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J. Biol. Chem., Vol. 282, Issue 18, 13746-13753, May 4, 2007

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Impaired Secretion of Apolipoprotein E2 from Macrophages^*

Daping Fan, Shenfeng Qiu, Cheryl D. Overton^¶, Patricia G. Yancey, Larry L. Swift^¶, W. Gray Jerome^¶, MacRae F. Linton^||, and Sergio Fazio^¶1

From the Atherosclerosis Research Unit, Division of Cardiology, Department of Medicine, Department of Molecular Physiology and Biophysics, ^¶Department of Pathology and ^||Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6300

Human apoE is a multifunctional and polymorphic protein synthesized and secreted by liver, brain, and tissue macrophages. Here we show that apoE isoforms and mutants expressed through lentiviral transduction display cell-specific differences in secretion efficiency. Whereas apoE3, apoE4, and a natural mutant of apoE4 (apoE-Cys¹⁴²) were efficiently secreted from macrophages, apoE2 and a non-natural apoE mutant (apoE-Cys¹¹²/Cys¹⁴²) were retained in the perinuclear region and only minimally secreted. The secretory block for apoE2 in macrophages was not affected by the ablation of LDLR (low density lipoprotein receptor), ABCA-1, or SR-BI (scavenger receptor class B type I) but was released in the absence of low density lipoprotein receptor related protein (LRP). In co-immunoprecipitation experiments, an anti-apoE antibody pulled down two times more LRP in apoE2-transduced macrophages than in apoE3-expressing macrophages. Non-reducing SDS-PAGE/Western blot analyses showed that macrophage apoE2 is mostly dimeric and multimeric, whereas apoE3 is predominantly monomeric. ApoE2 retention and multimer formation also occurred in human macrophages derived from the monocyte cell line THP-1. These results were specific for macrophages, as in transduced mouse primary hepatocytes: 1) ApoE2 was secreted as efficiently as apoE3 and apoE4; 2) all isoforms were exclusively in monomeric form; 3) there was no co-immunoprecipitation of apoE and LRP. A microsomal triglyceride transfer protein (MTP) inhibitor nearly deleted apoB100 secretion from hepatocytes without affecting apoE secretion. These data show that macrophages retain apoE2, a highly expressed protein carried by about 8% of the human population. Given the role of locally produced apoE in regulating cholesterol efflux, modulating inflammation, and controlling oxidative stress, this unique property of apoE2 may have important impacts on atherogenesis.

Received for publication, December 22, 2006 , and in revised form, March 2, 2007.

^* This work was supported by National Institutes of Health Grant HL-057986 (to S. F.) and an American Heart Association Postdoctoral Fellowship (to D. F.). This study was also supported in part by the Lipid, Lipoprotein, and Atherosclerosis Core of the Vanderbilt Mouse Metabolic Phenotyping Centers (National Institutes of Health Grant DK59637). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ To whom correspondence should be addressed: 2220 Pierce Ave., 383 PRB, Vanderbilt University, Nashville, TN 37232. Tel.: 615-936-1450; Fax: 615-936-3486; E-mail: sergio.fazio{at}vanderbilt.edu.

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