Essential involvement of 12‐lipoxygenase in regiospecific andstereospecific oxidation of low density lipoprotein by macrophages

Abstract

To establish a role of the 12-lipoxygenase on the generation of oxidized low density lipoprotein (LDL) in macrophages that leads to foam cell formation in atherosclerosis, we overexpressed 12-lipoxygenases in a macrophage-like cell line, J774A.1, that does not show intrinsic enzyme activity. When the 12-lipoxygenaseexpressing cells were incubated with 400 mg´mL 21 LDL in Dulbecco's modified Eagle's medium at 37 8C for 12 h, LDL oxidation, as determined by thiobarbituric acid reactive substance, was markedly increased compared with the mock-transfected cells. Oxygenated products in the modified LDL were examined by HPLC before and after alkaline hydrolysis. Most of the oxygenated derivatives were of an esterified form, and the major product was identified as 13S-hydroxyoctadeca-9Z,11E-dienoic acid. These results clearly demonstrate that esterified fatty acids in LDL are oxygenated by the 12-lipoxygenases expressed in the J774A.1 cells. Furthermore, the oxidized LDL generated by intracellular 12-lipoxygenases was recognized by a scavenger receptor as assessed by macrophage degradation assay.

Low Density Lipoprotein Receptor-related Protein-mediated Membrane Translocation of 12/15-Lipoxygenase Is Required for Oxidation of Low Density Lipoprotein by Macrophages

“…An anti-LDL receptor antibody was raised as described and purified to IgG using protein A-Sepharose (13). Human LDL was prepared from healthy volunteers and dialyzed against phosphate-buffered saline at 4°C for 24 h before each experiment as described previously (12,13). A murine macrophage-like cell line J774A.1 was kindly provided by Dr. Y. Saeki of Shiga University of Medical Science.…”

“…Cell Culture-J774A.1 cells permanently transfected with the pEF-BOS vector carrying porcine leukocyte 12/15-lipoxygenase cDNA and mock-transfected cells were establish as describe previously (12). The cells were cultured at 37°C with 5% CO 2 in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin G and 100 g/ml streptomycin sulfate, and subcultured every 2-3 days using a standard trypsin protocol.…”

“…12/15-Lipoxygenase activity was determined as described previously (12). Briefly, the cytosol and the membranes were incubated in a 200-l reaction mixture containing 50 mM Tris-HCl buffer at pH 7.4 and 25 M [1-…”

“…After 1 h the cells were incubated with 400 g/ml LDL in 100 l of DMEM containing the serum in the presence of L655238 for 12 h, and the culture medium was subjected to TBARS assay as described previously (12).…”

“…Using a macrophage-like cell line J774A.1, which did not have endogenous 12/15-lipoxygenase activity, we permanently transfected the cells with the 12/15-lipoxyganase cDNA and demonstrated that 12/15-lipoxygenase expressed in normal macrophages at a high level was required for LDL oxidation (12). However, the mechanism of extracellular LDL oxidation by intracellular 12/15-lipoxygenase has not been established.…”