Secretory Phospholipase A2 Activates the Cascade of Mitogen-activated Protein Kinases and Cytosolic Phospholipase A2 in the Human Astrocytoma Cell Line 1321N1
1998; Elsevier BV; Volume: 273; Issue: 1 Linguagem: Inglês
10.1074/jbc.273.1.606
ISSN1083-351X
AutoresMarita Hernández, Silvia Burillo, Mariano Sánchez Crespo, Marı́a Luisa Nieto,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe biological effects of type IIA 14-kDa phospholipase A2 (sPLA2) on 1321N1 astrocytoma cells were studied. sPLA2 induced a release of [3H]arachidonic acid ([3H]AA) similar to that elicited by lysophosphatidic acid (LPA), a messenger acting via a G-protein-coupled receptor and a product of sPLA2 on lipid microvesicles. In contrast, no release of [1-14C]oleate could be detected in cells labeled with this fatty acid. As these findings pointed to a selective mechanism of [3H]AA release, it was hypothesized that sPLA2 could act by a signaling mechanism involving the activation of cytosolic PLA2 (cPLA2), i.e. the type of PLA2 involved in the release of [3H]AA elicited by agonists. In keeping with this view, stimulation of 1321N1 cells with sPLA2 elicited the decrease in electrophoretic mobility that is characteristic of the phosphorylation of cPLA2, as well as activation of p42 mitogen-activated protein (MAP) kinase, c-Jun kinase, and p38 MAP kinase. Incubation with sPLA2 of quiescent 1321N1 cells elicited a mitogenic response as judged from an increased incorporation of [3H]thymidine. Attempts to correlate the effect of extracellular PLA2 with the generation of LPA were negative. Incubation with pertussis toxin prior to the addition of either sPLA2 or LPA only showed abrogation of the response to LPA, thus suggesting the involvement of pertussis-sensitive Gi-proteins in the case of LPA. Treatments with inhibitors of the catalytic effect of sPLA2 such asp-bromophenacyl bromide and dithiothreitol did not prevent the effect on cPLA2 activation. In contrast, preincubation of 1321N1 cells with the antagonist of the sPLA2 receptorp-aminophenyl-α-d-mannopyranoside-bovine serum albumin, blocked cPLA2 activation with a EC50 similar to that described for the inhibition of binding of sPLA2 to its receptor. Moreover, treatment of 1321N1 cells with the MAP kinase kinase inhibitor PD-98059 inhibited the activation of both cPLA2 and p42 MAP kinase produced by sPLA2. In summary, these data indicate the existence in astrocytoma cells of a signaling pathway triggered by engagement of a sPLA2-binding structure, that produces the release of [3H]AA by activating the MAP kinase cascade and cPLA2, and leads to a mitogenic response after longer periods of incubation. The biological effects of type IIA 14-kDa phospholipase A2 (sPLA2) on 1321N1 astrocytoma cells were studied. sPLA2 induced a release of [3H]arachidonic acid ([3H]AA) similar to that elicited by lysophosphatidic acid (LPA), a messenger acting via a G-protein-coupled receptor and a product of sPLA2 on lipid microvesicles. In contrast, no release of [1-14C]oleate could be detected in cells labeled with this fatty acid. As these findings pointed to a selective mechanism of [3H]AA release, it was hypothesized that sPLA2 could act by a signaling mechanism involving the activation of cytosolic PLA2 (cPLA2), i.e. the type of PLA2 involved in the release of [3H]AA elicited by agonists. In keeping with this view, stimulation of 1321N1 cells with sPLA2 elicited the decrease in electrophoretic mobility that is characteristic of the phosphorylation of cPLA2, as well as activation of p42 mitogen-activated protein (MAP) kinase, c-Jun kinase, and p38 MAP kinase. Incubation with sPLA2 of quiescent 1321N1 cells elicited a mitogenic response as judged from an increased incorporation of [3H]thymidine. Attempts to correlate the effect of extracellular PLA2 with the generation of LPA were negative. Incubation with pertussis toxin prior to the addition of either sPLA2 or LPA only showed abrogation of the response to LPA, thus suggesting the involvement of pertussis-sensitive Gi-proteins in the case of LPA. Treatments with inhibitors of the catalytic effect of sPLA2 such asp-bromophenacyl bromide and dithiothreitol did not prevent the effect on cPLA2 activation. In contrast, preincubation of 1321N1 cells with the antagonist of the sPLA2 receptorp-aminophenyl-α-d-mannopyranoside-bovine serum albumin, blocked cPLA2 activation with a EC50 similar to that described for the inhibition of binding of sPLA2 to its receptor. Moreover, treatment of 1321N1 cells with the MAP kinase kinase inhibitor PD-98059 inhibited the activation of both cPLA2 and p42 MAP kinase produced by sPLA2. In summary, these data indicate the existence in astrocytoma cells of a signaling pathway triggered by engagement of a sPLA2-binding structure, that produces the release of [3H]AA by activating the MAP kinase cascade and cPLA2, and leads to a mitogenic response after longer periods of incubation. Phospholipases A2 (phosphatidesn-2-acylhydrolases, EC 3.1.1.4) from mammalian tissues play a role in physiological functions such as defense mechanisms and the production of bioactive lipids (1Dennis E.A. Rhee S.G. Billah M.M. Hannun Y.A. FASEB J. 1991; 5: 2068-2077Crossref PubMed Scopus (476) Google Scholar, 2Mayer R.J. Marshall L.A. FASEB J. 1993; 7: 339-348Crossref PubMed Scopus (446) Google Scholar, 3Serhan C.N. Haeggstrom J.Z. Leslie C.C. FASEB J. 1996; 10: 1147-1158Crossref PubMed Scopus (372) Google Scholar). In the last years, purification and molecular cloning of phospholipases A2(PLA2) 1The abbreviations used are: PLA2, phospholipase A2; AA, arachidonic acid; BPB,p-bromophenacyl bromide; BSA, bovine serum albumin; cPLA2, cytosolic phospholipase A2; DTT, dithiothreitol; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; JNK, c-Jun N-terminal kinase; LPA, lysophosphatidic acid; MAP, mitogen-activated protein; mannose-BSA,p-aminophenyl-α-d-mannopyranoside-BSA; MEK, mitogen-activated protein kinase kinase; PAGE, polyacrylamide gel electrophoresis; PTX, pertussis toxin; sPLA2, secretory phospholipase A2. has allowed the characterization of several enzymes displaying significant differences in both structural and functional properties. On the one hand, the 14-kDa type IIA PLA2 (sPLA2) behaves as an acute phase protein whose production is induced in a variety of immunoinflammatory conditions, e.g. rheumatoid arthritis and endotoxemia (4Vadas P. Wasi S. Movat H. 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Chem. 1991; 266: 19139-19141Abstract Full Text PDF PubMed Google Scholar, 10Lambeau G. Ancian P. Barhanin J. Lazdunski M. J. Biol. Chem. 1994; 269: 1575-1578Abstract Full Text PDF PubMed Google Scholar) and the appearance of chronic epidermal hyperplasia and hyperkeratosis similar to those observed in human dermopathies in mice hyperexpressing the human type IIA PLA2 gene (11Grass D.S. Felkner R.H. Chiang M.Y. Wallace R.E. Nevalainen T.J. Bennet C.F. Swanson M.E. J. Clin. Invest. 1996; 97: 2233-2241Crossref PubMed Scopus (162) Google Scholar). A similar histological picture accompanied by inflammatory changes is produced by injection of sPLA2 in the skin of experimental animals (12Pruzanski W.P. Vadas P. Fornasier V. J. Invest. Dermatol. 1986; 86: 380-382Abstract Full Text PDF PubMed Scopus (69) Google Scholar, 13Nair X. Nettleton D. Clever D. Tramposch K.M. Ghosh S. Franson R.C. Inflammation. 1993; 17: 205-215Crossref PubMed Scopus (22) Google Scholar). 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(i) sPLA2 might interact with a binding structure on the outer leaflet of the cell membrane, or (ii) sPLA2 might generate both unesterified fatty acid and lysophospholipid, e.g. lysophosphatidate (LPA) and lysophosphatydylcholine, which could act on signaling either as cofactors for protein kinase C or, in the case of LPA, by acting on specific receptors. This poses as a likely possibility that sPLA2 might ultimately lead to the activation of cPLA2 by eliciting a signaling cascade mimicking the usual transducing mechanism conveyed by the physiological activators of this enzyme. In this connection, it should mentioned that cross-talk between cPLA2 and sPLA2 has been suggested in signal transduction events in polymorphonuclear leukocytes and macrophages (21Wijkander J. O'Flaherty J.T. Nixon A.B. Wykle R.L. J. Biol. Chem. 1995; 270: 26543-26549Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 22Balsinde J. Dennis E.A. J. Biol. 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Plasma from patients with septicemia was obtained from venous blood anticoagulated with heparin. [9,10-3H]Myristic acid (53 Ci/mmol), [1-14C]oleate (53, 9 mCi/mmol), and [3H]arachidonic acid (100 Ci/mmol) were from Amersham International, Bucks, United Kingdom. Essentially fatty acid-free BSA was from Miles Laboratories. Reagent for the measurement of proteins according to the method of Bradford (33Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) was purchased from Bio-Rad. Heparin-agarose type I,p-aminophenyl-α-d-mannopyranoside-BSA (mannose-BSA), and porcine pancreatic PLA2 were from Sigma. A C127 mouse fibroblast line stably transfected with the coding sequence of type IIA PLA2 from human placenta (34Pernas P. Masliah J. Olivier J.L. Salvat C. Bereziat G. Biochem. Biophys. Res. Commun. 1991; 178: 1298-1305Crossref PubMed Scopus (66) Google Scholar) was used as a source of human recombinant type IIA PLA2. Rabbit polyclonal anti-cPLA2 antibody was obtained as described (35deCarvalho M.S. McCormack F.X. Leslie C.C. Arch. Biochem. Biophys. 1993; 306: 534-540Crossref PubMed Scopus (41) Google Scholar). Mouse monoclonal anti-MAP kinase antibody reacting with both p42 and p44 MAP/ERK was from Zymed Laboratories Inc., San Francisco, CA. Rabbit polyclonal anti-p38 MAP kinase antibody was from Santa Cruz Biotechnology Inc., Santa Cruz, CA. Monoclonal anti-phosphotyrosine antibody clone 4G10 was from Upstate Biotechnology, Lake Placid, NY. The MAP kinase kinase (MEK) inhibitor PD-98059 was a gift from Dr. Alan R. Saltiel (Parke Davis Pharmaceutical Research, Ann Arbor, MI) (36Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2595) Google Scholar). The p38 MAP kinase inhibitor SB 203580 was a gift from Dr. John C. Lee (SmithKline Beecham Pharmaceuticals, King of Prussia, PA) (37Lee J.C. Young P.R. J. Leukocyte Biol. 1996; 59: 152-157Crossref PubMed Scopus (375) Google Scholar). GlutathioneS-transferase (GST) fusion protein with amino acids 1–223 of the N-terminal portion of c-Jun protein (a kind gift of Dr. Carmen Caelles, Instituto de Investigaciones Biomédicas, Madrid, Spain) was expressed in bacteria using a pGEX-2T plasmid (Pharmacia Biotech Inc.) and purified with glutathione-agarose beads from Sigma. sPLA2 was purified from both plasma of patients with septicemia and culture medium according to the protocol described in Ref. 38Gijón M.A. Pérez C. Méndez E. Sánchez Crespo M. Biochem. J. 1995; 306: 167-175Crossref PubMed Scopus (35) Google Scholar. Briefly, heparin-agarose was used to bind sPLA2 from plasma. Fractions showing PLA2 activity in the [1-14C]oleate-labeled Escherichia coli assay were concentrated and loaded into a HiLoad Superdex 75 column (Pharmacia LKB, Uppsala, Sweden). Fractions containing PLA2after this step were made in 0.1% trifluoroacetic acid, and applied into a C1/C8 reverse-phase FPLC column (ProRPC HR 5/2, Pharmacia LKB). Fractions showing PLA2 activity were pooled and evaporated to dryness in a Speed-Vac concentrator. Human recombinant type IIA phospholipase A2 was purified from cultures at superconfluence of line C127 mouse fibroblasts stably transfected with the coding sequence of type IIA PLA2 from human placenta (34Pernas P. Masliah J. Olivier J.L. Salvat C. Bereziat G. Biochem. Biophys. Res. Commun. 1991; 178: 1298-1305Crossref PubMed Scopus (66) Google Scholar). The assay was carried out in a total volume of 0.1 ml, according to the procedure of Elsbachet al. (39Elsbach P. Weiss J. Franson R.C. Beckerdite-Quagliata S. Schneider A. Harris L. J. Biol. Chem. 1979; 254: 11000-11009Abstract Full Text PDF PubMed Google Scholar). Samples were incubated with ≈5,000 dpm of [1-14C]oleate-labeled autoclaved E. coli of a K12 strain, containing 10–20 nmol of phospholipid, as assessed by the measurement of phospholipid-associated phosphate. The assay medium contained 0.1 m Tris/HCl, 1 mg/ml fatty acid-free BSA, and 0.5 mm CaCl2, pH 7.4. The reaction proceeded for 30 min and was stopped by addition of 0.04 ml of ice-cold 2n HCl and 0.02 ml of 10% BSA, followed by centrifugation for 5 min at 13,000 rpm in an Eppendorf microcentrifuge. The radioactivity released into the supernatant was assayed by liquid scintillation counting. 1321N1 astrocytoma cells were cultured in Dulbecco's modified Eagle's medium containing 5% fetal calf serum at 37 °C in an atmosphere containing 5% CO2. Labeling with [3H]AA was performed in cells in monolayer that had been deprived of fetal calf serum for 16 h to render them quiescent. Labeling with [3H]AA was carried out for 2 h in the presence of 0.3 μCi of [3H]AA/ml. Labeling with 0.3 μCi/ml [1-14C]oleate and 1 μCi/ml [3H]myristic acid was carried out under similar conditions but increasing the labeling period to overnight incubation. After labeling, cells were washed at 37 °C four or five times with medium, and finally allowed to equilibrate at 37 °C before addition of agonists or vehicle solution. The release of labeled [3H]AA and [1-14C]oleic acid acid was assessed in 0.2-ml aliquots of culture medium. Production of LPA was assessed from the incorporation of [3H]myristic acid into phosphatidic acid and was separated from the label incorporated in other phospholipid classes by two-dimensional chromatography using a system of solvents consisting of chloroform/methanol/28% ammonium hydroxide (6:4:1; v/v/v) in the first dimension and chloroform/acetone/methanol/acetic acid/water (6:8:2:2:1; v/v/v) in the second dimension (40Tokomura A. Harada K. Fukuzawa K. Tsukatani H. Biochim. Biophys. Acta. 1986; 875: 31-38Crossref PubMed Scopus (154) Google Scholar). Experiments were carried out with triplicate samples. Quiescent 1321N1 cells were treated in serum-free Dulbecco's modified Eagle's medium for 24 h with different agonists in the presence of 0.5 μCi/ml [3H]thymidine. At the end of this period, the incubation was terminated with three washes with ice-cold 0.1 mMgCl2, and the radioactivity incorporated into the trichloroacetic acid-precipitable fraction measured. Cell lysates from preconfluent 1321N1 cells were loaded into a 10% SDS-PAGE gel, and transferred to polyvinyldifluoride membrane (Immobilon P, Millipore Corp., Bedford, MA) using a liquid transfer module from CBS Laboratories. The membranes were blocked with dry milk for 2 h, washed with Tris-buffered saline, and used for immunoblot using a rabbit polyclonal anti-cPLA2. When the purpose of the experiments was the detection of p42 MAP kinase, a semidry transfer system was used and the membrane was incubated with mouse monoclonal antibody. This was followed by incubation with sheep anti-mouse IgG-horseradish peroxidase-conjugated antibody, and detection with the Amersham ECL system. For detection of tyrosine phosphorylation of p38 MAP kinase, the endogenous kinase was immunoprecipitated from cell lysates using anti-p38 MAP antibody. The immune complex was recovered using Gammabind G-Sepharose. After washing three times with Nonidet-P-40-buffer and twice with LiCl buffer, the beads were resuspended in Laemmli sample buffer and subjected to SDS-PAGE. The extent of tyrosine phosphorylation of the p38 MAP kinase immunoprecipitated was determined by immunoblot with anti-phosphotyrosine mouse monoclonal antibody. To obtain the substrate for the kinase assay as a GST-c-Jun fusion protein, the procedure of Smith and Corcoran (41Smith D.B. Corcoran L.M. Ausubel F.A. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1994: 16.7.1-16.7.7Google Scholar) was followed. For this purpose, transformed XL1-blue cells containing a pGEX-2T plasmid encoding residues 1–223 of the N-terminal portion of c-Jun protein were grown in LB/ampicillin medium. The expression of the fusion protein was induced by addition of 1 mm isopropyl-1-thio-β-d-galactoside. Cells were lysed using a probe sonicator and the fusion protein purified with glutathione-agarose beads. The cytosolic extracts for the kinase assay were obtained from the lysis of 5 × 106 1321N1 cells in 200 μl of a medium containing 25 mm Hepes, 0.3m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.1% Triton X-100, 1 mmphenylmethylsulfonyl fluoride, 100 mm orthovanadate, 20 mm β-glycerophosphate, 10 μg/ml leupeptin, and 10 μg/ml aprotinin, pH 7.7. After centrifugation at 12,000 rpm at 4 °C, the supernatant was diluted in 600 μl of the above mentioned medium without NaCl, and mixed with 10 μg of GST-c-Jun protein and glutathione-agarose beads. The mixture was incubated under continuous shaking for 3–5 h at 4 °C, and then washed to remove the fraction not associated to the glutathione-agarose beads. The kinase reaction was carried out with 20 mm ATP and 5 μCi of [γ-32P]ATP in a volume of 30 μl. The reaction was diluted in buffer and centrifugated to discard supernantant and then boiled in Laemmli SDS sample buffer and DTT. Phosphorylated GST-c-Jun was resolved by 10% SDS-PAGE and detected by autoradiography. Quantitation of the phosphorylation was carried out by densitometric scanning. Incubation of 1321N1 cells with sPLA2 at concentrations of 10 ng to 0.4 μg induced the release of [3H]AA into the medium (TableI, Fig.1 A). This release was similar to that produced by agonists acting on membrane receptors on this cell line, namely carbachol (25Bayón Y. Hernández M. Alonso A. Núñez L. Garcı́a-Sancho J. Leslie C.C. Sánchez Crespo M. Nieto M.L. Biochem. J. 1997; 323: 281-287Crossref PubMed Scopus (59) Google Scholar), thrombin (26Hernández M. Bayón Y. Sánchez Crespo M. Nieto M.L. Biochem. J. 1997; 328: 263-269Crossref PubMed Scopus (37) Google Scholar), and LPA (Fig.1 B). Astrocytes labeled with [1-14C]oleic acid were treated with sPLA2 under the same conditions used for the assay of [3H]AA release. As shown in Table I, no significant release of [1-14C]oleic acid was observed. Since sPLA2 produces mitogenesis in astrocytes (9Arita H. Hanasaki K. Hanako T. Oda S. Teraoka H. Matsumoto K. J. Biol. Chem. 1991; 266: 19139-19141Abstract Full Text PDF PubMed Google Scholar), we addressed whether this response was also elicited in quiescent 1321N1 cells, using 10% fetal calf serum as a positive control and thrombin as a prototypic mitogenic agonist of this cell line (42Post G.R. Collins L.R. Kennedy E.D. Moskowitz S.A. Aragay A.M. Goldstein D. Brown J.H. Mol. Biol. Cell. 1996; 7: 1679-1690Crossref PubMed Scopus (67) Google Scholar). As shown in Table II, sPLA2 behaved as a mitogenic agonist somewhat more potent than thrombin.Table IEffect of sPLA 2 on [3 H]AA and [1- 14 C]oleate release by 1321N1 cellssPLA2[3H]AA[1-14C]Oleateμg/mldpmdpm0876 ± 123289 ± 230.011435 ± 345267 ± 320.12139 ± 197278 ± 560.42646 ± 424301 ± 79 Open table in a new tab Table IIIncorporation of [3 H]thymidine into acid-precipitable materialStimulus[3H]Thymidine%None12 ± 4Serum100 ± 0Thrombin (0.5 unit/ml)45 ± 13sPLA2 (0.1 μg/ml)51 ± 7 Open table in a new tab Since cPLA2 is the most specific enzyme that releases arachidonate from phospholipids, and sPLA2 does not display selectivity for any unsaturated fatty acid on the sn-2 position of phospholipids (43Seilhamer J.J. Pruzanski W. Vadas P. Plant S. Miller J.A. Kloss J. Johnson L.K. J. Biol. Chem. 1989; 264: 5335-5338Abstract Full Text PDF PubMed Google Scholar, 44Kramer R.M. Hession C. Johansen B. Hayes G. McGray P. Chow E.P. Tizard R. Pepinsky R.B. J. Biol. Chem. 1989; 264: 5768-5775Abstract Full Text PDF PubMed Google Scholar), sPLA2 responses might a priori reflect either a direct consequence of its catalytic activity or recruitment of the arachidonate-selective enzyme cPLA2. Considering that 1321N1 cells contain cPLA2 as the main PLA2activity detected in cell-free homogenates and the implication of this activity in the mobilization of [3H]AA produced by receptor stimulation (25Bayón Y. Hernández M. Alonso A. Núñez L. Garcı́a-Sancho J. Leslie C.C. Sánchez Crespo M. Nieto M.L. Biochem. J. 1997; 323: 281-287Crossref PubMed Scopus (59) Google Scholar, 26Hernández M. Bayón Y. Sánchez Crespo M. Nieto M.L. Biochem. J. 1997; 328: 263-269Crossref PubMed Scopus (37) Google Scholar), we hypothesized that activation cPLA2 could explain the release of [3H]AA triggered by sPLA2. The increase in catalytic activity of the 85-kDa PLA2 has been linked to phosphorylation of the enzyme, which results in reduced mobility upon electrophoresis. As shown in Fig. 2 A, 0.1 μg/ml sPLA2 purified from the plasma of patients with septicemia induced phosphorylation of cPLA2. The activation of this protein shows a time course that clearly precedes [3H]AA release. Maximal amount of P-cPLA2 was achieved within 10–15 min and was maintained up to 30 min after cellular stimulation. Interestingly, phosphorylation of the p42 MAP kinase preceded cPLA2 phosphorylation, since it was near maximal values at 5 min (Fig. 2 A). In vitro kinase assay of c-Jun kinase activity in lysates from cells stimulated with sPLA2showed an increase of the activity that peaked about 10 min after addition of sPLA2 (Fig.3 A). Blotting with anti-phosphotyrosine antibody of the immunoprecipitate obtained with anti-p38 MAP antibody in lysates from 1321N1 cells, showed an increase of tyrosine phosphorylation of p38 MAP kinase of about 4-fold, 2 min after addition of sPLA2 (Fig. 3 B), thus suggesting that sPLA2 activates all the subgroups of the MAP kinase family following different time courses.Figure 3Effect of sPLA2 on c-Jun kinase activity 2nd on p38 MAP kinase phosphorylation. 1321N1 cells were incubated with sPLA2 for the times indicated, and at the end of these periods, cell lysates were collected and used for in vitro assay of c-Jun kinase activity using GST-c-Jun protein and glutathione-agarose beads. Heat shock (HS) was used as a positive control for c-Jun kinase activation. This was carried out by incubating the cells for 2 min in medium preheated at 45 °C, followed by incubation at 37 °C. Quantitative analysis of the phosphorylation of GST-c-Jun was obtained by densitometric scanning and is expressed as fold-increase of basal activity (A). The experiment shown in B was carried out by immunoprecipitation of p38 MAP kinase from cell lysates, SDS-PAGE separation of the immunoprecipitate, and blotting with anti-phosphotyrosine antibody. The histogram shows the densitometric scanning of the blots.P-Y-p38-MAP indicates p38 MAP kinase phosphorylated in tyrosine.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We also investigated the effect of human recombinant sPLA2isolated from permanent transfected C127 fibroblasts. Stimulation of astrocytes with concentrations of human recombinant sPLA2above 0.1 μg/ml also resulted in a shift of the electrophoretic mobility of cPLA2 (Fig. 4). Similarly, the addition of type I PLA2 (pancreatic PLA2, 0.8–8 μg/ml) to 1321N1 astrocytoma cells also increased the amount of P-cPLA2 detected upon electrophoresis (Fig. 4). To confirm that the observed increase of the cPLA2 phosphorylation was due to sPLA2 rather than linked to a possible lipopolysaccharide contamination in the sPLA2 preparation from septic patients, we treated our cells with 10 μg/ml lipopolysaccharide. SDS-PAGE revealed that lipopolysaccharide is not able to induce cPLA2phosphorylation (data not shown), thus ruling out the view that the observed activation of cPLA2 could be due to contamination by lipopolysaccharide. Having established that addition of either of the two secreted forms of PLA2 induced phosphorylation of cPLA2, we hypothesized two possible mechanisms either a direct action of sPLA2 on its receptor or an indirect effect through lipid mediators generated as a consequence of its catalytic activity. Since LPA is a mitogenic agonist (reviewed in Ref. 45Moolenaar W.H. J. Biol. Chem. 1995; 270: 12949-12952Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar) and a product of sPLA2 (14Fourcade O. Simon M.F. Viode C. Rugani N. Leballe F. Ragab A. Fournie B. Sarda L. Chap H. Cell. 1995; 80: 919-927Abstract Full Text PDF PubMed Scopus (496) Goog
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