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Genes to Cells (2009) 14, 191-204. doi:10.1111/j.1365-2443.2008.01261.x
© 2009 Blackwell Publishing or its licensors
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Retroendocytosis pathway of ABCA1/apoA-I contributes to HDL formation

 Yuya Azuma1, Mie Takada1, Hye-Won Shin2, Noriyuki Kioka1, Kazuhisa Nakayama2 and Kazumitsu Ueda1,3,*

1 Laboratory of Cellular Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
2 Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
3 Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto 606-8502, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
ATP-binding cassette protein A1 (ABCA1) mediates transfer of cellular free cholesterol and phospholipids to apolipoprotein A-I (apoA-I), an extracellular acceptor in plasma, to form high-density lipoprotein (HDL). It is currently unknown to what extent ABCA1 endocytosis and recycling contribute to the HDL formation. To address this issue, we expressed human ABCA1 constructs with either an extracellular HA tag or an intracellular GFP tag in cells, and used this system to characterize endocytosis and recycling of ABCA1 and apoA-I. Under basal conditions, ABCA1 and apoA-I are endocytosed via a clathrin- and Rab5-mediated pathway and recycled rapidly back to the cell surface, at least in part via a Rab4-mediated route; approximately 30% of the endocytosed ABCA1 is recycled back to the cell surface. When receptor-mediated endocytosis is inhibited, the level of ABCA1 at the cell surface increases and apoA-I internalization is blocked. Under these conditions, apoA-I mediated cholesterol efflux from cells that have accumulated lipoprotein-derived cholesterol is decreased, whereas efflux from cells without excess cholesterol is increased. These results suggest that the retroendocytosis pathway of ABCA1/apoA-I contributes to HDL formation when excess lipoprotein-derived cholesterol has accumulated in cells.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Disruption of cellular cholesterol homeostasis can lead to a variety of pathological conditions, including cardiovascular diseases (Oram & Vaughan 2006). ATP-binding cassette protein A1 (ABCA1), one of the key proteins in cholesterol homeostasis, mediates transfer of cellular free cholesterol and phospholipids to apolipoprotein A-I (apoA-I), an extracellular acceptor in plasma, to form high-density lipoprotein (HDL) (Abe-Dohmae et al. 2000; Tanaka et al. 2003). The formation of HDL is the only pathway by which excess cholesterol can be eliminated from peripheral cells. Defects in ABCA1 cause Tangier disease (Bodzioch et al. 1999; Brooks-Wilson et al. 1999; Rust et al. 1999), in which patients show a near-absence of circulating HDL, prominent cholesterol-ester accumulation in tissue macrophages, and premature atherosclerotic vascular lesions ( Singaraja et al. 2003; Oram & Vaughan 2006).

pite extensive studies, it remains uncertain how and where ABCA1 mediates transfer of cholesterol and phospholipids to apoA-I. The pioneering study of Takahashi and Smith (Takahashi & Smith 1999) showed that following internalization, apoA-I is recycled back to the cell surface to be re-secreted. Other studies showed that apoA-I and ABCA1 are co-localized in endosomal compartments, and that ABCA1 rapidly shuttles between intracellular compartments and the plasma membrane (Neufeld et al. 2001, 2004). Trapping ABCA1 on the plasma membrane by cyclosporine treatment reduces apoA-I-mediated cholesterol efflux (Le Goff et al. 2004). Deletion of a PEST sequence in the ABCA1 protein blocks its endocytosis and decreases apoA-I-mediated efflux of cholesterol derived from endocytosed lipoproteins (Chen et al. 2005). These results together support the idea that ABCA1-bound apoA-I is delivered to late endosomal and/or lysosomal compartments, where it forms nascent lipoprotein particles that are subsequently secreted from the cell.

However, several groups have recently examined internalization and recycling of apoA-I and concluded that the retroendocytosis pathway does not contribute significantly to HDL formation. The majority of internalized apoA-I is directly transported to late endosomes and lysosomes for degradation; furthermore, and blocking endocytosis does not decrease apoA-I-dependent cholesterol efflux from BHK cells expressing exogenous ABCA1 and RAW264.7 macrophages (Denis et al. 2008). Although apoA-I is specifically taken up by macrophages, only a small fraction of apoA-I is re-secreted from these cells. Furthermore, the majority of re-secreted apoA-I is degraded in the medium. These results suggest that the mass of retroendocytosed apoA-I is not sufficient to account for HDL produced by cholesterol efflux (Faulkner et al. 2008). The formation of novel apolipoprotein binding structures protruding from the cell surface may be an intermediate step in the pathway by which apolipoproteins remove excess cholesterol from the cells (Lin & Oram 2000; Vedhachalam et al. 2007).

In this study, we sought to characterize the endocytic and recycling routes of newly internalized ABCA1 molecules from the cell surface. To do so, we expressed human ABCA1 constructs with an extracellular HA tag and chased it by real-time observation. We found that internalized apoA-I co-localizes with cell surface-derived ABCA1 on endosomal compartments, and approximately 30% of the endocytosed ABCA1 is recycled to the cell surface. Our results also showed that clathrin-dependent endocytosis of ABCA1 and apoA-I contributes to HDL formation when excess lipoprotein-derived cholesterol has accumulated in cells.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Subcellular localization and function of ABCA1(207HA)

Endocytosis and recycling at the plasma membrane of ABCA1 has been proposed to be critical for HDL formation (Takahashi & Smith 1999; Neufeld et al. 2001; Ito et al. 2002). When the subcellular localization of ABCA1-GFP is observed, however, it is difficult to discriminate between ABCA1-GFP on newly internalized vesicles and protein residing in other intracellular vesicular compartments. To overcome this problem, and chase newly internalized ABCA1 molecules from the cell surface, we made use of a human ABCA1 construct, ABCA1(207HA), that bears an HA epitope sequence between residues 207 and 208 within its first extracellular domain (Fig. 1A).


Figure 1
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  		Figure 1  Subcellular localization of and cholesterol efflux by ABCA1(207HA). (A) The predicted secondary structure of ABCA1(207HA). The HA epitope sequence was inserted between residues 207 and 208 in the first extracellular domain of ABCA1. (B, C) Immunofluorescence detection of ABCA1(207HA). HEK293 cells stably expressing ABCA1(207HA) were stained with mouse monoclonal anti-HA antibody and Alexa488-anti-mouse IgG under non-permeabilized (B) and permeabilized (C) conditions. (D) Epifluorescence analysis of HEK293 cells stably expressing ABCA1-GFP. Bars, 10 µm. (E) Cholesterol efflux from control HEK293 cells (control), or those stably expressing wild-type untagged ABCA1, wild-type ABCA1(207HA), ABCA1(207HA)W590S, or ABCA1(207HA)MM in the presence of 10 µg/mL apoA-I at 37 °C for 24 h.

 
First, we examined whether the HA epitope insertion affected localization and function of ABCA1. When HEK293 cells stably expressing ABCA1(207HA) were incubated with a complex of anti-HA antibody and Alexa488-conjugated secondary antibody on ice for 30 min, the cell surface was specifically labeled (Fig. 1B); no fluorescent signals were detected with parental HEK293 cells (data not shown). When the ABCA1(207HA)-expressing cells were fixed, permeabilized and stained with the anti-HA antibody, fluorescent signals were detected not only on the plasma membrane but also on intracellular vesicular structures (Fig. 1C). The subcellular distribution of ABCA1(207HA) in permeabilized cells (Fig. 1C) was indistinguishable from that of ABCA1-GFP (Fig. 1D), suggesting that the insertion of the HA epitope sequence into the extracellular loop did not affect ABCA1 localization.

We then examined apoA-I-dependent cholesterol efflux from HEK293 cells stably expressing the ABCA1 constructs. As shown in Fig. 1E, ABCA1(207HA) mediated cholesterol efflux as efficiently as untagged ABCA1 did. A Tangier disease-type mutation (W590S) greatly reduced the cholesterol efflux activity of ABCA1(207HA), and lysine-to-methionine mutations (MM) in both of the two ATP-binding domains almost completely abolished activity (Fig. 1E), as we previously reported in the context of ABCA1 or ABCA1-GFP (Tanaka et al. 2003). As shown in Fig. S1 in Supporting Information/Supplementary material, the subcellular localization and cell surface expression of the W590S and MM mutants of ABCA1(207HA) (lower panels) were indistinguishable from those of the corresponding mutants of ABCA1-GFP. Taken together, the data presented so far indicate that the HA epitope insertion into the extracellular loop affects neither the subcellular localization nor the function of ABCA1.

Internalization of ABCA1 and apoA-I

To examine internalization of ABCA1(207HA), HEK293 cells stably expressing ABCA1(207HA) were labeled with a complex of anti-HA antibody and Alexa488-conjugated secondary antibody on ice. After unbound antibodies were removed, cells were incubated at 37 °C for up to 60 min in the presence or absence of apoA-I (Fig. 2A). Before starting the 37 °C incubation (0 min), the fluorescent signals were observed exclusively on the cell surface (top panel); no fluorescence signals were detected with parental HEK293 cells (data not shown). After incubation of the ABCA1(207HA)-expressing cells at 37 °C for 30 or 60 min, fluorescence signals were detected on intracellular vesicular structures, indicative of ABCA1(207HA) internalization. No difference in the signal distribution was discernible between the cells incubated with (bottom panels) or without (middle panels) apoA-I. These observations indicate that ABCA1(207HA) undergoes constitutive internalization, independent of apoA-I.


Figure 2
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  		Figure 2  Endocytosis of cell surface ABCA1, and co-localization of ABCA1 with internalized apoA-I. (A) HEK293 cells stably expressing ABCA1(207HA) was incubated with anti-HA antibody and Alexa488-anti-IgG on ice for 30 min (0 min), and then incubated at 37 °C for 30 or 60 min in the presence or absence of 10 µg/mL apoA-I. (B) HEK293 cells stably expressing ABCA1-GFP were incubated at 37 °C for 30 or 60 min with 10 µM monensin and 100 µg/mL cycloheximide in the presence or absence of 10 µg/mL apoA-I. Bar, 10 µm. (C, D) HEK293 cells stably expressing ABCA1(207HA) were incubated with anti-HA antibody and Alexa488-anti-IgG on ice for 30 min and then incubated at 37 °C with 10 µg/mL Alexa546-labeled apoA-I for 10 (C) or 30 min (D). Bar, 20 µm.

 
To exclude the possibility that binding of the HA antibody to the extracellular domain had triggered the ABCA1 internalization, we also examined internalization of ABCA1-GFP in the presence or absence of apoA-I (Fig. 2B). To follow the internalization of ABCA1-GFP, cells expressing ABCA1-GFP were treated with cycloheximide, to inhibit de novo protein synthesis and with monensin, which prevents delivery of proteins from the trans-Golgi network to the cell surface (Mollenhauer et al. 1990). Under these conditions, ABCA1-GFP gradually disappeared from the plasma membrane and instead appeared on intracellular vesicular structures, irrespective of the presence or absence of apoA-I. These observations agree well with those obtained using the ABCA1(207HA)-expressing cells (Fig. 2A) and support the model in which ABCA1 is constitutively internalized.

We next examined internalization of apoA-I in cells expressing the ABCA1(207HA) construct. After cells were incubated with Alexa546-conjugated apoA-I for 10 min, a significant apoA-I labeling was detected on the surface of cells expressing ABCA1(207HA) and ABCA1(207HA)W590S (Fig. 2C, top and middle row panels, respectively). By 30 min incubation, apoA-I was found on punctate endosomal structures, where it co-localized significantly with ABCA1 (Fig. 2D, top and middle row panels; also see magnified images on the right side). In marked contrast, neither cell-surface labeling nor internalization of apoA-I was observed in cells expressing ABCA1(207HA)MM (Fig. 2C,D, bottom row panels) or parental HEK293 cells (data not shown). These observations indicate that apoA-I is internalized in an ABCA1-dependent manner, and that while the ATPase activity of ABCA1 is required for binding of apoA-I on the cell surface, it is not required for internalization of ABCA1.

Recycling of ABCA1 to the cell surface

It has been shown that, once internalized, apoA-I reappears on the cell surface and is secreted into the medium (Takahashi & Smith 1999). However, it is not yet clear whether ABCA1 is also recycled to the cell surface. To address this issue, we took the following approach (Fig. 3A). Cells expressing ABCA1(207HA) were first incubated with anti-HA antibody on ice for 30 min, in order to label ABCA1(207HA) molecules on the cell surface (step 1), and were then incubated without the anti-HA antibody at 37 °C for 15 min to allow internalization of the ABCA1(207HA)-antibody complex (step 2). The cell surface level of ABCA1(207HA) was determined by measuring the amount of bound anti-HA antibody by ELISA (Fig. 3B). The cell surface level of ABCA1(207HA) at step 1 was defined as 100% (STEP1). Non-specific binding of the anti-HA antibody to parental HEK293 cells was estimated to be less than 10% of the binding of the anti-HA antibody to the ABCA1(207HA)-expressing cells (STEP1). After incubation at 37 °C for 15 min, 41.0 ± 4.6% of the bound anti-HA antibody remained on the cell surface (STEP2), suggesting that approximately half of the ABCA1 molecules were internalized or degraded on the cell surface at step 2.


Figure 3
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  		Figure 3  Recycling of internalized ABCA1 to the plasma membrane. (A) Schematic diagram of experimental procedures for detecting ABCA1 recycling to the cell surface. HEK293 cells stably expressing ABCA1(207HA) were incubated with anti-HA antibody on ice for 30 min (step 1) and at 37 °C for 15 min (step 2). The cells were washed with PBS+ (pH 3.2) to remove antibody from the cell surface (step 3) and incubated in the medium containing biotin-conjugated anti-mouse IgG for 60 min (step 4). Bound anti-mouse IgG was detected by ELISA. (B) Relative amounts of ABCA1(207HA) on the cell surface or recycled to the surface at step 5. HRP-conjugated streptavidin was added after cell permeabilization with 0.4% Triton X-100. Relative amount of cell surface ABCA1 was presented at step1, step2 and step3. The value of non-specific antibody binding and ELISA reaction with HEK293 cells was subtracted. Values are expressed as the percentage of the value at step 1 (100%).

 
The cells were then washed with acidic solution (pH 3.2) to remove the antibody remaining on the cell surface (step 3). As shown in Fig. 3B, more than 98% of the cell surface antibody was removed by the acid wash. Next, the cells were incubated in medium containing biotin-conjugated secondary antibody at 37 °C for 60 min (step 4). After this treatment, only ABCA1-anti-HA antibody complexes reappearing on the cell surface from inside the cell are expected to react with the secondary antibody in the medium. Finally, the cells were fixed and permeabilized, and the amount of the bound secondary antibody was determined by ELISA (step 5).

Specific antibody binding was estimated by subtracting the value of non-specific antibody binding to the parental HEK293 cells from that of the total binding (Fig. 3B). At step 5, 18.2 ± 1.9% of the initial cell surface ABCA1(207HA) at step 1 reacted with biotin-conjugated secondary antibody, corresponding to approximately 30% of the ABCA1(207HA) that disappeared from the cell surface at step 2. These results suggest that at least 30% of the internalized ABCA1(207HA) was able to be recycled to the cell surface in 60 min.

Endocytic route of ABCA1 and apoA-I

LDL receptor and transferrin receptor constitutively internalize and recycle to the plasma membrane via Rab5/Rab4-mediated endocytosis and recycling (Sonnichsen et al. 2000; McCaffrey et al. 2001; Lakadamyali et al. 2006). Rab5 regulates vesicular transport from the plasma membrane to early endosomes, whereas Rab4 directs recycling from early endosomes to the plasma membrane (Jordens et al. 2005). To determine whether ABCA1 follows a recycling pathway similar to that of LDL and transferrin receptors, we transiently expressed Rab proteins fused to the fluorescent protein in cells also expressing ABCA1(207HA) and compared the proteins’ subcellular localizations (Fig. 4). Twenty-four hours after transfection, the cells were incubated with a complex of anti-HA antibody and Alexa488-conjugated secondary antibody on ice for 30 min and then at 37 °C for up to 30 min to allow internalization of the ABCA1(207HA)-antibody complex. As shown in Fig. 4A, significant, albeit partial, co-localization of the internalized ABCA1(207HA) with GFP-Rab5a was observed up to 30 min at 37 °C, indicating that a fraction of internalized ABCA1 traverses early endosomes. Partial co-localization of internalized ABCA1(207HA) with YFP-Rab4a was also observed (Fig. 4B), suggesting direct recycling of ABCA1 through early endosomes to the cell surface. To further confirm the recycling of apoA-I by the same pathway, the localization of internalized apoA-I was compared with that of Rab4 using CHO-K1 cells, in which endogenous ABCA1 expression can be induced by treatment with TO901317 and 9-cis RA. As shown in Fig. 4C, Alexa546-labeled apoA-I co-localized with YFP-Rab4a, after 20 min internalization. Taken together, these observations indicate that ABCA1 and apoA-I are endocytosed and recycled to the cell surface through Rab5- and Rab4-positive endosomal compartments.


Figure 4
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  		Figure 4  Co-localization of ABCA1 with Rab GTPase. (A) HeLa cells were co-transfected with expression vectors for ABCA1(207HA) and GFP-Rab5a. Twenty-four hours after transfection, the cells were incubated with anti-HA antibody and Alexa546-conjugated anti-mouse IgG on ice, and then at 37 °C for 10, 20, or 30 min. The anti-HA antibody remaining on the cell surface was removed by acid wash (pH 2.0) to observe the internalized ABCA1(207HA). (B) CHO-K1 cells were co-transfected with expression vectors for ABCA1(207HA) and YFP-Rab4a. The internalized ABCA1 was visualized as described in (A). (C) CHO-K1 cells were transfected with an expression vector for YFP-Rab4a. Twenty-four hours after transfection, the cells were incubated with 5 µM TO901317 and 5 µM 9-cis-RA for 24 h to induce expression of ABCA1. The cells were then incubated with 10 µg/mL Alexa546-labeled apoA-I for 20 min. Bars, 10 µm.

 
We then examined whether Rab5 is involved in apoA-I endocytosis. As shown in Fig. 5A, GFP-Rab5a(WT) over-expression did not significantly affect apoA-I internalization in the induced CHO-K1 cells. In contrast, over-expression of GFP-Rab5a(S34N), a dominant-negative mutant, resulted in drastic suppression of apoA-I internalization; the majority of fluorescent apoA-I was found on the cell surface (Fig. 5B). In cells over-expressing GFP-Rab5a(Q79L), a constitutively active mutant, internalized apoA-I was accumulated in enlarged endosomal structures (Fig. 5C), which are generated by fusion of early endosomes induced by the active Rab5 mutant (Stenmark et al. 1994). These results indicate that internalization of apoA-I and its delivery to early endosomes of apoA-I are both dependent on Rab5.


Figure 5
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  		Figure 5  Effects of Rab5 mutants on apoA-I internalization. CHO-K1 cells were transfected with an expression vector for GFP-Rab5a(WT) (A), GFP-Rab5a(S34N) (B) or GFP-Rab5a(Q79L) (C). Twenty-four hours after transfection, the cells were incubated with 5 µM TO901317 and 5 µM 9-cis-RA for 24 h to induce expression of ABCA1. The cells were then incubated with 10 µg/mL Alexa546-labeled apoA-I for 60 min. Bars, 10 µm.

 
Clathrin-mediated internalization of ABCA1

We recently reported that ABCA1 resides in non-raft domains on the plasma membrane (Nagao et al. 2007); others have shown that apoA-I is internalized in a clathrin-dependent manner (Takahashi & Smith 1999). To determine whether endocytosis of ABCA1 is also a clathrin-mediated process, we investigated the effect of MDC on internalization of ABCA1 as well as apoA-I. MDC is a specific inhibitor of clathrin-mediated endocytosis, and has been reported to block internalization of various cell surface receptors (Schütze et al. 1999; Pierce et al. 2000; York et al. 2000). For this purpose, we used differentiated THP-1 macrophages, since these cells show endogenous expression of ABCA1 and can accumulate cholesterol in intracellular compartments following incubation with acetylated LDL. By treating THP-1 macrophages with MDC (100 µM), the cell surface level of ABCA1 was increased by approximately 1.5-fold (Fig. 6A,D) and apoA-I internalization was abolished (Fig. 6B,E), independent of prior incubation with acetylated LDL. These results indicate that, in the macrophages, internalization of ABCA1 and consequent ABCA1-mediated internalization of apoA-I are clathrin-dependent processes, irrespective of the intracellular cholesterol content.


Figure 6
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  		Figure 6  Effects of MDC on endocytosis of ABCA1 and apoA-I and on apoA-I-dependent cholesterol efflux from THP-1 cells. Human monocyte-derived THP-1 cells were incubated with 200 ng/ml PMA for 2 days, causing them to differentiate into macrophages. The resulting differentiated cells were then incubated with 3 µM TO901317 in the presence (D, E, F) or absence (A, B, C) of 50 µg/mL acetylated LDL for 24 h. (A, D) Cells pretreated with 100 µM MDC for 30 min or left untreated (control) were incubated with 10 µg/mL apoA-I for an additional 3 h in the continuous presence or absence of 100 µM MDC. Cell surface (biotinylated) and total ABCA1 in the lysate were detected by immunoblot analysis with anti-ABCA1 antibody. Vinculin was used as a control. The relative amounts of ABCA1 were determined (lower panels). (B, E) Cells pretreated with or without 100 µM MDC were incubated with 10 µg/mL Alexa546-labeled apoA-I for an additional 10 min in the continuous presence or absence of 100 µM MDC. Bars, 5 µm. (C, F) ApoA-I-dependent cholesterol efflux from cells preincubated with (F) or without (C) 50 µg/mL acetylated LDL for 3 h was measured in the presence or absence of 100 µM MDC.

 
On the other hand, the MDC treatment had differential effects on apoA-I-mediated cholesterol efflux (specifically, HDL formation) from THP-1 macrophages, depending on prior cholesterol accumulation resulting from incubation with acetylated LDL. The MDC treatment caused a 1.76-fold increase in the cholesterol efflux from macrophages not subjected to the acetylated LDL treatment (Fig. 6C). This increase was consistent with the increase in the cell surface level of ABCA1 observed following the MDC treatment (Fig. 6A; ~1.5-fold). Thus, when intracellular cholesterol content is limited, HDL formation does not involve clathrin-dependent endocytosis of either ABCA1 or apoA-I, but rather occur at the cell surface.

When the intracellular total and free cholesterol content of macrophages was increased by ~2.6-fold and ~2.1-fold, respectively, (data not shown) by prior incubation with acetylated LDL, apoA-I-mediated cholesterol efflux also increased by more than threefold compared with cells not subject to the acetylated LDL treatment. More importantly, the MDC treatment decreased the apoA-I-mediated cholesterol efflux by 35% relative to the cholesterol-preloaded macrophages (Fig. 6F), even if the cell surface level of ABCA1 was increased (Fig. 6D) and apoA-I internalization was blocked (Fig. 6E). To exclude the possibility that MDC impaired cholesterol trafficking from intracellular pools to the plasma membrane, we analyzed the effect of MDC on cholesterol extraction by MβCD, which strips cholesterol from the plasma membrane (Fig. S2 in Supporting Information/Supplementary Material). The amount of cholesterol available to MβCD was increased by 1.8-fold when cells were incubated with acetylated LDL, and the MDC treatment did not affect it. These results suggest that MDC does not affect intracellular cholesterol traffic to the plasma membrane. Furthermore, we examined the effect of hypertonic sucrose, which has been reported to inhibit endocytosis of receptors such as LDL receptor and β2-adrenergic receptor by blocking clathrin-coated pit formation (Heuser & Anderson 1989; Moore et al. 1995). Hypertonic sucrose blocked apoA-I internalization as expected (data not shown), and caused a 1.47-fold increase of the apoA-I-mediated cholesterol efflux from macrophages not subjected to the acetylated LDL treatment (Fig. S3 in Supporting Information/Supplementary Material). When the intracellular cholesterol content of macrophages was increased by prior incubation with acetylated LDL, hypertonic sucrose decreased the cholesterol efflux by 21%. Taken together, these data suggest that clathrin-mediated endocytosis of ABCA1 and apoA-I and possibly subsequent retroendocytosis of the ABCA1/apoA-I complex participate in HDL formation under conditions in which macrophages accumulate cholesterol intracellularly.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
Despite extensive studies on internalization and recycling of apoA-I (Denis et al. 2008; Faulkner et al. 2008; Hassan et al. 2008), internalization and recycling of ABCA1 have been poorly understood, and their contributions to HDL biogenesis have remained to be elucidated. In this study, we sought to characterize the endocytic route of ABCA1, and to determine the relative contribution of ABCA1/apoA-I retroendocytosis to cholesterol secretion. We find that, although it is dispensable under basal conditions, the retroendocytosis pathway contributes significantly to HDL formation when cells have accumulated excess lipoprotein-derived cholesterol in endosomes.

To estimate the contribution of endocytosis and recycling of ABCA1 to cholesterol secretion, we utilized MDC, a specific inhibitor of clathrin-mediated endocytosis (Fig. 6). By enhancing clathrin polymerization (Nandi et al. 1981) and thereby stabilizing clathrin cage assembly on the plasma membrane (Phonphok & Rosenthal 1991), MDC prevents internalization of various cell surface receptors, including tumor necrosis factor receptor (Schütze et al. 1999), β2-adrenergic receptor (Pierce et al. 2000), and nerve growth factor receptor TrkA (York et al. 2000). Treatment of differentiated THP-1 macrophages with MDC increased the cell-surface ABCA1 level by 1.5-fold and blocked apoA-I internalization. Unexpectedly, apoA-I-mediated cholesterol efflux was increased by 1.76-fold by the MDC treatment, under basal conditions when excess cholesterol had not accumulated in the cells (Fig. S3). The fold increase in the cholesterol efflux appeared to be parallel to the cell surface level of ABCA1. Thus, under these conditions, ABCA1 molecules on the cell surface make the major contribution to HDL formation; even though ABCA1 undergoes constitutive internalization and recycling, ABCA1 molecules in the intracellular compartments are dispensable for the HDL formation. These findings are consistent with recent reports suggesting that the ABCA1-mediated lipid transfer process occurs primarily at the membrane surface in macrophages (Denis et al. 2008; Faulkner et al. 2008).

In contrast, however, when THP-1 macrophages were subjected to prior incubation with acetylated LDL, causing them to accumulate cholesterol intracellularly, the apoA-I-mediated cholesterol efflux from THP-1 cells decreased by 35% in the presence of MDC (Fig. 6 and Fig. S3). As in the case of cells without incubation with acetylated LDL, the surface ABCA1 level was increased, and apoA-I internalization was blocked, as a result of the MDC treatment. If we assume that ABCA1-mediated lipid transfer process occurs primarily at the membrane surface in macrophages accumulate cholesterol, the apoA-I-mediated cholesterol efflux should be also increased by about 1.7-fold by the MDC treatment. However, conversely, it decreased by 35% in the presence of MDC. From these results, we may estimate that only about 40% (0.65/1.7 = 0.38) of HDL formation is mediated by ABCA1 on the membrane surface and that the internalized ABCA1/apoA-I is involved in 60% of HDL formation from macrophages accumulate cholesterol.

The similar effect was observed with hypertonic sucrose (Fig. S3), which inhibits endocytosis of receptors such as LDL receptor and β2-adrenergic receptor by blocking clathrin-coated pit formation (Heuser & Anderson 1989; Moore et al. 1995). Hypertonic sucrose caused a 1.47-fold increase of the apoA-I-mediated cholesterol efflux from macrophages not subjected to the acetylated LDL treatment. When the intracellular cholesterol content of macrophages was increased by prior incubation with acetylated LDL, hypertonic sucrose decreased the cholesterol efflux by 21%. Cholesterol accumulates in endosomal compartments in the free form, or in lipid droplets in its ester form (Maxfield & Tabas 2005). Because MDC did not affect MβCD-mediated cholesterol extraction from the plasma membrane, we conclude that this drug does not affect the cholesterol content in the plasma membrane. Tangier disease-type mutations in ABCA1 cause defects in late endocytic trafficking and accumulation of cholesterol and phospholipids in late endocytic vesicles (Neufeld et al. 2004). Taken together, these data suggest that clathrin-dependent endocytosis and subsequent retroendocytosis of the ABCA1/apoA-I complex is critical for HDL formation when cells accumulate excess lipoprotein-derived cholesterol.

Several lines of evidence from our study suggest constitutive internalization of ABCA1 in the absence of apoA-I. First, ABCA1(207HA) on the plasma membrane rapidly internalizes and translocates to endosomal compartments in the absence of apoA-I (Fig. 2A). Second, ABCA1-GFP on the cell surface translocates to intracellular vesicular structures in the absence of apoA-I when the cells are treated with monensin (Fig. 2B). Third, an ATPase-defective mutant of ABCA1, which is unable to interact with apoA-I, can internalize as efficiently as wild-type ABCA1 (Fig. 2C). Thus, apoA-I binding may not be a trigger for internalization of ABCA1, although endocytosis of ABCA1 is increased in the presence of apoA-I (Hassan et al. 2008). ApoA-I binding may alter the conformation of ABCA1 or the environment of surrounding lipids to prevent the proteolytic degradation of ABCA1; it is therefore possible that ABCA1 molecules that are not associated with apoA-I may be degraded faster than when they are associated with apoA-I. Indeed, more intense signals for ABCA1(207HA) and ABCA1-GFP were remained on the plasma membrane in the presence of apoA-I than in its absence (Fig. 2). ApoA-I association probably prevents degradation of ABCA1 on the plasma membrane, as reported previously (Arakawa & Yokoyama 2002; Wang et al. 2003), and consequently increases the amount of ABCA1 on the cell surface and in intracellular compartments. Most recently, it was reported that ABCA1 is internalized regardless of the presence of apoA-I, and that ABCA1, internalized without interacting with apoA-I, is intracellularly degraded by calpain (Lu et al. 2008).

Many membrane receptors, such as LDL receptor and transferrin receptor, constitutively internalize and recycle to the plasma membrane irrespective of whether they have bound their ligands. ABCA1 and apoA-I were endocytosed via a clathrin- and Rab5-mediated pathway and recycled rapidly to the cell surface, at least in part, via a Rab4-mediated route; approximately 30% of the endocytosed ABCA1 was recycled back to the cell surface. Based on these data, it is likely that ABCA1 follows essentially the same constitutive recycling pathway as the LDL receptor and transferrin receptor. Cholesterol secretion by ABCA1 is strongly dependent on the presence of extracellular acceptors such as lipid-free apoA-I. However, the role of apoA-I and the importance of the interaction of apoA-I and ABCA1 in HDL formation are still controversial. It is intriguing that ABCA1, involved in lipid secretion from cells, follows the same constitutive pathway as the LDL receptor, involved in cholesterol uptake.

To our knowledge, this is the first quantitative analysis of the fate of endocytosed ABCA1. We have reported here that the clathrin-mediated endocytosis and subsequent retroendocytosis of the ABCA1/apoA-I complex is likely to make a critical contribution to HDL formation under conditions where cells have accumulated excess lipoprotein-derived cholesterol in endosomal compartments. Recently, Faulkner et al. suggested that the retroendocytosis pathway of apoA-I does not contribute significantly to HDL formation (Faulkner et al. 2008). However, because the macrophages were labeled with cholesterol tracer, cholesterol could be selectively incorporated into the plasma membrane without passing through intracellular compartments. Besides the plasma membrane, endosomal compartments are the most important reservoirs of cellular cholesterol (Mukherjee et al. 1998). Our study sheds light on the importance of the endocytic pathway of ABCA1/apoA-I, and also on the role of endosomes as a source of cholesterol for HDL formation. The dynamics and function of ABCA1 in intracellular compartments should be analyzed more carefully and intensively in future studies.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Materials

We generated a mouse monoclonal anti-ABCA1 antibody, KM3110, which recognizes the C-terminal 20 amino acids of human ABCA1 (Munehira et al. 2004). Monoclonal mouse anti-HA antibody (F-7) was purchased from Santa Cruz Biotechnology. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was purchased from Bio-Rad. Biotin-conjugated anti-mouse IgG was purchased from DAKO. Alexa488- and Alexa546-conjugated anti-mouse IgG antibodies were purchased from Molecular Probes. HRP-conjugated streptavidin and sulfo-Sulfo-N-hydroxysuccinimidobiotin (NHS)-biotin were purchased from Pierce. TO901317 was purchased from Cayman Chemical. Monodansyl cadaverine (MDC), 9-cis retinoic acid (RA) and methyl-β-cyclodextrin (MβCD) were purchased from Sigma. Other chemicals were purchased from Sigma, GE Healthcare Biosciences, Wako Pure Chemical Industries, or Nacalai Tesque. Recombinant apoA-I (Nagao et al. 2007) and Alexa546-conjugated apoA-I (Neufeld et al. 2004) were prepared as described previously.

DNA construction

We constructed plasmids for expressing human wild-type ABCA1, ABCA1(W590S) and ABCA1(K939M,K1952M)(MM) bearing an insertion of the influenza virus hemagglutinin (HA) epitope sequence between residues 207 and 208 (within the first extracellular loop), using the bicistronic expression vector pHaMAIRESneo. We proceeded by fusing the ABCA1 cDNA to the aminoglycosidase phosphotransferase (neomycin resistance: neoR) gene with an internal ribosome entry site in order to co-express ABCA1 and neoR from a bicistronic transcript (Tanaka et al. 2003). We then established HEK293 cell lines stably expressing either WT or mutant ABCA1(207HA). Expression vectors for Rab protein with a fluorescent protein tag were described previously (Shin et al. 2004).

Cell culture and transfection

Human embryonic kidney (HEK)-293 cells and HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum (FBS) at 37 °C in 5% CO2. Chinese hamster ovary (CHO)-K1 cells were cultured in Ham's F-12 medium supplemented with 10% (v/v) FBS at 37 °C in 5% CO2. THP-1 monocytes were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS at 37 °C in 5% CO2. HEK293 cells stably expressing ABCA1-GFP was established as described previously (Tanaka et al. 2003). HeLa cells were transiently co-transfected with pcDNA3.1-ABCA1(207HA) and pEGFP-Rab5a to visualize ABCA1 and Rab5a. CHO-K1 cells were transiently co-transfected with pcDNA3.1-ABCA1(207HA) and pEYFP-Rab4a. Transient transfection was performed using LipofectAMINE Plus Reagent (Invitrogen) according to the manufacturer's instructions. To examine effects of Rab5 mutants on apoA-I trafficking, CHO-K1 cells were transfected with pEGFP-Rab5a (S34N or Q79L). Endogenous ABCA1 expression was induced by treatment with 5 µM TO901317 and 5 µM 9-cis RA.

Western blotting

Cells were washed with PBS and lyzed in RIPA buffer [20 mM Tris-NCl (pH 7.5), 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate] containing protease inhibitors, 100 µg/mL (p-amidinophenyl)methanesulfonyl fluoride, 2 µg/mL leupeptin, and 2 µg/mL aprotinin. Samples were electrophoresed on 7% SDS-polyacrylamide gel and detected with antibodies as indicated.

Confocal microscopic analysis of subcellular localization of ABCA1 and apoA-I

Cells stably expressing ABCA1(207HA) were grown on poly-L-lysine-coated coverslips. Cells were kept on ice for 10 min to inhibit intracellular trafficking and washed twice with ice cold PBS+ (phosphate-buffered saline containing 0.1 mg/mL CaCl2 and MgCl26H2O). Anti-HA antibody F-7 was pre-incubated with Alexa488-conjugated anti-mouse IgG at room temperature for 1 h before addition to the cell culture. The cells were incubated with anti-HA antibody F-7 (1 : 200) and anti-mouse IgG-Alexa488 (1 : 500) in PBS+ containing 1% bovine serum albumin (BSA) on ice for 30 min, washed three times with ice cold PBS+ to remove unbound antibodies, and incubated at 37 °C for the indicated times. The cells were then washed, fixed with 4% paraformaldehyde at room temperature for 30 min, and observed with a confocal microscope (LSM 510; Carl Zeiss). No fluorescence signals were detectable with control HEK293, HeLa and CHO-K1 cells (data not shown). For analyzing apoA-I localization, cells grown on collagen-coated coverslips were incubated with Alexa546-conjugated apoA-I (10 µg/mL) for the indicated times. To visualize co-localization of internalized ABCA1(207HA) and Rab GTPases on intracellular vesicles, the anti-HA antibody remaining on the cell surface after the incubation was removed by washing four times with 150 mM glycine (pH 2.0) .

Cellular lipid release assay

Prior to lipid release assay, THP-1 cells were treated with 200 ng/mL phorbol 12-myristate 13-acetate (PMA) for 2 days to facilitate differentiation into macrophages, and the adherent macrophages were incubated with 3 µM TO901317 to induce ABCA1 expression in the presence or absence of 50 µg/mL acetyl-LDL (Biomedical Technologies) for 24 h. THP-1 macrophages or HEK293 cells stably expressing an ABCA1 construct were washed twice with PBS, and then placed in growth medium containing 0.02% BSA. To analyze lipid release, 10 µg/mL recombinant apoA-I or 2 mM MβCD was added. The cholesterol content in the medium after the indicated time periods of incubation was determined using colorimetric enzyme assays as described previously (Abe-Dohmae et al. 2000).

Detection of recycled ABCA1

Cells stably expressing ABCA1(207HA) were seeded onto poly-L-lysine-coated 24-well plates at a density of 4.0 x 105 cells per well. After 24 h incubation, cells were kept on ice for 10 min and washed twice with ice-cold PBS+, and anti-HA antibody F-7 (1 : 800) was added in PBS+ containing 1% BSA on ice for 30 min. The cells were washed three times with ice cold PBS+ to remove unbound antibody, and incubated at 37 °C for 15 min. The cells were then washed twice with PBS+ (pH 3.2) for 30 s to remove cell surface antibodies, followed by a wash with PBS+. Next, the cells were incubated with growth medium supplemented with 10% FBS and biotin-conjugated anti-mouse IgG (1 : 20000; DAKO) for 1 h. The cells were fixed with 4% paraformaldehyde for 30 min, washed with PBS+, permeabilized in 0.4% Triton X-100 in PBS+, and blocked with 1% BSA in PBS+. To measure cell surface ABCA1 (at step1, step2 and step3), cells were fixed after incubation or acid wash, and then incubated with growth medium supplemented with 10% FBS and biotin-conjugated anti-mouse IgG for 1 h before permeabilization. The cells were incubated overnight at 4 °C with HRP-conjugated streptavidin (1 : 20000; Pierce) in PBS+ containing 1% BSA. The cells were washed twice with PBS+, and the amount of bound HRP-conjugated streptavidin was measured by enzyme-linked immunosorbent assay (ELISA); the enzymatic reaction was started by adding 300 µL of 0.1 M PBS (pH 6.0) containing 4 mM H2O2 and 4 mM o-phenylenediamine. Color development was stopped by the addition of 150 µL of 2.25 M H2SO4 after 8–16 min incubation at room temperature, and absorbance at 490 nm was measured. The amount of ABCA1(207HA) recycled to the cell surface was calculated by subtracting non-specific antibody binding and ELISA reaction with HEK293 cells from the total ELISA reaction with ABCA1(207HA).

Inhibition of clathrin-dependent endocytosis

Cells at approximately 20% confluence were washed and preincubated with growth medium containing 0.02% BSA in the presence or absence of 100 µM MDC for 30 min. The cells were incubated with growth medium containing 0.02% BSA and 10 µg/mL apoA-I in the presence or absence of 100 µM MDC for the indicated times. MDC at this concentration did not alter LDH release from the cells (data not shown). When the effect of hypertonic sucrose was examined, cells were preincubated with growth medium containing 0.02% BSA in the presence of 300 µM sucrose for 15 min, and then the cells were incubated with 10 µg/mL apoA-I in the presence of sucrose (300 µM) for 3 h.

Biotinylation of cell-surface proteins

Cell monolayers were kept on ice for 10 min, washed with ice-cold PBS+ and incubated with 0.5 mg/mL sulfo-NHS-biotin in PBS+ on ice for 30 min in the dark. The cells were washed with PBS+ to remove unbound sulfo-NHS-biotin and lyzed in RIPA buffer containing protease inhibitors. Immobilized monomeric avidin gel (Pierce) was added to the cell lysate to precipitate biotinylated proteins, and the bound proteins were electrophoresed on a 7% SDS-polyacrylamide gel and immunodetected.

Protein assay

Cells were incubated in 0.1 N NaOH for 1 h at room temperature. A protein assay was performed using the BCA protein assay reagent (PIERCE).

Statistical analysis

Values are presented as the means ± SE. Statistical significance was determined by Student's t-test. A value of P < 0.05 was considered statistically significant.


   Acknowledgements
 
This work was supported by Grant-in-aid for Scientific research (S) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and the program for promotion of fundamental studies in health sciences of the National Institute of Biomedical Innovation. This work was supported by the World Premier International Research Center Initiative (WPI initiative), MEXT Japan.


   Footnotes
 
Communicated by: Tadashi Yamamoto

* Correspondence: uedak{at}kais.kyoto-u.ac.jp


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 Experimental procedures
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Received: 8 July 2008
Accepted: 9 November 2008




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