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Am J Physiol Heart Circ Physiol 291: H677-H686, 2006. First published April 7, 2006; doi:10.1152/ajpheart.01092.2005
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Caveolin-1 and regulation of cellular cholesterol homeostasis

Philippe G. Frank,1,2,3 Michelle W.-C. Cheung,2 Stephanos Pavlides,2,3 Gemma Llaverias,2,3 David S. Park,2 and Michael P. Lisanti2,3

1Departments of Urology, 2Departments of Molecular Pharmacology and Medicine, and Albert Einstein Diabetes Research and Training Center, Albert Einstein College of Medicine, Bronx; New York; and 3Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania

Submitted 14 October 2005 ; accepted in final form 7 March 2006


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Caveolae are 50- to 100-nm cell surface plasma membrane invaginations present in terminally differentiated cells. They are characterized by the presence of caveolin-1, sphingolipids, and cholesterol. Caveolin-1 is thought to play an important role in the regulation of cellular cholesterol homeostasis, a process that needs to be properly controlled to limit and prevent cholesterol accumulation and eventually atherosclerosis. We have recently generated caveolin-1-deficient [Cav-1(–/–)] mice in which caveolae organelles are completely eliminated from all cell types, except cardiac and skeletal muscle. In the present study, we examined the metabolism of cholesterol in wild-type (WT) and Cav-1(–/–) mouse embryonic fibroblasts (MEFs) and mouse peritoneal macrophages (MPMs). We observed that Cav-1(–/–) MEFs are enriched in esterified cholesterol but depleted of free cholesterol compared with their wild-type counterparts. Similarly, Cav-1(–/–) MPMs also contained less free cholesterol and were enriched in esterified cholesterol on cholesterol loading. In agreement with this finding, caveolin-1 deficiency was associated with reduced free cholesterol synthesis but increased acyl-CoA:cholesterol acyl-transferase (ACAT) activity. In wild-type MPMs, we observed that caveolin-1 was markedly upregulated on cholesterol loading. Despite these differences, cellular cholesterol efflux from MEFs and MPMs to HDL was not affected in the Cav-1-deficient cells. Neither ATP-binding cassette transporter G1 (ABCG1)- nor scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux was affected. Cellular cholesterol efflux to apolipoprotein A-I was not significantly reduced in Cav-1(–/–) MPMs compared with wild-type MPMs. However, ABCA1-mediated cholesterol efflux was clearly more sensitive to the inhibitory effects of glyburide in Cav-1(–/–) MPMs versus WT MPMs. Taken together, these findings suggest that caveolin-1 plays an important role in the regulation of intracellular cholesterol homeostasis and can modulate the activity of other proteins that are involved in the regulation of intracellular cholesterol homeostasis.

high-density lipoprotein; lipoproteins; macrophages; atherosclerosis

CAVEOLAE are 50- to 100-nm cell surface plasma membrane invaginations that have proposed functions in the regulation of cell signaling, endocytosis, and cholesterol homeostasis. Together with their main protein marker caveolin-1, they are associated with several signaling proteins as well as proteins involved in cholesterol metabolism (54). For example, the scavenger receptor class B type I (SR-BI), which performs selective high-density lipoprotein (HDL)-cholesteryl ester (CE) uptake and cellular cholesterol efflux to HDL, was localized to caveolae (1, 21). We and others (16, 29, 51) have shown that caveolin-1 could inhibit selective HDL-CE uptake, but it was also shown that caveolin-1 had no effect on SR-BI mediated HDL cholesterol efflux.

Caveolin-1, which is responsible for the formation of caveolae, is also a high-affinity, cholesterol-binding protein (33). In fact, caveolae formation and caveolin-1 expression are highly dependent on the availability of cholesterol (9, 19, 28, 45). Therefore, it is likely to play a role in the regulation of the intracellular movement of cholesterol (8). In fact, Smart et al. (48, 49) have demonstrated that caveolin-1 could transfer newly synthesized cholesterol from the endoplasmic reticulum (ER) to the plasma membrane via a nonvesicular trafficking pathway. In addition, caveolin-1 interacts with sterol carrier protein-2 (SCP-2) (55), which has been involved in the transfer of cholesterol from ER to the plasma membrane (41). Thus a caveolin-1 deficiency may be associated with alterations in the intracellular movement of cholesterol and possibly lead to the accumulation of cholesterol in specific compartments. This hypothesis remains to be tested experimentally. Studies by Fielding et al. (10, 11) have also suggested that caveolin-1 may mediate cellular cholesterol efflux to extracellular acceptors. We and others have previously shown the lack of effect of caveolin-1 on cellular cholesterol efflux mediated by SR-BI. However, little is known about the role of caveolin-1 in ATP-binding cassette transporter-1 (ABCA1)-mediated cholesterol efflux.

We have recently shown that caveolin-1-deficient [Cav-1(–/–)] mice are remarkably less susceptible to atherosclerosis than wild-type (WT) mice in the apolipoprotein E null [ApoE(–/–)] background (14). This observation might be, at least in part, due to the role of caveolin-1 in the regulation of cellular cholesterol homeostasis (15). In the present study, we have therefore examined the role of caveolin-1 in the regulation of cellular cholesterol homeostasis by using mouse embryonic fibroblasts (MEFs) and peritoneal macrophages (MPMs) obtained from WT and Cav-1(–/–) mice. Our results suggest that caveolin-1 plays a novel and important role in the regulation of cellular cholesterol homeostasis but has minimal effects on cellular cholesterol efflux mediated by HDL or apolipoprotein A-I (apoA-I).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Antibodies and their sources were as follows: anti-caveolin-1 IgG (mAb 2297; gift of Dr. Roberto Campos-Gonzalez, BD Pharmingen) (47); anti-caveolin-2 IgG (mAb 65, gift of Dr. Roberto Campos-Gonzalez) (46); rabbit anti-SR-BI-1 and anti-ABCA1 (Novus Biological, Littleton, CO); anti-SREBP-1 (sterol regulatory element binding protein-1) antibody (Santa Cruz Biotechnologies, Santa Cruz, CA). All other reagents were analytical grade.

Animals. Cav-1(–/–) mice were as we previously described. All animals used in these studies were in the C57Bl/6J genetic background and were genotyped by PCR, as previously described (43). Housing and maintenance was provided by the Albert Einstein College of Medicine barrier facility; mice were kept on a 12-h:12-h light/dark cycle and had ad libitum access to food and water. All animal protocols used in this study were preapproved by the Albert Einstein College of Medicine Institute for Animal Studies.

Isolation and analysis of mouse peritoneal macrophages. Elicited mouse peritoneal macrophages (using sodium thioglycollate) were obtained and isolated as described by others (22a). Before incubation with appropriate media, the cells were washed with PBS. Then, the isolated macrophages were either incubated with DMEM-10% FBS or with DMEM containing 0.2% BSA and 75 µg/ml acetylated low-density lipoprotein (LDL) for 48 h.

Determination of cellular cholesterol content. Cellular cholesterol was extracted from cells by using isopropanol. Cholesterol content was determined by using colorimetric tests for total cholesterol (Sigma Aldrich, St. Louis, MO) and free cholesterol (Wako Chemical, Richmond, VA).

Cholesterol synthesis and esterification determination. Cells were seeded in six-well plates the day before the experiment. Labeling was performed for 6 h at 37°C using DMEM 10% FBS containing 5 µCi/well [1-14C]sodium acetate (American Radiolabeled Chemicals, St. Louis, MO) or 1 µCi/well [9,10-3H(N)]sodium oleate (PerkinElmer, Boston, MA). After the incubation, plates were placed on ice, and cells were washed with PBS and solubilized with 0.2 N NaOH overnight on a rocker. Lipids were extracted following the Bligh and Dyer method (4). The resulting extract was analyzed by thin-layer chromatography on ITLC SG plates (Gelman Sciences, Ann Arbor, MI) using a solvent system composed of hexane-diethylether-acetic acid (90:10:1 vol/vol/vol). The areas of the plates containing cholesterol and CE were scraped into vials, and radioactivity was determined by scintillation counting.

Efflux experiments. Mouse embryonic fibroblasts were prepared and cultured as we previously described (43). For cholesterol efflux experiments, MEFs were labeled as previously described (13). Briefly, cells were seeded in a six-well plate and labeled with 5 µCi [3H]cholesterol per well the following day. Cells were washed and incubated 24 h later with DMEM-0.2% BSA for 12 h. For the efflux experiments, cells were washed with DMEM-0.2% BSA and DMEM containing HDL3 (50 µg/ml) or lipid-free apoA-I (50 µg/ml) was added to the cells. Medium was collected as previously described (13). For macrophages, they were labeled during the cholesterol-loading period with 5 µCi [3H]cholesterol per well dispersed in 0.1% ethanol (% final volume of medium) for 24 h. After labeling was completed, a 12-h incubation period (DMEM containing 0.2% fatty acid free BSA) was performed to allow equilibration of the labeled cholesterol with intracellular cholesterol pools. For efflux experiments, cells were washed twice with DMEM alone, and efflux medium was added (50 µg/ml HDL) to the cells. Media aliquots were taken at different times of incubation and treated as previously described (13). 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.

Western blot analyses. The protein concentration was measured using the BCA protein assay (Bio-Rad Laboratories, Hercules, CA), with BSA as the protein standard. Equal amounts of protein for each sample were loaded and run on SDS-polyacrylamide 12% gels. After transfer to nitrocellulose, the expression levels of caveolin-1, SR-BI, ABCG1, and ABCA1 were examined by using specific antibodies. For SREBP-1 analysis, 2 h before the end of the experiment, cells were treated with N-acetyl-Leu-Leu-Nle-CHO (ALLN, 25 µg/ml).

Analysis of cells by immunofluorescence microscopy. The primary antibody was incubated for 1 h with the fixed cells in the presence of PBS, containing 0.2% BSA and 0.1% Triton X-100. After three washes, cells were incubated for 30 min with secondary antibody [Rhodamine red-X-labeled goat F(ab')2 anti-mouse IgG or FITC-labeled goat F(ab')2 anti-rabbit IgG; Jackson Immunoresearch Laboratory, West Grove, PA]. After being rinsed three times, the slides were mounted with Slow-Fade anti-fade reagent (Molecular Probes, Eugene, OR). Cells were observed by using a Bio-Rad Radiance 2000 Laser Scanning Confocal Microscope. 4, 4-Difluoro-1, 3, 5, 7, 8-pentamethyl-4-bora-3a-diaza-s-indacene (BODIPY 493/503; Molecular Probes) was used to detect intracellular neutral lipid droplets, as described by others (20).

Statistical analyses. Values are reported as the means (±SD). Comparisons between WT control and Cav-1(–/–) samples were performed using the Student t-test. P values of ≤0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Caveolin expression and localization in MEFs and macrophages. Confocal immunofluorescence microscopy was used to examine any possible change in the cellular distribution of caveolin-1 and caveolin-2 in MEFs on cholesterol loading (using methyl beta-cyclodextrin as a cholesterol carrier). Figure 1, A–C, illustrates the subcellular localization of caveolin-1 and caveolin-2 in control and cholesterol-loaded cells, respectively. In WT cells, cholesterol loading slightly affected caveolin-2 cellular localization by inducing its translocation to an intracellular compartment (Fig. 1C). Minimal effects were observed in the case of caveolin-1. In addition, caveolin-2 was partially associated with lipid droplets, surrounding them, as shown when cells were stained with BODIPY 493/503 (Fig. 1C). However, in the absence of caveolin-1 in control Cav-1(–/–) cells, caveolin-2 was associated with the Golgi compartment (Fig. 1D). Moreover, cholesterol loading of these cells induced a partial redistribution of caveolin-2 to the cytoplasm and to the plasma membrane (Fig. 1, C and E).


Figure 1
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  		Fig. 1. Immunofluorescent localization of caveolin-1 (Cav-1) and caveolin-2 (Cav-2) in wild-type (WT) and Cav-1(–/–) mouse embryonic fibroblasts (MEF) cells loaded with varying amounts of cholesterol. MEFs were seeded into 12-well plates containing glass coverslips. The following day, cells were incubated with or without of 50 µg/ml CD-cholesterol (cholesterol complexed with methyl-beta-cyclodextrin). Cells were then immunostained with antibodies directed against Cav-1 or Cav-2 and visualized by confocal fluorescence microscopy. Cholesterol-loaded cells were also stained with (BODIPY 493/503). Note that the pattern of Cav-1 localization is dramatically affected by cellular cholesterol loading. A: Cav-1 and Cav-2 in WT MEFs; B: Cav-1 and Bodipy in cholesterol-loaded WT MEFs; C: Cav-2 and Bodipy in cholesterol-loaded WT MEFs; D: Cav-2 in Cav-1(–/–) MEFs; E: Cav-2 and Bodipy in cholesterol-loaded Cav-1(–/–) MEFs. For merged image, inset shows a higher magnification of cell identified by the arrow.

 
The same experiments were also performed with MPMs (Fig. 2, A and B). MPMs were isolated after elicitation with sodium thioglycolate. In WT MPMs, caveolin-1 expression was relatively low compared with other cells types and, therefore, caveolin-2 was often found associated with the Golgi compartment (Fig. 2A). However, cholesterol loading (using acetylated LDL) of these cells resulted in a marked increase in caveolin-1 expression and a redistribution of caveolin-2 toward the plasma membrane (Fig. 2C). Similar to the results we obtained with caveolin-1 (–/–) MEFs, cholesterol loading of caveolin-1 (–/–) MPMs was associated with a redistribution of caveolin-2 from the cytoplasm to the plasma membrane (compare Fig. 2, B and D).


Figure 2
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  		Fig. 2. Immunofluorescent localization of Cav-1 and Cav-2 in WT and Cav-1(–/–) macrophages (MPMs). MPMs were seeded into 12-well plates containing glass coverslips. The following day, cells were incubated with or without of 75 µg/ml acetylated low-density lipoprotein (AcLDL). Cells were then immunostained with antibodies directed against Cav-1 or Cav-2 and visualized by confocal fluorescence microscopy. Cholesterol-loaded cells were also stained with Bodipy. Note that the pattern of caveolin-1 localization is dramatically affected by cellular cholesterol loading. A: Cav-1 and Cav-2 in WT MPMs; B: Cav-1 and Cav-2 in Cav-1(–/–) MPMs; C: Cav-1 and Cav-2 in cholesterol-loaded WT MPMs; D: Cav-1 and Cav-2 in cholesterol-loaded Cav-1(–/–) MPMs.

 
Western blot analysis was performed to validate these results and to examine the levels of three proteins that are critically involved in the regulation of cellular cholesterol homeostasis, namely, ABCA1, ABCGI, and SR-BI (Fig. 3). Caveolin-1 expression was shown to be remarkably upregulated in cholesterol-loaded MPMs. ABCA1 expression in macrophages plays a major role in the elimination of cellular cholesterol from cholesterol-loaded macrophages (7, 35). ABCA1 expression was also shown to be upregulated upon cholesterol loading of macrophages (7, 27). Expression of caveolin-1 has been demonstrated in primary cultures of macrophages (18, 25). As such, caveolin-1 may be involved in the regulation of cellular cholesterol metabolism. Therefore, we have examined the expression levels of ABCA1 in macrophages obtained from WT and Cav-1(–/–) mice. The results presented in Fig. 3 indicate that although ABCA1 levels remain lower in Cav-1(–/–) cells, cholesterol loading of macrophages stimulates ABCA1 expression to a similar extent in both WT and Cav-1(–/–) cells. SR-BI expression has also been associated with the ability of HDL to promote cholesterol efflux from cells. In these studies (Fig. 3), we show that SR-BI and ABCG1 expression are not affected by a caveolin-1 deficiency. It is important to note that the SR-BI protein was not detected in any of the cholesterol-loaded MPMs. In addition, ABCG1 upregulation in the presence of acetylated low-density lipoprotein (AcLDL) was similar in both WT and Cav-1(–/–) MPMs, as previously observed in the case of human macrophages (26).


Figure 3
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  		Fig. 3. Expression of Cav-1, scavenger receptor class B type 1 (SR-BI), ATP-binding cassette tranporter G1 (ABCG1), and ATP-binding cassette tranporter A1 (ABCA1) in mouse peritoneal macrophages (MPMs). MPMs were prepared and grown in the presence of M-CSF. MPMs from WT and Cav-1(–/–) were then cultured in DMEM 10% FBS, with or without 75 µg/ml AcLDL. Forty-eight hours after the incubation, cells were solubilized and 30 µg of protein were separated by SDS-PAGE. Cav-1, ABCA1, ABCG1, and SR-BI levels were assessed using specific antibodies.

 
Cellular cholesterol content of MEFs and peritoneal macrophages. Because caveolin-1 may affect cholesterol trafficking within cells, we next examined the cellular cholesterol composition of both WT and Cav-1(–/–) MEFs, under normal conditions (cells incubated with 10% serum only), or in cells loaded with cholesterol (in the presence of 10% serum and 50 µg/ml cholesterol complexed with cyclodextrin). Under both conditions (Table 1), we observed that Cav-1(–/–) MEFs had reduced free cholesterol but a slightly increased esterified-cholesterol content. These results suggest that caveolin-1 can regulate cholesterol trafficking within the cell, possibly by modifying the accessibility of cholesterol to acyl-CoA:cholesterol acyl-transferase (ACAT) or by affecting its function.


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  		Table 1. Cholesterol content of MEFs

 
In accordance with our results with Cav-1(–/–) MEFs, the free cholesterol content of Cav-1(–/–) macrophages (both control incubated with 10% serum or cholesterol loaded using AcLDL) was decreased compared with WT macrophages (Table 2). In addition, the esterified-cholesterol content was increased in Cav-1(–/–) macrophages. However, when cholesterol loaded using AcLDL, Cav-1(–/–) macrophages showed an increased total cellular cholesterol content. This difference observed between MEFs and macrophages may be due to the absence of serum, i.e, MEFs were loaded with CD-cholesterol (cholesterol complexed with methyl-beta-cyclodextrin) in the presence of serum.


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  		Table 2. Cholesterol content of macrophages

 
Cholesterol synthesis and esterification in MEFs. To determine whether cholesterol synthesis and/or cholesterol esterification were affected in Cav-1(–/–) MEFs, cells were labeled with [1-14C]sodium acetate or [9,10-3H(N)]sodium oleate for the quantification of cholesterol synthesis and cholesterol esterification, respectively. Cholesterol synthesis was reduced and cholesterol esterification was increased in Cav-1(–/–) cells (Fig. 4). These data suggest that caveolin-1 deficiency is associated with deregulation of the control of cholesterol homeostasis.


Figure 4
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  		Fig. 4. A Cav-1 deficiency leads to reduced free cholesterol synthesis (A) and increased cholesterol esterification (B). MEF cells were seeded in 6-well plates 1 day before the experiment. Labeling was performed for 6 h at 37°C using DMEM 10% FBS containing 5 µCi/well [1-14C]sodium acetate or 1 µCi/well [9,10-3H(N)]sodium oleate. Lipid extracts were then analyzed by thin-layer chromatography. Areas of the plates containing cholesterol and cholesteryl ester were scraped into vials, and radioactivity was determined by scintillation counting. Results were normalized to cellular protein content. Open bars, WT; closed bars, Cav-1(–/–) (KO, knockout).

 
Determination of endogenous SREBP-1 levels in MEFs. Previous studies have shown that caveolin-1 could mediate the transfer of newly synthesized cholesterol from the ER to the plasma membrane (48). SREBP-1 is a member of a family of transcription factors (6) that regulate the transcription of genes involved in cellular lipid homeostasis (23). Before being activated, full-length SREBP (125 kDa) is located in the ER but requires proteolysis for activation in the nucleus. This process necessitates the participation of the SREBP cleavage activation protein (SCAP) (6). SCAP is a sterol sensor protein that intiates the SREBP proteolytic cascade, leading to the transfer of the basic-helix-loop-helix-leucine zipper active fragment (68 kDa) to the nucleus. Therefore, a change in ER free-cholesterol content would be associated with a change in both the 125-kDa and the 67-kDa relative proportion in Cav-1(–/–) cells. Our results (Fig. 5) demonstrate that Cav-1(–/–) cells show increased levels of the full-length SREBP-1 form but reduced levels of the active nuclear SREBP-1 form. The data are consistent with an increase in the ER free cholesterol content in Cav-1(–/–) cells.


Figure 5
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  		Fig. 5. Sterol regulatory element binding protein-1 (SREBP-1) levels in WT and Cav-1(–/–) MEFs. MEFs were grown in DMEM 10% FBS. Two hours before the MEFs were solubilized, the cells were treated with N-acetyl-Leu-Leu-Nle-CHO (ALLN, 25 µg/ml). After solubilization, 30 µg of protein were separated using 8% SDS-PAGE. Levels of the full length (125 kDa) and of the activated (67 kDa) forms were then determined by Western blot analysis.

 
Cellular cholesterol efflux from MEFs. We have previously shown that caveolin-1 does not affect SR-BI-mediated cellular cholesterol efflux in HEK-293T cells (16). In the present study, we examined the ability of HDL3 to promote cellular cholesterol efflux from WT and Cav-1(–/–) MEFs. Interestingly, our observations suggest that loss of caveolin-1 expression does not affect the ability of HDL3 to promote cellular cholesterol efflux from MEF (Fig. 6).


Figure 6
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  		Fig. 6. Effects of a Cav-1 deficiency on cellular cholesterol efflux mediated by HDL3. MEFs were labeled in the presence of [3H]cholesterol. After an overnight equilibration in DMEM 0.2% BSA, HDL3 (50 µg/ml) was incubated with the cells. Aliquots of media were taken at indicated times and counted. Efflux is expressed as the percentage of [3H]cholesterol remaining in cells as a function of time. Experiments were performed in triplicate.

 
Cellular cholesterol efflux from MPMs. We have also examined the ability of HDL and apoA-I to promote cellular cholesterol efflux from macrophages. Figure 7 indicates that HDL can promote cellular cholesterol efflux equally well from both WT and Cav-1(–/–) macrophages. The same observation was made in cholesterol-loaded cells (data not shown). This finding is in agreement with the absence of effect of caveolin-1 on SR-BI expression in macrophages (Fig. 3). Because apoA-I has been shown to promote specific ABCA1-mediated cholesterol efflux, we next examined the ability of apoA-I to promote cellular cholesterol efflux from both WT and Cav-1(–/–) macrophages under normal or cholesterol-loaded conditions (Fig. 8). Whereas there were no differences between cells loaded with cholesterol, apoA-I-mediated cellular cholesterol efflux was slightly reduced under basal conditions (macrophages not loaded with cholesterol). These results correlate well with the expression of ABCA1 in the different conditions: maximum cholesterol efflux is observed with the highest levels of ABCA1 expression. Additionally, caveolin-1 regulates the expression of ABCA1 and may, therefore, directly affect cellular cholesterol efflux to apoA-I.


Figure 7
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  		Fig. 7. Effects of a Cav-1 deficiency on cellular cholesterol efflux mediated by HDL3. MPMs were labeled in the presence of [3H]cholesterol. After an overnight equilibration in DMEM 0.2% BSA, HDL3 (50 µg/ml) was incubated with the cells. Aliquots of media were taken at indicated times and counted. Efflux is expressed as the percentage of [3H]cholesterol remaining in cells as a function of time. Experiments were performed in triplicate.

 

Figure 8
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  		Fig. 8. Effects of a Cav-1 deficiency on cellular cholesterol efflux mediated by apolipoprotein A-I (apoA-I). Mouse peritoneal macrophages were labeled in the presence or absence of AcLDL (CTL) and with [3H]cholesterol. After an overnight equilibration in DMEM 0.2% BSA, apoA-I (50 µg/ml) was incubated with the cells. Aliquots of media were taken at indicated times and counted. Efflux is expressed as the percentage of [3H]cholesterol remaining in cells as a function of time. Experiments were performed in triplicate.

 
To determine the contribution of ABCA1 to apoA-I-mediated cholesterol efflux in MPMs, cholesterol-loaded cells were treated with the ABCA1 inhibitor glyburide (53). Under these conditions, we found that Cav-1(–/–) MPMs showed a more profound reduction in apoA-I-mediated cholesterol efflux (Fig. 9). These data suggest that caveolin-1 may normally participate, either directly or indirectly, in apoA-I-mediated cholesterol efflux from macrophages.


Figure 9
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  		Fig. 9. Inhibition of ABCA-1-mediated cholesterol efflux in cholesterol-loaded macrophages. MPMs were incubated in the absence or presence of AcLDL and labeled with [3H]cholesterol. After an overnight equilibration in DMEM 0.2% BSA, cells were preincubated for 5 h with DMEM 0.2% BSA, containing 100 µM glyburide (+) or vehicle alone (–). After a 5-h incubation period with apoA-I (50 µg/ml), radioactivity in the media and cell extracts was determined. Efflux is expressed as the percentage of efflux observed under control conditions. Experiments were performed in triplicate. *Statistical significance.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we have shown that a caveolin-1 deficiency leads to the dysregulation of the intracellular flux of cellular cholesterol. We show that a caveolin-1 deficiency is associated with reduced free cholesterol and increased esterified cholesterol. However, caveolin-1 deficiency does not have a major effect on cellular cholesterol efflux. Thus caveolin-1 may act as a intracellular cholesterol transporter, as previously suggested (48). In addition, a caveolin-1 deficiency also affects the subcellular localization of caveolin-2, in accordance with our previous findings (43).

Regulation of caveolin-1 expression by cellular cholesterol. It is now quite clear that caveolin-1 is regulated by cellular cholesterol levels. The CAV-1 promoter contains two sterol regulatory element-like sequences that can bind SREBP-1 and inhibit CAV-1 transcription (3). In addition, it has recently been shown that CAV-1 transcription can be regulated by the nuclear receptors peroxisome proliferator-activated receptor {gamma} and liver X receptor (28). Consistent with these findings, several groups have now shown that cellular cholesterol levels affect caveolin-1 protein levels. Whereas cholesterol loading of certain cell lines can lead to the upregulation of caveolin-1 protein levels (9, 16, 22), inhibition of cholesterol synthesis or reduction of cellular cholesterol levels with an extracellular acceptor are associated with lowered caveolin-1 protein levels (13, 22). Our current data validate these conclusions with macrophages loaded with cholesterol using AcLDL. Caveolin-1 expression has previously been demonstrated in macrophages (2, 18, 30). We now show for the first time that caveolin-1 expression is upregulated in cholesterol-loaded macrophages. In the case of MPMs, this finding is especially important because caveolin-1 protein levels are upregulated >10 times on cholesterol loading (Fig. 3). This finding may suggest an important role for caveolin-1 in the regulation of cellular cholesterol homeostasis. Indeed, numerous studies have now explored a potential role for caveolin-1 in the regulation of cellular cholesterol metabolism. Earlier studies suggested a role for caveolin-1 in the regulation of cellular cholesterol efflux to HDL (9, 11). However, later studies have not drawn the same conclusions (13, 16, 29, 51). The specificity of the cholesterol labeling, the type of extracellular cholesterol acceptors used, and/or the cell types used may explain some of the conflicts observed in the different studies. According to our results, we can conclude that caveolin-1 may play a role in the regulation of intracellular cholesterol metabolism and transport.

Caveolin-1 and the intracellular movement of free cholesterol. Previous studies have shown that caveolin-1 may mediate the transfer of newly synthesized cholesterol from the ER to the plasma membrane (48). This implies that a deficiency in caveolin-1 would be associated with the accumulation of cholesterol in the ER and potentially the reduced transfer of newly synthesized cholesterol from the ER to the plasma membrane. Accumulation of ER cholesterol would lead to increased ACAT activity and reduced free cholesterol synthesis. These predictions are in fact what we observed in Cav-1(–/–) cells (Tables 1 and 2, and Fig. 4). As a consequence, a caveolin-1 deficiency is associated with a significant increase in esterified-cholesterol (EC) and reduced free cholesterol (FC) under basal conditions (without cholesterol loading) and in cholesterol-loaded cells. This change in the EC-to-FC ratio may explain the reduced ABCA1 expression that we observed in Cav-1(–/–) macrophages (Fig. 3). A reduction in the transfer of cholesterol from the ER to the plasma membrane would also be associated with an inhibition of SREBP-1-mediated transcription of genes involved the metabolism of cholesterol. This is indeed what we observed in Cav-1(–/–) cells, which display reduced free cholesterol synthesis and increased ACAT activity. These findings are, in part, reminiscent of the work presented by another group (40). Pol et al. (40) showed that in BHK cells transfected with a dominant negative mutant of caveolin-3 (cav-3DGV), reduced free cholesterol synthesis was observed as well as reduced plasma membrane accumulation. However, they did not observe any increases in esterified-cholesterol levels. This last observation may be related to the fact the mutant protein could reduce access of ACAT to free cholesterol because caveolin presents a high affinity for cholesterol. In general, we believe that movement of cholesterol (newly synthesized or not) from the ER is affected in Cav-1(–/–) cells because increased esterified cholesterol was observed in Cav-1(–/–) cells incubated with CD-cholesterol. This hypothesis is also consistent with the association between caveolin-1 and SCP-2 recently demonstrated by Zhou et al. (55). Caveolin-1 may enhance the transfer of cholesterol to the plasma membrane already observed with SCP-2 alone (41).

Role of caveolin-1 in the regulation of cellular cholesterol efflux. In MEFs, caveolin-1 deficiency had no effect on the regulation of cellular cholesterol mediated by HDL3. Two important proteins that regulate cholesterol efflux from macrophages to HDL have been identified. They are SR-BI and ABCG1 (24, 34, 52). Because SR-BI is not detected in cholesterol-loaded macrophages, cholesterol efflux to HDL is more likely to be dependent on ABCG1 under these conditions. HDL-mediated cholesterol efflux from cholesterol-loaded macrophages was not affected in Cav-1(–/–) macrophages (not shown). This finding suggests that ABCG1-mediated cholesterol efflux is not affected in Cav-1(–/–) MPM. ABCG1 expression is in fact not affected in Cav-1(–/–) MPMs (Fig. 3). We also show that cholesterol efflux to HDL is unaffected in normal MPMs (Fig. 7). Taken together, these data suggest that a caveolin-1 deficiency does not affect HDL-mediated cholesterol efflux (via ABCG1 and SR-BI) in MEFs nor MPMs.

In macrophages, ABCA1 expression is in part regulated by cellular cholesterol levels (27). This observation may explain why, in Cav-1(–/–) MPMs, ABCA1 expression levels are reduced relative to those observed in WT MPMs. However, reduced ABCA1 expression in Cav-1 (–/–) MPMs is not associated with a reduced ability of apoA-I to promote cellular cholesterol efflux from MPMs (difference not significant). Under these conditions, caveolin-1 would have minimal effects, because its levels are relatively low (Fig. 3) and ABCA1 levels are therefore rate limiting for the efflux process. On the other hand, ABCA1 expression levels are upregulated in both WT and Cav-1(–/–) MPMs upon cholesterol-loading, although ABCA1 levels remain lower in Cav-1(–/–) MPMs. Cholesterol loading of macrophages stimulates ABCA1 expression to a similar extent in both WT and Cav-1(–/–) cells. However, cellular cholesterol efflux to apoA-I is not affected. This finding suggest that caveolin-1, which is remarkably upregulated in WT MPMs, can regulate ABCA1-mediated cellular cholesterol efflux to apoA-I because higher levels of ABCA1 are required in WT than in Cav-1(–/–) MPMs to promote equivalent levels of cellular cholesterol efflux to apoA-I. We also performed additional experiments in the presence of glyburide (Fig. 9). Our results show that, in Cav-1(–/–) MPMs, ABCA1 is more sensitive to the effects of glyburide than in WT cells. It is possible that caveolin-1 regulates ABCA1 function in WT cells. In this case, caveolin-1 may either directly participate in apoA-I-mediated cellular cholesterol efflux or may protect ABCA1, directly or indirectly, from the effect of glyburide.

The minor differences observed in terms of cholesterol efflux indicate a minimal role for caveolin-1 in the regulation of cellular cholesterol efflux to apoA-I and HDL. These findings are consistent with the fact that plasma HDL-cholesterol levels in Cav-1(–/–) mice remain similar to those observed in WT animals (42). This is in marked contrast to the findings made with ABCA1(–/–) mice, in which HDL particles are almost completely absent (31, 36). In addition, cholesterol efflux from fibroblasts obtained from Tangier disease patients (12, 44, 50) or from macrophages isolated from ABCA1(–/–) mice (36) have demonstrated a reduce ability to efflux cholesterol to apolipoprotein A-I.

Caveolin-1 and the formation of lipid droplets. Under specific conditions, caveolin-1 has been shown to interact with lipid droplets (17, 37, 40). Our current study also suggests that caveolin-2 may play a role in the formation of esterified-cholesterol containing lipid droplets, as previously suggested (17, 37, 40). Its role in this compartment has yet to be determined but, in the absence of caveolin-1, it may help facilitate the accessibility of esterified cholesterol to cholesteryl ester hydrolase and, therefore, compensate for the absence of caveolin-1 during cholesterol efflux.

Contrary to a previous study (18), we show that caveolin-2 localizes to the plasma membrane in the presence of caveolin-1. Our current study is also consistent with previous reports using nonmacrophage cell types (5, 32, 38, 43) and demonstrates that in the absence of caveolin-1, caveolin-2 remains in the Golgi complex (Figs. 1 and 2). Therefore, caveolin-2 requires the presence of caveolin-1 to be properly targeted to the plasma membrane in MEFs and MPMs.

Overall these findings suggest that caveolin-1 plays an important role in the regulation of intracellular cholesterol homeostasis, but that its role in the regulation of cellular cholesterol efflux is not as critical as previously suggested. These conclusions are also in agreement with our previous findings showing that caveolin-1-deficient mice are less susceptible to atherosclerosis than WT mice, both in the ApoE(–/–) background (14). Caveolin-1 deficiency in macrophages is, therefore, unlikely to play a crucial role in the protection against atherosclerosis observed in these mice.


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P. G. Frank was supported by a Scientist Development Grant from the American Heart Association and a grant from the Elsa U. Pardee Foundation. M. P. Lisanti was supported by grants from the National Institutes of Health and the American Heart Association. G. Llaverias was supported by a post doctoral fellowship from the Spanish Ministry of Education.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. G. Frank, Dept. of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson Univ, BLSB 933, 233 S. 10th St, Philadelphia, PA 19107 (e-mail: Philippe.Frank{at}jefferson.edu or Michael.Lisanti{at}jefferson.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.


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