The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway

Abstract

The ABC1 transporter was identified as the defect in Tangier disease by a combined strategy of gene expression microarray analysis, genetic mapping, and biochemical studies. Patients with Tangier disease have a defect in cellular cholesterol removal, which results in near zero plasma levels of HDL and in massive tissue deposition of cholesteryl esters. Blocking the expression or activity of ABC1 reduces apolipoprotein-mediated lipid efflux from cultured cells, and increasing expression of ABC1 enhances it. ABC1 expression is induced by cholesterol loading and cAMP treatment and is reduced upon subsequent cholesterol removal by apolipoproteins. The protein is incorporated into the plasma membrane in proportion to its level of expression. Different mutations were detected in the ABC1 gene of 3 unrelated patients. Thus, ABC1 has the properties of a key protein in the cellular lipid removal pathway, as emphasized by the consequences of its defect in patients with Tangier disease.

Figure 1

Gene expression microarray analysis. Six Gene Album™ microarrays, containing a total of 58,800 human cDNAs, were hybridized with cDNA labeled with Cy3 dye prepared from RNA from cAMP-treated TD1 cells, and with cDNA labeled with Cy5 dye from cAMP-treated normal cells as described in Methods. Spots indicate the relative expression of the 175 genes more than 2.5-fold under expressed in the TD cells compared with normal cells (above and to the left of the diagonal) and the 375 genes greater than 2.5-fold more abundantly expressed in the TD cells than in normal cells. For clarity, the other 58,250 spots that lie within the range depicted are not shown.

Figure 2

Effects of DIDS and BSP on apo A-I–mediated cholesterol efflux. Cholesterol-loaded and [³H]cholesterol–labeled normal fibroblasts were incubated for 6 hours with or without 5 μg/mL apo A-I and the indicated concentrations of DIDS or BSP. [³H]cholesterol efflux was measured as the percentage of total radiolabeled cholesterol appearing in the medium (y axis). Results are the mean ± SD (n = 3) of efflux in the presence of apo A-I after subtraction of values for apo A-I–free medium.

Figure 3

(a) Antisense inhibition of ABC1 reduces cholesterol efflux. Normal human skin fibroblasts were labeled with [³H]cholesterol as described in Methods. Cells were scrape-loaded in the presence of either 30 μM standard control Morpholino oligonucleotide (antisense complement of a β-globin thalassemic mRNA) or 30 μM ABC1 antisense Morpholino oligonucleotide, or were mock-loaded by scraping in the absence of oligonucleotide. Apo A-I–mediated efflux was measured after 12 hours as in the legend to Figure 2. Results presented are the mean ± SEM of 3 separate experiments, normalized to the value for apo A-I–specific efflux in the absence of oligonucleotide in each experiment. (b) Overexpression of ABC1 enhances apo A-I–mediated cholesterol efflux. Parental RAW 264.7 cells and clonal lines 3, 5, and 6 that had been stably transfected with a vector-expressing human ABC1 were cholesterol-loaded and labeled by incubation for 24 hours with 0.5 μCi/mL [³H]cholesterol and 50 μg/mL acetylated LDL, and efflux was measured as described in Methods. Results presented are the mean ± SEM of 3 separate experiments normalized to the value for apo A-I–specific efflux from parental RAW 264.7 cells within each experiment.

Figure 4

Regulation of ABC1 gene expression. Immortalized normal and TD1 and TD2 Tangier fibroblasts were incubated in serum-free medium plus albumin (filled bars), with the addition of 8-Br-cAMP (open bars), cholesterol (gray bars), or cholesterol followed by 18 hours of exposure to apo A-I (hatched bars). The defect in apo A-I–mediated cholesterol efflux described for TD cells is reflected by the lack of reduction in ABC1 mRNA after apo A-I treatment in TD1 and TD2 fibroblasts, which is apparent in normal cells (compare hatched bars with gray bars in each group).

Figure 5

Regulation of cell-surface ABC1 expression. Immortalized normal and TD1 fibroblasts were incubated in serum-free medium containing BSA, as follows: 18 hours with no additions (ctrl), 18 hours with 1 mM 8-Br-cAMP (cAMP), 48 hours with 30 μg/mL cholesterol plus 18 hours with no additions (chol), 48 hours with cholesterol plus 18 hours with 8-Br-cAMP (chol + cAMP). Cell-surface proteins were biotinylated with sulfo-NHS-biotin; ABC1 was isolated from detergent extracts by immunoprecipitation; and biotinylated ABC1 was identified with a streptavidin-horseradish peroxidase ECL detection assay as described in Methods.