1 Department of Molecular Pharmacology and 2 Marion Bessin Liver Research Center, Departments of Medicine and Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461; and 3 Lipoprotein and Atherosclerosis Group, University of Ottawa Heart Institute, Ottawa, Ontario, Canada K1Y 4W7
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ABSTRACT |
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Caveolin-1 is a principal structural component of caveolae membranes. These membrane microdomains participate in the regulation of signaling, transcytosis, and cholesterol homeostasis at the plasma membrane. In the present study, we determined the effect of caveolin-1 expression on cellular cholesterol efflux mediated by high-density lipoprotein (HDL). We evaluated this effect in parental NIH/3T3 cells as well as in two transformed NIH/3T3 cell lines in which caveolin-1 protein levels are dramatically downregulated. Compared with parental NIH/3T3 cells, these two transformed cell lines effluxed cholesterol more rapidly to HDL. In addition, NIH/3T3 cells harboring caveolin-1 antisense also effluxed cholesterol more rapidly to HDL. However, this effect was not due to changes in total cellular cholesterol content. We further showed that chronic HDL exposure reduced caveolin-1 protein expression in NIH/3T3 cells. HDL exposure also inhibited caveolin-1 promoter activity, suggesting a direct negative effect of HDL on caveolin-1 gene transcription. Moreover, we showed that HDL-induced downregulation of caveolin-1 prevents the uptake of oxidized low-density lipoprotein in human endothelial cells. These data suggest a novel proatherogenic role for caveolin-1, i.e., regarding the uptake and/or transcytosis of modified lipoproteins.
caveolin; oxidized low-density lipoprotein; sterol regulatory element-binding proteins
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INTRODUCTION |
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CAVEOLAE ARE PLASMA MEMBRANE invaginations (50-100 nm) that participate in cell signaling (40) and transcytosis (12, 76) as well as in the regulation of cellular cholesterol homeostasis (19). These subcompartments of the plasma membrane are characterized by the presence of caveolin proteins (29, 30). Several isoforms of caveolin have been identified (61, 62, 70, 74, 78). Caveolins are expressed principally in terminally differentiated cell types such as fibroblasts, adipocytes, smooth and striated muscle cells, and endothelial cells (63).
In nonmuscle cells, caveolin-1 is the main structural protein component of caveolae. Caveolin-1 drives caveolae formation through oligomerization (with itself and with caveolin-2) and by interacting with cholesterol (39, 53, 59). The formation of this functional assembly unit may facilitate targeting of numerous constituents to caveolae, including proteins involved in signal transduction (reviewed in Refs. 1, 42, and 67).
Caveolae have a very specific lipid composition; they are highly enriched in cholesterol and sphingomyelin. This has led to the hypothesis that caveolae may play a role in the regulation of cellular cholesterol homeostasis. The observation that caveolin-1 binds cholesterol specifically (47, 75) suggests that caveolin-1 may play a direct role in this process. Recently, Smart et al. (69) demonstrated that caveolin-1, coupled with other chaperone proteins, facilitates transport of cholesterol from the endoplasmic reticulum to the plasma membrane. Other studies (21) have indicated that free cholesterol (FC) selectively transferred from low-density lipoprotein (LDL) to cells appears first in clathrin-coated pits and is eventually transferred to caveolae via the trans-Golgi network. Furthermore, some investigators (22) also showed that plasma membrane caveolae can mediate cellular cholesterol efflux to plasma or high-density lipoprotein (HDL) using this specific cholesterol-labeling procedure. Additionally, Bist et al. (5) have recently demonstrated that caveolin-1 expression is under the positive control of cellular cholesterol levels.
The importance of HDL in cholesterol elimination has been suggested by several epidemiological studies that show an inverse correlation between the development of coronary artery disease and HDL cholesterol levels (9, 31, 32, 46, 79). This has led to the concept of reverse cholesterol transport, a process by which HDL removes excess peripheral cholesterol and transfers it to the liver for degradation and removal from the body (20). HDL particles are believed to function as the primary acceptors of cellular cholesterol. After cellular cholesterol efflux to HDL, cholesterol is esterified and transferred to apolipoprotein B-containing lipoproteins via the cholesterol ester transfer protein (20). However, the specific mechanisms involved in the removal of cellular cholesterol by apolipoprotein A-I-containing HDL particles are still poorly understood (24, 49).
The present study was designed to determine whether caveolin-1 protein expression specifically effects total cellular cholesterol efflux mediated by HDL. Our results indicate that caveolin-1 downregulation enhances cellular cholesterol efflux to HDL. Moreover, we show that chronic HDL exposure leads to the downregulation of caveolin-1 expression, a process that may be relevant to understanding the pathogenesis of coronary heart disease.
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MATERIALS AND METHODS |
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Materials.
Antibodies and their sources were as follows: anti-caveolin-1 IgG
[monoclonal antibody (MAb) 2297, gift of Dr. Roberto Campos-Gonzalez, Transduction Laboratories, Lexington, KY] (62);
anti-caveolin-2 IgG (MAb 65, gift of Dr. Roberto Campos-Gonzalez)
(60); antiactivated extracellularly regulated kinase (ERK)
1/2 (New England Biolabs, Beverly, MA); and anti-sterol regulatory
element-binding protein (SREBP)-1 (H-160; Santa Cruz Biotechnology,
Santa Cruz, CA). PD-98059 and U-0126 were purchased from Calbiochem and
Promega, respectively. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was obtained from Molecular Probes (Eugene, OR). The cDNA for
murine caveolin-1 was as we described previously (73).
[1,2
-3H]cholesterol was purchased from NEN (Boston,
MA). All other reagents were analytical grade.
Cell culture. Normal and transformed NIH/3T3 cells were cultured in a CO2 incubator at 37°C in DMEM high glucose, 10% donor calf serum (DCS), 4 mM glutamine, and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). NIH/3T3 cell lines harboring caveolin-1 antisense were as previously described (27) and were maintained in media containing 150 µg/ml hygromycin B to prevent spontaneous loss of the caveolin-1 antisense vector. The human microvascular endothelial cell line (HMEC-1; obtained from the Biological Products Branch of the Centers for Disease Control, Atlanta, GA) was grown in MCDB-131 containing 10% fetal bovine serum (FBS).
Isolation of lipoproteins. HDL3 and LDL from human plasma were purified by sequential ultracentrifugation, as previously described (65). Oxidized LDL (oxLDL) was prepared by incubating LDL with 10 µM CuSO4 at 37°C for 16 h. OxLDL was extensively dialyzed against PBS, and oxidation was monitored by submitting oxLDL to a precast 0.5% agarose gel electrophoresis (Lipogel; Beckman). Labeling with DiI was performed as described by others (71). Purified DiI-labeled oxLDL was obtained after ultracentrifugation and extensive dialysis.
Cellular cholesterol efflux. Cells in complete medium were seeded in six-well plates at a density of 2 × 104 cells/well. After 48 h, the medium was replaced with DMEM supplemented with 5% DCS and 5 µCi/ml [3H]cholesterol dispersed in 0.1% ethanol (% final volume of media) for 48 h. Before each efflux experiment, cells were washed three times with DMEM and then incubated with DMEM containing HDL3 (50 µg/ml) and 0.2% BSA. Media aliquots were taken at different times of incubation and treated as previously described (25). At the end of the experiment, cells were solubilized in 0.5 N NaOH to determine protein and [3H]cholesterol content. Results presented are expressed as the percentage of labeled cholesterol remaining in the cells as a function of time.
Determination of cellular cholesterol content. Cellular cholesterol was extracted from cells using isopropanol. Cholesterol content was determined using a colorimetric test (Sigma cholesterol determination kit).
Activation of p42/44 MAP kinase pathway. To determine the activation state of ERK1/2 (p42/44), cells were lysed in boiling SDS sample buffer and samples were then collected and boiled for another 5 min. After SDS-PAGE (12% polyacrylamide) and transfer to nitrocellulose (0.2 µm), blots were probed with primary antibodies (dilution 1:1,000; New England Biolabs) and the appropriate horseradish peroxidase-conjugated secondary antibody (dilution 1:5,000; Transduction Laboratories). Bound antibodies were visualized using enhanced chemiluminescence.
Caveolin-1 promoter construct. The caveolin gene promoter was cloned into pA3 Luc (16), a luciferase reporter plasmid (44, 81). In this study, we used a promoter construct that contains ~3 kb upstream of the caveolin-1 ATG, caveolin-1/exon 1, caveolin-1/intron 1, and a portion of caveolin-1/exon 2 (described as Pr-3 kb and Int 1 in Ref. 16).
Luciferase assays.
Transient transfections (using calcium phosphate precipitation) and
luciferase assays were performed essentially as we described previously
(13, 14). Briefly, 300,000 cells (HEK-293T cells) were
seeded in six-well plates 12-24 h before transfection. Each well
was transfected with 1 µg of each plasmid for experiments in which
two plasmids were cotransfected. Twelve hours posttransfection, the
cells were rinsed twice with PBS and incubated for 24 h in DMEM
containing 0.2% BSA with or without 500 µg/ml of HDL3.
The cells were then lysed in 200 µl of extraction buffer, of which 100 µl were used to measure luciferase activity, as described (50). Cotransfection with pSV--galactosidase was used
as an internal control to normalize for transfection efficiency.
Regulation of SREBP-1 cellular localization. NIH/3T3 cells were incubated with or without HDL3 for 24 h and fixed with PBS/2% paraformaldehyde. After permeabilization (PBS/0.1% Triton X-100 and 0.2% BSA), cells were incubated with anti-SREBP-1 polyclonal antibody (PAb). Detection of bound IgG was performed using a Rhodamine Red-X F(ab')2 goat anti-rabbit IgG (Jackson Immunoresearch Laboratories, West Grove, PA). Immunofluorescence was observed with a 12-bit Photometrics cooled charge-coupled device (CCD) camera mounted on an Olympus IX70 inverted microscope.
Cellular uptake of DiI-labeled oxLDL. For cellular uptake studies, HMEC-1 were seeded in 24-well plates containing coverslips. After 24 h in MCDB-131 media containing 10% FBS, cells were washed with media alone and incubated with either MCDB-131 or MCDB-131 containing 500 µg/ml HDL3 for 24 h. Media was then replaced with 50 µg/ml DiI-labeled oxLDL for 2 h at 37°C. At the end of the experiments, cells were washed with PBS. Immunofluorescence was observed with a 12-bit Photometrics cooled CCD camera mounted on an Olympus IX70 inverted microscope.
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RESULTS |
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Cholesterol efflux from normal and transformed NIH/3T3 cells.
Figure 1A shows loss of
caveolin-1 expression in cells transformed with the activated
oncogenes v-Abl and H-Ras (G12V). Caveolin-2 expression levels were
much less affected by cellular transformation (Fig. 1A).
Thus we examined the ability of HDL3 to promote cellular cholesterol efflux from these three different cell lines. Results presented in Fig. 1B show the percentage of cellular
cholesterol (radiolabeled) remaining in the cells upon incubation with
HDL3. The results indicate that in v-Abl and H-Ras
transformed cells, efflux is more active than in the parental cell
line. These differences are already significant after 0.5-1 h of
incubation.
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Effect of caveolin-1 downregulation on cellular cholesterol efflux
mediated by HDL3.
Because the NIH/3T3 cell lines used in Fig. 1 are transformed by
activated oncogenes, factors other than caveolin-1 downregulation could
mediate the observed increase in cellular cholesterol efflux to
HDL3. To evaluate the role of caveolin-1 expression
directly, we next employed an antisense-based approach. We previously
developed an NIH/3T3 cell line in which caveolin-1 protein expression
levels are downregulated by stable transfection with a caveolin-1
antisense vector (27). We therefore decided to examine the
ability of HDL3 to promote cellular cholesterol efflux from
this cell line [termed Cav-1-AS (27)] and compared it
with two other cells, parental NIH/3T3 cells and a revertant of
Cav-1-AS (named Rev-Cav-1-AS), which were grown in the absence of
selection marker (hygromycin) and have lost the antisense vector
(27), restoring normal caveolin-1 expression. This
revertant was shown to behave the same as the parental cell line.
Figure 2A shows that
caveolin-1 expression was clearly reduced in the antisense cell line
compared with other positive controls (parental NIH/3T3 and
Rev-Cav-1-AS), whereas caveolin-2 was not affected (Fig.
2A). Figure 2B shows that HDL3 promoted cellular cholesterol efflux from the parental and revertant cell lines with very similar efficiencies. However, cholesterol efflux
from the Cav-1-AS cell line was significantly enhanced compared with
the other cell lines.
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Total cellular cholesterol content is independent of caveolin-1
expression.
Previous studies (4) have indicated that cellular
cholesterol content influences the ability of HDL to promote cellular cholesterol efflux. Because caveolin-1 has been implicated in the
regulation of cellular cholesterol homeostasis, downregulation of this
protein could dramatically affect cellular cholesterol content. We
therefore determined the cholesterol content of the cell lines used in
this study. Table 1 shows that despite
greatly reduced caveolin-1 expression levels, the cellular cholesterol content of H-Ras- and v-Abl-transformed cells, as well as the Cav-1-AS
cell line, was similar to that of the parental and Rev-Cav-1-AS cell
lines.
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Chronic HDL3 exposure downregulates the expression of
caveolin-1 in NIH/3T3 cells.
To determine whether caveolin-1 expression levels were correlated with
cellular cholesterol content, NIH/3T3 cells were depleted of
cholesterol by incubation with varying HDL3 concentrations, and both caveolin-1 and -2 expression levels were determined after a
24-h incubation. Figure 3A
shows an immunoblot of the cellular extracts solubilized after
incubation with HDL3. It demonstrates that while caveolin-2
expression levels remained unchanged with or without HDL in the media,
caveolin-1 expression was downregulated by HDL.
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Relationship between HDL-mediated p42/44 MAP kinase activation and caveolin-1 expression. To determine whether HDL3 could act via the p42/44 MAP kinase pathway, NIH/3T3 cells were incubated with HDL3 for 24 h, and activation of p42/44 MAP kinase was assessed by determining the level of activated (phosphorylated) p42/44 proteins (ERK1 and ERK2), using phophospecific antibody probes (Fig. 3B). Results presented in Fig. 3B show that at high HDL3 concentration (>100 µg/ml), the p42/44 MAP kinase pathway is activated. However, downregulation of caveolin-1 at the lowest concentration of HDL3 does not appear to correlate with ERK1/2 activation (compare Fig. 3, A and B).
Activation of ERK1 and ERK2 is mediated by MEK1 and MEK2 and can be blocked by the inhibitor PD-98059. In previous studies, we have shown that caveolin-1 downregulation mediated by transformation with activated Ras (G12V) could be reversed by inhibiting MEK1/2 with PD-98059 (13, 27). To determine whether PD-98059 could reverse HDL3-mediated caveolin-1 downregulation, NIH/3T3 cells were incubated with HDL3 with or without the inhibitor. Results presented in Fig. 4 indicate that despite reduced activation of p42/44 MAP kinase, incubation of the cells with PD-98059 did not abolish caveolin-1 downregulation, suggesting that another mechanism of control for this protein is operating in the presence of HDL3, possibly via downregulation of cellular cholesterol content.
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Effects of chronic HDL exposure on caveolin-1 gene transcription.
The caveolin-1 promoter has been isolated and characterized by our
laboratory (16) and others (5). To determine
whether incubation of cells with HDL3 affects caveolin-1
promoter activity, we next incubated cells transfected with a
luciferase expression vector under the control of the caveolin-1
promoter (Fig. 6A) in the
presence or absence of HDL3. Our results demonstrate that under the conditions of the assay, HDL inhibits caveolin-1 promoter transcriptional activity by ~50% (Fig. 6B). Thus chronic
HDL exposure can negatively regulate caveolin-1 gene transcription.
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Incubation of NIH/3T3 cells with HDL3 induces
translocation of the active form of SREBP-1 to the nucleus.
To determine whether SREBP-1 might be involved in the downregulation of
caveolin-1, experiments were performed to analyze the effect of
HDL3 on SREBP-1 translocation to the nucleus. For this
purpose, we used a rabbit PAb directed against the SREBP-1 NH2-terminal domain, which is cleaved from the COOH
terminus and represents the active transcription factor transferred to
the nucleus (8). Figure 7
shows that incubation of NIH/3T3 cells with HDL3 induces
the translocation of SREBP-1 to the nucleus. Importantly, this
translocation event is not observed after treatment with media
alone (see Fig. 7A).
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HDL-induced downregulation of caveolin-1 prevents the uptake of
oxLDL in endothelial cells.
Numerous morphological studies have suggested that caveolae may be
involved in the transcytosis of modified lipoproteins across endothelial cells. Thus we next examined the effect of HDL-induced downregulation of caveolin-1 on the ability of endothelial cells to
take up oxLDL. As a prerequisite for these studies, we found that
caveolin-1 was downregulated after HDL3 treatment of
endothelial cells (Fig. 8A).
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DISCUSSION |
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Several studies have now implicated caveolin-1 in the regulation of cellular cholesterol homeostasis (18, 19, 21, 22, 33, 68, 69). These studies have indicated that FC selectively transferred from LDL to cells appears first in clathrin-coated pits and is eventually transferred to caveolae via the trans-Golgi network (21). Moreover, caveolin-1 is now known to bind cholesterol with a high affinity (47, 75), and it appears that its expression is under the positive control of cellular cholesterol levels (5).
In support of a role for caveolin-1 in cholesterol metabolism, it was shown that plasma membrane caveolae can mediate cellular cholesterol efflux to HDL or plasma (22). To directly determine whether caveolin-1 is involved in this process, we have used three different NIH/3T3 cell lines that lack normal levels of caveolin-1 expression (2 cell lines transformed with activated oncogenes, such as v-Abl and H-Ras, and another cell line harboring caveolin-1 antisense). Cellular cholesterol efflux mediated by HDL3 was examined in these cells and compared with parental NIH/3T3 cells. Our current results clearly demonstrate that caveolin-1 downregulation enhances cellular cholesterol efflux. Moreover, we also show that HDL3 can negatively regulate caveolin-1 expression, possibly by decreasing cellular cholesterol levels.
Mechanism of cellular cholesterol efflux mediated by HDL. Caveolae are plasma membrane domains that are enriched in cholesterol and sphingomyelin relative to the rest of the plasma membrane. Studies have indicated that transfer of cellular cholesterol to an acceptor may be a function of the existence of different microdomains with varying cholesterol content (2, 55). Of particular importance for this study is the fact that sphingomyelin preferentially interacts with cholesterol and has been shown to decrease sterol transfer between membranes (2). In agreement with this idea, we show in the present study that cholesterol efflux in cells that contain a decreased number of caveolae (36) and presumably reduced sphingomyelin/cholesterol-enriched plasma domains can transfer cholesterol to an acceptor faster than control cells. In this case, cholesterol could be transferred from intracellular stores to the plasma membrane via a pathway that may not involve caveolin-1. Numerous studies have demonstrated the existence of different cellular cholesterol pools that can efflux cholesterol with varying half-times (34, 43, 54, 55, 82). Our current results may reflect a reorganization of the different pools of cholesterol into faster effluxing pools when caveolin-1 is downregulated.
Earlier studies (18) found that downregulation of caveolin-1 leads to reduced cellular cholesterol efflux to media containing serum. This apparent contradiction with our study may suggest that cellular cholesterol efflux mediated by caveolae and caveolin-1 requires other plasma factors, such as 1) pre-Regulation of caveolin-1 expression: importance of HDL, cellular FC, and the MAP kinase pathway. In this study, we show for the first time that HDL can downregulate caveolin-1 expression levels without affecting caveolin-2. Consistent with the studies of Deeg et al. (11), we observed that HDL could activate the MAP kinase pathway through the activation of ERK1/2. Previous studies from our laboratory have demonstrated that activation of this pathway generally leads to the downregulation of caveolin-1 expression (15, 27). This result is consistent with that found by Furuchi and Anderson (26), who showed that short-term treatment with cyclodextrin could lead to activation of the MAP kinase pathway by specifically depleting caveolae cholesterol.
We have previously found that inhibition of the MAP kinase pathway using PD-98059 could prevent the downregulation of caveolin-1 in H-Ras (G12V)-transformed NIH/3T3 cells (15). However, when this inhibitor was added to the medium in the presence of HDL, caveolin-1 expression levels remained similar to those found in cells treated with HDL alone and lower than the control cells incubated without HDL (Fig. 4). This result suggests that caveolin-1 expression is also regulated by factors other than the p42/44 MAP kinase cascade. In support of this possibility, Bist et al. (5) have recently demonstrated that the promoter of the caveolin-1 gene contains two sterol regulatory-like elements. In the present study, we show that incubation of NIH/3T3 cells with HDL can induce the translocation of SREBP-1 into the nucleus. This result suggests that SREBP-1 can prevent CAV-1 transcription as previously shown (5). In addition, these investigators also showed that SREBP-1 can bind the CAV-1 promoter in gel shift analysis and DNase I footprinting experiments. More recent studies by Fra et al. (23) and Czarny et al. (10) have also demonstrated the importance of SREBP-1 in regulating caveolin-1 promoter transcriptional activity. Fra et al. (23) have shown that cells incubated with methylPhysiological significance: HDL-induced downregulation of caveolin-1 and the uptake of oxLDL. Endothelial cells express high levels of caveolin-1 and are, therefore, highly enriched in caveolae (42). Caveolae are involved in a number of cellular events, including transcytosis (12, 66). One of the earliest events that leads to the development of an atheroma may involve the transfer of oxLDL from the luminal side of the artery to the subendothelial space (66). Interestingly, SR-BI and CD36, both of which bind oxLDL, have been localized to caveolae and are expressed in endothelial cells (2, 42, 77).
In the present work, we have demonstrated that HDL can downregulate caveolin-1 expression and may, therefore, prevent the uptake of oxLDL. To test this hypothesis, we examined the effect of HDL3 on the ability of endothelial cells to take up oxLDL. For this purpose, we employed the ligand DiI-labeled oxLDL. We show that pretreatment of endothelial cells with HDL3 downregulates caveolin-1 expression and prevents uptake of oxidized LDL without inhibiting its binding to the plasma membrane. These data indicate that oxLDL receptors are still functional at the plasma membrane, but that ligand uptake is prevented when caveolin-1 is downregulated. These results provide further evidence that caveolae and caveolin-1 are part of a pathway that allows endothelial cells to take up and process oxLDL. Together, these findings may suggest a new mechanism to explain the antiatherogenic effects of HDL: HDL could reduce the transfer of atherogenic lipoproteins from the blood to the subendothelial space by reducing the number of caveolae available for transcytosis at the surface of endothelial cells. A direct test of this hypothesis in vivo will require the generation of a caveolin-1 null (Cav-1 ![]() |
ACKNOWLEDGEMENTS |
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This work was supported by grants from the National Institutes of Health, the Muscular Dystrophy Association, the Komen Breast Cancer Foundation, and the American Heart Association (to M. P. Lisanti). P. G. Frank was supported by postdoctoral fellowships from the Heart and Stroke Foundation of Canada and the Canadian Institutes of Health Research.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. P. Lisanti, Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: lisanti{at}aecom.yu.edu.).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 26 September 2000; accepted in final form 18 December 2000.
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