ABCG1 (ABC8), the human homolog of the Drosophila white gene, is a regulator of macrophage cholesterol and phospholipid transport Jochen Klucken*, Christa Bu¨ chler*, Evelyn Orso´ *, Wolfgang E. Kaminski*, Mustafa Porsch-Ozcu¨ ¨ ru¨ mez*, Gerhard Liebisch*, Michael Kapinsky*, Wendy Diederich*, Wolfgang Drobnik*, Michael Dean†, Rando Allikmets‡, and Gerd Schmitz*§ *Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, 93042 Regensburg, Germany; †National Cancer Institute, Laboratory of Genomic Diversity, Frederick, MD 21702-1201; and ‡Departments of Ophthalmology and Pathology, Columbia University, Eye Research Addition, New York, NY 10032 Edited by Jan L. Breslow, The Rockefeller University, New York, NY, and approved November 3, 1999 (received for review June 14, 1999) Excessive uptake of atherogenic lipoproteins such as modified low- lesterol transport. Although several effector molecules have been density lipoprotein complexes by vascular macrophages leads to proposed to participate in macrophage cholesterol efflux (6, 9), foam cell formation, a critical step in atherogenesis. Cholesterol efflux including endogenous apolipoprotein E (10) and the cholesteryl mediated by high-density lipoproteins (HDL) constitutes a protective ester transfer protein (11), the detailed molecular mechanisms mechanism against macrophage lipid overloading. The molecular underlying cholesterol export in these cells have not yet been mechanisms underlying this reverse cholesterol transport process are characterized. currently not fully understood. To identify effector proteins that are Recently, mutations of the ATP-binding cassette (ABC) trans- involved in macrophage lipid uptake and release, we searched for porter ABCA1 gene have been causatively linked to familial HDL genes that are regulated during lipid influx and efflux in human deficiency and Tangier disease (12–14). ABC transporters consti- macrophages using a differential display approach. We report here tute a family of transport molecules that couple the energy of ATP that the ATP-binding cassette (ABC) transporter ABCG1 (ABC8) is hydrolysis to the translocation of substrates (allokrites) across induced in monocyte-derived macrophages during cholesterol influx biologic membranes (15, 16). ABCG1, a recently cloned ABC MEDICAL SCIENCES mediated by acetylated low-density lipoprotein. Conversely, lipid transporter (also known as ABC8 or human white gene), is a efflux in cholesterol-laden macrophages, mediated by the cholesterol member of the subgroup of half-size transporters (17, 18). It exhibits acceptor HDL3, suppresses the expression of ABCG1. Immunocyto- high homology with the Drosophila white gene, which acts as a chemical and flow cytometric analyses revealed that ABCG1 is ex- regulator of tryptophan and guanine uptake in conjunction with the pressed on the cell surface and in intracellular compartments of scarlet and brown genes (19, 20). cholesterol-laden macrophages. Inhibition of ABCG1 protein expres- In the mammalian system, no definitive function has been sion using an antisense strategy resulted in reduced HDL3-dependent reported for ABCG1 to date. In the present study we identified efflux of cholesterol and choline-phospholipids. In a comprehensive genes that are involved in macrophage cholesterol transport and analysis of the expression and regulation of all currently known thus may be of potential relevance for the process of foam cell human ABC transporters, we identified an additional set of ABC genes formation. We report here the implication of ABCG1 in the whose expression is regulated by cholesterol uptake or HDL3-medi- regulation of macrophage cholesterol efflux. ated lipid release, suggesting a potential function for these trans- porters in macrophage lipid homeostasis. Our results demonstrating Materials and Methods a regulator function for ABCG1 in cholesterol and phospholipid Cultivation of Monocytes͞Macrophages. Monocytes were obtained transport define a biologic activity for ABC transporters in macro- from healthy normolipidemic volunteers by leukapheresis and were phages. purified by counterflow elutriation. Monocyte purity was Ͼ95% as confirmed by FACS analysis (Becton Dickinson). Aliquots of 1 ϫ holesterol homeostasis in macrophages involves a balance 106͞ml monocytes were cultured in macrophage serum-free me- Cbetween lipid influx and efflux. Deposition of excessive dium (GIBCO͞BRL). After 12 h, fresh macrophage medium was amounts of cholesteryl ester in macrophages of the vascular wall added supplemented with 50 ng͞ml human recombinant M-CSF (R leading to foam cell formation is a key event in atherogenesis (1). & D Systems). This medium was used in all experiments. The acquisition of cholesterol by macrophages is mediated by ϭ ͞ ϭ receptor-dependent and receptor-independent processes involving Lipoproteins. LDL (d 1.006–1.063 g ml) and HDL3 (d 1.125– both normal and modified lipoproteins. A number of macrophage 1.21 g͞ml) were prepared from human plasma of healthy donors cell surface receptors have been identified that can bind chemically using standard methods. LDL was acetylated through the repeated modified low-density lipoprotein (LDL) and include the class A addition of acetic anhydride followed by dialysis in PBS (21). scavenger receptors, CLA-1, class BI scavenger receptor, Fc␥RII (CD32), CD36, and CD68 (for review, see ref. 2). Determination of Cholesteryl Esters. Cellular lipids were extracted as ͞ Efficient reverse cholesterol transport from peripheral cells to described (22). In brief, samples were dissolved in CHCl3 MeOH the liver mediated by high-density lipoprotein (HDL) protects against the development of atherosclerosis (3). Sphingolipid-rich microdomains in the cell membrane such as caveolae and rafts (4), This paper was submitted directly (Track II) to the PNAS office. various receptor complexes including class BI scavenger receptors Abbreviations: LDL, low-density lipoprotein; HDL, high-density lipoprotein; ABC, ATP- (5, 6) and HB1͞2 (7), and GPI-linked proteins (e.g., CD36) (8) have binding cassette; acLDL, acetylated LDL. been suggested to be involved in the process of reverse cholesterol §To whom reprint requests should be addressed at: Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93042 transport. Moreover, apolipoproteins, lipid transfer proteins (cho- Regensburg, Germany. E-mail: [email protected]. lesteryl ester transfer protein, phospholipid transfer protein), and The publication costs of this article were defrayed in part by page charge payment. This lipoprotein processing enzymes (lecithin:cholesterol acyltrans- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. ferase, lipoprotein lipase, hepatic lipase) were implicated in cho- §1734 solely to indicate this fact. PNAS ͉ January 18, 2000 ͉ vol. 97 ͉ no. 2 ͉ 817–822 Downloaded by guest on September 29, 2021 and were separated by using external standards on 10- ϫ 20-cm separated on a 12% SDS͞PAGE and were transferred onto a silica gel HPTLC plates (Merck) in a 80:20:1 mixture of n-hexane, PVDF membrane (Fluorotrans transfer membrane, Pall). ABCG1 diethylether, and acetic acid (Ͼ99%). Plates were stained and was detected by using the chicken antibody (1:1,000 dilution) and quantified on a TLC scanner II (Camag, Berlin). a peroxidase-conjugated goat anti-chicken antibody (Sigma) (1:5,000). ABCA1 was visualized by using the polyclonal antiserum RNA Extraction and Northern Blot Analysis. Total RNA was isolated by ABC1͞18 (Pineda) recognizing the sequence GEESDEKSHPGS using the guanidine isothiocyanate method (23). The ABCG1 of ABCA1 (1:200). specific probe was generated from cDNA by using the primers 5ЈGATCAATCGCATTCATTTA3Ј (forward) and 5ЈTCCTTCT- Flow Cytometry. To assess ABCG1 surface expression, cultured TTGTTTGTTATAT3Ј (reverse). Filters were rehybridized with a monocytes͞macrophages were harvested, washed, and stained with glyceraldehyde-3-phosphate dehydrogenase-specific probe to verify a saturating concentration of the polyclonal ABCG1 antiserum that comparable RNA amounts were loaded. Tissue-specific ex- followed by incubation with a FITC-labeled affinity-purified don- pression of ABCG1 was assessed by using a multiple tissue key anti-chicken F(ab)2 fragment (Jackson ImmunoResearch). For poly(A)ϩ RNA master blot from various human tissues (Clontech). the determination of the intracellular expression of ABCG1, mac- The blots were quantitated densitometrically, and the individual rophages were fixed with 4% paraformaldehyde, were permeabil- signal densities were calculated relative to the mRNA expression in ized (0.1% saponin), and were stained as described above. Preim- the liver (set to 100%). mune chicken serum was used as an isotype control. Flow cyto- metric analyses were performed on a FACSCalibur flow cytometer Differential Display. Differential display screening for genes that are (Becton-Dickinson). regulated by cholesterol import or export was performed as follows: 0.2 ␮g of total RNA was reverse-transcribed by utilizing specific, Light Microscopic Immunocytochemistry. Cultured cells were fixed in anchored oligo(dT) primers (Metabion, Munich; GeneAmp RNA Zamboni’s fixative and were incubated in 1% NaBH4 after a PCR core kit, Perkin–Elmer). Oligo(dT) primers had two addi- washing step. Endogenous peroxidases were inhibited by using 3% Ј tional nucleotides at their respective 3 ends: an invariable A at the H2O2. A subset