Direct Interaction of Nuclear Liver X Receptor-β with ABCA1 Modulates Cholesterol Efflux

Abstract

Cholesterol is an essential component of eukaryotic cells; at the same time, however, hyperaccumulation of cholesterol is harmful. Therefore, the ABCA1 gene, the product of which mediates secretion of cholesterol, is highly regulated at both the transcriptional and post-transcriptional levels. The transcription of ABCA1 is regulated by intracellular oxysterol concentration via the nuclear liver X receptor (LXR)/retinoid X receptor (RXR); once synthesized, ABCA1 protein turns over rapidly with a half-life of 1-2 h. Here, we show that the LXR␤/RXR complex binds directly to ABCA1 on the plasma membrane of macrophages and modulates cholesterol secretion. When cholesterol does not accumulate, ABCA1-LXR␤/RXR localizes on the plasma membrane, but is inert. When cholesterol accumulates, oxysterols bind to LXR␤, and the LXR␤/RXR complex dissociates from ABCA1, restoring ABCA1 activity and allowing apoA-I-dependent cholesterol secretion. LXR␤ can exert an immediate post-translational response, as well as a rather slow transcriptional response, to changes in cellular cholesterol accumulation. Thus, we provide the first demonstration that protein-protein interaction suppresses ABCA1 function. Furthermore, we show that LXR␤ is involved in both the transcriptional and post-transcriptional regulation of the ABCA1 transporter.Maintenance of cellular cholesterol homeostasis is important for normal human physiology. Disruption of cellular cholesterol homeostasis leads to a variety of pathological conditions, including cardiovascular disease (1). ABCA1 (ATPbinding cassette protein A1), one of the key proteins in cholesterol homeostasis, mediates secretion of cellular free cholesterol and phospholipids to an extracellular acceptor in the plasma, apoA-I, to form high density lipoprotein (HDL) 3 (2, 3). HDL formation is the only known pathway that can eliminate excess cholesterol from peripheral cells. Defects in ABCA1 cause Tangier disease (4 -6), in which patients have a near absence of circulating HDL, prominent cholesterol ester accumulation in tissue macrophages, and premature atherosclerotic vascular disease (1, 7).ABCA1-mediated cholesterol efflux is highly regulated at both the transcriptional and post-transcriptional levels. When cholesterol accumulates in cells, intracellular concentrations of oxysterols increase; subsequently, the liver X receptor (LXR), activated via binding of oxysterols, stimulates the transcription of ABCA1 (8 -10). ABCA1 protein eliminates excess cellular cholesterol and turns over rapidly with a half-life of 1-2 h (11-15). Several proteins, including apoA-I, ␣1-syntrophin, and ␤1-syntrophin, have been reported to interact with ABCA1 and reduce the rate of ABCA1 protein degradation (13-16). Syntrophins play critical roles in regulating the apoA-I-dependent cholesterol efflux (and thus in lipid homeostasis) by suppressing protein degradation in brain (14) and liver (15). Because cholesterol is an essential component of cells, however, excessive elimination of cholesterol could result in cell death. Con...

Liver X Receptor β (LXRβ) Interacts Directly with ATP-binding Cassette A1 (ABCA1) to Promote High Density Lipoprotein Formation during Acute Cholesterol Accumulation

“…Cholesterol efflux by cells expressing mutant ABCA1 was not affected by the addition of TO901317 either in the presence or absence of LXR␤. Co-expression of LXR␤ doubled the plasma membrane levels of WT ABCA1 as shown previously (16), but surface levels of the mutants (L2247A, L2251A, and L2247/L2251A) were not affected by LXR␤ expression (Fig. 3C).…”

“…However, addition of TO901317 to the membrane fraction of cells co-expressing ABCA1 and LXR␤ led to photoaffinity labeling of ABCA1 (lane 8) comparable with that seen in the absence of LXR␤ (lane 6). Under the same conditions, the co-precipitation of ABCA1 with LXR␤ was abrogated (16). ABCA1-L2247A/L2251A double mutant was labeled as efficiently as WT ABCA1 (lane 9), but this was not inhibited by LXR␤ co-expression (lanes 11 and 12), suggesting that the interaction of LXR␤ with ABCA1 via Leu 2247 and Leu 2251 prevents tight ATP binding.…”

“…Antibodies-Rabbit anti-human ABCA1 polyclonal antiserum was generated against the linker region (amino acids 1134 -1345) of human ABCA1 as described previously (16). The rat anti-human ABCA1 monoclonal antibody KM3073 was generated against the first extracellular domain (amino acids 45-639) of human ABCA1 as described previously (13).…”

“…B and C, HEK293 cells were co-transfected with LXR␤ and WT or mutant ABCA1. At 28 h after transfection, membranes prepared by nitrogen cavitation were subjected to immunoprecipitation (IP) with anti-ABCA1 polyclonal antiserum (16). Cell lysates (10%) (B) and precipitated proteins (C) were subjected to immunoblotting with the anti-ABCA1 monoclonal antibody KM3073 or anti-LXR␤ monoclonal antibody.…”

“…Consequently, ABCA1-mediated cholesterol release is also regulated at the post-translational level. Several proteins, including syntrophins (13,14), JAK2 (15), and LXR␤ (16), have been reported to interact with ABCA1 and modulate its degradation and function. However, the precise mechanism(s) by which ABCA1 activity is regulated post-translationally remains unclear.…”