Apolipoprotein A-I in mouse cerebrospinal fluid derives from the liver and intestine via plasma high-density lipoproteins assembled by ABCA1 and LCAT.

Apolipoprotein (apo) A-I, the major structural protein of high-density lipoprotein (HDL), is present in human and mouse cerebrospinal fluid (CSF) despite its lack of expression in brain cells. To identify the origin of apoA-I in CSF, we generated intestine-specific and liver-specific Apoa1 knockout mice (Apoa1ΔInt and Apoa1Δliv mice, respectively). Lipoprotein profiles of Apoa1ΔInt and Apoa1ΔLiv mice resembled those of control littermates, whereas knockout of Apoa1 in both intestine and liver (Apoa1ΔIntΔLiv ) resulted in a 60-percent decrease in HDL-cholesterol levels, thus strongly mimicking the Apoa1-/- mice. Immunoassays revealed that mouse apoA-I was not present in the CSF of the Apoa1ΔIntΔLiv mice. Furthermore, apoA-I levels in CSF were highly correlated with plasma spherical HDL levels, which were regulated by ABCA1 and LCAT. Collectively, these results suggest that apoA-I protein in CSF originates in liver and small intestine and is taken up from the plasma.

The RXR agonists PA024 and HX630 have different abilities to activate LXR/RXR and to induce ABCA1 expression in macrophage cell lines.

Release of cellular cholesterol by ATP-binding cassette transporter (ABC)A1 and apolipoproteins is a major source of plasma high-density lipoprotein (HDL). Expression of ABC transporter A1 (ABCA1) is directly stimulated by liver X receptor (LXR)/retinoid X receptor (RXR) activation. We evaluated the abilities of two RXR agonists, PA024 and HX630, to increase ABCA1 expression. In differentiated THP-1 cells, the two agonists efficiently enhanced ABCA1 mRNA expression and apoA-I-dependent cellular cholesterol release. However, in RAW264 cells and undifferentiated THP-1 cells, PA024 was highly effective while HX630 was inactive in increasing ABCA1 mRNA. In parallel, the two agonists had different abilities to activate ABCA1 promoter in an LXR-responsive-element (LXRE)-dependent manner and to directly stimulate LXRalpha/RXR transactivation. The ability of HX630 to enhance ABCA1 expression was correlated closely with the cellular PPARgamma mRNA level. Moreover, HX630 was able to activate PPARgamma/RXR. Transfection of PPARgamma in RAW264 cells induced HX630-mediated activation of LXRE-dependent transcription and ABCA1 promoter, suggesting the ability of HX630 to activate PPARgamma-LXR-ABCA1 pathway. We conclude that RXR agonist PA024 and HX630 have different abilities to activate LXR/RXR, and that the cell-type-dependent effect of HX630 on ABCA1 expression and HDL generation is closely associated with this defect.

Sterol regulatory element-binding protein-2- and liver X receptor-driven dual promoter regulation of hepatic ABC transporter A1 gene expression: mechanism underlying the unique response to cellular cholesterol status.

ABC transporter A1 (ABCA1) mediates and rate-limits biogenesis of high density lipoprotein (HDL), and hepatic ABCA1 plays a major role in regulating plasma HDL levels. HDL generation is also responsible for release of cellular cholesterol. In peripheral cells ABCA1 is up-regulated by the liver X receptor (LXR) system when cell cholesterol increases. However, cholesterol feeding has failed to show a significant increase in hepatic ABCA1 gene expression, and its expression is up-regulated by statins (3-hydroy-3-methylglutaryl-CoA reductase inhibitors), suggesting distinct regulation. In this study we investigated the mechanism of regulation of the rat hepatic ABCA1 gene and identified two major ABCA1 transcripts and two corresponding promoter regions. Compactin activated the novel liver-type promoter in rat hepatoma McARH7777 cells by binding the sterol regulatory element-binding protein-2 (SREBP-2). In contrast, compactin repressed the previously identified peripheral-type promoter in an LXR-responsive element-dependent but not E-box-dependent manner. Thus, compactin increased the liver-type transcript and decreased the peripheral-type transcript. The same two transcripts were also dominant in human and mouse livers, whereas the intestine contains only the peripheral-type transcript. Treatment of rats with pravastatin and a bile acid binding resin (colestimide), which is known to activate SREBP-2 in the liver, caused a reduction in the hepatic cholesterol level and the same differential responses in vivo, leading to increases in hepatic ABCA1 mRNA and protein and plasma HDL levels. We conclude that the dual promoter system driven by SREBP-2 and LXR regulates hepatic ABCA1 expression and may mediate the unique response of hepatic ABCA1 gene expression to cellular cholesterol status.

Genetic etiology of isolated low HDL syndrome: incidence and heterogeneity of efflux defects.

OBJECTIVE: We have used a multitiered approach to identify genetic and cellular contributors to high-density lipoprotein (HDL) deficiency in 124 human subjects. METHODS AND RESULTS: We resequenced 4 candidate genes for HDL regulation and identified several functional nonsynonymous mutations including 2 in apolipoprotein A-I (APOA1), 4 in lecithin:cholesterol acyltransferase (LCAT), 1 in phospholipid transfer protein (PLTP), and 7 in the ATP-binding cassette transporter ABCA1, leaving 88% (110/124) of HDL deficient subjects without a genetic diagnosis. Cholesterol efflux assays performed using cholesterol-loaded monocyte-derived macrophages from the 124 low HDL subjects and 48 control subjects revealed that 33% (41/124) of low HDL subjects had low efflux, despite the fact that the majority of these subjects (34/41) were not carriers of dysfunctional ABCA1 alleles. In contrast, only 2% of control subjects presented with low efflux (1/48). In 3 families without ABCA1 mutations, efflux defects were found to cosegregate with low HDL. CONCLUSIONS: Efflux defects are frequent in low HDL syndromes, but the majority of HDL deficient subjects with cellular cholesterol efflux defects do not harbor ABCA1 mutations, suggesting that novel pathways contribute to this phenotype.

Purification of ATP-binding cassette transporter A1 and associated binding proteins reveals the importance of beta1-syntrophin in cholesterol efflux.

ATP-binding cassette transporter A1 (ABCA1) plays a critical role in HDL cholesterol metabolism, but the mechanism by which it transports lipid across membranes is poorly understood. Because growing evidence implicates accessory proteins in this process, we developed a method by which proteins interacting with the intact transporter could be identified. cDNAs encoding wild-type ABCA1 and a mutant lacking the C-terminal PDZ binding motif of ABCA1 were transfected into 293 cells, and the expressed proteins were solubilized using detergent conditions (0.75% CHAPS, 1 mg/ml phosphatidylcholine) predicted to retain high affinity protein-protein interactions. Proteins that co-purified with ABCA1 on an antibody affinity column were identified by liquid chromatographymass spectrometric analysis. A novel interaction with the PDZ protein beta1-syntrophin was identified using this approach, and this interaction was confirmed in human THP-1 macrophages and in mouse liver. Small interference RNA inhibition of beta1-syntrophin expression reduced cholesterol efflux from primary skin fibroblasts by 50% while decreasing efflux 30% in bone marrow-derived macrophages. Inhibition of beta1-syntrophin decreased ABCA1 protein levels, whereas overexpression of beta1-syntrophin increased ABCA1 cell-surface expression and stimulated efflux to apolipoprotein A-I. These findings indicate that beta1-syntrophin acts through a class-I PDZ interaction with the C terminus of ABCA1 to regulate the cellular distribution and activity of the transporter. The approach used to identify beta1-syntrophin as an ABCA1-binding protein should prove useful in elucidating other protein interactions upon which ABCA1 function depends.

Abca7 null mice retain normal macrophage phosphatidylcholine and cholesterol efflux activity despite alterations in adipose mass and serum cholesterol levels.

Mutations in the A class of ATP-binding cassette transporters (ABCA) are causally implicated in three human diseases: Tangier disease (ABCA1), Stargadt's macular degeneration (ABCA4), and neonatal respiratory failure (ABCA3). Both ABCA1 and ABCA4 have been shown to transport lipids across cellular membranes, and ABCA3 may play a similar role in transporting pulmonary surfactant. Although the functions of the other 10 ABCA class transporters identified in the human genome remain obscure, ABCA7-transfected cells have been shown to efflux lipids in response to stimulation by apolipoprotein A-I. In an effort to elucidate the physiologic role of ABCA7, we generated mice lacking this transporter (Abca7-/- mice). Homozygous null mice were produced from intercrosses of heterozygous null mice at the expected Mendelian frequency and developed normally without any obvious phenotypic abnormalities. Cholesterol and phospholipid efflux stimulated by apolipoprotein A-I from macrophages isolated from wild type and Abca7-/- mice did not differ, suggesting that these activities may not be central to the physiological role of the transporter in vivo. Abca7-/- females, but not males, had significantly less visceral fat and lower total serum and high density lipoprotein cholesterol levels than wild type, gender-matched littermates. ABCA7 expression was detected in hippocampal and cortical neurons by in situ hybridization and in brain and white adipose tissue by Western blotting. Induction of adipocyte differentiation from 3T3 fibroblasts in culture led to a marked increase in ABCA7 expression. These studies suggest that ABCA7 plays a novel role in lipid and fat metabolism that Abca7-/- mice can be used to elucidate.

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.

Potential involvement of dissociated apoA-I in the ABCA1-dependent cellular lipid release by HDL.

Helical apolipoproteins of high density lipoprotein (HDL) remove phospholipid and cholesterol from cells and generate HDL particles being mediated by ATP binding cassette transporter A1 (ABCA1). In murine macrophage cell line RAW264 cells, cAMP induced expression of ABCA1, release of cellular phospholipid and cholesterol by apolipoprotein A-I (apoA-I), and reversible binding of apoA-I to the cell. The apoA-I-dependent lipid release was directly proportional to the cAMP-induced binding of apoA-I, and was inhibited 70% by a monoclonal antibody selective to lipid-free apoA-I, 725-1E2. In contrast, apparent cellular cholesterol release to HDL was substantial even without ABCA1 induction, and it was increased only by 27% after the cAMP treatment. The antibody inhibited this increment by 70%. Lipid-free apoA-II liberated apoA-I from HDL by displacement and thereby markedly expanded the cAMP-induced part of the cholesterol release to the HDL-containing medium, and the antibody inhibited this part also by 70%. Binding experiments of the double-labeled reconstituted HDL showed that cAMP induced reversible binding of apoA-I but not the association of cholesteryl ester with the cells. The effect of the antibody on the cellular cholesterol release to the reconstituted HDL was similar to that of the HDL-mediated release. The data implicated that the ABCA1-dependent cholesterol release to HDL is mediated by apoA-I dissociated from HDL.

Human ABCA7 supports apolipoprotein-mediated release of cellular cholesterol and phospholipid to generate high density lipoprotein.

Apolipoprotein-mediated release of cellular cholesterol and phospholipids was induced in HEK293 cells by expressing human ATP-binding cassette transporter A7 (ABCA7) and ABC transporter A1 (ABCA1) proteins, whether transient or stable, to generate cholesterol-rich high density lipoprotein (HDL). Green fluorescent protein (GFP) attached at their C termini did not influence the lipid release reactions. Transfected ABCA7-GFP induced apolipoprotein-mediated assembly of cholesterol-containing HDL also in L929 cells, which otherwise generate only cholesterol-deficient HDL with their endogenous ABCA1. Time-dependent release of cholesterol and phospholipid by apolipoprotein A (apoA)-I was parallel both with ABCA1 and with ABCA7 when highly expressed in HEK293 cells, but dose-dependent profiles of lipid release on apoA-I and apoA-II were somewhat different between ABCA1 and ABCA7. Analyses of the stable clones with ABCA1-GFP (293/2c) and ABCA7-GFP (293/6c) by using the same vector indicated some differences in regulation of their activities by protein kinase modulators. Dibutyryl cyclic AMP increased ABCA1-GFP and the release of cholesterol and phospholipid in 293/2c but increased neither ABCA7-GFP nor the lipid release in 293/6c. Expression of ABCA1-GFP- and apoA-I-mediated lipid release were enhanced in parallel by phorbol 12-myristate 13-acetate (PMA) in 293/2c cells. In contrast, the same treatment of 293/6c increased ABCA7-GFP, but apoA-I-mediated lipid release was significantly suppressed. Despite these different responses to PMA, all of the effects of PMA were reversed by a specific protein kinase C inhibitor Gö6976, suggesting that the changes were in fact due to protein kinase C activation. A thiol protease inhibitor, N-acetyl-Leu-Leu-norleucinal, increased the protein levels of ABCA1-GFP in 293/2c and ABCA7-GFP in 293/6c, indicating their common degradation pathway. The data indicated that human ABCA7 would compensate the function of ABCA1 for release of cell cholesterol in a certain condition(s), but post-transcriptional regulation of their activity is different.

Effects of mutations of ABCA1 in the first extracellular domain on subcellular trafficking and ATP binding/hydrolysis.

ABCA1 mediates release of cellular cholesterol and phospholipid to form high density lipoprotein (HDL). The three different mutants in the first extracellular domain of human ABCA1 associated with Tangier disease, R587W, W590S, and Q597R, were examined for their subcellular localization and function by using ABCA1-GFP fusion protein stably expressed in HEK293 cells. ABCA1-GFP expressed in HEK293 was fully functional for apoA-I-mediated HDL assembly. Immunostaining and confocal microscopic analyses demonstrated that ABCA1-GFP was mainly localized to the plasma membrane (PM) but also substantially in intracellular compartments. All three mutant ABCA1-GFPs showed no or little apoA-I-mediated HDL assembly. R587W and Q597R were associated with impaired processing of oligosaccharide from high mannose type to complex type and failed to be localized to the PM, whereas W590S did not show such dysfunctions. Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the dysfunction in the W590S mutant. Photoaffinity labeling of W590S with 8-azido-[alpha-(32)P]ATP was stimulated by adding ortho-vanadate in the presence of Mn(2+) as much as in the presence of wild-type ABCA1. These results suggest that the defect of HDL assembly in R587W and Q597R is due to the impaired localization to the PM, whereas W590S has a functional defect other than the initial ATP binding and hydrolysis.