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Originally published In Press as doi:10.1074/jbc.M505685200 on August 23, 2005

J. Biol. Chem., Vol. 280, Issue 43, 35890-35895, October 28, 2005
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Serum Amyloid A Promotes Cholesterol Efflux Mediated by Scavenger Receptor B-I*

Deneys R. van der Westhuyzen{ddagger}12, Lei Cai{ddagger}1, Maria C. de Beer§, and Frederick C. de Beer{ddagger}

From the Departments of {ddagger}Internal Medicine and §Physiology, Graduate Center for Nutrition Sciences, the University of Kentucky Medical Center, Lexington, Kentucky 40536 and the Department of Veterans Affairs Medical Center, Lexington, Kentucky 40511

Received for publication, May 24, 2005 , and in revised form, August 18, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Serum amyloid A (SAA) is an acute phase protein whose expression is markedly up-regulated during inflammation and infection. The physiological function of SAA is unclear. In this study, we reported that SAA promotes cellular cholesterol efflux mediated by scavenger receptor B-I (SR-BI). In Chinese hamster ovary cells, SAA promoted cellular cholesterol efflux in an SR-BI-dependent manner, whereas apoA-I did not. Similarly, SAA, but not apoA-I, promoted cholesterol efflux from HepG2 cells in an SR-BI-dependent manner as shown by using the SR-BI inhibitor BLT-1. When SAA was overexpressed in HepG2 cells using adenovirus-mediated gene transfer, the endogenously expressed SAA promoted SR-BI-dependent efflux. To assess the effect of SAA on SR-BI-mediated efflux to high density lipoprotein (HDL), we compared normal HDL, acute phase HDL (AP-HDL, prepared from mice injected with lipopolysaccharide), and AdSAA-HDL (HDL prepared from mice overexpressing SAA). Both AP-HDL and AdSAA-HDL promoted 2-fold greater cholesterol efflux than normal HDL. Lipid-free SAA was shown to also stimulate ABCA1-dependent cholesterol efflux in fibroblasts, in line with an earlier report (Stonik, J. A., Remaley, A. T., Demosky, S. J., Neufeld, E. B., Bocharov, A., and Brewer, H. B. (2004) Biochem. Biophys. Res. Commun. 321, 936–941). When added to cells together, SAA and HDL exerted a synergistic effect in promoting ABCA1-dependent efflux, suggesting that SAA may remodel HDL in a manner that releases apoA-I or other efficient ABCA1 ligands from HDL. SAA also facilitated efflux by a process that was independent of SR-BI and ABCA1. We conclude that the acute phase protein SAA plays an important role in HDL cholesterol metabolism by promoting cellular cholesterol efflux through a number of different efflux pathways.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Serum amyloid A (SAA)3 is an acute phase protein whose concentration increases markedly following bacterial infection, tissue damage, and inflammation (2, 3). SAA synthesis is induced by inflammatory stimuli mainly in the liver, although induction of SAA synthesis also occurs in adipocytes, intestinal epithelial cells, myocytes, and macrophages (2). During the acute phase response, SAA synthesis accounts for as much as 2.5% of total protein production in the liver, and plasma SAA levels can reach 1 mg/ml, suggesting a beneficial role for SAA in host defense. SAA is present in plasma mainly as an HDL apolipoprotein and can replace apoA-I as the major HDL protein (4, 5). SAA shares certain structural features with other apolipoproteins, including amphipathic helices, a motif that appears responsible for SAA binding to HDL (6).

The biological function of SAA remains uncertain (reviewed in Refs. 1 and 2). For example, various studies have suggested that SAA plays a role in inflammatory processes, probably acting via the G-protein-coupled receptor FPRL1 (7, 8). As an HDL apolipoprotein, it is plausible that SAA influences HDL function, including the role of HDL in cholesterol transport. SAA has been reported in some (9, 10) but not all (11) studies to facilitate cholesterol efflux from macrophages to HDL. More importantly, SAA was recently shown to promote specifically both ABCA1-dependent and ABCA1-independent cholesterol and phospholipids efflux from cells (1). Together, these results suggest that SAA may promote cholesterol removal from cells and perhaps extracellular lipid deposits at sites of inflammation and tissue repair.

Scavenger receptor SR-BI is an HDL receptor that mediates cellular uptake of cholesterol ester from HDL by a mechanism known as selective lipid uptake (12, 13). SR-BI-dependent selective lipid uptake in the liver plays a key role in HDL cholesterol clearance, thereby facilitating reverse cholesterol transport from the periphery to the liver. The receptor exhibits a broad ligand binding specificity and binds low density lipoprotein, very low density lipoprotein, and oxidized lipoproteins in addition to HDL (12). SR-BI also binds anionic phospholipids as well as the apolipoproteins A-I, A-II, C-III, and E, either as lipid-bound or as free apolipoproteins. In addition to mediating selective uptake, SR-BI facilitates the efflux of cellular free cholesterol to HDL. We reported recently that SAA is a high affinity ligand for SR-BI that exerts an inhibitory effect on HDL binding and selective lipid uptake (14).

In this study we demonstrate that SAA, unlike apoA-I, promotes both SR-BI-dependent and SR-BI-independent cholesterol efflux from cells and that the presence of SAA on HDL is associated with increased cholesterol efflux to HDL. In addition, SAA was shown to function in a synergistic manner with HDL in promoting ABCA1-dependent efflux.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Human apoA-I and recombinant human SAA (corresponding to human SAA1{alpha} except for the presence of an N-terminal methionine and substitution of asparagine for aspartic acid at position 60 and arginine for histidine at position 71) were purchased from PeproTech (Rocky Hill, NJ). [3H]Cholesterol was purchased from Amersham Biosciences. BLT-1 was kindly provided by Dr. G. H. Rothblat (University of Pennsylvania). HDL3 was prepared as described previously (15).

Animals—C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mouse SAA overexpression was obtained by tail vein injection of a second generation recombinant adenovirus expressing the mouse CE/J isoform of SAA that closely resembles both SAA1 and SAA2 (16). The SAA adenovirus, Ad-SAA, was prepared as described previously (17). 4 x 1011 particles of Ad-SAA adenovirus or Ad-Null virus (a virus containing no transgene) were injected into each mouse. Animals were sacrificed 72 h after virus infusion; blood was collected by cardiac puncture, and HDL was isolated as described previously (14). Acute phase-HDL (AP-HDL) was obtained from C57BL/6 mice injected with lipopolysaccharide (25 µg/mice) and sacrificed 24 h later. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (Veterans Affairs Medical Center, Lexington, KY).

Cell Culture—Stable transfectants of CHO cells expressing human SR-BI (CHO-SRBI cells) were generated as described elsewhere (18) and cultured in medium containing 0.25 g/liter geneticin (Invitrogen). CHO cells were grown in Ham's F-12 medium supplemented with 5% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 50 units/ml penicillin G, and 50 µg/ml streptomycin. Clones were screened for expression of the scavenger receptors by immunoblotting with a rabbit polyclonal antibody recognizing the extracellular domain of SR-BI (18). For biochemical assays, CHO cells were grown in 12-well clusters (Corning Corp., Corning, NY). Human skin fibroblasts and Tangier disease fibroblasts were kindly provided by Dr. John Oram (Washington University, Seattle). Fibroblasts were grown in DMEM containing 10% (v/v) heat-inactivated fetal bovine serum (BSA), 2 mM L-glutamine, 50 units/ml penicillin G, and 50 µg/ml streptomycin. HepG2 cells were grown in minimum essential medium (Eagle, from ATCC) with 10% (v/v) fetal bovine serum, 0.1 mM nonessential amino acid, 2 mM L-glutamine, and 1.0 mM sodium pyruvate. Adenoviral mediated gene overexpression of SAA, apoE3, and apoA-I in HepG2 cells was performed by addition of Ad-Null, Ad-apoE3, Ad-SAA, or Ad-apoA-I at a viral dose of 6 x 103 particles per cell. The production of replication-defective adenoviruses expressing human apoA-I and apoE3 was described previously (19).

Cholesterol Efflux Determination—Cellular cholesterol efflux was determined essentially as described (20). For cholesterol efflux experiments, cells (about 70% confluent) in 12-well plates were labeled with 0.2 µCi/ml [3H]cholesterol (35–50 Ci/mmol, Amersham Biosciences) in medium for 48 h. Cells were then washed five times with PBS containing 1 mg/ml BSA and equilibrated in serum-free medium containing 0.2% fatty acid-free BSA for 16 h. Thereafter, cells were incubated for 5 h at 37 °C in media with or without lipoproteins or free apolipoproteins, as indicated in the legends to figures. Following incubation, the medium was collected, and cells were washed three times with PBS containing 1 mg/ml fatty acid-free BSA and then three times with PBS only at 4 °C. Radioactivity in the media was measured directly in a Packard {beta} liquid scintillation counter. Cellular lipid was extracted with hexane/isopropyl alcohol (3:2 v/v) for 30 min at room temperature and counted for radioactivity. Lipid-extracted cells were solubilized in 0.1 N NaOH for protein determination. Efflux of cellular [3H]cholesterol to media is expressed as a percentage of total radioactivity in media and cells. Efflux was calculated as the percentage of counts in the medium to the counts in the medium and cells together. SR-BI-specific values were calculated as the difference between the efflux values in CHO-SRBI cells and control CHO-A7 cells. In fibroblast experiments, ABCA1 expression was up-regulated following 3H labeling by treating cells for 48 h with 30 µg/ml free cholesterol in DMEM containing 0.2% fatty acid-free BSA. Efflux experiments were carried out in BSA-containing medium with or without ligands for 16 h.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The ability of SAA to function as an acceptor for SR-BI-mediated cholesterol efflux from cells was tested using purified lipid-free SAA. Like apoA-I, SAA was shown recently to bind to SR-BI as a high affinity ligand (14, 21). Fig. 1A shows that lipid-free SAA (10 and 30 µg/ml) mediated cellular cholesterol efflux in an SR-BI-dependent manner from CHO cells expressing human SR-BI. SR-BI-specific values are shown for a 5-h efflux period and calculated as the difference in efflux from CHO-SRBI and control CHO-A7 cells. Values for efflux in control cells represented 20–30% of the efflux from SR-BI-expressing cells. SR-BI-mediated cholesterol efflux was less than that observed to human HDL, although greater than the low efflux observed to apoA-I. Efflux to SAA was concentration-dependent and tended to reach a maximum at the higher SAA concentrations used (Fig. 1B). At higher concentrations, cholesterol efflux to SAA approached the value for HDL. SAA also facilitated cholesterol efflux in a manner that was SR-BI independent, as indicated by the efflux values in the control cells not expressing SR-BI.



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  		FIGURE 1.
SR-BI-dependent cholesterol efflux to SAA. CHO-SRBI and control CHO-A7 cells were labeled with 0.2 µCi/ml [3H]cholesterol for 48 h, equilibrated in medium in the absence of cholesterol for 16 h, and then incubated with SAA, apoA-I, or human HDL3 at 37 °C for 5 h for the determination of cellular efflux as described under "Experimental Procedures." Medium was collected and assayed for [3H]cholesterol. Efflux of free cholesterol into the media is expressed as percentage of the total radioactivity in the media and cells ([3H]cholesterol in the medium x 100)/([3H]cholesterol in medium + [3H]cholesterol in the cells). SR-BI-specific values were calculated as the difference between the efflux values in CHO-SRBI cells and control CHO-A7 cells. Values represent the average of triplicate determinations (mean ± S.D.). Similar results were obtained in three separate experiments. A, SR-BI-specific cholesterol efflux to SAA, apoA-I, and HDL. B, SR-BI-mediated cholesterol efflux to SAA and apoA-I in a concentration-dependent manner.

 
The ability of SAA to facilitate cholesterol efflux in hepatocytes was addressed by using HepG2 cells. These cells express endogenous SR-BI that mediates selective lipid uptake from HDL. As shown for CHO-SRBI cells, SAA mediated significant cholesterol efflux from HepG2 cells (Fig. 2A). In contrast, efflux to apoA-I was, as for CHO-SRBI cells, considerably lower than efflux to SAA. To assess the role of SR-BI in cholesterol efflux from HepG2 cells, BLT-1, an inhibitor of SR-BI-mediated efflux (22), was tested. BLT-1 inhibited SAA-dependent efflux from HepG2 cells by ~60–70%, and HDL-mediated efflux by about 30–40% (Fig. 2B). The same inhibitor reduced SR-BI-dependent cholesterol efflux in CHO cells by about 70% (Fig. 2C). These results indicate that in HepG2 cells the bulk of cellular efflux to SAA is mediated by SR-BI.

To assess whether SAA produced endogenously in cells is able to facilitate cholesterol efflux from cells, SAA was overexpressed in HepG2 cells by means of adenoviral mediated gene transfer, and cholesterol efflux was then measured. For comparison, apoA-I and apoE3 were also examined. In each case, gene transfer resulted in the cellular expression and secretion of apolipoprotein into the medium. Apolipoprotein quantification by Western blotting using purified protein standards showed similar levels of each of the apolipoproteins in the cell media at the end of the efflux period (SAA, 9 µg/ml; apoA-I, 11 µg/ml; apoE3, 10 µg/ml). Overexpression of SAA increased cholesterol efflux ~1.5-fold (Fig. 3). ApoE3 overexpression similarly increased cholesterol efflux about 1.7-fold. ApoA-I overexpression, on the other hand, had no effect on efflux, in line with the results shown in Figs. 1 and 2.



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  		FIGURE 2.
Cholesterol efflux to SAA in HepG2 cells. HepG2 cells were labeled with [3H]cholesterol for 48 h, equilibrated in medium in the absence of cholesterol for 16 h, and then incubated with SAA, apoA-I, or human HDL3 at 37 °C for 5 h. For BLT-1 treatments, BLT-1 (5 µM) was added together with acceptor and incubated at 37 °C for 5 h for determination of cellular efflux as described under "Experimental Procedures." Medium was collected and analyzed for [3H]cholesterol. Efflux of free cholesterol into the media is expressed as a percentage of the total radioactivity in the media and cells. A, cholesterol efflux to SAA, apoA-I, and HDL. B, cholesterol efflux to SAA (30 µg/ml) or HDL (40 µg/ml) with BLT-1 treatment. C, SR-BI-dependent cholesterol efflux to HDL (40 µg/ml) with BLT-1 treatment in CHO cells.

 
To assess whether the presence of SAA on HDL influences the ability of HDL to serve as an acceptor for SR-BI-mediated cholesterol efflux, SAA-HDL, AP-HDL, and control mouse HDL were compared in their ability to facilitate efflux. SAA-HDL was isolated from mice overexpressing SAA through adenoviral mediated gene transfer (17). AP-HDL was isolated from mice following lipopolysaccharide injection. Compared with control normal mouse HDL, both AdSAA-HDL and AP-HDL showed significantly greater (2–3-fold) efflux from CHO-SRBI cells (Fig. 4A). Similar results showing increased cholesterol efflux to the SAA-containing HDLs were obtained when efflux to the three ligands was measured in HepG2 cells (Fig. 4B).



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  		FIGURE 3.
Cholesterol efflux to endogenously expressed SAA in HepG2 cells. HepG2 cells were labeled with 0.2 µCi/ml [3H]cholesterol in medium for 48 h, washed five times with PBS containing 0.1% fatty acid-free BSA, and then incubated for 18 h at 37 °C with 0.2% BSA minimum Eagle's medium containing the indicated adenovirus at a viral dose of 6 x 103 particle per cell. Medium was collected and assayed for efflux as described in Fig. 1. Efflux of free cholesterol into the media is expressed as the percentage of the total radioactivity in the media and cells. Values represent the average of triplicate determinations (mean ± S.D.). Similar results were obtained in three separate experiments.

 
In contrast to SR-BI-mediated cholesterol efflux, efflux-mediated by ABCA1, the well studied cholesterol transporter, was not influenced by the SAA enrichment of HDL (Fig. 5A). Most interestingly, neither SAA-HDL nor AP-HDL showed a greater ability than normal HDL to facilitate ABCA1-dependent cholesterol efflux from fibroblasts. To investigate the ability of SAA to function in cellular cholesterol efflux facilitated by ABCA1, we measured cholesterol efflux to lipid-free SAA or apoA-I in normal human fibroblasts and fibroblasts lacking functional ABCA1 obtained from a patient with Tangier disease. SAA, like apoA-I, was able to function as an acceptor of cellular cholesterol (Fig. 5B). Although at higher concentrations the two apolipoproteins appeared similar in their ability to mediate efflux, at lower concentrations apoA-1 was a more efficient ligand than SAA. SAA can displace apoA-I from HDL and become the major apolipoprotein of HDL (4, 5). To test the hypothesis that SAA might indirectly affect ABCA1-mediated cholesterol efflux by displacing apoA-I from HDL, cholesterol efflux to HDL was measured in the presence or absence of SAA. As shown in Fig. 6, both lipid-free apoA-I and SAA functioned as acceptors for cellular cholesterol. As expected (Fig. 5B), SAA served as a less efficient acceptor than apoA-I. The addition of apoA-I to HDL resulted in an additive increase in efflux. Most interestingly, the addition of SAA to HDL results in a synergistic type of increase in efflux so that efflux in the presence of both SAA and HDL always exceeded the sum of the efflux values for SAA and HDL when added alone. These results support the hypothesis that SAA affects ABCA1 efflux by displacing apoA-I or other apoA-I-containing fractions that are efficient ABCA1 ligands, from HDL.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we report that the acute phase protein SAA, like HDL, facilitates SR-BI-dependent efflux of cholesterol from both CHO cells and the hepatocyte cell line HepG2. The ability of lipid-free SAA to mediate cholesterol efflux was significantly greater than that of apoA-I in each cell type. Enhanced cellular cholesterol efflux was also observed in response to SAA produced within cells, as shown using adenovirus-mediated overexpression of the protein. Enrichment of HDL with SAA also significantly increased SR-BI-dependent cholesterol efflux to HDL. Lipid-free SAA was shown to also stimulate ABCA1-dependent cholesterol efflux, in line with an earlier report (1). Most interestingly, when added to cells together with HDL, SAA exerted a synergistic effect in increasing ABCA1-dependent efflux, suggesting that SAA may remodel HDL in a manner that releases apoA-I, an efficient ABCA1 ligand, from HDL. In addition to SR-BI- and ABCA1-dependent cholesterol efflux, SAA also facilitated a degree of efflux that was independent of these two receptors.



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  		FIGURE 4.
Cholesterol efflux to AP-HDL and AdSAA-HDL in CHO-SRBI and HepG2 cells. Efflux experiments were carried out at 37 °C for 5 h in 0.2% BSA medium containing AP-HDL and AdSAA-HDL (40 µg/ml) as described in Fig. 1. Efflux to normal mouse HDL is shown for comparison. SR-BI-specific efflux was calculated as described in Fig. 1. Values represent the average of triplicate determinations (mean ± S.D.). Similar results were obtained in three separate experiments. A, SR-BI-specific cholesterol efflux in CHO cells. B, cholesterol efflux in HepG2 cells.

 
Efflux of cholesterol from cells occurs by two main mechanisms, namely passive diffusion from the plasma membrane and an active efflux mediated mainly by ABCA1 to lipoproteins (23). SR-BI also facilitates cellular cholesterol efflux to HDL (24), and evidence suggests that this receptor acts by allowing bi-directional cholesterol flux according to the cholesterol concentration gradient between the plasma membrane and phospholipid-containing acceptor particles (25). Phospholipid-enriched HDLs generally function as the most effective acceptors for SR-BI-dependent efflux. This is in clear contrast to ABCA1-dependent efflux, for which the best acceptors are lipid-free or lipid-depleted apolipoproteins (26). In the case of SR-BI-dependent efflux, there is some uncertainty if efflux requires HDL binding or docking to the receptor, as suggested by some studies (27), or if efflux is independent of direct HDL binding and promoted rather through an ability of SR-BI to alter cellular cholesterol distribution and to increase cholesterol concentration in an accessible pool in the plasma membrane outer leaflet (28). In the present study we show that lipid-free SAA, but not apoA-I, is an effective acceptor for SR-BI-dependent efflux. The mechanism of such efflux is not yet understood. We recently reported that SAA is a high affinity ligand for SR-BI (14). Such binding to SR-BI might facilitate the transfer of membrane cholesterol to SAA, a protein known to bind cholesterol with a 1:1 stoichiometry (29). However, the calculated molar amount of cholesterol effluxed from cells exceeds that of SAA in the medium by ~10-fold, indicating that direct binding of cholesterol to SAA could only account for a small fraction of the effluxed cholesterol in the medium. Another possibility is that SR-BI-dependent efflux does not require direct binding of SAA to SR-BI but rather is the consequence of SR-BI expanding the accessible membrane cholesterol pool (28). In this case, SAA may promote efflux through a detergent-like extraction process as has been suggested by Stonik et al. (1) in the case of ABCA1-independent cholesterol efflux to SAA. In the case of SR-BI-dependent efflux, SAA binding to the receptor at the cell surface may serve to promote such an extraction or microsolubilization process. In contrast to SAA, apoA-I is much less efficient in promoting both SR-BI-dependent and SR-BI-independent cholesterol efflux. This may relate to a higher affinity of cholesterol for SAA than for apoA-I and greater interfacial surface binding properties of SAA compared with apoA-I (5). SAA can displace apoA-I from HDL, suggesting a greater lipid binding affinity of SAA than apoA-I.



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  		FIGURE 5.
SAA promotes ABCA1-dependent cholesterol efflux in human skin fibroblasts. ABCA1-dependent cholesterol efflux was determined in skin fibroblasts isolated from normal and Tangier disease patients. Human skin fibroblast cells were labeled with 0.2 µCi/ml [3H]cholesterol in medium for 48 h. Cellular ABCA1 expression was stimulated with free cholesterol loading by incubating fibroblasts with 30 µg/ml cholesterol in medium containing 0.2% fatty acid-free BSA for 48 h. Efflux experiments were performed at 37 °C by incubating cells with AP-HDL, AdSAA-HDL, or control AdNull-HDL for 16 h in DMEM containing 0.2% BSA. ABCA1-specific efflux was calculated by subtracting the value obtained in Tangier disease fibroblasts from the value in normal fibroblasts. Values represent the average of triplicate determinations (mean ± S.D.). Similar results were obtained in three separate experiments. A, cholesterol efflux to AP-HDL and AdSAA-HDL and normal mouse HDL (40 µg/ml). B, concentration-dependent cholesterol efflux to apoA-I and SAA in ABCA1 and control Tangier cells lacking ABCA1.

 



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  		FIGURE 6.
SAA and HDL exert a synergistic effect in promoting ABCA1-dependent cholesterol efflux. ABCA1-dependent efflux was carried out in human skin fibroblasts as described in Fig. 5. ABCA1-dependent efflux was performed at 37 °C for 16 h by incubating cells with apoA-I, SAA, human HDL3, or SAA and HDL3 together. Values represent the average of triplicate determinations (mean ± S.D.). Similar results were obtained in three separate experiments.

 
To test how the presence of SAA on HDL may influence SR-BI-dependent cholesterol efflux, we overexpressed SAA in C57BL/6 mice using adenoviral mediated gene transfer (17). HDL from these mice was enriched in SAA, as is typical for acute phase HDL, with SAA being the second most abundant apolipoprotein (5, 17). As an alternative approach, we also assessed SAA-enriched HDL isolated from acute phase mice following the injection of lipopolysaccharide. For both types of HDL, SR-BI-dependent efflux in CHO cells as well as HepG2 cells was significantly increased compared with control mouse HDL. Increased efflux occurred despite increased levels of unesterified cholesterol in both types of SAA-enriched particles (14). These results are in line with earlier reports showing that the presence of SAA on HDL increased efflux from cholesterol-loaded macrophages, although the receptors involved in those studies were not identified (30). The mechanism by which SAA on HDL stimulates efflux is not clear but apparently does not relate to changes in HDL binding to SR-BI or changes in HDL phospholipid content, both of which are relatively unchanged in the SAA-enriched HDL (14). Most interestingly, SAA-enriched particles were not more effective than control HDL in mediating ABCA1-dependent efflux, indicating that SAA influences these two efflux pathways differently.

Lipid-free SAA has been shown previously to promote ABCA1-dependent as well as ABCA1-independent efflux, and our results using normal and Tangier disease fibroblasts confirm these findings (1). To assess the relative effectiveness of the two apolipoproteins more closely, we compared ABCA1-dependent efflux rates at different ligand concentrations. ApoA-I functioned more effectively at lower concentrations than SAA, but at higher concentrations the two proteins exhibited similar activity. SAA also promoted efflux from fibroblasts independently of ABCA1, whereas apoA-I was ineffective in the absence of ABCA1. In additional studies we made the interesting observation that when added to HDL, SAA, but not apoA-I, resulted in a level of efflux that was more than additive compared with efflux to the two acceptors added individually. This finding suggests that SAA may promote efflux directly through ABCA1 as well as indirectly through its interaction with HDL. SAA can displace apoA-I from HDL (11), and such lipid-free apoA-I would serve as an efficient ABCA1 ligand. Alternatively, it is possible that the addition of SAA to HDL may result in the remodeling of HDL particles with the generation of highly efficient ABCA1 ligands such as pre-{beta} HDL.

The stimulation of cholesterol efflux by SAA through ABCA1- and SR-BI-dependent mechanisms, as well as by a receptor-independent process, provides evidence that SAA plays a physiological role in promoting cholesterol efflux from cells and reverse cholesterol transport during inflammation and the acute phase response. SAA appears to be well placed for such a role. SAA synthesis is markedly induced (up to 1000-fold) during the acute phase response (2). Although the liver is the major site of SAA synthesis, SAA is also produced in macrophages, adipocytes, enterocytes, and myocytes (2). SAA has also been found in atherosclerotic plaques (31). The endocytosis of SAA has been proposed to explain the increased ability of acute phase HDL to promote cellular cholesterol efflux from cholesterol-laden macrophages (30), although not from non-lipid-loaded macrophages (30). These studies did not identify the receptor(s) responsible for SAA-induced efflux. Internalization of SAA by macrophages has been shown, and the SAA2.1 isoform or SAA2.1-derived peptides have been reported to regulate inversely the intracellular acyl-CoA:cholesterol O-acyltransferase and cholesterol esterase activities in a manner that elevates cellular free cholesterol and consequently cholesterol efflux (10). SR-BI is expressed in activated macrophages (32) and also in atherosclerotic lesions (33), and we have reported that SR-BI internalizes SAA (14). However, the contribution of SR-BI to SAA uptake in macrophages is not known.

The liver represents the major site of SAA synthesis. The finding that SAA facilitates cholesterol efflux through ABCA1- and SR-BI-dependent pathways, as well as a nonreceptor mechanism, suggests that SAA may serve to promote cholesterol efflux also from the liver. Based on our findings, efflux to SAA-enriched HDL through SR-BI would be expected to increase. Lipid-free SAA would also increase efflux. Although the bulk of SAA in plasma is associated with lipoproteins, largely HDL, it is possible that sufficient SAA may exist in lipid-free form to promote significant efflux (7, 34). In addition, evidence suggests that newly synthesized SAA is secreted in a lipid-free form from hepatocytes (35), and we propose that within the confines of the space of Disse in the liver, secreted SAA would serve to facilitate cholesterol efflux during the acute phase response when SAA is subject to marked induction. Infection and inflammation are associated with a general reduction in HDL and cholesterol levels (36), and although it has been thought that SAA in HDL might be responsible for reduced HDL, recent evidence indicates that this is not the case. It is clear that the decrease in HDL is rapid and precedes the increase in SAA (37). Furthermore, an increased expression of SAA does not decrease plasma HDL in the absence of infection or inflammation (38). Our results suggest that SAA functions rather to contribute to the maintenance of HDL and cholesterol levels during the acute phase by decreasing SR-BI-mediated HDL cholesterol ester uptake (14) or, on the other hand, by promoting liver cholesterol efflux.

In conclusion, our findings indicate that SAA promotes cellular cholesterol efflux through SR-BI- and ABCA1-dependent pathways as well as a nonreceptor process. During the acute phase response, SAA influences cellular and HDL cholesterol metabolism through its effects on HDL cholesterol ester uptake by SR-BI and its effects on cellular cholesterol efflux through different efflux pathways.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants HL-63763, HL-65730 (to D. R. v. d. W.), and AG-17237 (to F. C. d. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: University of Kentucky, Graduate Center for Nutritional Sciences, Wethington Health Science Bldg. 541, 900 S. Limestone St., Lexington, KY 40536-0200. Tel.: 859-323-4933 (ext. 81397); Fax: 859-257-3646; E-mail: dvwest1{at}uky.edu.

3 The abbreviations used are: SAA, serum amyloid A; SR-BI, scavenger receptor B-I; ABCA1, ATP-binding cassette transporter A1; AP-HDL, acute phase-high density lipoprotein; BSA, bovine serum albumin; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank Wei Shi, Nathan Whitaker, and Xin Shi for excellent technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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