Molecular structure of a novel cholesterol-responsive A subclass ABC transporter,ABCA9

We recently identified a novel ABC A subclass transporter, ABCA6, in human macrophages. Here, we report the molecular cloning of an additional ABC A subfamily transporter from macrophages denoted ABCA9. The identified coding sequence is 4.9 kb in size and codes for a 1624 amino acid protein product. In accordance with the proposed nomenclature, the novel transporter was designated ABCA9. The putative full-length ABC transporter polypeptide consists of two transmembrane domains and two nucleotide binding folds and thus conforms to the group of full-size ABC transporters. We identified alternative ABCA9 mRNA variants in human macrophages that predict the existence of three truncated forms of the novel transporter. Among the human ABC A subfamily transporters, ABCA9 exhibits the highest amino acid sequence homology with ABCA8 (72%) and ABCA6 (60%), respectively. The striking amino acid sequence similarity between these transporter molecules supports the notion that they represent an evolutionary more recently emerged subgroup within the family of ABC A transporters, which we refer to as "ABCA6-like transporters." ABCA9 mRNA is ubiquitously expressed with the highest mRNA levels in heart, brain, and fetal tissues. Analysis of the genomic structure revealed that the ABCA9 gene consists of 39 exons that are located within a genomic region of approximately 85 kb size on chromosome 17q24.2. In human macrophages, ABCA9 mRNA is induced during monocyte differentiation into macrophages and suppressed by cholesterol import indicating that ABCA9, like other known ABC A subfamily transporters, is a cholesterol-responsive gene. Based on this information, ABCA9 is likely involved in monocyte differentiation and macrophage lipid homeostasis.     

Cloning of human and rat ABCA5/Abca5 and detection of a human splice variant

We presently report the cloning of cDNA sequences encoding the novel rat ATP-binding cassette (ABC) transporter Abca5 and the orthologous human transporter, recently designated as ABCA5. Furthermore, the existence of a novel non-translated exon of the ABCA5 gene, previously assigned to an ABCA gene cluster in the chromosomal region 17q24.2-3, is demonstrated. Abca5 and ABCA5 cDNAs are predicted to give rise to proteins of 1642 amino acids which exhibit the typical domain arrangement of ABC full transporters and share 90% identity within the amino acid sequences. A cDNA representing an ABCA5 mRNA splice variant was cloned which would result in a truncated protein equivalent to an ABC half transporter. Northern blot analyses revealed expression of ABCA5 or Abca5 mRNA in several tissues, but particularly high Abca5 mRNA expression was observed in rat testis. Up-regulation of Abca5 mRNA expression during culture of primary rat hepatocytes suggests that hepatocyte cultures should provide a basis for investigation of Abca5 gene regulation and elucidation of Abca5 function.     

ATP binding cassette transporter ABCA1 modulates the secretion of apolipoprotein E from human monocyte-derived macrophages.

Apolipoprotein E (apoE) produced by macrophages in the arterial wall protects against atherosclerosis, but the regulation of its secretion by these cells is poorly understood. Here we investigated the contribution of the adenosine triphosphate binding cassette transporters ABCA1 and ABC8 to the secretion of apoE from either primary human monocyte-derived macrophages (HMDM) or human THP1 macrophages. During incubations of up to 6 h, apoE secretion from both THP1 macrophages and HMDM was stimulated by 8-Br-cAMP, which activates ABCA1 expression. The putative ABCA1 inhibitor glyburide and antisense oligonucleotides directed against ABCA1 mRNA significantly reduced apoE secretion from THP1 macrophages and HMDM. Antisense oligonucleotides directed against ABC8 mRNA also inhibited apoE secretion, although this inhibition was less pronounced and consistent than in the case of ABCA1. ApoE secretion from HMDM of ABCA1-deficient patients with Tangier disease was also decreased. ApoE mRNA expression was not affected by inhibition of ABCA1 or ABC8 in normal HMDM or the lack of functional ABCA1 in HMDM from Tangier disease patients. Inhibition of ABCA1 in HMDM prevented the occurrence of anti-apoE-immunoreactive granular structures in the plasma membrane. We conclude that ABCA1 and, to a lesser extent, ABC8 both promote secretion of apoE from human macrophages.     

Molecular cloning of the human ATP-binding cassette transporter 1 (hABC1): evidence for sterol-dependent regulation in macrophages

We have cloned the full-length cDNA for the human ATP binding cassette transporter 1 (hABC1). The 6603-bp open reading frame encodes a polypeptide of 2201 amino acids resulting in a deduced molecular weight of 220 kDa. The hABC1 cDNA is highly homologous (62%) to the human rim ABC transporter (ABCR). hABC1 is expressed in a variety of human tissues with highest expression levels found in placenta, liver, lung, adrenal glands, and fetal tissues. We demonstrate that the hABC1 expression is induced during differentiation of human monocytes into macrophages in vitro. In macrophages, both the hABC1 mRNA and protein expression are upregulated in the presence of acetylated low-density lipoprotein (AcLDL). The AcLDL-induced increase in hABC1 expression is reversed by cholesterol depletion mediated by the addition of high-density lipoprotein (HDL3). Our data, demonstrating sterol-dependent regulation of hABC1 in human monocytes/macrophages, suggest a novel role for this transporter molecule in membrane lipid transport.     

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.     

Phospholipase A2-Modified Low-Density Lipoprotein Activates Liver X Receptor in Human Macrophages

Macrophages respond to cholesterol accumulation by increasing cholesterol efflux, which is mediated by activation of the nuclear liver X receptor (LXR) and ATP binding cassette (ABC) transporters. In the present study, we investigated whether foam cell formation induced by phospholipase A(2)-modified low-density lipoprotein (PLA-LDL) influences LXR activity and cholesterol efflux in primary human monocyte-derived macrophages (MDMs). Macrophages were treated with PLA-LDL and expression of the LXR target genes ABCA1 and ABCG1 was analyzed by quantitative PCR and western blot. PLA-LDL time-dependently up-regulated ABCA1 and ABCG1 mRNA and protein. Removal of non-esterified fatty acids from PLA-LDL particles did not influence the induction of ABC transporters. A role of LXR in PLA-LDL-stimulated ABCG1 expression was verified by LXR-knockdown and luciferase reporter assays using a construct containing a LXR response element from the ABCG1 gene. Functionally, cholesterol efflux to apolipoprotein A-I and high-density lipoprotein was higher in PLA-LDL treated cells compared to controls. Together, these results demonstrate that in primary human MDMs PLA-LDL induces ABC transporter expression via LXR activation. A concomitantly increased cholesterol efflux may prevent excessive cholesterol accumulation and thus, attenuate foam cell formation.     

ABC transporters in cellular lipid trafficking

ATP-binding cassette (ABC) transporters constitute a group of evolutionary highly conserved cellular transmembrane transport proteins. Recent work has implicated ABC transporters in cellular transmembrane lipid transport and hereditary diseases have been causatively linked to defective ABC transporters translocating lipid compounds. The emerging concept that a defined subset of ABC transporters is intimately involved in cellular lipid trafficking has recently been substantiated convincingly by the finding that ABCA1 plays a central role in the regulation of HDL metabolism and macrophage targeting to the RES or the vascular wall. Differentiation dependent expression of a large number of ABC transporters in monocytes/macrophages and their regulation by sterol flux render these transporter molecules potentially critical players in atherogenesis and other chronic inflammatory diseases.     

ABCA1 is the cAMP-inducible apolipoprotein receptor that mediates cholesterol secretion from macrophages

Lipid-poor high density lipoprotein apolipoproteins remove cholesterol and phospholipids from cells by an active secretory pathway controlled by an ABC transporter called ABCA1. This pathway is induced by cholesterol and cAMP analogs in a cell-specific manner. Here we provide evidence that increased plasma membrane ABCA1 accounts for the enhanced apolipoprotein-mediated lipid secretion from macrophages induced by cAMP analogs. Treatment of RAW264 macrophages with 8-bromo-cAMP caused parallel increases in apoA-I-mediated cholesterol efflux, ABCA1 mRNA and protein levels, incorporation of ABCA1 into the plasma membrane, and binding of apoA-I to cell-surface ABCA1. All of these parameters declined to near base-line values within 6 h after removal of 8-bromo-cAMP, indicating that ABCA1 is highly unstable and is degraded rapidly in the absence of inducer. Thus, ABCA1 is likely to be the cAMP-inducible apolipoprotein receptor that promotes removal of cholesterol and phospholipids from macrophages.     

Functional analysis of ABCA8, a new drug transporter

We examined the transport capacity in Xenopus laevis oocytes of human EST KIAA0822/ABCA8, a member of the ABC superfamily. Substrates of ABCC2/MRP-2 such as [14C]estradiol-beta-glucuronide, taurocholate, and LTC4, and of organic anion transporter (OAT), such as para-aminohippuric acid, ochratoxin-A, were significantly accumulated while tetraethylammonium and doxorubicin were not. The transport of [14C]estradiol-beta-glucuronide was ATP-dependent and K(m) and V(max) values of 30.4microM and 66.9pmol/h/egg, respectively, were estimated. The transport of [14C]estradiol-beta-glucuronide was inhibited by substrates/inhibitors of ABCC2/MRP-2, but not by those of the organic cation transporter and multidrug resistance protein (MDR)-1. KIAA0822/ABCA8 possesses two ATP-binding sites and fourteen transmembrane domains. Northern blot analysis revealed expression in most organs, especially in heart, skeletal muscle, and liver. Thus, ABCA8 is a new member of the xenobiotic transporter ABC-subfamily.