Phosphatidylcholine transfer protein promotes apolipoprotein A-I-mediated lipid efflux in Chinese hamster ovary cells.

Phosphatidylcholine transfer protein (PC-TP) is a cytosolic protein of unknown function that catalyzes intermembrane transfer of phosphatidylcholines in vitro. Using stably transfected CHO cells, we explored the influence of PC-TP on apolipoprotein A-I- and high density lipoprotein 3 (HDL(3))-mediated lipid efflux. In proportion to its cellular level of expression, PC-TP accelerated apolipoprotein A-I-mediated phospholipid and cholesterol efflux as pre-beta-HDL particles. PC-TP increased rates of efflux of both lipids by >2-fold but did not affect mRNA levels or the activity of ATP-binding cassette A1, a plasma membrane protein that regulates apolipoprotein A-I-mediated lipid efflux. Overexpression of PC-TP was associated with only slight increases in HDL(3)-mediated phospholipid efflux and no changes in cholesterol efflux. In scavenger receptor BI-overexpressing cells, PC-TP expression minimally influenced apolipoprotein A-I- or HDL(3)-mediated lipid efflux. PC-TP did not affect cellular phospholipid compositions, phosphatidylcholine contents, or phosphatidylcholine synthetic rates. These findings suggest that a physiological function of PC-TP is to replenish the plasma membrane with phosphatidylcholines that are removed during pre-beta-HDL particle formation due to the activity of ATP-binding cassette A1.

Phosphatidylcholine transfer protein (PC-TP) 1 is a 25-kDa cytosolic protein that promotes intermembrane exchange and net transfer of phosphatidylcholines but no other phospholipid class (1). The gene encoding PC-TP is highly conserved among species (2)(3)(4) and is widely expressed in human (4) and mouse (2) tissues, with the highest levels present in liver. Although unrelated to other cytosolic lipid transfer proteins or to the plasma phospholipid transfer protein, PC-TP has been identified as a steroidogenic acute regulatory protein-related (START) domain (5) using a recently developed algorithm for protein data base analysis (6). Whereas steroidogenic acute regulatory protein appears to function as a lipid transfer protein in steroidogenic tissues to promote delivery of cholesterol from the outer to inner mitochondrial membrane (7), the function of PC-TP remains unknown (8).
Reverse cholesterol transport is the metabolic pathway for movement of cholesterol from peripheral tissues to liver (9), which depends critically upon metabolism of high density lipoproteins (HDL). Formation of HDL begins when apolipoprotein A-I (apoA-I) molecules interact with the plasma membrane to form particles with pre-␤ mobility on agarose gels (10 -14). Assembly of pre-␤-HDL requires the presence on the plasma membrane of the Tangier disease gene product, ATP-binding cassette A1 (ABCA1) (15,16). ABCA1 appears to play a critical role in the molecular organization of phospholipids within the plasma membrane bilayer (17)(18)(19), which could explain earlier observations that phospholipids in pre-␤-HDL particles formed by exposing cultured cells to apoA-I are enriched (Ͼ70%) in phosphatidylcholines (20 -24). Mechanistic studies have further suggested that ABCA1-mediated incorporation of phospholipids into pre-␤-HDL precedes efflux of cellular cholesterol to these particles (11,12,24,25).
To preserve its steady state lipid composition, the plasma membrane is presumably replenished with the same lipids that are incorporated into pre-␤-HDL. This study was undertaken to explore the hypothesis that PC-TP may promote apoA-Imediated lipid efflux by supplying the plasma membrane with phosphatidylcholines. Using stably transfected Chinese hamster ovary (CHO) cells, we demonstrate that PC-TP increases apoA-I-mediated efflux of both phospholipids and cholesterol in proportion to its cellular expression level. [1,[2][3] H]cholesterol (45 Ci/mmol) and [methyl-3 H]choline chloride (75 Ci/mmol) were purchased from PerkinElmer Life Sciences. D-␣-[5-methyl- 14 C]tocopherol (57 mCi/mmol) was from Amersham Biosciences. Reagent grade organic solvents and chemicals were purchased from Fisher. Recombinant human PC-TP was expressed in Escherichia coli and purified (Ͼ98%) as previously described (26). A polyclonal rabbit anti-human recombinant PC-TP antibody was prepared as previously described (27). Purified human apoA-I (21) was generously provided by Dr. Michael C. Phillips (Children's Hospital of Philadelphia) and yielded a single band by SDS-PAGE. Prior to use, apoA-I was solubilized in 6 M guanidine HCl and dialyzed extensively against Tris-HCl buffer (10 mM Tris-HCl, 150 mM NaCl, 1.0 mM EDTA, pH 8.2) using 10,000 molecular weight cut-off Slide-A-Lyzer dialysis cartridges (Pierce). Human HDL 3 was purified by ultracentrifugation (28). A cDNA encoding mouse ABCA1 was a generous gift from Dr. Richard M. Green (Northwestern University, Chicago, IL). Human liver cytosol was kindly provided by Dr. Richard Stockert (Albert Einstein College of Medicine).

Preparation of CHO Cell Lines That Overexpress PC-TP
A cDNA encoding human PC-TP (4) was cloned into pcDNA 3.1(Ϫ)/ CMV (Invitrogen, La Jolla, CA), which contained a hygromycin resistance gene. CHO cells were seeded in 37-mm wells of six-well plates at a density of 5 ϫ 10 5 cells/well and grown overnight. The following day, 2 g of vector DNA was mixed with 6 l of LipofectAMINE Plus™ reagent (Invitrogen) in 100 l of serum-free/antibiotic-free IMDM for 15 min at room temperature. This was added to a mixture containing 4 l of LipofectAMINE Plus TM in 100 l of serum-free/antibiotic-free IMDM. After 15 min, the volume was adjusted to 1 ml using serum-free/ antibiotic-free IMDM and added together with 2 ml of serum-free/ antibiotic-free IMDM to a monolayer that had been washed twice with phosphate-buffered saline (PBS). Following incubation for 5 h at 37°C, cells were washed twice with PBS prior to overnight incubation with 3 ml of antibiotic-free IMDM supplemented with 10% serum. The following day, cells were washed twice with PBS, and then selection medium (3 ml) composed of IMDM containing 400 units/ml hygromycin B (Calbiochem) was added. Selected clones were purified by limiting dilution. In preliminary experiments, overexpression of PC-TP did not affect the morphological appearance of CHO cells by light microscopy, nor did it alter their growth kinetics.

Measurements of Cellular Lipid Efflux
Cellular phospholipids or cholesterol were radiolabeled essentially as described by Gillotte et al. (21) by incubation of cells with [ 3 H]choline chloride or [ 3 H]cholesterol, respectively. Cellular ␣-tocopherol was radiolabeled as described by Oram et al. (30). Briefly, cells were seeded at a density of 2-3 ϫ 10 5 cells in 22-mm wells (12-well plates) or 4 -5 ϫ 10 5 cells in 37-mm wells (six-well plates). When the monolayers reached 80% confluence (ϳ2 days), the medium was replaced by IMDM plus 1% fetal bovine serum containing either 3-5 Ci/ml [ 3 H]choline chloride or 2-3 Ci/ml [ 3 H]cholesterol or IMDM plus 10% fetal bovine serum containing 1 Ci/ml [ 14 C]␣-tocopherol. Radiolabeled compounds were added to the medium as concentrated ethanolic stock solutions so that final concentrations of ethanol were less than 1% (by volume). Following 48 -72-h labeling periods, cells were washed three times with IMDM containing Hepes (50 mM) (IMDM-Hepes). Lipid efflux was initiated by the addition to IMDM-Hepes of apoA-I (0 -100 g/ml) or HDL 3 (0 -200 g/ml). During experiments, the temperature was maintained at 37°C by using prewarmed media and by floating covered tissue culture plates in a temperature-controlled water bath. For time course experiments, small aliquots (100 -200 l) of medium were sampled at specified intervals, whereas saturation curves were established by removing a single aliquot at 6 h.
To remove detached cells and cellular debris prior to measurements of radioactivity by liquid scintillation counting, the medium was filtered through a 0.45-m filter using a 96-well vacuum manifold (Millipore Corp., Bedford, MA). For measurements of phospholipid in the medium, [ 3 H]choline-containing lipids were separated from unincorporated [ 3 H]choline by extraction into chloroform (31). The chloroform phase was washed three times with methanol/H 2 O (20:19 by volume), dried under nitrogen, and resuspended in 5 ml of Ecolume liquid scintillation fluid (National Diagnostics U.S.A., Atlanta, GA). Cholesterol was determined by the addition of filtered media directly to the scintillation fluid. To quantify radiolabeled lipids that remained in the monolayer, cells were washed three times with PBS and solubilized overnight in isopropyl alcohol (32). Solutions were dried under nitrogen and resolubilized in scintillation fluid. Unincorporated [ 3 H]choline was removed by organic extraction prior to scintillation counting. Efflux of phospholipid or cholesterol at each time point was quantified as the percentage of total [ 3 H]cholesterol or organic [ 3 H]choline counts (i.e. counts in the medium plus those in the monolayer) that were present in the medium after subtracting counts released nonspecifically into the medium. Counts released nonspecifically were determined by incubating radio-labeled cell monolayers with media that lacked extracellular lipid acceptors under otherwise identical experimental conditions. Nonspecific efflux accounted for 5-15% of counts released in the presence of lipid acceptors.

Characterization of Lipoprotein Particles
Fast Protein Liquid Chromatography (FPLC)-Aliquots of media were applied to a prepacked Superose 6 HR 10/30 size exclusion column (Amersham Pharmacia Biotech) equilibrated with PBS. The flow rate was 0.3 ml/min, and samples were collected at 1-min intervals. Distributions of phospholipid and cholesterol among fractions were determined by liquid scintillation counting. The distribution of apoA-I was determined by slot blot analysis. Briefly, fractions were transferred to a polyvinylidene difluoride membrane using a slot blot apparatus (Invitrogen). The membranes were then probed using a rabbit anti-apoA-I primary antibody (Biodesign, Saco, ME). Detection was by enhanced chemiluminescence (PerkinElmer Life Sciences) with a goat anti-rabbit secondary antibody. Relative band intensities were quantified by laserscanning densitometry/image analysis using ImageQuant™ software (Molecular Dynamics, Inc., Sunnyvale, CA).
Agarose Gel Electrophoresis-Samples of media were electrophoresed together with purified human lipoprotein standards for 30 min at 100 V on preformed lipogels using a Beckman Paragon electrophoresis system (Beckman Coulter, Fullerton, CA). To visualize neutral lipids, gels were fixed, dried, and stained with Sudan Black according to the manufacturer's recommended protocol. Protein components of the particles were analyzed by staining with Coomassie Brilliant Blue. Specific localization of apoA-I was accomplished by transferring particles within the lipogel to a nitrocellulose membrane (Amersham Biosciences) followed by immunodetection as described above.

Lipid Analyses
Cellular Phospholipid Contents-Cell monolayers were extracted overnight using isopropyl alcohol. Lipids were further extracted into chloroform (31) and dried under nitrogen. Phospholipid mass was quantified by an inorganic phosphorus procedure (33). Lipid contents were normalized to cellular protein contents of lysates prepared from identically treated tissue culture dishes. Lysates were prepared by solubilizing monolayers in a single detergent lysis buffer (50 mM Hepes, 200 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% Nonidet P-40, 1 mM dithiothreitol, 2 g/ml leupeptin, 0.1 mM sodium orthovanadate, and 0.1 mM phenylmethylsulfonyl fluoride), and protein contents were determined by the method of Bradford (34) using a Bio-Rad protein assay reagent (Bio-Rad) and bovine serum albumin as a standard.
Phospholipid Classes-Phospholipid classes were quantified by thin layer chromatography (TLC) using silica plates (Whatman), with standards purchased from Avanti Polar Lipids (Alabaster, AL). To determine relative contents of phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, and sphingomyelins, lipids were extracted into chloroform, loaded as 0.5-cm bands on TLC plates, and separated using methyl acetate/n-propanol/chloroform/methanol/ 0.25% aqueous potassium chloride (25:25:25:10:9 by volume) as the solvent system (35). Plates were dried and sprayed with a 0.05% solution in acetone/water (80:20 by volume) of the lipid-binding fluorescent dye primulin (Sigma) (36). An image of the silica plate was generated by laser-excited fluorescence detection scanning using a STORM 840 Phos-phorImager (Molecular Dynamics), and ImageQuant™ software was used to integrate intensities of individual bands. Separately loaded standards were used to correct for phospholipid class-dependent variations in primulin fluorescence.
To determine cellular phosphatidylcholine mass, monolayers were solubilized in 10 mM Hepes, 1 mM EDTA, 2 mM dithiothreitol, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 0.025% NaN 3 , pH 7.4. Phospholipid classes were separated by TLC using chloroform/methanol/ ammonium hydroxide (65:25:5 by volume) as the mobile phase (37). The phosphatidylcholine band was identified using standards, scraped from the silica plate, and eluted from the silica with chloroform/methanol (1:2 by volume). Phosphatidylcholine mass was determined based on lipid phosphorus (33) and expressed as g of lipid phosphorus/mg of cell protein.

Phosphatidylcholine Synthetic Rates
Cells were plated at 10 6 /well in six-well plates and cultured overnight. The following day, cells were pulsed with 0.5 Ci/ml [ 3 H]choline chloride for 30 min, washed, and then incubated in media containing 100 mM unlabeled choline chloride. As functions of time, cellular lipids were extracted (40) and separated by TLC on silica plates with chloroform/methanol/ammonium hydroxide (65:25:5 by volume) as the mobile phase. Radiolabeled phosphatidylcholine was identified based on comigration with standards and was quantified by scraping and liquid scintillation counting. Raw data were corrected for extraction and counting efficiencies and were normalized for cell protein in units of dpm/g of cell protein.

Stable Expression of Human PC-TP in CHO Cells-
To test the influence of PC-TP on cellular lipid efflux in a cell culture model, we prepared CHO cells that overexpress PC-TP. By Western blot analysis (Fig. 1A), there was trace expression of PC-TP in vector-transfected CHO cells. In contrast, expression at low and high levels was readily detected in two independently derived PC-TP-transfected clones. To ensure that the low level of PC-TP in vector-transfected cells by immunoblotting was not attributable to a lack of antibody cross-reactivity with hamster PC-TP, Northern blot analysis was performed at low stringency using a full-length human PC-TP cDNA (Fig. 1B). This demonstrated abundant mRNA expression in PC-TPtransfected, but not in vector-transfected CHO cells. Because apoA-I-mediated lipid efflux is regulated by ABCA1, we also examined whether overexpression of PC-TP influenced ABCA1 expression in CHO cells. Northern blot analysis of vector-transfected and CHO cells expressing PC-TP at high levels revealed no difference in steady state ABCA1 mRNA levels (Fig. 1B). As shown in Fig. 1C, cytosolic levels of PC-TP in low expressing CHO cells were equivalent to human liver cytosol. Fig. 2A shows the influence of PC-TP expression on time-dependent efflux of phospholipid in the presence of apoA-I (50 g/ml). For each cell line, regression analysis showed that phospholipid efflux from CHO cells to apoA-I increased linearly over the 6-h time period. The slopes of these lines represent the rates of phospholipid efflux and are plotted as a function of PC-TP expression in the inset to Fig. 2A. Compared with vector-transfected controls, rates for CHO cells expressing high levels of PC-TP increased by over 2-fold.

PC-TP Expression in CHO Cells Enhances Rates of Phospholipid and Cholesterol Efflux to ApoA-I-
The influence of PC-TP on time-dependent cholesterol efflux from CHO cells is displayed in Fig. 2B. As was observed for phospholipids, efflux of cholesterol increased linearly over the 6-h time course of these experiments in all three cell lines. Rates of cholesterol efflux obtained from linear regression analysis are plotted in the inset to Fig. 2B. Cholesterol efflux rates also increased as a function of PC-TP expression to exceed vector-transfected cells by more than 2-fold. Fig. 3 shows saturation curves for apoA-I-mediated phospholipid and cholesterol efflux, which permitted calculation of apparent K m and apparent V max values by computer fitting the data to a single rectangular, 2-parameter hyperbolic (Michaelis-Menten type) equation using SigmaPlot software (SPSS Science, Chicago, IL). Efflux of phospholipids increased sharply as functions of apoA-I concentration, leveling off between 25 and 50 g/ml. PC-TP appeared to decrease the apparent K m of phospholipid efflux by 2-fold (inset to Fig. 3A). Although this was a consistent observation (n ϭ 3), statistical significance was not achieved (p ϭ 0.11) by two-tailed t test. There was a significant (p Ͻ 0.05) increase in apparent V max by 1.5-fold (inset to Fig. 3A). In addition to characterizing kinetics of lipid efflux from PC-TP-transfected CHO cells in these experiments, we also examined the effects of SR-BI overexpression. Without changing steady state mRNA levels of ABCA1 by Northern blot analysis (not shown), SR-BI overexpression markedly diminished apparent V max for phospholipid efflux to apoA-I by 5-fold from 2.1% for vector-transfected CHO cells to 0.4% for cells that overexpressed SR-BI. We co-expressed PC-TP in SR-BIoverexpressing cells, achieving PC-TP expression at levels similar to high expressing CHO cells in Fig. 1A. The presence of PC-TP was not associated with a significant change in apparent V max (0.3%) and did not affect ABCA1 mRNA levels or SR-BI protein expression (not shown). Fig. 3B demonstrates that PC-TP expression also influenced the kinetics of apoA-I-mediated cholesterol efflux. As was observed for phospholipids, apparent V max increased significantly (1.4-fold, p Ͻ 0.05) as functions of increasing PC-TP expression levels (inset to Fig. 3B). PC-TP expression at low or high levels did not influence the apparent K m . As was observed for phospholipids, overexpression of SR-BI nearly eliminated apoA-Imediated efflux of cholesterol, reducing apparent V max values to 0.5%.
Influence of PC-TP Expression on HDL 3 -mediated Lipid Efflux- Fig. 4A shows the time course of phospholipid efflux in the presence of HDL 3 (100 g/ml). In contrast to phospholipid efflux to apoA-I (Fig. 3A), the observed increase in HDL 3mediated phospholipid efflux was nonlinear over the 6-h time course. High level PC-TP expression was associated with only a slight increase in phospholipid efflux. Time-dependent cholesterol efflux (Fig. 4B) was also nonlinear and was unaffected by PC-TP. Fig. 5 displays the influence of HDL 3 concentration on lipid efflux. Because efflux of phospholipid and cholesterol were not linear functions of time (Fig. 4), application of a Michaelis-Menten type equation for quantification of kinetic parameters was not feasible. Nevertheless, it is apparent that high level PC-TP expression modestly enhanced phospholipid efflux from CHO cells compared with vector-transfected controls (Fig. 5A). Overexpression of SR-BI was associated with reduced phospholipid efflux to HDL 3 , and this was unaffected by PC-TP. The influence of PC-TP on cholesterol efflux to HDL 3 is shown in Fig. 5B. PC-TP expression had no effect on cholesterol release from CHO cells. In contrast, overexpression of SR-BI was associated with a marked increase in HDL 3 -mediated cholesterol efflux. This increase was modestly attenuated by PC-TP.
Characterization of Nascent Particles Following Incubation of CHO Cells with ApoA-I- Fig. 6 shows Superose 6 FPLC elution profiles of particles formed following 6-h incubations of CHO cells with purified human apoA-I (50 g/ml). Phospholipid was predominantly (Ͼ88%) incorporated into two closely associated peaks, with elution volumes (V e ) of 12.8 -15.2 ml and 15.2-16.4 ml (Fig. 6A). The larger of these two peaks eluted near the position of a purified human HDL 3 standard. A small amount (Ͻ12%) of phospholipid released to the medium eluted at the void volume (V o ) as larger particles. The inset to Fig. 6A plots as functions of PC-TP expression, cpm representing total phospholipid as well as phospholipid associated with the small (V e ϭ 12.8 -16.4 ml) and large particles (V o ). Compared with vector-transfected controls, there was a 1.2-and 1.7-fold increase in total apoA-I-mediated phospholipid efflux from low and high PC-TP-expressing CHO cells, respectively. Phospholipid associated with small particles paralleled that of total phospholipid, whereas its incorporation into large particles at V o remained unchanged.
Whereas phospholipids were predominantly associated with small particles, cholesterol was distributed more evenly between the large particles eluting at V o and small particles with V e ranging from 12.8 to 16.4 ml. The inset to Fig. 6B demonstrates that PC-TP expression principally increased the concentration of small particles. Compared with vector-transfected controls, we observed a 2.0-fold increase in small particles for CHO cells expressing PC-TP at low levels and a 2.5-fold increase for the clone expressing PC-TP at high levels. PC-TP expression was associated with only slight (20 -40%) increases in cholesterol efflux to large particles. As shown in Fig. 6C, a single apoA-I peak was resolved by Superose 6 chromatography. The elution volume of the apoA-I peak partially over-lapped with the peaks of phospholipid (Fig. 6A) and cholesterol (Fig. 6B) that were associated with small particle sizes.
To further characterize particles formed by incubation of CHO cells with apoA-I, we analyzed electrophoretic mobility by agarose gel electrophoresis. Following a 6-h incubation period with apoA-I (50 g/ml), medium was collected, filtered, concentrated, and subjected to electrophoresis. Sudan Black staining showed that lipids migrated with pre-␤ mobility, and Coomassie Brilliant Blue staining identified a single band with pre-␤ mobility for the protein component, which was confirmed to be apoA-I by immunoblotting (data not shown).
Lipid Efflux to ApoA-I Is Not Increased by Conditioned Media or by Media Supplemented with Recombinant Human PC-TP-PC-TP is a cytosolic protein and is not known to be secreted from cells. In our experiments, however, enhancement of apoA-I-mediated lipid efflux from PC-TP-transfected CHO cells could have been due to the action of extracellular PC-TP if it was present in media due to secretion or to spillage of the protein from senescent cells. To explore this possibility we 1) tested media for the presence of PC-TP, 2) measured apoA-Imediated lipid efflux from vector-transfected CHO cells using conditioned media obtained from CHO cells that express low or high levels of PC-TP, and 3) measured lipid efflux from vectortransfected controls using media to which we added a high concentration (10 g/ml) of purified recombinant PC-TP (26).
PC-TP release from CHO cells expressing low and high levels of PC-TP was examined by immunoblotting. Monolayers of vector-transfected CHO cells, as well as cells expressing low and high levels of PC-TP were grown to 80% confluence and prepared for lipid efflux experiments, with the exception that radioisotopes were omitted. Cells were then washed three times with IMDM, after which they were incubated in serumfree IMDM-Hepes for 8 h at 37°C. The conditioned medium was collected and filtered to remove any cells. PC-TP was undetectable by Western blot analysis using 40-fold concentrated media (Centricon 10 filter; Millipore) from CHO cells expressing either low or high levels of PC-TP. In separate experiments, conditioned medium was tested on vector-transfected CHO cells for an effect on lipid efflux. Radiolabeled monolayers of vector-transfected CHO cells were exposed for 6 h at 37°C to conditioned medium that was supplemented with apoA-I at a final concentration of 50 g/ml. No differences in efflux of phospholipid or cholesterol were observed due to the use of conditioned media (data not shown). Finally, lipid efflux was measured from vector-transfected CHO cells using nonconditioned medium that contained both apoA-I (50 g/ml) and purified recombinant human PC-TP (10 g/ml). The addition of PC-TP to the medium had no effect on apoA-I-mediated efflux of phospholipid or cholesterol (data not shown).

Expression of PC-TP in CHO Cells Does Not Alter Cellular
Phosphatidylcholine Mass, Rate of Phosphatidylcholine Synthesis, or Phospholipid Composition in Cells or Media-As illustrated in Fig. 7A, PC-TP expression was not associated with changes in steady state cellular levels of phosphatidylcholines. The influence of PC-TP on the distribution of the major phospholipid classes both in CHO cells and media following incubation with apoA-I was determined by TLC. In three independent experiments, no differences were observed in the relative composition of the major cellular phospholipid classes for vector-transfected cells and cells expressing PC-TP at high levels: 50% phosphatidylcholine, 25% phosphatidylethanolamine, 10% phosphatidylinositol, 10% phosphatidylserine, and 15% sphingomyelin. Similarly, there was no influence of PC-TP expression on the cellular distribution of phosphatidylcholine molecular species (i.e. acyl chain composition) as determined by HPLC (data not shown). The distribution of the phospholipid classes released to apoA-I in the medium was similarly unaffected by PC-TP expression in CHO cells: 70% phosphatidylcholine, 20% phosphatidylethanolamine, and 10% sphingomyelin. Under the current experimental conditions, insufficient mass of phosphatidylcholines was released into the medium for determination of acyl chain composition.

DISCUSSION
Although the biochemical properties of purified native (1) and recombinant (26,27) PC-TP have been well characterized, a physiological function for PC-TP has yet to be elucidated (8). The current study was designed to explore whether PC-TP might participate in export of lipids from cells. We examined the influence of PC-TP on lipid efflux when CHO cells were exposed to two types of lipid acceptors that exploit distinct cellular mechanisms for removal of phospholipid and cholesterol. ApoA-I interacts with ABCA1 and the plasma membrane to form pre-␤-HDL by promoting phospholipid-dependent cholesterol efflux from cells (17,24), whereas HDL 3 particles are rich in phospholipids and promote cholesterol efflux principally by an aqueous diffusion-mediated mechanism (42). PC-TP increased rates of apoA-I-but not HDL 3 -mediated lipid efflux in proportion to its level of expression in CHO cells.
Stable transfection of CHO cells allowed us to examine the effects of PC-TP at levels that ranged from trace (vector-transfected cells) to high and included cells with expression levels equivalent to human liver (Fig. 1C). We found no evidence that PC-TP was present in the medium, that conditioned medium from PC-TP-expressing cells enhanced lipid efflux from vectortransfected CHO cells, or that purified recombinant PC-TP increased lipid efflux when added to the medium. These findings indicated that PC-TP acted intracellularly. Expression of ABCA1 is required for apoA-I-mediated efflux of cellular phospholipids and cholesterol (16,43), and mRNA levels for ABCA1 correlate with rates of cholesterol efflux in a variety of cell types (44,45). Therefore, an important initial consideration was whether PC-TP in CHO cells up-regulated expression of ABCA1. We found that steady state ABCA1 mRNA levels were unaffected by overexpression of PC-TP (Fig. 1B).
Although the mechanism(s) by which ABCA1 functions to promote cellular lipid efflux is not known with certainty, accumulating evidence suggests that its catalytic activity involves transmembrane translocation of phospholipids (17)(18)(19). Moreover, it appears that the initial step in pre-␤-HDL formation is ABCA1-dependent assembly of apoA-I-phospholipid com- plexes, which in turn facilitate efflux of cholesterol (24). Because phospholipid and cholesterol were linear functions of time over a 6-h time period (Fig. 2), we used a kinetic analysis of apoA-I saturation curves (Fig. 3) to gain mechanistic insights into PC-TP function. As evidenced by increased values of apparent V max for both phospholipids and cholesterol, PC-TP enhanced the capacity for lipid efflux in proportion to its expression level. Consistent with a primary role in increasing ABCA1-mediated transfer of phospholipids to apoA-I, PC-TP tended to lower apparent K m values for efflux of phospholipid without affecting the apparent K m for cholesterol efflux.
To gain further mechanistic insights, we analyzed the cell culture media to determine the types and compositions of lipid aggregates formed following incubation of cells with apoA-I. By FPLC (Fig. 6), cellular expression of PC-TP increased the number (i.e. peak areas) of smaller, HDL 3 -sized particles and only slightly increased particle sizes (i.e. elution volumes). Although FPLC appeared to resolve two populations of HDL-sized particles, agarose gel electrophoresis revealed only one particle type. In contrast to Forte et al. (23), who demonstrated the presence of ␣-migrating particles following prolonged (24-h) incubation of CHO cells with apoA-I, we observed only pre-␤ migrating particles following shorter (6-h) incubation times. Whereas two separate populations of pre-␤-HDL particles may have been formed in our experiments, the apparent separation of pre-␤ particles into two sizes by FPLC may represent an artifact of gel filtration. ApoA-I is an amphiphilic protein, which solubilizes phospholipids and cholesterol in aggregates that co-exist in equilibrium with monomers of apoA-I (46). As a result of dilution during FPLC, sizes and compositions of lipid aggregates within the column may have been altered due to partial dissociation of the amphiphiles from the particles (47). This would also explain the slightly higher elution volume of apoA-I compared with the elution volumes of phospholipids and cholesterol in Fig. 6 (47). The larger particles that eluted near V o may have been vesicles that were shed from cells or that formed due to nonspecific interactions of apoA-I with the plasma membrane. Because only cholesterol is capable of equilibrating with cellular lipids by aqueous diffusion, these could become preferentially enriched in radiolabeled cholesterol (Fig.  6B) but not radiolabeled phospholipid (Fig. 6A).
Apparent limitations of FPLC analysis notwithstanding, these results taken together with less perturbing agarose gel electrophoresis measurements indicate that PC-TP promoted lipid efflux by increasing pre-␤-HDL particle formation rather than by altering the type of particles formed. In a recent study, Oram et al. have demonstrated that ABCA1 also regulates apoA-I-mediated cellular efflux of ␣-tocopherol (30). We measured efflux of ␣-tocopherol to apoA-1 and observed no influence of PC-TP expression level (data not shown). This finding indicates that PC-TP expression did not affect the intrinsic activity of ABCA1.
To determine whether PC-TP specifically influenced apoA-Imediated lipid efflux, we also examined phospholipid and cholesterol efflux using HDL 3 as a lipid acceptor. Because HDL 3 is a phospholipid-rich particle, it acts as a lipid sink to promote cellular cholesterol efflux by an aqueous diffusion-controlled mechanism, which is modulated by SR-BI (42). In the absence of SR-BI overexpression, PC-TP had no effect on HDL 3 -mediated cholesterol efflux (Figs. 4 and 5). However, we did observe a modest increase in phospholipid efflux at 6 h. In this connection, Wang et al. (48) reported a small increase in HDL 3mediated phospholipid efflux in HEK 293 cells following overexpression of ABCA1. This suggests that the increase in phospholipid efflux to HDL 3 in PC-TP-transfected cells may have been due to phospholipid trafficking via ABCA1 rather than SR-BI.
Whereas CHO cells express ABCA1 at high levels compared with several cell lines (45), they express only modest levels of SR-BI (49). We therefore compared the influence of PC-TP on HDL 3 -mediated lipid efflux in a CHO cell line that overexpresses murine SR-BI (29). Overexpression of SR-BI and/or PC-TP did not alter ABCA1 mRNA levels, and overexpression of PC-TP did not affect SR-BI. Consistent with previous findings in CHO cells (49), SR-BI overexpression enhanced HDL 3mediated cholesterol efflux (Fig. 5). Compared with CHO cells that overexpressed only SR-BI, overexpression of both SR-BI and PC-TP slightly attenuated cholesterol efflux to HDL 3 . Overexpression of SR-BI with or without PC-TP was associated with a decrease in phospholipid efflux to similar levels. Although cells that overexpress SR-BI were derived from a CHO cell line in which the low density lipoprotein receptor was deleted (50), the absence of this receptor was unlikely in our experiments to account for differences in lipid efflux. We therefore conclude that PC-TP does not promote HDL 3 -mediated lipid efflux via SR-BI.
A recent study has demonstrated that overexpression of SR-BI inhibits ABCA1-mediated cholesterol efflux in RAW and HEK 293 cells (51). To further explore the specificity of PC-TP for promoting lipid efflux via ABCA1, we tested the effects of SR-BI on apoA-I-mediated lipid efflux (Fig. 3). We observed that apoA-I-mediated efflux of both phospholipids and choles-  2). B, phosphatidylcholine synthetic rates were determined using a pulse-chase strategy as described under "Experimental Procedures" for vector-transfected CHO cells (‚) and cells expressing PC-TP at high levels (f). Slope values from linear regression analyses represent the phosphatidylcholine synthetic rates. Rates were the same for vector-transfected (0.116 Ϯ 0.016 dpm/mg/min) and PC-TP overexpressing (0.139 Ϯ 0.010 dpm/mg/min) CHO cells. Data points represent triplicate determinations in a representative experiment (n ϭ 3). terol was markedly diminished by SR-BI overexpression, irrespective of the presence of PC-TP. Decreased cholesterol efflux to apoA-I in the setting of SR-BI overexpression is consistent with the findings of Chen et al. (51), who presented evidence that SR-BI suppresses apoA-I-mediated cholesterol efflux by promoting the reuptake of cholesterol that was secreted via ABCA1. In contrast to our findings in CHO cells, these investigators found no effect of SR-BI overexpression on apoA-Imediated phospholipid efflux in RAW cells (51) and postulated that this was due to a lack of SR-BI-mediated reuptake of these molecules. Although we have not investigated lipid uptake under these conditions, SR-BI has been shown to mediate efficient uptake of phospholipids in other cell types (52,53). Alternatively, SR-BI may inhibit ABCA1 activity in CHO cells by another mechanism, such as reorganization of the plasma membrane (42).
PC-TP has been reported to stimulate choline phosphotransferase in vitro (54), suggesting a role in phosphatidylcholine synthesis. However, we observed no effect of PC-TP in CHO cells on the rate of phosphatidylcholine synthesis, cellular phosphatidylcholine mass, or the relative content of phospholipid classes or molecular species in CHO cells. This argues against the possibility that PC-TP may have increased the availability of phospholipid for efflux by promoting synthesis.
The most straightforward interpretation of our data is that PC-TP functions as an intracellular phosphatidylcholine shuttle that replenishes the plasma membrane in response to cellular lipid efflux. Because significant phospholipid efflux was observed in vector-transfected CHO cells, PC-TP expression is not an absolute requirement and other mechanisms are presumably available for intracellular phosphatidylcholine transport. More likely, a physiological function of PC-TP may be to enhance the lipid efflux capacity of tissues in which it is expressed.