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Apolipoprotein A-1 interaction with plasma membrane lipid rafts controls cholesterol export from macrophages ¹

KATHARINA GAUS, LEONARD KRITHARIDES^,, GERD SCHMITZ^#, ALFRED BOETTCHER^#, WOLFGANG DROBNIK^#, THOMAS LANGMANN^#, CARMEL M. QUINN, ALISON DEATH, ROGER T. DEAN^|| and WENDY JESSUP^,2

Macrophage Biology Group, Centre for Vascular Research, School of Medical Sciences, University of New South Wales, Australia;
Department of Cardiology, Concord Hospital, NSW, Australia;
^# Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany;
Department of Medicine, University of Sydney, Australia; and
^|| University of Canberra, Australia

²Correspondence: Centre for Vascular Research, School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia. E-mail: w.jessup{at}unsw.edu.au

SPECIFIC AIMS

ApoA-1-mediated export of cholesterol is important in the removal of excess cholesterol from peripheral cells in the first step of its transport to the liver for catabolism to bile acids, yet the mechanism(s) involved are still unclear. To examine how apolipoprotein A-1 (apoA-1) mediates cholesterol removal from macrophage foam cells (cholesterol-loaded cells found in atherosclerotic lesions), we studied how 7-ketocholesterol (7KC) interferes with the process. We previously reported that cellular accumulation of 7KC prevents apoA-1 dependent cholesterol efflux, used kinetic studies to show that 7KC decreases the size of a minor membrane cholesterol pool involved in initial, rapid efflux of cholesterol to apoA-1, and hypothesized that the physical location of this pool may be plasma membrane (PM) lipid rafts and that its mobilization is essential for mass cholesterol export.

PRINCIPAL FINDINGS

1. Inhibition of cellular cholesterol efflux by 7KC is associated with decreased apoA-1 binding to the cell surface but is independent of ABCA1 expression
Differentiated human THP-1 macrophages were loaded with excess cholesterol acquired by uptake of acetylated low-density lipoprotein (AcLDL). Where required, 7KC was incorporated into LDL before acetylation and exposure to the cells. Macrophage cholesterol was increased 2.5-fold (from 40 to 100 nmol/mg cell protein) ± 20 nmol/mg 7KC. We used these cells to measure cholesterol export to apoA-1, showing (as before) that efflux was inhibited when 7KC was present (4.3±1.1%/3 h vs. 9.7±1.3%/3 h, with and without 7KC, respectively) even though their cholesterol contents were identical.

7KC supplementation did not affect on PM cholesterol content, but caused a large (2.5-fold) loss in cell surface apoA-1 specific binding capacity (B_max 501±233 ng/mg vs. 1299±258ng/mg at 4°C). Similar results were found using primary human monocyte-derived macrophages (HMDM).

ApoA-1 binding and cholesterol efflux are dependent on ABCA1, a membrane lipid transporter. ABCA1 mRNA and protein expression were unaffected by 7KC supplementation, indicating that the inhibitory action of 7KC is independent of direct effects on ABCA1 expression.

2. 7KC depletes lipid raft cholesterol and inhibits apoA-1 binding to lipid raft domains in the plasma membrane
On the basis of previous kinetic analysis of cholesterol efflux stimulated by apoA-1, we defined two independent pathways from distinct PM pools: a rapid initial (0–1 h) efflux from a minor pool, then a slower sustained (>1.5 h) efflux from a larger pool, from which the majority of cholesterol export occurs. Cholesterol mobilization from this fast pool is required to stimulate subsequent efflux from the major slow pathway. We hypothesized that fast cholesterol efflux is derived from lipid raft regions of the PM and that 7KC decreases cholesterol availability in this pool. To test this, we isolated PM and separated "lipid raft" and "non-raft" domains by sonication and density gradient centrifugation. Lipid rafts contained 20% total PM cholesterol and sphingomyelin was the major phospholipid (PL). THP-1 and HMDM raft compositions were similar, except that HMDM lacked detectable caveolin-1. 7KC supplementation of cholesterol-loaded cells caused a significant loss of cholesterol from lipid rafts (445±62 vs. 776±113 nmol/mg protein, with and without 7KC, respectively) while having no effect on non-raft domain cholesterol (101±38 vs. 91±43 nmol/mg protein).

To determine the extent of apoA-1 binding to lipid raft and non-raft PM domains and the influence of 7KC, ¹²⁵I-apoA-1 was first bound to intact cells at 4°C before the domains were isolated. Most PM-associated apoA-1 binds to non-raft domains, but a small and significant proportion (10% total) binds to lipid rafts. 7KC supplementation reduced binding to both domains, but proportionally affected binding more to lipid rafts (8-fold) than to non-raft domains (2.8-fold).

3. Replenishment of lipid raft cholesterol restores apoA-1 raft binding and cholesterol efflux
Normally macrophages esterify 40% cholesterol (and 7KC, where added), but this can be prevented by incorporation of an ACAT inhibitor (S-58035) during sterol loading. Under these conditions, cholesterol efflux increased (16.1±0.8%/3 h vs. 9.7±1.3%/3 h for cells loaded with cholesterol with and without S-58035, respectively). The inhibitory effect of 7KC on cholesterol efflux was no longer observed (15.0±0.4%/3 h vs. 16.1±0.8%/3 h for cells with and without 7KC, respectively).

S-58035 increased cholesterol levels in the PM, in both lipid raft and non-raft domains. It prevented the cholesterol-depleting effect of 7KC on lipid raft cholesterol (1102±87 vs. 1198±168 nmol cholesterol/mg protein, with and without 7KC, respectively) and reversed its inhibition of apoA-1 binding to lipid rafts.

4. Cyclodextrin mediated depletion of raft cholesterol also specifically inhibits raft apoA-1 binding and cholesterol efflux
Correlations between 7KC-induced changes in lipid raft cholesterol content, apoA-1 binding and overall cholesterol efflux are consistent with our hypothesis that direct apoA-1/lipid raft interaction is necessary for cholesterol efflux. However an additional independent method for lipid raft cholesterol depletion was applied, in which cells were preincubated with cyclodextrin (CD) for 1 h. Under these conditions, there is significant loss of cholesterol from lipid rafts but little effect on total sterol loading (Fig. 1 ). CD selectively depleted lipid raft cholesterol 50% and inhibited subsequent apoA-1 binding to lipid rafts significantly (5-fold) while corresponding non-raft parameters were unaffected. Significantly, apoA-1 induced cholesterol efflux was inhibited 50%. Similar results were found for HMDM (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 1. Cyclodextrin pretreatment lowers lipid raft cholesterol levels, cholesterol efflux and apoA-1 binding to lipid rafts. AcLDL-loaded cells were pretreated for 1 h at 37°C with medium containing BSA (1 mg/mL) alone or plus hydroxypropyl-ß-cyclodextrin (1 mg/mL; hpCD) or trimethyl-b-cyclodextrin (3 mg/mL; tmCD). A) Cholesterol in lipid rafts after CD treatment. B) After the above preincubations, cholesterol efflux to fresh medium containing apoA-1 (50 µg/mL) was measured. The effect of CD preincubation on 125I-ApoA-1 binding at 4°C to lipid raft (C) and non-raft (D) membrane domains. Total apoA-1 binding in these experiments was 558 ± 42, 426 ± 13, and 523 ± 120 ng/mg cell protein control, hpCD, and tmCD pretreatments, respectively. E) Kinetics of cholesterol efflux to apoA-1 over the first 3 h after BSA (), hpCD () and tmCD () pretreatment. A, C D) Data are means of 2 separate experiments (±range); B, E) data are from 3 separate experiments (±SD); *statistical difference from control of P < 0.05.

CONCLUSIONS AND SIGNIFICANCE

We propose a model for the mechanism of apoA-1 mediated cholesterol efflux that involves two discrete interactions with microdomains in the plasma membrane (Fig. 2 ). First, apoA-1 binds to lipid rafts. This stimulates an initial fast efflux of a small membrane cholesterol pool originating from rafts. More important, the interaction of apoA-1 with lipid rafts permits initiation of cholesterol efflux from larger and more slowly released pools. The bulk of cholesterol efflux, including that derived from cholesterol ester hydrolysis, occurs by this slower pathway. We suggest that this second pathway is the ABCA1-dependent process and involves interactions between apoA-1 and non-raft domains of the plasma membrane. This model is supported by binding studies, kinetic analysis of cholesterol efflux, and simultaneous perturbation of both apoA-1 binding to rafts and of cholesterol efflux by treatments (7KC or cyclodextrins) that deplete lipid rafts of cholesterol. The lack of caveolin-1 in HMDM indicates that caveolae are not required.

View larger version (20K): [in this window] [in a new window]   Figure 2. Model for cholesterol efflux to apoA-1.

We considered an alternative model in which apoA-1 is lipidated by raft-derived lipids to become a more efficient acceptor for subsequent efflux of cholesterol by the slow pathway. This was excluded by our earlier work in which "conditioning" apoA-1 by preexposure to cholesterol-loaded macrophages did not accelerate onset of the slow efflux pathway when applied to a fresh set of cells. The relative proportions of individual PL extracted by apoA-1 more closely resemble the composition of non-raft PM domains. We suggest that lipid rafts are less likely to act as sources of lipid but, rather, to provide a functional platform whose engagement appears to be essential for apoA-1 stimulated, ABCA1-dependent lipid export. Lipid rafts are the sites of initiation of some signaling cascades, suggesting that apoA-1 may trigger such a pathway required to stimulate cholesterol export. The nature of such a mechanism and its possible interaction with ABCA1 remain to be determined. Recent studies suggesting the ABCA1 undergoes phosphorylation point to this as a possible target in apoA-1/lipid raft stimulation of mass cholesterol efflux.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0486fje;

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