Impaired development of atherosclerosis in Abcg1-/- Apoe-/- mice: identification of specific oxysterols that both accumulate in Abcg1-/- Apoe-/- tissues and induce apoptosis.

OBJECTIVE: To generate Abcg1(-/-) Apoe(-/-) mice to understand the mechanism and cell types involved in changes in atherosclerosis after loss of ABCG1. METHODS AND RESULTS: ABCG1 is highly expressed in macrophages and endothelial cells, 2 cell types that play important roles in the development of atherosclerosis. Abcg1(-/-) Apoe(-/-) and Apoe(-/-) mice and recipient Apoe(-/-) mice that had undergone transplantation with bone marrow from Apoe(-/-) or Abcg1(-/-) Apoe(-/-) mice were fed a Western diet for 12 or 16 weeks before quantification of atherosclerotic lesions. These studies demonstrated that loss of ABCG1 from all tissues, or from only hematopoietic cells, was associated with significantly smaller lesions that contained increased numbers of TUNEL- and cleaved caspase 3-positive apoptotic Abcg1(-/-) macrophages. We also identified specific oxysterols that accumulate in the brains and macrophages of the Abcg1(-/-) Apoe(-/-) mice. These oxysterols promoted apoptosis and altered the expression of proapoptotic genes when added to macrophages in vitro. CONCLUSIONS: Loss of ABCG1 from all tissues or from only hematopoietic cells results in smaller atherosclerotic lesions populated with increased apoptotic macrophages, by processes independent of ApoE. Specific oxysterols identified in tissues of Abcg1(-/-) Apoe(-/-) mice may be critical because they induce macrophage apoptosis and the expression of proapoptotic genes.

Differential expression and function of ABCG1 and ABCG4 during development and aging.

ABCG1 and ABCG4 are highly homologous members of the ATP binding cassette (ABC) transporter family that regulate cellular cholesterol homeostasis. In adult mice, ABCG1 is known to be expressed in numerous cell types and tissues, whereas ABCG4 expression is limited to the central nervous system (CNS). Here, we show significant differences in expression of these two transporters during development. Examination of beta-galactosidase-stained tissue sections from Abcg1(-/-)LacZ and Abcg4(-/-)LacZ knockin mice shows that ABCG4 is highly but transiently expressed both in hematopoietic cells and in enterocytes during development. In contrast, ABCG1 is expressed in macrophages and in endothelial cells of both embryonic and adult liver. We also show that ABCG1 and ABCG4 are both expressed as early as E12.5 in the embryonic eye and developing CNS. Loss of both ABCG1 and ABCG4 results in accumulation in the retina and/or brain of oxysterols, in altered expression of liver X receptor and sterol-regulatory element binding protein-2 target genes, and in a stress response gene. Finally, behavioral tests show that Abcg4(-/-) mice have a general deficit in associative fear memory. Together, these data indicate that loss of ABCG1 and/or ABCG4 from the CNS results in changes in metabolic pathways and in behavior.

Emerging new paradigms for ABCG transporters.

Every cell is separated from its external environment by a lipid membrane. Survival depends on the regulated and selective transport of nutrients, waste products and regulatory molecules across these membranes, a process that is often mediated by integral membrane proteins. The largest and most diverse of these membrane transport systems is the ATP binding cassette (ABC) family of membrane transport proteins. The ABC family is a large evolutionary conserved family of transmembrane proteins (>250 members) present in all phyla, from bacteria to Homo sapiens, which require energy in the form of ATP hydrolysis to transport substrates against concentration gradients. In prokaryotes the majority of ABC transporters are involved in the transport of nutrients and other macromolecules into the cell. In eukaryotes, with the exception of the cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7), ABC transporters mobilize substrates from the cytoplasm out of the cell or into specific intracellular organelles. This review focuses on the members of the ABCG subfamily of transporters, which are conserved through evolution in multiple taxa. As discussed below, these proteins participate in multiple cellular homeostatic processes, and functional mutations in some of them have clinical relevance in humans.

The ABCs of sterol transport.

Mammalian cells have developed various responses to minimize accumulation of unesterified cholesterol, as the latter can result in cell toxicity and death [reviewed in this edition by Björkhem (Björkhem, I. 2009. Are side-chain oxidized oxysterols regulators also in vivo? J. Lipid Res. In press)]. These responses include esterification to sequester excess sterol in intracellular lipid droplets, repression of both cholesterol synthesis and LDL receptor expression (thus reducing endocytosis of LDL), and induction of a panoply of genes that promote sterol efflux and affect lipid metabolism. The nuclear receptor liver-X-receptor (LXR) functions as a cellular "sterol sensor" and plays a critical role in these latter transcriptional changes [reviewed in this edition by Glass (Shibata, N., and Glass C, K. 2009. Regulation of macrophage function in inflammation and atherosclerosis. J. Lipid Res. In press)]. Activation of LXR by either endogenous oxysterols or synthetic agonists induces the expression of many genes, including those encoding ATP-binding cassette (ABC) transporters ABCA1, ABCG1, ABCG5, and ABCG8. As discussed below, these four proteins function to promote sterol efflux from cells.

Expression and regulation of multiple murine ATP-binding cassette transporter G1 mRNAs/isoforms that stimulate cellular cholesterol efflux to high density lipoprotein.

The murine Abcg1 gene is reported to consist of 15 exons that encode a single mRNA (herein referred to as Abcg1-a) and protein. We now demonstrate that (i) the murine gene contains two additional coding exons downstream of exon 1, (ii) transcription involves the use of multiple promoters, and (iii) the RNA undergoes alternative splicing reactions. As a result, three mRNAs are expressed that encode three putative protein isoforms that differ at their amino terminus. ABCG1 transcripts are induced in vivo in multiple tissues in response to the liver X receptor ligand T0901317. Identification and characterization of four liver X receptor response elements in the intron downstream of exon 2 provides a mechanism by which this induction occurs. Importantly, cholesterol efflux to high density lipoprotein was stimulated following transfection of Hek293 cells with plasmids encoding individual ABCG1 isoforms. In situ hybridization studies in embryonic day 11.5-15.5 mouse embryos revealed strong expression of ABCG1 transcripts in the olfactory epithelium, hind brain, eye, and dorsal root ganglia. The relatively high levels of expression in neuronal tissues and the eye suggest that ABCG1-dependent cholesterol efflux may be critical for normal neuronal function in addition to its role in macrophages.