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

2001; Elsevier BV; Volume: 276; Issue: 39 Linguagem: Inglês

10.1074/jbc.m105093200

ISSN

1083-351X

Autores

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

Tópico(s)

Peroxisome Proliferator-Activated Receptors

Resumo

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. 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. low density lipoprotein LDL receptor-related protein Dulbecco's modified Eagle's medium 3,3′-dioctadecylindocarbocyanine reverse transcription polymerase chain reaction thiobarbituric acid reactive substance glyceraldehyde-3-phosphate dehydrogenase Lipoxygenases are a class of enzymes that incorporate one molecular oxygen into unsaturated fatty acids giving rise to their hydroperoxy derivatives. There are 5-, 8-, 12-, and 15-lipoxygenases in mammalian tissues, named according to the number of carbon atoms of arachidonic acid to be oxygenated (1Yoshimoto T. Yamamoto S. J. Lipid Mediat. Cell Signal. 1995; 12: 195-212Crossref PubMed Scopus (40) Google Scholar, 2Funk C.D. Biochim. Biophys. Acta. 1996; 1304: 65-84Crossref PubMed Scopus (238) Google Scholar, 3Brash A.R. J. Biol. Chem. 1999; 274: 23679-23682Abstract Full Text Full Text PDF PubMed Scopus (1155) Google Scholar, 4Kuhn H. Thiele B.J. FEBS Lett. 1999; 449: 7-11Crossref PubMed Scopus (285) Google Scholar). The 12-lipoxygenase subfamily includes leukocyte, platelet, and epidermal isoforms. 15-Lipoxygenase-1 was first isolated from reticulocytes, and 15-lipoxygenase-2 was cloned from hair follicles (3Brash A.R. J. Biol. Chem. 1999; 274: 23679-23682Abstract Full Text Full Text PDF PubMed Scopus (1155) Google Scholar). Because the leukocyte 12-lipoxygenase and 15-lipoxygenase-1 are highly related in their primary structures and enzymological properties and are abundant in various tissues of many species, these enzymes are called collectively 12/15-lipoxygenases (2Funk C.D. Biochim. Biophys. Acta. 1996; 1304: 65-84Crossref PubMed Scopus (238) Google Scholar,5Sun D. Funk C.D. J. Biol. Chem. 1996; 271: 24055-24062Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Although the pathophysiological functions of the 12/15-lipoxygenases are still a subject of investigation and discussion, recent research progress has revealed the involvement of the enzymes in the development of atherosclerosis (6Berliner J.A. Heinecke J.W. Free Radic. Biol. Med. 1996; 20: 707-727Crossref PubMed Scopus (1278) Google Scholar, 7Kuhn H. Chan L. Curr. Opin. Lipidol. 1997; 8: 111-117Crossref PubMed Scopus (91) Google Scholar, 8Cathcart M.K. Folcik V.A. Free Radic. Biol. Med. 2000; 28: 1726-1734Crossref PubMed Scopus (98) Google Scholar). The oxidative modification of low density lipoprotein (LDL)1 has been implicated in the early stage of atherosclerosis (9Brown M.S. Goldstein J.L. Nature. 1990; 343: 508-509Crossref PubMed Scopus (160) Google Scholar, 10Steinberg D. Parthasarathy S. Carew T.E. Khoo J.C. Witztum J.L. N. Engl. J. Med. 1989; 320: 915-924Crossref PubMed Google Scholar). Macrophages are likely candidates to mediate in vivo LDL oxidation, because they are accumulated in the atherosclerosis lesions and are capable ofin vitro LDL oxidation in culture medium free of metal ion additives (11Chisolm G.M. Hazen S.L. Fox P.L. Cathcart M.K. J. Biol. Chem. 1999; 274: 25959-25962Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). A number of evidences suggest that the 12/15-lipoxygenase present in monocyte-macrophage contributes to the cell-mediated LDL oxidation (12Sigal E. Laughton C.W. Mulkins M.A. Ann. N. Y. Acad. Sci. 1994; 714: 211-224Crossref PubMed Scopus (36) Google Scholar, 13Kim J.A. Gu J.L. Natarajan R. Berliner J.A. Nadler J.L. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 942-948Crossref PubMed Scopus (81) Google Scholar). Incubation of LDL with 12/15-lipoxygenase led to significant oxidation of LDL (14Sparrow C.P. Parthasarathy S. Steinberg D. J. Lipid Res. 1988; 29: 745-753Abstract Full Text PDF PubMed Google Scholar, 15Cathcart M.K. McNally A.K. Chisolm G.M. J. Lipid Res. 1991; 32: 63-70Abstract Full Text PDF PubMed Google Scholar). The 12/15-lipoxygenase and oxidized fatty acids colocalized with oxidized LDL in fatty streak lesions (16Yla-Herttuala S. Rosenfeld M.E. Parthasarathy S. Sigal E. Sarkioja T. Witztum J.L. Steinberg D. J. Clin. Invest. 1991; 87: 1146-1152Crossref PubMed Scopus (343) Google Scholar, 17Folcik V.A. Nivar-Aristy R.A. Krajewski L.P. Cathcart M.K. J. Clin. Invest. 1995; 96: 504-510Crossref PubMed Scopus (219) Google Scholar, 18Kuhn H. Heydeck D. Hugou I. Gniwotta C. J. Clin. Invest. 1997; 99: 888-893Crossref PubMed Scopus (149) Google Scholar). A disruption of the 12/15-lipoxygenase gene diminished atherosclerosis in apoE-deficient mice (19Cyrus T. Witztum J.L. Rader D.J. Tangirala R. Fazio S. Linton M.F. Funk C.D. J. Clin. Invest. 1999; 103: 1597-1604Crossref PubMed Scopus (466) Google Scholar), and overexpression of 12/15-lipoxygenase facilitated atherosclerosis in the LDL receptor-deficient mice (20Harats D. Shaish A. George J. Mulkins M. Kurihara H. Levkovitz H. Sigal E. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2100-2105Crossref PubMed Scopus (212) Google Scholar). We previously demonstrated that 12/15-lipoxygenase overexpressed in mouse macrophage-like J774A.1 cells was involved essentially in the oxidation of LDL based upon the stereospecific oxygenation of esterified unsaturated fatty acid in LDL (21Sakashita T. Takahashi Y. Kinoshita T. Yoshimoto T. Eur. J. Biochem. 1999; 265: 825-831Crossref PubMed Scopus (35) Google Scholar). This fact suggests direct interaction of the enzyme with LDL or the transfer of cellular lipids oxygenated by the enzyme to LDL. Secretion or leakage of the 12/15-lipoxygenase to the medium was ruled out (21Sakashita T. Takahashi Y. Kinoshita T. Yoshimoto T. Eur. J. Biochem. 1999; 265: 825-831Crossref PubMed Scopus (35) Google Scholar). As the mechanism of the cell-mediated oxidation of extracellular LDL, we postulate that binding of native LDL to cell surface receptors is the first step in the 12/15-lipoxygenase-expressing cells. Among such receptors, the LDL receptor plays an important role in LDL metabolism in liver and steroidogenic tissues. However, the LDL receptor is not expressed in the intima of normal or atherosclerotic arteries (22Hiltunen T.P. Yla-Herttuala S. Atherosclerosis. 1998; 137: S81-S88Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Importantly, the LDL receptor is not required in the cell-mediated LDL oxidation as shown by in vitro experiments (23Cathcart M.K. Li Q. Chisolm G.M. J. Lipid Res. 1995; 36: 1857-1865Abstract Full Text PDF PubMed Google Scholar) and LDL receptor-deficient mice studies (20Harats D. Shaish A. George J. Mulkins M. Kurihara H. Levkovitz H. Sigal E. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2100-2105Crossref PubMed Scopus (212) Google Scholar, 24Tangirala R.K. Mol M.J. Steinberg D. J. Lipid Res. 1996; 37: 835-843Abstract Full Text PDF PubMed Google Scholar). The native LDL also binds to LDL receptor-related protein (LRP) and scavenger receptor BI (22Hiltunen T.P. Yla-Herttuala S. Atherosclerosis. 1998; 137: S81-S88Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The former is also referred to as α2-macroglobulin receptor or CD91 and the latter as high density lipoprotein receptor. It was demonstrated that the both receptors were expressed on the cell surface of macrophages in atherosclerotic lesions (22Hiltunen T.P. Yla-Herttuala S. Atherosclerosis. 1998; 137: S81-S88Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 25Moestrup S.K. Gliemann J. Pallesen G. Cell Tissue Res. 1992; 269: 375-382Crossref PubMed Scopus (359) Google Scholar), and LRP internalized the ligand into the cell by endocytosis (26Willnow T.E. Sheng Z. Ishibashi S. Herz J. Science. 1994; 264: 1471-1474Crossref PubMed Scopus (255) Google Scholar). It is of importance to identify the receptor involved in the initial step of LDL oxidation that leads to the development of atherosclerosis. In the present study, we demonstrate for the first time the essential requirement of LRP for the cell-mediated oxidation of LDL by 12/15-lipoxygenase. 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), [α-32P]dCTP (110 TBq/mmol), Megaprime DNA labeling system and Hybond N+ 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 3,3′-dioctadecylindocarbocyanine (DiI)-LDL from Molecular Probes (Junction City, OR). Oligodeoxyribonucleotides for reverse transcription-polymerase chain reaction (RT-PCR) were synthesized by Hokkaido System Science (Sapporo, Japan). Antisense and sense oligodeoxyribonucleotides synthesized by the phosphorothioate approach using tetraethylthiuram disulfate (27Yonekura H. Migita H. Sakurai S. Wang H. Harada S. Abedin M.J. Yamagishi S. Yamamoto H. Nucleic Acids Res. 1999; 27: 2591-2600Crossref PubMed Scopus (20) Google Scholar) were obtained from Greiner (Tokyo, Japan). A murine macrophage-like cell line, J774A.1, was provided kindly by Dr. Y. Saeki of Shiga University of Medical Science. An expression vector, pEF-BOS (28Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5322Crossref PubMed Scopus (1499) Google Scholar), having a powerful elongation factor-1α promoter was provided kindly by Dr. S. Nagata of Osaka University. An anti-LRP antibody (29Kowal R.C. Herz J. Goldstein J.L. Esser V. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5810-5814Crossref PubMed Scopus (457) Google Scholar) was a generous gift from Dr. Michael R. Brown of University of Texas Southwestern Medical Center. J774A.1 cells transfected with the pEF-BOS vector carrying porcine leukocyte 12/15-lipoxygenase cDNA and mock-transfected cells were established as described previously (21Sakashita T. Takahashi Y. Kinoshita T. Yoshimoto T. Eur. J. Biochem. 1999; 265: 825-831Crossref PubMed Scopus (35) Google Scholar). The cells were maintained in a humidified incubator at 37 °C with 5% CO2 in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 100 μg/ml streptomycin sulfate. The cells were subcultured every 2–3 days using a standard trypsin protocol. Human LDL (relative density = 1.019–1.063) was prepared from fresh plasma of healthy volunteers by sequential floating ultracentrifugation in potassium bromide density gradient (21Sakashita T. Takahashi Y. Kinoshita T. Yoshimoto T. Eur. J. Biochem. 1999; 265: 825-831Crossref PubMed Scopus (35) Google Scholar, 30Havel R.J. Eder H.A. Bragdon J.H. J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6498) Google Scholar). Approximately 20 mg of protein of LDL was obtained from 50 ml of plasma. LDL was stored at 4 °C in the dark and used within 2 weeks. EDTA was removed from the LDL by dialysis against phosphate-buffered saline at 4 °C for 24 h before each experiment. A synthetic peptide, CDSDRDCLDGSDE, was conjugated to keyhole limpet hemocyanin (Calbiochem, San Diego, CA). The peptide represents 106–118 amino acid residues in the third cysteine-rich repeat of the ligand binding domain of the human LDL receptor and shares 92% identity with the mouse receptor. Rabbits were immunized with the conjugate in Freund's complete adjuvants and subsequently received six booster injections at 2-week intervals. IgG was purified from rabbit serum by ammonium sulfate precipitation and protein A-Sepharose chromatography according to manufacturer protocol. Specificity of the anti-LDL receptor antibody was confirmed by Western blotting using a membrane fraction from Chinese hamster ovary cells expressing the human LDL receptor and HepG2 cells. The 12/15-lipoxygenase-expressing cells (2 × 105) were incubated with 400 μg/ml of LDL in 100 μl of DMEM without serum for 12 h, and the culture medium was subjected to TBARS assay as described previously (21Sakashita T. Takahashi Y. Kinoshita T. Yoshimoto T. Eur. J. Biochem. 1999; 265: 825-831Crossref PubMed Scopus (35) Google Scholar). The protein concentration was determined by the method of Lowry et al. (31Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) using bovine serum albumin as a standard. Total RNA was isolated from 12/15-lipoxygenase-expressing cells using Sepasol according to manufacturer instructions. cDNA was synthesized using oligo(dT)12–18 as a primer and SuperScript II reverse transcriptase. PCR was carried out with ExTaq DNA polymerase and primers as shown in Table I under the following conditions: denaturation at 94 °C for 1 min, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min. An equal amount of aliquots from 15 and 20 thermocycles was electrophoresed in 2% agarose gel. The amplified DNAs were transferred to a nylon membrane and fixed by UV irradiation. The membrane was hybridized in QuikHyb hybridization solution with cDNA for the LDL receptor, LRP, scavenger receptor BI, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which had been labeled with [α-32P]dCTP using the Megaprime DNA labeling system. After washing the membrane, radioactivity was analyzed by a Fujix BAS1000 imaging analyzer (Tokyo, Japan). 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).Table IPrimers of PCRPrimerSequenceLength of PCR productbase pairsGAPDH-1F5′-GAGAACGGGAAGCTTGTCATCAATGG-3′339GAPDH-2R5′-AGTTGAGTCCTTCCACGATACCAAAG-3′LDL receptor-1F5′-GAAGACTCATGCAGCAGGAACG-3′468LDL receptor-2R5′-CTCATCGGAGCCGTCAACACAG-3′LRP-1F5′-GTATCTCAAAGGGCTGGCGGTG-3′619LRP-2R5′-TGCACCCAGCATACGGTCTC-3′Scavenger receptor BI-1F5′-CTCGGCGTTGTCATGATCCTCAT-3′912Scavenger receptor BI-2R5′-GCCAGACTCTCGGCATGGGCAGA-3′ Open table in a new tab The full-length cDNA for hexahistidine-tagged (underlined below) heparin-binding protein 44 (32Furukawa T. Ozawa M. Huang R.P. Muramatsu T. J. Biochem. (Tokyo). 1990; 108: 297-302Crossref PubMed Scopus (47) Google Scholar) was prepared with total RNA from J774A.1 cells by RT-PCR using the following primers: upstream, 5′-TCTAGAATGGGGGGTTCTCATCATCATCATCATCATTACTCGCGAGAGAAGAACGAGCC-3′, and downstream, 5′-TCTAGATCAGAGCTCATTGTGCCGAGCCC-3′. The cDNA was subcloned in pCR2.1 and then ligated to the pEF-BOS vector. The resultant plasmid was introduced into COS-7 cells by the DEAE-dextran method (33Qiao N. Takahashi Y. Takamatsu H. Yoshimoto T. Biochim. Biophys. Acta. 1999; 1438: 131-139Crossref PubMed Scopus (12) Google Scholar). After incubation for 48 h, the cells were harvested and sonicated on ice in 50 mm Tris-HCl buffer, pH 7.4, 150 mm NaCl, 1 mmMgCl2, 1 mm CaCl2, and 1 mm phenylmethanesulfonyl fluoride. β-Octylglucopyranoside at 50 mm was added, and the lysate was incubated on ice for 1 h (34Ashcom J.D. Tiller S.E. Dickerson K. Cravens J.L. Argraves W.S. Strickland D.K. J. Cell Biol. 1990; 110: 1041-1048Crossref PubMed Scopus (207) Google Scholar). After centrifugation at 250,000 × gat 4 °C for 30 min, the supernatant was subjected to nickel-nitrilotriacetate agarose column chromatography using manufacturer instructions. The histidine-tagged heparin-binding protein 44 was eluted with 0.2 m histidine in phosphate-buffered saline at pH 7.4. Sequences of sense and antisense oligodeoxyribonucleotides for the LDL receptor, LRP, and scavenger receptor BI are shown in Table II. To destroy secondary structure the oligodeoxyribonucleotides were heated at 95 °C for 5 min and then chilled on ice. 12/15-Lipoxygenase-expressing cells were incubated with 25 μm of the oligodeoxyribonucleotides in DMEM supplemented with 1% fetal bovine serum for 7 days (27Yonekura H. Migita H. Sakurai S. Wang H. Harada S. Abedin M.J. Yamagishi S. Yamamoto H. Nucleic Acids Res. 1999; 27: 2591-2600Crossref PubMed Scopus (20) Google Scholar). The cells were incubated further for 12 h with 400 μg/ml LDL followed by TBARS assay. The oligodeoxyribonucleotides-treated cells were also subjected to RT-PCR analysis for the LDL receptor, LRP, and scavenger receptor BI.Table IIAntisense repertoiresOligodeoxyribonucleotidesSequenceLDL receptor sense5′-ATGAGCACCGCGGATCTGAT-3′LDL receptor anti-sense5′-ATCAGATCCGCGGTGCTCAT-3′LRP sense5′-ATGCTGACCCCGCCGTTGCT-3′LRP anti-sense5′-AGCAACGGCGGGGTCAGCAT-3′Scavenger receptor BI sense5′-ATGGGCGGCAGCTCCAGGGC-3′Scavenger receptor BI anti-sense5′-GCCCTGGAGCTGCCGCCCAT-3′ Open table in a new tab The 12/15-lipoxygenase-expressing cells were incubated at 37 °C for 12 h in DMEM supplemented with 10% fetal bovine serum. DiI-LDL at 5 μg of protein/ml was added, and the cells were incubated further for 2 h. After washing the cells three times at 4 °C, photographs were taken using an Olympus BX-50/BX-FLA fluorescent microscope (Tokyo, Japan) equipped with an appropriate excitation filter (35Stephan Z.F. Yurachek E.C. J. Lipid Res. 1993; 34: 325-330Abstract Full Text PDF PubMed Google Scholar). For flow-cytometric analysis, the cells were dispersed with trypsin, washed twice with Hanks' balanced salt solution, and suspended in phosphate-buffered saline. The 6 × 103 cells were analyzed in each experiment using a FACScalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ) equipped with an argon ion laser that emits at 488 nm. Forward and side scatter were adjusted to exclude debris and dead cells. The mean fluorescence intensity was determined after subtracting the auto fluorescence obtained from the cells incubated in the absence of DiI-LDL. 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 (21Sakashita T. Takahashi Y. Kinoshita T. Yoshimoto T. Eur. J. Biochem. 1999; 265: 825-831Crossref PubMed Scopus (35) Google Scholar). As shown in the control of Fig.1, the oxidation of LDL by the 12/15-lipoxygenase-expressing cells was 3.7 times higher than that by mock-transfected cells as determined by TBARS generation. To explore mechanisms of the LDL oxidation caused by intracellular 12/15-lipoxygenase, we examined the possible involvement of the LDL receptor expressed on the surface of 12/15-lipoxygenase-expressing cells. It is known that expression of the LDL receptor is down-regulated by the incubation of the cells with LDL or cholesterol and up-regulated by the incubation of lipoprotein-deficient serum (36Brown M.S. Goldstein J.L. Cell. 1975; 6: 307-316Abstract Full Text PDF PubMed Scopus (317) Google Scholar). In fact, as shown in Fig. 2, incubation of the 12/15-lipoxygenase-expressing cells with LDL or cholesterol decreased the mRNA level of the LDL receptor by 77 and 58%, respectively, as determined by RT-PCR using each set of primers shown in Table I. In contrast, incubation of the cells with lipoprotein-deficient serum increased the mRNA of the LDL receptor by 2.6 times under our experimental conditions. We examined whether the LDL oxidation would be changed by the above-mentioned preincubations. As shown in Fig. 1, TBARS generation by the 12/15-lipoxygenase-expressing cells was reduced by 39 and 19% after the preincubation of the cells with LDL and cholesterol, respectively. On the other hand, an ∼2-fold increase in TBARS generation was observed after the preincubation of the cells with lipoprotein-deficient serum. There were no apparent changes of TBARS generation in mock-transfected cells preincubated with LDL, cholesterol, or lipoprotein-deficient serum (Fig. 1, open bars). These results suggest that the LDL oxidation by the 12/15-lipoxygenase-expressing 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. It was reported that LRP and scavenger receptor BI bound to native LDL and expressed in J774A.1 cells (22Hiltunen T.P. Yla-Herttuala S. Atherosclerosis. 1998; 137: S81-S88Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 37Wu S.M. Pizzo S.V. Arch. Biochem. Biophys. 1996; 326: 39-47Crossref PubMed Scopus (24) Google Scholar). We then examined whether expression levels of these receptors would be altered by the different culture conditions. RT-PCR was carried out to analyze the mRNA level of these receptors in the 12/15-lipoxygenase-expressing cells preincubated with LDL, cholesterol, or lipoprotein-deficient serum. Primers for GAPDH were also included as internal control for RNA quantity and integrity. As shown in Fig. 2A, aliquots from 15 and 20 cycles were analyzed by agarose gel electrophoresis to verify the linearity of PCR amplification. The LRP mRNA level of the 12/15-lipoxygenase-expressing cells was decreased by preincubation with LDL and cholesterol to 65 and 62% of the control, respectively. The LRP expression was up-regulated 1.3-fold after the preincubation of the cells with lipoprotein-deficient serum (Fig. 2B). On the other hand, scavenger receptor BI mRNA expression apparently did not change under the same preincubation conditions.Figure 2Changes of expression level of LDL-binding receptors under different culture conditions. A, the 12/15-lipoxygenase-expressing cells were incubated at 37 °C for 48 h, and abbreviations are as described in the Fig. 1 legend. Total RNA from the treated cells was subjected to RT-PCR analysis for the LDL receptor (LDLR), LRP, and scavenger receptor BI (SR-BI); bp, base pairs. GAPDH (339 base pairs) was also amplified as an internal standard. B, radioactivity was quantified and normalized with that of GAPDH. The ratios to control are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Heparin-binding protein 44, a mouse homologue of human LRP receptor-associated protein, is a universal antagonist of the LDL receptor family including the LDL receptor and LRP and inhibits the binding of LDL (38Strickland D.K. Ashcom J.D. Williams S. Battey F. Behre E. McTigue K. Battey J.F. Argraves W.S. J. Biol. Chem. 1991; 266: 13364-13369Abstract Full Text PDF PubMed Google Scholar, 39Medh J.D. Fry G.L. Bowen S.L. Pladet M.W. Strickland D.K. Chappell D.A. J. Biol. Chem. 1995; 270: 536-540Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 40Herz J. Goldstein J.L. Strickland D.K. Ho Y.K. Brown M.S. J. Biol. Chem. 1991; 266: 21232-21238Abstract Full Text PDF PubMed Google Scholar). To examine the effect of heparin-binding protein 44 on the 12/15-lipoxygenase-mediated LDL oxidation, the hexahistidine-tagged heparin-binding protein 44 was expressed in COS-7 cells and purified by affinity chromatography. The 12/15-lipoxygenase-expressing or mock-transfected cells were incubated with LDL in the presence of the purified heparin-binding protein 44. As shown in Fig. 3A, heparin-binding protein 44 inhibited TBARS generation in a concentration-dependent fashion, and a maximal inhibition was observed at a concentration as low as 2 μg/ml. These results suggest that the LDL receptor and/or LRP are involved in the LDL oxidation by intracellular 12/15-lipoxygenase. To examine whether the LDL receptor and LRP are involved in the cell-mediated LDL oxidation, we employed antibodies against the LDL receptor (this study) and LRP (29Kowal R.C. Herz J. Goldstein J.L. Esser V. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5810-5814Crossref PubMed Scopus (457) Google Scholar), both of which blocked the LDL binding to the cells. The LDL binding to the LDL receptor was inhibited almost completely by 10 μg/ml IgG against the LDL receptor (data not shown). The 12/15-lipoxygenase-expressing cells and mock-transfected cells then were incubated with 400 μg/ml LDL in the serum-free DMEM in the presence of the anti-LDL receptor IgG at 37 °C for 12 h. As shown in Fig. 3B, the 12/15-lipoxygenase-mediated TBARS generation was not inhibited by the antibody at concentrations up to 50 μg/ml. It should be noted that the anti-LRP antibody suppressed TBARS generation by the 12/15-lipoxygenase-expressing cells in a dose-dependent manner (Fig. 3C). The TBARS generation was inhibited by more than 90% with 10 μl/ml antiserum. The anti-LRP antibody had no effects on the LDL oxidation by CuSO4 or mock-transfected cells. On the other hand, the nonimmunized rabbit serum was without effect on the TBARS generation. The results strongly suggest that LRP is at least one of the receptors required for the LDL oxidation by 12/15-lipoxygenase-expressing cells. To confirm the role of LRP in cell-mediated LDL oxidation, antisense oligodeoxyribonucleotides complementary to the 5′ region of the mouse LRP, LDL receptor, and scavenger receptor BI mRNA containing the initiator AUG codon were synthesized as well as the corresponding sense oligodeoxyribonucleotides (Table II). After incubation of the 12/15-lipoxygenase-expressing cells with the antisense or sense oligodeoxyribonucleotides for 7 days, RT-PCR analysis was carried out. As shown in Fig.4A, the antisense oligodeoxyribonucleotides inhibited the mRNA expression of respective receptors, whereas the sense oligodeoxyribonucleotides did not inhibit the receptor expression. We performed Western blotting for LRP and the LDL receptor, but the specific bands were not observed probably because of the low expression of these receptors. We then examined the TBARS generation by the 12/15-lipoxygenase cells after the treatment with the antisense or sense oligodeoxyribonucleotides. As shown in Fig. 4B, the antisense oligodeoxyribonucleotides against LRP suppressed TBARS generation in the culture medium by the 12/15-lipoxygenase cells by 67% as compared with control incubation, whereas sense oligodeoxyribonucleotides did not inhibit the TBARS generation. As anticipated from the data in Fig. 3B, antisense oligodeoxyribonucleotides against the LDL receptor or scavenger receptor BI did not affect the TBARS generation by the 12/15-lipoxygenase cells. These results taken together indicate that LRP is responsible for the 12/15-lipoxygenase cell-mediated LDL oxidation, and neither the LDL receptor nor scavenger receptor BI is involved in this process. To confirm the specific binding of native LDL to LRP, fluorescence-labeled LDL, DiI-LDL, was incubated with the 12/15-lipoxygenase-expressing cells. After a 2-h incubation, bright fluorescence was observed in most of the cells (Fig.5A), whereas little or no fluorescence in the presence of a 100-fold excess of unlabeled LDL (Fig. 5D). When the LRP (Fig. 5B) or the LDL receptor (Fig. 5C) was blocked by co-incubation of the antibodies with DiI-LDL, much less fluorescence was observed in the 12/15-lipoxygenase-expressing cells as compared with the control incubation. The means of the fluorescence intensity as determined by the flow-cytometric analysis was decreased by 39, 41, and 78% in the presence of anti-LRP antibody at 10 μl/ml, anti-LDL receptor IgG at 50 μg/ml, and a 100-fold excess of unlabeled LDL, respectively. This result indicates that LRP as well as the LDL receptor is responsible for the specific binding of LDL to the 12/15-lipoxygenase-expressing cells. LRP was shown to be expressed in the 12/15-lipoxygenase-expressing J774A.1 cells and responsible for cell-mediated LDL oxidation by these cells. The LRP expression was down-regulated by incubations with LDL or cholesterol and up-regulated by lipoprotein-deficient serum (Fig. 2). A previous study showed that LRP expression was not changed significantly by cholesterol loading in the LDL receptor-deficient human fibroblasts (29Kowal R.C. Herz J. Goldstein J.L. Esser V. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5810-5814Crossref PubMed Scopus (457) Google Scholar). The reasons for apparently different response of LRP expression to cholesterol are not known and may be the difference of cell types used in the experiments. It should be emphasized that changes of both LDL oxidation and LRP expression of the 12/15-lipoxygenase-expressing cells coincided under the above culture conditions (Figs. 1 and 2). The change of LDL oxidation was not caused by the alteration of 12/15-lipoxygenase expression, because the enzyme activity did not change significantly under these culture conditions (data not shown). Involvement of LRP in the LDL oxidation by 12/15-lipoxygenase-expressing cells is also supported by the experiment with heparin-binding protein 44. This protein is a mouse homologue of the human LRP receptor-associated protein, is a universal antagonist of the LDL receptor family, and inhibits the binding of LDL to the LDL receptor and LRP (38Strickland D.K. Ashcom J.D. Williams S. Battey F. Behre E. McTigue K. Battey J.F. Argraves W.S. J. Biol. Chem. 1991; 266: 13364-13369Abstract Full Text PDF PubMed Google Scholar, 39Medh J.D. Fry G.L. Bowen S.L. Pladet M.W. Strickland D.K. Chappell D.A. J. Biol. Chem. 1995; 270: 536-540Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 40Herz J. Goldstein J.L. Strickland D.K. Ho Y.K. Brown M.S. J. Biol. Chem. 1991; 266: 21232-21238Abstract Full Text PDF PubMed Google Scholar, 41Willnow T.E. J. Mol. Med. 1999; 77: 306-315Crossref PubMed Scopus (127) Google Scholar). The LDL oxidation caused by the 12/15-lipoxygenase-expressing cells was blocked by heparin-binding protein 44, confirming that LDL binding to cell surface receptors was necessary for the cell-mediated LDL oxidation (Fig. 3A). The LDL oxidation by 12/15-lipoxygenase-expressing cells was blocked by an anti-LRP antibody in a dose-dependent manner (Fig.3C). Furthermore, antisense oligodeoxyribonucleotides against LRP that suppressed the mRNA expression of LRP also inhibited the LDL oxidation (Fig. 4). We tested whether LRP could mediate LDL oxidation by thioglycollate-induced peritoneal macrophages of C57BL/6 mice (42Zhuang D. Kawajiri H. Takahashi Y. Yoshimoto T. J. Biochem. (Tokyo). 2000; 127: 451-456Crossref PubMed Scopus (10) Google Scholar). The anti-LRP serum at 10 μl/ml inhibited LDL oxidation by 56.3%. The LDL oxidation as determined by TBARS of 1 × 105 peritoneal macrophages was 0.39 ± 0.03 (n = 4) nmol of malondialdehyde/mg of LDL, and that of 2 × 105 12/15-lipoxygenase-expressing J774A.1 cells was 0.26 ± 0.07 (n = 4) nmol of malondialdehyde/mg of LDL. When the 12/15-lipoxygenase activity of these cells was determined using 25 μm exogenous arachidonic acid as substrate (21Sakashita T. Takahashi Y. Kinoshita T. Yoshimoto T. Eur. J. Biochem. 1999; 265: 825-831Crossref PubMed Scopus (35) Google Scholar), it was 74.8 and 37.2 nmol/10 min/mg of protein, respectively. Our results taken together suggest that LRP, at least in part, mediates the LDL oxidation not only by 12/15-lipoxygenase-expressing J774A.1 cells but also by normal macrophages, which express 12/15-lipoxygenase at a high level and accumulate in atherosclerotic lesions (16Yla-Herttuala S. Rosenfeld M.E. Parthasarathy S. Sigal E. Sarkioja T. Witztum J.L. Steinberg D. J. Clin. Invest. 1991; 87: 1146-1152Crossref PubMed Scopus (343) Google Scholar, 22Hiltunen T.P. Yla-Herttuala S. Atherosclerosis. 1998; 137: S81-S88Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The very low level of LDL oxidation by mock-transfected cells that hardly expressed 12/15-lipoxygenase was not affected by heparin-binding protein 44 or an anti-LRP antibody, suggesting 12/15-lipoxygenase-independent LDL oxidation, if any, was not mediated by LRP. We could not detect specific bands of LRP or the LDL receptor by Western blotting, suggesting that the expression level of these receptors in the 12/15-lipoxygenase-expressing cells was not high. However, antibodies against these two receptors significantly inhibited binding of DiI-LDL to these cells as assessed by flow-cytometric analysis, indicating that functional receptors were definitely expressed in these cells. Small fluorescence was still observed even when both antibodies were added at the same time to the cells incubated with DiI-LDL (data not shown). The results agree with the expression of scavenger receptor BI in these cells and also suggest the presence of other LDL-binding receptors. The LDL receptor did not seem to be involved in the cell-mediated LDL oxidation (Figs. 3B and 4B). This is in good agreement with the previous reports showing that macrophages prepared from the LDL receptor-deficient mouse could oxidize LDL to the same extent as the wild-type mouse (24Tangirala R.K. Mol M.J. Steinberg D. J. Lipid Res. 1996; 37: 835-843Abstract Full Text PDF PubMed Google Scholar). In fact, the LDL receptor processes native LDL via receptor-mediated endocytosis in which the LDL particle is delivered to lysosomes, in which cholesteryl ester is hydrolyzed to free cholesterol for use by the cells (43Goldstein J.L. Brown M.S. Annu. Rev. Biochem. 1977; 46: 897-930Crossref PubMed Scopus (1616) Google Scholar). LRP is a multiligand receptor, and the binding of ligands to the receptor is usually followed by receptor-mediated endocytosis and degradation of the ligands in lysosomes (26Willnow T.E. Sheng Z. Ishibashi S. Herz J. Science. 1994; 264: 1471-1474Crossref PubMed Scopus (255) Google Scholar). However, a recent paper reported that LRP also mediated the selective uptake of cholesteryl ester in LDL, which is transferred to the plasma membrane without internalization and degradation of LDL particles (44Swarnakar S. Beers J. Strickland D.K. Azhar S. Williams D.L. J. Biol. Chem. 2001; 276: 21121-21128Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). It is possible that the cholesteryl ester in the plasma membrane is oxygenated directly by the intracellular 12/15-lipoxygenase followed by reincorporation to the LDL particles (45Kuhn H. Belkner J. Suzuki H. Yamamoto S. J. Lipid Res. 1994; 35: 1749-1759Abstract Full Text PDF PubMed Google Scholar, 46Takahashi Y. Glasgow W.C. Suzuki H. Taketani Y. Yamamoto S. Anton M. Kuhn H. Brash A.R. Eur. J. Biochem. 1993; 218: 165-171Crossref PubMed Scopus (78) Google Scholar). Scavenger receptor BI is an 82-kDa protein that binds high density lipoprotein, LDL, modified LDL, and very low density lipoprotein (47de Villiers W.J. Smart E.J. J. Leukoc. Biol. 1999; 66: 740-746Crossref PubMed Scopus (140) Google Scholar). The receptor is expressed on the surface of macrophage in atherosclerotic lesions (22Hiltunen T.P. Yla-Herttuala S. Atherosclerosis. 1998; 137: S81-S88Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). It was reported that scavenger receptor BI, similar to LRP, mediates the selective uptake of cholesteryl ester in LDL and high density lipoprotein (48Swarnakar S. Temel R.E. Connelly M.A. Azhar S. Williams D.L. J. Biol. Chem. 1999; 274: 29733-29739Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 49Stangl H. Hyatt M. Hobbs H.H. J. Biol. Chem. 1999; 274: 32692-32698Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 50Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2011) Google Scholar). However, mRNA expression of scavenger receptor BI was not altered by the preincubation with LDL, cholesterol, or lipoprotein-deficient serum under our experimental conditions (Fig. 2). Furthermore, no inhibition of LDL oxidation by antisense oligodeoxyribonucleotides against scavenger receptor BI suggests a trivial role of this receptor (Fig.4). The reason that scavenger receptor BI is not responsible for the cell-mediated LDL oxidation is not known, but this receptor could be involved in the LDL oxidation in the other experimental systems (48Swarnakar S. Temel R.E. Connelly M.A. Azhar S. Williams D.L. J. Biol. Chem. 1999; 274: 29733-29739Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). LRP was reported to bind to a variety of ligands including LDL, α2-macroglobulin, very low density lipoprotein remnants, plasminogen activator, and so on (41Willnow T.E. J. Mol. Med. 1999; 77: 306-315Crossref PubMed Scopus (127) Google Scholar). Thus, the receptor has been postulated to participate in a number of diverse physiological and pathological processes such as the homeostasis of plasma lipoproteins and atherosclerosis, fibrinolysis, and neuronal regeneration (41Willnow T.E. J. Mol. Med. 1999; 77: 306-315Crossref PubMed Scopus (127) Google Scholar). A recent study revealed the role of the receptor in clearance of chylomicron remnants by inducible disruption of “hepatic” LRP in mice (51Rohlmann A. Gotthardt M. Hammer R.E. Herz J. J. Clin. Invest. 1998; 101: 689-695Crossref PubMed Scopus (403) Google Scholar). Our study implicates a novel function of “macrophage” LRP in the 12/15-lipoxygenase-mediated LDL oxidation as the initial trigger of the progression of atherosclerosis. Further investigations are needed to explore the LRP-mediated LDL oxidation in detail in relation to other receptors and cellular factors. We are indebted to Drs. H. Yonekura, O. Amano, and A. Yachie of Kanazawa University and Dr. T. Yamamoto of Tohoku University for valuable technical assistance. We thank Professor Y. Saeki of Shiga University for the generous gift of J774A.1 cells, Dr. S. Nagata of Osaka University for providing the pEF-BOS vector, and Dr. Michael R. Brown of University of Texas Southwestern Medical Center for providing the anti-LRP antibody.

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