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Serum Amyloid a promotes ABCA1-dependent and ABCA1-independent lipid efflux from cells
BBRC Biochemical and Biophysical Research Communications 321 (2004) 936–941 www.elsevier.com/locate/ybbrc
Serum Amyloid a promotes ABCA1-dependent and ABCA1-independent lipid efflux from cells John A. Stonik a, Alan T. Remaley a,*, Steve J. Demosky a, Edward B. Neufeld a, Alexander Bocharov b, H. Bryan Brewer a a
National Institutes of Health Molecular Disease Branch, National Heart, Lung, and Blood Institute, Bethesda, MD, USA b National Institutes of Health, National Institute of Diabetes and Kidney Diseases, Bethesda, MD, USA Received 8 July 2004 Available online 28 July 2004
Abstract Serum amyloid A (SAA) is an acute phase protein that associates with HDL. In order to examine the role of SAA in reversecholesterol transport, lipid efflux was tested to SAA from HeLa cells before and after transfection with the ABCA1 transporter. ABCA1 expression increased efflux of cholesterol and phospholipid to SAA by 3-fold and 2-fold, respectively. In contrast to apoA-I, SAA also removed lipid without ABCA1; cholesterol efflux from control cells to SAA was 10-fold higher than for apoA-I. Furthermore, SAA effluxed cholesterol from Tangier disease fibroblasts and from cells after inhibition of ABCA1 by fixation with paraformaldehyde. In summary, SAA can act as a lipid acceptor for ABCA1, but unlike apoA-I, it can also efflux lipid without ABCA1, by most likely a detergent-like extraction process. These results suggest that SAA may play a unique role as an auxiliary lipid acceptor in the removal of lipid from sites of inflammation. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Serum amyloid A; Apolipoprotein A-I; ABCA1 transporter; Cholesterol; Atherosclerosis
Serum amyloid A (SAA) is an acute phase protein that is produced by the liver and is induced over 1000fold during inflammation [1–4]. In the circulation, SAA is mostly bound to HDL [1,4,5]. During acute infections, SAA can reach protein concentrations as high as apoA-I and can replace apoA-I as the major protein on HDL [5]. Like apoA-I, SAA has several amphipathic helices [6,7], a structural motif that facilitates lipid binding by proteins [8]. The physiologic role of SAA has not been clearly established, but numerous and diverse possible functions have been proposed [1–4]. Because of its association with HDL [1,4,5], and its presence in atherosclerotic plaques [9], one hypothesis is that SAA may play a role in
*
Corresponding author. Fax: 1-301-402-1885. E-mail address: [email protected] (A.T. Remaley).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.07.052
lipoprotein metabolism and in the pathogenesis of atherosclerosis [10]. The level of HDL is markedly reduced, during the acute phase response, and SAA has been suggested to increase HDL catabolism [1,4,11]. The expression, however, of SAA by adenovirus in mice, in the absence of inflammation, did not significantly alter HDL levels [12]. It has also been proposed that SAA may alter the ability of HDL to efflux excess cholesterol from cells [1–4], the first step in the reverse cholesterol transport pathway. Several studies have shown that the enrichment of SAA on HDL decreases the ability of HDL to efflux cholesterol from cells [13,14], and promotes the binding of HDL to macrophages [13–16], and HDL–cholesterol uptake by cells [13,14,17,18]. This has led to the hypothesis that SAA may mediate the delivery of lipid to regenerating cells at the site of inflammation or tissue injury [13,14,17,18]. Under some conditions, however, SAA may instead increase the removal of
J.A. Stonik et al. / Biochemical and Biophysical Research Communications 321 (2004) 936–941
excess lipid from sites of inflammation or tissue injury [10,19,20]. During tissue repair, macrophages phagocytose cell debris from apoptotic and necrotic cells and are transformed into foam cells [10]. Recently, it has been shown, if macrophages are first loaded with cholesterol, SAA-enriched HDL has greater capacity for removing cholesterol from cells than normal HDL [19,20]. Cells efflux excess cholesterol to HDL by either an active process, involving the ABCA1 transporter, or by the passive diffusion of cholesterol from cells to extracellular lipoproteins [21,22]. Mutations in the ABCA1 transporter result in Tangier disease [21,23,24], which is characterized by low HDL and the accumulation of cholesterol in macrophages. Unlike the passive diffusion pathway, lipid-free or lipid-poor apolipoproteins are believed to be the principal acceptor of lipid by the ABCA1 transporter and initiate lipid efflux after first binding to cells [21,22,25]. A key structural motif that is necessary for a protein to act as a lipid acceptor from the ABCA1 transporter is the amphipathic helix [25,26]. In addition to apoA-I, all of the exchangeable type apolipoproteins [25], as well as synthetic peptides [26], such as the 37pA peptide, which contains two amphipathic helices, can also promote ABCA1-dependent lipid efflux. Because SAA contains several amphipathic helices [6,7] and is known to be associated with HDL [1,4,5], it is possible that SAA may also promote to efflux lipid by the ABCA1 transporter. In this report, the ability of lipid-free SAA to promote lipid efflux from cells before and after transfection with the human ABCA1 transporter was examined. The results from this study show that SAA can mediate cholesterol and phospholipid efflux by ABCA1, but unlike apoA-I, it can also promote lipid efflux from cells by an ABCA1-independent pathway, which most likely involves a detergent-like extraction mechanism. These results suggest that SAA may act as an auxiliary lipid acceptor for the reverse cholesterol transport pathway. Because of its unique ability to also efflux lipid without the ABCA1 transporter, SAA may help mobilize lipid from apoptotic or necrotic cells, which cannot support the energy-dependent ABCA1 transport of lipid.
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(control cells) were produced, as previously described [25]. Skin fibroblasts were grown in 24-well plates with a-MEM plus 10% FCS and pen/strep. The ABCA1 transporter molecular defect in the Tangier disease fibroblast cell line has been previously described [24]. Lipid efflux studies. Cholesterol efflux was performed on cells radiolabelled with [3H]cholesterol, and phospholipid efflux was performed on cells radiolabelled with [3H]choline, as previously described [30]. Percentage efflux was calculated, after subtracting the radioactive counts in the blank media (a-MEM plus 1 mg/mL of BSA), and expressed as the percentage of total radioactive counts removed from the cells, during an 18 h period. Cell fixation was performed by a 10 min treatment with 3% paraformaldehyde in phosphate buffered saline, followed by three washes with blank media.
Results In Fig. 1, the ability of phosphatidylcholine (PC) vesicles, lipid-free apoA-I (A1), a synthetic amphipathic helical peptide (37pA) [26], and lipid-free SAA to efflux cholesterol from HeLa cells before and after stable transfection with human ABCA1 cDNA was examined. The level of cholesterol efflux to PC vesicles, which removes cholesterol by the passive diffusion pathway [21,22], was not significantly different between ABCA1 transfected cells and control HeLa cells. In contrast, ABCA1 expression did markedly increase the level of cholesterol efflux to apoA-I, as previously described [25,26]. ABCA1 expression also increased the level of cholesterol efflux to 37pA and SAA, which suggests that SAA can also serve as a lipid acceptor from the ABCA1 transporter. Unlike apoA-I, SAA, as well as the 37pA peptide [26], also effluxed significant amounts of cholesterol from the control cells, which suggests that SAA may also work by an ABCA1-independent pathway [26]. In Fig. 2, a dose–response study for cholesterol and phospholipid efflux to SAA was performed for ABCA1 transfected cells and control cells. A greater amount of
Materials and methods Materials. Purified recombinant SAA, which is a hybrid of human SAA1.1 and SAA2.1 proteins [17], was obtained from Pepro Tech (Rocky Hill, NJ). Prior to use in lipid efflux studies, lyophilized apoA-I and SAA were delipidated with chloroform:methanol (2:1) and dissolved in PBS at a concentration of 1 mg/mL, as previously described [27,28]. Phosphatidylcholine vesicles were prepared by sonication [29]. The 37pA peptide was synthesized by a solid-pase procedure, using Fmoc/DIC/HOBt protocol on a Biosearch 9600 peptide synthesizer [26]. Cell culture. HeLa cells were grown in 24-well plates with a-MEM plus 10% FCS and 100 lg/mL hygromycin. A stably transfected HeLa cell line expressing the human ABCA1 transporter and a control HeLa cell line transfected with only a hygromycin-resistant control plasmid
Fig. 1. Lipid efflux by ABCA1-transfected cells and control cells to SAA. ABCA1-transfected cells (solid bars) and control cells (open bars) were examined for their ability to efflux cholesterol to 50 lg/mL PC vesicles (PC), 10 lg/mL apoA-I (A-1), 10 lg/mL (37pA), and 10 lg/ mL (SAA). Results represent means of triplicates ± 1 SD.
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Fig. 3. Lipid efflux to SAA from normal and Tangier disease skin fibroblasts. Normal skin fibroblasts (open bars) and Tangier disease skin fibroblasts (solid bars) were examined for their ability to efflux cholesterol to 10 lg/mL apoA-I (AI), 50 lg/mL PC vesicles (PC), and 10 lg/mL SAA (SAA). Results are expressed as means of triplicates ± 1 SD.
Fig. 2. Dose–response curve for lipid efflux by ABCA1 transfected cells and control cells to SAA. ABCA1 transfected cells (s) and control cells ( ) were examined for their ability to efflux cholesterol (A) and phospholipid (B) to SAA at the concentrations indicated on the X-axis. Results represent means of triplicates ± 1 SD.
cholesterol and phospholipid efflux occurred from ABCA1 expressing cells, thus confirming the ability of SAA to promote both cholesterol and phospholipid efflux by the ABCA1 transporter. Similar to what was observed in Fig. 1, SAA also stimulated significant cholesterol and phospholipid efflux from the control cells, which do not express the ABCA1 transporter [25,26]. As can be seen in Fig. 1, and as previously described [25,26], apoA-I, in contrast, is almost completely dependent upon the presence of the ABCA1 transporter to mediate cholesterol and phospholipid efflux. Interestingly, ABCA1 expression increased cholesterol efflux to SAA at a greater extent than phospholipid efflux (Fig. 2). In order to establish that cholesterol efflux from the control HeLa cells to SAA was not due to a low level of endogenous ABCA1 expression, a Tangier disease fibroblast cell line, with a non-functional ABCA1 transporter [24] was also studied (Fig. 3). ApoA-I, PC, and SAA all effluxed cholesterol from normal skin fibroblasts, but as expected, apoA-I did not efflux detectable
amounts of cholesterol from the Tangier disease cells. The level of passive cholesterol efflux to PC vesicles was, however, similar for the normal and Tangier disease cells. SAA also promoted cholesterol efflux from normal fibroblasts, but unlike apoA-I, SAA was still able, albeit at a reduced level, to promote cholesterol efflux from the Tangier disease cells (Fig. 3). To further examine the mechanism for SAA lipid efflux, control cells and ABCA1 transfected cells were tested for cholesterol efflux to SAA before and after fixation with paraformaldehyde (Fig. 4), which renders the cells nonviable and blocks energy-dependent transport by ABCA1 [26]. As expected, cell fixation blocked cholesterol efflux to apoA-I from the ABCA1-transfected cell line (Fig. 4A). Cell fixation, however, had only a modest negative effect on cholesterol efflux by passive diffusion to PC vesicles from either the ABCA1 transfected cells or control cells. In case of SAA, cell fixation of ABCA1 transfected cells partially decreased but did not completely eliminate cholesterol efflux to SAA, thus confirming the ability of SAA to work independent of the ABCA1 transporter. Interestingly, cell fixation did not significantly decrease cholesterol efflux to SAA from the control cells (Fig. 4B), and the level of cholesterol efflux to the control cells before and after fixation was similar to the level observed from ABCA1 transfected cells after fixation. This result is consistent with a model whereby lipid efflux by SAA from ABCA1-transfected cells occurs by both an ABCA1-dependent and an ABCA1-independent pathway, whereas lipid efflux from the control cells only occurs by the ABCA1-independent pathway.
Discussion An important finding from this study, in regard to the possible physiologic function of SAA, is that it can act
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Fig. 4. Effect of cell fixation on cholesterol efflux to SAA. ABCA1transfected cells (A) and control cells (B) were examined for their ability to efflux cholesterol to 10 lg/mL apoA-I (AI), 50 lg/mL PC vesicles (PC), and 10 lg/mL SAA (SAA) before (open bars) and after fixation (solid bars) with 3% paraformaldehyde. Results are expressed as means of triplicates ± 1 SD.
as an effective lipid acceptor from the ABCA1 transporter (Figs. 1 and 2), and thus, it can participate in the first step of reverse cholesterol transport pathway. Recently, it was shown that SAA-enriched HDL compared to normal HDL has greater ability to efflux cholesterol from J744 macrophages after treatment with cAMP [20]. Because cAMP increases the expression of the ABCA1 transporter, this finding is consistent with a role of SAA as a lipid acceptor from the ABCA1 transporter. Treatment of macrophages with cAMP, however, results in numerous changes in gene expression [31], as well as changes in many cell signalling pathways. The present study, which utilized a well-defined experimental system, namely ABCA1-transfected cells and ABCA1-negative control cells, thus firmly establishes that SAA can be acceptor for lipid efflux by the ABCA1 transporter. All of the previous studies [13–20] have also used SAA-enriched HDL or SAA reconstituted with phospholipid, instead of the lipid-free SAA, which makes it difficult to differentiate between an effect of SAA on cholesterol efflux by the passive diffusion pathway versus the ABCA1 transporter pathway. The mechanism of action of the ABCA1 transporter is not fully understood, but it has been shown that amphipathic helices are a key structural motif for enabling proteins to act as lipid acceptors from the transporter [25,26]. In addition to apoA-I, all of the other exchangeable type apolipoproteins, which all contain
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amphipathic helices, have been shown to be suitable lipid acceptors for the ABCA1 transporter [26]. SAA contains at least two amphipathic helices, which account for its lipid binding ability [6,7]. In addition, SAA can displace apoA-I from HDL [32], and it has been proposed that SAA may even have higher lipid affinity than apoA-I. This is supported by studies that show that SAA has higher affinity for cholesterol than apoA-I and has greater interfacial surface binding properties [17,18]. SAA also has several other characteristics that may facilitate its ability to promote reverse cholesterol transport. Unlike apoA-I, which is constitutively expressed, SAA can be induced over 1000-fold [1–4], and may, therefore, increase reverse cholesterol transport, during periods of inflammation. It has been hypothesized that there may be increased need for cholesterol efflux from macrophages, during the acute phase response, in order to reduce intracellular lipids that have accumulated from the phagocytosis of cell membrane fragments at sites of inflammation [10]. The fact that SAA, unlike apoA-I, is also synthesized in extrahepatic tissue [3] may make SAA ideally suited for such a role. SAA has been found in atherosclerotic plaques and can be produced by not only macrophages but also by endothelial cells and smooth muscle cells [9], all of which are prone to lipid accumulation during atherogenesis. The localized production of SAA at sites of inflammation may, therefore, facilitate the efflux of lipid from these cells. Another interesting feature of SAA is that that it has several cell binding motifs, for ligands such as fibronectin, laminin, and a heparin [3], which may facilitate lipid efflux by promoting the initial binding of SAA to cells. Finally, SAA appears to be internalized by cells and can activate neutral cholesterol hydrolase and inhibit acylcoA:cholesterol acyltransferase [10,19,20], which could potentially increase the available pool of free cholesterol for efflux from cells. The finding that SAA can stimulate cholesterol and phospholipid efflux from HeLa cells, even in the absence of the ABCA1 transporter (Figs. 1 and 2), may also be relevant for reverse cholesterol transport from sites of inflammation and or tissue damage. This result was confirmed by showing that SAA, unlike apoA-I, promotes cholesterol efflux from Tangier disease skin fibroblasts (Fig. 3). The fact that SAA can even stimulate cholesterol efflux from non-viable fixed cells (Fig. 4) indicates that SAA must be able to remove lipid from the plasma membrane of cells by a passive physical process. Synthetic amphipathic helical peptides with very high lipid affinity, such as the 37pA peptide [26], have been shown to remove lipid from ABCA1-negative cells by a detergentlike extraction process [26]. The relatively high lipid affinity of SAA [17,18,32] and/or its ability to bind to cells without the ABCA1 transporter via other cell recognition sequences [3] may possibly account for its ability to efflux lipid independent of the ABCA1 transporter.
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The results in Fig. 4 are, therefore, consistent with a possible role for SAA in the mobilization of lipid from areas of inflammation that may contain necrotic or apoptotic cells that are unable to support the energy-dependent efflux of lipid by the ABCA1 transporter. In summary, SAA was shown to be an effective acceptor for cholesterol and phospholipid from the ABCA1 transporter. In addition, SAA can also promote lipid efflux by a passive energy-independent process that does not involve the ABCA1 transporter. A model is proposed whereby SAA may serve as an ancillary lipid acceptor, during states in which there is an increased need for reverse cholesterol transport, such as during inflammation. The ability of SAA to promote lipid efflux independent of the ABCA1 transporter suggests that it may be more effective than apoA-I in removing excess lipid from injured cells at sites of inflammation and/or possibly from extracellular lipid deposits.
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Serum Amyloid a promotes ABCA1-dependent and ABCA1-independent lipid efflux from cells John A. Stonik a, Alan T. Remaley a,*, Steve J. Demosky a, Edward B. Neufeld a, Alexander Bocharov b, H. Bryan Brewer a a
National Institutes of Health Molecular Disease Branch, National Heart, Lung, and Blood Institute, Bethesda, MD, USA b National Institutes of Health, National Institute of Diabetes and Kidney Diseases, Bethesda, MD, USA Received 8 July 2004 Available online 28 July 2004
Abstract Serum amyloid A (SAA) is an acute phase protein that associates with HDL. In order to examine the role of SAA in reversecholesterol transport, lipid efflux was tested to SAA from HeLa cells before and after transfection with the ABCA1 transporter. ABCA1 expression increased efflux of cholesterol and phospholipid to SAA by 3-fold and 2-fold, respectively. In contrast to apoA-I, SAA also removed lipid without ABCA1; cholesterol efflux from control cells to SAA was 10-fold higher than for apoA-I. Furthermore, SAA effluxed cholesterol from Tangier disease fibroblasts and from cells after inhibition of ABCA1 by fixation with paraformaldehyde. In summary, SAA can act as a lipid acceptor for ABCA1, but unlike apoA-I, it can also efflux lipid without ABCA1, by most likely a detergent-like extraction process. These results suggest that SAA may play a unique role as an auxiliary lipid acceptor in the removal of lipid from sites of inflammation. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Serum amyloid A; Apolipoprotein A-I; ABCA1 transporter; Cholesterol; Atherosclerosis
Serum amyloid A (SAA) is an acute phase protein that is produced by the liver and is induced over 1000fold during inflammation [1–4]. In the circulation, SAA is mostly bound to HDL [1,4,5]. During acute infections, SAA can reach protein concentrations as high as apoA-I and can replace apoA-I as the major protein on HDL [5]. Like apoA-I, SAA has several amphipathic helices [6,7], a structural motif that facilitates lipid binding by proteins [8]. The physiologic role of SAA has not been clearly established, but numerous and diverse possible functions have been proposed [1–4]. Because of its association with HDL [1,4,5], and its presence in atherosclerotic plaques [9], one hypothesis is that SAA may play a role in
*
Corresponding author. Fax: 1-301-402-1885. E-mail address: [email protected] (A.T. Remaley).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.07.052
lipoprotein metabolism and in the pathogenesis of atherosclerosis [10]. The level of HDL is markedly reduced, during the acute phase response, and SAA has been suggested to increase HDL catabolism [1,4,11]. The expression, however, of SAA by adenovirus in mice, in the absence of inflammation, did not significantly alter HDL levels [12]. It has also been proposed that SAA may alter the ability of HDL to efflux excess cholesterol from cells [1–4], the first step in the reverse cholesterol transport pathway. Several studies have shown that the enrichment of SAA on HDL decreases the ability of HDL to efflux cholesterol from cells [13,14], and promotes the binding of HDL to macrophages [13–16], and HDL–cholesterol uptake by cells [13,14,17,18]. This has led to the hypothesis that SAA may mediate the delivery of lipid to regenerating cells at the site of inflammation or tissue injury [13,14,17,18]. Under some conditions, however, SAA may instead increase the removal of
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excess lipid from sites of inflammation or tissue injury [10,19,20]. During tissue repair, macrophages phagocytose cell debris from apoptotic and necrotic cells and are transformed into foam cells [10]. Recently, it has been shown, if macrophages are first loaded with cholesterol, SAA-enriched HDL has greater capacity for removing cholesterol from cells than normal HDL [19,20]. Cells efflux excess cholesterol to HDL by either an active process, involving the ABCA1 transporter, or by the passive diffusion of cholesterol from cells to extracellular lipoproteins [21,22]. Mutations in the ABCA1 transporter result in Tangier disease [21,23,24], which is characterized by low HDL and the accumulation of cholesterol in macrophages. Unlike the passive diffusion pathway, lipid-free or lipid-poor apolipoproteins are believed to be the principal acceptor of lipid by the ABCA1 transporter and initiate lipid efflux after first binding to cells [21,22,25]. A key structural motif that is necessary for a protein to act as a lipid acceptor from the ABCA1 transporter is the amphipathic helix [25,26]. In addition to apoA-I, all of the exchangeable type apolipoproteins [25], as well as synthetic peptides [26], such as the 37pA peptide, which contains two amphipathic helices, can also promote ABCA1-dependent lipid efflux. Because SAA contains several amphipathic helices [6,7] and is known to be associated with HDL [1,4,5], it is possible that SAA may also promote to efflux lipid by the ABCA1 transporter. In this report, the ability of lipid-free SAA to promote lipid efflux from cells before and after transfection with the human ABCA1 transporter was examined. The results from this study show that SAA can mediate cholesterol and phospholipid efflux by ABCA1, but unlike apoA-I, it can also promote lipid efflux from cells by an ABCA1-independent pathway, which most likely involves a detergent-like extraction mechanism. These results suggest that SAA may act as an auxiliary lipid acceptor for the reverse cholesterol transport pathway. Because of its unique ability to also efflux lipid without the ABCA1 transporter, SAA may help mobilize lipid from apoptotic or necrotic cells, which cannot support the energy-dependent ABCA1 transport of lipid.
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(control cells) were produced, as previously described [25]. Skin fibroblasts were grown in 24-well plates with a-MEM plus 10% FCS and pen/strep. The ABCA1 transporter molecular defect in the Tangier disease fibroblast cell line has been previously described [24]. Lipid efflux studies. Cholesterol efflux was performed on cells radiolabelled with [3H]cholesterol, and phospholipid efflux was performed on cells radiolabelled with [3H]choline, as previously described [30]. Percentage efflux was calculated, after subtracting the radioactive counts in the blank media (a-MEM plus 1 mg/mL of BSA), and expressed as the percentage of total radioactive counts removed from the cells, during an 18 h period. Cell fixation was performed by a 10 min treatment with 3% paraformaldehyde in phosphate buffered saline, followed by three washes with blank media.
Results In Fig. 1, the ability of phosphatidylcholine (PC) vesicles, lipid-free apoA-I (A1), a synthetic amphipathic helical peptide (37pA) [26], and lipid-free SAA to efflux cholesterol from HeLa cells before and after stable transfection with human ABCA1 cDNA was examined. The level of cholesterol efflux to PC vesicles, which removes cholesterol by the passive diffusion pathway [21,22], was not significantly different between ABCA1 transfected cells and control HeLa cells. In contrast, ABCA1 expression did markedly increase the level of cholesterol efflux to apoA-I, as previously described [25,26]. ABCA1 expression also increased the level of cholesterol efflux to 37pA and SAA, which suggests that SAA can also serve as a lipid acceptor from the ABCA1 transporter. Unlike apoA-I, SAA, as well as the 37pA peptide [26], also effluxed significant amounts of cholesterol from the control cells, which suggests that SAA may also work by an ABCA1-independent pathway [26]. In Fig. 2, a dose–response study for cholesterol and phospholipid efflux to SAA was performed for ABCA1 transfected cells and control cells. A greater amount of
Materials and methods Materials. Purified recombinant SAA, which is a hybrid of human SAA1.1 and SAA2.1 proteins [17], was obtained from Pepro Tech (Rocky Hill, NJ). Prior to use in lipid efflux studies, lyophilized apoA-I and SAA were delipidated with chloroform:methanol (2:1) and dissolved in PBS at a concentration of 1 mg/mL, as previously described [27,28]. Phosphatidylcholine vesicles were prepared by sonication [29]. The 37pA peptide was synthesized by a solid-pase procedure, using Fmoc/DIC/HOBt protocol on a Biosearch 9600 peptide synthesizer [26]. Cell culture. HeLa cells were grown in 24-well plates with a-MEM plus 10% FCS and 100 lg/mL hygromycin. A stably transfected HeLa cell line expressing the human ABCA1 transporter and a control HeLa cell line transfected with only a hygromycin-resistant control plasmid
Fig. 1. Lipid efflux by ABCA1-transfected cells and control cells to SAA. ABCA1-transfected cells (solid bars) and control cells (open bars) were examined for their ability to efflux cholesterol to 50 lg/mL PC vesicles (PC), 10 lg/mL apoA-I (A-1), 10 lg/mL (37pA), and 10 lg/ mL (SAA). Results represent means of triplicates ± 1 SD.
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Fig. 3. Lipid efflux to SAA from normal and Tangier disease skin fibroblasts. Normal skin fibroblasts (open bars) and Tangier disease skin fibroblasts (solid bars) were examined for their ability to efflux cholesterol to 10 lg/mL apoA-I (AI), 50 lg/mL PC vesicles (PC), and 10 lg/mL SAA (SAA). Results are expressed as means of triplicates ± 1 SD.
Fig. 2. Dose–response curve for lipid efflux by ABCA1 transfected cells and control cells to SAA. ABCA1 transfected cells (s) and control cells ( ) were examined for their ability to efflux cholesterol (A) and phospholipid (B) to SAA at the concentrations indicated on the X-axis. Results represent means of triplicates ± 1 SD.
cholesterol and phospholipid efflux occurred from ABCA1 expressing cells, thus confirming the ability of SAA to promote both cholesterol and phospholipid efflux by the ABCA1 transporter. Similar to what was observed in Fig. 1, SAA also stimulated significant cholesterol and phospholipid efflux from the control cells, which do not express the ABCA1 transporter [25,26]. As can be seen in Fig. 1, and as previously described [25,26], apoA-I, in contrast, is almost completely dependent upon the presence of the ABCA1 transporter to mediate cholesterol and phospholipid efflux. Interestingly, ABCA1 expression increased cholesterol efflux to SAA at a greater extent than phospholipid efflux (Fig. 2). In order to establish that cholesterol efflux from the control HeLa cells to SAA was not due to a low level of endogenous ABCA1 expression, a Tangier disease fibroblast cell line, with a non-functional ABCA1 transporter [24] was also studied (Fig. 3). ApoA-I, PC, and SAA all effluxed cholesterol from normal skin fibroblasts, but as expected, apoA-I did not efflux detectable
amounts of cholesterol from the Tangier disease cells. The level of passive cholesterol efflux to PC vesicles was, however, similar for the normal and Tangier disease cells. SAA also promoted cholesterol efflux from normal fibroblasts, but unlike apoA-I, SAA was still able, albeit at a reduced level, to promote cholesterol efflux from the Tangier disease cells (Fig. 3). To further examine the mechanism for SAA lipid efflux, control cells and ABCA1 transfected cells were tested for cholesterol efflux to SAA before and after fixation with paraformaldehyde (Fig. 4), which renders the cells nonviable and blocks energy-dependent transport by ABCA1 [26]. As expected, cell fixation blocked cholesterol efflux to apoA-I from the ABCA1-transfected cell line (Fig. 4A). Cell fixation, however, had only a modest negative effect on cholesterol efflux by passive diffusion to PC vesicles from either the ABCA1 transfected cells or control cells. In case of SAA, cell fixation of ABCA1 transfected cells partially decreased but did not completely eliminate cholesterol efflux to SAA, thus confirming the ability of SAA to work independent of the ABCA1 transporter. Interestingly, cell fixation did not significantly decrease cholesterol efflux to SAA from the control cells (Fig. 4B), and the level of cholesterol efflux to the control cells before and after fixation was similar to the level observed from ABCA1 transfected cells after fixation. This result is consistent with a model whereby lipid efflux by SAA from ABCA1-transfected cells occurs by both an ABCA1-dependent and an ABCA1-independent pathway, whereas lipid efflux from the control cells only occurs by the ABCA1-independent pathway.
Discussion An important finding from this study, in regard to the possible physiologic function of SAA, is that it can act
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Fig. 4. Effect of cell fixation on cholesterol efflux to SAA. ABCA1transfected cells (A) and control cells (B) were examined for their ability to efflux cholesterol to 10 lg/mL apoA-I (AI), 50 lg/mL PC vesicles (PC), and 10 lg/mL SAA (SAA) before (open bars) and after fixation (solid bars) with 3% paraformaldehyde. Results are expressed as means of triplicates ± 1 SD.
as an effective lipid acceptor from the ABCA1 transporter (Figs. 1 and 2), and thus, it can participate in the first step of reverse cholesterol transport pathway. Recently, it was shown that SAA-enriched HDL compared to normal HDL has greater ability to efflux cholesterol from J744 macrophages after treatment with cAMP [20]. Because cAMP increases the expression of the ABCA1 transporter, this finding is consistent with a role of SAA as a lipid acceptor from the ABCA1 transporter. Treatment of macrophages with cAMP, however, results in numerous changes in gene expression [31], as well as changes in many cell signalling pathways. The present study, which utilized a well-defined experimental system, namely ABCA1-transfected cells and ABCA1-negative control cells, thus firmly establishes that SAA can be acceptor for lipid efflux by the ABCA1 transporter. All of the previous studies [13–20] have also used SAA-enriched HDL or SAA reconstituted with phospholipid, instead of the lipid-free SAA, which makes it difficult to differentiate between an effect of SAA on cholesterol efflux by the passive diffusion pathway versus the ABCA1 transporter pathway. The mechanism of action of the ABCA1 transporter is not fully understood, but it has been shown that amphipathic helices are a key structural motif for enabling proteins to act as lipid acceptors from the transporter [25,26]. In addition to apoA-I, all of the other exchangeable type apolipoproteins, which all contain
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amphipathic helices, have been shown to be suitable lipid acceptors for the ABCA1 transporter [26]. SAA contains at least two amphipathic helices, which account for its lipid binding ability [6,7]. In addition, SAA can displace apoA-I from HDL [32], and it has been proposed that SAA may even have higher lipid affinity than apoA-I. This is supported by studies that show that SAA has higher affinity for cholesterol than apoA-I and has greater interfacial surface binding properties [17,18]. SAA also has several other characteristics that may facilitate its ability to promote reverse cholesterol transport. Unlike apoA-I, which is constitutively expressed, SAA can be induced over 1000-fold [1–4], and may, therefore, increase reverse cholesterol transport, during periods of inflammation. It has been hypothesized that there may be increased need for cholesterol efflux from macrophages, during the acute phase response, in order to reduce intracellular lipids that have accumulated from the phagocytosis of cell membrane fragments at sites of inflammation [10]. The fact that SAA, unlike apoA-I, is also synthesized in extrahepatic tissue [3] may make SAA ideally suited for such a role. SAA has been found in atherosclerotic plaques and can be produced by not only macrophages but also by endothelial cells and smooth muscle cells [9], all of which are prone to lipid accumulation during atherogenesis. The localized production of SAA at sites of inflammation may, therefore, facilitate the efflux of lipid from these cells. Another interesting feature of SAA is that that it has several cell binding motifs, for ligands such as fibronectin, laminin, and a heparin [3], which may facilitate lipid efflux by promoting the initial binding of SAA to cells. Finally, SAA appears to be internalized by cells and can activate neutral cholesterol hydrolase and inhibit acylcoA:cholesterol acyltransferase [10,19,20], which could potentially increase the available pool of free cholesterol for efflux from cells. The finding that SAA can stimulate cholesterol and phospholipid efflux from HeLa cells, even in the absence of the ABCA1 transporter (Figs. 1 and 2), may also be relevant for reverse cholesterol transport from sites of inflammation and or tissue damage. This result was confirmed by showing that SAA, unlike apoA-I, promotes cholesterol efflux from Tangier disease skin fibroblasts (Fig. 3). The fact that SAA can even stimulate cholesterol efflux from non-viable fixed cells (Fig. 4) indicates that SAA must be able to remove lipid from the plasma membrane of cells by a passive physical process. Synthetic amphipathic helical peptides with very high lipid affinity, such as the 37pA peptide [26], have been shown to remove lipid from ABCA1-negative cells by a detergentlike extraction process [26]. The relatively high lipid affinity of SAA [17,18,32] and/or its ability to bind to cells without the ABCA1 transporter via other cell recognition sequences [3] may possibly account for its ability to efflux lipid independent of the ABCA1 transporter.
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The results in Fig. 4 are, therefore, consistent with a possible role for SAA in the mobilization of lipid from areas of inflammation that may contain necrotic or apoptotic cells that are unable to support the energy-dependent efflux of lipid by the ABCA1 transporter. In summary, SAA was shown to be an effective acceptor for cholesterol and phospholipid from the ABCA1 transporter. In addition, SAA can also promote lipid efflux by a passive energy-independent process that does not involve the ABCA1 transporter. A model is proposed whereby SAA may serve as an ancillary lipid acceptor, during states in which there is an increased need for reverse cholesterol transport, such as during inflammation. The ability of SAA to promote lipid efflux independent of the ABCA1 transporter suggests that it may be more effective than apoA-I in removing excess lipid from injured cells at sites of inflammation and/or possibly from extracellular lipid deposits.
[13]
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