ando A.
      • Chavanieu A.
      • Calas B.
      • Grigorescu F.
      • Nishiyama M.
      • Waterfield M.D.
      • Kasuga M.
      ). Wortmannin, obtained from Kyowa Medex Co. (Tokyo), was dissolved in dimethyl sulfoxide at 10 mM, stored in the dark at 4°C, and diluted in buffer just before use. BAPTA/AM and Fura-2/AM were purchased from Dojin (Kumamoto, Japan), ML-7 and ML-9 from Seikagaku Co. (Tokyo), and pertussis toxin (PTX) from Funakoshi (Tokyo). BIOTRAK(™) MAPK assay kit and [γ-32P]ATP were purchased from Amersham International plc. Anti-PI 3-K antibodies, mouse recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), and fetal bovine serum were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). PAF (C-16) provided by Cayman Chemicals (Ann Harbor, MI). RPMI 1640 medium from Nissui Pharmaceutical Co. (Tokyo). Fatty acid-free bovine serum albumin (BSA), phospholipids, and cytochrome c were obtained from Sigma. Hygromycin B from Wako Pure Chemical Industries (Osaka, Japan) and geneticin from Life Technologies, Inc.

      Cell Culture and Cell Preparation

      P388D1 cells were grown in RPMI 1640 medium containing 10% fetal bovine serum (heat-inactivated) at 37°C in a humidified atmosphere (5% CO2) to ∼70% confluence before passage (or alternatively experimental use). Adherent cells were recovered by scraping with a silicon cell scraper. Experiments were done on serum-starved (12-16 h) cells suspended in Hanks' balanced salt solution (HBSS) supplemented with 10 mM Hepes-NaOH (pH 7.4) and 2 mg/ml BSA. Guinea pig peritoneal neutrophils were harvested and prepared as described (
      • Okada T.
      • Sakuma L.
      • Fukui Y.
      • Hazeki O.
      • Ui M.
      ).

      Inducible Expression of Δp85 in P388D1

      Bovine p85α with a deletion of 34 amino acids in the p110 binding region from 479-513 and insertion of other amino acids (Ser-Arg) in this deleted region (
      • Hara K.
      • Yonezawa K.
      • Sakaue H.
      • Ando A.
      • Kotani K.
      • Kitamura Y.
      • Ueda H.
      • Stephens L.
      • Jacsorn T.R.
      • Hawkins R.T.
      • Dharn R.
      • Clark A.E.
      • Holman G.D.
      • Waterfield M.D.
      • Kasuga M.
      ,
      • Kotani K.
      • Yonezawa K.
      • Hara K.
      • Ueda H.
      • Kitamura Y.
      • Sakaue H.
      • Ando A.
      • Chavanieu A.
      • Calas B.
      • Grigorescu F.
      • Nishiyama M.
      • Waterfield M.D.
      • Kasuga M.
      ) were subcloned into the expression vector pOPRSVICAT (pOPRSVI-Δp85). This vector contains a geneticin resistance gene and Lac operator sequences that control expression of the downstream gene by IPTG. First, cells were transfected by electroporation with the lac repressor expressing vector p3′SS containing a hygromycin resistance gene. Transformants were selected by maintaining cells in medium supplemented with 200 μg/ml hygromycin B and isolated with iron cylinders and examined for Lac repressor expression by Northern blotting. Two clones exhibiting the highest Lac repressor expression were transfected by electroporation with the pOPRSVI-Δp85 plasmid, and the Neo-resistant clones were selected by growth in 600 μg/ml geneticin. Clones were examined for IPTG-induced p85 expression by enzyme-linked immunosorbent assay (ELISA) method (see below). Three clones consistently displaying high IPTG-induced p85 expression were selected for further experiments, denoted Δp85a, Δp85b, and Δp85c. After selection, clones were maintained in medium supplemented with 400 μg/ml geneticin and 150 μg/ml hygromycin B.

      ELISA for Quantitation of p85 Expression

      Basically performed by standard procedures(
      • Eager K.B.
      • Kennett R.H.
      ). Ninety-six-well assay plates (Corning, low stringency polystyrene plates) were coated with anti-rat polyclonal p85 (PI 3-K) antibody (1/1,000 dilution) in phosphate-buffered saline at 4°C overnight, rinsed well, and blocked with 20% fetal bovine serum in phosphate-buffered saline overnight. After a mild rinse, cell lysates (50 μl/well) were applied and incubated for 2 h at room temperature. Plates were rinsed three times with Tris-buffered saline (TBS) containing 0.05% Tween 20. Anti-mouse monoclonal p85 antibody (1/1,000 dilution) was added to the wells and left for 2 h at room temperature. After several rinses with TBS containing 0.05% Tween 20, wells were incubated with peroxidase-labeled anti-mouse IgG antisera (1/3,000 dilution) for 2 h at room temperature, followed by extensive washing in TBS and measurement of peroxidase activity.

      MAPK Assay

      Cell aliquots (1 × 106 cells) were challenged with ligand at 37°C and the reaction terminated by directly adding the lysis buffer containing 20 mM Tris-HCl (pH 8.0), 20 mM β-glycerophosphate, 1 mM sodium orthovanadate, 2 mM EGTA, 2 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 0.1% Triton X-100 (final concentrations) in a total volume of 200 μl. Aliquots (15 μl) were then assayed for MAPK activity as described previously(
      • Ferby I.M.
      • Waga I.
      • Sakanaka C.
      • Kume K.
      • Shimizu T.
      ). When examining the effect of PTX, cells were incubated overnight with 100 ng/ml PTX.

      Measurement of PI 3-K Activity

      Cells were lysed in 145 mM NaCl, 20 mM Tris-HCl (pH 7.4), 5 mM EDTA, 200 μM sodium orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40, and 10 μg/ml aprotinin and cell debris removed by centrifugation. Precleared lysates were immunoprecipitated with a polyclonal p85 antibody overnight. After extensive washing, immunoprecipitates were subjected to PI 3-K assay as described previously(
      • Stephens L.
      • Jackson T.
      • Hawkins P.T.
      ). Briefly, precipitates were incubated for 20 min at 25°C in 7 mM Tris-HCl (pH 7.4), 75 mM NaCl, 2.5 mM EDTA (pH 7.4), 50 μM sodium orthovanadate, 10 mM MgCl2 10 μg of PI and 10 μg of phosphatidylserine and 50 μM [γ-32P]ATP (25 μCi) in a total volume of 100 μl (including precipitate), reaction terminated by addition of 20 μl of 6 N HCl, phospholipids extracted with 160 μl of chloroform/methanol (1:1, v/v) spotted on a 1.2% potassium oxalate-preactivated TLC plate (Silica Gel-60, Merck) and developed in chloroform/methanol/acetone/acetic acid/water (7:5:2:2:2, v/v). PI 3-phosphate (PIP) spots (RF about 0.7) were quantitated with a Fuji-BAS 2000 Image Analyzer. For the in vivo PI 3-K assay, cells were labeled with [32P]orthophosphate for 1 h, challenged with 100 nM PAF, and subjected to PI 3-K assay as described previously(
      • Okada T.
      • Sakuma L.
      • Fukui Y.
      • Hazeki O.
      • Ui M.
      ).

      Depletion of Intracellular Ca2+

      P388D1 cells (5 × 106/ml) were suspended in Ca2+-free HBSS supplemented with 10 mM Hepes-NaOH (pH 7.4) and 2 mg/ml BSA and were incubated for 30 min at 25°C in the presence of 20 μM BAPTA/AM, washed twice, and resuspended in the same buffer without BAPTA/AM(
      • Ferby I.M.
      • Waga I.
      • Sakanaka C.
      • Kume K.
      • Shimizu T.
      ).

      Measurement of Intracellular Ca2+ Concentrations

      Cells suspended in Ca2+-containing HBSS were loaded with 4 μM Fura-2/AM at room temperature for 20 min, washed twice, resuspended in HBSS to a concentration of 106 cells/ml, and measured for intracellular Ca2+, as described(
      • Ferby I.M.
      • Waga I.
      • Sakanaka C.
      • Kume K.
      • Shimizu T.
      ,
      • Waga I.
      • Nakamura M.
      • Honda Z.
      • Ferby I.
      • Toyoshima S.
      • Ishiguro S.
      • Shimizu T.
      ). After a 5-min equilibration at 37°C under gentle stirring, ligand was added, and elevations of intracellular Ca2+ concentrations were measured on a spectrofluorometer (model CAF-100 or CAM-230, JASCO, Tokyo), with emission wavelength set at 510 nm and excitation wavelengths pendeling between 340 and 380 nm.

      Superoxide Production and β-Glucuronidase Release

      Superoxide released from cells into the surrounding medium was detected as reduction of horse heart cytochrome c as described(
      • Okada T.
      • Sakuma L.
      • Fukui Y.
      • Hazeki O.
      • Ui M.
      ,
      • Pinchard R.N.
      • Showell H.J.
      • Castillo R.
      • Lear C.
      • Breslow R.
      • McManus L.M.
      • Woodard D.S.
      • Ludwig J.C.
      ), and released β-glucuronidase activity was measured as described(
      • Pinchard R.N.
      • Showell H.J.
      • Castillo R.
      • Lear C.
      • Breslow R.
      • McManus L.M.
      • Woodard D.S.
      • Ludwig J.C.
      ).

      RESULTS

      Differential Effects of Wortmannin on MAPK and PI 3-K Activation

      We have shown previously that wortmannin partially inhibits MAPK activated by PAF in guinea pig neutrophils(
      • Ferby I.M.
      • Waga I.
      • Sakanaka C.
      • Kume K.
      • Shimizu T.
      ). First, we compared the potency by which wortmannin inhibits MAPK, PI 3-K, and various biological functional responses. Pretreatment of guinea pig neutrophils with various doses of wortmannin for 10 min at 37°C, dose-dependently inhibited PAF (100 nM)-induced activation of MAPK and PI 3-K, production of superoxide, and release of β-glucuronidase (Fig. 1). While wortmannin inhibited PI 3-K activation, enzyme release and superoxide production with similar IC50 values varying from 5 to 20 nM, doses around 300 nM were required for half-maximal inhibition of MAPK activation. The discrepancy raised a possibility that PI 3-K, in particular a conventional isoform, p85-dependent PI 3-K, is not a target of wortmannin in the inhibitory process of PAF-induced MAPK activation. The following experiments were done, therefore, to determine at the molecular level whether p85-dependent PI 3-K is a target of wortmannin effect.
      Figure thumbnail gr1
      Figure 1:Dose-dependent inhibition by wortmannin of MAPK, PI 3-K, and functional responses in PAF-stimulated guinea pig neutrophils. MAPK (▪) and in vivo PI 3-K (×) activity, release of β-glucuronidase (○), and production of superoxide (•) was measured on guinea pig peritoneal neutrophils, pretreated with various doses of wortmannin for 10 min at 37°C, prior to stimulation with 100 nM PAF. Samples for MAPK assay were stimulated for 1 min (100% activation correspond to a 5.2-fold increase over basal 1.1 × 104 cpm). Cells were stimulated for 30 s, and the samples were assayed for in vivo PI 3-K activity (100% activation correspond to a 2.7-fold increase in phosphatidyl(
      • Ingraham L.M.
      • Coates T.D.
      • Allen J.M.
      • Higgins C.P.
      • Baehner R.L.
      • Boxer L.A.
      ,
      • Hayashi H.
      • Kudo I.
      • Nojima S.
      • Inoue K.
      ,
      • Asmis R.
      • Randriamampita C.
      • Tsien R.Y.
      • Dennis E.A.
      ) -trisphosphate production). Cells (5 × 106/ml) measured for superoxide production and β-glucuronidase release assays were stimulated for 2 min before extracellular medium was recovered and subjected to measurements. 100% activation of superoxide production correspond to a 5.6-fold over basal (5 nM superoxide) and for β-glucuronidase release to a 2.9-fold increase. See “Experimental Procedures” for assay procedures. Symbols and vertical bars represent the mean and S.D. of three experiments.

      Activation of MAPK in P388D1 Cells and Inhibition by Wortmannin

      Murine macrophage-like P388D1 cells were examined for activation of MAPK, following addition of 100 nM PAF. PAF evoked a rapid and transient activation that peaked 1 min after stimulation, with a 2-3-fold activation of MAPK (Fig. 2A). Pretreatment of cells with 1 μM wortmannin for 10 min at 37°C resulted in a partial (50-60%) inhibition of MAPK activation in response to PAF (Fig. 2B), correlating with our previous finding that wortmannin partially inhibits PAF-induced MAPK activation in guinea pig neutrophils ((
      • Ferby I.M.
      • Waga I.
      • Sakanaka C.
      • Kume K.
      • Shimizu T.
      ) and Fig. 1). Treatment with wortmannin alone had no effects on PAF-evoked MAPK activity (data not shown). To determine which kind of G-protein(s) coupled with MAPK and calcium mobilization, cells were treated overnight with 100 ng/ml PTX, either response was not altered (data not shown), suggesting that PAF couples with PTX-insensitive G-protein(s) in P388D1 cells.
      Figure thumbnail gr2
      Figure 2:Activation of MAPK by PAF and inhibition by wortmannin in P388D1 cells. A, P388D1 cells (2 × 106 cells/ml) were challenged with 100 nM PAF for indicated times and MAPK activity measured on cell lysates as described under “Experimental Procedures.” Basal radioactivity was 9 × 103 cpm. B, cells were preincubated with 1 μM wortmannin or vehicle for 10 min at 37°C, prior to stimulation. Column 1, nonstimulated cells (1.1 × 104 cpm); column 2, cells stimulated with 100 nM PAF for 1 min; column 3, wortmannin-pretreated cells stimulated with PAF. Filled circles/columns and vertical bars denote the mean and S.D., respectively, of three experiments.

      Inducible Expression of Dominant-negative p85, Δp85, in P388D1 Cells

      We obtained three independent P388D1 cell lines exhibiting high expression of Δp85 upon induction by IPTG, as measured by ELISA. IPTG (2 mM) induced a 5-15-fold increase of Δp85 expression over the basal level (nontransfected cells) (Fig. 3A) that reached a maximum level after 5-6 h induction time and remained unaltered for at least 24 h (data not shown). IPTG at doses higher than 2 mM did not enhance the induction level (not shown).
      Figure thumbnail gr3
      Figure 3:Inducible expression of dominant-negative p85 (Δp85) in P388D1 cells. A, wild-type and Δp85a-c cells (∼70% confluence in 24-well plates) were incubated for 6 h in medium in the absence (black columns) or presence (shaded columns) of 2 mM IPTG, medium removed, cells lysed in 200 μl of lysis buffer/well, and subjected to p85-ELISA as described under “Experimental Procedures.” p85 expression is expressed as peroxidase activity (absorbance at 450 nm); B, cell aliquots (2 × 106 cells) of wild-type (lanes 1-5) and Δp85a (lanes 6-10) P388D1 clones were challenged with ligand, lysed, immunoprecipitated with polyclonal p85 antibody, and subjected to in vitro PI 3-K assay as described under “Experimental Procedures.” Lanes 1 and 6, nonstimulated cells; lanes 2 and 7, cells stimulated with 500 nM PAF for 1 min; lanes 4, 5, and 8-10, cells stimulated with 50 ng/ml GM-CSF for 3 min; lane 5, GM-CSF-stimulated cells pretreated with 1 μM wortmannin for 10 min; lane 9, GM-CSF-stimulated cells pretreated with 2 mM IPTG for 6 h. Experiments are repeated three times, and means (column) ± S.D. are shown. ∗, p < 0.05;∗∗, p < 0.01 as compared with the basal level (lanes 1 and 6). C, increase of intracellular Ca2+ induced by 100 nM PAF (arrows), as monitored by Fura-2, in wild-type and Δp85a-c P388D1 cells.
      Wild-type and Δp85a cells stimulated with 100 nM PAF, or 50 ng/ml GM-CSF, were lysed, immunoprecipitated with anti-p85 antiserum, and subjected to in vitro PI 3-K assay (Fig. 3B). PAF induced only a very slight increase in the PI 3-K activity, while GM-CSF on the other hand evoked a 3-5-fold increased production of PIP. Wortmannin (500 nM) completely blocked the response by PAF or GM-CSF. In Δp85a cells, the basal as well as PAF- or GM-CSF-induced activities were lowered ∼2-fold, indicating that the lac repressor system is somewhat leaky, allowing some Δp85 expression, even without induction with IPTG. However, in IPTG-induced Δp85a cells, GM-CSF totally failed to induce PIP production. Similar results were obtained for the Δp85b and Δp85c clones (data not shown).
      Wild-type and Δp85-expressing P388D1 clones were examined for Ca2+ influx elicited by PAF, as monitored by Fura-2 (Fig. 3C). PAF caused an immediate and transient, ∼3-fold increase of intracellular Ca2+ in wild-type as well as Δp85-expressing clones induced with IPTG for 6 h, thus indicating that Δp85 expression and differences between the individual clone did not cause any major changes in PAF receptor-mediated calcium signaling.

      Inhibition of PAF-induced MAPK Activity by Wortmannin in Wild-type versus Δp85-transfected Cells

      In the following, we examined the effect of dominant-negative p85 expression on PAF-induced MAPK activity. Preincubating cells with 500 nM to 1 μM wortmannin for 10 min at 37°C inhibited MAPK activation induced by PAF (100 nM) with ∼50% in wild-type as well as Δp85-expressing cells, subjected to IPTG induction (2 mM) for 6 h (Fig. 4). Although some differences were observed in magnitude of the PAF-induced response between the individual clones, all were inhibited by wortmannin to approximately the same extent. The results clearly demonstrated that Δp85 did not affect the same target as wortmannin involved in PAF-mediated MAPK activation.
      Figure thumbnail gr4
      Figure 4:PAF-induced MAPK activity and its inhibition by wortmannin in wild-type and Δp85a-c. MAPK activity induced by 100 nM PAF was measured in wild-type and IPTG-treated Δp85a-c clones as described under “Experimental Procedures.” Black columns represent nonstimulated samples; hatched columns, PAF-stimulated samples; and shaded columns, PAF-stimulated samples preincubated 10 min with 1 μM wortmannin. The basal activities of wild-type cells were 8,407 ± 129 cpm; for Δp85a, 7,230 ± 1,820 cpm; for Δp85b, 9,208 ± 239 cpm; and for Δp85c, 6,642 ± 582 cpm. Columns and vertical bars denote the mean and S.D., respectively, of three experiments.
      Since 1 μM wortmannin only partially inhibited PAF-induced MAPK activity in wild-type and Δp85-expressing P388D1 clones, we next examined the effect of calcium depletion on the wortmannin-insensitive part of the response in both wild-type and Δp85-transfected cells. Preloading cells with BAPTA/AM under conditions that were shown previously to abolish PAF-induced calcium response in guinea pig neutrophils (
      • Ferby I.M.
      • Waga I.
      • Sakanaka C.
      • Kume K.
      • Shimizu T.
      ) caused a ∼50% inhibition of the PAF-induced MAPK response in wild-type P388D1 cells. When combining BAPTA/AM loading with wortmannin treatment, a complete inhibition occurred (Fig. 5A). When the Δp85a clone, induced for Δp85 expression with IPTG for 6 h, was used in the same experiment, essentially the same result was obtained (Fig. 5B). Neither BAPTA/AM nor wortmannin use alone had effect on MAPK activity (data not shown).
      Figure thumbnail gr5
      Figure 5:Additive inhibition by BAPTA/AM and wortmannin of PAF-induced MAPK activation. Wild-type (lanes 1-4) and Δp85a (lanes 5-8) cells were treated with or without 20 μM BAPTA/AM for 30 min at room temperature and/or 1 μM wortmannin for 10 min at 37°C. Columns 1 and 5, control cells; column 2 and 6, cells stimulated with 100 nM PAF for 1 min; column 3 and 7, BAPTA/AM-pretreated cells; column 4 and 8, pretreatment with both BAPTA/AM and wortmannin. Basal activity for wild-type and Δp85a samples were 3,706 ± 450 cpm and 5,178 ± 354 cpm, respectively. Neither BAPTA/AM alone nor wortmannin alone has effect on MAPK activity. Columns and vertical bars denote the mean and S.D., respectively, of three experiments.

      Effect of MLCK Inhibitors on PAF-induced MAPK Activation

      Since p85/PI 3-K apparently is not the target of wortmannin involved in PAF-induced MAPK activation, we next examined whether another reported target of wortmannin, MLCK is instead involved in PAF-mediated activation of MAPK. Preincubation of P388D1 cells with 50 μM ML-7 or 100 μM ML-9 for 30 min at 37°C, concentrations to specifically inhibit MLCK(
      • Hashimoto Y.
      • Sasaki H.
      • Togo M.
      • Tsukamoto K.
      • Horie Y.
      • Fukuta H.
      • Watanabe T.
      • Kurokawa K.
      ,
      • Makishima M.
      • Honma Y.
      • Hozumi M.
      • Nagata N.
      • Motoyoshi K.
      ), did not have any significant effects on the basal nor PAF (100 nM)-induced MAPK activity (Table 1).
      Tabled 1
      Table thumbnail grt1
      All these results indicate that p85-dependent PI 3K is not a target of wortmannin in the inhibitory process of MAPK activation through Ca2+-independent pathway.

      DISCUSSION

      Recent studies from our laboratory and others (
      • Ferby I.M.
      • Waga I.
      • Sakanaka C.
      • Kume K.
      • Shimizu T.
      ,
      • Nishioka N.
      • Hirai S.
      • Mizuno K.
      • Osada S.
      • Suzuki A.
      • Kosaka K.
      • Ohno S.
      ) have demonstrated the involvement of a wortmannin-sensitive target in G-protein-mediated MAPK activation. PAF-induced MAPK activation in guinea pig neutrophils (
      • Ferby I.M.
      • Waga I.
      • Sakanaka C.
      • Kume K.
      • Shimizu T.
      ) and CHO cells carrying the PAF receptor
      C. Sakanaka and T. Shimizu, unpublished observation.
      were partially inhibited by wortmannin, while on the other hand, wortmannin blocked completely somatostatin-induced MAPK activation in CHO cells expressing a cloned somatostatin receptor (SSTR4)(
      • Sakanaka C.
      • Ferby I.
      • Waga I.
      • Bito H.
      • Shimizu T.
      ). SSTR4 does not cause any Ca2+ signaling in the cells, but potently activates MAPK (
      • Sakanaka C.
      • Ferby I.
      • Waga I.
      • Bito H.
      • Shimizu T.
      ). These findings raised a reasonable possibility that PI 3-K might be involved in G-protein-dependent MAPK activation in a Ca2+-independent pathway. In rat 3Y1 fibroblasts stimulated with vasopressin, a similar additive effect was observed with wortmannin treatment and protein kinase C down-regulation(
      • Nishioka N.
      • Hirai S.
      • Mizuno K.
      • Osada S.
      • Suzuki A.
      • Kosaka K.
      • Ohno S.
      ).
      The mechanism by which G-protein-coupled receptors induce PI 3-K activation is still unclear. Partially purified PI 3-K from platelets (
      • Thomason P.A.
      • James S.R.
      • Casey P.J.
      • Downes C.P.
      ) and neutrophils (
      • Stephens L.
      • Smrcka A.
      • Cooke F.T.
      • Jackson T.R.
      • Sternweis P.C.
      • Hawkins P.T.
      ) activated by the βγ-subunit of G-proteins might result from a p85-p110 interaction (
      • Thomason P.A.
      • James S.R.
      • Casey P.J.
      • Downes C.P.
      ) or might involve a distinct G-protein-specific PI 3-K isotype(
      • Stephens L.
      • Smrcka A.
      • Cooke F.T.
      • Jackson T.R.
      • Sternweis P.C.
      • Hawkins P.T.
      ). The present study was, therefore, undertaken to determine whether or not p85-dependent PI 3-K is actually involved in the activation process of MAPK. A more than one order of discrepancy in the dose required to inhibit PAF-induced MAPK activation and PI 3-K is observed in guinea pig neutrophils (Fig. 1). Inhibition by wortmannin of common neutrophil functional responses appears to follow the dose dependence for inhibition of PI 3-K. The discrepancy raises the possibility that a molecule other than PI 3-K is involved in PAF-induced MAPK activation.
      Among a panel of myeloid cell lines, the murine P388D1 macrophage-like cell line displayed a relatively strong (2-3-fold) activation of MAPK by PAF (Fig. 2) and was thus chosen for further studies. We established three independent P388D1 transfectants, inducibly expressing a dominant-negative mutant of p85, Δp85. Although, these clones upon induction exhibited unaltered PAF-induced Ca2+ response, they failed to activate PI 3-K toward PAF or GM-CSF (positive control) (Fig. 3).
      Induced dominant-negative p85 expression did not affect the PAF-induced activation of MAPK (Fig. 4). Despite Δp85 expression, wortmannin was still inhibitory to MAPK activation by PAF in all three independent Δp85 clones approximately to the same extent. Furthermore, Ca2+ depletion by BAPTA/AM loading partially (∼50%) inhibited PAF-induced MAPK activation, while combined treatment with BAPTA/AM and wortmannin completely abolished the activity, in wild-type as well as in a Δp85-expressing clone (Fig. 5). Neither BAPTA/AM alone nor wortmannin alone has effect on MAPK activity. These results indicate that wortmannin inhibits a target involved in a Ca2+-independent pathway and further supports that p85-dependent PI 3-K is not involved in neither Ca2+-dependent nor -independent activation of MAPK by PAF.
      Although characterized as a Ca2+-independent enzyme, an alternative possible target of wortmannin was MLCK, shown to be inhibited by higher doses of wortmannin, correlating with the doses required to inhibit the PAF-induced MAPK response. However, the structurally related MLCK inhibitors ML-7 and ML-9 did not affect PAF-induced MAPK activation (Table 1).
      In conclusion, wortmannin inhibits MAPK activation by PAF, on a target(s) other than p85-dependent PI 3-K or MLCK in P388D1 cells. One remaining possibility is that a distinct G-protein-activated PI 3-K isotype is the true target of wortmannin involved. Indeed, a PI 3-K activated by the α- and βγ-subunits of G-proteins, p110γ, activated independently of p85 has most recently been cloned(
      • Stoyanov B.
      • Volinia S.
      • Hanck T.
      • Rubio I.
      • Loubtchenkov M.
      • Malek D.
      • Stoyanova S.
      • Vanhaesebroeck B.
      • Dhand R.
      • Nurnberg B.
      • Waterfield M.D.
      • Wetzker R.
      ). Future investigation will have to reveal whether this or a similar enzyme is the true target in G-protein-dependent, but calcium-protein kinase C-independent, pathway of MAPK activation.

      Acknowledgments

      We are grateful to Dr. M. Kasuga at Kobe University for providing the dominant-negative PI 3-K cDNA and Drs. T. Okada, O. Hazeki, and T. Katada at the University of Tokyo for discussion.

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