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.     

Accumulation of cardiolipin and lysocardiolipin in fibroblasts from Tangier disease subjects

Tangier disease (TD) is an inherited disorder of lipid metabolism characterized by very low high density lipoprotein (HDL) plasma levels, cellular cholesteryl ester accumulation and reduced cholesterol excretion in response to HDL apolipoproteins. Molecular defects in the ATP binding cassette transporter 1 (ABCA1) have recently been identified as the cause of TD. ABCA1 plays a key role in the translocation of cholesterol across the plasma membrane, and defective ABCA1 causes cholesterol storage in TD cells. Not only cholesterol efflux, but also phospholipid efflux was shown to be impaired in TD cells. By use of thin layer chromatography, high performance liquid chromatography and time-of-flight secondary ion mass spectrometry, we characterized the cellular phospholipid content in fibroblasts from three homozygous TD patients. The cellular content of the major phospholipids was not found to be significantly altered in TD fibroblasts. However, the two phospholipids cardiolipin and lysocardiolipin, which make up minute amounts in normal cells, were at least 3–5-fold enriched in fibroblasts from TD subjects. A structurally closely related phospholipid (lysobisphosphatidic acid) has recently been shown to be enriched in Niemann–Pick type C, another lipid storage disorder. Altogether these data may indicate that the role of these phospholipids is a regulatory one rather than that of a bulk mediator of cholesterol solubilization in sterol trafficking and efflux.     

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.     

Serum amyloid A promotes ABCA1-dependent and ABCA1-independent lipid efflux from cells

Serum amyloid A (SAA) is an acute phase protein that associates with HDL. In order to examine the role of SAA in reverse-cholesterol transport, lipid efflux was tested to SAA from HeLa cells before and after transfection with the ABCA1 transporter. ABCA1 expression increased efflux of cholesterol and phospholipid to SAA by 3-fold and 2-fold, respectively. In contrast to apoA-I, SAA also removed lipid without ABCA1; cholesterol efflux from control cells to SAA was 10-fold higher than for apoA-I. Furthermore, SAA effluxed cholesterol from Tangier disease fibroblasts and from cells after inhibition of ABCA1 by fixation with paraformaldehyde. In summary, SAA can act as a lipid acceptor for ABCA1, but unlike apoA-I, it can also efflux lipid without ABCA1, by most likely a detergent-like extraction process. These results suggest that SAA may play a unique role as an auxiliary lipid acceptor in the removal of lipid from sites of inflammation.     

Molecular interactions between apoE and ABCA1: impact on apoE lipidation

Apolipoprotein E (apoE)/ABCA1 interactions were investigated in human intact fibroblasts induced with 22(R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Here, we show that purified human plasma apoE3 forms a complex with ABCA1 in normal fibroblasts. Lipid-free apoE3 inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than reconstituted HDL particles (IC(50) = 2.5 +/- 0.4 microg/ml vs. 12.3 +/- 1.3 microg/ml). ApoE isoforms showed similar binding for ABCA1 and exhibited identical kinetics in their abilities to induce ABCA1-dependent cholesterol efflux. Mutation of ABCA1 associated with Tangier disease (C1477R) abolished both apoE3 binding and apoE3-mediated cholesterol efflux. Analysis of apoE3-containing particles generated during the incubation of lipid-free apoE3 with stimulated normal cells showed nascent apoE3/cholesterol/phospholipid complexes that exhibited prebeta-electrophoretic mobility with a particle size ranging from 9 to 15 nm, whereas lipid-free apoE3 incubated with ABCA1 mutant (C1477R) cells was unable to form such particles. These results demonstrate that 1). apoE association with lipids reduced its ability to interact with ABCA1; 2). apoE isoforms did not affect apoE binding to ABCA1; 3). apoE-mediated ABCA1-dependent cholesterol efflux was not affected by apoE isoforms in fibroblasts; and 4). the lipid translocase activity of ABCA1 generates apoE-containing high density-sized lipoprotein particles. Thus, ABCA1 is essential for the biogenesis of high density-sized lipoprotein containing only apoE particles in vivo.     

The ABCA1 transporter modulates late endocytic trafficking: insights from the correction of the genetic defect in Tangier disease

We have previously established that the ABCA1 transporter, which plays a critical role in the lipidation of extracellular apolipoprotein acceptors, traffics between late endocytic vesicles and the cell surface (Neufeld, E. B., Remaley, A. T., Demosky, S. J., Jr., Stonik, J. A., Cooney, A. M., Comly, M., Dwyer, N. K., Zhang, M., Blanchette-Mackie, J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) J. Biol. Chem. 276, 27584-27590). The present study provides evidence that ABCA1 in late endocytic vesicles plays a role in cellular lipid efflux. Late endocytic trafficking was defective in Tangier disease fibroblasts that lack functional ABCA1. Consistent with a late endocytic protein trafficking defect, the hydrophobic amine U18666A retained NPC1 in abnormally tubulated, cholesterol-poor, Tangier disease late endosomes, rather than cholesterol-laden lysosomes, as in wild type fibroblasts. Consistent with a lipid trafficking defect, Tangier disease late endocytic vesicles accumulated both cholesterol and sphingomyelin and were immobilized in a perinuclear localization. The excess cholesterol in Tangier disease late endocytic vesicles retained massive amounts of NPC1, which traffics lysosomal cholesterol to other cellular sites. Exogenous apoA-I abrogated the cholesterol-induced retention of NPC1 in wild type but not in Tangier disease late endosomes. Adenovirally mediated ABCA1-GFP expression in Tangier disease fibroblasts corrected the late endocytic trafficking defects and restored apoA-I-mediated cholesterol efflux. ABCA1-GFP expression in wild type fibroblasts also reduced late endosome-associated NPC1, induced a marked uptake of fluorescent apoA-I into ABCA1-GFP-containing endosomes (that shuttled between late endosomes and the cell surface), and enhanced apoA-I-mediated cholesterol efflux. The combined results of this study suggest that ABCA1 converts pools of late endocytic lipids that retain NPC1 to pools that can associate with endocytosed apoA-I, and be released from the cell as nascent high density lipoprotein.     

Cellular cholesterol efflux is modulated by phospholipid-derived signaling molecules in familial HDL deficiency/Tangier disease fibroblasts

Familial HDL deficiency (FHD) is the heterozygous form of Tangier disease (TD). Mutations of the ABCA1 gene cause FHD and TD. FHD/TD cells are unable to normally efflux cholesterol onto nascent HDL particles, which are rapidly catabolized. TD fibroblasts have an abnormal pattern of PLC and PLD activation following cell stimulation with HDL(3) or apolipoprotein A-I (apoA-I). We examined cellular cholesterol efflux in FHD and TD fibroblasts by phospholipid-derived-molecules, compared with that of normal cells. We used the PKC agonist 1,2-dioctanoylglycerol (DOG) and phorbol myristate acetate (PMA) to activate PKC, calphostin C, and GO 6976, as inhibitors of PKC; phosphatidic acid (PA), which is the product of PLD, and lysophosphatidic acid (LPA), phosphatidylcholine, sphingomyelin, and beta-cyclodextrin to investigate their potential effect in modulating cellular cholesterol efflux in [(3)H]cholesterol-labeled and cholesterol-loaded fibroblasts. Phosphatidylcholine, sphingomyelin, and beta-cyclodextrin promoted cholesterol efflux in an identical fashion in control, FHD, or TD fibroblasts. In a dose-dependent fashion, DOG (0-200 microM) increased apoA-I-mediated cellular cholesterol efflux by 40% in controls, 71% in FHD, and 242% in TD cells. PMA similarly increased cholesterol efflux to a maximum of 256% in controls, 182% in FHD, and 191% in TD cells. This effect was inhibited by calphostin C. PA (100 microM) also increased cholesterol efflux by 25% in control, 44% in FHD, and 100% in TD cells. Conversely, LPA reduced cholesterol efflux in a dose-dependent fashion in control and FHD cells (-50%, 200 microM) but not in TD cells, where efflux was increased by 140%. Propranolol (100 microM) significantly increased cholesterol efflux at 24 h in all three cell lines. n-Butanol partially decreased the DOG-mediated increase in cholesterol efflux. The inhibitory effect of calphostin C on DOG-stimulated cholesterol efflux could be partially overcome by propranolol, suggesting that PA is a downstream mediator of PKC-stimulated cholesterol efflux.We conclude that PLC and PLD activities are required for apoA-I-mediated cellular cholesterol efflux, and modulating cellular PA concentration can correct, at least partially, the cholesterol efflux defect in FHD and TD.     

Accumulation of RhoA, RhoB, RhoG, and Rac1 in fibroblasts from Tangier disease subjects suggests a regulatory role of Rho family proteins in cholesterol efflux

Tangier disease (TD) is an inherited disorder of lipid metabolism characterized by very low high density lipoprotein (HDL) plasma levels, cellular cholesteryl ester accumulation and reduced cholesterol excretion in response to HDL apolipoproteins. Molecular defects in the ATP binding cassette transporter 1 (ABCA1) have recently been identified as the cause of TD. ABCA1 plays a key role in the translocation of cholesterol across the plasma membrane, and defective ABCA1 causes cholesterol storage in TD cells. However, the exact relationship of many of the biochemical and morphological abnormalities in TD to ABCA1 is unknown. Since small GTP-binding proteins are important regulators of many cellular functions, we characterized these proteins in normal and TD fibroblasts using the [alpha-32P]GTP overlay technique and Western blotting of SDS and isoelectric focusing gels. Our results indicate that GTP-binding proteins of the Rho family (RhoA, RhoB, RhoG, Rac-1) are enriched in fibroblasts from TD patients. The accumulation of small G proteins may have potential implications for the TD phenotype and the regulation of cholesterol excretion in TD cells.     

ATP binding cassette transporter A1 - key roles in cellular lipid transport and atherosclerosis

ATP-binding cassette transporter A1 (ABCA1) was recently recognized as the mutant molecule responsible for Tangier disease with low HDL levels, accumulation of cholesteryl esters in tissues, and increased risk of cardiovascular disease. Extensive studies for the past 2 years have recognized the critical role of ABCA1 in cholesterol and phospholipid trafficking. Since the removal of cholesterol from tissues is a key step in the prevention of atherosclerosis, significant attention has been focused on this molecule. Natural ABCA1 mutations in Tangier disease (TD) patients and WHAM chickens together with induced mutation in ABCA1 knock-out mice unequivocally established the important role of ABCA1 in maintaining circulating HDL levels and promoting cholesterol efflux from the arterial wall. Mice lacking ABCA1 showed similar phenotypes observed in Tangier disease patients with low levels of HDL. Further understanding of the roles of ABCA1 in lipid transport and atherosclerosis became clear from studies with ABCA1 transgenic mice. These mice showed enhanced cholesterol efflux from macrophages and reduced atherosclerotic lesion formation. The promoter of the ABCA1 gene has been mapped to a large extent, with the exception of cAMP response element. The present review summarizes recent developments on the role of ABCA1 in cholesterol efflux and prevention of atherosclerosis. Given the antiatherogenic properties of ABCA1, this molecule can serve as an appropriate target for developing drugs to treat individuals with low levels of HDL.     

Characterization of Cholesterol Homeostasis in Telomerase-immortalized Tangier Disease Fibroblasts Reveals Marked Phenotype Variability

We compared the consequences of an ABCA1 mutation that produced an apparent lack of atherosclerosis (Tangier family 1, N935S) with an ABCA1 mutation with functional ABCA1 knockout that was associated with severe atherosclerosis (Tangier family 2, Leu⁵⁴⁸:Leu⁵⁷⁵-End), using primary and telomerase-immortalized fibroblasts. Telomerase-immortalized Tangier fibroblasts of family 1 (TT1) showed 30% residual cholesterol efflux capacity in response to apolipoprotein A-I, whereas telomerase-immortalized Tangier fibroblasts of family 2 (TT2) showed only 20%. However, there were a number of secondary differences that were often stronger and may help to explain the more rapid development of atherosclerosis in family 2. First, the total cellular cholesterol content increase was 2–3-fold and 3–5-fold in TT1 and TT2 cells, respectively. The corresponding increase in esterified cholesterol concentration was 10- and 40-fold, respectively. Second, 24-, 25-, and 27-hydroxycholesterol concentrations were moderately increased in TT1 cells, but were increased as much as 200-fold in TT2 cells. Third, cholesterol biosynthesis was moderately decreased in TT1 cells, but was markedly decreased in TT2 cells. Fourth, potentially atheroprotective LXR-dependent SREBP1c signaling was normal in TT1, but was rather suppressed in TT2 cells. Cultivated primary Tangier fibroblasts were characterized by premature aging in culture and were associated with less obvious biochemical differences. In summary, these results may help to understand the differential atherosclerotic susceptibility in Tangier disease and further demonstrate the usefulness of telomerase-immortalized cells in studying this cellular phenotype. The data support the contention that side chain-oxidized oxysterols are strong suppressors of cholesterol biosynthesis under specific pathological conditions in humans.     

Sodium taurocholate-dependent lipid efflux by ABCA1: effects of W590S mutation on lipid translocation and apolipoprotein A-I dissociation

ABCA1 plays a major role in HDL metabolism. Cholesterol secretion by ABCA1 is dependent on the presence of extracellular acceptors, such as lipid-free apolipoprotein A-I (apoA-I). However, the importance of the direct interaction between apoA-I and ABCA1 in HDL formation remains unclear. In contrast, ABCB4 mediates the secretion of phospholipids and cholesterol in the presence of sodium taurocholate (NaTC) but not in the presence of apoA-I. In this study, we analyzed apoA-I binding and NaTC-dependent lipid efflux by ABCA1. ABCA1 mediated the efflux of cholesterol and phospholipids in the presence of NaTC as well as in the presence of apoA-I in an ATP-dependent manner. The Tangier disease mutation W590S, which resides in the extracellular domain and impairs apoA-I-dependent lipid efflux, greatly decreased NaTC-dependent cholesterol and phospholipid efflux. However, the W590S mutation did not impair apoA-I binding and, conversely, retarded the dissociation of apoA-I from ABCA1. These results suggest that the W590S mutation impairs ATP-dependent lipid translocation and that lipid translocation or possibly lipid loading, facilitates apoA-I dissociation from ABCA1. NaTC is a good tool for analyzing ABCA1-mediated lipid efflux and allows dissection of the steps of HDL formation by ABCA1.     

ATP-binding cassette transporter A7 (ABCA7) binds apolipoprotein A-I and mediates cellular phospholipid but not cholesterol efflux

ATP-binding cassette transporter 1 (ABCA1), the defective transporter in Tangier disease, binds and promotes cellular cholesterol and phospholipid efflux to apolipoprotein I (apoA-I). Based on a high degree of sequence homology between ABCA1 and ABCA7, a transporter of unknown function, we investigated the possibility that ABCA7 might be involved in apolipoprotein binding and lipid efflux. Similarly to cells expressing ABCA1, HEK293 cells overexpressing ABCA7 showed specific binding and cross-linking of lipid-poor apoA-I. ABCA7 expression increased cellular phosphatidylcholine and sphingomyelin efflux to apoA-I in a manner similar to ABCA1 but had no effect on cholesterol efflux. Western analysis showed a high protein level of ABCA7 in mouse spleen, lung, adrenal, and brain but low expression in liver. In contrast to ABCA1, ABCA7 showed moderate basal mRNA and protein levels in macrophages and lymphocytes but no induction by liver X receptor activation. These studies show that ABCA7 has the ability to bind apolipoproteins and promote efflux of cellular phospholipids without cholesterol, and they suggest a possible role of ABCA7 in cellular phospholipid metabolism in peripheral tissues.     

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   

ATP-binding cassette transporter A1 contains a novel C-terminal VFVNFA motif that is required for its cholesterol efflux and ApoA-I binding activities

The stimulation of cellular cholesterol and phospholipid efflux by apolipoprotein A-I is mediated by the activity of the ATP-binding cassette transporter A1 (ABCA1). Individuals with Tangier disease harbor loss-of-function mutations in this transporter that have proven useful in illuminating its activity. Here, we analyze a mutation that deletes the last 46 residues of the 2261 amino acid transporter (Delta46) and eliminates its lipid efflux. As the final four amino acids of the C terminus represent a putative PDZ-binding motif, we initially characterized deletion mutants lacking only these residues. Although a moderate decline in lipid efflux was detected, this decline was not as profound as that seen in the Delta46 mutant. Subsequent systematic analysis of the ABCA1 C terminus revealed a novel, highly conserved motif (VFVNFA) that was required for lipid efflux. Alteration of this motif, which is present in some but not all members of the ABCA family, did not prevent trafficking of the transporter to the plasma membrane but did eliminate its binding of apoA-I. Chimeric transporters, generated by substituting the C termini of either ABCA4 or ABCA7 for the endogenous terminus, demonstrated that ABCA1 could stimulate cholesterol efflux without its PDZ-binding motif but not without the VFVNFA motif. When a peptide containing the VFVNFA sequence was introduced into ABCA1-expressing cells, ABCA1-mediated lipid efflux was also markedly inhibited. These results indicate that the C-terminal VFVNFA motif of ABCA1 is essential for its lipid efflux activity. The data also suggest that this motif participates in novel protein-protein interactions that may be shared among members of the ABCA family.     

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.     

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.     

The impact of severe LDL receptor mutations on SREBP-pathway regulation in homozygous familial hypercholesterolemia (FH)

Familial hypercholesterolemia (FH), Niemann-Pick disease type C (NPC) and Tangier disease (TD) are genetic inherited disorders with impaired processing of cholesterol, caused by mutations in genes that regulate cellular uptake, intracellular movement and transport of cholesterol. Various studies have shown a crucial regulatory role of the SREBP-pathway for cellular cholesterol homeostasis in these diseases. Since cholesterol is an essential structural component of cells, we assessed the impact of a severe FH causing LDLR mutation (FH p.W556R) on the SREBP pathway in primary FH fibroblasts. Primary FH fibroblasts derived from patients with the LDL receptor mutation p.W556R were used for gene expression experiments. Gene expression studies revealed increased expressions of SREBP regulated genes HMGCR, LDLR, SREBP-2, SREBP-1, SR-BI, INSIG-1, but interestingly not SCAP. In contrast expression of ABCA1, was strongly decreased in homozygous, but not in heterozygous p.W556R fibroblasts. Gene expression experiments with LDL receptor lacking primary FH fibroblasts, revealed that SR-BI and ABCA1 are important regulators for cholesterol acquisition in FH cells, consistent with findings in cells from NPC and TD patients.     

Two novel missense mutations in ABCA1 result in altered trafficking and cause severe autosomal recessive HDL deficiency

Extremely low concentrations of high density lipoprotein (HDL)-cholesterol and apolipoprotein (apo) AI are features of Tangier disease caused by autosomal recessive mutations in ATP-binding cassette transporter A1 (ABCA1). Less deleterious, but dominantly inherited mutations cause HDL deficiency. We investigated causes of severe HDL deficiency in a 42-year-old female with progressive coronary disease. ApoAI-mediated efflux of cholesterol from the proband's fibroblasts was less than 10% of normal and nucleotide sequencing revealed inheritance of two novel mutations in ABCAI, V1704D and L1379F. ABCA1 mRNA was approximately 3-fold higher in the proband's cells than in control cells; preincubation with cholesterol increased it 5-fold in control and 8-fold in the proband's cells, but similar amounts of ABCA1 protein were present in control and mutant cells. When transiently transfected into HEK293 cells, confocal microscopy revealed that both mutant proteins were retained in the endoplasmic reticulum, while wild-type ABCA1 was located at the plasma membrane. Severe HDL deficiency in the proband was caused by two novel autosomal recessive mutations in ABCA1, one (V1704D) predicted to lie in a transmembrane segment and the other (L1379F) in a large extracellular loop. Both mutations prevent normal trafficking of ABCA1, thereby explaining their inability to mediate apoA1-dependent lipid efflux.     

Sphingomyelin Depletion Impairs Anionic Phospholipid Inward Translocation and Induces Cholesterol Efflux

The phosphatidylserine (PS) floppase activity (outward translocation) of ABCA1 leads to plasma membrane remodeling that plays a role in lipid efflux to apolipoprotein A-I (apoAI) generating nascent high density lipoprotein. The Tangier disease W590S ABCA1 mutation has defective PS floppase activity and diminished cholesterol efflux activity. Here, we report that depletion of sphingomyelin by inhibitors or sphingomyelinase caused plasma membrane remodeling, leading to defective flip (inward translocation) of PS, higher PS exposure, and higher cholesterol efflux from cells by both ABCA1-dependent and ABCA1-independent mechanisms. Mechanistically, sphingomyelin was connected to PS translocation in cell-free liposome studies that showed that sphingomyelin increased the rate of spontaneous PS flipping. Depletion of sphingomyelin in stably transfected HEK293 cells expressing the Tangier disease W590S mutant ABCA1 isoform rescued the defect in PS exposure and restored cholesterol efflux to apoAI. Liposome studies showed that PS directly increased cholesterol accessibility to extraction by cyclodextrin, providing the mechanistic link between cell surface PS and cholesterol efflux. We conclude that altered plasma membrane environment conferred by depleting sphingomyelin impairs PS flip and promotes cholesterol efflux in ABCA1-dependent and -independent manners.     

Structural modification of plasma HDL by phospholipids promotes efficient ABCA1-mediated cholesterol release

It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apoA-I). Here, we show that treatment of plasma with dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles generates prebeta(1)-apoA-I-containing lipoproteins (LpA-I)-like particles similar to those of native plasma. Isolated prebeta(1)-LpA-I-like particles inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than HDL(3) (IC(50) = 2.20 +/- 0.35 vs. 37.60 +/- 4.78 microg/ml). We next investigated the ability of DMPC-treated plasma to promote phospholipid and unesterified (free) cholesterol efflux from J774 macrophages stimulated or not with cAMP. At 2 mg DMPC/ml plasma, both phospholipid and free cholesterol efflux were increased ( approximately 50% and 40%, respectively) in cAMP-stimulated cells compared with unstimulated cells. Similarly, both phospholipid and free cholesterol efflux to either isolated native prebeta(1)-LpA-I and prebeta(1)-LpA-I-like particles were increased significantly in stimulated cells. Furthermore, glyburide significantly inhibited phospholipid and free cholesterol efflux to DMPC-treated plasma. Removal of apoA-I-containing lipoproteins from normolipidemic plasma drastically reduced free cholesterol efflux mediated by DMPC-treated plasma. Finally, treatment of Tangier disease plasma with DMPC affected the amount of neither prebeta(1)-LpA-I nor free cholesterol efflux. These results indicate that DMPC enrichment of normal plasma resulted in the redistribution of apoA-I from alpha-HDL to prebeta-HDL, allowing for more efficient ABCA1-mediated cellular lipid release. Increasing the plasma prebeta(1)-LpA-I level by either pharmacological agents or direct infusions might prevent foam cell formation and reduce atherosclerotic vascular disease.