title: Epidermal growth factor enhances a microsomal 12-lipoxygenase activity in A431 cells.
abstract: 12-Hydroxyeicosatetraenoic acid (12-HETE) is formed from arachidonic acid either by 12-lipoxygenase or by a cytochrome P450 monooxygenase. 12-Lipoxygenase is generally localized in the soluble cytosolic fraction, and the cytochrome P450 monooxygenase is a microsomal enzyme. In this study, 12-HETE biosynthesis and the regulation of 12-HETE biosynthesis by epidermal growth factor (EGF) in A431 cells were investigated. 12-HETE was biosynthesized from arachidonic acid by the microsomal fraction of A431 cells, but not by the cytosolic fraction. The formation of 12-HETE was inhibited by 5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid, and caffeic acid. Nordihydroguaiaretic acid at 10(-4) M and 5,8,11,14-eicosatetraynoic acid at 10(-5) M almost completely inhibited its formation. However, the formation of 12-HETE was not affected by the presence of an NADPH-generating system, carbon monoxide, or SKF 525A. The biosynthetic 12-HETE was analyzed by chiral stationary phase high performance liquid chromatography and was highly enriched in (12S)-HETE. We therefore concluded that the enzyme responsible for the formation of (12S)-HETE in the microsomes of A431 cells is a 12-lipoxygenase. The microsomal 12-lipoxygenase of A431 cells belongs to the "leukocyte-type" enzyme as determined by substrate specificity and enzyme kinetics studies. The microsomal 12-lipoxygenase oxygenated linoleic acid much faster than the cytosolic platelet 12-lipoxygenase and is a "self-catalyzed inactivation" enzyme. Treatment of cells with 50 ng/ml EGF significantly induced microsomal 12-lipoxygenase activity. The lag period for the expression of the stimulatory effect of EGF on 12-lipoxygenase activity was approximately 10 h. The stimulatory effect of EGF on 12-lipoxygenase activity was completely blocked by treatment with 35 microM cycloheximide, indicating a requirement for de novo protein biosynthesis. Furthermore, the presence of the endogenous inhibitor of 12-lipoxygenase (which masked (12S)-HETE biosynthesis in intact cells) was identified in the cytosolic fraction of A431 cells. The putative inhibitor was enzyme-selective. It inhibited the leukocyte-type 12-lipoxygenase, but not the "platelet-type" enzyme.
authors.affiliation: Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China.
authors.forename: W C
authors.initials: WC
authors.lastname: Chang
authors.forename: C C
authors.initials: CC
authors.lastname: Ning
authors.forename: M T
authors.initials: MT
authors.lastname: Lin
authors.forename: J D
authors.initials: JD
authors.lastname: Huang
mesh_terms: D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D015117:5,8,11,14-Eicosatetraynoic Acid / Q000494:pharmacology; D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism; D001241:Aspirin / Q000494:pharmacology; D002109:Caffeic Acids / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003513:Cycloheximide / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism*; D007213:Indomethacin / Q000494:pharmacology; D016859:Lipoxygenase Inhibitors / Q000378:metabolism; D009637:Masoprocol / Q000494:pharmacology; D051379:Mice; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
chemical_list: D002109:Caffeic Acids; D006893:Hydroxyeicosatetraenoic Acids; D016859:Lipoxygenase Inhibitors; D015117:5,8,11,14-Eicosatetraynoic Acid; D016718:Arachidonic Acid; D009249:NADP; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D004815:Epidermal Growth Factor; D009637:Masoprocol; D003513:Cycloheximide; D001092:Arachidonate 12-Lipoxygenase; D001241:Aspirin; C040048:caffeic acid; D007213:Indomethacin