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.     

Blockade of cholesterol absorption by ezetimibe reveals a complex homeostatic network in enterocytes

Enterocyte cholesterol homeostasis reflects aggregated rates of sterol synthesis, efflux, and uptake from plasma and gut lumen. Cholesterol synthesis and LDL uptake are coordinately regulated by sterol regulatory element-binding proteins (SREBP), whereas sterol efflux is regulated by liver X receptors (LXR). How these processes are coordinately regulated in enterocytes, the site of cholesterol absorption, is not well understood. Here, we treat mice with ezetimibe to investigate the effect of blocking cholesterol absorption on intestinal SREBPs, LXRs, and their effectors. Ezetimibe increased nuclear SREBP-2 8-fold. HMG-CoA reductase (HMGR) and LDL receptor (LDLR) mRNA levels increased less than 3-fold, whereas their protein levels increased 30- and 10-fold, respectively. Expression of inducible degrader of LDLR (IDOL), an LXR-regulated gene that degrades LDLRs, was reduced 50% by ezetimibe. Coadministration of ezetimibe with the LXR agonist T0901317 abolished the reduction in IDOL and prevented the increase in LDLR protein. Ezetimibe-stimulated LDLR expression was independent of proprotein convertase subtilisin/kexin type 9 (PSCK9), a protein that degrades LDLRs. To maintain cholesterol homeostasis in the face of ezetimibe, enterocytes boost LDL uptake by increasing LDLR number, and they boost sterol synthesis by increasing HMGR and other cholesterologenic genes. These studies reveal a hitherto undescribed homeostatic network in enterocytes triggered by blockade of cholesterol absorption.     

SR-BI inhibits ABCG1-stimulated net cholesterol efflux from cells to plasma HDL

This study compares the roles of ABCG1 and scavenger receptor class B type I (SR-BI) singly or together in promoting net cellular cholesterol efflux to plasma HDL containing active LCAT. In transfected cells, SR-BI promoted free cholesterol efflux to HDL, but this was offset by an increased uptake of HDL cholesteryl ester (CE) into cells, resulting in no net efflux. Coexpression of SR-BI with ABCG1 inhibited the ABCG1-mediated net cholesterol efflux to HDL, apparently by promoting the reuptake of CE from medium. However, ABCG1-mediated cholesterol efflux was not altered in cholesterol-loaded, SR-BI-deficient (SR-BI(-/-)) macrophages. Briefly cultured macrophages collected from SR-BI(-/-) mice loaded with acetylated LDL in the peritoneal cavity did exhibit reduced efflux to HDL. However, this was attributable to reduced expression of ABCG1 and ABCA1, likely reflecting increased macrophage cholesterol efflux to apolipoprotein E-enriched HDL during loading in SR-BI(-/-) mice. In conclusion, cellular SR-BI does not promote net cholesterol efflux from cells to plasma HDL containing active LCAT as a result of the reuptake of HDL-CE into cells. Previous findings of increased atherosclerosis in mice transplanted with SR-BI(-/-) bone marrow probably cannot be explained by a defect in macrophage cholesterol efflux.     

Effects of miR-33a-5P on ABCA1/G1-Mediated Cholesterol Efflux under Inflammatory Stress in THP-1 Macrophages

The present study is to investigate whether inflammatory cytokines inhibit ABCA1/ABCG1-mediated cholesterol efflux by regulating miR-33a-5P in THP-1 macrophages. We used interleukin-6 and tumor necrosis factor-alpha in the presence or absence of native low density lipoprotein (LDL) to stimulate THP-1 macrophages. THP-1 macrophages were infected by either control lentivirus vectors or lentivirus encoding miR-33a-5P or antisense miR-33a-5P. The effects of inflammatory cytokines, miR-33a-5P and antisense miR-33a-5P on intracellular lipids accumulation and intracellular cholesterol contents were assessed by oil red O staining and quantitative intracellular cholesterol assay. ApoA-I-mediated cholesterol efflux was examined using the fluorescent sterol (BODIPY-cholesterol). The gene and protein expressions of the molecules involved in cholesterol trafficking were examined using quantitative real-time polymerase chain reaction and Western blotting. Inflammatory cytokines or miR-33a-5P increased intracellular lipid accumulation and decreased apoA-I-mediated cholesterol efflux via decreasing the expression of ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. However, antisense miR-33a-5P reversed the effects of inflammatory cytokines on intracellular lipid accumulation, cholesterol efflux, and the expression of miR-33a-5P, ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. This study indicated that inflammatory cytokines inhibited ABCA1/ABCG1-mediated cholesterol efflux by up-regulating miR-33a-5P in THP-1 macrophages.     

LXR/RXR activation enhances basolateral efflux of cholesterol in CaCo-2 cells

Regulation of gene expression of ATP-binding cassette transporter (ABC)A1 and ABCG1 by liver X receptor/retinoid X receptor (LXR/RXR) ligands was investigated in the human intestinal cell line CaCo-2. Neither the RXR ligand, 9-cis retinoic acid, nor the natural LXR ligand 22-hydroxycholesterol alone altered ABCA1 mRNA levels. When added together, ABCA1 and ABCG1 mRNA levels were increased 3- and 7-fold, respectively. T0901317, a synthetic non-sterol LXR agonist, increased ABCA1 and ABCG1 gene expression 11- and 6-fold, respectively. ABCA1 mass was increased by LXR/RXR activation. T0901317 or 9-cis retinoic acid and 22-hydroxycholesterol increased cholesterol efflux from basolateral but not apical membranes. Cholesterol efflux was increased by the LXR/RXR ligands to apolipoprotein (apo)A-I or HDL but not to taurocholate/phosphatidylcholine micelles. Actinomycin D prevented the increase in ABCA1 and ABCG1 mRNA levels and the increase in cholesterol efflux induced by the ligands. Glyburide, an inhibitor of ABCA1 activity, attenuated the increase in basolateral cholesterol efflux induced by T0901317. LXR/RXR activation decreased the esterification and secretion of cholesterol esters derived from plasma membranes. Thus, in CaCo-2 cells, LXR/RXR activation increases gene expression of ABCA1 and ABCG1 and the basolateral efflux of cholesterol, suggesting that ABCA1 plays an important role in intestinal HDL production and cholesterol absorption.     

Cholesterol secretion and homeostasis in chondrocytes: a liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein A1 expression.

Cholesterol is required for chondrocyte differentiation and bone formation. Apolipoprotein A1 (apoA-1) plays a major role in lipoprotein clearance and cholesterol redistribution. We report here that apoA-1 is expressed during chondrocyte differentiation in vitro and in vivo. In differentiating chondrocytes, the expression of the liver X receptor (LXR) is modulated and its expression correlates to the expression of apoA-1. The expression of other LXR target genes related to cholesterol homeostasis such as ABCA1 cholesterol transporter and sterol regulatory element-binding protein 1 (SREBP1) is similarly regulated. Small molecule ligands activating either LXR or retinoid X receptor (RXR) lead to a dramatic increase in apoA-1 mRNA and protein expression in cultured chondrocytes. These ligands strongly induce ABCA1 cholesterol transporter expression and effectively mediate cholesterol efflux from hypertrophic chondrocytes. In addition, we report that, in the same cells, the ligands down modulate Serum Amyloid A expression induced by bacterial lipopolysaccharide. Our studies provide evidence that LXR/RXR mediate a fine regulation of cholesterol homeostasis in differentiating chondrocytes.     

Phytosterols differentially influence ABC transporter expression, cholesterol efflux and inflammatory cytokine secretion in macrophage foam cells

Phytosterol supplements lower low-density lipoprotein (LDL) cholesterol, but accumulate in vascular lesions of patients and limit the anti-atherosclerotic effects of LDL lowering in apolipoprotein E (Apo E)-deficient mice, suggesting that the cholesterol-lowering benefit of phytosterol supplementation may not be fully realized. Individual phytosterols have cell-type specific effects that may be either beneficial or deleterious with respect to atherosclerosis, but little is known concerning their effects on macrophage function. The effects of phytosterols on ABCA1 and ABCG1 abundance, cholesterol efflux and inflammatory cytokine secretion were determined in cultured macrophage foam cells. Among the commonly consumed phytosterols, stigmasterol increased expression of ABCA1 and ABCG1 and increased efflux of cholesterol to apolipoprotein (Apo) AI and high-density lipoprotein (HDL). Campesterol and sitosterol had no effect on ABCA1 or ABCG1 levels. Sitosterol had no effect on cholesterol efflux to Apo AI or HDL, whereas campesterol had a modest but significant reduction in cholesterol efflux to HDL in THP-1 macrophages. Whereas stigmasterol blunted aggregated LDL (agLDL) induced increases in tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β secretion, sitosterol exacerbated these effects. The presence of campesterol had no effect on agLDL-induced inflammatory cytokine secretion from THP-1 macrophages. In conclusion, the presence of stigmasterol in modified lipoproteins promoted cholesterol efflux and suppressed inflammatory cytokine secretion in response to lipid loading in macrophage foam cells. While campesterol was largely inert, the presence of sitosterol increased the proinflammatory cytokine secretion.     

Sterol-responsive element-binding protein (SREBP) 2 down-regulates ATP-binding cassette transporter A1 in vascular endothelial cells: a novel role of SREBP in regulating cholesterol metabolism

ATP-binding cassette transporter A1 (ABCA1) is a pivotal regulator of cholesterol efflux from cells to apolipoproteins, whereas sterol-responsive element-binding protein 2 (SREBP2) is the key protein regulating cholesterol synthesis and uptake. We investigated the regulation of ABCA1 by SREBP2 in vascular endothelial cells (ECs). Our results showed that sterol depletion activated SREBP2 and increased its target, low density lipoprotein receptor mRNA, with a concurrent decrease in the ABCA1 mRNA. Transient transfection analysis revealed that sterol depletion decreased the ABCA1 promoter activity by 50%, but low density lipoprotein receptor promoter- and the sterol-responsive element-driven luciferase activities were increased. Overexpression of the N terminus of SREBP2 (SREBP2(N)), an active form of SREBP2, also inhibited the ABCA1 promoter activity. Functionally adenovirus-mediated SREBP2(N) expression increased cholesterol accumulation and decreased apoA-I-mediated cholesterol efflux. The conserved E-box motif was responsible for the SREBP2(N)-mediated inhibition since mutation of the E-box increased the basal activity of the ABCA1 promoter and abolished the inhibitory effect of SREBP2(N). Furthermore sterol depletion and SREBP2(N) overexpression induced the binding of SREBP2(N) to both consensus and ABCA1-specific E-box. Chromatin immunoprecipitation assay demonstrated that serum starvation enhanced the association of SREBP2 and the ABCA1 promoter in ECs. To correlate this mechanism pathophysiologically, we found that oscillatory flow caused the activation of SREBP2 and therefore attenuated ABCA1 promoter activity in ECs. Thus, this SREBP-regulated mechanism may control the efflux of cholesterol, which is a newly defined function of SREBP2 in ECs in addition to its role in cholesterol uptake and biosynthesis.     

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.     

LXR-Mediated ABCA1 Expression and Function Are Modulated by High Glucose and PRMT2

High cholesterol and diabetes are major risk factors for atherosclerosis. Regression of atherosclerosis is mediated in part by the Liver X Receptor (LXR) through the induction of genes involved in cholesterol transport and efflux. In the context of diabetes, regression of atherosclerosis is impaired. We proposed that changes in glucose levels modulate LXR-dependent gene expression. Using a mouse macrophage cell line (RAW 264.7) and primary bone marrow derived macrophages (BMDMs) cultured in normal or diabetes relevant high glucose conditions we found that high glucose inhibits the LXR-dependent expression of ATP-binding cassette transporter A1 (ABCA1), but not ABCG1. To probe for this mechanism, we surveyed the expression of a host of chromatin-modifying enzymes and found that Protein Arginine Methyltransferase 2 (PRMT2) was reduced in high compared to normal glucose conditions. Importantly, ABCA1 expression and ABCA1-mediated cholesterol efflux were reduced in Prmt2 ^-/- compared to wild type BMDMs. Monocytes from diabetic mice also showed decreased expression of Prmt2 compared to non-diabetic counterparts. Thus, PRMT2 represents a glucose-sensitive factor that plays a role in LXR-mediated ABCA1-dependent cholesterol efflux and lends insight to the presence of increased atherosclerosis in diabetic patients.     

PLK1 promotes cholesterol efflux and alleviates atherosclerosis by up-regulating ABCA1 and ABCG1 expression via the AMPK/PPARγ/LXRα pathway

Polo-like kinase 1 (PLK1) is a serine/threonine kinase involving lipid metabolism and cardiovascular disease. However, its role in atherogenesis has yet to be determined. The aim of this study was to observe the impact of PLK1 on macrophage lipid accumulation and atherosclerosis development and to explore the underlying mechanisms. We found a significant reduction of PLK1 expression in lipid-loaded macrophages and atherosclerosis model mice. Lentivirus-mediated overexpression of PLK1 promoted cholesterol efflux and inhibited lipid accumulation in THP-1 macrophage-derived foam cells. Mechanistic analysis revealed that PLK1 stimulated the phosphorylation of AMP-activated protein kinase (AMPK), leading to activation of the peroxisome proliferator-activated receptor γ (PPARγ)/liver X receptor α (LXRα) pathway and up-regulation of ATP binding cassette transporter A1 (ABCA1) and ABCG1 expression. Injection of lentiviral vector expressing PLK1 increased reverse cholesterol transport, improved plasma lipid profiles and decreased atherosclerotic lesion area in apoE-deficient mice fed a Western diet. PLK1 overexpression also facilitated AMPK and HSL phosphorylation and enhanced the expression of PPARγ, LXRα, ABCA1, ABCG1 and LPL in the aorta. In summary, these data suggest that PLK1 inhibits macrophage lipid accumulation and mitigates atherosclerosis by promoting ABCA1- and ABCG1-dependent cholesterol efflux via the AMPK/PPARγ/LXRα pathway.     

ABCG1 mediated oxidized LDL-derived oxysterol efflux from macrophages

Objectives

The uptake of oxidized LDL (oxLDL) by macrophages is a key initial event in atherogenesis, and the removal of oxidized lipids from artery wall via reverse cholesterol transport is considered antiatherogenic. The aims of this study were to investigate the pathways mediating the removal of oxysterols from oxLDL-loaded macrophages, and the subsequent uptake of the oxysterols by hepatocytes.

Methods

LDL was labeled with [3H]cholesterol, and LDL-[3H]cholesterol was oxidized by copper using a standard method. [3H]oxysterol formation in oxLDL was analyzed by thin layer chromatography. oxLDL-[3H]sterol was incubated with macrophages, allowing the uptake of [3H]sterol by macrophages. [3H]sterol efflux from macrophages mediated by ATP binding cassette transporters (ABCA1, ABCG1), or scavenger receptor class B type I (SR-BI) was measured. The subsequent uptake of the [3H]sterol by hepatocytes was also determined.

Results

7-Ketocholesterol was the major oxysterol formed in oxLDL, and it was significantly higher in oxLDL compared with that in native LDL (naLDL). oxLDL-derived sterol efflux to HDL from macrophages was significantly increased compared with naLDL-derived sterol, and it was mainly mediated by ABCG1, but not by ABCA1 or SR-BI. Moreover, although HDL dose-dependently induced sterol efflux from macrophages, only the exported sterol by ABCG1 pathway was efficiently taken up by hepatocytes.

Conclusions

ABCG1 mediates oxysterol efflux from oxLDL-loaded macrophages, and the exported oxysterol by ABCG1 pathway can be selectively taken up by hepatocytes.     

Correction of apolipoprotein A-I-mediated lipid efflux and high density lipoprotein particle formation in human Niemann-Pick type C disease fibroblasts

Impaired cell cholesterol trafficking in Niemann-Pick type C (NPC) disease results in the first known instance of impaired regulation of the ATP-binding cassette transporter A1 (ABCA1), a lipid transporter mediating the rate-limiting step in high density lipoprotein (HDL) formation, as a cause of low plasma HDL-cholesterol in humans. We show here that treatment of human NPC1(-/-) fibroblasts with the liver X receptor (LXR) agonist TO-901317 increases ABCA1 expression and activity in human NPC1(-/-) fibroblasts, as indicated by near normalization of efflux of radiolabeled phosphatidylcholine and a marked increase in efflux of cholesterol mass to apoA-I. LXR agonist treatment prior to and during apoA-I incubation resulted in reduction in filipin staining of unesterified cholesterol in late endosomes/lysosomes, as well as cholesterol mass, in NPC1(-/-) cells. HDL species in human NPC disease plasma showed the same pattern of diminished large, cholesterol-rich alpha-1 HDL particles as seen in isolated heterozygous ABCA1 deficiency. Incubating NPC1(-/-) fibroblasts with the LXR agonist normalized the pattern of HDL particle formation by these cells. ABCG1, another LXR target gene involved in cholesterol efflux to HDL, also showed diminished expression in NPC1(-/-) fibroblasts and increased expression upon LXR agonist treatment. These results suggest that NPC1 mutations can be largely bypassed and that NPC1 protein function is non-essential for the trafficking and removal of cellular cholesterol if the down-stream defects in ABCA1 and ABCG1 regulation in NPC disease cells are corrected using an LXR agonist.     

Expression of ABCG1, but not ABCA1, correlates with cholesterol release by cerebellar astroglia

Central nervous system lipoproteins mediate the exchange of cholesterol between cells and support synaptogenesis and neuronal growth. The primary source of lipoproteins in the brain is astroglia cells that synthesize and secrete apolipoprotein (apo) E in high density lipoprotein-like particles. Small quantities of apoA1, derived from the peripheral circulation, are also present in the brain. In addition to the direct secretion of apoE-containing lipoproteins from astroglia, glia-derived lipoproteins are thought to be formed by cholesterol efflux to extracellular apolipoproteins via ATP-binding cassette (ABC) transporters. We used cultured cerebellar murine astroglia to investigate the relationship among cholesterol availability, apoE secretion, expression of ABCA1 and ABCG1, and cholesterol efflux. In many cell types, cholesterol content, ABCA1 expression, and cholesterol efflux are closely correlated. In contrast, cholesterol enrichment of glia failed to increase ABCA1 expression, although ABCG1 expression and cholesterol efflux to apoA1 were increased. Moreover, the liver X receptor (LXR) agonist TO901317 up-regulated ABCA1 and ABCG1 expression in glia without stimulating cholesterol efflux. Larger lipoproteins were generated when glia were enriched with cholesterol, whereas treatment with the LXR agonist produced smaller particles that were eliminated when the glia were loaded with cholesterol. We also used glia from ApoE(-/-) mice to distinguish between direct lipoprotein secretion and the extracellular generation of lipoproteins. Our observations indicate that partially lipidated apoE, secreted directly by glia, is likely to be the major extracellular acceptor of cholesterol released from glia in a process mediated by ABCG1.