Activation of ATP-binding cassette transporter A1 transcription by chromatin remodeling complex.

OBJECTIVE: Liver X receptor (LXR) regulates the transcription of ATP-binding cassette transporter A1 (ABCA1) by binding to the DR-4 promoter element as a heterodimer with retinoid X receptor (RXR). The role of chromatin remodeling complex in LXR or ABCA1 activation has not been established previously. In this study, we investigated the activation of ABCA1 by brahma-related gene 1 (BRG-1) and brahma, members of the SWI/SNF (mating type switching/sucrose nonfermenting) chromatin remodeling complex. METHODS AND RESULTS: Overexpression of wild-type BRG-1 in SW-13 cells, but not a catalytically inactive mutant, increased ABCA1 mRNA levels determined by RT-PCR. These effects were enhanced by LXR and RXR agonists. In 293T (epithelial kidney cell line) and Hep3B (hepatocyte cell line) cells, small interfering RNA against BRG-1/brm also affected ABCA1 mRNA levels. Synergistic activation of ABCA1 was obtained after coexpressing BRG-1 and SRC-1, a coactivator of LXR. Luciferase assays showed that this activation of ABCA1 was dependent on the promoter DR-4 element. Coimmunoprecipitation and chromatin immunoprecipitation studies indicated that the mechanism of BRG-1-mediated activation of ABCA1 involved interaction of LXR/RXR with BRG-1 and binding of this complex to ABCA1 promoter. CONCLUSIONS: Catalytic subunits of SWI/SNF chromatin remodeling complex, BRG-1 and brahma, play significant roles in enhancing LXR/RXR-mediated transcription of ABCA1 via the promoter DR-4 element.

Relationship between cholesterol trafficking and signaling in rafts and caveolae.

Caveolae and lipid rafts are two distinct populations of free cholesterol, sphingolipid (FC/SPH)-rich cell surface microdomains. They differ in stability, shape, and the presence or absence of caveolin (present in caveolae) or GPI-anchored proteins (enriched in lipid rafts). In primary cells, caveolae and rafts support the assembly of different signaling complexes, though signal transduction from both is strongly dependent on the presence of FC. It was initially thought that FC promoted the formation of inactive reservoirs of signaling proteins. Recent data supports the concept of a more dynamic role for FC in caveolae and probably, also lipid rafts. It is more likely that the FC content of these domains is actively modulated as protein complexes are formed and, following signal transduction, disassembled. In transformed cell lines with few caveolae, little caveolin and a preponderance of rafts, complexes normally assembled on caveolae may function in rafts, albeit with altered kinetics. However, caveolae and lipid rafts appear not to be interconvertible. The presence of non-caveolar pools of caveolin in recycling endosomes (RE), the trans-Golgi network (TGN) and in mobile chaperone complexes is now recognized. A role in the uptake of microorganisms by cells ascribed to caveolae now seems more likely to be mediated by cell surface rafts.

Role of p160 coactivator complex in the activation of liver X receptor.

OBJECTIVE: Liver X receptor (LXR) is a member of a nuclear receptor family regulating the expression of several key proteins involved in lipid metabolism and inflammation. In contrast to several other nuclear receptors, very little is known about the coactivators needed for the agonist-mediated transactivation by LXR. In this study, we have investigated the role of p160 coactivator complex in the regulation of ATP-binding transporter A1 (ABCA1), a clinically important gene transcriptionally upregulated by LXR/RXR (retinoid X receptor) heterodimer. METHODS AND RESULTS: Overexpression of LXRalpha, SRC-1, and p300, either alone or in combination, increased the luciferase activity driven by the wild-type ABCA1 promoter. The same coactivators bound to the ABCA1 promoter on oxysterol induction in chromatin immunoprecipitation assays. To the contrary, CARM-1 and P/CAF had no effect on ABCA1 transactivation, nor do they bind the promoter. When the DR-4 element was mutated from the ABCA1 promoter, only p300 was able to activate ABCA1 transcription in a ligand-independent manner. CONCLUSIONS: The p160 coactivator complex members SRC-1 and p300, but not CARM-1 and P/CAF, coactivate LXR-mediated transcription of ABCA1 gene. In addition, p300 activates ABCA1 transcription independently of DR-4 element and LXR/RXR.

Bone morphogenetic protein-1 (BMP-1) cleaves human proapolipoprotein A1 and regulates its activation for lipid binding.

Apolipoprotein A1 (apo A1), the major protein of high-density lipoprotein, is secreted as a proprotein and then cleaved by an uncharacterized metalloproteinase. Here this enzyme is identified as C-terminal procollagen endoproteinase/bone morphogenetic protein-1 (BMP-1). Studies with recombinant BMP-1, BMP-1 antibody, and BMP-1 siRNA establish this proteinase as the major or only apo A1-converting activity secreted by human liver-derived (HepG2) cells and CHO cells stably expressing human apo A1. BMP-1 stimulates the conversion of newly secreted proapo A1 to its phospholipid- (PL-) binding form. In this way it promotes formation of functional HDL and reverse cholesterol transport, while inhibiting filtration and clearance of uncleaved proprotein. Alpha2-macroglobulin, a protease inhibitor secreted as part of the innate immune response, inhibits BMP-1 activity and blocks the maturation of proapo A1. The decrease in circulating apo A1 levels that is characteristic of the response to inflammation and infection may be mediated, at least in part, via BMP-1 by this novel mechanism.

Mechanism of prebeta-HDL formation and activation.

The mechanism of formation of functional high-density lipoprotein (HDL) from secreted lipid-free apolipoprotein A1 (apo A1) was determined using human liver-derived (HepG2) cells, human intestine-derived (CaCO2) cells, and CHO cells stably expressing full-length human apo A1 (CHO-A1 cells). In each cell line, a significant proportion of secreted apo A1 had a Stokes radius of 2.6 nm and was inactive in binding phospholipids (PL) or free cholesterol (FC). Extracellularly, in a reaction dependent on membrane transporter ABCA1, prealpha-migrating 2.6 nm apo A1 was converted to a prebeta-migrating product that was able to bind PL. Both forms were reactive with mAb55201, a monoclonal antibody specific for native plasma lipid-poor (prebeta1) HDL [Nakamura, Y., et al. (2004) Biochemistry 43, 14311-14318]. The physical properties of precursor and product apo A1 suggested that both are monomers, with Stokes radii of 2.6 and 3.6 nm, respectively, consistent with the absence of intermolecular cross-linking of apo A1 in lipid-poor HDL, reported previously. Product but not precursor apo A1 promoted reverse cholesterol transport (RCT) from human aortic smooth muscle cells. These studies suggest an important contribution of secreted lipid-free apo A1 to HDL formation.

Preparation and biochemical evaluation of fluorenone-containing lipid analogs.

Syntheses are described of fatty acid analogs 5 and 6, and cholesterol (2) analogs 7 and 8 containing fluorenone groups, which are both photoactivable and fluorescent. The potential of the analogs of 2 as biochemical research tools has been demonstrated by the findings that 7 and 8 can replace 2 in apolipoprotein A-I-induced cellular efflux of 2 and that fluorescence is easily visible at the surface of smooth muscle cells equilibrated with 8.

Regulation of ATP-binding cassette transporter A1 transcription by thyroid hormone receptor.

Transcriptional regulation of the ATP-binding cassette transporter (ABCA1) gene is complex. It involves multiple transcription start sites and the binding of several different transcription factors to the ABCA1 promoter region. Cholesterol- and oxysterol-mediated up-regulation of ABCA1 transcription includes the binding of the liver X receptor and retinoid X receptor (LXR/RXR) heterodimer to the DR-4 element of the ABCA1 promoter. In this study we show that another nuclear hormone receptor, thyroid hormone receptor (TR), can suppress ABCA1 transcription. Electrophoretic mobility shift assays using both purified proteins and isolated nuclear extracts from primary human fibroblasts and 293T cells demonstrate that the TR/RXR heterodimer is able to bind to the DR-4 element of the ABCA1 promoter. This binding is also demonstrated in vivo by chromatin immunoprecipitation studies. Luciferase assays from 293T cells transfected with TRbeta or LXRalpha expression plasmids show that TR, together with its ligand T3, suppresses ABCA1 transcriptional activity, even in the presence of LXR-activating oxysterols. Finally, competition between TR/RXR and LXR/RXR heterodimers to suppress or activate ABCA1 transcription is shown to be dynamic and dependent on the amount of nuclear receptor present in the cells. These data identify a novel regulatory mechanism for ABCA1 and suggest new strategies to modify its expression.

Mechanism of platelet-derived growth factor-dependent caveolin-1 phosphorylation: relationship to sterol binding and the role of serine-80.

In human vascular smooth muscle cells, inhibitors of protein kinase C activity reduced serine phosphorylation of caveolin-1 and increased sterol binding by this protein. This was measured after immunoprecipitation of caveolin-1 from cells labeled with tritiated cholesterol or the photoactivable cholesterol analogue FCBP [Fielding et al. (2002) Biochemistry 41, 4929-4937]. At the same time cellular sterol efflux was inhibited. Mutagenesis within a caveolin-1 central domain (residues 80-104) suggested a major role for serine-80 in mediating both of these effects. To perturb sterol binding, platelet-derived growth factor was added to the cells, leading to a transient loss of caveolin-1-associated sterol. Under these conditions, sterol efflux was stimulated, and caveolin-1 phosphorylation at tyrosine(14), assayed with a selective antibody, was substantially increased above baseline levels. These changes were also blocked by inhibitors of protein kinase C activity. Selective inhibitors of the platelet-derived growth factor receptor and downstream kinases were used to show that loss of sterol from caveolin-1 preceded tyrosine phosphorylation, but relipidation was dependent on phosphotyrosine hydrolysis.

Molecular mechanism of reverse cholesterol transport: reaction of pre-beta-migrating high-density lipoprotein with plasma lecithin/cholesterol acyltransferase.

A 70-75 kDa high-density lipoprotein (HDL) particle with pre-beta-electrophoretic migration (pre-beta(1)-HDL) has been identified in several studies as an early acceptor of cell-derived cholesterol. However, the further metabolism of this complex has not been determined. Here we sought to identify the mechanism by which cell-derived cholesterol was esterified and converted to mature HDL as part of reverse cholesterol transport (RCT). Human plasma selectively immunodepleted of pre-beta(1)-HDL was used to study factors regulating pre-beta(1)-HDL production. A major role for phospholipid transfer protein (PLTP) in the recycling of pre-beta(1)-HDL was identified. Cholesterol binding, esterification by lecithin/cholesterol acyltransferase (LCAT) and transfer by cholesteryl ester transfer protein (CETP) were measured using (3)H-cholesterol-labeled cell monolayers. LCAT bound to (3)H-free cholesterol (FC)-labeled pre-beta(1)-HDL generated cholesteryl esters at a rate much greater than the rest of HDL. The cholesteryl ester produced in pre-beta(1)-HDL in turn became the preferred substrate of CETP. Selective LCAT-mediated reactivity with pre-beta(1)-HDL represents a novel mechanism increasing the efficiency of RCT.

Dynamic regulation of alternative ATP-binding cassette transporter A1 transcripts.

The ATP-binding cassette transporter A1 (ABCA1) gene, a major regulator of high-density lipoprotein (HDL) metabolism, utilizes several transcriptional start sites. The pattern of transcripts among tissues and cells differed significantly. The longest (class 1) transcripts were abundant in adult brain and fetal tissues. Class 2 transcripts predominated in most other tissues. The shortest (class 3) transcripts were present mainly in adult liver and lung. To study the biochemical significance of changes in transcript distribution, two cell models were compared. In primary human fibroblasts, upregulation of mRNA levels by oxysterols and retinoic acid increased the relative proportion of class 2 transcript compared to class 1. Phorbol ester stimulated human macrophage-derived THP-1 cells increased the abundance of class 1 transcripts relative to class 2. In both cell lines class 3 transcript levels were minimal and unchanged. It is shown here for the first time that the regulation of ABCA1 mRNA levels exploits the use of alternative transcription start sites.

Benzophenone-containing cholesterol surrogates: synthesis and biological evaluation.

Eight analogs of cholesterol (1) containing a benzophenone group have been synthesized as prospective photoaffinity labels for studies in cellular sterol efflux and HDL formation. Six of these compounds (4-9) have the photophore replacing different portions of the cholesterol alkyl side chain, and two (10 and 11) have it attached via nitrogen at carbon 3. The suitability of these analogs as cholesterol surrogates was determined by examining their ability to replace [3H]1 in fibroblasts preequilibrated with [3H]1. All eight analogs were effective in replacing natural 1 in competition with [3H]1 for apolipoprotein A-I-induced efflux. These are the first compounds shown to replace cholesterol successfully in a complex pathway of multiple intracellular steps. The results suggest an unexpected tolerance of biological membranes regarding the incorporation of sterols of differing chemical structure.