ABCA1 mediates high-affinity uptake of 25-hydroxycholesterol by membrane vesicles and rapid efflux of oxysterol by intact cells

Abstract

Binding Cassette (ABC) transporter, ABCA1, plays a pivotal role in reverse cholesterol transport by mediating the cellular efflux of phospholipid and cholesterol. Studies using intact cells strongly suggest that ABCA1 acts as a phospholipid floppase, but there has been no direct demonstration that the protein is a primary active sterol transporter. Using membrane vesicles from insect Sf21 cells, we found that ABCA1 mediated ATP-dependent uptake of [ 3 H]25-hydroxycholesterol with an apparent K m of 0.7 M. Consistent with this high apparent affinity, expression of ABCA1 in human embryonic kidney cells both increased rapid efflux of 25-hydroxcholesterol and prevented oxysterol-mediated repression of low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase mRNAs. Comparison of wild-type and ABCA1 Ϫ/Ϫ murine fibroblasts indicates that 25-hydroxycholesterol is effluxed ϳ5-fold more rapidly by wild-type cells. In addition, the rate of efflux from the wild-type but not the ABCA1 Ϫ/Ϫ fibroblasts is increased a further twofold by inducers of ABCA1 expression. Thus under the experimental conditions employed, endogenous ABCA1 is a major contributor to 25-hydroxycholesterol efflux from wild-type fibroblasts. Evidence from in vitro studies indicates that oxysterols are potent inducers of genes involved in cellular cholesterol efflux and metabolism, including the ABCA1 gene, and repressors of genes involved in cholesterol synthesis or uptake. Our observations raise the possibility that efflux of oxysterols by ABCA1 could contribute to a homeostatic mechanism, which both attenuates oxysterol-induced expression of its cognate gene and alleviates repression of genes encoding proteins, such as HMG-CoA reductase and LDL receptor. active transport; cholesterol homeostasis THE ATP BINDING CASSETTE (ABC) protein, ABCA1, was identified during a search for novel ABC proteins expressed in macrophages and at the time of its discovery, its function was unknown (34). Defects in the ABCA1 gene were subsequently shown to be the cause of Tangier disease, which is characterized by a lack, or abnormally low level, of high-density lipoprotein (HDL) and a markedly increased risk of coronary artery disease (5,7,11,56). Studies (2, 6, 14) using knockout mice have confirmed that ABCA1 plays a pivotal role in reverse cholesterol transport and in vitro, increased expression of the protein in several cell types results in elevated net efflux of cellular cholesterol and phospholipids. This efflux is dependent on the presence of an acceptor such as lipid-poor HDL or apolipoprotein A (apoA)-I (19, 70). Other proteins can also act as acceptors although their physiological relevance is presently unclear (46).The mechanism by which ABCA1 mediates efflux of cholesterol and phospholipids remains poorly defined. ABC transporters typically use the energy of ATP binding and hydrolysis to drive the transport of substrate across cellular membranes (26). In the case of hydrophobic compounds, transport is thought to involve ...

Niemann-Pick C1 protects against atherosclerosis in mice via regulation of macrophage intracellular cholesterol trafficking

“…In Npc1 -/-macrophages, decreased oxysterol synthesis by the mitochondrial sterol 27-hydroxylase resulted in failure to activate LXR-dependent gene expression and cholesterol efflux, further exacerbating free cholesterol-induced mitochondrial dysfunction and promoting oxidative stress. The accumulation of cholesterol autoxidation products in the Npc1 -/-macrophages may be caused not only by the highly oxidative intracellular milieu, but also by decreased oxysterol export by the LXR-regulated ABCA1 sterol transporter (31). An unexpected finding in our study was that treatment with the synthetic LXR ligand attenuated ROS generation.…”

“…The intracellular accumulation of oxysterols may reflect impaired oxysterol efflux due to reduced expression of LXR-activated sterol transporters. Recent reports showing that 7-ketocholesterol, 25-HC, and 27-HC accumulate in ABCG1-, ABCA1-, and ABCG1/ABCG4-deficient cells, respectively, support such a mechanism (31)(32)(33). Whether the elevated levels of nonenzymatic cholesterol autoxidation products, which have been shown to be cytotoxic in vitro, also contribute to atherogenesis in vivo, or are simply markers of oxidative stress, is not known.…”

Distinct pathways of cholesterol biosynthesis impact on insulin secretion

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Adaptation of neuronal cells to chronic oxidative stress is associated with altered cholesterol and sphingolipid homeostasis and lysosomal function

“…Oxysterols are potent repressors of genes involved in cholesterol synthesis or uptake (Tam et al 2006;Bjorkhem 2008). Therefore, we determined mRNA levels of the ratelimiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase by real-time PCR.…”