Effect of the modulation of the membrane lipid composition on the localization and function of P-glycoprotein in MDR1-MDCK cells

Summary Multidrug resistance (MDR) is a major obstacle in cancer therapy. It results from different mechanisms; among them is P-glycoprotein (P-gp)-mediated drug efflux out of cells. The mechanism of action remains elusive. The membrane lipid surrounding of P-gp, especially cholesterol, has been postulated to play an important role. To determine the effect of cholesterol depletion on P-gp, Madin Darby canine kidney (MDCK) cells, transfected with the mdr1 gene (MDR1-MDCK cells), were treated with methyl-β-cyclodextrin (MβCD). The localization and function of P-gp were analyzed using confocal laser scanning microscopy. Treatment with 100 mM MβCD did not affect viability but altered the structural appearance of the cells and abolished efflux of rhodamine 123, a P-gp substrate. The MβCD treatment released P-gp from intact cells into the supernatant and reduced the amount of P-gp in total membrane preparations. The P-gp was shifted from the raft fractions (1% Triton X-100, 4° C) to higher density fractions in MβCD-treated cells. The amount of cholesterol was significantly decreased in the raft fractions. Treatment of cells with 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, a glucosylceramide synthase inhibitor, also led to a shift of P-gp to higher density fractions. These results show that removal of cholesterol modulates the membrane lipid composition, changes the localization of P-gp, and results in loss of P-gp function.     

Enhanced apoA-I-dependent cholesterol efflux by ABCA1 from sphingomyelin-deficient Chinese hamster ovary cells

ATP binding cassette protein A1 (ABCA1) plays a major role in cholesterol homeostasis and high density lipoprotein (HDL) metabolism. It is proposed that ABCA1 reorganizes the plasma membrane and generates more loosely packed domains that facilitate apoA-I-dependent cholesterol efflux. In this study, we examined the effects of the cellular sphingomyelin level on HDL formation by ABCA1 by using a Chinese hamster ovary-K1 mutant cell line, LY-A, which has a missense mutation in the ceramide transfer protein CERT. When LY-A cells were cultured in Nutridoma-BO medium and sphingomyelin content was reduced, apoA-I-dependent cholesterol efflux by ABCA1 from LY-A cells increased 1.65-fold compared with that from LY-A/CERT cells stably transfected with human CERT cDNA. Exogenously added sphingomyelin significantly reduced the apoA-I-dependent efflux of cholesterol from LY-A cells, confirming that the decrease in sphingomyelin content in the plasma membrane stimulates cholesterol efflux by ABCA1. The amount of cholesterol available to cold methyl-beta-cyclodextrin (MbetaCD) extraction from LY-A cells was increased by 40% by the expression of ABCA1 and was 1.6-fold higher than that from LY-A/CERT cells. This step in ABCA1 function, making cholesterol available to cold MbetaCD, was independent of apoA-I. These results suggest that the function of ABCA1 could be divided into two steps: (i) a flopping step to move phosphatidylcholine and cholesterol from the inner to outer leaflet of the plasma membrane, where cholesterol becomes available to cold MbetaCD extraction, and (ii) a loading step to load phosphatidylcholine and cholesterol onto apoA-I to generate HDL.     

Membrane lipid domains distinct from cholesterol/sphingomyelin-rich rafts are involved in the ABCA1-mediated lipid secretory pathway

Efflux of excess cellular cholesterol mediated by lipid-poor apolipoproteins occurs by an active mechanism distinct from passive diffusion and is controlled by the ATP-binding cassette transporter ABCA1. Here we examined whether ABCA1-mediated lipid efflux involves the selective removal of lipids associated with membrane rafts, plasma membrane domains enriched in cholesterol and sphingomyelin. ABCA1 was not associated with cholesterol and sphingolipid-rich membrane raft domains based on detergent solubility and lack of colocalization with marker proteins associated with raft domains. Lipid efflux to apoA-I was accounted for by decreases in cellular lipids not associated with cholesterol/sphingomyelin-rich membranes. Treating cells with filipin, to disrupt raft structure, or with sphingomyelinase, to digest plasma membrane sphingomyelin, did not impair apoA-I-mediated cholesterol or phosphatidylcholine efflux. In contrast, efflux of cholesterol to high density lipoproteins (HDL) or plasma was partially accounted for by depletion of cholesterol from membrane rafts. Additionally, HDL-mediated cholesterol efflux was partially inhibited by filipin and sphingomyelinase treatment. Apo-A-I-mediated cholesterol efflux was absent from fibroblasts with nonfunctional ABCA1 (Tangier disease cells), despite near normal amounts of cholesterol associated with raft domains and normal abilities of plasma and HDL to deplete cholesterol from these domains. Thus, the involvement of membrane rafts in cholesterol efflux applies to lipidated HDL particles but not to lipid-free apoA-I. We conclude that cholesterol and sphingomyelin-rich membrane rafts do not provide lipid for efflux promoted by apolipoproteins through the ABCA1-mediated lipid secretory pathway and that ABCA1 is not associated with these domains.     

Binding of high density lipoproteins to cell receptors promotes translocation of cholesterol from intracellular membranes to the cell surface

Cultured cells have on their cell surface a specific high-affinity binding site (receptor) for high density lipoproteins (HDL) which appears to promote cholesterol efflux. In this study we characterized the cellular mechanisms involved in HDL receptor-mediated transport of cholesterol from cultured human fibroblasts and bovine aortic endothelial cells. HDL3, chemically modified by tetranitromethane (TNM-HDL3), is not recognized by this receptor and was used as a control for efflux not mediated by HDL receptor binding. HDL3 and TNM-HDL3 were found to be equally effective in causing efflux of plasma membrane cholesterol radiolabeled with [3H]cholesterol. However, HDL3 was much more effective than TNM-HDL3 in causing efflux of [3H]cholesterol associated with intracellular membranes. By measuring movement of endogenously synthesized [3H]cholesterol to the plasma membrane, and into the medium, we found that HDL3 induced a rapid movement of [3H]cholesterol from a preplasma membrane compartment to the plasma membrane that preceded [3H]cholesterol efflux. This effect was not observed with TNM-HDL3. Thus, receptor binding of HDL3 appears to facilitate removal of cellular cholesterol from specific intracellular pools by initiation of translocation of intracellular cholesterol to the plasma membrane.     

Membrane proteins and phospholipids as effectors of reverse cholesterol transport

The review highlights the membrane aspect of cholesterol efflux from cell membranes to high density lipoproteins (HDL), an initial stage of reverse cholesterol transport to liver. In addition to traditional viewpoints considering cholesterol transport as the step of sequential lipoprotein transformation, which involves blood plasma apoproteins and proteins transporters, employment of proteomic approaches has shown the active role of cell plasma membranes as cholesterol donors and plasma membrane bound proteins in cholesterol transport. These include ATP-binding ABC-A1 transporter and membrane receptor SR-B1. There is experimental and clinical evidence that impairment of genes encoding these proteins cause impairments of reverse cholesterol transport (e.g. Tangier disease and genetic manipulations with experimental animals.) Although precise mechanism involving these membrane proteins remains unknown it is suggested that ABC-AI with free plasma apoA1 facilitates the efflux of membrane phospholipids and formation of their complex with apoAI. This complex accepts membrane cholesterol, with simultaneous formation of a full HDL particle. In certain cells there is correlation between cholesterol efflux into HDL and expression of SR-BI, which reversibly binds to HDL. This receptor protein may influence molecular organization of membrane phospholipids and cholesterol, facilitating cholesterol efflux. The review also deals with properties of ABC-A1 and SR-B1, putative mechanisms of their effects, the role of these proteins in reverse cholesterol transport and their functional coupling to the phospholipid matrix of biomembranes.     

Cholesterol efflux from macrophage foam cells is enhanced by active phospholipid transfer protein through generation of two types of acceptor particles

Phospholipid transfer protein (PLTP) is expressed by macrophage-derived foam cells in human atherosclerotic lesions, suggesting a regulatory role for PLTP in cellular cholesterol homeostasis. However, the exact role of PLTP in the reverse cholesterol transport pathway is not known. PLTP is present in plasma as two forms, a highly active (HA-PLTP) and a lowly active (LA-PLTP) form. In this study we clarify the role of the two forms of PLTP in cholesterol efflux from [3H]cholesterol oleate-acetyl-LDL-loaded THP-1 macrophages. Incubation of HDL in the presence of HA-PLTP resulted in the formation of two types of acceptor particles, prebeta-HDL and large fused HDL. HA-PLTP increased prebeta-HDL formation and caused a 42% increase in [3H]cholesterol efflux to HDL, while LA-PLTP neither formed prebeta-HDL nor increased cholesterol efflux. Removal of the formed prebeta-HDL by immunoprecipitation decreased cholesterol efflux by 47%. Neither HA- nor LA-PLTP enhanced cholesterol efflux to lipid-free apoA-I. Importantly, also the large fused HDL particles formed during incubation of HDL with HA-PLTP acted as efficient cholesterol acceptors. These observations demonstrate that only HA-PLTP increases macrophage cholesterol efflux, via formation of efficient cholesterol acceptors, prebeta-HDL and large fused HDL particles.     

Efflux of sphingomyelin, cholesterol, and phosphatidylcholine by ABCG1

Cholesterol and phospholipids are essential to the body, but an excess of cholesterol or lipids is toxic and a risk factor for arteriosclerosis. ABCG1, one of the half-type ABC proteins, is thought to be involved in cholesterol homeostasis. To explore the role of ABCG1 in cholesterol homeostasis, we examined its subcellular localization and function. ABCG1 and ABCG1-K120M, a WalkerA lysine mutant, were localized to the plasma membrane in HEK293 cells stably expressing ABCG1 and formed a homodimer. A stable transformant expressing ABCG1 exhibited efflux of cholesterol and choline phospholipids in the presence of BSA, and the cholesterol efflux was enhanced by the presence of HDL, whereas cells expressing ABCG1-K120M did not, suggesting that ATP binding and/or hydrolysis is required for the efflux. Mass and TLC analyses revealed that ABCG1 and ABCA1 secrete several species of sphingomyelin (SM) and phosphatidylcholine (PC), and SMs were preferentially secreted by ABCG1, whereas PCs were preferentially secreted by ABCA1. These results suggest that ABCA1 and ABCG1 mediate the lipid efflux in different mechanisms, in which different species of phospholipids are secreted, and function coordinately in the removal of cholesterol and phospholipids from peripheral cells.     

Scavenger receptor BI (SR-BI) mediates free cholesterol flux independently of HDL tethering to the cell surface

In addition to its effect on high density lipoprotein (HDL) cholesteryl ester (CE) uptake, scavenger receptor BI (SR-BI) was recently reported to stimulate free cholesterol (FC) flux from Chinese hamster ovary (CHO) cells stably expressing mouse SR-BI, a novel function of SR-BI that may play a role in cholesterol removal from the vessel wall where the receptor can be found. It is possible that SR-BI stimulates flux simply by tethering acceptor HDL particles in close apposition to the cell surface thereby facilitating the movement of cholesterol between the plasma membrane and HDL. To test this, we used transiently transfected cells and compared the closely related class B scavenger receptors mouse SR-BI and rat CD36 for their ability to stimulate cholesterol efflux as both receptors bind HDL with high affinity. The results showed that, although acceptor binding to SR-BI may contribute to efflux to a modest extent, the major stimulation of FC efflux occurs independently of acceptor binding to cell surface receptors. Instead our data indicate that SR-BI mediates alterations to membrane FC domains which provoke enhanced bidirectional FC flux between cells and extracellular acceptors.     

Protein kinase C as a mediator of high density lipoprotein receptor-dependent efflux of intracellular cholesterol.

The interaction of high density lipoproteins (HDL) with the HDL receptor stimulates the translocation of cholesterol from intracellular pools to the plasma membrane where the cholesterol becomes available for removal by appropriate acceptors. The role of signal transduction through protein kinase C in HDL receptor-dependent cholesterol translocation and efflux was examined using cholesterol-loaded cultured human skin fibroblasts. Treatment of cells with HDL3 activated protein kinase C, demonstrated by a transient increase in membrane associated kinase activity. Kinase activation appeared to be dependent on binding of HDL3 to the HDL receptor, since tetranitromethane-modified HDL3, which does not bind to the receptor, was without effect. Translocation of intracellular sterol to the plasma membrane was stimulated by treatment of cells with the protein kinase C activators, dioctanoylglycerol and phorbol myristic acetate, and the calcium ionophore A23187. Conversely, treatment of cells with sphingosine, a protein kinase C inhibitor, reduced HDL3-mediated translocation and efflux of intracellular sterols. However, sphingosine had no effect on efflux of labeled cholesterol derived from the plasma membrane. Down-regulation of cellular protein kinase C activity by long term incubation with phorbol esters also inhibited HDL3-mediated efflux of intracellular sterols and abolished the ability of sphingosine to further inhibit HDL3-mediated efflux. These studies support the conclusion that HDL receptor-mediated translocation and efflux of intracellular cholesterol occurs through activation of protein kinase C.     

ABCG1 has a critical role in mediating cholesterol efflux to HDL and preventing cellular lipid accumulation

Here we demonstrate that the ABC transporter ABCG1 plays a critical role in lipid homeostasis by controlling both tissue lipid levels and the efflux of cellular cholesterol to HDL. Targeted disruption of Abcg1 in mice has no effect on plasma lipids but results in massive accumulation of both neutral lipids and phospholipids in hepatocytes and in macrophages within multiple tissues following administration of a high-fat and -cholesterol diet. In contrast, overexpression of human ABCG1 protects murine tissues from dietary fat-induced lipid accumulation. Finally, we show that cholesterol efflux to HDL specifically requires ABCG1, whereas efflux to apoA1 requires ABCA1. These studies identify Abcg1 as a key gene involved in both cholesterol efflux to HDL and in tissue lipid homeostasis.     

Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1

Mutations of the ABC1 transporter have been identified as the defect in Tangier disease, characterized by low HDL and cholesterol ester accumulation in macrophages. A full-length mouse ABC1 cDNA was used to investigate the mechanisms of lipid efflux to apoA-I or HDL in transfected 293 cells. ABC1 expression markedly increased cellular cholesterol and phospholipid efflux to apoA-I but had only minor effects on lipid efflux to HDL. The increased lipid efflux appears to involve a direct interaction between apoA-I and ABC1, because ABC1 expression substantially increased apoA-I binding at the cell surface, and chemical cross-linking and immunoprecipitation analysis showed that apoA-I binds directly to ABC1. In contrast to scavenger receptor BI (SR-BI), another cell surface molecule capable of facilitating cholesterol efflux, ABC1 preferentially bound lipid-free apoA-I but not HDL. Immunofluorescence confocal microscopy showed that ABC1 is primarily localized on the cell surface. In the absence of apoA-I, cells overexpressing ABC1 displayed a distinctive morphology, characterized by plasma membrane protrusions and resembling echinocytes that form when there are excess lipids in the outer membrane hemileaflet. The studies provide evidence for a direct interaction between ABC1 and apoA-I, but not HDL, indicating that free apoA-I is the metabolic substrate for ABC1. Plasma membrane ABC1 may act as a phospholipid/cholesterol flippase, providing lipid to bound apoA-I, or to the outer membrane hemileaflet.     

Host Cell P-glycoprotein Is Essential for Cholesterol Uptake and Replication of Toxoplasma gondii

P-glycoprotein (P-gp) is a membrane-bound efflux pump that actively exports a wide range of compounds from the cell and is associated with the phenomenon of multidrug resistance. However, the role of P-gp in normal physiological processes remains elusive. Using P-gp-deficient fibroblasts, we showed that P-gp was critical for the replication of the intracellular parasite Toxoplasma gondii but was not involved in invasion of host cells by the parasite. Importantly, we found that the protein participated in the transport of host-derived cholesterol to the intracellular parasite. T. gondii replication in P-gp-deficient host cells not only resulted in reduced cholesterol content in the parasite but also altered its sphingolipid metabolism. In addition, we found that different levels of P-gp expression modified the cholesterol metabolism in uninfected fibroblasts. Collectively our findings reveal a key and previously undocumented role of P-gp in host-parasite interaction and suggest a physiological role for P-gp in cholesterol trafficking in mammalian cells.     

HDLs induce raft domain vanishing in heterogeneous giant vesicles

Cholesterol efflux from the plasma membrane to HDLs is essential for cell cholesterol homeostasis. Recently, cholesterol-enriched ordered membrane domains, i.e. lipid rafts have been proposed to play an important role in this process. Here we introduce a new method to investigate the role of HDL interactions with the raft lipid phase and to directly visualize the effects of HDL-induced cholesterol efflux on rafts in model membranes. Addition of HDLs to giant lipid vesicles containing raft-type domains promoted decrease in size and disappearance of such domains as visualized by fluorescence microscopy. This was interpreted as resulting from cholesterol efflux from the vesicles to the HDLs. The raft vanishing rate was directly related to the HDL concentration. Evidence for a direct interaction of HDLs with the membrane was obtained by observing mutual adhesion of vesicles. It is suggested that the present method can be used to study the selective role of the bilayer lipid phase (raft and non-raft) in cholesterol efflux and membrane-HDL interaction and their underlying mechanisms. Such mechanisms may contribute to cholesterol efflux in vivo.     

Role of the hepatic ABCA1 transporter in modulating intrahepatic cholesterol and plasma HDL cholesterol concentrations

The current model for reverse cholesterol transport proposes that HDL transports excess cholesterol derived primarily from peripheral cells to the liver for removal. However, recent studies in ABCA1 transgenic mice suggest that the liver itself may be a major source of HDL cholesterol (HDL-C). To directly investigate the hepatic contribution to plasma HDL-C levels, we generated an adenovirus (rABCA1-GFP-AdV) that targets expression of mouse ABCA1-GFP in vivo to the liver. Compared with mice injected with control AdV, infusion of rABCA1-GFP-AdV into C57Bl/6 mice resulted in increased expression of mouse ABCA1 mRNA and protein in the liver. ApoA-I-dependent cholesterol efflux was increased 2.6-fold in primary hepatocytes isolated 1 day after rABCA1-GFP-AdV infusion. Hepatic ABCA1 expression in C57Bl/6 mice (n = 15) raised baseline levels of TC, PL, FC, HDL-C, apoE, and apoA-I by 150-300% (P < 0.05 all). ABCA1 expression led to significant compensatory changes in expression of genes that increase hepatic cholesterol, including HMG-CoA reductase (3.5-fold), LDLr (2.1-fold), and LRP (5-fold) in the liver. These combined results demonstrate that ABCA1 plays a key role in hepatic cholesterol efflux, inducing pathways that modulate cholesterol homeostasis in the liver, and establish the liver as a major source of plasma HDL-C.     

Stimulation of lipolysis enhances the rate of cholesterol efflux to HDL in adipocytes

Adipose tissue constitutes a major location for cholesterol storage and, as such, it may play a role in the regulation of circulating cholesterol levels. A possible metabolic link between the lipolytic activity of adipocytes and their ability to release cholesterol to reconstituted human high density lipoprotein, HDL, was investigated in 3T3-L1 adipocytes. In the presence of HDL, composed of human apoA-I and phosphatidylcholine, adipocytes release cholesterol in a lipoprotein-dose and time dependent fashion. β-adrenergic activation of the lipolysis promotes a 22% increase in the extent of cholesterol efflux to reconstituted discoidal HDL particles. Activation of lipolysis promotes a rapid decrease in the cholesterol content of the plasma membrane and a concomitant increase in lipid droplet cholesterol. This change is independent of the presence of HDL. Activation of the lipolysis does not affect the levels of ABCA1 and SR-BI. Therefore, the enhancement of cholesterol efflux is not due to the level of plasma membrane cholesterol, or to the levels of the cholesterol transporters ABCA1 and scavenger receptor SR-BI. Brefeldin A did not affect the rate of cholesterol efflux under basal lipolytic conditions, but it abolished the lipolysis-dependent enhancement of cholesterol efflux to HDL. This study suggests that activation of lipolysis is accompanied by an increase in BFA-sensitive vesicular transport that in turn enhances cholesterol efflux to HDL. The study supports a metabolic link between the lipolytic activity of adipocytes and the rate of cellular cholesterol efflux to HDL.