Serum amyloid A promotes ABCA1-dependent and ABCA1-independent lipid efflux from cells

Serum amyloid A (SAA) is an acute phase protein that associates with HDL. In order to examine the role of SAA in reverse-cholesterol transport, lipid efflux was tested to SAA from HeLa cells before and after transfection with the ABCA1 transporter. ABCA1 expression increased efflux of cholesterol and phospholipid to SAA by 3-fold and 2-fold, respectively. In contrast to apoA-I, SAA also removed lipid without ABCA1; cholesterol efflux from control cells to SAA was 10-fold higher than for apoA-I. Furthermore, SAA effluxed cholesterol from Tangier disease fibroblasts and from cells after inhibition of ABCA1 by fixation with paraformaldehyde. In summary, SAA can act as a lipid acceptor for ABCA1, but unlike apoA-I, it can also efflux lipid without ABCA1, by most likely a detergent-like extraction process. These results suggest that SAA may play a unique role as an auxiliary lipid acceptor in the removal of lipid from sites of inflammation.     

The establishment of telomerase-immortalized Tangier disease cell lines indicates the existence of an apolipoprotein A-I-inducible but ABCA1-independent cholesterol efflux pathway

Tangier disease (TD) is a human genetic disorder associated with defective apolipoprotein-I-induced lipid efflux and increased atherosclerotic susceptibility. It has been linked to mutations in the ATP-binding cassette protein A1 (ABCA1). Here we describe the establishment of permanent Tangier cell lines using telomerase. Ectopic expression of the catalytic subunit of human telomerase extended the life span of control and TD skin fibroblasts, and (in contrast to immortalization procedures using viral oncogenes) did not impair apolipoprotein A-I-induced lipid efflux. The key characteristics of TD fibroblasts (reduced cholesterol and phospholipid efflux) were observed both in primary and telomerase-immortalized fibroblasts from two unrelated homozygous patients. Surprisingly, the apolipoprotein-inducible cholesterol efflux in TD cells was significantly improved after immortalization (up to 40% of normal values). In contrast to ABCA1-dependent cholesterol efflux, this efflux was not inhibited by brefeldin A, glybenclamide, or intracellular ATP depletion but was inhibited in the presence of cytochalasin D. Apolipoprotein A-I-dependent cholesterol efflux was inversely correlated with the population doubling number in cell culture and was inhibited up to 40% in near-senescent normal diploid fibroblasts. This inhibition was completely reversed by telomerase. Thus ectopic expression of telomerase is a way to circumvent the lack of critical experimental material and represents a major improvement for studying cholesterol efflux pathways in lipid disorders. Our findings indicate the existence of an ABCA1-independent but cytoskeleton-dependent cholesterol removal pathway that may help to prevent early atherosclerosis in Tangier disease but may also be sensitive to aging phenomena ex vivo and possibly in vivo.     

ATP binding cassette transporter A1 - key roles in cellular lipid transport and atherosclerosis

ATP-binding cassette transporter A1 (ABCA1) was recently recognized as the mutant molecule responsible for Tangier disease with low HDL levels, accumulation of cholesteryl esters in tissues, and increased risk of cardiovascular disease. Extensive studies for the past 2 years have recognized the critical role of ABCA1 in cholesterol and phospholipid trafficking. Since the removal of cholesterol from tissues is a key step in the prevention of atherosclerosis, significant attention has been focused on this molecule. Natural ABCA1 mutations in Tangier disease (TD) patients and WHAM chickens together with induced mutation in ABCA1 knock-out mice unequivocally established the important role of ABCA1 in maintaining circulating HDL levels and promoting cholesterol efflux from the arterial wall. Mice lacking ABCA1 showed similar phenotypes observed in Tangier disease patients with low levels of HDL. Further understanding of the roles of ABCA1 in lipid transport and atherosclerosis became clear from studies with ABCA1 transgenic mice. These mice showed enhanced cholesterol efflux from macrophages and reduced atherosclerotic lesion formation. The promoter of the ABCA1 gene has been mapped to a large extent, with the exception of cAMP response element. The present review summarizes recent developments on the role of ABCA1 in cholesterol efflux and prevention of atherosclerosis. Given the antiatherogenic properties of ABCA1, this molecule can serve as an appropriate target for developing drugs to treat individuals with low levels of HDL.     

The impact of severe LDL receptor mutations on SREBP-pathway regulation in homozygous familial hypercholesterolemia (FH)

Familial hypercholesterolemia (FH), Niemann-Pick disease type C (NPC) and Tangier disease (TD) are genetic inherited disorders with impaired processing of cholesterol, caused by mutations in genes that regulate cellular uptake, intracellular movement and transport of cholesterol. Various studies have shown a crucial regulatory role of the SREBP-pathway for cellular cholesterol homeostasis in these diseases. Since cholesterol is an essential structural component of cells, we assessed the impact of a severe FH causing LDLR mutation (FH p.W556R) on the SREBP pathway in primary FH fibroblasts. Primary FH fibroblasts derived from patients with the LDL receptor mutation p.W556R were used for gene expression experiments. Gene expression studies revealed increased expressions of SREBP regulated genes HMGCR, LDLR, SREBP-2, SREBP-1, SR-BI, INSIG-1, but interestingly not SCAP. In contrast expression of ABCA1, was strongly decreased in homozygous, but not in heterozygous p.W556R fibroblasts. Gene expression experiments with LDL receptor lacking primary FH fibroblasts, revealed that SR-BI and ABCA1 are important regulators for cholesterol acquisition in FH cells, consistent with findings in cells from NPC and TD patients.     

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.     

A novel splicing mutation in the ABCA1 gene,causing Tangier disease and familial HDL deficiency in a large family

Tangier disease is a rare disorder of lipoprotein metabolism that presents with extremely low levels of HDL cholesterol and apoprotein A-I. It is caused by mutations in the ATP-binding cassette transporter A1 (ABCA1) gene. Clinical heterogeneity and mutational pattern of Tangier disease are poorly characterized. Moreover, also familial HDL deficiency may be caused by mutations in ABCA1 gene.ATP-binding cassette transporter A1 (ABCA1) gene mutations in a patient with Tangier disease, who presented an uncommon clinical history, and in his family were found and characterized. He was found to be compound heterozygous for two intronic mutations of ABCA1 gene, causing abnormal pre-mRNAs splicing. The novel c.1510-1G?>?A mutation was located in intron 12 and caused the activation of a cryptic splice site in exon 13, which determined the loss of 22 amino acids of exon 13 with the introduction of a premature stop codon. Five heterozygous carriers of this mutation were also found in proband's family, all presenting reduced HDL cholesterol and ApoAI (0.86?±?0.16?mmol/L and 92.2?±?10.9?mg/dL respectively), but not the typical features of Tangier disease, a phenotype compatible with the diagnosis of familial HDL deficiency. The other known mutation c.1195-27G?>?A was confirmed to cause aberrant retention of 25 nucleotides of intron 10 leading to the insertion of a stop codon after 20 amino acids of exon 11. Heterozygous carriers of this mutation also showed the clinical phenotype of familial HDL deficiency.Our study extends the catalog of pathogenic intronic mutations affecting ABCA1 pre-mRNA splicing. In a large family, a clear demonstration that the same mutations may cause Tangier disease (if in compound heterozygosis) or familial HDL deficiency (if in heterozygosis) is provided.     

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.     

Two novel missense mutations in ABCA1 result in altered trafficking and cause severe autosomal recessive HDL deficiency

Extremely low concentrations of high density lipoprotein (HDL)-cholesterol and apolipoprotein (apo) AI are features of Tangier disease caused by autosomal recessive mutations in ATP-binding cassette transporter A1 (ABCA1). Less deleterious, but dominantly inherited mutations cause HDL deficiency. We investigated causes of severe HDL deficiency in a 42-year-old female with progressive coronary disease. ApoAI-mediated efflux of cholesterol from the proband's fibroblasts was less than 10% of normal and nucleotide sequencing revealed inheritance of two novel mutations in ABCAI, V1704D and L1379F. ABCA1 mRNA was approximately 3-fold higher in the proband's cells than in control cells; preincubation with cholesterol increased it 5-fold in control and 8-fold in the proband's cells, but similar amounts of ABCA1 protein were present in control and mutant cells. When transiently transfected into HEK293 cells, confocal microscopy revealed that both mutant proteins were retained in the endoplasmic reticulum, while wild-type ABCA1 was located at the plasma membrane. Severe HDL deficiency in the proband was caused by two novel autosomal recessive mutations in ABCA1, one (V1704D) predicted to lie in a transmembrane segment and the other (L1379F) in a large extracellular loop. Both mutations prevent normal trafficking of ABCA1, thereby explaining their inability to mediate apoA1-dependent lipid efflux.     

The ABCA1 transporter modulates late endocytic trafficking: insights from the correction of the genetic defect in Tangier disease

We have previously established that the ABCA1 transporter, which plays a critical role in the lipidation of extracellular apolipoprotein acceptors, traffics between late endocytic vesicles and the cell surface (Neufeld, E. B., Remaley, A. T., Demosky, S. J., Jr., Stonik, J. A., Cooney, A. M., Comly, M., Dwyer, N. K., Zhang, M., Blanchette-Mackie, J., Santamarina-Fojo, S., and Brewer, H. B., Jr. (2001) J. Biol. Chem. 276, 27584-27590). The present study provides evidence that ABCA1 in late endocytic vesicles plays a role in cellular lipid efflux. Late endocytic trafficking was defective in Tangier disease fibroblasts that lack functional ABCA1. Consistent with a late endocytic protein trafficking defect, the hydrophobic amine U18666A retained NPC1 in abnormally tubulated, cholesterol-poor, Tangier disease late endosomes, rather than cholesterol-laden lysosomes, as in wild type fibroblasts. Consistent with a lipid trafficking defect, Tangier disease late endocytic vesicles accumulated both cholesterol and sphingomyelin and were immobilized in a perinuclear localization. The excess cholesterol in Tangier disease late endocytic vesicles retained massive amounts of NPC1, which traffics lysosomal cholesterol to other cellular sites. Exogenous apoA-I abrogated the cholesterol-induced retention of NPC1 in wild type but not in Tangier disease late endosomes. Adenovirally mediated ABCA1-GFP expression in Tangier disease fibroblasts corrected the late endocytic trafficking defects and restored apoA-I-mediated cholesterol efflux. ABCA1-GFP expression in wild type fibroblasts also reduced late endosome-associated NPC1, induced a marked uptake of fluorescent apoA-I into ABCA1-GFP-containing endosomes (that shuttled between late endosomes and the cell surface), and enhanced apoA-I-mediated cholesterol efflux. The combined results of this study suggest that ABCA1 converts pools of late endocytic lipids that retain NPC1 to pools that can associate with endocytosed apoA-I, and be released from the cell as nascent high density lipoprotein.     

Tangier disease and ABCA1

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

Possibility of increasing cholesterol efflux by adiponectin and its receptors through the ATP binding cassette transporter A1 in HEK293T cells

A decrease in adiponectin secretion leads to the early stage of atherosclerosis. Discoidal high-density lipoproteins (HDL) accept the cholesterol that effluxes from cells expressing the ATP binding cassette transporter A1 (ABCA1) in the first step of reverse cholesterol transport (RCT). Recently, a new therapeutic strategy involving reconstituted (r)HDL has been shown to enhance RCT. Therefore, we hypothesized that adiponectin may increase the efflux associated with ABCA1 and also enhance rHDL-induced efflux in human embryonic kidney 293 (HEK293T) cells. We transfected adiponectin receptor 1 and 2 (AdipoR1 and AdipoR2) cDNA into cells. The transfected cells were labeled with [³H]cholesterol following cholesterol loading with or without adiponectin for 24 h. The levels of cholesterol efflux were analyzed using a liquid scintillation counter. Treatment with adiponectin was associated with significantly higher levels of efflux in AdipoR1- and AdipoR2-transfected cells. Interestingly, rHDL-induced cholesterol efflux was enhanced in the presence of adiponectin. The down-regulation of adiponectin receptors using short-hairpin RNA decreased rHDL-induced cholesterol efflux with the down-regulation of ABCA1. In summary, adiponectin and its receptors increased cholesterol efflux and also enhanced rHDL-induced efflux at least partially through an ABCA1 pathway. These results suggest that adiponectin may enhance the RCT system and induce an anti-atherogenic effect.     

Accumulation of cardiolipin and lysocardiolipin in fibroblasts from Tangier disease subjects

Tangier disease (TD) is an inherited disorder of lipid metabolism characterized by very low high density lipoprotein (HDL) plasma levels, cellular cholesteryl ester accumulation and reduced cholesterol excretion in response to HDL apolipoproteins. Molecular defects in the ATP binding cassette transporter 1 (ABCA1) have recently been identified as the cause of TD. ABCA1 plays a key role in the translocation of cholesterol across the plasma membrane, and defective ABCA1 causes cholesterol storage in TD cells. Not only cholesterol efflux, but also phospholipid efflux was shown to be impaired in TD cells. By use of thin layer chromatography, high performance liquid chromatography and time-of-flight secondary ion mass spectrometry, we characterized the cellular phospholipid content in fibroblasts from three homozygous TD patients. The cellular content of the major phospholipids was not found to be significantly altered in TD fibroblasts. However, the two phospholipids cardiolipin and lysocardiolipin, which make up minute amounts in normal cells, were at least 3–5-fold enriched in fibroblasts from TD subjects. A structurally closely related phospholipid (lysobisphosphatidic acid) has recently been shown to be enriched in Niemann–Pick type C, another lipid storage disorder. Altogether these data may indicate that the role of these phospholipids is a regulatory one rather than that of a bulk mediator of cholesterol solubilization in sterol trafficking and efflux.     

Molecular interactions between apoE and ABCA1: impact on apoE lipidation

Apolipoprotein E (apoE)/ABCA1 interactions were investigated in human intact fibroblasts induced with 22(R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Here, we show that purified human plasma apoE3 forms a complex with ABCA1 in normal fibroblasts. Lipid-free apoE3 inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than reconstituted HDL particles (IC(50) = 2.5 +/- 0.4 microg/ml vs. 12.3 +/- 1.3 microg/ml). ApoE isoforms showed similar binding for ABCA1 and exhibited identical kinetics in their abilities to induce ABCA1-dependent cholesterol efflux. Mutation of ABCA1 associated with Tangier disease (C1477R) abolished both apoE3 binding and apoE3-mediated cholesterol efflux. Analysis of apoE3-containing particles generated during the incubation of lipid-free apoE3 with stimulated normal cells showed nascent apoE3/cholesterol/phospholipid complexes that exhibited prebeta-electrophoretic mobility with a particle size ranging from 9 to 15 nm, whereas lipid-free apoE3 incubated with ABCA1 mutant (C1477R) cells was unable to form such particles. These results demonstrate that 1). apoE association with lipids reduced its ability to interact with ABCA1; 2). apoE isoforms did not affect apoE binding to ABCA1; 3). apoE-mediated ABCA1-dependent cholesterol efflux was not affected by apoE isoforms in fibroblasts; and 4). the lipid translocase activity of ABCA1 generates apoE-containing high density-sized lipoprotein particles. Thus, ABCA1 is essential for the biogenesis of high density-sized lipoprotein containing only apoE particles in vivo.     

Sphingomyelin Depletion Impairs Anionic Phospholipid Inward Translocation and Induces Cholesterol Efflux

The phosphatidylserine (PS) floppase activity (outward translocation) of ABCA1 leads to plasma membrane remodeling that plays a role in lipid efflux to apolipoprotein A-I (apoAI) generating nascent high density lipoprotein. The Tangier disease W590S ABCA1 mutation has defective PS floppase activity and diminished cholesterol efflux activity. Here, we report that depletion of sphingomyelin by inhibitors or sphingomyelinase caused plasma membrane remodeling, leading to defective flip (inward translocation) of PS, higher PS exposure, and higher cholesterol efflux from cells by both ABCA1-dependent and ABCA1-independent mechanisms. Mechanistically, sphingomyelin was connected to PS translocation in cell-free liposome studies that showed that sphingomyelin increased the rate of spontaneous PS flipping. Depletion of sphingomyelin in stably transfected HEK293 cells expressing the Tangier disease W590S mutant ABCA1 isoform rescued the defect in PS exposure and restored cholesterol efflux to apoAI. Liposome studies showed that PS directly increased cholesterol accessibility to extraction by cyclodextrin, providing the mechanistic link between cell surface PS and cholesterol efflux. We conclude that altered plasma membrane environment conferred by depleting sphingomyelin impairs PS flip and promotes cholesterol efflux in ABCA1-dependent and -independent manners.     

ABCA1. The gatekeeper for eliminating excess tissue cholesterol

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

Apolipoprotein A-I activates cellular cAMP signaling through the ABCA1 transporter

It has been suggested that the signal transduction pathway initiated by apoA-I activates key proteins involved in cellular lipid efflux. We investigated apoA-I-mediated cAMP signaling in cultured human fibroblasts induced with (22R)-hydroxycholesterol and 9-cis-retinoic acid (stimulated cells). Treatment of stimulated fibroblasts with apoA-I for short periods of time (相似文献   

Accumulation of RhoA, RhoB, RhoG, and Rac1 in fibroblasts from Tangier disease subjects suggests a regulatory role of Rho family proteins in cholesterol efflux

Tangier disease (TD) is an inherited disorder of lipid metabolism characterized by very low high density lipoprotein (HDL) plasma levels, cellular cholesteryl ester accumulation and reduced cholesterol excretion in response to HDL apolipoproteins. Molecular defects in the ATP binding cassette transporter 1 (ABCA1) have recently been identified as the cause of TD. ABCA1 plays a key role in the translocation of cholesterol across the plasma membrane, and defective ABCA1 causes cholesterol storage in TD cells. However, the exact relationship of many of the biochemical and morphological abnormalities in TD to ABCA1 is unknown. Since small GTP-binding proteins are important regulators of many cellular functions, we characterized these proteins in normal and TD fibroblasts using the [alpha-32P]GTP overlay technique and Western blotting of SDS and isoelectric focusing gels. Our results indicate that GTP-binding proteins of the Rho family (RhoA, RhoB, RhoG, Rac-1) are enriched in fibroblasts from TD patients. The accumulation of small G proteins may have potential implications for the TD phenotype and the regulation of cholesterol excretion in TD cells.     

ABCA1-dependent mobilization of lysosomal cholesterol requires functional Niemann-Pick C2 but not Niemann-Pick C1 protein

Niemann-Pick disease type C (NPC) is caused by mutations leading to loss of function of NPC1 or NPC2 proteins, resulting in accumulation of unesterified cholesterol in late endosomes and lysosomes. We previously reported that expression of the ATP-binding cassette transporter A1 (ABCA1) is impaired in human NPC1^−/− fibroblasts, resulting in reduced HDL particle formation and providing a mechanism for the reduced plasma HDL cholesterol seen in the majority of NPC1 patients. We also found that treatment of NPC1^−/− fibroblasts with an agonist of liver X-receptor corrects ABCA1 expression and HDL formation and reduces lysosomal cholesterol accumulation. We have confirmed that ABCA1 expression is also reduced in NPC2^−/− cells, and found that α-HDL particle formation is impaired in these cells. To determine whether selective up-regulation of ABCA1 can correct lysosomal cholesterol accumulation in NPC disease cells and HDL particle formation, we produced and infected NPC1^−/− and NPC2^−/− fibroblasts with an adenovirus expressing full-length ABCA1 and enhanced green fluorescent protein (AdABCA1-EGFP). ABCA1-EGFP expression in NPC1^−/− fibroblasts resulted in normalization of cholesterol efflux to apolipoprotein A-I (apoA-I) and α-HDL particle formation, plus a marked reduction in filipin staining of unesterified cholesterol in late endosomes/lysosomes. In contrast, AdABCA1-EGFP treatment of NPC2^−/− fibroblasts to normalize ABCA1 expression had no effect on cholesterol efflux to apoA-I or accumulation of excess cholesterol in lysosomes, and only partially corrected α-HDL formation by these cells. These results suggest that correction of ABCA1 expression can bypass the mutation of NPC1 but not NPC2 to mobilize excess cholesterol from late endosomes and lysosomes in NPC disease cells. Expression of ABCA1-EGFP in NPC1^−/− cells increased cholesterol available for esterification and reduced levels of HMG-CoA reductase protein, effects that were abrogated by co-incubation with apoA-I. A model can be generated in which ABCA1 is able to mobilize cholesterol, to join the intracellular regulatory pool or to be effluxed for HDL particle formation, either directly or indirectly from the lysosomal membrane, but not from the lysosomal lumen. 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).     

Endocytosis is enhanced in Tangier fibroblasts: possible role of ATP-binding cassette protein A1 in endosomal vesicular transport

A human genetic disorder, Tangier disease, has been linked recently to mutations in ATP-binding cassette protein A1 (ABCA1). In addition to its function in apoprotein A-I-mediated lipid removal, ABCA1 was also shown to be a phosphatidylserine (PS) translocase that facilitates PS exofacial flipping. This PS translocation is crucial for the plasma membrane to produce protrusions enabling the engulfment of apoptotic cells. In this report, we show that ABCA1 also plays a role in endocytosis. Receptor-mediated endocytosis, probed by both transferrin and low density lipoprotein, is up-regulated by more than 50% in homozygous Tangier fibroblasts in comparison with controls. Fluid-phase uptake is increased similarly. We also demonstrate that bulk membrane flow, including lipid endocytosis and exocytosis, is accelerated greatly in Tangier cells. Moreover, endocytosis is similarly enhanced in normal fibroblasts when ABCA1 function is inhibited by glyburide, whereas glyburide has no effect on endocytosis in Tangier cells. In addition, we demonstrate a decreased annexin V binding in Tangier fibroblasts as compared with controls, supporting the notion that PS transmembrane distribution is indeed defective in the presence of ABCA1 mutations. Furthermore, adding a PS analog to the exofacial leaflet of the plasma membrane normalizes endocytosis in Tangier cells. Taken together, these data demonstrate that ABCA1 plays an important role in endocytosis. We speculate that this is related to the PS translocase function of ABCA1. A loss of functional ABCA1, as in the case of Tangier cells, enhances membrane inward bending and facilitates endocytosis.     

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.