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Mitogen-activated Protein (MAP) Kinase Regulates Production of Tumor Necrosis Factor-α and Release of Arachidonic Acid in Mast Cells

1997; Elsevier BV; Volume: 272; Issue: 20 Linguagem: Inglês

10.1074/jbc.272.20.13397

1083-351X

Cheng Zhang, Rudolf A. Baumgartner, Koji Yamada, Michael A. Beaven,

PI3K/AKT/mTOR signaling in cancer

Aggregation of the high affinity IgE receptor (FcεRI) in a mast cell line resulted in activation of the p42 and the stress-activated p38 mitogen-activated protein (MAP) kinases. Selective inhibition of these respective kinases with PD 098059 and SB 203580 indicated that p42 MAP kinase, but not p38 MAP kinase, contributed to the production of the cytokine, tumor necrosis factor-α, and the release of arachidonic acid in these cells. Neither kinase, however, was essential for FcεRI-mediated degranulation or constitutive production of tumor growth factor-β. Studies with SB 203580 and the p38 MAP kinase activator anisomycin also revealed that p38 MAP kinase negatively regulated activation of p42 MAP kinase and the responses mediated by this kinase. Aggregation of the high affinity IgE receptor (FcεRI) in a mast cell line resulted in activation of the p42 and the stress-activated p38 mitogen-activated protein (MAP) kinases. Selective inhibition of these respective kinases with PD 098059 and SB 203580 indicated that p42 MAP kinase, but not p38 MAP kinase, contributed to the production of the cytokine, tumor necrosis factor-α, and the release of arachidonic acid in these cells. Neither kinase, however, was essential for FcεRI-mediated degranulation or constitutive production of tumor growth factor-β. Studies with SB 203580 and the p38 MAP kinase activator anisomycin also revealed that p38 MAP kinase negatively regulated activation of p42 MAP kinase and the responses mediated by this kinase. Stimulation of mast cells by aggregation of membrane IgE receptors (FcεRI), leads to recruitment of the tyrosine kinase Syk and activation of Syk-dependent signaling cascades (1Scharenberg A.M. Lin S. Cuenod B. Yamamura H. Kinet J.P. EMBO. J. 1995; 14: 3385-3394Crossref PubMed Scopus (143) Google Scholar, 2Hirasawa N. Scharenberg A. Yamamura H. Beaven M.A. Kinet J.-P. J. Biol. Chem. 1995; 270: 10960-10967Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). These cascades include activation of phospholipase C and sphingosine kinase for mobilization of calcium ions and PKC 1The abbreviations used are: PKC, protein kinase C; PLA2, phospholipase A2; cPLA2, cytosolic PLA2; MAP, mitogen-activated protein; FcεRI, high affinity receptor for IgE; DNP-BSA, antigen consisting of 24 molecules of O-dinitrophenol conjugated with one molecule of bovine serum albumin; TNFα and -β, tumor necrosis factor-α and -β, respectively; TGFβ, transforming growth factor-β; PIPES, 1,4-piperazinediethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; MEK, MAP kinase kinase. (3Choi O.H. Kim J.-H. Kinet J.-P. Nature. 1996; 380: 634-636Crossref PubMed Scopus (385) Google Scholar, 4Beaven M.A. Curr. Biol. 1996; 6: 798-801Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and the activation of p42 MAP kinase cascade through Ras (2Hirasawa N. Scharenberg A. Yamamura H. Beaven M.A. Kinet J.-P. J. Biol. Chem. 1995; 270: 10960-10967Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 5Jabril-Cuenod B. Zhang C. Scharenberg A.M. Paolini R. Numerof R. Beaven M.A. Kinet J.-P. J. Biol. Chem. 1996; 271: 16268-16272Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). These cascades lead ultimately to secretion of intracellular granules, a response primarily driven by the increase in [Ca2+]i and activation of PKC (6Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Abstract Full Text PDF PubMed Google Scholar), and a cPLA2-mediated release of arachidonic acid. The activation of cPLA2 is dependent on increase of [Ca2+]i and phosphorylation by MAP kinase (2Hirasawa N. Scharenberg A. Yamamura H. Beaven M.A. Kinet J.-P. J. Biol. Chem. 1995; 270: 10960-10967Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 7Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1995; 154: 5391-5402PubMed Google Scholar,8Nakatani Y. Murakami M. Kudo I. Inoue K. J. Immunol. 1994; 153: 796-803PubMed Google Scholar). Stimulated mast cells also produce a variety of cytokines that include interleukins 1, 3, 4, 5, and 6 as well as TNFα and granulocyte-macrophage colony-stimulating factor (9Baumgartner R.A. Beaven M.A. Herzenberg L.A. Herzenberg L. Weir D.M. Blackwell C. Handbook of Experimental Immunology. 5th Ed. 4. Blackwell Science, Inc., Cambridge, MA1996: 213.1-213.8Google Scholar, 10Galli S.J. N. Eng. J. Med. 1993; 328: 257-265Crossref PubMed Scopus (911) Google Scholar). Typically, increased expression of cytokine mRNA and protein is detectable 30 min to several hours after the addition of stimulant (11Gordon J.R. Burd P.R. Galli S.J. Immunol. Today. 1990; 11: 458-464Abstract Full Text PDF PubMed Scopus (173) Google Scholar). These cytokines, particularly TNFα, are thought to mediate pathologic inflammatory reactions (10Galli S.J. N. Eng. J. Med. 1993; 328: 257-265Crossref PubMed Scopus (911) Google Scholar) and protective responses to bacterial infection (12Galli S.J. Wershil B.K. Nature. 1996; 381: 21-22Crossref PubMed Scopus (177) Google Scholar). The production and release of TNFα are regulated through signals transduced by calcium and PKC, although there are indications that additional FcεRI-mediated signals may operate for optimal production of TNFα in cultured RBL-2H3 mast cells. Compared with antigen, other stimulants are relatively weak inducers of TNFα production when doses of stimulants are matched for maximal stimulation of degranulation (13Baumgartner R.A. Yamada K. Deramo V.A. Beaven M.A. J. Immunol. 1994; 153: 2609-2617PubMed Google Scholar). Also, concentrations of Ro31–7549 that block PKC, secretion of granules, and release of TNFα only partially block production of TNFα (13Baumgartner R.A. Yamada K. Deramo V.A. Beaven M.A. J. Immunol. 1994; 153: 2609-2617PubMed Google Scholar). The present objective was to determine whether stimulation of MAP kinases induces additional signals for production of TNFα. A linkage between these events has not been established in mast cells. Antigen-induced stimulation of p42 MAP kinase coincides with the activation of its upstream regulators, Ras, Raf, and MEK-1 (2Hirasawa N. Scharenberg A. Yamamura H. Beaven M.A. Kinet J.-P. J. Biol. Chem. 1995; 270: 10960-10967Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 5Jabril-Cuenod B. Zhang C. Scharenberg A.M. Paolini R. Numerof R. Beaven M.A. Kinet J.-P. J. Biol. Chem. 1996; 271: 16268-16272Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), and persists through the period when production of TNFα would be most apparent (14Zhang C Hirasawa N Beaven M. A J. Immunol. in press, 1997: 158Google Scholar). As noted in this paper, however, RBL-2H3 cells also possess the mammalian homologue of the yeast HOG-1 protein kinase, p38 MAP kinase. We have utilized the MEK-1 inhibitor, PD 098059 (15Dudley 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 (2593) Google Scholar, 16Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3256) Google Scholar), and the p38 MAP kinase inhibitor, SB 203580 (17Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Stickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3138) Google Scholar), to evaluate the role of these MAP kinases in the production of TNFα and, for comparison, the release of arachidonic acid, degranulation, and production of TGFβ. Release of arachidonic acid is thought to be dependent on phosphorylation of cPLA2 by MAP kinase, although the identity of the MAP kinase is uncertain (18Kramer R.M. Roberts E.F. Strifler B.A. Johnstone E.M. J. Biol. Chem. 1995; 270: 27395-27398Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Degranulation and TGFβ production were assumed to be MAP-kinase-independent responses (7Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1995; 154: 5391-5402PubMed Google Scholar, 19Baumgartner R.A. Deramo V.A. Beaven M.A. J. Immunol. 1996; 157: 4087-4093PubMed Google Scholar). We show that, while p42 MAP kinase regulated production of both TNFα and arachidonic acid, p38 MAP kinase negatively regulated the activation of p42 MAP kinase and the responses mediated by this kinase. Reagents were obtained from the following sources: all reagents for cell culture from Life Technologies, Inc.; ATP from Boehringer Mannheim; adenosine 5′-[γ-32P]triphosphate tetra(triethyl-ammonium) salt and [14C]arachidonic acid from DuPont NEN; phenyl-Sepharose from Pharmacia (Uppsala, Sweden); MAP kinase substrates (myelin basic protein and a myelin basic peptide, residues 94–102), polyclonal antibodies against the COOH-terminal peptide of rat MAP kinase R2 (erk1-CT), anti-phosphotyrosine monoclonal antibody, 4G10, and p42 MAP kinase glutathione S-transferase fusion protein from Upstate Biotechnology Inc. (Lake Placid, NY); polyclonal antibodies against p38 MAP kinase, MEK, and cPLA2 from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); TGFβ 1 kit and Factor-testTM Mouse TNF-α from Genzyme Corp. (Cambridge, MA). Ro31–7549 was obtained from LC Laboratories. The compounds PD 098059 and SB 203580 were synthesized in the Tsukuba Research Laboratories, Eisai Co., according to the procedures of Bridges et al. (20Bridge A.J. Saltiel A.R. et al.U. S. Patent.. 1995; 5: 525-625Google Scholar) and Adams et al. (21Adams, J. L. (1993) International Patent Application WO 93/14081Google Scholar), respectively, and purified by column chromatography and recrystallization. These compounds were determined to be >95% pure on the basis of high performance liquid chromatography and NMR analysis. The antigen, DNP-BSA, and O-dinitrophenol-specific monoclonal IgE were kindly supplied by Dr. Henry Metzger (NIAMS, National Institutes of Health, Bethesda, MD). The RBL-2H3 cell line was maintained in complete growth medium (minimum essential medium) supplemented with 15% fetal calf serum, glutamine, antibiotic, and antimycotic agents. Trypsinized cells were plated into 150-mm culture dishes or six-well Costar cluster plates and were incubated overnight in complete growth medium withO-dinitrophenol-specific IgE (0.5 μg/ml) and, for measurement of arachidonic acid release, [14C]arachidonic acid (0.1 μCi/ml). Cultures were washed the next day and replenished with the required medium. For the assay of hexosaminidase or [14C]arachidonic acid, experiments were performed in a PIPES-buffered medium (25 mm PIPES, pH 7.2, 159 mm NaCl, 5 mm KCl, 0.4 mmMgCl2, 1.0 mm CaCl2, 5.6 mm glucose, and 0.1% fatty acid-free fraction V bovine serum albumin). For [32P]phosphorylation of proteins, cultures were incubated for 90 min with 32P-labeled orthophosphate in PIPES-buffered medium exactly as described (7Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1995; 154: 5391-5402PubMed Google Scholar). For all other assays, experiments were performed in complete growth medium supplemented with 15% fetal calf serum (for measurement of TNFα), 5% fetal calf serum (for measurement of TGFβ), or 0.1% bovine serum albumin and 25 mm Hepes, pH 7.2 (for assay of MAP kinases and separation of proteins by immunoprecipitation and electrophoresis). The inhibitors were added either 30 min (PD 098059) or 15 min (SB 203580 and indomethacin) before stimulation of cultures with antigen (DNP-BSA) as described in the figure legends. Release of the granule marker, hexosaminidase, was determined by colorimetric assay of medium and cell lysates by previously described procedures (6Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Abstract Full Text PDF PubMed Google Scholar). For measurement of release of arachidonic acid, cells were labeled to equilibrium with [14C]arachidonic acid before the addition of inhibitors and antigen as described above. Reactions were terminated by placing cultures on ice and rapidly removing medium. The medium was briefly centrifuged (Beckman Microfuge for 30 s) to remove extraneous cells. Both medium and cell lysates (in 0.1% Triton X-100) were assayed for hexosaminidase (6Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Abstract Full Text PDF PubMed Google Scholar) and radiolabel (22Collado-Escobar D. Cunha-Melo J.R. Beaven M.A. J. Immunol. 1990; 144: 244-250PubMed Google Scholar). Values were expressed as the percentage of intracellular hexosaminidase or radiolabel that was released into the external medium, and they were corrected for spontaneous release from unstimulated cells. It should be noted that, in RBL-2H3 cells, arachidonic acid is metabolized in part to leukotriene C4/B4 and prostaglandin D2 via the 5-lipoxygenase and cyclooxygenase pathways, respectively (23McGivney A. Morita Y. Crews F.T. Hirata F. Axelrod J. Siraganian R.P. Arch. Biochem. Biophys. 1981; 212: 572-580Crossref PubMed Scopus (68) Google Scholar, 24Igarashi Y. Lundgren J.D. Shelhamer J.H. Kaliner M.A. White M.V. Immunopharmacology. 1993; 25: 131-144Crossref PubMed Scopus (12) Google Scholar). Release of radiolabel, as measured in this paper, was an estimate of total release of [14C]arachidonic acid and its metabolites. The cytokines were assayed as described elsewhere (19Baumgartner R.A. Deramo V.A. Beaven M.A. J. Immunol. 1996; 157: 4087-4093PubMed Google Scholar). Whole cell lysates were prepared by freezing and thawing the cultures three times. TGFβ was assayed with a human TGFβ enzyme-linked immunosorbent assay kit, which utilized a mouse monoclonal anti-human antibody that cross-reacted with rat TGFβ. TNFα was assayed with a murine TNFα enzyme-linked immunosorbent assay kit, which utilized a monoclonal hamster anti-murine antibody that reacted with mouse or rat TNFα and -β. The limits of detection for these assays were 25 pg of TGFβ/106 cells and 6 pg of TNFα/106 cells. Values were corrected for spontaneous release in the absence of stimulant (≤3% for release of hexosaminidase, ≤2% for release of arachidonic acid, and undetectable release of TNFα) except for TGFβ, which was produced constitutively in RBL-2H3 cells (19Baumgartner R.A. Deramo V.A. Beaven M.A. J. Immunol. 1996; 157: 4087-4093PubMed Google Scholar). After stimulation of cultures in six-well cluster plates, the cultures were washed once, and the medium was removed. The plates were then placed on ice before the addition of 510 μl of a Tris buffer (25 mmTris, pH 7.5, 25 mm NaCl, 1 mmNa3VO4, 2 mm EGTA, 1.5 mm dithiothreitol, 2.5 mm p-nitrophenyl phosphate, and 20 μg/ml leupeptin and aprotinin). Cells were disrupted by freezing and thawing three times. The lysate was centrifuged (15,800 × g for 10 min), and 450 μl of the supernatant fraction was mixed with 50 μl of ethylene glycol and 80 μl of washed phenyl-Sepharose. The phenyl-Sepharose was washed beforehand with 300 μl of the Tris buffer. The mixture was kept on ice for 5 min for binding of MAP kinase to the beads. After centrifugation, the phenyl-Sepharose beads were washed with 1 ml of 10% (v/v) ethylene glycol and then with 30% (v/v) ethylene glycol. Finally, MAP kinase was eluted by incubating the beads with 75 μl of 60% ethylene glycol for 5 min on ice. After centrifugation of the suspension, 15 μl of supernatant was incubated (15 min, 30 °C) in a solution that contained 50 mm Tris (pH 7.5), 10 mm MgCl2, [γ-32P]ATP (10 Ci/mmol, 37 kBq/tube), and 25 μg of MAP kinase substrate peptide (peptide 94–102 of bovine myelin basic protein). The phosphorylated peptide was isolated by centrifugation of the incubation mixture through phosphocellulose membrane (SpinZyme; Pierce), which was then washed twice with 500 μl of 75 mmH3PO4 for the assay of radioactivity. p42 and p38 MAP kinases were immunoprecipitated with the appropriate polyclonal antibodies by procedures described elsewhere (2Hirasawa N. Scharenberg A. Yamamura H. Beaven M.A. Kinet J.-P. J. Biol. Chem. 1995; 270: 10960-10967Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). Equal amounts of immunoprecipitated proteins from 5 × 106 cells were incubated in a MOPS buffer (25 mm β-glycerol phosphate, 1 mmEGTA, 1 mm sodium orthovanadate, 1 mmdithiothreitol, and 25 mm MOPS, pH 7.2) with Mg2+-[γ-32P]ATP (10 μCi in 150 μm cold ATP, 25 μm MgCl2) and 5 μm myelin basic protein (18 kDa) as substrate in a final volume of 30 μl. The mixture was incubated at 30 °C for 12 min. The reaction was terminated by the addition of 30 μl of 2 × SDS sample buffer. MEK was immunoprecipitated with anti-MEK antibody and assayed similarly except that p42 MAP kinase glutathioneS-transferase fusion protein (1 μg/assay) was used as substrate for phosphorylation. Proteins were separated by 12% SDS-PAGE. Radioactive proteins were detected by autoradiography. The preparation of cell lysates and immunoprecipitates, analysis of proteins by SDS-PAGE, and transfer to nitrocellulose paper were performed as described elsewhere (2Hirasawa N. Scharenberg A. Yamamura H. Beaven M.A. Kinet J.-P. J. Biol. Chem. 1995; 270: 10960-10967Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 7Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1995; 154: 5391-5402PubMed Google Scholar) with the following exception: cPLA2 was separated on NOVEX 10% Tris/glycine gels for 3 h at 35 mA and 4 °C as described by Kramer and co-workers (25Kramer R.M. Roberts E.F. Um S.L. Borsch-Haubold A.G. Watson S.P. Fisher M.J. Jakubowski J.A. J. Biol. Chem. 1996; 271: 27723-27729Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Previously described procedures were used for isolation and detection of [32P]MEK (7Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1995; 154: 5391-5402PubMed Google Scholar). Otherwise, proteins were detected by the immunoblotting technique with antibodies against MEK, p42 MAP kinase, cPLA2, or anti-phosphotyrosine. Secondary antibodies included horseradish peroxidase-conjugated antibody against rabbit IgG or mouse IgG. Finally, proteins were visualized by the ECL System (Amersham Corp.) or by autoradiography. As shown in Fig. 1, the MEK inhibitor PD 098059 attenuated antigen-induced [32P]phosphorylation of MEK (panel A) and the activation of MEK as determined by in vitroassay of immunoprecipitated MEK (panel B). Activation of p42 MAP kinase was also attenuated, as indicated by the change in electrophoretic migration of p42 MAP kinase (panel C) or by the assay of MAP kinase activity of immunoprecipitated p42 MAP kinase (panel D). The extent of these inhibitions was dependent on the concentration of PD 098059. As shown in Fig. 2, the suppression of MAP kinase activation by PD 098059 (panel A) was associated with similar dose-dependent suppression of arachidonic acid release (panel B) and TNFα production (panel C). The suppression of the latter two responses was highly correlated (r > 0.95). All three responses were inhibited by ∼50% with 10 μm PD 098059. As will be described later, activation of cPLA2 was also inhibited by PD 098059. These results suggested that release of arachidonic acid and production of TNFα were both regulated by p42 MAP kinase.Figure 2Suppression of MAP kinase-dependent responses by PD 098059. A, cultures were incubated with the indicated concentrations of PD 098059 for 30 min and then either left unstimulated (Unstim.) or stimulated for 5 min with 20 ng/ml DNP-BSA. MAP kinase activity was assayed in whole cell extracts as described under "Materials and Methods." B, cultures were incubated with the indicated concentrations of PD 098059 for 30 min and then stimulated with 20 ng/ml DNP-BSA for 15 min for measurement of release of arachidonic acid. C, cultures were incubated for 10 min with PD098059 and then stimulated for 135 min with 20 ng/ml DNP-BSA for measurement of production of TNFα (C) as described under "Materials and Methods." The values for this and all other figures were mean (±S.E.) values from three separate experiments (two cultures in each).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To test the selectivity of PD 098059, we next examined the effects of this compound on antigen-stimulated degranulation and the constitutive production of TGFβ, which are thought not to be regulated by MAP kinase (7Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1995; 154: 5391-5402PubMed Google Scholar, 19Baumgartner R.A. Deramo V.A. Beaven M.A. J. Immunol. 1996; 157: 4087-4093PubMed Google Scholar). PD 098059 had only minimal effects on stimulated release of the granule constituent, hexosaminidase (Fig.3 A) and the production of TGFβ in unstimulated cells (Fig. 3 B). The only significant effect was partial inhibition (<30%) of degranulation at 50 μmPD 098059 (Fig. 3 A). Antigen stimulation also resulted in increased activity of p38 MAP kinase (Fig.4 A, compare lanes 1 and2). The p38 kinase inhibitor, SB 203580, inhibited this activation (Fig. 4 A, lanes 3 and 4). Interestingly, antigen activation of p42 MAP kinase was enhanced significantly by SB 203580. This enhancement was apparent when cells were stimulated with 20 or 200 ng/ml antigen (Fig. 4 B). The latter concentration of antigen was known to elicit maximal activation of p42 MAP kinase. 2C. Zhang, R. A. Baumgartner, K. Yamada, and M. A. Beaven, unpublished data. These results suggested that p38 MAP kinase negatively regulates p42 MAP kinase and that this regulation is alleviated by SB 203580. The enhanced activation of p42 MAP kinase in the presence of SB 203580 was associated with increased release of arachidonic acid (Fig.5 A) and production of TNFα (Fig.5 B). In the experiment shown in Fig. 5 B, cells were stimulated with a low concentration of antigen (6 ng/ml) to maximize enhancement of the TNFα response (250% increase in Fig.5 B). At optimal doses of antigen enhancement of TNFα production was less (40–80% increase) but still significant (data not shown). Because pyridinyl imidazoles that are closely related to SB 203580 have cyclooxygenase-inhibitory activity (25Kramer R.M. Roberts E.F. Um S.L. Borsch-Haubold A.G. Watson S.P. Fisher M.J. Jakubowski J.A. J. Biol. Chem. 1996; 271: 27723-27729Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 26Lee J.C. Badger A.M. Griswold D.E. Dunnington D. Truneh A. Votta B. White J.R. Young P.R. Bender P.E. Ann. N. Y. Acad. Sci. 1993; 696: 149-170Crossref PubMed Scopus (154) Google Scholar), experiments were conducted to determine whether blockade of cyclooxygenase activity with indomethacin (27Vane J.R. Nat. New. Biol. 1971; 231: 232-235Crossref PubMed Scopus (7348) Google Scholar) altered accumulation of radiolabel in the medium by suppressing metabolism [14C]arachidonate via this enzyme. Unlike SB 203580, 10 μm indomethacin did not significantly alter release of radiolabel from antigen-stimulated cells (7.8 ± 0.4% release over 15 min versus 7.2 ± 0.2% release in the absence of indomethacin; mean ± S.E. in eight cultures from two experiments). It seemed probable, therefore, that SB 203580 enhanced release rather than the accumulation of [14C]arachidonic acid in the medium. In contrast to the increased release of arachidonic acid and production of TNFα, SB 203580 had no significant effect on antigen-induced degranulation (Fig. 5 C) or constitutive production of TGFβ (Fig. 5 D). Collectively, these results provided further evidence for the notion that release of arachidonic acid and TNFα production are both regulated by p42 MAP kinase. In addition, the results suggested that p38 MAP kinase negatively modulates these responses through p42 MAP kinase. The above results suggested that release of arachidonic acid, as well as production of TNFα, was regulated by p42 MAP kinase. As in other systems (25Kramer R.M. Roberts E.F. Um S.L. Borsch-Haubold A.G. Watson S.P. Fisher M.J. Jakubowski J.A. J. Biol. Chem. 1996; 271: 27723-27729Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar, 28Lin L.L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1657) Google Scholar), the phosphorylation of cPLA2 in stimulated RBL-2H3 cells leads to decreased electrophoretic mobility of the enzyme (7Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1995; 154: 5391-5402PubMed Google Scholar). The connection between p42 MAP kinase and the release of arachidonic acid via cPLA2 was further demonstrated by the finding that the antigen-induced retardation of electrophoretic migration of cPLA2 (25Kramer R.M. Roberts E.F. Um S.L. Borsch-Haubold A.G. Watson S.P. Fisher M.J. Jakubowski J.A. J. Biol. Chem. 1996; 271: 27723-27729Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar) was suppressed by PD 098059 but not by SB 203580 (Fig. 6). The p38 MAP kinase activator, anisomycin, markedly activated this kinase (Fig. 7 A) but much less so p42 MAP kinase (Fig. 7 B). The combination of anisomycin and antigen revealed inhibitory communication between these two kinase. For example, the combination of stimulants resulted in less activation of p38 MAP kinase (Fig. 7 C, lane 3) than that induced by antigen (Fig. 7 C, lane 2) or anisomycin (Fig. 7 A, lane 2) alone. The combination also caused less activation of p42 MAP kinase (Fig.7 D, lane 3) than that by antigen alone (Fig.7 D, lane 2). Thus, stimulation of p42 MAP kinase by antigen appeared to block activation of p38 MAP kinase by anisomycin, and conversely stimulation of p38 MAP kinase by anisomycin appeared to partially block activation of p42 MAP kinase by antigen. Consistent with the latter situation, anisomycin partially suppressed antigen-induced release of arachidonic acid (25 ± 4% reduction, mean of three experiments). This reduction corresponded to an approximately 25% reduction in p42 MAP kinase activation as determined by densitometric analysis of the blots shown in Fig. 7 D and two other experiments. Anisomycin almost totally blocked (by 83 ± 4%) antigen-induced production of TNFα, probably as a consequence, however, of its known inhibitory actions on protein synthesis at the translation step (29Vazquez D. Mol. Biol. Biochem. Biophys. 1979; 130: 513-527Google Scholar). Presumably, de novo synthesis of TNFα would be especially sensitive to inhibitors of protein synthesis. Past studies have shown that the responses evoked by antigen in RBL-2H3 cells were dependent on calcium and signals generated through PKC or MAP kinase. These studies indicated, for example, that PKC regulated degranulation (6Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Abstract Full Text PDF PubMed Google Scholar) as well as the production and secretion of TNFα, although it appeared likely that additional FcεRI-mediated signals facilitated TNFα production (13Baumgartner R.A. Yamada K. Deramo V.A. Beaven M.A. J. Immunol. 1994; 153: 2609-2617PubMed Google Scholar). Activation of p42 MAP kinase, in contrast, was associated with phosphorylation of cPLA2 and release of arachidonic acid (2Hirasawa N. Scharenberg A. Yamamura H. Beaven M.A. Kinet J.-P. J. Biol. Chem. 1995; 270: 10960-10967Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 7Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1995; 154: 5391-5402PubMed Google Scholar). These studies, however, did not address the issue of whether other MAP kinases, such as p38 MAP kinase, regulated cPLA2. The present results demonstrate that both p38 and p42 MAP kinases are activated in antigen-stimulated cells. Activation of the latter kinase appears to be most closely related to release of arachidonic acid and production of TNFα. All three events are inhibited by the MEK inhibitor, PD 098059 (Fig. 2), and enhanced by the p38 MAP kinase inhibitor, SB 203580 (Figs. 4 and 5). Both compounds are reported to be selective inhibitors of MEK (i.e. PD 098059) and p38 MAP kinase (i.e. SB 203580) when tested against a wide range of kinases (15Dudley 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 (2593) Google Scholar, 16Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3256) Google Scholar, 17Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Stickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3138) Google Scholar). In addition, the enhancement of responses in the presence of SB 203580, in contrast to the attenuation of p42 MAP kinase activation by the p38 MAP kinase activator, anisomycin (Fig. 7), suggest that p38 MAP kinase negatively regulates activation of p42 MAP kinase and its associated responses. Antigen-stimulated degranulation and the constitutive production of TGFβ in RBL-2H3 cells are minimally affected by the inhibitors (Figs. 3 and 5). Collectively, the results support the notion that p42 MAP kinase regulates release of arachidonic acid and promotes an additional signal for stimulating TNFα production but does not regulate degranulation. Interestingly, the p38 MAP kinase inhibitor, SB 203580, was first identified as an inhibitor of cytokine biosynthesis in lipopolysaccharide-stimulated human monocytes (17Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Stickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3138) Google Scholar) and was subsequently shown to suppress TNFα production in lipopolysaccharide-injected mice (30Badger A.M. Bradbeer J.N. Votta B. Lee J.C. Adams J.L. Griswold D.E. J. Pharmacol. Exp. Ther. 1996; 279: 1453-1461PubMed Google Scholar). The compound also possessed anti-inflammatory activity in mouse models of arthritis (collagen- and adjuvant-induced), whereas cellular immune responses measured ex vivo were unaffected (30Badger A.M. Bradbeer J.N. Votta B. Lee J.C. Adams J.L. Griswold D.E. J. Pharmacol. Exp. Ther. 1996; 279: 1453-1461PubMed Google Scholar). It is possible, therefore, that different MAP kinase pathways are utilized for activating gene transcription for cytokine synthesis when synthesis is induced by inflammatory agents or through multimeric immunologic receptors such as FcεRI. The question has been raised whether p38 rather than p42 MAP kinase is responsible for the activation of cPLA2 (18Kramer R.M. Roberts E.F. Strifler B.A. Johnstone E.M. J. Biol. Chem. 1995; 270: 27395-27398Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 25Kramer R.M. Roberts E.F. Um S.L. Borsch-Haubold A.G. Watson S.P. Fisher M.J. Jakubowski J.A. J. Biol. Chem. 1996; 271: 27723-27729Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). cPLA2 is phosphorylated by both kinases in thrombin-stimulated platelets, although the phosphorylation by p38 MAP kinase does not appear to activate cPLA2 (25Kramer R.M. Roberts E.F. Um S.L. Borsch-Haubold A.G. Watson S.P. Fisher M.J. Jakubowski J.A. J. Biol. Chem. 1996; 271: 27723-27729Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). Our results indicate that p42 MAP kinase regulates phosphorylation of cPLA2 and release of arachidonic acid and suggest, therefore, that this kinase is the activator of cPLA2 at least in RBL-2H3 cells. Mast cells, including RBL-2H3 cells, also contain a low molecular weight secreted form (type II) of PLA2 (31Murakami M. Kudo I. Umeda M. Matsuzawa A. Takeda M. Komada M. Fujimoi Y. Takahashi K. Inoue K. J. Biochem. ( Tokyo ). 1992; 111: 175-181Crossref PubMed Scopus (71) Google Scholar) in secretory granules, and this form is presumably released along with other granule constituents in activated cells (32Chock S.P. Schmauder-Chock E.A. Cordella-Miele E. Miele L. Mukherjee A.B. Biochem. J. 1994; 300: 619-622Crossref PubMed Scopus (43) Google Scholar). A role for secreted PLA2 is unlikely, however, because total suppression of degranulation by selective inhibitors of PKC, such as Ro31–7549 and calphostin C, minimally affects release of arachidonic acid in RBL-2H3 cells (Refs. 7Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1995; 154: 5391-5402PubMed Google Scholar and 33Yamada K. Jelsema C.L. Beaven M.A. J. Immunol. 1992; 149: 1031-1037PubMed Google Scholar and see below). 3In addition to the cytosolic (cPLA2) and secreted (sPLA2) PLA2, a calcium-independent form of PLA2 (iPLA2) has been described in myocytes and smooth muscle cells (40Wolf M.J. Wang J. Turk J. Gross R.W. J. Biol. Chem. 1997; 272: 1522-1526Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). iPLA2 is inhibited by bromoenol lactone (41Ackermann E.J. Conde-Frieboes K. Dennis E.A. J. Biol. Chem. 1995; 270: 445-450Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar) and activated by depletion of intracellular calcium stores or by the calmodulin inhibitor, W7 (40Wolf M.J. Wang J. Turk J. Gross R.W. J. Biol. Chem. 1997; 272: 1522-1526Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). By use of the same experimental strategies, we find no evidence for the presence of iPLA2 in RBL-2H3 cells (T. Hundley and M. A. Beaven, unpublished data). In addition to the correlations between activation of the p42 MAP kinase/cPLA2 pathway and release of arachidonic acid noted here with PD 098059 and SB 203580, similar correlations have been noted in previous studies with less specific MAP kinase inhibitors. Activation of the entire Raf/MEK/p42 MAP kinase pathway and release of arachidonic acid were suppressed equally by the glucorticoid, dexamethasone, and the kinase inhibitor, quercetin, while effects on degranulation were apparent only at relatively high concentrations of these agents (7Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1995; 154: 5391-5402PubMed Google Scholar, 34Rider L.G. Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1996; 157: 2374-2380PubMed Google Scholar). Correlations were noted with the PKC inhibitor, Ro31–7549. This inhibitor transiently delayed activation of p42 MAP kinase in antigen-stimulated RBL-2H3 cells. There was a corresponding transient delay in the release of arachidonic acid, although the cumulative release eventually equaled that observed in the absence of Ro31–7549 (14Zhang C Hirasawa N Beaven M. A J. Immunol. in press, 1997: 158Google Scholar). On the basis of these and other results, we have suggested that the p42 MAP kinase/cPLA2 pathway, although transiently activated by PKC, was predominantly activated by Ras through recruitment of Shc/Grb2/Sos or Vav by FcεRI (14Zhang C Hirasawa N Beaven M. A J. Immunol. in press, 1997: 158Google Scholar). Others have reported that fatty acids, particularly arachidonic acid, activate p42 and p44 MAP kinases through PKC (35Rao G.N. Baas A.S. Glasgow W.C. Eling T.E. Runge M.S. Alexander R.W. J. Biol. Chem. 1994; 269: 32586-32591Abstract Full Text PDF PubMed Google Scholar). This scenario is unlikely in antigen-stimulated RBL-2H3 cells, however, because of the predominance of the PKC-independent (i.e. Ro31–7549-resistant) pathway in RBL-2H3 cells. The present data extend previous findings on the regulation of TNFα synthesis and release. This cytokine is synthesized de novoand subsequently secreted via Golgi in a PKC- and calcium-dependent manner (13Baumgartner R.A. Yamada K. Deramo V.A. Beaven M.A. J. Immunol. 1994; 153: 2609-2617PubMed Google Scholar). The PKC inhibitor, Ro31–7549, blocks secretion of TNFα but only partially suppresses synthesis of TNFα (6Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Abstract Full Text PDF PubMed Google Scholar, 13Baumgartner R.A. Yamada K. Deramo V.A. Beaven M.A. J. Immunol. 1994; 153: 2609-2617PubMed Google Scholar, 19Baumgartner R.A. Deramo V.A. Beaven M.A. J. Immunol. 1996; 157: 4087-4093PubMed Google Scholar). Thus, additional signals may be necessary for optimal stimulation of TNFα synthesis. Antigen is a particularly potent stimulant of TNFα production when compared with the combination of calcium ionophore and PKC agonist, phorbol 12-myristate 13-acetate (13Baumgartner R.A. Yamada K. Deramo V.A. Beaven M.A. J. Immunol. 1994; 153: 2609-2617PubMed Google Scholar, 19Baumgartner R.A. Deramo V.A. Beaven M.A. J. Immunol. 1996; 157: 4087-4093PubMed Google Scholar). These observations and the present studies with the MAP kinase inhibitors suggest that optimal production of TNFα is achieved through activation of both PKC and p42 MAP kinase. The present findings may explain why antigen-induced activation of p42 MAP kinase (34Rider L.G. Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1996; 157: 2374-2380PubMed Google Scholar), release of arachidonic acid (22Collado-Escobar D. Cunha-Melo J.R. Beaven M.A. J. Immunol. 1990; 144: 244-250PubMed Google Scholar, 34Rider L.G. Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1996; 157: 2374-2380PubMed Google Scholar) and production of TNFα (36Baumgartner R.A. Hirasawa N. Ozawa K. Gusovsky F. Beaven M.A. J. Immunol. 1996; 157: 1625-1629PubMed Google Scholar) exhibit similar sensitivity to dexamethasone. All three responses are totally suppressed in RBL-2H3 cells that have been treated with 10 nm dexamethasone, whereas antigen-stimulated hydrolysis of phosphoinositides, increase in [Ca2+]i, and degranulation (22Collado-Escobar D. Cunha-Melo J.R. Beaven M.A. J. Immunol. 1990; 144: 244-250PubMed Google Scholar, 34Rider L.G. Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1996; 157: 2374-2380PubMed Google Scholar) are only partially suppressed by treatment of cells with 100 nmdexamethasone. Dexamethasone, as noted above, inhibits the entire Raf/MEK/p42 MAP kinase/cPLA2 pathway at nanomolar concentrations (34Rider L.G. Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1996; 157: 2374-2380PubMed Google Scholar). Therefore, if p42 MAP kinase is the common regulator of TNFα production and arachidonic acid release, the similar sensitivities to dexamethasone would be expected. The connections between the p42 MAP kinase pathway and cytokine production are unknown for mast cells, but recent reports indicate the following connections in other types of cells. The overexpression of Raf1 (37Reimann T. Buscher D. Hipskind R.A. Krautwald S. Lohmann-Matthes M.L. Baccarini M. J. Immunol. 1994; 153: 5740-5749PubMed Google Scholar, 38Li S. Sedivy J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9247-9251Crossref PubMed Scopus (195) Google Scholar) or p42 MAP kinase (39Park J.H. Levitt L. Blood. 1993; 82: 2470-2477Crossref PubMed Google Scholar) results in enhanced expression of a variety of cytokine genes in T cells and macrophages (37Reimann T. Buscher D. Hipskind R.A. Krautwald S. Lohmann-Matthes M.L. Baccarini M. J. Immunol. 1994; 153: 5740-5749PubMed Google Scholar, 39Park J.H. Levitt L. Blood. 1993; 82: 2470-2477Crossref PubMed Google Scholar), the inactivation of IκB (38Li S. Sedivy J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9247-9251Crossref PubMed Scopus (195) Google Scholar), and the enhanced binding activity of cytokine transcription factors such as NF-κB and AP-1 (39Park J.H. Levitt L. Blood. 1993; 82: 2470-2477Crossref PubMed Google Scholar). In conclusion, the above results provide the first indication that p42 MAP kinase regulates antigen-mediated production of TNFα in a mast cell line and that p38 MAP kinase may negatively regulate the p42 MAP kinase/cytokine pathway. We can, for the first time, broadly define the regulatory pathways for all three functional responses of mast cells to antigen as follows. Along with elevated [Ca2+]i, the additional primary signals are as follows: for degranulation (6Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Abstract Full Text PDF PubMed Google Scholar) and secretion of newly formed TNFα (13Baumgartner R.A. Yamada K. Deramo V.A. Beaven M.A. J. Immunol. 1994; 153: 2609-2617PubMed Google Scholar), activation of PKC (6Ozawa K. Szallasi Z. Kazanietz M.G. Blumberg P.M. Mischak H. Mushinski J.F. Beaven M.A. J. Biol. Chem. 1993; 268: 1749-1756Abstract Full Text PDF PubMed Google Scholar); for cPLA2-mediated release of arachidonic acid, activation of p42 MAP kinase (Refs. 2Hirasawa N. Scharenberg A. Yamamura H. Beaven M.A. Kinet J.-P. J. Biol. Chem. 1995; 270: 10960-10967Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 7Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1995; 154: 5391-5402PubMed Google Scholar, and 34Rider L.G. Hirasawa N. Santini F. Beaven M.A. J. Immunol. 1996; 157: 2374-2380PubMed Google Scholar, and this paper); and for production of TNFα, the coactivation of PKC and p42 MAP kinase (Ref. 13Baumgartner R.A. Yamada K. Deramo V.A. Beaven M.A. J. Immunol. 1994; 153: 2609-2617PubMed Google Scholar and this paper).