ABCA1 is required for normal central nervous system ApoE levels and for lipidation of astrocyte-secreted apoE

ABCA1 is an ATP-binding cassette protein that transports cellular cholesterol and phospholipids onto high density lipoproteins (HDL) in plasma. Lack of ABCA1 in humans and mice causes abnormal lipidation and increased catabolism of HDL, resulting in very low plasma apoA-I, apoA-II, and HDL. Herein, we have used Abca1-/- mice to ask whether ABCA1 is involved in lipidation of HDL in the central nervous system (CNS). ApoE is the most abundant CNS apolipoprotein and is present in HDL-like lipoproteins in CSF. We found that Abca1-/- mice have greatly decreased apoE levels in both the cortex (80% reduction) and the CSF (98% reduction). CSF from Abca1-/- mice had significantly reduced cholesterol as well as small apoE-containing lipoproteins, suggesting abnormal lipidation of apoE. Astrocytes, the primary producer of CNS apoE, were cultured from Abca1+/+, +/-, and -/- mice, and nascent lipoprotein particles were collected. Abca1-/- astrocytes secreted lipoprotein particles that had markedly decreased cholesterol and apoE and had smaller apoE-containing particles than particles from Abca1+/+ astrocytes. These findings demonstrate that ABCA1 plays a critical role in CNS apoE metabolism. Since apoE isoforms and levels strongly influence Alzheimer's disease pathology and risk, these data suggest that ABCA1 may be a novel therapeutic target.     

The LXR agonist GW3965 increases apoA-I protein levels in the central nervous system independent of ABCA1

Lipoprotein metabolism in the central nervous system (CNS) is based on high-density lipoprotein-like particles that use apoE as their predominant apolipoprotein rather than apoA-I. Although apoA-I is not expressed in astrocytes and microglia, which produce CNS apoE, apoA-I is reported to be expressed in porcine brain capillary endothelial cells and also crosses the blood-brain barrier (BBB). These mechanisms allow apoA-I to reach concentrations in cerebrospinal fluid (CSF) that are approximately 0.5% of its plasma levels. Recently, apoA-I has been shown to enhance cognitive function and reduce cerebrovascular amyloid deposition in Alzheimer's Disease (AD) mice, raising questions about the regulation and function of apoA-I in the CNS. Peripheral apoA-I metabolism is highly influenced by ABCA1, but less is known about how ABCA1 regulates CNS apoA-I. We report that ABCA1 deficiency leads to greater retention of apoA-I in the CNS than in the periphery. Additionally, treatment of symptomatic AD mice with GW3965, an LXR agonist that stimulates ABCA1 expression, increases apoA-I more dramatically in the CNS compared to the periphery. Furthermore, GW3965-mediated up-regulation of CNS apoA-I is independent of ABCA1. Our results suggest that apoA-I may be regulated by distinct mechanisms on either side of the BBB and that apoA-I may serve to integrate peripheral and CNS lipid metabolism. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

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.     

The carboxy-terminal region of apoA-I is required for the ABCA1-dependent formation of alpha-HDL but not prebeta-HDL particles in vivo

ATP-binding cassette transporter A-1 (ABCA1)-mediated lipid efflux to lipid-poor apolipoprotein A-I (apoA-I) results in the gradual lipidation of apoA-I. This leads to the formation of discoidal high-density lipoproteins (HDL), which are subsequently converted to spherical HDL by the action of lecithin:cholesterol acyltransferase (LCAT). We have investigated the effect of point mutations and deletions in the carboxy-terminal region of apoA-I on the biogenesis of HDL using adenovirus-mediated gene transfer in apoA-I-deficient mice. It was found that the plasma HDL levels were greatly reduced in mice expressing the carboxy-terminal deletion mutants apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)], shown previously to diminish the ABCA1-mediated lipid efflux. The HDL levels were normal in mice expressing the WT apoA-I, the apoA-I[Delta(232-243)] deletion mutant, or the apoA-I[E191A/H193A/K195A] point mutant, which promote normal ABCA1-mediated lipid efflux. Electron microscopy and two-dimensional gel electrophoresis showed that the apoA-I[Delta(185-243)] and apoA-I[Delta(220-243)] mutants formed mainly prebeta-HDL particles and few spherical particles enriched in apoE, while WT apoA-I, apoA-I[Delta(232-243)], and apoA-I[E191A/H193A/K195A] formed spherical alpha-HDL particles. The findings establish that (a) deletions that eliminate the 220-231 region of apoA-I prevent the synthesis of alpha-HDL but allow the synthesis of prebeta-HDL particles in vivo, (b) the amino-terminal segment 1-184 of apoA-I can promote synthesis of prebeta-HDL-type particles in an ABCA1-independent process, and (c) the charged residues in the 191-195 region of apoA-I do not influence the biogenesis of HDL.     

Testing the role of apoA-I,HDL, and cholesterol efflux in the atheroprotective action of low-level apoE expression

Low levels of transgenic mouse apolipoprotein E (apoE) suppress atherosclerosis in apoE knockout (apoE-/-) mice without normalizing plasma cholesterol. To test whether this is due to facilitation of cholesterol efflux from the vessel wall, we produced apoA-I-/-/apoE-/- mice with or without the transgene. Even without apoA-I and HDL, apoA-I-/-/apoE-/- mice had the same amount of aorta cholesteryl ester as apoE-/- mice. Low apoE in the apoA-I-/-/apoE-/- transgenic mice reduced aortic lesions by 70% versus their apoA-I-/-/apoE-/- siblings. To define the free cholesterol (FC) efflux capacity of lipoproteins from the various genotypes, sera were assayed on macrophages expressing ATP-binding cassette transporter A1 (ABCA1). Surprisingly, ABCA1 FC efflux was twice as high to sera from the apoA-I-/-/apoE-/- or apoE-/- mice compared with wild-type mice, and this activity correlated with serum apoA-IV. Immunodepletion of apoA-IV from apoA-I-/-/apoE-/- serum abolished ABCA1 FC efflux, indicating that apoAI-V serves as a potent acceptor for FC efflux via ABCA1. With increasing apoE expression, apoA-IV and FC acceptor capacity decreased, indicating a reciprocal relationship between plasma apoE and apoA-IV. Low plasma apoE (1-3 x 10(-8) M) suppresses atherosclerosis by as yet undefined mechanisms, not dependent on the presence of apoA-I or HDL or an increased capacity of serum acceptors for FC efflux.     

ABCA1-mediated cholesterol efflux generates microparticles in addition to HDL through processes governed by membrane rigidity

ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-poor apolipoprotein A-I (apoA-I) and generates HDL. Here, we demonstrate that ABCA1 also directly mediates the production of apoA-I free microparticles. In baby hamster kidney (BHK) cells and RAW macrophages, ABCA1 expression led to lipid efflux in the absence of apoA-I and released large microparticles devoid of apoB and apoE. We provide evidence that these microparticles are an integral component of the classical cholesterol efflux pathway when apoA-I is present and accounted for approximately 30% of the total cholesterol released to the medium. Furthermore, microparticle release required similar ABCA1 activities as was required for HDL production. For instance, a nucleotide binding domain mutation in ABCA1 (A937V) that impaired HDL generation also abolished microparticle release. Similarly, inhibition of protein kinase A (PKA) prevented the release of both types of particles. Interestingly, physical modulation of membrane dynamics affected HDL and microparticle production, rigidifying the plasma membrane with wheat germ agglutinin inhibited HDL and microparticle release, whereas increasing the fluidity promoted the production of these particles. Given the established role of ABCA1 in expending nonraft or more fluid-like membrane domains, our results suggest that both HDL and microparticle release is favored by a more fluid plasma membrane. We speculate that ABCA1 enhances the dynamic movement of the plasma membrane, which is required for apoA-I lipidation and microparticle formation.     

Cross-inhibition of SR-BI- and ABCA1-mediated cholesterol transport by the small molecules BLT-4 and glyburide

Scavenger receptor class B type I (SR-BI) and ABCA1 are structurally dissimilar cell surface proteins that play key roles in HDL metabolism. SR-BI is a receptor that binds HDL with high affinity and mediates both the selective lipid uptake of cholesteryl esters from lipid-rich HDL to cells and the efflux of unesterified cholesterol from cells to HDL. ABCA1 mediates the efflux of unesterified cholesterol and phospholipids from cells to lipid-poor apolipoprotein A-I (apoA-I). The activities of ABCA1 and other ATP binding cassette superfamily members are inhibited by the drug glyburide, and SR-BI-mediated lipid transport is blocked by small molecule inhibitors called BLTs. Here, we show that one BLT, [1-(2-methoxy-phenyl)-3-naphthalen-2-yl-urea] (BLT-4), blocked ABCA1-mediated cholesterol efflux to lipid-poor apoA-I at a potency similar to that for its inhibition of SR-BI (IC(50) approximately 55-60 microM). Reciprocally, glyburide blocked SR-BI-mediated selective lipid uptake and efflux at a potency similar to that for its inhibition of ABCA1 (IC(50) approximately 275-300 microM). As is the case with BLTs, glyburide increased the apparent affinity of HDL binding to SR-BI. The reciprocal inhibition of SR-BI and ABCA1 by BLT-4 and glyburide raises the possibility that these proteins may share similar or common steps in their mechanisms of lipid transport.     

Ethanol induces cholesterol efflux and up-regulates ATP-binding cassette cholesterol transporters in fetal astrocytes

Cholesterol plays an important role during brain development, since it is involved in glial cell proliferation, neuronal survival and differentiation, and synaptogenesis. Astrocytes produce large amounts of brain cholesterol and produce and release lipoproteins containing apoE that can extract cholesterol from CNS cells for elimination. We hypothesized that some of the deleterious effects of ethanol in the developing brain may be due to the disruption of cholesterol homeostasis in astrocytes. This study investigates the effect of ethanol on cholesterol efflux mediated by ATP-binding cassette (ABC) cholesterol transporters. In fetal rat astrocytes in culture, ethanol caused a concentration-dependent increase in cholesterol efflux and increased the levels of ABCA1 starting at 25 mm. Similar effects of ethanol on cholesterol efflux and ABCA1 were also observed in fetal human astrocytes. In addition, ABCA1 levels were increased in the brains of 7-day-old pups treated for 3 days with 2, 4, or 6 g/kg ethanol. Ethanol also increased apoE release from fetal rat astrocytes, and conditioned medium prepared from ethanol-treated astrocytes extracted more cholesterol than conditioned medium from untreated cells. In addition, ethanol increased the levels of another cholesterol transporter, ABCG1. Ethanol did not affect cholesterol synthesis and reduced the levels of intracellular cholesterol in rat astrocytes. Retinoic acid, which induces teratogenic effects similarly to ethanol, also caused up-regulation of ABCA1 and ABCG1.     

Differential Generation of High-Density Lipoprotein by Endogenous and Exogenous Apolipoproteins in Cultured Fetal Rat Astrocytes

Most peripheral cells generate cholesterol-rich high-density lipoprotein (HDL) with exogenous apolipoprotein as one of the mechanisms for the maintenance of cellular cholesterol homeostasis. Astrocytes isolated from fetal rat brain showed a unique behavior in this reaction. Consistent with previous findings, the astrocytes synthesized apolipoprotein (apo) E and generated cholesterol-rich pre-beta-HDL-like lipoprotein with this apoE, and cellular cholesterol and phospholipids. When exogenous apoA-I and E were added to the medium, they caused generation of additional HDL with cellular phospholipid. It is interesting that this additional part was very poor in cholesterol except for the generation of relatively cholesterol-rich HDL only in the initial few hours of the incubation. The mobilization of intracellular cholesterol for this reaction was also very limited, reflecting the poor cholesterol incorporation into the HDL. Thus, the results demonstrated a unique profile of HDL generation and cholesterol efflux by apolipoproteins in rat astrocytes, with endogenous apoE producing cholesterol-rich HDL and exogenous apolipoproteins producing cholesterol-poor HDL. These lipoproteins may play differential roles in cholesterol transport in the CNS.     

ATP-binding cassette transporter A7 regulates processing of amyloid precursor protein in vitro

ATP-binding cassette transporter A7 (ABCA7) is expressed in the brain and, like its closest homolog ABCA1, belongs to the ABCA subfamily of full-length ABC transporters. ABCA1 promotes cellular cholesterol efflux to lipid-free apolipoprotein acceptors and also inhibits the production of neurotoxic β-amyloid (Aβ) peptides in vitro . The potential functions of ABCA7 in the brain are unknown. This study investigated the ability of ABCA7 to regulate cholesterol efflux to extracellular apolipoprotein acceptors and to modulate Aβ production. The transient expression of ABCA7 in human embryonic kidney cells significantly stimulated cholesterol efflux (fourfold) to apolipoprotein E (apoE) discoidal lipid complexes but not to lipid-free apoE or apoA-I. ABCA7 also significantly inhibited Aβ secretion from Chinese hamster ovary cells stably expressing human amyloid precursor protein (APP) or APP containing the Swedish K670M671→N670L671 mutations when compared with mock-transfected cells. Studies with fluorogenic substrates indicated that ABCA7 had no impact on α-, β-, or γ-secretase activities. Live cell imaging of Chinese hamster ovary cells expressing APP-GFP indicated an apparent retention of APP in a perinuclear location in ABCA7 co-transfected cells. These studies indicate that ABCA7 has the capacity to stimulate cellular cholesterol efflux to apoE discs and regulate APP processing resulting in an inhibition of Aβ production.     

Apolipoprotein A-I-stimulated apolipoprotein E secretion from human macrophages is independent of cholesterol efflux

Apolipoprotein A-I (apoA-I)-mediated cholesterol efflux involves the binding of apoA-I to the plasma membrane via its C terminus and requires cellular ATP-binding cassette transporter (ABCA1) activity. ApoA-I also stimulates secretion of apolipoprotein E (apoE) from macrophage foam cells, although the mechanism of this process is not understood. In this study, we demonstrate that apoA-I stimulates secretion of apoE independently of both ABCA1-mediated cholesterol efflux and of lipid binding by its C terminus. Pulse-chase experiments using (35)S-labeled cellular apoE demonstrate that macrophage apoE exists in both relatively mobile (E(m)) and stable (E(s)) pools, that apoA-I diverts apoE from degradation to secretion, and that only a small proportion of apoA-I-mobilized apoE is derived from the cell surface. The structural requirements for induction of apoE secretion and cholesterol efflux are clearly dissociated, as C-terminal deletions in recombinant apoA-I reduce cholesterol efflux but increase apoE secretion, and deletion of central helices 5 and 6 decreases apoE secretion without perturbing cholesterol efflux. Moreover, a range of 11- and 22-mer alpha-helical peptides representing amphipathic alpha-helical segments of apoA-I stimulate apoE secretion whereas only the C-terminal alpha-helix (domains 220-241) stimulates cholesterol efflux. Other alpha-helix-containing apolipoproteins (apoA-II, apoA-IV, apoE2, apoE3, apoE4) also stimulate apoE secretion, implying a positive feedback autocrine loop for apoE secretion, although apoE4 is less effective. Finally, apoA-I stimulates apoE secretion normally from macrophages of two unrelated subjects with genetically confirmed Tangier Disease (mutations C733R and c.5220-5222delTCT; and mutations A1046D and c.4629-4630insA), despite severely inhibited cholesterol efflux. We conclude that apoA-I stimulates secretion of apoE independently of cholesterol efflux, and that this represents a novel, ABCA-1-independent, positive feedback pathway for stimulation of potentially anti-atherogenic apoE secretion by alpha-helix-containing molecules including apoA-I and apoE.     

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.     

The role of the ABCA1 transporter and cholesterol efflux in familial hypoalphalipoproteinemia

Defects in the gene encoding for the ATP binding cassette (ABC) transporter A1 (ABCA1) were shown to be one of the genetic causes for familial hypoalphalipoproteinemia (FHA). We investigated the role of ABCA1-mediated cholesterol efflux in Dutch subjects suffering from FHA. Eighty-eight subjects (mean HDL cholesterol levels 0.63 +/- 0.21 mmol/l) were enrolled. Fibroblasts were cultured and loaded with [3H]cholesterol. ABCA1 and non-ABCA1-mediated efflux was studied by using apolipoprotein A-I (apoA-I), HDL, and methyl-beta-cyclodextrin as acceptors. Efflux to apoA-I was decreased in four patients (4/88, 4.5%), and in all cases, a mutation in the ABCA1 gene was found. In the remaining 84 subjects, no correlation between efflux and apoA-I or HDL cholesterol was found. Efflux to both HDL and cyclodextrin, in contrast, did correlate with HDL cholesterol plasma levels (r = 0.34, P = 0.01; and r = 0.27, P = 0.008, respectively). The prevalence of defects in ABCA1-dependent cholesterol efflux in Dutch FHA patients is low. The significant correlation between plasma HDL cholesterol levels and methyl-beta-cyclodextrin-mediated efflux in the FHA patients with normal ABCA1 function suggests that non-ABCA1-mediated efflux might also be important for plasma HDL cholesterol levels in these individuals.     

Differential effects of HDL subpopulations on cellular ABCA1- and SR-BI-mediated cholesterol efflux

Our objective was to evaluate the associations of individual apolipoprotein A-I (apoA-I)-containing HDL subpopulation levels with ABCA1- and scavenger receptor class B type I (SR-BI)-mediated cellular cholesterol efflux. HDL subpopulations were measured by nondenaturing two-dimensional gel electrophoresis from 105 male subjects selected with various levels of apoA-I in pre-beta-1, alpha-1, and alpha-3 HDL particles. ApoB-containing lipoprotein-depleted serum was incubated with [(3)H]cholesterol-labeled cells to measure efflux. The difference in efflux between control and ABCA1-upregulated J774 macrophages was taken as a measure of ABCA1-mediated efflux. SR-BI-mediated efflux was determined using cholesterol-labeled Fu5AH hepatoma cells. Fractional efflux values obtained from these two cell systems were correlated with the levels of individual HDL subpopulations. A multivariate analysis showed that two HDL subspecies correlated significantly with ABCA1-mediated efflux: small, lipid-poor pre-beta-1 particles (P=0.0022) and intermediate-sized alpha-2 particles (P=0.0477). With regard to SR-BI-mediated efflux, multivariate analysis revealed significant correlations with alpha-2 (P=0.0004), alpha-1 (P=0.0030), pre-beta-1 (P=0.0056), and alpha-3 (P=0.0127) HDL particles. These data demonstrate that the small, lipid-poor pre-beta-1 HDL has the strongest association with ABCA1-mediated cholesterol even in the presence of all other HDL subpopulations. Cholesterol efflux via the SR-BI pathway is associated with several HDL subpopulations with different apolipoprotein composition, lipid content, and size.     

The C-terminal lipid-binding domain of apolipoprotein E is a highly efficient mediator of ABCA1-dependent cholesterol efflux that promotes the assembly of high-density lipoproteins

This study was undertaken to identify the alpha-helical domains of human apoE that mediate cellular cholesterol efflux and HDL assembly via ATP-binding cassette transporter A1 (ABCA1). The C-terminal (CT) domain (residues 222-299) of apoE was found to stimulate ABCA1-dependent cholesterol efflux in a manner similar to that of intact apoE2, -E3, and -E4 in studies using J774 macrophages and HeLa cells. The N-terminal (NT) four-helix bundle domain (residues 1-191) was a relatively poor mediator of cholesterol efflux. On a per molecule basis, the CT domain stimulated cholesterol efflux with the same efficiency (Km approximately 0.2 microM) as intact apoA-I and apoE. Gel filtration chromatography of conditioned medium from ABCA1-expressing J774 cells revealed that, like the intact apoE isoforms, the CT domain promoted the assembly of HDL particles with diameters of 8 and 13 nm. Removal of the CT domain abolished the formation of HDL-sized particles, and only larger particles eluting in the void volume were formed. Studies with CT truncation mutants of apoE3 and peptides indicated that hydrophobic helical segments governed the efficiency of cellular cholesterol efflux and that conjoined class A and G amphipathic alpha-helices were required for optimal efflux activity. Collectively, the data suggest that the CT lipid-binding domain of apoE encompassing amino acids 222-299 is necessary and sufficient for mediating ABCA1 lipid efflux and HDL particle assembly.     

Apolipoprotein A-I induces translocation of cholesterol, phospholipid, and caveolin-1 to cytosol in rat astrocytes.

Intercellular cholesterol transport in the brain is carried by high density lipoprotein (HDL) generated in situ by cellular interaction with the apolipoprotein apoE, which is mainly synthesized by astrocytes, and with apoA-I secreted by cells such as endothelial cells. Rat astrocytes in fact generate HDL with extracellular apoA-I in addition to releasing HDL with endogenously synthesized apoE, seemingly by the same mechanism as the HDL assembly for systemic circulation. Relating to this reaction, apoA-I induced translocation of newly synthesized cholesterol and phospholipid to the cytosol prior to extracellular assembly of HDL, accompanied by an increase of caveolin-1 in the cytosol, activation of sterol regulatory element-binding protein, and enhancement of cholesterol synthesis. The lipid translocated into the cytosol was recovered in the fraction with a density of 1.09-1.16 g/ml as well as caveolin-1 and cyclophilin A. Cyclosporin A inhibited these apoA-I-mediated reactions and suppressed apoA-I-mediated cholesterol release. The findings suggest that such translocation of cholesterol and phospholipid into the cytosol is related to the apo A-I-mediated HDL assembly in astrocytes through functional association with caveolin-1 and a cyclosporin A-sensitive cyclophilin protein(s).     

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.     

ABCA1 promotes the de novo biogenesis of apolipoprotein CIII-containing HDL particles in vivo and modulates the severity of apolipoprotein CIII-induced hypertriglyceridemia

In this study, the ability of the lipid transporter ABCA1 and apolipoprotein CIII (apoCIII) to promote the de novo biogenesis of apoCIII-containing HDL in vivo and the role of this HDL in apoCIII-induced hypertriglyceridemia were investigated, using adenovirus-mediated gene transfer in apoE (-/-) x apoA-I (-/-) mice or ABCA1 (-/-) mice. Injection of apoE (-/-) x apoA-I (-/-) mice with 8 x 10 (8) pfu of an adenovirus expressing the wild-type human apoCIII (AdGFP-CIII g) generated HDL-like particles and triggered only a modest increase in plasma cholesterol and triglyceride levels of these mice, 3-5 days postinfection. Plasma human apoCIII was distributed among HDL, VLDL/IDL, and LDL in these mice. In contrast, ABCA1 (-/-) mice treated similarly failed to form HDL particles and developed severe hypertriglyceridemia which could be alleviated by coinfection with an adenovirus expressing human LpL, while their plasma cholesterol levels remained unchanged 3-5 days postinfection with AdGFP-CIII g. Human apoCIII in these mice accumulated exclusively on VLDL. Control experiments confirmed that the differences between apoE (-/-) x apoA-I (-/-) and ABCA1 (-/-) mice expressing human apoCIII were not due to differences in apoCIII expression. Overall, these data show that ABCA1 and human apoCIII promote the formation of apoCIII-containing HDL-like particles that are distinct from classical apoE- or apoA-I-containing HDL. Formation of apoCIII-containing HDL prevents excess accumulation of plasma apoCIII on VLDL and allows for the efficient lipolysis of VLDL triglycerides by LpL. Furthermore, the data establish that ABCA1 and apoCIII-containing HDL play key roles in the prevention of apoCIII-induced hypertriglyceridemia in mice.     

ABCA1-dependent lipid efflux to apolipoprotein A-I mediates HDL particle formation and decreases VLDL secretion from murine hepatocytes

High levels of expression of the ATP binding cassette transporter A1 (ABCA1) in the liver and the need to over- or underexpress hepatic ABCA1 to impact plasma HDL levels in mice suggest a major role of the liver in HDL formation and in determining circulating HDL levels. Cultured murine hepatocytes were used to examine the role of hepatic ABCA1 in mediating the lipidation of apolipoprotein A-I (apoA-I) for HDL particle formation. Exogenous apoA-I stimulated cholesterol efflux to the medium from wild-type hepatocytes, but not from ABCA1-deficient (abca1(-/-)) hepatocytes. ApoA-I induced the formation of new HDL particles and enhanced the lipidation of endogenously secreted murine apoA-I in ABCA1-expressing but not abca1(-/-) hepatocytes. ABCA1-dependent cholesterol mobilization to apoA-I increased new cholesterol synthesis, indicating depletion of the regulatory pool of hepatocyte cholesterol during HDL formation. Secretion of triacylglycerol and apoB was decreased following apoA-I incubation with ABCA1-expressing but not abca1(-/-) hepatocytes. These results support a major role for hepatocyte ABCA1 in generating a critical pool of HDL precursor particles that enhance further HDL generation and passive cholesterol mobilization in the periphery. The results also suggest that diversion of hepatocyte cholesterol into the "reverse" cholesterol transport pathway diminishes cholesterol availability for apoB-containing lipoprotein secretion by the liver.     

Cholesterol homeostasis in rat astrocytoma cells GA-1

Astrocytes play a key role in cholesterol metabolism in central nervous system. We have shown that fetal rat astrocytes in primary culture secrete cholesterol-rich HDL with the endogenous apolipoprotein (apo) E and generate cholesterol-poor HDL with exogenous apoE and apoA-I [Ito et al. (1999) J. Neurochem. 72, 2362]. In order to study these reactions in relation to the stage of cell differentiation, we examined generation of HDL by rat astrocytoma cells. Lack of apoE secretion was found in three astrocytoma cell lines, human T98G, rat C6, and GA-1 [Kano-Tanaka et al. (1986) Proc. Jpn. Acad. Ser. B 62, 109]. GA-1 produced apoE at very low level and therefore generated much less HDL by itself than the astrocytes in primary culture. In contrast, GA-1 interacted with exogenous apoE and apoA-I to produce cholesterol-rich HDL while the astrocytes produced cholesterol-poor HDL with these apolipoproteins. Cholesterol biosynthesis rate measured from mevalonate was higher and down-regulated more by LDL in the astrocytes than GA-1. On the other hand, the cellular cholesterol level, uptake of LDL, and cyclodextrin-mediated non-specific diffusion of cholesterol from cell surface were same between these two cells. Treatment of GA-1 with acidic fibroblast growth factor influenced neither the production of apoE nor the baseline lipid secretion, but increased the cholesterol synthesis from mevalonate and the magnitude of its down-regulation by LDL, and decreased cholesterol content in the HDL produced by exogenous apoA-I. In conclusion, suppression of apoE biosynthesis in the undifferentiated astrocytes GA-1 resulted in poor secretion of cholesterol-rich HDL and in turn more production of HDL with exogenous apolipoprotein. Cellular cholesterol homeostasis was altered accordingly.