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.     

Janus kinase 2 modulates the apolipoprotein interactions with ABCA1 required for removing cellular cholesterol

ATP-binding cassette transporter A1 (ABCA1) mediates transport of cellular cholesterol and phospholipids to high density lipoprotein (HDL) apolipoproteins, such as apoA-I. ABCA1 mutations can cause a severe HDL deficiency and atherosclerosis. Here we show that the protein-tyrosine kinase (TK) Janus kinase 2 (JAK2) modulates the apolipoprotein interactions with ABCA1 required for removing cellular lipids. The protein kinase A (PKA) inhibitor H89, the TK inhibitor genistein, and the JAK2 inhibitor AG490 suppressed apoA-I-mediated cholesterol and phospholipid efflux from ABCA1-expressing cells without altering the membrane ABCA1 content. Whereas PKA inhibition had no effect on apoA-I binding to cells or to ABCA1, TK and JAK2 inhibition greatly reduced these activities. Conversely, PKA but not JAK2 inhibition significantly reduced the intrinsic cholesterol translocase activity of ABCA1. Mutant cells lacking JAK2 had a severely impaired apoA-I-mediated cholesterol and phospholipid efflux and apoA-I binding despite normal ABCA1 protein levels and near normal cholesterol translocase activity. Thus, although PKA modulates ABCA1 lipid transport activity, JAK2 appears to selectively modulate apolipoprotein interactions with ABCA1. TK-mediated phosphorylation of ABCA1 was undetectable, implicating the involvement of another JAK2-targeted protein. Acute incubation of ABCA1-expressing cells with apoA-I had no effect on ABCA1 phosphorylation but stimulated JAK2 autophosphorylation. These results suggest that the interaction of apolipoproteins with ABCA1-expressing cells activates JAK2, which in turn activates a process that enhances apolipoprotein interactions with ABCA1 and lipid removal from cells.     

The C-terminal domain of apolipoprotein A-I is involved in ABCA1-driven phospholipid and cholesterol efflux

ABCA1, a member of the ATP-binding cassette family, mediates the efflux of cellular lipids to free apolipoproteins, mainly apoA-I. The role of the C-terminal domain of apoA-I in this process has been evaluated by measuring the efflux capacity of a truncated form (apoA-I-(1-192)) versus intact apoA-I in different cellular models. In stimulated J774 macrophages, cholesterol efflux to apoA-I-(1-192) was remarkably lower than that to the intact apoA-I. The truncated apoA-I, lacking an important lipid-binding domain, was also significantly less efficient in removing phospholipids from stimulated macrophages. No difference was detected with stimulated Tangier fibroblasts that do not express functional ABCA1. The C-terminal domain of apoA-I is clearly involved in ABCA1-driven lipid efflux. Independent of the interaction with the cell surface, it may be the decreased ability of the truncated apoA-I to recruit membrane phospholipids that impairs its capacity to promote cell cholesterol efflux.     

The Macrophage Cholesterol Exporter ABCA1 Functions as an Anti-inflammatory Receptor

ATP-binding cassette transporter A1 (ABCA1) is a cell membrane protein that exports excess cholesterol from cells to apolipoprotein (apo) A-I, the major protein in high density lipoproteins. Genetic studies have shown that ABCA1 protects against cardiovascular disease. The interaction of apoA-I with ABCA1 promotes cholesterol removal and activates signaling molecules, such as Janus kinase 2 (JAK2), that optimize the lipid export activity of ABCA1. Here we show that the ABCA1-mediated activation of JAK2 also activates STAT3, which is independent of the lipid transport function of ABCA1. ABCA1 contains two candidate STAT3 docking sites that are required for the apoA-I/ABCA1/JAK2 activation of STAT3. The interaction of apoA-I with ABCA1-expressing macrophages suppressed the ability of lysopolysaccaride to induce the inflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α, which was reversed by silencing STAT3 or ABCA1. Thus, the apoA-I/ABCA1 pathway in macrophages functions as an anti-inflammatory receptor through activation of JAK2/STAT3. These findings implicate ABCA1 as a direct molecular link between the cardioprotective effects of cholesterol export from arterial macrophages and suppressed inflammation.     

Membrane lipid domains distinct from cholesterol/sphingomyelin-rich rafts are involved in the ABCA1-mediated lipid secretory pathway

Efflux of excess cellular cholesterol mediated by lipid-poor apolipoproteins occurs by an active mechanism distinct from passive diffusion and is controlled by the ATP-binding cassette transporter ABCA1. Here we examined whether ABCA1-mediated lipid efflux involves the selective removal of lipids associated with membrane rafts, plasma membrane domains enriched in cholesterol and sphingomyelin. ABCA1 was not associated with cholesterol and sphingolipid-rich membrane raft domains based on detergent solubility and lack of colocalization with marker proteins associated with raft domains. Lipid efflux to apoA-I was accounted for by decreases in cellular lipids not associated with cholesterol/sphingomyelin-rich membranes. Treating cells with filipin, to disrupt raft structure, or with sphingomyelinase, to digest plasma membrane sphingomyelin, did not impair apoA-I-mediated cholesterol or phosphatidylcholine efflux. In contrast, efflux of cholesterol to high density lipoproteins (HDL) or plasma was partially accounted for by depletion of cholesterol from membrane rafts. Additionally, HDL-mediated cholesterol efflux was partially inhibited by filipin and sphingomyelinase treatment. Apo-A-I-mediated cholesterol efflux was absent from fibroblasts with nonfunctional ABCA1 (Tangier disease cells), despite near normal amounts of cholesterol associated with raft domains and normal abilities of plasma and HDL to deplete cholesterol from these domains. Thus, the involvement of membrane rafts in cholesterol efflux applies to lipidated HDL particles but not to lipid-free apoA-I. We conclude that cholesterol and sphingomyelin-rich membrane rafts do not provide lipid for efflux promoted by apolipoproteins through the ABCA1-mediated lipid secretory pathway and that ABCA1 is not associated with these domains.     

ATP-binding cassette transporter A1 (ABCA1) functions as a cholesterol efflux regulatory protein

ABCA1, an ATP-binding cassette transporter mutated in Tangier disease, promotes cellular phospholipid and cholesterol efflux by loading free apoA-I with these lipids. This process involves binding of apoA-I to the cell surface and phospholipid translocation by ABCA1. The goals of this study were to examine the relationship between ABCA1-mediated lipid efflux and apolipoprotein binding and to determine whether phospholipid and cholesterol efflux are coupled. Inhibition of lipid efflux by glybenclamide treatment or by mutation of the ATP-binding cassette of ABCA1 showed a close correlation between lipid efflux, the binding of apoA-I to cells, and cross-linking of apoA-I to ABCA1. The data suggest that a functionally important apoA-I binding site exists on ABCA1 and that the binding site could also involve lipids. After using cyclodextrin preincubation to deplete cellular cholesterol, ABCA1-mediated cholesterol efflux was abolished but phospholipid efflux and the binding of apoA-I were unaffected. The conditioned media from cyclodextrin-pretreated, ABCA1-expressing cells readily promoted cholesterol efflux when added to fresh cells not expressing ABCA1, indicating that cholesterol efflux can be dissociated from phospholipid efflux. Further, using a photoactivatable cholesterol analog, we showed that ABCA1 did not bind cholesterol directly, even though several other cholesterol-binding proteins specifically bound the cholesterol analog. The data suggest that the binding of apoA-I to ABCA1 leads to the formation of phospholipid-apoA-I complexes, which subsequently promote cholesterol efflux in an autocrine or paracrine fashion.     

Phospholipid transfer protein interacts with and stabilizes ATP-binding cassette transporter A1 and enhances cholesterol efflux from cells

Phospholipid lipid transfer protein (PLTP) is ubiquitously expressed in animal tissues and plays multiple roles in lipoprotein metabolism, but the function of peripheral PLTP is still poorly understood. Here we show that one of its possible functions is to transport cholesterol and phospholipids from cells to lipoprotein particles by a process involving PLTP interactions with cellular ATP-binding cassette transporter A1 (ABCA1). When ABCA1 was induced in murine macrophages or ABCA1-transfected baby hamster kidney cells, PLTP gained the ability to promote cholesterol and phospholipid efflux from cells. Although PLTP alone had lipid efflux activity, its maximum activity was observed in the presence of high density lipoprotein particles. Pulsechase studies showed that the interaction of PLTP with ABCA1-expressing cells played a role in promoting lipid efflux. Overexpression of ABCA1 dramatically increased binding of both PLTP and apoA-I to common sites on the cell surface. Both PLTP and apoA-I were covalently cross-linked to ABCA1, each protein blocked cross-linking of the other, and both PLTP and apoA-I stabilized ABCA1 protein. These results are consistent with PLTP and apoA-I binding to ABCA1 at the same or closely related sites. Thus, PLTP mimics apolipoproteins in removing cellular lipids by the ABCA1 pathway, except that PLTP acts more as an intermediary in the transfer of cellular lipids to lipoprotein particles.     

Caveolin-1-mediated apolipoprotein A-I membrane binding sites are not required for cholesterol efflux

Caveolin-1 (Cav1), a structural protein required for the formation of invaginated membrane domains known as caveolae, has been implicated in cholesterol trafficking and homeostasis. Here we investigated the contribution of Cav1 to apolipoprotein A-I (apoA-I) cell surface binding and intracellular processing using mouse embryonic fibroblasts (MEFs) derived from wild type (WT) or Cav1-deficient (Cav1(-/-)) animals. We found that cells expressing Cav1 have 2.6-fold more apoA-I binding sites than Cav1(-/-) cells although these additional binding sites are not associated with detergent-free lipid rafts. Further, Cav1-mediated binding targets apoA-I for internalization and degradation and these processes are not correlated to cholesterol efflux. Despite lower apoA-I binding, cholesterol efflux from Cav1(-/-) MEFs is 1.7-fold higher than from WT MEFs. Stimulation of ABCA1 expression with an LXR agonist enhances cholesterol efflux from both WT and Cav1(-/-) cells without increasing apoA-I surface binding or affecting apoA-I processing. Our results indicate that there are at least two independent lipid binding sites for apoA-I; Cav1-mediated apoA-I surface binding and uptake is not linked to cholesterol efflux, indicating that membrane domains other than caveolae regulate ABCA1-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.     

Apolipoprotein A-I activates Cdc42 signaling through the ABCA1 transporter

It has been suggested that the signal transduction initiated by apolipoprotein A-I (apoA-I) activates key proteins involved in cholesterol efflux. ABCA1 serves as a binding partner for apoA-I, but its participation in apoA-I-induced signaling remains uncertain. We show that the exposure of human fibroblasts to ABCA1 ligands (apolipoproteins and amphipathic helical peptides) results in the generation of intracellular signals, including activation of the small G-protein Cdc42, protein kinases (PAK-1 and p54JNK), and actin polymerization. ApoA-I-induced signaling was abrogated by glyburide, an inhibitor of the ABC transporter family, and in fibroblasts from patients with Tangier disease, which do not express ABCA1. Conversely, induction of ABCA1 expression with the liver X receptor agonist, T0901317, and the retinoid X receptor agonist, R0264456, potentiated apoA-I-induced signaling. Similar effects were observed in HEK293 cells overexpressing ABCA1-green fluorescent protein (GFP) fusion protein, but not ABCA1-GFP (K939M), which fails to hydrolyze ATP, or a nonfunctional ABCA1-GFP with a truncated C terminus. We further found that Cdc42 coimmunoprecipitates with ABCA1 in ABCA1-GFP-expressing HEK293 cells exposed to apoA-I but not in cells expressing ABCA1 mutants. We conclude that ABCA1 transduces signals from apoA-I by complexing and activating Cdc42 and downstream kinases and, therefore, acts as a full apoA-I receptor.     

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.     

Potential involvement of dissociated apoA-I in the ABCA1-dependent cellular lipid release by HDL

Helical apolipoproteins of high density lipoprotein (HDL) remove phospholipid and cholesterol from cells and generate HDL particles being mediated by ATP binding cassette transporter A1 (ABCA1). In murine macrophage cell line RAW264 cells, cAMP induced expression of ABCA1, release of cellular phospholipid and cholesterol by apolipoprotein A-I (apoA-I), and reversible binding of apoA-I to the cell. The apoA-I-dependent lipid release was directly proportional to the cAMP-induced binding of apoA-I, and was inhibited 70% by a monoclonal antibody selective to lipid-free apoA-I, 725-1E2. In contrast, apparent cellular cholesterol release to HDL was substantial even without ABCA1 induction, and it was increased only by 27% after the cAMP treatment. The antibody inhibited this increment by 70%. Lipid-free apoA-II liberated apoA-I from HDL by displacement and thereby markedly expanded the cAMP-induced part of the cholesterol release to the HDL-containing medium, and the antibody inhibited this part also by 70%. Binding experiments of the double-labeled reconstituted HDL showed that cAMP induced reversible binding of apoA-I but not the association of cholesteryl ester with the cells. The effect of the antibody on the cellular cholesterol release to the reconstituted HDL was similar to that of the HDL-mediated release. The data implicated that the ABCA1-dependent cholesterol release to HDL is mediated by apoA-I dissociated from HDL.     

Roles of ATP binding cassette transporters A1 and G1, scavenger receptor BI and membrane lipid domains in cholesterol export from macrophages

PURPOSE OF REVIEW: The initial steps of reverse cholesterol transport involve export of cholesterol from peripheral cells to plasma lipoproteins for subsequent delivery to the liver. The review discusses recent developments in our understanding of how these steps occur, with particular emphasis on the macrophage, the major site of cellular cholesterol accumulation in atherosclerosis. RECENT FINDINGS: ATP binding cassette transporter (ABC) A1 exports cholesterol and phospholipid to lipid-free apolipoproteins, while ATP binding cassette transporter G1 and scavenger receptor BI export cholesterol to phospholipid-containing acceptors. ABCA1-dependent cholesterol export involves an initial interaction of apolipoprotein AI with lipid raft membrane domains, although ABCA1 and most exported cholesterol are not raft associated. ABCG1 exports cholesterol to HDL and other phospholipid-containing acceptors. These include particles generated during lipidation of apoAI by ABCA1, suggesting that the two transporters cooperate in cholesterol export. Scavenger receptor BI is atheroprotective, mediating clearance of HDL cholesterol by the liver. The relative contributions of scavenger receptor BI and ABCG to cholesterol export to HDL from macrophages is unclear and may depend on cellular cholesterol status and the cholesterol gradient between cell and acceptor. SUMMARY: The presence of distinct pathways for cholesterol efflux to lipid-free apolipoprotein AI and phospholipid-containing HDL species clarifies our understanding of reverse cholesterol transport, and provides new opportunities for its therapeutic manipulation.     

Lysine residues of ABCA1 are required for the interaction with apoA-I

ATP-binding cassette protein A1 (ABCA1) plays a pivotal role in cholesterol homeostasis by generating high-density lipoprotein (HDL). Apolipoprotein A-I (apoA-I), a lipid acceptor for ABCA1, reportedly interacts with ABCA1. However, it has also been proposed that apoA-I interacts with ABCA1-generated special domains on the plasma membrane, but apart from ABCA1, and solubilizes membrane lipids. To determine the importance of the apoA-I-ABCA1 interaction in HDL formation, the electrostatic interaction between apoA-I and ABCA1, which mediates the interaction between apoB100 in low-density lipoprotein particles (LDL) and LDL receptor, was analyzed. The apoA-I binding to ABCA1 and the cross-linking between them were inhibited by the highly charged molecules heparin and poly-L-lysine. Treating cells with membrane impermeable reagents that specifically react with primary amino groups abolished the interaction between apoA-I and ABCA1. However, these reagents did not affect the characteristic tight ATP binding to ABCA1. These results suggest that lysine residues in the extracellular domains of ABCA1 contribute to the interaction with apoA-I. The electrostatic interaction between ABCA1 and apoA-I is predicted to be the first step in HDL formation. This article is part of a Special Issue entitled Advances in high density lipoprotein formation and metabolism: a tribute to John F. Oram (1945-2010).     

The interaction of ApoA-I and ABCA1 triggers signal transduction pathways to mediate efflux of cellular lipids

Reverse cholesterol transport (RCT) has been characterized as a crucial step for antiatherosclerosis, which is initiated by ATP-binding cassette A1 (ABCA1) to mediate the efflux of cellular phospholipids and cholesterol to lipid-free apolipoprotein A-I (apoA-I). However, the mechanisms underlying apoA-I/ABCA1 interaction to lead to the lipidation of apoA-I are poorly understood. There are several models proposed for the interaction of apoA-I with ABCA1 as well as the lipidation of apoA-I mediated by ABCA1. ApoA-I increases the levels of ABCA1 protein markedly. In turn, ABCA1 can stabilize apoA-I. The interaction of apoA-I with ABCA1 could activate signaling molecules that modulate posttranslational ABCA1 activity or lipid transport activity. The key signaling molecules in these processes include protein kinase A (PKA), protein kinase C (PKC), Janus kinase 2 (JAK2), Rho GTPases and Ca²⁺, and many factors also could influence the interaction of apoA-I with ABCA1. This review will summarize these mechanisms for the apoA-I interaction with ABCA1 as well as the signal transduction pathways involved in these processes.     

Specific docking of apolipoprotein A-I at the cell surface requires a functional ABCA1 transporter

The identification of defects in ABCA1 as the molecular basis of Tangier disease has highlighted its crucial role in the loading with phospholipids and cholesterol of nascent apolipoprotein particles. Indeed the expression of ABCA1 affects apolipoprotein A-I (apoA-I)-mediated removal of lipids from cell membranes, and the possible role of ABCA1 as an apoA-I surface receptor has been recently suggested. In the present study, we have investigated the role of the ABCA1 transporter as an apoA-I receptor with the analysis of a panel of transfectants expressing functional or mutant forms of ABCA1. We provide experimental evidence that the forced expression of a functional ABCA1 transporter confers surface competence for apoA-I binding. This, however, appears to be dependent on ABCA1 function. Structurally intact but ATPase-deficient forms of the transporter fail to elicit a specific cell association of the ligand. In addition the diffusion parameters of membrane-associated apoA-I indicate an interaction with membrane lipids rather than proteins. These results do not support a direct molecular interaction between ABCA1 and apoA-I, but rather suggest that the ABCA1-induced modification of the lipid distribution in the membrane, evidenced by the phosphatidylserine exofacial flopping, generates a biophysical microenvironment required for the docking of apoA-I at the cell surface.     

The E3 ubiquitin ligase,HECTD1, is involved in ABCA1-mediated cholesterol export from macrophages

The ABC lipid transporters, ABCA1 and ABCG1, are essential for maintaining lipid homeostasis in cells such as macrophages by exporting excess cholesterol to extracellular acceptors. These transporters are highly regulated at the post-translational level, including protein ubiquitination. Our aim was to investigate the role of the E3 ubiquitin ligase HECTD1, recently identified as associated with ABCG1, on ABCG1 and ABCA1 protein levels and cholesterol export function. Here, we show that HECTD1 protein is widely expressed in a range of human and murine primary cells and cell lines, including macrophages, neuronal cells and insulin secreting β-cells. siRNA knockdown of HECTD1 unexpectedly decreased overexpressed ABCG1 protein levels and cell growth, but increased native ABCA1 protein in CHO-K1 cells. Knockdown of HECTD1 in unloaded THP-1 macrophages did not affect ABCG1 but significantly increased ABCA1 protein levels, in wild-type as well as THP-1 cells that do not express ABCG1. Cholesterol export from macrophages to apoA-I over time was increased after knockdown of HECTD1, however these effects were not sustained in cholesterol-loaded cells. In conclusion, we have identified a new candidate, the E3 ubiquitin ligase HECTD1, that may be involved in the regulation of ABCA1-mediated cholesterol export from unloaded macrophages to apoA-I. The exact mechanism by which this ligase affects this pathway remains to be elucidated.