title: Low density lipoprotein receptor-related protein-mediated membrane translocation of 12/15-lipoxygenase is required for oxidation of low density lipoprotein by macrophages.
abstract: Oxidation of low density lipoprotein (LDL) is the key step for the development of atherosclerosis. The 12/15-lipoxygenase expressed in macrophages is capable of oxygenating linoleic acid esterified to cholesterol in the LDL particle, and thus this enzyme is presumed to initiate LDL oxidation. We recently reported that LDL receptor-related protein (LRP) was required for the enzyme-mediated LDL oxidation by macrophages and suggested the selective uptake of cholesterol ester from LDL to the plasma membrane (Xu, W., Takahashi, Y., Sakashita, T., Iwasaki, T., Hattori, H., and Yoshimoto. T. (2001) J. Biol. Chem. 276, 36454-36459). To elucidate precise mechanisms of lipoxygenase-mediated LDL oxidation, we investigated the intracellular localization of 12/15-lipoxygenase. The 12/15-lipoxygenase was predominantly detected in cytosol of resting peritoneal macrophages and of macrophage-like J774A.1 cells permanently transfected with the cDNA for the enzyme. When the cells were treated with LDL and subjected to subcellular fractionation, the 12/15-lipoxygenase was detected in the membranes with a concomitant decrease in cytosol as shown by Western blot analysis. The levels of the enzyme associated with the membrane reached maximum in 15 min after LDL addition and then decreased. However, the enzymatic activity of 12/15-lipoxygenase in the membrane fraction was very weak even after LDL treatment. This fact supports the suicide inactivation of the enzyme by the oxygenation of cholesterol ester transferred from the LDL particle to the plasma membrane. Immunohistochemical analysis using an antibody against 12/15-lipoxygenase revealed that the plasma membrane was the major site of the enzyme translocation by the LDL treatment. LDL-dependent 12/15-lipoxygenase translocation was inhibited by a blocking antibody against LRP. Furthermore, an enzyme translocation inhibitor, L655238, inhibited the LDL oxidation caused by the 12/15-lipoxygenase. We propose that cholesterol ester selectively transferred from the LDL particle to the plasma membrane via LRP is oxygenated by 12/15-lipoxygenase translocated to this membrane.
authors.affiliation: Department of Molecular Pharmacology, Kanazawa University Graduate School of Medical Science, Kanazawa 920-8640, Japan.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhu
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Wanpeng
authors.initials: W
authors.lastname: Xu
authors.forename: Hiroo
authors.initials: H
authors.lastname: Kawajiri
authors.forename: Takashi
authors.initials: T
authors.lastname: Murakami
authors.forename: Miyuki
authors.initials: M
authors.lastname: Yamamoto
authors.forename: Shoichi
authors.initials: S
authors.lastname: Iseki
authors.forename: Tadao
authors.initials: T
authors.lastname: Iwasaki
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Hattori
authors.forename: Tanihiro
authors.initials: T
authors.lastname: Yoshimoto
mesh_terms: D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000378:metabolism*; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002788:Cholesterol Esters / Q000378:metabolism; D005822:Genetic Vectors; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D010084:Oxidation-Reduction; D021381:Protein Transport; D011973:Receptors, LDL / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine
chemical_list: C475064:12-15-lipoxygenase; D002788:Cholesterol Esters; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D011994:Recombinant Proteins; C099252:oxidized low density lipoprotein; D001092:Arachidonate 12-Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase