ABCA1 Protein Enhances Toll-like Receptor 4 (TLR4)-stimulated Interleukin-10 (IL-10) Secretion through Protein Kinase A (PKA) Activation

Nonresolving inflammatory response from macrophages is a major characteristic of atherosclerosis. Macrophage ABCA1 has been previously shown to suppress the secretion of proinflammatory cytokine. In the present study, we demonstrate that ABCA1 also promotes the secretion of IL-10, an anti-inflammatory cytokine critical for inflammation resolution. ABCA1^+/+ bone marrow-derived macrophages secrete more IL-10 but less proinflammatory cytokines than ABCA1^−/− bone marrow-derived macrophages, similar to alternatively activated (M2) macrophages. We present evidence that ABCA1 activates PKA and that this elevated PKA activity contributes to M2-like inflammatory response from ABCA1^+/+ bone marrow-derived macrophages. Furthermore, cholesterol lowering by statins, methyl-β-cyclodextrin, or filipin also activates PKA and, consequently, transforms macrophages toward M2-like phenotype. Conversely, cholesterol enrichment suppresses PKA activity and promotes M1-like inflammatory response. As the primary function of ABCA1 is cholesterol removal, our results suggest that ABCA1 activates PKA by regulating cholesterol. Indeed, forced cholesterol enrichment in ABCA1-expressing macrophages suppresses PKA activation and elicits M1-like response. Collectively, these findings reveal a novel protective process by ABCA1-activated PKA in macrophages. They also suggest cholesterol lowering in extra-hepatic tissues by statins as an anti-inflammation strategy.     

Role of the ABC transporters A1 and G1, key reverse cholesterol transport proteins,in atherosclerosis

Atherosclerosis is one of the most common causes of death worldwide. Epidemiology studies firmly established an inverse relationship between atherogenesis and distorted lipid metabolism, in particular, higher levels of total cholesterol, an accumulation of CH-laden macrophages (foam cells), and lower plasma levels of antiatherogenic high density lipoprotein (HDL). It is believed that the reverse cholesterol transport, a process that removes excess cholesterol from peripheral tissues/cells including macrophages to circulating HDL, is one of the main mechanisms responsible for anti-atherogenic properties of HDL. The key proteins of reverse cholesterol transport—ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1)—mediate the cholesterol efflux from macrophages and prevent their transformation into foam cells. This review focuses on the role of ABC transporters A1 and G1 in the pathogenesis of atherosclerosis.     

Absence of endogenous phospholipid transfer protein impairs ABCA1-dependent efflux of cholesterol from macrophage foam cells

In vitro experiments have demonstrated that exogenous phospholipid transfer protein (PLTP), i.e. purified PLTP added to macrophage cultures, influences ABCA1-mediated cholesterol efflux from macrophages to HDL. To investigate whether PLTP produced by the macrophages (i.e., endogenous PLTP) is also part of this process, we used peritoneal macrophages derived from PLTP-knockout (KO) and wild-type (WT) mice. The macrophages were transformed to foam cells by cholesterol loading, and this resulted in the upregulation of ABCA1. Such macrophage foam cells from PLTP-KO mice released less cholesterol to lipid-free apolipoprotein A-I (apoA-I) and to HDL than did the corresponding WT foam cells. Also, when plasma from either WT or PLTP-KO mice was used as an acceptor, cholesterol efflux from PLTP-KO foam cells was less efficient than that from WT foam cells. After cAMP treatment, which upregulated the expression of ABCA1, cholesterol efflux from PLTP-KO foam cells to apoA-I increased markedly and reached a level similar to that observed in cAMP-treated WT foam cells, restoring the decreased cholesterol efflux associated with PLTP deficiency. These results indicate that endogenous PLTP produced by macrophages contributes to the optimal function of the ABCA1-mediated cholesterol efflux-promoting machinery in these cells. Whether macrophage PLTP acts at the plasma membrane or intracellularly or shuttles between these compartments needs further study.     

Statins downregulate ATP-binding-cassette transporter A1 gene expression in macrophages

The ATP-binding-cassette transporter A1 (ABCA1) plays an essential role in cellular cholesterol efflux and helps prevent macrophages from becoming foam cells. The statins are widely used as cholesterol-lowering agents and have other anti-atherogenic actions. We tested the effects of four different statins (fluvastatin, atorvastatin, simvastatin, and lovastatin) on ABCA1 expression in macrophages in vitro. The statins suppressed ABCA1 mRNA expression in RAW246.7 and THP-1 macrophage cell lines and in mouse peritoneal macrophages. The effect was time- and dose-dependent and was abolished by the addition of the post-reductase product, mevalonate. These findings imply that there is a possible modulation of the well-known beneficial effects of the statins on the reverse cholesterol transport pathway.     

Secretory vesicular transport from the Golgi is altered during ATP-binding cassette protein A1 (ABCA1)-mediated cholesterol efflux

Apolipoprotein AI (apoAI)-mediated cholesterol efflux is a process by which cells export excess cellular cholesterol to apoAI to form high density lipoprotein. ATP-binding cassette protein A1 (ABCA1) has recently been identified as the key regulator of this process. The pathways of intracellular cholesterol transport during efflux are largely unknown nor is the molecular mechanism by which ABCA1 governs cholesterol efflux well understood. Here, we report that, in both macrophages and fibroblasts, the secretory vesicular transport changes in response to apoAI-mediated cholesterol efflux. Vesicular transport from the Golgi to the plasma membrane increased 2-fold during efflux. This increase in vesicular transport during efflux was observed in both raft-poor and raft-rich vesicle populations originated from the Golgi. Importantly, enhanced vesicular transport in response to apoAI is absent in Tangier fibroblasts, a cell type with deficient cholesterol efflux due to functional ABCA1 mutations. These findings are consistent with an efflux model whereby cholesterol is transported from the storage site to the plasma membrane via the Golgi. ABCA1 may influence cholesterol efflux in part by enhancing vesicular trafficking from the Golgi to the plasma membrane.     

Protein kinase A site-specific phosphorylation regulates ATP-binding cassette A1 (ABCA1)-mediated phospholipid efflux

ATP-binding cassette A1 (ABCA1) is a key mediator of cholesterol and phospholipid efflux to apolipoprotein particles. We show that ABCA1 is a constitutively phosphorylated protein in both RAW macrophages and in a human embryonic kidney cell line expressing ABCA1. Furthermore, we demonstrate that phosphorylation of ABCA1 is mediated by protein kinase A (PKA) or a PKA-like kinase in vivo. Through site-directed mutagenesis studies of consensus PKA phosphorylation sites and in vitro PKA kinase assays, we show that Ser-1042 and Ser-2054, located in the nucleotide binding domains of ABCA1, are major phosphorylation sites for PKA. ApoA-I-dependent phospholipid efflux was decreased significantly by mutation of Ser-2054 alone and Ser-1042/Ser-2054 but was not significantly impaired with Ser-1042 alone. The mechanism by which ABCA1 phosphorylation affected ApoA-I-dependent phospholipid efflux did not involve either alterations in ApoA-I binding or changes in ABCA1 protein stability. These studies demonstrate a novel serine (Ser-2054) on the ABCA1 protein crucial for PKA phosphorylation and for regulation of ABCA1 transporter activity.     

Wogonin promotes cholesterol efflux by increasing protein phosphatase 2B-dependent dephosphorylation at ATP-binding cassette transporter-A1 in macrophages

Wogonin, one component in Scutellaria baicalensis Georgi extracts, has several beneficial properties for cancers and inflammatory diseases. However, the efficacy of wogonin in cholesterol metabolism of macrophages remains unknown. In macrophages, cholesterol uptake is controlled by scavenger receptors (SR-A and CD36) and cholesterol efflux by SR-BI, ATP-binding cassette transporter-A1 (ABCA1) and ABCG1. In the present study, we investigated the effect and underlying molecular mechanism of wogonin on the formation of macrophage foam cells by murine J774.A1 macrophages. Wogonin attenuated oxidized low-density lipoprotein (oxLDL)-induced cholesterol accumulation in macrophages. The binding of oxLDL to macrophages and protein expression of SR-A and CD36 were not affected by wogonin. Wogonin enhanced cholesterol efflux and increased the protein level of ABCA1 without affecting the protein expression of SR-BI or ABCG1. Inhibition of ABCA1 by pharmacological inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt or neutralizing antibody abolished this suppressive effect of wogonin on lipid accumulation. Moreover, the up-regulation of ABCA1 protein by wogonin resulted from a decrease in degradation rate of ABCA1 protein, with no effect on ABCA1 mRNA expression. This reduction in ABCA1 degradation was due to increased protein phosphatase 2B (PP2B)-mediated ABCA1 dephosphorylation, as evidenced by increased interaction between ABCA1 and PP2B; pharmacological inhibition of PP2B would prevent wogonin-induced ABCA1 protein expression, dephosphorylation and attenuation of lipid accumulation. Collectively, wogonin increases the protein stability of ABCA1 via PP2B-mediated dephosphorylation, thus leading to reduced cholesterol accumulation in macrophage foam cells.     

Interleukin-6 protects human macrophages from cellular cholesterol accumulation and attenuates the proinflammatory response

Cholesterol-laden monocyte-derived macrophages are phagocytic cells characteristic of early and advanced atherosclerotic lesions. Interleukin-6 (IL-6) is a macrophage secretory product that is abundantly expressed in atherosclerotic plaques but whose precise role in atherogenesis is unclear. The capacity of macrophages to clear apoptotic cells, through the efferocytosis mechanism, as well as to reduce cellular cholesterol accumulation contributes to prevent plaque progression and instability. By virtue of its capacity to promote cellular cholesterol efflux from phagocyte-macrophages, ABCA1 was reported to reduce atherosclerosis. We demonstrated that lipid loading in human macrophages was accompanied by a strong increase of IL-6 secretion. Interestingly, IL-6 markedly induced ABCA1 expression and enhanced ABCA1-mediated cholesterol efflux from human macrophages to apoAI. Stimulation of ABCA1-mediated cholesterol efflux by IL-6 was, however, abolished by selective inhibition of the Jak-2/Stat3 signaling pathway. In addition, we observed that the expression of molecules described to promote efferocytosis, i.e. c-mer proto-oncogene-tyrosine kinase, thrombospondin-1, and transglutaminase 2, was significantly induced in human macrophages upon treatment with IL-6. Consistent with these findings, IL-6 enhanced the capacity of human macrophages to phagocytose apoptotic cells; moreover, we observed that IL-6 stimulates the ABCA1-mediated efflux of cholesterol derived from the ingestion of free cholesterol-loaded apoptotic macrophages. Finally, the treatment of human macrophages with IL-6 led to the establishment of an anti-inflammatory cytokine profile, characterized by an increased secretion of IL-4 and IL-10 together with a decrease of that of IL-1β. Taken together, our results indicate that IL-6 favors the elimination of excess cholesterol in human macrophages and phagocytes by stimulation of ABCA1-mediated cellular free cholesterol efflux and attenuates the macrophage proinflammatory phenotype. Thus, high amounts of IL-6 secreted by lipid laden human macrophages may constitute a protective response from macrophages to prevent accumulation of cytotoxic-free cholesterol. Such a cellular recycling of free cholesterol may contribute to reduce both foam cell formation and the accumulation of apoptotic bodies as well as intraplaque inflammation in atherosclerotic lesions.     

Interleukin-10 Facilitates Both Cholesterol Uptake and Efflux in Macrophages

Foam cell formation is a hallmark event during atherosclerosis. The current paradigm is that lipid uptake by scavenger receptor in macrophages initiates the chronic proinflammatory cascade and necrosis core formation that characterize atherosclerosis. We report here that a cytokine considered to be anti-atherogenic, interleukin-10 (IL10), promotes cholesterol uptake from modified lipoproteins in macrophages and its transformation into foam cells by increasing the expression of scavenger receptor CD36 and scavenger receptor A. Although uptake of modified lipoproteins is considered proatherogenic, we found that IL10 also increases cholesterol efflux from macrophages to protect against toxicity of free cholesterol accumulation in the cell. This process was PPARγ-dependent and was mediated through up-regulation of ABCA1 (ATP-binding cassette transporter A1) protein expression. Importantly, expression of inflammatory molecules, such as tumor necrosis factor-α, intercellular adhesion molecule-1, and MMP9 as well as apoptosis were dramatically suppressed in lipid-laden foam cells treated with IL10. The notion that IL10 can mediate both the uptake of cholesterol from modified lipoproteins and the efflux of stored cholesterol suggests that the process of foam cell formation is not necessarily detrimental as long as mechanisms of cholesterol efflux and transfer to an exogenous acceptor are functioning robustly. Our results present a comprehensive antiatherogenic role of IL10 in macrophages, including enhanced disposal of harmful lipoproteins, inhibition of inflammatory molecules, and reduced apoptosis.     

Apolipoprotein E4 Is Deficient in Inducing Macrophage ABCA1 Expression and Stimulating the Sp1 Signaling Pathway

ATP binding cassette A1 (ABCA1) is a membrane protein that promotes cellular cholesterol efflux. Using RAW 264.7 macrophages, we studied the relative effects of apolipoprotein (apo) E3 and apoE4 on ABCA1 and on the signaling pathway that regulates its expression. Both lipid-associated and lipid-free apoE4 forms induced ∼30% lower levels of ABCA1 protein and mRNA than apoE3 forms. Phosphorylated levels of phosphoinositol 3-kinase (PI3K), protein kinase Cζ (PKCζ) and specificity protein 1 (Sp1) were also lower when treated with apoE4 compared to apoE3. The reduced ability of apoE4 to induce ABCA1 expression, PKCζ and Sp1 phosphorylation were confirmed in human THP-1 monocytes/macrophages. Sequential phosphorylation of PI3K, PKCζ and Sp1 has been suggested as a mechanism for upregulation of ABCA1 expression. Both apoE3 and apoE4 reduced total cholesterol and cholesterol esters in lipid-laden RAW 264.7 cells, and induced apoAI-mediated cholesterol efflux. However, the cholesterol esters and cholesterol efflux in apoE4-treated cells were ∼50% and ∼24% lower, respectively, compared to apoE3-treated cells. Accumulation of cholesterol esters in macrophages is a mechanism for foam cell formation. Thus the reduced ability of apoE4 to activate the PI3K-PKCζ-Sp1 signaling pathway and induce ABCA1 expression likely impairs cholesterol ester removal, and increases foam cell formation.     

An analysis of the role of a retroendocytosis pathway in ABCA1-mediated cholesterol efflux from macrophages

The ATP binding cassette transporter A-1 (ABCA1) is critical for apolipoprotein-mediated cholesterol efflux, an important mechanism employed by macrophages to avoid becoming lipid-laden foam cells, the hallmark of early atherosclerotic lesions. It has been proposed that lipid-free apolipoprotein A-I (apoA-I) enters the cell and is resecreted as a lipidated particle via a retroendocytosis pathway during ABCA1-mediated cholesterol efflux from macrophages. To determine the functional importance of such a pathway, confocal microscopy was used to characterize the internalization of a fully functional apoA-I cysteine mutant containing a thiol-reactive fluorescent probe in cultured macrophages. ApoA-I was also endogenously labeled with (35)S-methionine to quantify cellular uptake and to determine the metabolic fate of the internalized protein. It was found that apoA-I was specifically taken inside macrophages and that a small amount of intact apoA-I was resecreted from the cells. However, a majority of the label that reappeared in the media was degraded. We estimate that the mass of apoA-I retroendocytosed is not sufficient to account for the HDL produced by the cholesterol efflux reaction. Furthermore, we have demonstrated that lipid-free apoA-I-mediated cholesterol efflux from macrophages can be pharmacologically uncoupled from apoA-I internalization into cells. On the basis these findings, we present a model in which the ABCA1-mediated lipid transfer process occurs primarily at the membrane surface in macrophages, but still accounts for the observed specific internalization of apoA-I.     

Lipid efflux by the ATP-binding cassette transporters ABCA1 and ABCG1

Plasma levels of high-density lipoproteins (HDL) and apolipoprotein A-I (apoA-I) are inversely correlated with the risk of cardiovascular disease. One major atheroprotective mechanism of HDL and apoA-I is their role in reverse cholesterol transport, i.e., the transport of excess cholesterol from foam cells to the liver for secretion. The ATP-binding cassette transporters ABCA1 and ABCG1 play a pivotal role in this process by effluxing lipids from foam cells to apoA-I and HDL, respectively. In the liver, ABCA1 activity is one rate-limiting step in the formation of HDL. In macrophages, ABCA1 and ABCG1 prevent the excessive accumulation of lipids and thereby protect the arteries from developing atherosclerotic lesions. However, the mechanisms by which ABCA1 and ABCG1 mediate lipid removal are still unclear. Particularly, three questions remain controversial and are discussed in this review: (1) Do apoA-I and HDL directly interact with ABCA1 and ABCG1, respectively? (2) Does cholesterol efflux involve retroendocytosis of apoA-I or HDL? (3) Which lipids are directly transported by ABCA1 and ABCG1?     

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.     

Phospholipase A2-Modified Low-Density Lipoprotein Activates Liver X Receptor in Human Macrophages

Macrophages respond to cholesterol accumulation by increasing cholesterol efflux, which is mediated by activation of the nuclear liver X receptor (LXR) and ATP binding cassette (ABC) transporters. In the present study, we investigated whether foam cell formation induced by phospholipase A(2)-modified low-density lipoprotein (PLA-LDL) influences LXR activity and cholesterol efflux in primary human monocyte-derived macrophages (MDMs). Macrophages were treated with PLA-LDL and expression of the LXR target genes ABCA1 and ABCG1 was analyzed by quantitative PCR and western blot. PLA-LDL time-dependently up-regulated ABCA1 and ABCG1 mRNA and protein. Removal of non-esterified fatty acids from PLA-LDL particles did not influence the induction of ABC transporters. A role of LXR in PLA-LDL-stimulated ABCG1 expression was verified by LXR-knockdown and luciferase reporter assays using a construct containing a LXR response element from the ABCG1 gene. Functionally, cholesterol efflux to apolipoprotein A-I and high-density lipoprotein was higher in PLA-LDL treated cells compared to controls. Together, these results demonstrate that in primary human MDMs PLA-LDL induces ABC transporter expression via LXR activation. A concomitantly increased cholesterol efflux may prevent excessive cholesterol accumulation and thus, attenuate foam cell formation.     

Regulation of cholesterol homeostasis in macrophages and consequences for atherosclerotic lesion development

Foam cell formation due to excessive accumulation of cholesterol by macrophages is a pathological hallmark of atherosclerosis. Macrophages cannot limit the uptake of cholesterol and therefore depend on cholesterol efflux pathways for preventing their transformation into foam cells. Several ABC-transporters, including ABCA1 and ABCG1, facilitate the efflux of cholesterol from macrophages. These transporters, however, also affect membrane lipid asymmetry which may have important implications for cellular endocytotic pathways. We propose that in addition to the generally accepted role of these ABC-transporters in the prevention of foam cell formation by induction of cholesterol efflux from macrophages, they also influence the macrophage endocytotic uptake.     

Effect of apolipoprotein A-I on ATP binding cassette transporter A1 degradation and cholesterol efflux in THP-1 macrophage-derived foam cells

The accumulation of lipoprotein cholesterol in theartery wall is thought to be an important factor in thedevelopment of atherosclerosis. After retentionand modi-fication in arteries, atherogenic lipoproteins are taken upby macrophages, bringing about macrophage-derived foamcells. High-density lipoprotein (HDL) plays a role in trans-porting cholesterol from peripheral tissues to the liver.The elevated level of HDL is associated with a decreasein atherosclerosis and the apolipoproteins to remo…