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

Wanpeng Xu,Yoshitaka Takahashi,Toshiki Sakashita,Tadao Iwasaki,Hiroaki Hattori,Tanihiro Yoshimoto

doi:10.1074/jbc.m105093200

Abstract

The oxidative modification of low density lipoprotein (LDL) has been implicated in the early stage of atherosclerosis through multiple potential pathways, and 12/15-lipoxygenase is suggested to be involved in this oxidation process. We demonstrated previously that the 12/15-lipoxygenase overexpressed in mouse macrophage-like J774A.1 cells was required for the cell-mediated LDL oxidation. However, the mechanism of the oxidation of extracellular LDL by the intracellular 12/15-lipoxygenase has not yet been elucidated. In the present study, we found that not only the LDL receptor but also LDL receptor-related protein (LRP), both of which are cell surface native LDL-binding receptors, were down-regulated by the preincubation of the cells with cholesterol or LDL and up-regulated by lipoprotein-deficient serum. Moreover, 12/15-lipoxygenase-expressing cell-mediated LDL oxidation was decreased by the preincubation of the cells with LDL or cholesterol and increased by the preincubation with lipoprotein-deficient serum. Heparin-binding protein 44, an antagonist of the LDL receptor family, also suppressed the cell-mediated LDL oxidation in a dose-dependent manner. The cell-mediated LDL oxidation was dose-dependently blocked by an anti-LRP antibody but not by an anti-LDL receptor antibody. Furthermore, antisense oligodeoxyribonucleotides against LRP reduced the cell-mediated LDL oxidation under the conditions in which the expression of LRP was decreased. The results taken together indicate that LRP was involved essentially for the cell-mediated LDL oxidation by 12/15-lipoxygenase expressed in J774A.1 cells, suggesting an important pathophysiological role of this receptor-enzyme system as the initial trigger of the progression of atherosclerosis.

Highlights

Lipoxygenases are a class of enzymes that incorporate one molecular oxygen into unsaturated fatty acids giving rise to their hydroperoxy derivatives
Effect of Up-regulation and Down-regulation of low density lipoprotein (LDL)-binding Receptors on LDL Oxidation—We previously reported that the 12/15-lipoxygenase of porcine leukocytes overexpressed in macrophage-like J774A.1 cells was responsible for oxidative modification of LDL in the medium [21]
These results suggest that the LDL oxidation by the 12/15-lipoxygenaseexpressing cells is mediated by either the LDL receptor or other cell surface proteins that bind to native LDL and are down- and up-regulated by the culture conditions described above

Summary

EXPERIMENTAL PROCEDURES

Materials—Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Nissui (Tokyo, Japan), fetal bovine serum from JRH biosciences (Lenexa, KS), Sepasol from Nacalai (Kyoto, Japan), SuperScript II reverse transcriptase from Life Technologies, Inc., ExTaq DNA polymerase from Takara (Kyoto, Japan), [␣-32P]dCTP (110 TBq/mmol), Megaprime DNA labeling system and Hybond Nϩ nylon membrane from Amersham Pharmacia Biotech, QuikHyb hybridization solution from Stratagene (La Jolla, CA), lipoprotein-deficient serum and calf thymus DNA from Sigma, restriction enzymes from Toyobo (Osaka, Japan), 2-thiobarbituric acid and 1,1,3,3-tetramethoxypropane (malondialdehyde bis) from Wako (Osaka, Japan), nickel-nitrilotriacetate agarose from Qiagen (Hilden, Germany), and 3,3Ј-dioctadecylindocarbocyanine (DiI)-LDL from Molecular Probes (Junction City, OR). Thiobarbituric Acid Reactive Substance (TBARS) Assay—The 12/15lipoxygenase-expressing cells (2 ϫ 105) were incubated with 400 ␮g/ml of LDL in 100 ␮l of DMEM without serum for 12 h, and the culture medium was subjected to TBARS assay as described previously [21]. Nucleotide sequences of the LDL receptor, LRP, scavenger receptor BI, GAPDH, and heparin-binding protein 44 (see below) were determined by dRhodamine terminator cycle sequencing kit using an automated DNA sequencer ABI PRISM 310 (PerkinElmer Life Sciences). 12/15-Lipoxygenase-expressing cells were incubated with 25 ␮M of the oligodeoxyribonucleotides in DMEM supplemented with 1% fetal bovine serum for 7 days [27]. The oligodeoxyribonucleotides-treated cells were subjected to RT-PCR analysis for the LDL receptor, LRP, and scavenger receptor BI. DiI-LDL Uptake—The 12/15-lipoxygenase-expressing cells were incubated at 37 °C for 12 h in DMEM supplemented with 10% fetal bovine serum. The mean fluorescence intensity was determined after subtracting the auto fluorescence obtained from the cells incubated in the absence of DiI-LDL

RESULTS

TABLE I Primers of PCR

DISCUSSION

TABLE II Antisense repertoires