ABCA1 protein is involved in lipid metabolism in several regards. This protein promotes reverse cholesterol transport from the peripheral tissues. ABCA1, being located on the basolateral surface of hepatocytes and intestinal cells, has been shown to play a crucial role in the hepatic and intestinal secretion of HDL . In the present study we generated and characterized a model system for the quantitative assessment of the plasma membrane level of ABCA1. Inclusion of an extracellular hemagglutinin epitope into ABCA1 enabled monitoring of its cell surface expression by flow cytometry and confocal microscopy. By using retroviral transduction we established mammalian cell lines stably expressing functional and non-functional HA-tagged ABCA1 variants. This experimental tool allowed us to investigate the effect of different drugs on cell surface expression of ABCA1.
In previous studies, the protein expression level and subcellular localization of wild type ABCA1 and non-functional mutant forms were found to be identical [27, 31]. Similarly, in our model system, both the overall and the plasma membrane expression levels of each HA-tagged ABCA1 variant were comparable, as determined by Western blotting and cell surface labeling (Figures 2B, 3). ABCA1 has been reported to be localized to the basolateral membrane in polarized cells . We found proper subcellular localization for each HA-tagged ABCA1 variant (Figures 4, 5) in accordance with previous reports on the untagged versions of the transporter .
Since the applicability and reproducibility of a cellular model system requires stable protein expression levels, we investigated the persistence of our transgene in various cell lines. We found that ABCA1 expression remains stable in MDCKII and HEK293H cells up to 50 and 20 passages, respectively. Thus, our experimental test system substantially surpasses previous attempts to generate cellular models with ABCA1, since in those cases protein expression rapidly declined after a few passages . We demonstrated the applicability of our system to assess the cell surface appearance of ABCA1 by using BFA and ALLN, which have been reported to influence ABCA1 trafficking and degradation (Figure 7A, B).
In agreement with previous reports [35, 36], we demonstrated the stabilizing effect of apoA-I on the HA-tagged ABCA1 at the cell surface (Figure 7C). Furthermore, we found that the cell surface levels of the Walker motif mutant ABCA1 (MM), which fails to bind apoA-I (Figure 6B) [28, 29], was unaffected by apoA-I (Figure 7D). This observation is in line with previous findings demonstrating that binding of apoA-I is required for the apoA-I-mediated increase of ABCA1 expression [29, 37].
In clinical trials, statins were highly effective in reducing LDL-cholesterol, but only moderate effects were seen on the HDL levels. Among a number of different statins, atorvastatin was the most effective and induced redistribution in HDL subpopulations by increasing the fraction of larger HDL particles . Conflicting results were published on the effect of statins on ABCA1 expression and lipid efflux in various cellular systems. Wong et al. demonstrated down-regulation of ABCA1 mRNA and protein levels as well as reduction in the apoA-I-mediated cholesterol efflux in response to statin treatment of PMA-induced Thp-1 cells [14, 15]. In contrast, another study found that atorvastatin increased mRNA expression of ABCA1 and promoted cholesterol efflux in the same cell type . In hepatoma cells, statins induced ABCA1 expression resulting in cholesterol and phospholipid efflux to apoA-I . These observations leave the question open, whether statins influence apoA-I-dependent cholesterol efflux solely by changing ABCA1 expression levels or by modifying its subcellular trafficking and cell surface appearance. Since our model system excludes transcriptional regulation, our results revealed that atorvastatin has no effect on the plasma membrane expression of ABCA1.
Niacin has been reported to be one of the most potent agents increasing HDL cholesterol levels , and to stimulate ABCA1 expression and function in monocytic cell lines via influencing the cAMP/PKA pathway . By investigating the effect of niacin in our system, we observed no direct effect on cell surface appearance of ABCA1 in accordance with previous observations on the regulatory mechanism of niacin.
Calcium channel blockers have also been reported to influence ABCA1 expression. Hasegawa et al. demonstrated the role of different signaling pathways including PKA, tyrosine kinase, and Janus kinase in ABCA1 expression modulation in response to various CCBs. They found that some of the CCBs, e.g., aranidipine and efonidipine, increase ABCA1 protein expression without altering the mRNA level . In contrast, Suzuki et al. demonstrated a dose-dependent stimulatory effect of other CCBs, such as verapamil, nifedipine, and nicardipine, on both the mRNA and protein expression levels of ABCA1, resulting in elevated cellular lipid release . Our results do not support these findings, since we observed no significant effect on the plasma membrane level of ABCA1 in a wide concentration range of verapamil and nifedipine.
Membrane cholesterol has been implicated to regulate many membrane proteins, including several ABC transporters, e.g. ABCB1 and ABCG2 [47, 52, 53]. Membrane microdomains with high cholesterol content have been suggested to play a role in such regulation . However, neither cholesterol overload nor depletion influenced ABCA1 cell surface expression in our model system. It is noteworthy that treatment with methyl-β-cyclodextrin (CD) reduced, whereas cholesterol-loaded CD significantly increased cell viability. The most plausible explanation for this observation is that CD treatment makes the cell membrane fragile and more sensitive to mechanical stress. This notion should be taken in account in future studies on ABCA1-expressing cells treated with CD.
We studied the effect of glyburide and CsA, which have previously been reported to influence the plasma membrane level or function of ABCA1 [29, 38]. In accordance with previous observations, glyburide had no effect on ABCA1 plasma membrane appearance in our model system , whereas CsA greatly increased the cell surface level of ABCA1 (Figure 7C). ALLN elevated both ABCA1 cell surface expression and apoA-I binding, whereas CsA reduced apoA-I binding despite the increased plasma membrane level of ABCA1. This observation is line with a previous report suggesting that CsA sequesters ABCA1 in the plasma membrane . The observation that cell surface expression of the non-functional ABCA1 mutant (MM) is also increased by CsA pretreatment clearly indicates that the trapping effect of this drug is independent from ABCA1 function.
Ezetimibe, a drug which is known to block the intestinal cholesterol absorption, has not only an inhibitory effect on several brush border membrane proteins, including Niemann-Pick C1-like 1, scavenger receptor B-I and CD36 , but has also been reported to lower the mRNA level of several other genes involved in lipid metabolism. The transcription of ABCA1 is also reduced by EZ , but no study has yet been reported on its effect on the cell surface expression of ABCA1. Our model system allows us to investigate the plasma membrane expression of ABCA1 independently from its transcriptional regulation. Strikingly, we found that EZ significantly lowers the cell surface expression of wt HA-ABCA1 (Figure 7C). Since no reduction in the cell surface expression of the MM HA-ABCA1 was seen in response to EZ treatment (Figure 7D), the effect of EZ occurs only with a functional ABCA1, thus, its effect is likely connected to a functional form of the protein.
There are several possibilities to explain the lack of effect of EZ on cell surface expression of the non-functional MM-ABCA1 variant. It is theoretically possible but very unlikely that EZ inhibits the trafficking of only the de novo synthesized functional proteins. It is more plausible that EZ influences the protein internalization and recycling to the plasma membrane, since ABCA1 has been shown to undergo a relatively rapid internalization and recycling to the plasma membrane, which process is widely accepted to require protein function [39, 56]. Therefore, if EZ either accelerates protein internalization or inhibits recycling, and in the meantime MM-ABCA1 has a lower internalization and recycling turnover compared to wild type, EZ causes a smaller effect on the cell surface appearance of the mutant transporter. This hypothesis is supported by the observation that the effects of ALLN were also smaller in HA-MM-ABCA1-expressing cells, as compared to wild type HA-ABCA1. It is also possible that EZ accelerates ABCA1 degradation, since the internalization itself is dependent on the transporter function. This idea is not supported by our recent observations using confocal microscopy, when we found that following EZ treatment the wt HA-ABCA1, but not the MM variant, accumulated intracellularly in vesicles but not in lysosomes (data not shown). Nevertheless, further studies are needed to reveal the mechanism of EZ effect on the cell surface expression of ABCA1.