Serum amyloid A generates high density lipoprotein with cellular lipid in an ABCA1- or ABCA7-dependent manner

Serum amyloid A (SAA) is an amphiphilic helical protein that is found associated with plasma HDL in various pathological conditions, such as acute or chronic inflammation. Cellular lipid release and generation of HDL by this protein were investigated, in comparison with the reactions by apolipoprotein A-I (apoA-I) and several types of cells that appear with various specific profiles of cholesterol and phospholipid release. SAA mediated cellular lipid release from these cells with the same profile as apoA-I. Upregulation of cellular ABCA1 protein by liver X receptor/retinoid X receptor agonists resulted in an increase of cellular lipid release by apoA-I and SAA. SAA reacted with the HEK293-derived clones that stably express human ABCA1 (293/2c) or ABCA7 (293/6c) to generate cholesterol-containing HDL in a similar manner to apoA-I. Dibutyryl cyclic AMP and phorbol 12-myristate 13-acetate, which differentiate apoA-I-mediated cellular lipid release between 293/2c and 293/6c, also exhibited the same differential effects on the SAA-mediated reactions. No evidence was found for the ABCA1/ABCA7-independent lipid release by SAA. Characterization of physicochemical properties of the HDL revealed that SAA-generated HDL particles had higher density, larger diameter, and slower electrophoretic mobility than those generated by apoA-I. These results demonstrate that SAA generates cholesterol-containing HDL directly with cellular lipid and that the reaction is mediated by ABCA1 and ABCA7.     

Effects of mutations of ABCA1 in the first extracellular domain on subcellular trafficking and ATP binding/hydrolysis

ABCA1 mediates release of cellular cholesterol and phospholipid to form high density lipoprotein (HDL). The three different mutants in the first extracellular domain of human ABCA1 associated with Tangier disease, R587W, W590S, and Q597R, were examined for their subcellular localization and function by using ABCA1-GFP fusion protein stably expressed in HEK293 cells. ABCA1-GFP expressed in HEK293 was fully functional for apoA-I-mediated HDL assembly. Immunostaining and confocal microscopic analyses demonstrated that ABCA1-GFP was mainly localized to the plasma membrane (PM) but also substantially in intracellular compartments. All three mutant ABCA1-GFPs showed no or little apoA-I-mediated HDL assembly. R587W and Q597R were associated with impaired processing of oligosaccharide from high mannose type to complex type and failed to be localized to the PM, whereas W590S did not show such dysfunctions. Vanadate-induced nucleotide trapping was examined to elucidate the mechanism for the dysfunction in the W590S mutant. Photoaffinity labeling of W590S with 8-azido-[alpha-(32)P]ATP was stimulated by adding ortho-vanadate in the presence of Mn(2+) as much as in the presence of wild-type ABCA1. These results suggest that the defect of HDL assembly in R587W and Q597R is due to the impaired localization to the PM, whereas W590S has a functional defect other than the initial ATP binding and hydrolysis.     

Intracellular cholesterol mobilization involved in the ABCA1/apolipoprotein-mediated assembly of high density lipoprotein in fibroblasts

Differential regulation has been suggested for cellular cholesterol and phospholipid release mediated by apolipoprotein A-I (apoA-I)/ABCA1. We investigated various factors involved in cholesterol mobilization related to this pathway. ApoA-I induced a rapid decrease of the cellular cholesterol compartment that is in equilibrium with the ACAT-accessible pool in cells that generate cholesterol-rich HDL. Pharmacological and genetic inactivation of ACAT enhanced the apoA-I-mediated cholesterol release through upregulation of ABCA1 and through cholesterol enrichment in the HDL generated. Pharmacological activation of protein kinase C (PKC) also decreased the ACAT-accessible cholesterol pool, not only in the cells that produce cholesterol-rich HDL by apoA-I (i.e., human fibroblast WI-38 cells) but also in the cells that generate cholesterol-poor HDL (mouse fibroblast L929 cells). In L929 cells, the PKC activation caused an increase in apoA-I-mediated cholesterol release without detectable change in phospholipid release and in ABCA1 expression. These results indicate that apoA-I mobilizes intracellular cholesterol for the ABCA1-mediated release from the compartment that is under the control of ACAT. The cholesterol mobilization process is presumably related to PKC activation by apoA-I.     

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

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

Heterogeneity of high density lipoprotein generated by ABCA1 and ABCA7

The assembly of HDL by helical apolipoprotein and cellular lipid was studied using HEK293 cells to which ecdysone-inducible human ABCA1 or human ABCA7 was transfected. Expression of both ABCA1 and ABCA7 was induced linearly proportional to ponasterone A concentration in the medium. In the experimental conditions used, the ABC protein expression levels limited the rate of lipid release when the apolipoprotein concentration was high, and the apolipoprotein concentration was rate-limiting when the ABC protein expression levels were high. When ABCA1 expression increased in conditions in which it was rate-limiting, relative cholesterol content to phospholipid increased in the HDL produced. In contrast, it was constant when ABCA7 expression increased. To investigate the background mechanism, the HDL particles were analyzed by density gradient ultracentrifugation and high performance lipid chromatography. The ABCA1-mediated reaction produced two distinct HDLs, large cholesterol-rich and small cholesterol-poor particles, and the ABCA7-mediated reaction generated mostly small cholesterol-poor particles. The increase of HDL assembly with the increase of ABCA1 expression was predominant in large cholesterol-rich particles, whereas only small cholesterol-poor HDL increased as ABCA7 expression increased. We conclude that ABCA1 generates cholesterol-rich and cholesterol-poor HDL and that the former is more prominently dependent on the increase of ABCA1 expression. ABCA7 produces this HDL subfraction only as a very minor component.     

An inhibitor of acylCoA: cholesterol acyltransferase increases expression of ATP-binding cassette transporter A1 and thereby enhances the ApoA-I-mediated release of cholesterol from macrophages

The effect of inhibition of acylCoA: cholesterol acyltransferase (ACAT) was studied on high density lipoprotein (HDL) metabolism. An inhibitor of ACAT, MCC-147, was given mouse peritoneal macrophages and expression of ATP-binding cassette transporter A1 (ABCA1) was examined. ABCA1 was increased both at the mRNA and protein levels, only when the cells are cholesterol-loaded and thereby the inhibitor decreased esterified cholesterol and increased unesterified cholesterol. In this condition, the ACAT inhibitor increased reversible binding of apoA-I to the cells and enhanced apoA-I-mediated release of cellular cholesterol and phospholipid, but did not influence nonspecific cellular cholesterol efflux to lipid microemulsion. It was therefore concluded that the ACAT inhibitor increased the release of cholesterol from the cholesterol-loaded macrophages by increasing the expression of ABCA1, putatively through shifting cholesterol distribution from the esterified to the free compartments.     

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.     

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   

ATP-binding cassette transporter A1 mediates cellular secretion of alpha-tocopherol

Alpha-tocopherol (alpha-TOH) is associated with plasma lipoproteins and accumulates in cell membranes throughout the body, suggesting that lipoproteins play a role in transporting alpha-TOH between tissues. Here we show that secretion of alpha-TOH from cultured cells is mediated in part by ABCA1, an ATP-binding cassette protein that transports cellular cholesterol and phospholipids to lipid-poor high density lipoprotein (HDL) apolipoproteins such as apoA-I. Treatment of human fibroblasts and murine RAW264 macrophages with cholesterol and/or 8-bromo-cyclic AMP, which induces ABCA1 expression, enhanced apoA-I-mediated alpha-TOH efflux. ApoA-I lacked the ability to remove alpha-TOH from Tangier disease fibroblasts that have a nonfunctional ABCA1. BHK cells that lack an active ABCA1 pathway markedly increased secretion of alpha-TOH to apoA-I when forced to express ABCA1. ABCA1 also mediated a fraction of the alpha-TOH efflux promoted by lipid-containing HDL particles, indicating that HDL promotes alpha-TOH efflux by both ABCA1-dependent and -independent processes. Exposing apoA-I to ABCA1-expressing cells did not enhance its ability to remove alpha-TOH from cells lacking ABCA1, consistent with this transporter participating directly in the translocation of alpha-TOH to apolipoproteins. These studies provide evidence that ABCA1 mediates secretion of cellular alpha-TOH into the HDL metabolic pathway, a process that may facilitate vitamin transport between tissues and influence lipid oxidation.     

Cholesterol transport via ABCA1: New insights from solid-phase binding assay

It is now well established that the ATP-binding cassette transporter A1 (ABCA1) plays a pivotal role in HDL metabolism, reverse cholesterol transport and net efflux of cellular cholesterol and phospholipids. We aimed to resolve some uncertainties related to the putative function of ABCA1 as a mediator of lipid transport by using a methodology developed in the laboratory to isolate a protein and study its interactions with other compounds. ABCA1 was tagged with the 1D4 peptide at the C terminus and expressed in human HEK 293 cells. Preliminary experiments showed that the tag modified neither the protein expression/localization within the cells nor the ability of ABCA1 to promote cholesterol cellular efflux to apolipoprotein A-I. ABCA1-1D4 was then purified and reconstituted in liposomes. ABCA1 displayed an ATPase activity in phospholipid liposomes that was significantly decreased by cholesterol. Finally, interactions with either cholesterol or apolipoprotein A-I were assessed by binding experiments with protein immobilized on an immunoaffinity matrix. Solid-phase binding assays showed no direct binding of cholesterol or apolipoprotein A-I to ABCA1. Overall, our data support the hypothesis that ABCA1 is able to mediate the transport of cholesterol from cells without direct interaction and that apo A-I primarily binds to membrane surface or accessory protein(s).     

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.     

Apolipoprotein A-I induces translocation of cholesterol, phospholipid, and caveolin-1 to cytosol in rat astrocytes.

Intercellular cholesterol transport in the brain is carried by high density lipoprotein (HDL) generated in situ by cellular interaction with the apolipoprotein apoE, which is mainly synthesized by astrocytes, and with apoA-I secreted by cells such as endothelial cells. Rat astrocytes in fact generate HDL with extracellular apoA-I in addition to releasing HDL with endogenously synthesized apoE, seemingly by the same mechanism as the HDL assembly for systemic circulation. Relating to this reaction, apoA-I induced translocation of newly synthesized cholesterol and phospholipid to the cytosol prior to extracellular assembly of HDL, accompanied by an increase of caveolin-1 in the cytosol, activation of sterol regulatory element-binding protein, and enhancement of cholesterol synthesis. The lipid translocated into the cytosol was recovered in the fraction with a density of 1.09-1.16 g/ml as well as caveolin-1 and cyclophilin A. Cyclosporin A inhibited these apoA-I-mediated reactions and suppressed apoA-I-mediated cholesterol release. The findings suggest that such translocation of cholesterol and phospholipid into the cytosol is related to the apo A-I-mediated HDL assembly in astrocytes through functional association with caveolin-1 and a cyclosporin A-sensitive cyclophilin protein(s).     

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

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

ABCA1-mediated cholesterol efflux generates microparticles in addition to HDL through processes governed by membrane rigidity

ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-poor apolipoprotein A-I (apoA-I) and generates HDL. Here, we demonstrate that ABCA1 also directly mediates the production of apoA-I free microparticles. In baby hamster kidney (BHK) cells and RAW macrophages, ABCA1 expression led to lipid efflux in the absence of apoA-I and released large microparticles devoid of apoB and apoE. We provide evidence that these microparticles are an integral component of the classical cholesterol efflux pathway when apoA-I is present and accounted for approximately 30% of the total cholesterol released to the medium. Furthermore, microparticle release required similar ABCA1 activities as was required for HDL production. For instance, a nucleotide binding domain mutation in ABCA1 (A937V) that impaired HDL generation also abolished microparticle release. Similarly, inhibition of protein kinase A (PKA) prevented the release of both types of particles. Interestingly, physical modulation of membrane dynamics affected HDL and microparticle production, rigidifying the plasma membrane with wheat germ agglutinin inhibited HDL and microparticle release, whereas increasing the fluidity promoted the production of these particles. Given the established role of ABCA1 in expending nonraft or more fluid-like membrane domains, our results suggest that both HDL and microparticle release is favored by a more fluid plasma membrane. We speculate that ABCA1 enhances the dynamic movement of the plasma membrane, which is required for apoA-I lipidation and microparticle formation.     

The ABCA1 transporter functions on the basolateral surface of hepatocytes

ABCA1 on the cell surface and in endosomes plays an essential role in the cell-mediated lipidation of apoA-I to form nascent HDL. Our previous studies of transgenic mice overexpressing ABCA1 suggested that ABCA1 in the liver plays a major role in regulating plasma HDL levels. The site of function of ABCA1 in the polarized hepatocyte was currently assessed by expression of an adenoviral construct encoding a human ABCA1-GFP fusion protein in the polarized hepatocyte-like WIF-B cell line. Consistent with localization of ABCA1 at the basolateral (vascular) cell surface, expression of ABCA1-GFP stimulated apoA-I mediated efflux of WIF-B cell cholesterol into the culture medium. Confocal fluorescence microscopy revealed that ABCA1-GFP was expressed solely on the basolateral surface and associated endocytic vesicles. These findings suggest an important role for hepatocyte basolateral membrane ABCA1 in the regulation of the levels of intracellular hepatic cholesterol, as well as plasma HDL.     

cAMP induces ABCA1 phosphorylation activity and promotes cholesterol efflux from fibroblasts

ATP-binding cassette transporter A1 (ABCA1) plays a crucial role in apoA-I lipidation, a key step in reverse cholesterol transport. cAMP induces apoA-I binding activity and promotes cellular cholesterol efflux. We investigated the role of the cAMP/protein kinase A (PKA) dependent pathway in the regulation of cellular cholesterol efflux. Treatment of normal fibroblasts with 8-bromo-cAMP (8-Br-cAMP) increased significantly apoA-I-mediated cholesterol efflux, with specificity for apoA-I, but not for cyclodextrin. Concomitantly, 8-Br-cAMP increased ABCA1 phosphorylation in a time-dependent manner. Maximum phosphorylation was reached in <10 min, representing a 260% increase compared to basal ABCA1 phosphorylation level. Forskolin, a known cAMP regulator, increased both cellular cholesterol efflux and ABCA1 phosphorylation. In contrast, H-89 PKA inhibitor reduced cellular cholesterol efflux by 70% in a dose-dependent manner and inhibited almost completely ABCA1 phosphorylation. To determine whether naturally occurring mutants of ABCA1 may affect its phosphorylation activity, fibroblasts from subjects with familial HDL deficiency (FHD, heterozygous ABCA1 defect) and Tangier disease (TD, homozygous/compound heterozygous ABCA1 defect) were treated with 8-Br-cAMP or forskolin. Cellular cholesterol efflux and ABCA1 phosphorylation were increased in FHD but not in TD cells. Taken together, these findings provide evidence for a link between the cAMP/PKA-dependent pathway, ABCA1 phosphorylation, and apoA-I mediated cellular cholesterol efflux.     

Cellular localization and trafficking of the human ABCA1 transporter

ABCA1, the ATP-binding cassette protein mutated in Tangier disease, mediates the efflux of excess cellular sterol to apoA-I and thereby the formation of high density lipoprotein. The intracellular localization and trafficking of ABCA1 was examined in stably and transiently transfected HeLa cells expressing a functional human ABCA1-green fluorescent protein (GFP) fusion protein. The fluorescent chimeric ABCA1 transporter was found to reside on the cell surface and on intracellular vesicles that include a novel subset of early endosomes, as well as late endosomes and lysosomes. Studies of the localization and trafficking of ABCA1-GFP in the presence of brefeldin A or monensin, agents known to block intracellular vesicular trafficking, as well as apoA-I-mediated cellular lipid efflux, showed that: (i) ABCA1 functions in lipid efflux at the cell surface, and (ii) delivery of ABCA1 to lysosomes for degradation may serve as a mechanism to modulate its surface expression. Time-lapse fluorescence microscopy revealed that ABCA1-GFP-containing early endosomes undergo fusion, fission, and tubulation and transiently interact with one another, late endocytic vesicles, and the cell surface. These studies establish a complex intracellular trafficking pathway for human ABCA1 that may play important roles in modulating ABCA1 transporter activity and cellular cholesterol homeostasis.