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.     

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.     

Cultured gallbladder epithelial cells synthesize apolipoproteins A-I and E

Gallbladder epithelial cells (GBEC) are exposed to high and fluctuating concentrations of biliary cholesterol on their apical (AP) surface. GBEC absorb and efflux cholesterol, but the mechanisms of cholesterol uptake, intracellular trafficking, and efflux in these cells are not known. We previously reported that ATP binding cassette (ABC)A1 mediates basolateral (BL) cholesterol efflux in cultured polarized GBEC. In addition, the nuclear hormone receptors liver X receptor (LXR)alpha and retinoid X receptor (RXR) mediate both AP and BL cholesterol efflux. An interesting finding from our previous study was that apolipoprotein (apo)A-I applied to the AP surfaces of cells elicited BL ABCA1-mediated cholesterol efflux. Because ABCA1-mediated cholesterol efflux requires the presence of a cholesterol acceptor, we hypothesized that GBEC synthesize and secrete endogenous apo into the BL compartment. Here, we demonstrate that cholesterol loading of cells with model bile and AP apoA-I treatment is associated with an increase in the synthesis of apoE mRNA and protein. Furthermore, apoE is secreted into the BL compartment. LXRalpha/RXR ligands stimulate the synthesis of endogenous apoA-I mRNA and protein, as well as apoE mRNA. BL secretion of apoA-I is elicited by LXRalpha/RXR ligands. Therefore, GBEC synthesize apoA-I and -E and efflux cholesterol using ABCA1- and non-ABCA1- mediated pathways. These processes may alter gallbladder biliary cholesterol concentrations and thereby influence gallstone formation.     

Regulation of ABCA1 expression in human keratinocytes and murine epidermis

Keratinocytes require abundant cholesterol for cutaneous permeability barrier function; hence, the regulation of cholesterol homeostasis is of great importance. ABCA1 is a membrane transporter responsible for cholesterol efflux and plays a pivotal role in regulating cellular cholesterol levels. We demonstrate that ABCA1 is expressed in cultured human keratinocytes (CHKs) and murine epidermis. Liver X receptor (LXR) activation markedly stimulates ABCA1 mRNA and protein levels in CHKs and mouse epidermis. In addition to LXR, activators of peroxisome proliferator-activated receptor (PPAR)alpha, PPARbeta/delta, and retinoid X receptor (RXR), but neither PPARgamma nor retinoic acid receptor, also increase ABCA1 expression in CHKs. Increases in cholesterol supply induced by LDL or mevalonate stimulate ABCA1 expression, whereas inhibiting cholesterol synthesis with statins or cholesterol sulfate decreases ABCA1 expression in CHKs. After acute permeability barrier disruption by either tape-stripping or acetone treatment, ABCA1 expression declines, and this attenuates cellular cholesterol efflux, making more cholesterol available for regeneration of the barrier. In addition, during fetal epidermal development, ABCA1 expression decreases at days 18-22 of gestation (term = 22 days), leaving more cholesterol available during the critical period of barrier formation. Together, our results show that ABCA1 is expressed in keratinocytes, where it is negatively regulated by a decrease in cellular cholesterol levels or altered permeability barrier requirements and positively regulated by activators of LXR, PPARs, and RXR or increases in cellular cholesterol levels.     

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.     

24(S)‐hydroxycholesterol is actively eliminated from neuronal cells by ABCA1

High cholesterol turnover catalyzed by cholesterol 24‐hydroxylase is essential for neural functions, especially learning. Because 24(S)‐hydroxycholesterol (24‐OHC), produced by 24‐hydroxylase, induces apoptosis of neuronal cells, it is vital to eliminate it rapidly from cells. Here, using differentiated SH‐SY5Y neuron‐like cells as a model, we examined whether 24‐OHC is actively eliminated via transporters induced by its accumulation. The expression of ABCA1 and ABCG1 was induced by 24‐OHC, as well as TO901317 and retinoic acid, which are ligands of the nuclear receptors liver X receptor/retinoid X receptor (LXR/RXR). When the expression of ABCA1 and ABCG1 was induced, 24‐OHC efflux was stimulated in the presence of high‐density lipoprotein (HDL), whereas apolipoprotein A‐I was not an efficient acceptor. The efflux was suppressed by the addition of siRNA against ABCA1, but not by ABCG1 siRNA. To confirm the role of each transporter, we analyzed human embryonic kidney 293 cells stably expressing human ABCA1 or ABCG1; we clearly observed 24‐OHC efflux in the presence of HDL, whereas efflux in the presence of apolipoprotein A‐I was marginal. Furthermore, the treatment of primary cerebral neurons with LXR/RXR ligands suppressed the toxicity of 24‐OHC. These results suggest that ABCA1 actively eliminates 24‐OHC in the presence of HDL as a lipid acceptor and protects neuronal cells.     

A liver X receptor and retinoid X receptor heterodimer mediates apolipoprotein E expression, secretion and cholesterol homeostasis in astrocytes

Apolipoprotein E (apoE) is an important protein involved in lipoprotein clearance and cholesterol redistribution. ApoE is abundantly expressed in astrocytes in the brain and is closely linked to the pathogenesis of Alzheimer's disease (AD). We report here that small molecule ligands that activate either liver X receptors (LXR) or retinoid X receptor (RXR) lead to a dramatic increase in apoE mRNA and protein expression as well as secretion of apoE in a human astrocytoma cell line (CCF-STTG1 cells). Examination of primary mouse astrocytes also revealed significant induction of apoE mRNA, and protein expression and secretion following incubation with LXR/RXR agonists. Moreover, treatment of mice with a specific synthetic LXR agonist T0901317 resulted in up-regulation of apoE mRNA and protein in both hippocampus and cerebral cortex, indicating that apoE expression in brain can be up-regulated by LXR agonists in vivo. Along with a dramatic induction of ABCA1 cholesterol transporter expression, these ligands effectively mediate cholesterol efflux in both CCF-STTG1 cells and mouse astrocytes in the presence or absence of apolipoprotein AI (apoAI). Our studies provide strong evidence that small molecule LXR/RXR agonists can effectively mediate apoE synthesis and secretion as well as cholesterol homeostasis in astrocytes. LXR/RXR agonists may have significant impact on the pathogenesis of multiple neurological diseases, including AD.     

Hormone-sensitive lipase deficiency affects the expression of SR-BI,LDLr, and ABCA1 receptors/transporters involved in cellular cholesterol uptake and efflux and disturbs fertility in mouse testis

Hormone-sensitive lipase (HSL) hydrolyse acylglycerols, cholesteryl and retinyl esters. HSL is a key lipase in mice testis, as HSL deficiency results in male sterility. The present work study the effects of the deficiency and lack of HSL on the localization and expression of SR-BI, LDLr, and ABCA1 receptors/transporters involved in uptake and efflux of cholesterol in mice testis, to determine the impact of HSL gene dosage on testis morphology, lipid homeostasis and fertility. The results of this work show that the lack of HSL in mice alters testis morphology and spermatogenesis, decreasing sperm counts, sperm motility and increasing the amount of Leydig cells and lipid droplets. They also show that there are differences in the localization of HSL, SR-BI, LDLr and ABCA1 in HSL^+/+, HSL^+/− and HSL^−/− mice. The deficiency or lack of HSL has effects on protein and mRNA expression of genes involved in lipid metabolisms in mouse testis. HSL^−/− testis have augmented expression of SR-BI, LDLr, ABCA1 and LXRβ, a critical sterol sensor that regulate multiple genes involved in lipid metabolism; whereas LDLr expression decreased in HSL^+/− mice. Plin2, Abca1 and Ldlr mRNA levels increased; and LXRα (Nr1h3) and LXRβ (Nr1h2) decreased in testis from HSL^−/− compared with HSL^+/+; with no differences in Scarb1. Together these data suggest that HSL deficiency or lack in mice testis induces lipid homeostasis alterations that affect the cellular localization and expression of key receptors/transporter involved in cellular cholesterol uptake and efflux (SR-BI, LDRr, ABCA1); alters normal cellular function and impact fertility.     

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.     

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.     

Nascent HDL formation in hepatocytes and role of ABCA1, ABCG1, and SR-BI

To study the mechanisms of hepatic HDL formation, we investigated the roles of ABCA1, ABCG1, and SR-BI in nascent HDL formation in primary hepatocytes isolated from mice deficient in ABCA1, ABCG1, or SR-BI and from wild-type (WT) mice. Under basal conditions, in WT hepatocytes, cholesterol efflux to exogenous apoA-I was accompanied by conversion of apoA-I to HDL-sized particles. LXR activation by T0901317 markedly enhanced the formation of larger HDL-sized particles as well as cellular cholesterol efflux to apoA-I. Glyburide treatment completely abolished the formation of 7.4 nm diameter and greater particles but led to the formation of novel 7.2 nm-sized particles. However, cells lacking ABCA1 failed to form such particles. ABCG1-deficient cells showed similar capacity to efflux cholesterol to apoA-I and to form nascent HDL particles compared with WT cells. Cholesterol efflux to apoA-I and nascent HDL formation were slightly but significantly enhanced in SR-BI-deficient cells compared with WT cells under basal but not LXR activated conditions. As in WT but not in ABCA1-deficient hepatocytes, 7.2 nm-sized particles generated by glyburide treatment were also detected in ABCG1-deficient and SR-BI-deficient hepatocytes. Our data indicate that hepatic nascent HDL formation is highly dependent on ABCA1 but not on ABCG1 or SR-BI.     

Genetic deletion of low density lipoprotein receptor impairs sterol-induced mouse macrophage ABCA1 expression. A new SREBP1-dependent mechanism

Low density lipoprotein receptor (LDLR) mutations cause familial hypercholesterolemia and early atherosclerosis. ABCA1 facilitates free cholesterol efflux from peripheral tissues. We investigated the effects of LDLR deletion (LDLR(-/-)) on ABCA1 expression. LDLR(-/-) macrophages had reduced basal levels of ABCA1, ABCG1, and cholesterol efflux. A high fat diet increased cholesterol in LDLR(-/-) macrophages but not wild type cells. A liver X receptor (LXR) agonist induced expression of ABCA1, ABCG1, and cholesterol efflux in both LDLR(-/-) and wild type macrophages, whereas expression of LXRalpha or LXRbeta was similar. Interestingly, oxidized LDL induced more ABCA1 in wild type macrophages than LDLR(-/-) cells. LDL induced ABCA1 expression in wild type cells but inhibited it in LDLR(-/-) macrophages in a concentration-dependent manner. However, lipoproteins regulated ABCG1 expression similarly in LDLR(-/-) and wild type macrophages. Cholesterol or oxysterols induced ABCA1 expression in wild type macrophages but had little or inhibitory effects on ABCA1 expression in LDLR(-/-) macrophages. Active sterol regulatory element-binding protein 1a (SREBP1a) inhibited ABCA1 promoter activity in an LXRE-dependent manner and decreased both macrophage ABCA1 expression and cholesterol efflux. Expression of ABCA1 in animal tissues was inversely correlated to active SREBP1. Oxysterols inactivated SREBP1 in wild type macrophages but not in LDLR(-/-) cells. Oxysterol synergized with nonsteroid LXR ligand induced ABCA1 expression in wild type macrophages but blocked induction in LDLR(-/-) cells. Taken together, our studies suggest that LDLR is critical in the regulation of cholesterol efflux and ABCA1 expression in macrophage. Lack of the LDLR impairs sterol-induced macrophage ABCA1 expression by a sterol regulatory element-binding protein 1-dependent mechanism that can result in reduced cholesterol efflux and lipid accumulation in macrophages under hypercholesterolemic conditions.     

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.     

Dominant expression of ATP-binding cassette transporter-1 on basolateral surface of Caco-2 cells stimulated by LXR/RXR ligands

ATP-binding cassette transporter-1 (ABCA1) is a cause of Tangier disease, which is a familial deficiency of plasma high density lipoproteins (HDL). This molecule is known to be expressed in the multiple tissues and organs including small intestines, liver, and macrophages in the blood vessels. Recent in vivo studies suggested that ABCA1 plays some roles in the flux of cholesterol in the intestines. One of the major questions to understand the roles of ABCA1 in the intestines is the expression pattern in the intestinal epithelial cells. To address this issue, we have investigated the expression and regulation of ABCA1 in Caco-2 cells cultured on Transwell as a model, especially focusing on possible polarized expression of ABCA1. The expression of ABCA1 was up-regulated during the differentiation and under the stimulation of LXR/RXR by the addition of 9-cis-retinoic acid (9-cis-RA) and 22-R-hydroxycholesterol (22-OH). Apolipoprotein-AI-mediated cholesterol efflux was dominant toward the basolateral side of polarized cells when stimulated by 9-cis-RA and 22-OH. The cell surface biotinylation experiment followed by Western blot analyses demonstrated a markedly dominant expression of ABCA1 on the basolateral surface, which was clearly confirmed by the confocal laser scanning microscopy. In conclusion, the present study demonstrates that ABCA1 is dominantly expressed on the basolateral surface of Caco-2 cells tested, suggesting that this molecule may play a role in the basolateral movement of cholesterol at least when stimulated by LXR/RXR ligands.