www.clinsci.org Clinical Science (2010) 119, 265–272 (Printed in Great Britain) doi:10.1042/CS20100266 265 ACCELERATED PUBLICATION Overexpression of STARD3 in human monocyte/macrophages induces an anti-atherogenic lipid phenotype Faye BORTHWICK, Anne-Marie ALLEN, Janice M. TAYLOR and Annette GRAHAM Vascular Biology Group, Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Glasgow G4 0BA, U.K. ABSTRACT Dysregulated macrophage cholesterol homoeostasis lies at the heart of early and developing atheroma, and removal of excess cholesterol from macrophage foam cells, by efficient transport mechanisms, is central to stabilization and regression of atherosclerotic lesions. The present study demonstrates that transient overexpression of STARD3 {START [StAR (steroidogenic acute regulatory protein)-related lipid transfer] domain 3; also known as MLN64 (metastatic lymph node 64)}, an endosomal cholesterol transporter and member of the ‘START’ family of lipid trafficking proteins, induces significant increases in macrophage ABCA1 (ATP-binding cassette transporter A1) mRNA and protein, enhances [3H]cholesterol efflux to apo (apolipoprotein) AI, and reduces biosynthesis of cholesterol, cholesteryl ester, fatty acids, triacylglycerol and phospholipids from [14C]acetate, compared with controls. Notably, overexpression of STARD3 prevents increases in cholesterol esterification in response to acetylated LDL (low-density lipoprotein), blocking cholesteryl ester deposition. Thus enhanced endosomal trafficking via STARD3 induces an anti- atherogenic macrophage lipid phenotype, positing a potentially therapeutic strategy. Clinical Science INTRODUCTION and stabilization, and can be orchestrated, at least in vitro, by ABC (ATP-binding cassette) lipid trans- Dysregulated macrophage cholesterol homoeostasis lies porters such as ABCA1, ABCG1 and ABCG4, and at the heart of early and developing atheroma, the apo (apolipoprotein) acceptors, such as apoAI and apoE principal cause of coronary heart disease. Macrophage [2]. Efficient intracellular cholesterol transport is pivotal ‘foam cells’, laden with cholesterol and cholesteryl ester, in marshalling appropriate cholesterol homoeostasis result from unregulated uptake of modified lipoproteins mechanisms, regulating sterol-responsive transcription by macrophage scavenger receptors (CD36, CD68 and factors, such as LXRα/β (liver X receptors α/β)and SR-AI/AII), and influence both plaque stability and pro- SREBPs (sterol-regulatory-element-binding proteins), gression [1]. Removal of excess cholesterol from and controlling cholesterol content of organelles, macrophage ‘foam’ cells is central to lesion regression lipid rafts and membranes, and storage as cytosolic Key words: atherosclerosis, cholesterol efflux, lipid transport, macrophage foam cell, steroidogenic acute regulatory protein-related lipid transfer domain 3/metastatic lymph node 64 (STARD3/MLN64). Abbreviations: ABC, ATP-binding cassette; ACAT, acyl-CoA:cholesterol acyltransferase; apo, apolipoprotein; CHO, Chinese- hamster ovary; CYP, cytochrome P450; ER, endoplasmic reticulum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDL, high-density lipoprotein; LDL, low-density lipoprotein; AcLDL, acetylated LDL; LXR, liver X receptor; MLN64, metastatic lymph node 64; MENTAL domain, MLN64 N-terminal domain; NPC, Niemann–Pick type C; Q-PCR, quantitative real-time PCR; SREBP, sterol-regulatory-element-binding protein; StAR, steroidogenic acute regulatory protein; START, StAR-related lipid transfer; STARD, START domain. Correspondence: Professor Annette Graham (email [email protected]). © 2010 The Author(s) The author(s) has paid for this article to be freely available under the terms of the Creative Commons AttributionC Non-CommercialThe Authors Journal Licence compilation (http://creativecommons.org/licenses/by-nc/2.5/)C 2010 Biochemical Society which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited. 266 F. Borthwick and others droplets of cholesteryl ester [2,3]. Despite this, the (AbCAM), primers and fluorescent probes (Eurogentec), proteins involved in non-vesicular cholesterol transport and LDL (low-density lipoprotein) (Athens Research), mechanisms remain poorly understood. acetylated as described previously [14]. Peripheral human The START [StAR (steroidogenic acute regulatory monocyte/macrophages were purchased from Lonza, protein)-related lipid transfer] domain is a 210 amino and human heart aorta RNA, derived from four to seven acid conserved ‘helix-grip’ fold, providing an adaptable human heart aortae, was purchased from Clontech. binding site for lipids such as cholesterol, oxysterols, phospholipids and ceramides. In humans, START Cellular studies domains are found in 15 distinct proteins (STARD1– Human (THP-1) monocytes (ECACC 880812101) were STARD15), implicated in non-vesicular lipid transport, maintained in RPMI 1640 medium containing 10% (v/v) cell signalling and lipid metabolism [4,5]. The prototypic FBS (fetal bovine serum), as described previously [14,15]. member of this family, StAR (STARD1), delivers STARD3, STARD4, STARD5 and empty vector control cholesterol from the outer to the inner mitochondrial (pCMV; 0.5 μg of DNA) were delivered to THP-1 membrane, to the CYP (cytochrome P450) side- monocytes (107 cells) using an Amaxa Human Monocyte chain-cleavage enzyme involved in steroidogenesis [6]. Nucleofector® kit (VPA-1007). Transfection efficiency Overexpression of StAR can also decrease macrophage using protocol Y001 (78.5%) was determined using lipid content, inflammatory status and arterial cholesterol the proprietary pmaxGFP® vector provided by Amaxa levels [7,8], and increase cholesterol efflux to apoAI and flow cytometric analysis of 50000 cells. Transfected via a mechanism dependent upon mitochondrial sterol human THP-1 monocytes (1.5 × 106 cells/well) were 27-hydroxylase (CYP27A1), LXR activation and in- differentiated into macrophages by addition of 100 nM creases in ABCA1 mRNA and protein expression [9]. PMA. Cellular lipids, RNA and cell protein lysates were However, one ‘downside’ of StAR overexpression in collected 72 h post-transfection. Macrophage foam cell macrophages is the induction of lipogenesis [9], possibly prevention experiments were initiated 48 h after transfec- mediated by LXR-dependent increases in SREBP1c tion. Macrophages were radiolabelled with [3H]oleic acid expression, a problem also associated with non-sterol (1 μCi/ml; 10 μM) in the presence or absence of AcLDL LXR agonists. (acetylated LDL; 50 μg/ml) and with or without an The other member of the STARD1 subfamily ACAT (acyl-CoA:cholesterol acyltransferase) inhibitor STARD3 [also known as MLN64 (metastatic lymph (447C88; 10 μM) for 24 h. Wild-type macrophages were node 64)] is a 54 kDa protein, co-amplified within incubated with progesterone (10 μM; 24 h) or U18666A chromosome band 17q12, a region containing the potent (25 μM; 24 h) to inhibit endosomal trafficking, using oncogene ERBB2 in human breast carcinoma [10–12]. ethanol vehicle (<0.1%). Cellular viability was assessed STARD3 is a late endosomal protein with two distinct by conversion of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- conserved cholesterol-binding domains: a region of four diphenyl-2H-tetrazolium bromide] into formazan. The transmembrane helices with three short intervening caspase-Glo® 3/7 assay system (Promega) was used to loops, called the MENTAL domain (MLN64 N-terminal detect apoptosis. domain), and the C-terminal ‘START’ domain [13]. The MENTAL domain may maintain cholesterol at the late Lipid analyses endosomal membrane, prior to its shuttle to cytoplasmic Flux of [3H]oleic acid (1 μCi/ml; 10 μM) into the cho- acceptor(s) via the START domain. lesteryl ester pool and of [14C]acetate (1μCi/ml) into cho- Our previous studies have shown that cholesterol lesterol, cholesteryl ester, fatty acid, triacylglycerol and loading represses STARD3 expression, implicating this phospholipid pools were assessed by TLC, as described protein in dysfunctional cholesterol pathologies [14]. In previously [14,15]. Efflux of [3H]cholesterol (0.5 μCi/ml) the present study, we sought to establish a functional to apoA1 (10 μg/ml) and HDL (high-density lipoprotein; role for this endosomal cholesterol trafficking protein in 10 μg/ml) were measured as described previously [13,15]. macrophage lipid homoeostasis, testing the hypothesis Mass of macrophage total cholesterol, triacylglycerol that enhanced expression of STARD3 may be useful in and choline-containing phospholipids were measured the prevention of foam cell formation. using InfinityTM and Phospholipids-B colorimetric assays (Alpha Labs) [14,15]. MATERIALS AND METHODS Gene and protein expression Materials Total RNA (Tri-Reagent; Sigma–Aldrich) was isolated Tissue culture reagents were purchased from Lonza; from macrophages and reverse-transcribed to cDNA other reagents include pCMV.Script, and STARD4 and utilizing the MMLV (Moloney-murine-leukaemia virus) STARD5 clones (Origene), STARD3 clone (Stratagene), reverse transcriptase (Bioline). Expression of STARD3, Amaxa monocyte/macrophage transfection reagent, STARD4, STARD5, APOE, ABCA1, ABCG1, ABCG4, NuPAGE gels and buffers (Invitrogen), antibodies NR1H3 (encoding LXRα), SREBP1 and SREBP2 © 2010 The Author(s) The author(s) has paidC The for Authorsthis article Journal to be compilationfreely availableC under2010 Biochemicalthe terms of Societythe Creative Commons Attribution Non-Commercial Licence