The ATP-binding cassette transporter-2 (ABCA2) regulates esterification of plasma membrane cholesterol by modulation of sphingolipid metabolism

Abstract

The ATP-binding cassette transporters are a large family (~48 genes divided into seven families A-G) of proteins that utilize the energy of ATP-hydrolysis to pump substrates across lipid bilayers against a concentration gradient. The ABC "A" subfamily is comprised of 13 members and transport sterols, phospholipids and bile acids. ABCA2 is the most abundant ABC transporter in human and rodent brain with highest expression in oligodendrocytes, although it is also expressed in neurons. Several groups have studied a possible connection between ABCA2 and Alzheimer's disease as well as early atherosclerosis. ABCA2 expression levels have been associated with changes in cholesterol and sphingolipid metabolism. In this paper, we hypothesized that ABCA2 expression level may regulate esterification of plasma membrane-derived cholesterol by modulation of sphingolipid metabolism. ABCA2 overexpression in N2a neuroblastoma cells was associated with an altered bilayer distribution of the sphingolipid ceramide that inhibited acylCoA:cholesterol acyltransferase (ACAT) activity and cholesterol esterification. In contrast, depletion of endogenous ABCA2 in the rat schwannoma cell line D6P2T increased esterification of plasma membrane cholesterol following treatment with exogenous bacterial sphingomyelinase. These findings suggest that control of ABCA2 expression level may be a key locus of regulation for esterification of plasma membrane-derived cholesterol through modulation of sphingolipid metabolism.

Keywords: ABCA2; Ceramide; Cholesterol; Esterification; Sphingomyelin; Transporter.

Figure 1. ABCA2 protein expression in N2a and D6P2T cells

A. A representative Western blot is shown of ABCA2 protein expression in parental N2a cells and human ABCA2 overexpressing A2.1 cells. B. A representative Western blot is shown of ABCA2 protein expression in control D6P2TL (lamin-specific RNAi) cells and ABCA2-depleted D6P2TA (ABCA2 RNAi-specific) cells. The ABCA2-specific c-terminal antibody detects human, rat and mouse ABCA2 protein.

Figure 2. ABCA2 expression modulates cholesterol esterification in N2a cells

A. Cells were metabolically radiolabeled to equilibrium with [³H]cholesterol for 24 hours. Bacterial sphingomyelinase (0.5 units), 25-hydroxycholesterol (20 μg/ml), or both were added and the cells were cultured for 6 hours before lipid extraction and TLC. B. Cells were radiolabeled as described above and treated with U18666a (3 mg/ml) for 4 hours before addition of ± 25-hydroxycholesterol (20 μg/ml) and the cells were cultured for an additional 4 hours before lipid extraction in hexane/isopropanol (3:2) and thin-layer chromatography (TLC) in hexane/ethyl ether/acetic acid (80:18.5:1.5). [³H]cholesteryl ester levels were determined using a Bioscan 2000 thin layer chromatography scanner.

Figure 3. Depletion of endogenous ABCA2 increases cholesterol esterification in D6P2T cells

Cells were metabolically radiolabeled to equilibrium with [³H]cholesterol for 24 hours. Bacterial sphingomyelinase (0.5 units) was added and the cells were cultured for 5 hours before lipid extraction and TLC performed as described in Methods.

Figure 4. Distribution of cholesterol and sphingomyelin in lipid raft compartments in D6P2T cells

Cells were cultured as described in Methods and cell homogenates were prepared by solubilization of D6P2TL and D6P2TA cells in Triton X-100 and Lubrol WX in MBS buffer, followed by sucrose density gradient ultracentrifugation as described in Methods. Twelve fractions were recovered and probed by Western blot with antibodies to flotillin-1 (raft) and calnexin (non-raft). Cholesterol and sphingomyelin levels were determined on an aliquot of each fraction using fluorimetric assays as described in Methods.

Figure 5. Plasma membrane sphingomyelin-dependent lysenin toxicity in D6P2T cells

Cells were treated with 200 ng/ml of the plasma membrane sphingomyelin-specific toxin lysenin for 2 hours before cells were counted and re-plated at a density of 1.2 × 10⁶ cells per ml. Cell viability was measured after addition of the WST-1 cell proliferation reagent, culture for 2 hours at 37° C and measurement of absorbance at 350 nm.

Figure 6. In vitro sphingomyelin synthase activity is modulated by ABCA2 expression

Cells were cultured as described in Methods. Cell pellets were lysed by N₂ cavitation and 100 μg of cell homogenate were incubated with 2 μg/ml of C6-NBD ceramide and 10 μg of L-a-phosphatidylcholine for 2 hours at 37° C. Lipids were extracted by method of Bligh and Dyer and TLC was performed in chloroform/methanol/ammonium hydroxide (70:30:5). Fluorescent NBD-lipids were detected using a Storm phosphorimager and quantified using ImageQuant software.

Figure 7. ABCA2 expression modulates de novo sphingomyelin synthesis in intact cells

Cells were cultured as described in Methods. After addition of 2 μCi of [methyl-¹⁴C]chloride, the cells were cultured for 4 hours before lipid extraction in hexane/isopropanol (3:2) and TLC performed in chloroform/methanol/ammonium hydroxide (70:30:5). Formation of [¹⁴C]sphingomyelin was quantified using a Bioscan 2000 thin layer chromatography scanner and normalized to total cell protein.

Figure 8. Plasma membrane sphingomyelin synthase-2 expression in N2a and D6P2T cells

Whole cell homogenates were prepared from N2a and A2.1 cells or D6P2TL and D6P2TA cells and probed for SMS2 expression. Shown is a representative Western blot with a low-amplification signal to detect SMS2 expression in D6P2T cells and high-amplification signal to detect SMS2 expression in N2a cells.

Figure 9. Transbilayer transport of ceramide in the plasma membrane in N2a cells

Cells were cultured as described in Methods. C6-NBD-ceramide complexed to BSA was added to a final concentration of 5 μM and the cells were incubated at 4° C for 30 min. in the dark and then warmed to 37° C for 2 hours. Cells were counted and lipid extraction was performed on equal numbers of cells with chloroform/methanol/50 mM Tris-HCl, pH 8.5 (2:1:0.3) as described in Methods. Fluorescent NBD-C1P was recovered from the aqueous phase and measured in a plate reader at ex:em 460:515–580. TLC confirmed that the fluorescent product was NBD-C1P.

Figure 10. Back-extraction of NBD-lipids from the plasma membrane in N2a cells

Cells were cultured as described in Methods. C6-NBD-ceramide, complexed to BSA was added to a final concentration of 5 μM and the cells were incubated at 4° C for 30 min. in the dark and then warmed to 37° C for 2 hours. After the incubation, period cells were washed and NBD-lipids were removed from the outer surface of the plasma membrane with ± 3% BSA at 4° C. Cells were counted and lipid extraction was performed on equal numbers of cells with chloroform/methanol/0.01 N HCl (1:2:0.6). Cell-associated NBD-lipids were dried under N₂ and TLC performed in chloroform/methanol/ammonium hydroxide (70:30:5) followed by fluorescence imaging using a Storm phosphorimager as described above.

Figure 11. Elevation of endogenous ceramide by D-erythro MAPP decreases cholesterol esterification in N2a cells

Cells were cultured as described in Methods. D-erythro-MAPP (50 μg/ml) was added and cells were cultured for 5 hours. Lipids were extracted in hexane/isopropanol (3:2) and TLC performed in hexane/ethyl ether/acetic acid (80:18.5:1.5) to determine [³H]cholesteryl ester levels.

Fig. 12. Analysis of ceramide and sphingomyelin levels in N2a, A2.1, D6P2TL and D6P2TA cells

Total lipids were extracted from the cells and subjected to electrospray ionization/mass spectrometry analysis as described in Methods. The levels of ceramide (A) and sphingomyelin B were determined and normalized to Pi concentrations. Results are the mean ± SD. Statistical significance was determined using the Student’s t test, p < 0.05.

Fig. 13. Model of ABCA2 function in esterification of plasma membrane cholesterol

A. Ceramide moves between inner and outer leaflet of the lipid bilayer. ABCA2 overexpression in A2.1 cells alters the bilayer distribution, favoring the outer leaflet. ABCA2 promotes NBD-ceramide distribution in the outer leaflet and BSA extraction from cells. Sphingomyelinase hydrolysis of membrane sphingomyelin or inhibition of ceramidase activity, both elevate membrane ceramide levels. In ABCA2-overexpressing cells, elevated ceramide in the outer leaflet inhibits ACAT activity and conversion of plasma membrane cholesterol to cholesteryl esters. B. Depletion of endogenous ABCA2 by RNAi in D6P2TA cells is associated with decrease sphingomyelin synthase-2 (SMS2) expression, the enzyme responsible for synthesis of plasma membrane sphingomyelin. D6P2TA cells also show increased lysenin toxicity, an agent that specifically binds sphingomyelin in the outer leaflet of the plasma membrane. Sphingomyelinase hydrolysis of plasma membrane sphingomyelin releases associated cholesterol for trafficking to the endoplasmic reticulum for esterification.