Over-activation of NMDA receptors promotes ABCA1 degradation and foam cell formation

ATP-binding cassette transporter A1 (ABCA1) is an essential regulator of intracellular cholesterol efflux. Secreted cholesterol binds to lipid-free apolipoprotein A-I (apoA-I) in peripheral blood to constitute high-density lipoprotein cholesterol (HDL) complexes. ABCA1 protein on the surface of macrophages acts as a crucial controller in preventing cholesterol accumulation. Importantly, ABCA1 is unstable and easily degraded via a series of biochemical activities, including but not limited to calpain-mediated and ubiquitin-proteasome system-mediated processes. How accelerated ABCA1 degradation impacts disordered lipid metabolism in macrophages and foam cell formation is unclear. N-methyl d-aspartate receptors (NMDARs) are ionotropic glutamate receptors with high calcium permeability. Calcium influx via NMDARs activates downstream signaling pathways. Over-activation of NMDARs stimulated by NMDA contributes to dysfunctional lipid metabolism in macrophages and foam cell formation via promotion of calpain-mediated ABCA1 proteolysis. However, increased NMDAR activity does not affect liver X receptor expression or ABCA1 mRNA levels. Following NMDA receptor silencing or calpain inhibition, NMDA treatment did not reduce ABCA1 protein levels, nor caused lipid accumulation in macrophages. In addition, NMDAR over-activation activates NF-κB signaling to promote IL-1β and IL-6 macrophage marker expression. However, NMDAR silencing and calpain inhibition reduce inflammatory macrophage responses. In summary, our study suggests that NMDAR activation reduces surface ABCA1 protein, promotes lipid accumulation, and induces the production and secretion of many inflammatory mediators in macrophages, possibly through enhanced calpain-mediated ABCA1 protein degradation. Thus, the NMDAR receptor may be a novel pharmacologic target for atherosclerosis therapy.     

Tangier disease and ABCA1

Tangier disease is an autosomal recessive genetic disorder characterized by a severe high-density lipoprotein (HDL) deficiency, sterol deposition in tissue macrophages, and prevalent atherosclerosis. Mutations in the ATP binding cassette transporter ABCA1 cause Tangier disease and other familial HDL deficiencies. ABCA1 controls a cellular pathway that secretes cholesterol and phospholipids to lipid-poor apolipoproteins. This implies that an inability of newly synthesized apolipoproteins to acquire cellular lipids by the ABCA1 pathway leads to their rapid degradation and an over-accumulation of cholesterol in macrophages. Thus, ABCA1 plays a critical role in modulating flux of tissue cholesterol and phospholipids into the reverse cholesterol transport pathway, making it an important therapeutic target for clearing excess cholesterol from macrophages and preventing atherosclerosis.     

Helical apolipoproteins of high-density lipoprotein enhance phagocytosis by stabilizing ATP-binding cassette transporter A7

We previously reported that the endogenous ATP-binding cassette transporter (ABC)A7 strongly associates with phagocytic function rather than biogenesis of high-density lipoprotein (HDL), being regulated by sterol-regulatory element binding protein (SREBP)2. Phagocytic activity was found enhanced by apolipoprotein (apo)A-I and apoA-II more than twice the maximum in J774 and mouse peritoneal macrophages. Therefore we investigated the molecular basis of this reaction in association with the function of ABCA7. Similar to ABCA1, ABCA7 was degraded, likely by calpain, and apoA-I and apoA-II stabilize ABCA7 against degradation. Cell surface biotinylation experiments demonstrated that endogenous ABCA7 predominantly resides on the cell surface and that the apolipoproteins increase the surface ABCA7. The increase of phagocytosis by apolipoproteins was retained in the J774 cells treated with ABCA1 siRNA and in the peritoneal macrophages from ABCA1-knockout mice, but it was abolished in the J774 cells treated with ABCA7 siRNA and in the peritoneal macrophages from ABCA7-knockout mice. Phagocytosis was decreased in the cells in the peritoneal cavity of the ABCA7-knockout mouse compared with the wild-type control. We thus concluded that extracellular helical apolipoproteins augment ABCA7-associated phagocytosis by stabilizing ABCA7. The results demonstrated direct enhancement of the host defense system by HDL components.     

Distinct sites on ABCA1 control distinct steps required for cellular release of phospholipids

The loss of ABCA1 function leads to Tangier dyslipidemia in humans and to a Tangier-like phenotype in mice, by impairing the transformation of nascent apolipoproteins into mature HDL particles. Mechanistically this ensues from the inability of cells to release membrane lipids and cholesterol. Whereas the ability of ABCA1 to promote phospholipid effluxes, surface binding of apolipoproteins and outward flip of membrane lipids has been documented, the relationship between this series of ABCA1-dependent events is still elusive. Here we provide evidence that i) lipid effluxes require both flip of membrane lipids and binding of apolipoproteins to the cell surface, ii) apolipoprotein A-I binding depends on structural determinants on ABCA1, and iii) phospholipid effluxes can be modulated by engineered mutations on the structural determinants identified on ABCA1.     

Helical domains that mediate lipid solubilization and ABCA1-specific cholesterol efflux in apolipoproteins C-I and A-II

Many of the apolipoproteins in HDL can elicit cholesterol efflux via ABCA1, a critical initial step in HDL formation. Recent work has indicated that omnipresent amphipathic helices play a critical role, and these have been studied intensively in the most common HDL protein, apolipoprotein (apo)A-I. However, little information exists about helical domain arrangement in other apolipoproteins. We studied two of the smallest apolipoproteins known to interact with ABCA1, human apoA-II and apoC-I, in terms of ability to reorganize phospholipid (PL) bilayers and to promote ABCA1-mediated cholesterol. We found that both proteins contained helical domains that were fast and slow with respect to solubilizing PL. ABCA1-medated efflux required a minimum of a bihelical polypeptide comprised of at least one each of a slow and fast lipid reorganizing domain. In both proteins, the fast helix was located at the C terminus preceded by a slow helix. Helical placement in apoC-I was not critical for ABCA1 activity, but helix swaps in apoA-II dramatically disrupted cholesterol efflux, indicating that the tertiary structure of the longer apolipoprotein is important for the pathway. This work has implications for a more complete molecular understanding of apolipoprotein-mediated cholesterol efflux.     

ABCA1 redistributes membrane cholesterol independent of apolipoprotein interactions

ATP binding cassette transporter A1 (ABCA1) mediates the transport of phospholipids and cholesterol from cells to lipid-poor HDL apolipoproteins. Cholesterol loading of cells induces ABCA1, implicating cholesterol as its major physiologic substrate. It is believed, however, that ABCA1 is primarily a phospholipid transporter and that cholesterol efflux occurs by diffusion to ABCA1-generated phospholipid-rich apolipoproteins. Here we show that overexpression of ABCA1 in baby hamster kidney cells in the absence of apolipoproteins redistributed membrane cholesterol to cell-surface domains accessible to treatment with the enzyme cholesterol oxidase. The cholesterol removed by apolipoprotein A-I (apoA-I), but not by HDL phospholipids, was derived exclusively from these domains. ABCA1 overexpression also increased cholesterol esterification, which was prevented by addition of apoA-I, suggesting that some of the cell-surface cholesterol not removed by apolipoproteins is transported to the intracellular esterifying enzyme acyl-CoA:cholesterol acyltransferase. ABCA1 expression was essential for cholesterol efflux even when apolipoproteins had already acquired phospholipids during prior exposure to ABCA1-expressing cells.These studies show that ABCA1 redistributes cholesterol to cell-surface domains, where it becomes accessible for removal by apolipoproteins, consistent with a direct role of ABCA1 in cholesterol transport.     

ABCA1. The gatekeeper for eliminating excess tissue cholesterol

It is widely believed that HDL functions to transport cholesterol from peripheral cells to the liver by reverse cholesterol transport, a pathway that may protect against atherosclerosis by clearing excess cholesterol from arterial cells. A cellular ATP-binding cassette transporter (ABC) called ABCA1 mediates the first step of reverse cholesterol transport: the transfer of cellular cholesterol and phospholipids to lipid-poor apolipoproteins. Mutations in ABCA1 cause Tangier disease (TD), a severe HDL deficiency syndrome characterized by accumulation of cholesterol in tissue macrophages and prevalent atherosclerosis. Studies of TD heterozygotes revealed that ABCA1 activity is a major determinant of plasma HDL levels and susceptibility to CVD. Drugs that induce ABCA1 in mice increase clearance of cholesterol from tissues and inhibit intestinal absorption of dietary cholesterol. Multiple factors related to lipid metabolism and other processes modulate expression and tissue distribution of ABCA1.Therefore, as the primary gatekeeper for eliminating tissue cholesterol, ABCA1 has a major impact on cellular and whole body cholesterol metabolism and is likely to play an important role in protecting against cardiovascular disease.     

Janus kinase 2 modulates the lipid-removing but not protein-stabilizing interactions of amphipathic helices with ABCA1

ABCA1 mediates the transport of cellular cholesterol and phospholipids to HDL apolipoproteins. Apolipoprotein A-I (apoA-I) interactions with ABCA1-expressing cells elicit several responses, including removing cellular lipids, stabilizing ABCA1 protein, and activating Janus kinase 2 (JAK2). Here, we used synthetic apolipoprotein-mimetic peptides to characterize the relationship between these responses. Peptides containing one amphipathic helix of L- or D-amino acids (2F, D-2F, or 4F) and a peptide containing two helices (37pA) all promoted ABCA1-dependent cholesterol efflux, competed for apoA-I binding to ABCA1-expressing cells, blocked covalent cross-linking of apoA-I to ABCA1, and inhibited ABCA1 degradation. 37pA was cross-linked to ABCA1, confirming the direct binding of amphipathic helices to ABCA1. 2F, 4F, 37pA, and D-37pA all stimulated JAK2 autophosphorylation. Inhibition of JAK2 greatly reduced peptide-mediated cholesterol efflux, peptide binding to ABCA1-expressing cells, and peptide cross-linking to ABCA1, indicating that these processes require an active JAK2. In contrast, apoA-I and peptides stabilized ABCA1 protein even in the absence of an active JAK2, implying that this process is independent of JAK2 and lipid efflux-promoting binding of amphipathic helices to ABCA1. These findings show that amphipathic helices coordinate the activity of ABCA1 by several distinct mechanisms that are likely to involve different cell surface binding sites.     

Unsaturated fatty acids phosphorylate and destabilize ABCA1 through a phospholipase D2 pathway

Abnormal high density lipoprotein (HDL) metabolism among patients with diabetes and insulin resistance may contribute to their increased risk of atherosclerosis. ATP-binding cassette transporter ABCA1 mediates the transport of cholesterol and phospholipids from cells to HDL apolipoproteins and thus modulates HDL levels and atherogenesis. Unsaturated fatty acids, which are elevated in diabetes, impair the ABCA1 pathway in cultured cells by destabilizing ABCA1 protein. Here we examined the cellular pathway that mediates the ABCA1 destabilizing effects of fatty acids. The long-chain acyl-CoA synthetase inhibitor triacsin C completely reversed fatty acid-induced ABCA1 destabilization, indicating that fatty acids need to be activated to their CoA derivatives to enhance ABCA1 degradation. Unsaturated but not saturated fatty acids stimulated phospholipase D (PLD) activity, the PLD inhibitor 1-butanol prevented the unsaturated fatty acid-induced reduction in ABCA1 levels, and the PLD2 activator mastoparan markedly reduced ABCA1 protein levels, implicating a role for PLD2 in the ABCA1 destabilizing effects of fatty acids. Unsaturated fatty acids and mastoparan increased phosphorylation of ABCA1 serines. PLD2 small interfering RNA abolished the ability of unsaturated fatty acids to inhibit lipid transport activity, to reduce protein levels, and to increase serine phosphorylation of ABCA1. The diacylglycerol analog oleoylacetylglycerol also reduced ABCA1 protein levels and increased its serine phosphorylation, suggesting that PLD2-generated diacylglycerols promote the destabilizing phosphorylation of ABCA1. These data provide evidence that intracellular unsaturated acyl-CoA derivatives destabilize ABCA1 by activating a PLD2 signaling pathway.     

Inhibition of ERK1/2 and Activation of Liver X Receptor Synergistically Induce Macrophage ABCA1 Expression and Cholesterol Efflux

ATP-binding cassette transporter A1 (ABCA1), a molecule mediating free cholesterol efflux from peripheral tissues to apoAI and high density lipoprotein (HDL), inhibits the formation of lipid-laden macrophage/foam cells and the development of atherosclerosis. ERK1/2 are important signaling molecules regulating cellular growth and differentiation. The ERK1/2 signaling pathway is implicated in cardiac development and hypertrophy. However, the role of ERK1/2 in the development of atherosclerosis, particularly in macrophage cholesterol homeostasis, is unknown. In this study, we investigated the effects of ERK1/2 activity on macrophage ABCA1 expression and cholesterol efflux. Compared with a minor effect by inhibition of other kinases, inhibition of ERK1/2 significantly increased macrophage cholesterol efflux to apoAI and HDL. In contrast, activation of ERK1/2 reduced macrophage cholesterol efflux and ABCA1 expression. The increased cholesterol efflux by ERK1/2 inhibitors was associated with the increased ABCA1 levels and the binding of apoAI to cells. The increased ABCA1 by ERK1/2 inhibitors was due to increased ABCA1 mRNA and protein stability. The induction of ABCA1 expression and cholesterol efflux by ERK1/2 inhibitors was concentration-dependent. The mechanism study indicated that activation of liver X receptor (LXR) had little effect on ERK1/2 expression and activation. ERK1/2 inhibitors had no effect on macrophage LXRα/β expression, whereas they did not influence the activation or the inhibition of the ABCA1 promoter by LXR or sterol regulatory element-binding protein (SREBP). However, inhibition of ERK1/2 and activation of LXR synergistically induced macrophage cholesterol efflux and ABCA1 expression. Our data suggest that ERK1/2 activity can play an important role in macrophage cholesterol trafficking.     

ATP-binding cassette A1-mediated lipidation of apolipoprotein A-I occurs at the plasma membrane and not in the endocytic compartments

ATP-binding cassette transporter (ABC) A1 is required for the lipidation of apolipoprotein A-I to generate high density lipoprotein (HDL). This process is proposed to occur through a retro-endocytosis pathway in which apoA-I internalizes with ABCA1 and generates HDL from the endosomal compartments before resecretion. The aim of this study was to determine the route of apoA-I endocytosis and whether endocytosis contributes to HDL biogenesis. Using confocal microscopy, we found that internalized apoA-I only transiently colocalized with transferrin, a retro-endocytosis marker. Instead, apoA-I perfectly colocalized with a bulk phase uptake marker (fluorescein isothiocyanate-dextran) and, at later time points, with LysoTracker in several cell models including macrophages, fibroblasts, and baby hamster kidney cells. ABCA1 colocalized poorly with internalized apoA-I. To determine the contribution of internalized apoA-I to HDL biogenesis, we specifically removed apoA-I from the cell surface and analyzed the fate of internalized apoA-I. We found that 23% of cell-associated apoA-I was internalized at steady state. Of internalized apoA-I, only 20% was converted to HDL, and the rest was degraded, consistent with a lysosomal destination. We also found that apoA-I was released approximately five times faster from the plasma membrane than from the intracellular compartments. From these kinetic parameters, we estimated that approximately 5.6% of apoA-I that interacts with cells is degraded and that internalized apoA-I contributes to approximately 1.4% of total HDL production. We also found that blocking endocytosis with sucrose or cytochalasin D did not decrease cholesterol efflux or HDL biogenesis. We therefore conclude that the plasma membrane is the main platform where ABCA1-mediated lipidation of apoA-I occurs.     

Deficiency of ABCA1 impairs apolipoprotein E metabolism in brain

ABCA1 is a cholesterol transporter that is widely expressed throughout the body. Outside the central nervous system (CNS), ABCA1 functions in the biogenesis of high-density lipoprotein (HDL), where it mediates the efflux of cholesterol and phospholipids to apolipoprotein (apo) A-I. Deficiency of ABCA1 results in lack of circulating HDL and greatly reduced levels of apoA-I. ABCA1 is also expressed in cells within the CNS, but its roles in brain lipid metabolism are not yet fully understood. In the brain, glia synthesize the apolipoproteins involved in CNS lipid metabolism. Here we demonstrate that glial ABCA1 is required for cholesterol efflux to apoA-I and plays a key role in facilitating cholesterol efflux to apoE, which is the major apolipoprotein in the brain. In both astrocytes and microglia, ABCA1 deficiency reduces lipid efflux to exogenous apoE. The impaired ability to efflux lipids in ABCA1-/- glia results in lipid accumulation in both astrocytes and microglia under normal culture conditions. Additionally, apoE secretion is compromised in ABCA1-/- astrocytes and microglia. In vivo, deficiency of ABCA1 results in a 65% decrease in apoE levels in whole brain, and a 75-80% decrease in apoE levels in hippocampus and striatum. Additionally, the effect of ABCA1 on apoE is selective, as apoJ levels are unchanged in brains of ABCA1-/- mice. Taken together, these results show that glial ABCA1 is a key influence on apoE metabolism in the CNS.