title: Regulation of eicosanoid biosynthesis in the macrophage. Involvement of protein tyrosine phosphorylation and modulation by selective protein tyrosine kinase inhibitors.
abstract: The protein tyrosine kinase (PTK) inhibitor genistein has been demonstrated to inhibit platelet-activating factor-stimulated prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-primed P388D1 macrophage-like cells (Glaser et al., J Biol Chem 265: 8658-8664, 1990). Therefore, the role of PTK in eicosanoid biosynthesis was investigated in murine resident peritoneal macrophages using genistein and tyrphostin-25, selective PTK inhibitors. Genistein, a competitive inhibitor of ATP binding on PTK, inhibited PGE2 production (IC50 = 20 microM) in response to zymosan, calcium ionophore A23187, and phorbol myristate acetate stimulation. Genistein also inhibited leukotriene C4 (LTC4) production in response to zymosan and calcium ionophore A23187 (IC50 = 10 and 15 microM, respectively) stimulation. Tyrphostin-25, a competitive inhibitor of substrate binding on PTK, inhibited zymosan-stimulated PGE2 and LTC4 production, IC50 = 20 and 7 microM, respectively. Neither genistein nor tyrophostin-25 had any effect on human synovial fluid phospholipase A2 (PLA2) activity in vitro or on cyclooxygenase activity in the intact macrophage; however, tyrphostin-25 did affect 5-lipoxygenase activity (determined from the metabolism of exogenously applied arachidonic acid). These results suggest PTK-mediated phosphorylation as a common event in the signal transduction mechanisms of different stimuli which activate PLA2 for arachidonic acid release and subsequent eicosanoid biosynthesis. Immunoblot analyses of zymosan-stimulated peritoneal exudate cells with the phosphotyrosine monoclonal antibody clone 4G10 demonstrated an increase in protein phosphotyrosine levels in eight major protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis: p59, 71, 76, 90, 100, 112, 125 and 150. Maximal phosphorylation of these protein substrates occurred after 1-2 min stimulation. Zymosan and LPS stimulation of peritoneal exudate cells produced similar patterns of protein tyrosine phosphorylation. Zymosan-stimulated tyrosine phosphorylation was inhibited by tyrphostin-25 in a concentration-dependent manner between 10 and 60 microM, demonstrating a similar concentration response between effects on tyrosine phosphorylation and eicosanoid biosynthesis in the murine peritoneal macrophage. The use of selective PTK inhibitors suggests a common role for PTK and tyrosine phosphorylation in eicosanoid biosynthesis in the murine peritoneal macrophage.
authors.affiliation: Division of Immunopharmacology, Wyeth-Ayerst Research, Princeton, NJ 08543-8000.
authors.forename: K B
authors.initials: KB
authors.lastname: Glaser
authors.forename: A
authors.initials: A
authors.lastname: Sung
authors.forename: J
authors.initials: J
authors.lastname: Bauer
authors.forename: B M
authors.initials: BM
authors.lastname: Weichman
mesh_terms: D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism; D001202:Ascitic Fluid / Q000166:cytology; D015232:Dinoprostone / Q000096:biosynthesis; D015777:Eicosanoids / Q000096:biosynthesis*; D019833:Genistein; D007529:Isoflavones / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D051379:Mice; D009570:Nitriles / Q000494:pharmacology*; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D010766:Phosphorylation / Q000187:drug effects; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D013189:SRS-A / Q000096:biosynthesis; D014443:Tyrosine / Q000378:metabolism*; D020032:Tyrphostins; D015054:Zymosan / Q000494:pharmacology
chemical_list: D015777:Eicosanoids; D007529:Isoflavones; D008070:Lipopolysaccharides; D009570:Nitriles; D013189:SRS-A; D020032:Tyrphostins; C079836:tyrphostin 25; D014443:Tyrosine; D015054:Zymosan; D019833:Genistein; D001094:Arachidonate 5-Lipoxygenase; D011451:Prostaglandin-Endoperoxide Synthases; D011505:Protein-Tyrosine Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D015232:Dinoprostone