cAMP induces ABCA1 phosphorylation activity and promotes cholesterol efflux from fibroblasts

ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in apoA-I lipidation, a key step in reverse cholesterol transport. cAMP induces apoA-I binding activity and promotes cellular cholesterol efflux. We investigated the role of the cAMP/protein kinase A (PKA) dependent pathway in the regulation of cellular cholesterol efflux. Treatment of normal fibroblasts with 8-bromo-cAMP (8-Br-cAMP) increased significantly apoA-I-mediated cholesterol efflux, with specificity for apoA-I, but not for cyclodextrin. Concomitantly, 8-Br-cAMP increased ABCA1 phosphorylation in a time-dependent manner. Maximum phosphorylation was reached in <10 min, representing a 260% increase compared to basal ABCA1 phosphorylation level. Forskolin, a known cAMP regulator, increased both cellular cholesterol efflux and ABCA1 phosphorylation. In contrast, H-89 PKA inhibitor reduced cellular cholesterol efflux by 70% in a dose-dependent manner and inhibited almost completely ABCA1 phosphorylation. To determine whether naturally occurring mutants of ABCA1 may affect its phosphorylation activity, fibroblasts from subjects with familial HDL deficiency (FHD, heterozygous ABCA1 defect) and Tangier disease (TD, homozygous/compound heterozygous ABCA1 defect) were treated with 8-Br-cAMP or forskolin. Cellular cholesterol efflux and ABCA1 phosphorylation were increased in FHD but not in TD cells. Taken together, these findings provide evidence for a link between the cAMP/PKA-dependent pathway, ABCA1 phosphorylation, and apoA-I mediated cellular cholesterol efflux.     

Janus kinase 2 modulates the apolipoprotein interactions with ABCA1 required for removing cellular cholesterol

ATP-binding cassette transporter A1 (ABCA1) mediates transport of cellular cholesterol and phospholipids to high density lipoprotein (HDL) apolipoproteins, such as apoA-I. ABCA1 mutations can cause a severe HDL deficiency and atherosclerosis. Here we show that the protein-tyrosine kinase (TK) Janus kinase 2 (JAK2) modulates the apolipoprotein interactions with ABCA1 required for removing cellular lipids. The protein kinase A (PKA) inhibitor H89, the TK inhibitor genistein, and the JAK2 inhibitor AG490 suppressed apoA-I-mediated cholesterol and phospholipid efflux from ABCA1-expressing cells without altering the membrane ABCA1 content. Whereas PKA inhibition had no effect on apoA-I binding to cells or to ABCA1, TK and JAK2 inhibition greatly reduced these activities. Conversely, PKA but not JAK2 inhibition significantly reduced the intrinsic cholesterol translocase activity of ABCA1. Mutant cells lacking JAK2 had a severely impaired apoA-I-mediated cholesterol and phospholipid efflux and apoA-I binding despite normal ABCA1 protein levels and near normal cholesterol translocase activity. Thus, although PKA modulates ABCA1 lipid transport activity, JAK2 appears to selectively modulate apolipoprotein interactions with ABCA1. TK-mediated phosphorylation of ABCA1 was undetectable, implicating the involvement of another JAK2-targeted protein. Acute incubation of ABCA1-expressing cells with apoA-I had no effect on ABCA1 phosphorylation but stimulated JAK2 autophosphorylation. These results suggest that the interaction of apolipoproteins with ABCA1-expressing cells activates JAK2, which in turn activates a process that enhances apolipoprotein interactions with ABCA1 and lipid removal from cells.     

Effect of apolipoprotein A-I on ATP binding cassette transporter A1 degradation and cholesterol efflux in THP-1 macrophage-derived foam cells

The accumulation of lipoprotein cholesterol in theartery wall is thought to be an important factor in thedevelopment of atherosclerosis. After retentionand modi-fication in arteries, atherogenic lipoproteins are taken upby macrophages, bringing about macrophage-derived foamcells. High-density lipoprotein (HDL) plays a role in trans-porting cholesterol from peripheral tissues to the liver.The elevated level of HDL is associated with a decreasein atherosclerosis and the apolipoproteins to remo…     

An analysis of the role of a retroendocytosis pathway in ABCA1-mediated cholesterol efflux from macrophages

The ATP binding cassette transporter A-1 (ABCA1) is critical for apolipoprotein-mediated cholesterol efflux, an important mechanism employed by macrophages to avoid becoming lipid-laden foam cells, the hallmark of early atherosclerotic lesions. It has been proposed that lipid-free apolipoprotein A-I (apoA-I) enters the cell and is resecreted as a lipidated particle via a retroendocytosis pathway during ABCA1-mediated cholesterol efflux from macrophages. To determine the functional importance of such a pathway, confocal microscopy was used to characterize the internalization of a fully functional apoA-I cysteine mutant containing a thiol-reactive fluorescent probe in cultured macrophages. ApoA-I was also endogenously labeled with (35)S-methionine to quantify cellular uptake and to determine the metabolic fate of the internalized protein. It was found that apoA-I was specifically taken inside macrophages and that a small amount of intact apoA-I was resecreted from the cells. However, a majority of the label that reappeared in the media was degraded. We estimate that the mass of apoA-I retroendocytosed is not sufficient to account for the HDL produced by the cholesterol efflux reaction. Furthermore, we have demonstrated that lipid-free apoA-I-mediated cholesterol efflux from macrophages can be pharmacologically uncoupled from apoA-I internalization into cells. On the basis these findings, we present a model in which the ABCA1-mediated lipid transfer process occurs primarily at the membrane surface in macrophages, but still accounts for the observed specific internalization of apoA-I.     

Cyclic AMP-specific phosphodiesterase 4 inhibitors promote ABCA1 expression and cholesterol efflux.

ATP cassette binding protein 1 (ABCA1) controls the apolipoprotein-mediated cholesterol efflux pathway and determines plasma HDL levels. Although cAMP is known to promote ABCA1 expression and cholesterol efflux from cells, it has not been determined whether cyclic nucleotide phosphodiesterase (PDE) isoforms regulate this pathway. We show that rolipram and cilomilast, inhibitors of cAMP-specific PDE4, increase apolipoprotein A-I (apoA-I)-mediated cholesterol efflux up to 80 and 140% in human THP-1 and mouse J774.A1 macrophages, respectively, concomitant with an elevation of cAMP levels. The EC(50) value was estimated to be 1 to 2 microM for both inhibitors. Rolipram and cilomilast also increase ABCA1 protein expression in THP-1 and J774.A1 macrophages. Thus, PDE4 inhibitors cause parallel increases in cAMP levels, ABCA1 expression and apoA-I-mediated cholesterol efflux. PDE4 inhibitors may provide a novel strategy for the treatment of cardiovascular disease by mobilizing cholesterol from atherosclerotic lesions.     

The C-terminal domain of apolipoprotein A-I is involved in ABCA1-driven phospholipid and cholesterol efflux

ABCA1, a member of the ATP-binding cassette family, mediates the efflux of cellular lipids to free apolipoproteins, mainly apoA-I. The role of the C-terminal domain of apoA-I in this process has been evaluated by measuring the efflux capacity of a truncated form (apoA-I-(1-192)) versus intact apoA-I in different cellular models. In stimulated J774 macrophages, cholesterol efflux to apoA-I-(1-192) was remarkably lower than that to the intact apoA-I. The truncated apoA-I, lacking an important lipid-binding domain, was also significantly less efficient in removing phospholipids from stimulated macrophages. No difference was detected with stimulated Tangier fibroblasts that do not express functional ABCA1. The C-terminal domain of apoA-I is clearly involved in ABCA1-driven lipid efflux. Independent of the interaction with the cell surface, it may be the decreased ability of the truncated apoA-I to recruit membrane phospholipids that impairs its capacity to promote cell cholesterol efflux.     

An inhibitor of acylCoA: cholesterol acyltransferase increases expression of ATP-binding cassette transporter A1 and thereby enhances the ApoA-I-mediated release of cholesterol from macrophages

The effect of inhibition of acylCoA: cholesterol acyltransferase (ACAT) was studied on high density lipoprotein (HDL) metabolism. An inhibitor of ACAT, MCC-147, was given mouse peritoneal macrophages and expression of ATP-binding cassette transporter A1 (ABCA1) was examined. ABCA1 was increased both at the mRNA and protein levels, only when the cells are cholesterol-loaded and thereby the inhibitor decreased esterified cholesterol and increased unesterified cholesterol. In this condition, the ACAT inhibitor increased reversible binding of apoA-I to the cells and enhanced apoA-I-mediated release of cellular cholesterol and phospholipid, but did not influence nonspecific cellular cholesterol efflux to lipid microemulsion. It was therefore concluded that the ACAT inhibitor increased the release of cholesterol from the cholesterol-loaded macrophages by increasing the expression of ABCA1, putatively through shifting cholesterol distribution from the esterified to the free compartments.     

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.     

ATP-binding cassette transporter A1 (ABCA1) functions as a cholesterol efflux regulatory protein

ABCA1, an ATP-binding cassette transporter mutated in Tangier disease, promotes cellular phospholipid and cholesterol efflux by loading free apoA-I with these lipids. This process involves binding of apoA-I to the cell surface and phospholipid translocation by ABCA1. The goals of this study were to examine the relationship between ABCA1-mediated lipid efflux and apolipoprotein binding and to determine whether phospholipid and cholesterol efflux are coupled. Inhibition of lipid efflux by glybenclamide treatment or by mutation of the ATP-binding cassette of ABCA1 showed a close correlation between lipid efflux, the binding of apoA-I to cells, and cross-linking of apoA-I to ABCA1. The data suggest that a functionally important apoA-I binding site exists on ABCA1 and that the binding site could also involve lipids. After using cyclodextrin preincubation to deplete cellular cholesterol, ABCA1-mediated cholesterol efflux was abolished but phospholipid efflux and the binding of apoA-I were unaffected. The conditioned media from cyclodextrin-pretreated, ABCA1-expressing cells readily promoted cholesterol efflux when added to fresh cells not expressing ABCA1, indicating that cholesterol efflux can be dissociated from phospholipid efflux. Further, using a photoactivatable cholesterol analog, we showed that ABCA1 did not bind cholesterol directly, even though several other cholesterol-binding proteins specifically bound the cholesterol analog. The data suggest that the binding of apoA-I to ABCA1 leads to the formation of phospholipid-apoA-I complexes, which subsequently promote cholesterol efflux in an autocrine or paracrine fashion.     

Preferential ATP-binding cassette transporter A1-mediated cholesterol efflux from late endosomes/lysosomes

Recently, ATP-binding cassette transporter A1 (ABCA1), the defective molecule in Tangier disease, has been shown to stimulate phospholipid and cholesterol efflux to apolipoprotein A-I (apoA-I); however, little is known concerning the cellular cholesterol pools that act as the source of cholesterol for ABCA1-mediated efflux. We observed a higher level of isotopic and mass cholesterol efflux from mouse peritoneal macrophages labeled with [(3)H]cholesterol/acetyl low density lipoprotein (where cholesterol accumulates in late endosomes and lysosomes) compared with cells labeled with [(3)H]cholesterol with 10% fetal bovine serum, suggesting that late endosomes/lysosomes act as a preferential source of cholesterol for ABCA1-mediated efflux. Consistent with this idea, macrophages from Niemann-Pick C1 mice that have an inability to exit cholesterol from late endosomes/lysosomes showed a profound defect in cholesterol efflux to apoA-I. In contrast, phospholipid efflux to apoA-I was normal in Niemann-Pick C1 macrophages, as was cholesterol efflux following plasma membrane cholesterol labeling. These results suggest that cholesterol deposited in late endosomes/lysosomes preferentially acts as a source of cholesterol for ABCA1-mediated cholesterol efflux.     

Serum amyloid A promotes cholesterol efflux mediated by scavenger receptor B-I

Serum amyloid A (SAA) is an acute phase protein whose expression is markedly up-regulated during inflammation and infection. The physiological function of SAA is unclear. In this study, we reported that SAA promotes cellular cholesterol efflux mediated by scavenger receptor B-I (SR-BI). In Chinese hamster ovary cells, SAA promoted cellular cholesterol efflux in an SR-BI-dependent manner, whereas apoA-I did not. Similarly, SAA, but not apoA-I, promoted cholesterol efflux from HepG2 cells in an SR-BI-dependent manner as shown by using the SR-BI inhibitor BLT-1. When SAA was overexpressed in HepG2 cells using adenovirus-mediated gene transfer, the endogenously expressed SAA promoted SR-BI-dependent efflux. To assess the effect of SAA on SR-BI-mediated efflux to high density lipoprotein (HDL), we compared normal HDL, acute phase HDL (AP-HDL, prepared from mice injected with lipopolysaccharide), and AdSAA-HDL (HDL prepared from mice overexpressing SAA). Both AP-HDL and AdSAA-HDL promoted 2-fold greater cholesterol efflux than normal HDL. Lipid-free SAA was shown to also stimulate ABCA1-dependent cholesterol efflux in fibroblasts, in line with an earlier report (Stonik, J. A., Remaley, A. T., Demosky, S. J., Neufeld, E. B., Bocharov, A., and Brewer, H. B. (2004) Biochem. Biophys. Res. Commun. 321, 936-941). When added to cells together, SAA and HDL exerted a synergistic effect in promoting ABCA1-dependent efflux, suggesting that SAA may remodel HDL in a manner that releases apoA-I or other efficient ABCA1 ligands from HDL. SAA also facilitated efflux by a process that was independent of SR-BI and ABCA1. We conclude that the acute phase protein SAA plays an important role in HDL cholesterol metabolism by promoting cellular cholesterol efflux through a number of different efflux pathways.     

Endoplasmic reticulum stress impairs cholesterol efflux and synthesis in hepatic cells

Metabolic disorders such as type 2 diabetes cause hepatic endoplasmic reticulum (ER) stress, which affects neutral lipid metabolism. However, the role of ER stress in cholesterol metabolism is incompletely understood. Here, we show that induction of acute ER stress in human hepatic HepG2 cells reduced ABCA1 expression and caused ABCA1 redistribution to tubular perinuclear compartments. Consequently, cholesterol efflux to apoA-I, a key step in nascent HDL formation, was diminished by 80%. Besides ABCA1, endogenous apoA-I expression was reduced upon ER stress induction, which contributed to reduced cholesterol efflux. Liver X receptor, a key regulator of ABCA1 in peripheral cells, was not involved in this process. Despite reduced cholesterol efflux, cellular cholesterol levels remained unchanged during ER stress. This was due to impaired de novo cholesterol synthesis by reduction of HMG-CoA reductase activity by 70%, although sterol response element-binding protein-2 activity was induced. In mice, ER stress induction led to a marked reduction of hepatic ABCA1 expression. However, HDL cholesterol levels were unaltered, presumably because of scavenger receptor class B, type I downregulation under ER stress. Taken together, our data suggest that ER stress in metabolic disorders reduces HDL biogenesis due to impaired hepatic ABCA1 function.     

Trypsin-sensitive and lipid-containing sites of the macrophage extracellular matrix bind apolipoprotein A-I and participate in ABCA1-dependent cholesterol efflux

A unique property of the extracellular matrix of J774 and THP-1 cells has been identified, which contributes to the ability of these cells to promote cholesterol efflux. We demonstrate high level apolipoprotein (apo) A-I binding to macrophage cells (THP-1 and J774) and to their extracellular matrix (ECM). However, high level apoA-I binding is not observed on fibroblasts, HepG2 cells, or U937 cells (a macrophage cell line that does not efflux cholesterol to apoA-I or bind apoA-I on their respective ECM). Binding to the ECM of THP-1 or J774 macrophages depends on the presence of apoA-I C-terminal helices and is markedly reduced with a mutant lacking residues 187-243 (apoA-I Delta(187-243)), suggesting that the hydrophobic C terminus forms a hydrophobic interaction with the ECM. ApoA-I binding is lost upon trypsin treatment or with Triton X-100, a preparation method that de-lipidates the ECM. However, binding is recovered with re-lipidation, and is preserved with ECM prepared using cytochalasin B, which conserves the endogenous phospholipid levels of the ECM. We also demonstrate that specific cholesterol efflux to apoA-I is much reduced in cells released from their native ECM, but fully restored when ECM-depleted cells are added back to ECM in the presence of apoA-I. The apoA-I-mediated efflux is deficient in plated or suspension U937 macrophages, but is restored to high levels when the suspension U937 cells are reconstituted with the ECM of J774 cells. The ECM-dependent activity was much reduced in the presence of glyburide, indicating participation of ABCA1 (ATP-binding cassette transporter 1) in the efflux mechanism. These studies establish a novel binding site for apoA-I on the macrophage ECM that may function together with ABCA1 in promoting cholesterol efflux.     

Apolipoprotein A-I activates cellular cAMP signaling through the ABCA1 transporter

It has been suggested that the signal transduction pathway initiated by apoA-I activates key proteins involved in cellular lipid efflux. We investigated apoA-I-mediated cAMP signaling in cultured human fibroblasts induced with (22R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Treatment of stimulated fibroblasts with apoA-I for short periods of time (相似文献   

Caveolin-1-mediated apolipoprotein A-I membrane binding sites are not required for cholesterol efflux

Caveolin-1 (Cav1), a structural protein required for the formation of invaginated membrane domains known as caveolae, has been implicated in cholesterol trafficking and homeostasis. Here we investigated the contribution of Cav1 to apolipoprotein A-I (apoA-I) cell surface binding and intracellular processing using mouse embryonic fibroblasts (MEFs) derived from wild type (WT) or Cav1-deficient (Cav1(-/-)) animals. We found that cells expressing Cav1 have 2.6-fold more apoA-I binding sites than Cav1(-/-) cells although these additional binding sites are not associated with detergent-free lipid rafts. Further, Cav1-mediated binding targets apoA-I for internalization and degradation and these processes are not correlated to cholesterol efflux. Despite lower apoA-I binding, cholesterol efflux from Cav1(-/-) MEFs is 1.7-fold higher than from WT MEFs. Stimulation of ABCA1 expression with an LXR agonist enhances cholesterol efflux from both WT and Cav1(-/-) cells without increasing apoA-I surface binding or affecting apoA-I processing. Our results indicate that there are at least two independent lipid binding sites for apoA-I; Cav1-mediated apoA-I surface binding and uptake is not linked to cholesterol efflux, indicating that membrane domains other than caveolae regulate ABCA1-mediated cholesterol efflux.     

Effects of enrichment of fibroblasts with unesterified cholesterol on the efflux of cellular lipids to apolipoprotein A-I.

This study elucidates the factors underlying the enhancement in efflux of human fibroblast unesterified cholesterol and phospholipid (PL) by lipid-free apolipoprotein (apo) A-I that is induced by cholesterol enrichment of the cells. Doubling the unesterified cholesterol content of the plasma membrane by incubation for 24 h with low density lipoprotein and lipid/cholesterol dispersions increases the pools of PL and cholesterol available for removal by apoA-I from about 0.8-5%; the initial rates of mass release of cholesterol and PL are both increased about 6-fold. Expression of the ATP binding cassette transporter A1 (ABCA1) is critical for this increased efflux of lipids, and cholesterol loading of the fibroblasts over 24 h increases ABCA1 mRNA about 12-fold. The presence of more ABCA1 and cholesterol in the plasma membrane results in a 2-fold increase in the level of specific binding of apoA-I to the cells with no change in binding affinity. Characterization of the species released from either control or cholesterol-enriched cells indicates that the plasma membrane domains from which lipids are removed are cholesterol-enriched with respect to the average plasma membrane composition. Cholesterol enrichment of fibroblasts also affects PL synthesis, and this leads to enhanced release of phosphatidylcholine (PC) relative to sphingomyelin (SM); the ratios of PC to SM solubilized from control and cholesterol-enriched fibroblasts are approximately 2/1 and 5/1, respectively. Biosynthesis of PC is critical for this preferential release of PC and the enhanced cholesterol efflux because inhibition of PC synthesis by choline depletion reduces cholesterol efflux from cholesterol-enriched cells. Overall, it is clear that enrichment of fibroblasts with unesterified cholesterol enhances efflux of cholesterol and PL to apoA-I because of three effects, 1) increased PC biosynthesis, 2) increased PC transport via ABCA1, and 3) increased cholesterol in the plasma membrane.     

Characterization and properties of pre beta-HDL particles formed by ABCA1-mediated cellular lipid efflux to apoA-I

The contribution of ABCA1-mediated efflux of cellular phospholipid (PL) and cholesterol to human apolipoprotein A-I (apoA-I) to the formation of pre beta 1-HDL (or lipid-poor apoA-I) is not well defined. To explore this issue, we characterized the nascent HDL particles formed when lipid-free apoA-I was incubated with fibroblasts in which expression of the ABCA1 was upregulated. After a 2 h incubation, the extracellular medium contained small apoA-I/PL particles (pre beta 1-HDL; diameter = 7.5 +/- 0.4 nm). The pre beta 1-HDL (or lipid-poor apoA-I) particles contained a single apoA-I molecule and three to four PL molecules and one to two cholesterol molecules. An apoA-I variant lacking the C-terminal alpha-helix did not form such particles when incubated with the cell, indicating that this helix is critical for the formation of lipid-poor apoA-I particles. These pre beta 1-HDL particles were as effective as lipid-free apoA-I molecules in mediating both the efflux of cellular lipids via ABCA1 and the formation of larger, discoidal HDL particles. In conclusion, pre beta 1-HDL is both a product and a substrate in the ABCA1-mediated reaction to efflux cellular PL and cholesterol to apoA-I. A monomeric apoA-I molecule associated with three to four PL molecules (i.e., lipid-poor apoA-I) has similar properties to the lipid-free apoA-I molecule.     

Lysosomal acid lipase deficiency impairs regulation of ABCA1 gene and formation of high density lipoproteins in cholesteryl ester storage disease

ATP-binding cassette transporter A1 (ABCA1) mediates the rate-limiting step in high density lipoprotein (HDL) particle formation, and its expression is regulated primarily by oxysterol-dependent activation of liver X receptors. We previously reported that ABCA1 expression and HDL formation are impaired in the lysosomal cholesterol storage disorder Niemann-Pick disease type C1 and that plasma HDL-C is low in the majority of Niemann-Pick disease type C patients. Here, we show that ABCA1 regulation and activity are also impaired in cholesteryl ester storage disease (CESD), caused by mutations in the LIPA gene that result in less than 5% of normal lysosomal acid lipase (LAL) activity. Fibroblasts from patients with CESD showed impaired up-regulation of ABCA1 in response to low density lipoprotein (LDL) loading, reduced phospholipid and cholesterol efflux to apolipoprotein A-I, and reduced α-HDL particle formation. Treatment of normal fibroblasts with chloroquine to inhibit LAL activity reduced ABCA1 expression and activity, similar to that of CESD cells. Liver X receptor agonist treatment of CESD cells corrected ABCA1 expression but failed to correct LDL cholesteryl ester hydrolysis and cholesterol efflux to apoA-I. LDL-induced production of 27-hydroxycholesterol was reduced in CESD compared with normal fibroblasts. Treatment with conditioned medium containing LAL from normal fibroblasts or with recombinant human LAL rescued ABCA1 expression, apoA-I-mediated cholesterol efflux, HDL particle formation, and production of 27-hydroxycholesterol by CESD cells. These results provide further evidence that the rate of release of cholesterol from late endosomes/lysosomes is a critical regulator of ABCA1 expression and activity, and an explanation for the hypoalphalipoproteinemia seen in CESD patients.     

ATP-binding cassette transporter A1 mediates cellular secretion of alpha-tocopherol

Alpha-tocopherol (alpha-TOH) is associated with plasma lipoproteins and accumulates in cell membranes throughout the body, suggesting that lipoproteins play a role in transporting alpha-TOH between tissues. Here we show that secretion of alpha-TOH from cultured cells is mediated in part by ABCA1, an ATP-binding cassette protein that transports cellular cholesterol and phospholipids to lipid-poor high density lipoprotein (HDL) apolipoproteins such as apoA-I. Treatment of human fibroblasts and murine RAW264 macrophages with cholesterol and/or 8-bromo-cyclic AMP, which induces ABCA1 expression, enhanced apoA-I-mediated alpha-TOH efflux. ApoA-I lacked the ability to remove alpha-TOH from Tangier disease fibroblasts that have a nonfunctional ABCA1. BHK cells that lack an active ABCA1 pathway markedly increased secretion of alpha-TOH to apoA-I when forced to express ABCA1. ABCA1 also mediated a fraction of the alpha-TOH efflux promoted by lipid-containing HDL particles, indicating that HDL promotes alpha-TOH efflux by both ABCA1-dependent and -independent processes. Exposing apoA-I to ABCA1-expressing cells did not enhance its ability to remove alpha-TOH from cells lacking ABCA1, consistent with this transporter participating directly in the translocation of alpha-TOH to apolipoproteins. These studies provide evidence that ABCA1 mediates secretion of cellular alpha-TOH into the HDL metabolic pathway, a process that may facilitate vitamin transport between tissues and influence lipid oxidation.     

Intracellular cholesterol mobilization involved in the ABCA1/apolipoprotein-mediated assembly of high density lipoprotein in fibroblasts

Differential regulation has been suggested for cellular cholesterol and phospholipid release mediated by apolipoprotein A-I (apoA-I)/ABCA1. We investigated various factors involved in cholesterol mobilization related to this pathway. ApoA-I induced a rapid decrease of the cellular cholesterol compartment that is in equilibrium with the ACAT-accessible pool in cells that generate cholesterol-rich HDL. Pharmacological and genetic inactivation of ACAT enhanced the apoA-I-mediated cholesterol release through upregulation of ABCA1 and through cholesterol enrichment in the HDL generated. Pharmacological activation of protein kinase C (PKC) also decreased the ACAT-accessible cholesterol pool, not only in the cells that produce cholesterol-rich HDL by apoA-I (i.e., human fibroblast WI-38 cells) but also in the cells that generate cholesterol-poor HDL (mouse fibroblast L929 cells). In L929 cells, the PKC activation caused an increase in apoA-I-mediated cholesterol release without detectable change in phospholipid release and in ABCA1 expression. These results indicate that apoA-I mobilizes intracellular cholesterol for the ABCA1-mediated release from the compartment that is under the control of ACAT. The cholesterol mobilization process is presumably related to PKC activation by apoA-I.