Liver X receptor inhibits the synthesis and secretion of apolipoprotein A1 by human liver-derived cells.

The liver X receptor (LXR) agonist TO901317 inhibited the synthesis of apolipoprotein A1 (apo A1) by human liver-derived cells, including the formation of lipid-poor, prebeta-migrating high-density lipoprotein (HDL). Despite activation of the lipid transporter ABCA1 under these conditions, cellular efflux of PL and cholesterol from liver cells was also reduced. By assaying transcription from full-length and truncated promoters and by site-directed mutagenesis, the effect of LXR and its ligand was localized to a binding site for hepatic nuclear factor-4 (HNF4) in the proximal apo A1 promoter (-132/-119 bp). Chromatin immunoprecipitation analysis of apo A1 transcription complexes from control and ligand-activated cells showed an increase in the binding of reported apo A1 transcriptional inhibitor COUP-TF, which competes with HNF4 for DNA binding. It also identified LXR in the apo A1 transcription complex of TO901317-treated cells. Displacement of HNF4 from the -132/-119 bp promoter DNA sequence in the presence of TO901317 was confirmed by gel shift analysis. These data indicate that LXR can be a significant negative regulator of apo A1 transcription and HDL synthesis.

Membrane cholesterol and the regulation of signal transduction.

The plasma membrane of mammalian cells consists of microdomains differing in lipid and protein composition. Two distinct classes of cholesterol/sphingolipid microdomain (caveolae and lipid rafts) are assembly points for transmembrane signalling complexes. Recent evidence suggests that transient changes in cholesterol content may be important in regulating signal transduction.

Sterol efflux to apolipoprotein A-I originates from caveolin-rich microdomains and potentiates PDGF-dependent protein kinase activity.

The kinetics of sterol efflux from human aortic smooth muscle cells equilibrated with a [(3)H]benzophenone-modified photoactivable free cholesterol analogue ((3)H-FCBP) did not differ significantly from those labeled with free cholesterol ((3)H-FC). Trypsin digestion of caveolin cross-linked by photoactivation of FCBP led to association of radiolabel in a single low molecular weight fraction, indicating relative structural homogeneity of caveolin-bound sterol. These findings were used to investigate the organization of sterols in caveolae and the ability of these domains to transfer sterols to apolipoprotein A-I (apo A-I), the major protein of human plasma high-density lipoproteins (HDL). During long-term (4-5 h) incubation with apo A-I, caveolin-associated (3)H-FC and (3)H-FCBP decreased, in parallel with an increase in apo A-I-associated sterol. Assay of caveolin-associated labeled sterols indicated that caveolae were a major source of sterol lost from the cells during HDL formation. Short-term changes of sterol distribution in caveolae were assayed using platelet-derived growth factor (PDGF). PDGF was without effect on FC efflux in the absence of apo A-I, but when apo A-I was present, PDGF increased FC efflux approximately 3-fold beyond the efflux rate catalyzed by apo A-I alone. At the same time, caveolin-associated FC decreased, and PDGF-dependent protein kinase activity was stimulated. Parallel results were obtained with (3)H-FCBP-equilibrated cells, in which apo A-I potentiated a PDGF-mediated reduction of radiolabel cross-linked to caveolin following photoactivation. These results suggest that sterols within caveolae are mobile and selectively transferred to apo A-I. They also suggest a novel role for sterol efflux in amplifying PDGF-mediated signal transduction.

Cellular cholesterol efflux.

Efflux of free cholesterol (FC) continues even when cellular FC mass is unchanged. This reflects a recirculation of preformed FC between cells and extracellular fluids which has multiple functions in cell biology including receptor recycling and signaling as well as cellular FC homeostasis. Total FC efflux is heterogeneous. Simple diffusion to mature high density lipoprotein (HDL), mainly via albumin as intermediate, initiates FC net transport driven by plasma lecithin:cholesterol acyltransferase activity. A second major efflux component reflects protein-facilitated transport from cell surface domains (caveolae, rafts) driven by FC binding to lipid-poor, pre-beta-migrating HDL (pre-beta-HDL). Facilitated efflux from caveolae, unlike simple diffusion, is highly regulated. Neither ABC1 (the protein defective in Tangier disease) nor other ATP-dependent transporters now appear likely to contribute directly to FC efflux. Their role is limited to the initial formation of a particle precursor to circulating pre-beta-HDL, which recycles without further lipid input from ATP-dependent transporter proteins. Lipid-free apolipoprotein A-I, previously considered a surrogate for pre-beta-HDL, has a reactivity much lower than that of native lipoprotein FC acceptors.

Caveolae and intracellular trafficking of cholesterol.

Caveolae, free cholesterol (FC)-rich microdomains of the plasma membrane, are both a terminus for the intracellular transit of newly synthesized and recycling cellular FC, and a site for FC efflux to the extracellular medium. The same domains play key roles as locations for the assembly of signaling complexes and for the endocytosis of selected ligands. Caveolin, the major structural protein of caveolae, plays a regulatory role in growth, the cell cycle, and cell adhesion. Each of these functions is FC-dependent. Caveolae appear to act as both sensors and regulators of cellular FC content, and in this way mediate an array of membrane-dependent cell functions.

Cholesterol and caveolae: structural and functional relationships.

Caveolae are free cholesterol (FC)- and sphingolipid-rich surface microdomains abundant in most peripheral cells. Caveolin, a FC binding protein, is a major structural element of these domains. Caveolae serve as portals to regulate cellular FC homeostasis, possibly via their association with ancillary proteins including scavenger receptor B1. The FC content of caveolae regulates the transmission of both extracellular receptor-mediated and endogenous signal transduction via changes in the composition of caveolin-associated complexes of signaling intermediates. By controlling surface FC content, reporting membrane changes by signal transduction to the nucleus, and regulating signal traffic in response to extracellular stimuli, caveolae exert a multifaceted influence on cell physiology including growth and cell division, adhesion, and hormonal response. Cell surface lipid 'rafts' may assume many of the functions of caveolae in cells with low levels of caveolin.

A two-step mechanism for free cholesterol and phospholipid efflux from human vascular cells to apolipoprotein A-1.

Smooth muscle and endothelial cells in vivo are quiescent yet exposed to high levels of lipoprotein lipids. Phospholipid (PL) and free cholesterol (FC) efflux maintain homeostasis. Smooth muscle cells (SMC) expressed high levels of ABC-1 transporter mRNA, and glyburide-dependent PL and FC efflux to apolipoprotein A-1 (apo A-1), the major protein of high-density lipoprotein. FC efflux was inhibited by vanadate and okadaic acid, while PL efflux was not. Phosphatidylcholine was the major PL transferred by both cell types. Stimulation of phosphatidylserine efflux, redistributed within the membrane by this transporter, was only minimally increased. Umbilical vein and aortic endothelial cells expressed little ABC-1 mRNA, nor did these cells promote either PL or FC efflux in response to the presence of apo A-1. To investigate the mechanism of ABC-1-dependent lipid efflux from these cells, apo A-1 was preincubated in the presence of unlabeled SMC or fibroblasts, and the conditioned medium was then transferred to endothelial cells. This medium catalyzed the efflux of FC but not of PL from endothelial cells. Such FC efflux was resistant to glyburide but inhibited by okadaic acid and vanadate. The data suggest that ABC-1-dependent PL efflux precedes FC efflux to apo A-1 and that the complex of apo A-1 and PL is a much better acceptor of FC than apo A-1 itself. Inhibition of FC but not PL efflux by vanadate and okadaic acid suggests these transfers involve different mechanisms.

p53 regulates caveolin gene transcription, cell cholesterol, and growth by a novel mechanism.

Transcription of the human caveolin gene, directed by a TATA-less promoter, is downregulated in actively dividing cells during S-phase, together with free cholesterol (FC) efflux. It is upregulated by medium low density lipoprotein FC levels in quiescent cells. In this study, a common mechanism has been identified to coordinate the growth- and FC-dependent expression of caveolin. In human skin fibroblasts, transcription factors E2F/DP-1 and Sp1 bound to adjacent consensus sites at -151 to -138 bp of the caveolin promoter DNA sequence in a complex stabilized by tumor suppressor protein p53. Wild-type p53 also bound directly to DNA to a caveolin promoter sequence containing two consensus half-sites (-292 to -283 bp and -273 to -264 bp) for this transcription factor. SREBP-1, previously identified as a transcriptional regulator of caveolin expression in response to FC, mediated its effect via the same E2F/Sp1 site. Overexpression of E2F or p53 increased E2F binding to the -148 to -141 bp site, increased FC efflux, and inhibited cell division. The mutant protein p53(143V-->A) was inactive. Okadaic acid, previously shown to inhibit growth, FC efflux, and caveolin expression, inhibited E2F/Sp1 binding, while higher concentrations of extracellular FC increased it. The present findings provide a molecular link between the cell cycle and FC homeostatic effects of caveolin. These results also describe a novel mechanism of action for p53 in a TATA-less gene promoter and provide further evidence for a significant regulatory role for FC in cell cycle progression.

Intracellular cholesterol transport in synchronized human skin fibroblasts.

Normal human skin fibroblasts maintained in serum-containing medium were synchronized with aphidicolin. After removal of inhibitor, free cholesterol (FC) homeostasis was determined at intervals during the following cell cycle. FC mass per cell doubled following S-phase, and reached its maximum well before mitosis. This increase was mainly the result of stimulation of the rate of selective uptake of FC from medium lipoproteins, and reduction of FC efflux. Rates of cholesterol synthesis, endocytosis of intact low-density lipoprotein, and HDL receptor (CLA-1) activity were relatively low and little changed during the cell cycle. The expression of caveolin (structural protein of cell surface caveolae) and caveolar FC were decreased along with FC efflux. To test the hypothesis that regulation of caveolin expression could contribute to changes in FC efflux during cell division, cells were transfected with human caveolin cDNA, synchronized with aphidicolin, and then allowed to divide. In the transfected cells, caveolar FC and FC efflux were both increased. FC accumulation and entry into mitosis were markedly inhibited compared to controls. The contribution of transcriptional regulation to caveolin mRNA levels was determined with a 705 bp caveolin 5'-flanking sequence ligated to the pGL3 luciferase expression vector. Expression of the reporter gene was downregulated at S-phase of synchronized cells. Deletion of a hybrid E2F/ Sp1-like site between -139 and -150 bp abolished this downregulation. These data are consistent with a role for caveolin in cell cycle kinetics, which may be mediated, at least in part, at the transcriptional level.

Two sterol regulatory element-like sequences mediate up-regulation of caveolin gene transcription in response to low density lipoprotein free cholesterol.

Caveolae form the terminus for a major pathway of intracellular free cholesterol (FC) transport. Caveolin mRNA levels in confluent human skin fibroblasts were up-regulated following increased uptake of low density lipoprotein (LDL) FC. The increase induced by FC was not associated with detectable change in mRNA stability, indicating that caveolin mRNA levels were mediated at the level of gene transcription. A total of 924 bp of 5' flanking region of the caveolin gene were cloned and sequenced. The promoter sequence included three G+C-rich potential sterol regulatory elements (SREs), a CAAT sequence and a Sp1 consensus sequence. Deletional mutagenesis of individual SRE-like sequences indicated that of these two (at -646 and -395 bp) were essential for the increased transcription rates mediated by LDL-FC, whereas the third was inconsequential. Gel shift analysis of protein binding from nuclear extracts to these caveolin promoter DNA sequences, together with DNase I footprinting, confirmed nucleoprotein binding to the SRE-like elements as part of the transcriptional response to LDL-FC. A supershift obtained with antibody to SRE-binding protein 1 (SPEBP-1) indicated that this protein binds at -395 bp. There was no reaction at -395 bp with anti-Sp1 antibody nor with either antibody at -646 bp. The cysteine protease inhibitor N-acetyl-leu-leu-norleucinal (ALLN), which inhibits SREBP catabolism, superinhibited caveolin mRNA levels regardless of LDL-FC. This finding suggests that SREBP inhibits caveolin gene transcription in contrast to its stimulating effect on other promoters. The findings of this study are consistent with the postulated role for caveolin as a regulator of cellular FC homeostasis in quiescent peripheral cells, and the coordinate regulation by SREBP of FC influx and efflux.

Intracellular cholesterol transport.

Recent data on the roles of vesicle- and 'raft'-mediated pathways in intracellular free cholesterol (FC) transport are reviewed. Cholesterol internalized from plasma lipoproteins is transferred via endocytic vesicles to the trans-Golgi network (TGN), consistent with prior data indicating a key role for this organelle in protein and lipid sorting and transport. Newly synthesized and lipoprotein-derived FC are returned to the cell surface by a common raft-dependent pathway. Intracellular FC transport promotes the delivery of GPI-anchored proteins to the cell surface; it is also an additional mechanism to regulate cell FC content. Many peripheral cells express caveolin, an FC-binding protein localized to plasma membrane caveolae. FC delivery to cell surface caveolae is accelerated by caveolin. Caveolar FC becomes targeted to small, lipid-poor (prebeta-) high density lipoprotein particles. Caveolin may protect quiescent cells, regulating FC efflux more efficiently in response to changing medium lipoprotein concentrations. Overall, these recent findings suggest that cell FC content can be regulated at the levels of both influx and efflux, and indicate key roles for the TGN and in cells expressing caveolin, cell-surface caveolae.

Caveolin mRNA levels are up-regulated by free cholesterol and down-regulated by oxysterols in fibroblast monolayers.

In confluent fibroblast monolayers, an increase in the selective uptake of free cholesterol (FC) from plasma low density lipoprotein (LDL) was accompanied by an increase in FC efflux. The rate of FC efflux was proportional to the FC content of the cell surface caveolae and to mRNA levels of caveolin, an FC-binding protein of caveolae. Inhibitors of LDL-FC internalization reduced the increase in caveolin mRNA levels and FC efflux. Oxysterols reduced caveolin mRNA levels, as well as transport of FC to the cell surface and FC efflux. DNA antisense to caveolin reduced caveolin mRNA and inhibited FC efflux. These data suggest that regulation of FC efflux can contribute to cellular FC homeostasis when LDL levels are modified over the physiological range, and they link this regulation to caveolin.

Intracellular transport of low density lipoprotein derived free cholesterol begins at clathrin-coated pits and terminates at cell surface caveolae.

Free cholesterol (FC) is selectively internalized from low-density lipoprotein (LDL) by confluent fibroblast monolayers (Fielding & Fielding (1995) Biochemistry 34, 14237-14244). The kinetics of transport of LDL-derived 3H-FC within the cell were studied by density-gradient ultracentrifugal fractionation and in terms of the effects of inhibitors of endocytosis and intracellular transport. By these criteria, the initial uptake of LDL-FC was mediated by the cell-surface clathrin-coated pits. FC label then appeared in clathrin-coated dense vesicles. Uncoating of clathrin from these vesicles led to the appearance of label in a light density fraction and, subsequently, in an intermediate density fraction coincident with protein markers of the trans-Golgi network in these cells. 3H-FC was finally transported to the plasma membrane via a temperature-sensitive, probably microtubule-dependent pathway. These data are consistent with a role for the trans-Golgi network as an intermediate compartment in intracellular FC transport. They provide further evidence of a role for cell-surface caveolae in FC efflux.

Two-dimensional nondenaturing electrophoresis of lipoproteins: applications to high-density lipoprotein speciation.

No single technique is able to separate each of the many HDL species present in native plasma. Some are present in only trace proportions. Some HDL have no obvious independent metabolic role, beyond perhaps serving as reservoirs of apoproteins active in metabolic events in other lipoproteins. The choice of HDL analytical technique depends mainly on the problem under study. Two-dimensional nondenaturing electrophoresis has been useful in studies of plasma cholesterol metabolism and cholesterol transport from cells, because it separates intermediates in these processes.

Plasma membrane caveolae mediate the efflux of cellular free cholesterol.

Caveolae are clathrin-free cell-surface organelles implicated in transmembrane transport. A fibroblast caveolar membrane fraction was isolated by sucrose density gradient ultracentrifugation and its identity confirmed by protein markers (caveolin, annexin II). When 3H-labeled free cholesterol was selectively transferred to the cells from labeled low density lipoprotein to increase cell free cholesterol approximately 15%, there was a 6-fold increase in label in the caveolar fraction above baseline levels. Subsequent incubation of these cells with unlabeled native plasma or plasma high density lipoprotein selectively unloaded caveolar free cholesterol into the medium. Okadaic acid, which decreased caveolar activity as measured by cholera toxin binding and uptake, decreased cholesterol efflux in parallel. Cholesterol newly synthesized from [3H]mevalonate was also preferentially incorporated into the caveolar fraction and selectively released by plasma into the medium. Together these data indicate that caveolae represent a major site of efflux of both newly synthesized and low density lipoprotein-derived free cholesterol in these cells.

Role of an N-ethylmaleimide-sensitive factor in the selective cellular uptake of low-density lipoprotein free cholesterol.

Low-density lipoprotein (LDL) was the major contributor to an influx of free sterol from plasma which balances high-density lipoprotein (HDL)-mediated efflux from cultured skin fibroblasts. When HDL was absent, the uptake of LDL free cholesterol was associated with an increase in total cell cholesterol, due in part to accumulation of esterified cholesterol. This influx was mediated by a high-capacity, low-affinity pathway whose magnitude was similar in normal and LDL receptor-deficient cells. In the presence of HDL, some of the interiorized labeled LDL free cholesterol became available for HDL-mediated efflux and some was interiorized, as a result of a transport mechanism which was sensitive to N-ethylmaleimide (NEM) and nitrate ion but resistant to progesterone, azide, or vanadate. We suggest that normal free cholesterol homeostasis in these cells includes the initial binding of LDL followed by the selective transfer of free cholesterol to a compartment from which it is either returned to the membrane for efflux or internalized for storage or further metabolism within the cell. In the presence of NEM, LDL-derived free cholesterol remained mostly accessible for efflux from the cell surface. This free cholesterol pathway may function physiologically to stabilize plasma membrane cholesterol levels against the effect of varying concentrations of HDL and LDL.

Molecular physiology of reverse cholesterol transport.

Reverse cholesterol transport (RCT) is the pathway by which peripheral cell cholesterol can be returned to the liver for catabolism. Evidence of specific functions for molecular structures within individual plasma lipoprotein species has rapidly accumulated from recent studies using molecular and cellular physiology techniques. The removal of cholesterol from cells, like its delivery, appears to be specific and well regulated. Although further research will be needed, RCT can now be understood in molecular terms.

Unique epitope of apolipoprotein A-I expressed in pre-beta-1 high-density lipoprotein and its role in the catalyzed efflux of cellular cholesterol.

The ability of mouse anti-apolipoprotein A-I (apo A-I) monoclonal antibodies to recognize pre-beta-HDL species in native plasma was determined. An antibody identifying residues 137-144 of the mature protein uniquely recognized pre-beta-1 HDL, an HDL species of low molecular weight implicated in early cholesterol transport from cell membranes to plasma [Castro, G. R., & Fielding, C. J. (1988) Biochemistry 27, 25-29]. Incubation of plasma with this antibody significantly inhibited the efflux of labeled cholesterol from cultured fibroblast monolayers. A second antibody, binding to residues 93-99 of apo A-I, recognized a second pre-beta-HDL species (pre-beta-2 HDL) but not pre-beta-1 HDL and did not inhibit cholesterol efflux. Several other antibodies had broad specificity for HDL (including pre-beta-1 HDL). This research suggests that apo A-I residues 137-144 are adjacent to or part of a structural site in pre-beta-1 HDL active in promoting the efflux of cellular cholesterol and that this site is not exposed in other HDL species.

Quantitation of pre beta-HDL-dependent and nonspecific components of the total efflux of cellular cholesterol and phospholipid.

Both receptor-mediated and diffusional processes have been proposed as mechanisms for the efflux of cellular cholesterol to plasma. The depletion of a minor high-density lipoprotein subfraction (pre beta-1-HDL) from plasma by incubation was associated with a proportional reduction in up to 58% of cholesterol and lecithin efflux from cultured fibroblasts. Pre beta-HDL-dependent efflux was blocked by protease pretreatment of the cells, while residual ("nonspecific") efflux was protease-insensitive. The whole of cholesterol efflux from blood erythrocytes was both pre beta-1-HDL-and protease-independent. These data suggest that two distinct pathways contribute to total efflux from fibroblast monolayers; one of these is directly proportional to plasma pre beta-1-HDL concentration and may involve a cell-surface protein.

Regulation of the concentration of pre beta high-density lipoprotein in normal plasma by cell membranes and lecithin-cholesterol acyltransferase activity.

A minor fraction of plasma high-density lipoprotein (pre beta-1 HDL) has been shown to promote cholesterol efflux from peripheral cell membranes [Castro, G. R., & Fielding, C. J. (1988) Biochemistry 27, 25-29]. When isolated native plasma is incubated at 37 degrees C, this fraction is specifically decreased. On the other hand, the level of plasma pre beta-1 HDL is fully protected in the presence of even very low levels of fibroblasts, vascular smooth muscle cells, or macrophages. Blood cells were completely inactive in maintaining plasma pre beta-1 HDL levels in the absence of peripheral cells, even at the relatively high levels present in whole blood. The loss of pre beta-1 observed in isolated plasma was dependent upon lecithin-cholesterol acyltransferase (LCAT) activity. These data suggest that reverse cholesterol transport catalyzed by pre beta-1 HDL, and subsequent LCAT-mediated cholesterol esterification, is directly dependent upon the interaction between this HDL species and competent peripheral cells.