©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Compartmental Isolation of Cholesterol Participating in the Cytoplasmic Cholesteryl Ester Cycle in Chinese Hamster Ovary 25-RA Cells (*)

(Received for publication, July 17, 1995; and in revised form, November 30, 1995)

Judeth J. Klansek (§) Gregory J. Warner William J. Johnson Jane M. Glick (¶)

From the Medical College of Pennsylvania and Hahnemann University, Department of Biochemistry, Philadelphia, Pennsylvania 19129

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
APPENDIX
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Using the Chinese hamster ovary cell line, 25-RA, we have demonstrated that lipoprotein-derived cholesterol and endogenously synthesized cholesterol are selectively differentiated with respect to their cellular locations. These cells lack sterol-mediated regulation, spontaneously storing large amounts of esterified cholesterol, which turns over with a half-time of 7.5 h. When [^3H]cholesterol was provided to the cells in serum to trace cellular cholesterol, the specific activities of cellular free and esterified cholesterol (6238 ± 273 and 5128 ± 277 cpm/µg, respectively) failed to equilibrate, indicating that bulk cellular free cholesterol is isolated from that participating in the cholesteryl ester cycle. Using [^3H]acetate to trace the fate of endogenously synthesized cholesterol, a failure of equilibration was also observed (specific activities of free and esterified cholesterol = 280 ± 37 and 458 ± 8 cpm/µg, respectively). The lower specific activity of the precursor indicates that endogenously synthesized cholesterol is preferentially esterified. When cells radiolabeled with [^3H]acetate were post-incubated in the absence of radiolabel, the specific activity of the esterified cholesterol pool remained significantly higher than that of the free cholesterol, suggesting that cholesterol derived from hydrolysis of esterified cholesterol is preferentially re-esterified.


INTRODUCTION

Many investigators have used cell culture models for studying cholesterol metabolism to examine the pathology of atherosclerosis at the cellular level (for review see (1) ). Chinese hamster ovary (CHO) (^1)cells are fibroblast-like cells that have been used extensively to examine the somatic cell genetics of cholesterol metabolism(2) . They are particularly useful because of their haploid phenotype, which allows one to obtain cells deficient in gene products of interest in order to elucidate specific pathways involved in cell processes(3) . One such mutant CHO cell line (CHO 25-RA), selected for resistance to 25-hydroxycholesterol, spontaneously stores large amounts of cholesteryl ester, making it similar in appearance to the foam cells found in the atherosclerotic plaque. When exposed to 25-hydroxycholesterol, normal cells are subject to down-regulation of endogenous cholesterol biosynthesis and the low density lipoprotein (LDL) receptor pathway, and thus the cells normally maintain low cholesterol levels. The 25-RA cells, which are resistant to the actions of 25-hydroxycholesterol, continue to take in cholesterol via LDL receptors and to synthesize cholesterol via biosynthetic pathways(4, 5) . In characterizing the cell line, Chang and Limanek (4) documented that the expression of the first four cholesterogenic enzymes, as well as the activity of the LDL receptor, is unregulated. Although the precise molecular defect in 25-RA cells remains to be established, the phenotype is similar to another CHO line that has been shown to have a mutant sterol regulatory element binding protein 2(6) . The lack of sterol regulation proved useful for the studies presented here on intracellular cholesterol trafficking.

Intracellular movement of cholesterol involves a complex set of pathways (for reviews see (7, 8, 9) ). In a mammalian cell, cholesterol, existing in either the unesterified or stored (esterified) form, can be derived from two distinct sources, either de novo synthesis or exogenous, lipoprotein-derived cholesterol. The LDL receptor-mediated internalization of cholesterol has been well characterized. LDL binds to a cell surface-associated receptor and is internalized, and the lipoprotein cholesteryl ester is degraded to unesterified cholesterol in the lysosomes(10) . At this point, the LDL-derived cholesterol is thought to enter a pool that can either incorporate into the plasma membrane or move to intracellular membranes to be esterified by acyl CoA:cholesterol acyltransferase (ACAT) for storage as cholesteryl esters. Xu and Tabas (11) concluded that trafficking of LDL-derived cholesterol to the plasma membrane precedes the utilization of cholesterol for storage in cholesteryl esters and have demonstrated that in macrophages the esterification of cholesterol occurs only after the free cholesterol pool expands 25% above basal levels. Similar trafficking patterns have been proposed by other investigators(7, 12) .

The bulk of cholesterol synthesis is thought to occur in the endoplasmic reticulum (ER) because the biosynthetic enzymes co-fractionate with ER markers(13, 14) . In mammalian cells, cholesterol is important for new membrane synthesis and is found mainly in the plasma membrane(15) . Alternatively, especially when cellular cholesterol levels rise, cholesterol may become a substrate for ACAT, another enzyme found in the endoplasmic reticulum. In a recent review, Liscum and Faust (8) suggest that the majority of cholesterol esterified by ACAT is endogenously synthesized; however, definitive data demonstrating this are lacking. The concept is complementary to the data of Xu and Tabas (11) suggesting that exogenous cholesterol is channeled away from ACAT and appears to reside initially in the plasma membrane. Although these studies suggested that there is selective trafficking of cholesterol derived from either exogenous or endogenous sources, the intact regulation of cholesterol synthesis in these cell systems has precluded rigorous examination.

The 25-RA cell line, having lost its regulatory sensitivity to increasing cholesterol levels, seemed an ideal system to use to examine this compartmentalization, because synthesis continues at high levels regardless of the influx of lipoproteins. Using radiolabeled compounds to trace exogenous and endogenous cholesterol, we observed differences in specific activities that can best be explained by a specific compartmentalization of the pools of cholesterol in the cell. The data presented here demonstrate that not only are these pathways distinct but that a compartmentalization of endogenous cholesterol exists within the cholesteryl ester cycle. These studies using 25-RA cells as a foam cell model provide new information on the preferential handling of endogenous and exogenous cellular cholesterol that may contribute to the process of foam cell formation in atherosclerosis.


EXPERIMENTAL PROCEDURES

Materials

Unesterified cholesterol, 5-cholesten-3beta-ol 3-methyl ether (cholesterol methyl ether), heat-inactivated fetal bovine serum, bovine serum albumin (BSA, essentially fatty acid free), progesterone, mevalonate, and gentamicin were purchased from Sigma. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine was purchased from Avanti (Alabaster, AL). Reagent grade organic solvents were obtained from Fisher. Chemicals for saponification (tetrachloroethylene, ethylpropionate, and tetramethylammonium hydroxide) were purchased from Eastman Kodak Company. Instant thin layer chromatography plates were obtained from Gelman Sciences (Ann Arbor, MI). Precoated Anasil G (250 µm) silica gel plates were obtained from Analabs (North Haven, CT). [1,2-^3H]Cholesterol (51.3 Ci/mmol) and [^3H]acetate, sodium salt (4.2 Ci/mmol) were purchased from DuPont NEN. Human LDL was prepared by ultracentrifugation (1.019-1.063 g/ml)(16) , prior to acetylation by the method of Basu et al.(17) . Tissue culture flasks and plates were obtained from Falcon (Lincoln, NJ) and Corning Glass Works (Corning, NY), respectively. Cell culture reagents and media were purchased from Life Technologies Inc. Sandoz compound 58035 (3-[decyldimethylsilyl]-N-[2(4-methylphenyl)-1-phenylethyl] propanamide) was a gift from Dr. John Heider of Sandoz, Inc. (East Hanover, NJ). Mevinolin was kindly provided by Dr. Peter Gillies of DuPont Merck Pharmaceutical Co.

Analytic Procedures

Unesterified and total cholesterol masses were quantitated by gas liquid chromatography using cholesterol methyl ether as an internal standard(18, 19) . Phosphorous content of free cholesterol lipid dispersions was determined by the method of Sokoloff and Rothblat(20) . Cell protein content of the monolayers was determined by the procedure of Lowry et al.(21) as modified by Markwell et al.(22) using BSA as a standard. Radioactivity was measured using a Beckman LS 5000 liquid scintillation counter and Scintiverse BD mixture (Fisher). Delipidized calf serum protein was prepared as described previously(18) . The protein was dissolved in Ham's F-12 medium at a final concentration of 1 mg protein/ml; after sterilization by passage through a 0.45-µm filter, this solution was stored at 4 °C. Sonicated free cholesterol-rich dispersions containing free cholesterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (molar ratio, >2) were prepared as described previously(23) .

Cell Culture

The mutant Chinese hamster ovary cell line (CHO 25-RA) was obtained from Dr. T. Y. Chang (Dartmouth College, NH). CHO 25-RA cells were grown as monolayers in Ham's F-12 medium (24) supplemented with 10% fetal bovine serum (FBS) (v/v) and 50 µg/ml gentamicin as described(25) . Rat Fu5AH hepatoma cells and mouse macrophage J774 cells were grown and loaded with excess cholesterol as described(26) . For experiments, CHO cells from stock cultures were plated in growth medium (Ham's F-12, 10% FBS) on 35-mm dishes at a density of 1 times 10^5 cells/ml. Lipid and protein contents of the cell monolayers were determined as described(25) .

Radiolabeling of Cellular Cholesterol

The metabolic fate of exogenous free cholesterol was monitored by growing cells in 35-mm wells with growth medium supplemented with 0.5 µCi/ml of [1,2-^3H]cholesterol for 3 days. The distribution of radiolabel between free and esterified cholesterol was determined by thin layer chromatography on instant thin layer chromatography plates (SA, Gelman Sciences, Ann Arbor, MI) developed in petroleum ether:diethyl ether:glacial acetic acid (90:10:1, v/v/v). Migration of specific lipids was identified by comparison of lipid standards; bands were cut, and the radioactivity in the bands was quantitated by liquid scintillation counting.

CHO 25-RA cells grown on 10% FBS or 1 mg/ml delipidized calf serum protein were labeled with [^3H]acetate to trace endogenously synthesized cholesterol. The cells were plated on 100-mm culture dishes and allowed to grow for 24 h. Fresh media containing [^3H]acetate (5 µCi/plate) were added, and the cells were incubated for 3 days. The media were removed, and the monolayers were washed three times with phosphate-buffered saline. Isopropanol was added to the monolayers overnight to extract the lipids. Dried isopropanol extracts were partitioned using the method of Bligh and Dyer(27) . Individual lipid classes were separated by thin layer chromatography on glass-backed Anasil-G 250-µm plates developed in petroleum ether:diethyl ether:glacial acetic acid (90:10:1, v/v/v). The areas of the plate corresponding to the major lipid subclasses, as identified by the migration of standards and by scanning for radioactivity using a Radiomatic Imaging TLC Scanner, were scraped into test tubes. The lipids were extracted from the silica gel by the procedure of Folch et al.(28) as modified by Bamberger et al.(29) . The individual lipids then were saponified in 10% alcoholic potassium hydroxide for 1 h at 60 °C and then extracted three times with petroleum ether and twice with diethyl ether. The collected extractions were dried under nitrogen and washed twice with deionized water. The final sample was analyzed for radioactivity and cholesterol mass. Specific activities of the free and esterified cholesterol pools were calculated as the number of cpm/µg of cholesterol.

Statistical Analysis

Statistical comparisons were made using a standard two-tailed, paired Student's t test (GraphPAD InStat t(m), GraphPAD Software V2.02)).


RESULTS

CHO 25-RA cells, maintained in 10% fetal bovine serum, accumulate extremely high levels of cellular cholesterol. Over a 3-day period, cells maintain fairly constant free cholesterol levels of about 20 µg/mg cell protein while storing approximately 120-130 µg of esterified cholesterol/mg of cell protein (data not shown). Even in the absence of an exogenous source of cholesterol, cells continue to maintain excess cholesterol. As shown in Fig. 1, when 25-RA cells were incubated in lipid-free medium for 3 days, cellular esterified cholesterol decreased, but the cells were by no means depleted of cholesteryl esters, because approximately 60% of the cellular cholesterol remained in the esterified form. The 25-RA stock cultures used for some of the experiments described below, which were maintained on delipidized serum protein for several weeks, continued to contain 40-50% of total cholesterol in the esterified form. The decline in cellular cholesterol upon incubation with lipid-free medium was not due to efflux of cholesterol to the medium but rather was a function of the normalization of the data for cholesterol content as a function of the monolayer protein content, which increased over time (Fig. 1, inset).


Figure 1: Effect of lipid-free medium in CHO 25-RA cells. CHO 25-RA cells were plated in growth medium (Ham's F-12/10% FBS) and allowed to grow for 3 days. The cells were then rinsed with serum-free medium, and fresh Ham's F-12 medium containing delipidized serum calf protein at a concentration of 1 mg/ml was added to the wells. On each day thereafter, a set of cultures was removed and the lipids were extracted from the cell monolayers with isopropanol as described under ``Experimental Procedures.'' Each value for esterified cholesterol () and free cholesterol (bullet) represents the average of duplicate cultures. Shown in the inset are the protein contents of the cell monolayer (up triangle).



It was of interest to compare the changes in cellular cholesterol contents of 25-RA cells incubated with lipid-free medium with those that occur when the cholesteryl ester cycle was interrupted using the ACAT inhibitor, Sandoz 58035. This compound specifically blocks the synthetic arm of the cholesteryl ester cycle(30) , allowing direct assessment of hydrolase activity through the measurement of the disappearance of cellular esterified cholesterol(31) . As shown in Fig. 2, cellular cholesteryl ester content declined much more rapidly and to a much greater extent in the presence of the ACAT inhibitor than in the absence of exogenous lipoprotein. Because the ACAT inhibitor prevents the reesterification of free cholesterol generated by cholesteryl ester hydrolysis, cellular free cholesterol contents rose to high levels. The differences in the rates of disappearance of esterified cholesterol under the two conditions indicates that the cholesteryl ester cycle remained active in 25-RA cells even in the absence of an exogenous source of cholesterol. Because the decline in cholesteryl ester content of cells incubated with the ACAT inhibitor was so rapid, we performed an additional experiment to examine in more detail the initial kinetics of cholesteryl ester hydrolysis in the presence of the ACAT inhibitor. Using the data shown in Fig. 3, we calculated a half-time of 7.5 h for cholesteryl ester hydrolysis, which is assumed to reflect the turnover of the cholesteryl ester cycle(31) . Additional experiments indicated that this hydrolysis was extra-lysosomal; chloroquine, an inhibitor of lysosomal function(32) , was found to have no inhibitory effect on the cholesteryl ester hydrolysis, whereas umbelliferyl diethyl phosphate, a selective inhibitor of cytoplasmic lipid esterases(33, 34) , blocked hydrolysis (data not shown).


Figure 2: Effect of inhibiting acyl CoA:cholesterol acyltransferase. CHO 25-RA cells were plated and harvested as described in the legend to Fig. 1. Values for esterified cholesterol () and free cholesterol (bullet) contents represent the average of duplicate cultures normalized to cell protein. Control cells (A) were supplemented with 0.25% Me(2)SO (v/v), a concentration equal to that used as vehicle for S-58035 in the experiment shown in B. A final concentration of S-58035 utilized in these studies was 1 µg/ml.




Figure 3: Kinetics of cholesteryl ester hydrolysis. CHO 25-RA cells were plated as described in the legend to Fig. 1. After 3 days, the monolayers were rinsed with serum-free medium, and fresh medium containing 10% FBS, 1 µg/ml S-58035, and 0.25% Me(2)SO (v/v) was added. At indicated intervals, cultures were harvested and analyzed as described under ``Experimental Procedures.'' The values are the average of duplicate wells. The dotted lines indicate the time in which 50% of the cellular cholesteryl ester was hydrolyzed.



Because cholesteryl esters are derived from free cholesterol and exhibit a turnover with a half-time of approximately 7.5 h, both the free and esterified cholesterol pools would be expected to become uniformly radiolabeled quickly in the presence of radiolabeled cholesterol if the cellular free cholesterol is in a single freely equilibrating pool. To determine whether this is the case in CHO 25-RA cells, cells were exposed to [^3H]cholesterol, and the percentage of esterified cholesterol as determined by mass measurements was compared with the percentage of esterified cholesterol determined after separating radiolabeled free and esterified cholesterol by TLC. If there were complete equilibration of all cellular free cholesterol, these values should converge to a similar level relatively quickly after addition of exogenous radiolabeled cholesterol. Shown in Fig. 4are data from an experiment in which [^3H]cholesterol was supplied in growth medium and cells were harvested daily over the course of 5 days. The percentage of esterified cholesterol by mass remained higher than that by radiolabel over the entire 5-day period (Fig. 4A). Fig. 4B, which shows data from the same experiment expressed in terms of specific activities, confirms that the specific activity of the free cholesterol remained higher throughout the 5-day period. This implies that the exogenous, radiolabeled cholesterol is not in rapid equilibrium with the free cholesterol in the cholesteryl ester cycle.


Figure 4: Incorporation of exogenous radiolabeled cholesterol into esterified cholesterol. CHO 25-RA cells were plated at 1 times 10^5 cells/ml, 2 ml/35-mm dish in standard growth medium (Ham's F-12/10% FBS) containing 0.5 µCi/ml [1,2-^3H]cholesterol. On each of 5 days after plating the cells, a set of cultures was analyzed for esterified lipid content as described under ``Experimental Procedures.'' Shown in A are data expressed as the percentage of esterified cholesterol by mass (box) and by radiolabel (). The same data are expressed as specific activity in B where free cholesterol (bullet) and esterified cholesterol () specific activities are expressed as cpm/µg cholesterol. The values are means and standard deviations of triplicate cultures.



One potential explanation for the differences in specific activities is the continuous presence of exogenous radiolabeled free cholesterol. Because at the outset the cells had a very large unlabeled esterified cholesterol pool, it was possible that equilibration might be observed only when the source of radiolabeled precursor was removed. To examine this, 25-RA cells were plated in the presence of exogenous radiolabeled cholesterol, as in the previous experiment, for 3 days to label the cellular cholesterol pools. Medium containing radiolabel was removed, and the cells were incubated with lipid-free medium containing 1% BSA. Fig. 5shows the specific activities of the free and esterified cholesterol of the cultures harvested at 0, 12, 18, and 24 h. Again, the specific activity of free cholesterol at the beginning of the equilibration period was twice that of esterified cholesterol. Although the specific activity of free cholesterol decreased over time, a significant difference (p < 0.05) was maintained between the two specific activities throughout this time period. Using theoretical concepts involving a two compartmental system (see ``Appendix''), the rate of cholesteryl ester cycle turnover and the masses of the free and esterified cholesterol, we calculated that complete equilibration should have occurred by 12 h. The data presented in Fig. 5, where the cells are placed on label-free, lipid-free medium, show a decline in the specific activity of the free cholesterol, which is expected, because the cells are still synthesizing unlabeled free cholesterol. However, there is no decline in the specific activity of the esterified cholesterol pool, suggesting compartmentalization of the free cholesterol that is part of the cholesteryl ester cycle.


Figure 5: Specific activity determination during equilibration with 1% BSA. CHO 25-RA cells were plated in growth medium containing 0.5 µCi/ml [1,2-^3H]cholesterol and allowed to grow for 3 days. Cultures were then rinsed with serum-free medium and refed with Ham's F-12 medium containing 1% BSA. Cultures were harvested at the indicated times, and lipids were extracted and analyzed as described under ``Experimental Procedures.'' Specific activities, expressed as cpm/µg cholesterol, for free (bullet) and esterified () cholesterol are the mean and standard deviations of triplicate cultures. The asterisks indicate significant differences (t test) between FC SA and EC SA at p < 0.01 (**) and p < 0.005 (***).



The next set of experiments examined the fate of synthesized cholesterol. CHO 25-RA cells maintained on 10% FBS were plated in growth medium and incubated for 24 h. The cells were then exposed to [^3H]acetate in fresh growth medium for 3 days, followed by a 24-h chase period using fresh, unlabeled medium. At various time intervals, cellular lipids were extracted with isopropanol and separated on TLC plates, and the separate lipid classes were isolated. Cholesteryl esters were subjected to an additional saponification step to remove the fatty acyl moiety and ensure that we were examining only the radiolabeled sterol derived from the cholesteryl ester. Specific activities of free and esterified cholesterol at various time points are shown in Fig. 6. As in prior experiments, esterified cholesterol mass levels in the cells on 10% serum comprised approximately 75% of the total cholesterol. At the end of the 3-day labeling period, the specific activity of esterified cholesterol was 1.6 times greater that of free cholesterol (EC SA = 1550 ± 41; FC SA = 945 ± 18 cpm/µg cholesterol). Because esterified cholesterol is a product derived from free cholesterol, the higher specific activity of esterified cholesterol demonstrates that endogenously synthesized cholesterol is preferentially esterified. At the first determination after changing the medium (4 h), a small increase was seen in the specific activities of free and esterified cholesterol that can probably be attributed to continued incorporation of radiolabeled precursor into cholesterol after removal of the radiolabeled medium. Beyond 4 h, the specific activities of free and esterified cholesterol appeared to drop in parallel. However, the decrease was significant only for free cholesterol (p < 0.05), and the specific activity of esterified cholesterol remained high throughout the chase period, even though the source of radioisotope was removed. The differences seen in specific activity during chase period again suggest that there is a specific compartmentalization of the cholesterol that is part of the cholesteryl ester cycle; once cholesterol becomes part of the cycle, it tends to remain there.


Figure 6: Endogenous labeling of endogenously synthesized cholesterol: effect of a chase period. CHO 25-RA cells maintained on 10% serum were plated on 100-mm culture dishes (10 ml/plate) and allowed to grow for 24 h. The media were then changed to fresh Ham's F-12/10% FBS containing [^3H]acetate (1 µCi/ml), and the cells were incubated for 3 days. To begin the chase period, cultures were rinsed with phosphate-buffered saline, and fresh growth medium (Ham's F-12/10% FBS) was added to the plates. At each time point, cultures were harvested, and cellular lipids were extracted and saponified as described under ``Experimental Procedures.'' Specific activities are calculated to be cpm/µg cholesterol. Final SA shown for esterified cholesterol () and free cholesterol (bullet) are the mean and standard deviations of triplicate cultures. The asterisks indicate significant differences (t test) between FC SA and EC SA at p < 0.005 (***).



In the previous experiment, cells had large stores of cholesteryl ester because they had been previously maintained on 10% FBS. It was possible that such cells might not exhibit equilibration of cholesterol pools within the time frame examined due to the large amount of unlabeled cholesterol initially present in the cells. Therefore, a similar experiment was conducted wherein cells maintained on delipidized serum protein for several weeks to lower their cholesteryl ester stores were exposed to [^3H]acetate in lipid-free medium. For this experiment, cells were exposed to the radiolabel for 3 days to produce significantly different specific activities in free and esterified cholesterol. The cells were then exposed to medium containing 1% BSA, and cultures were harvested at intervals over the next 24 h. As shown in Fig. 7, whereas the specific activities of both free and esterified cholesterol decreased with time, that of esterified cholesterol remained higher at all points, again supporting our conclusion that endogenously synthesized cholesterol is preferentially esterified and cholesterol derived from hydrolysis of cholesteryl ester is recaptured and recycled in the cholesteryl ester cycle. If the failure to achieve equal specific activities in free and esterified cholesterol is indeed a consequence of the elevated, unregulated synthesis of cholesterol, one would predict that inhibition of cholesterol synthesis would result in equilibration of radiolabel between the two cholesterol pools. Shown in Fig. 8are the specific activities of free and esterified cholesterol from 25-RA cells cultured in the presence of radiolabeled cholesterol, mevinolin, and mevalonate for a period of 5 days. As predicted, an equilibration of specific activities was achieved but not until 5 days of culture. The extended delay in equilibration supports our conclusion about the relative compartmentalization of cholesteryl ester and is likely due to the large store of unlabeled cholesteryl ester present at the beginning of the experiment.


Figure 7: Endogenous labeling of endogenously synthesized cholesterol in CHO 25-RA lipid-depleted cells: effect of a chase in cholesterol-free medium. CHO 25-RA cells maintained on lipid-free medium (Ham's F-12/1 mg/ml delipidized serum calf protein) were plated and treated as described in the legend to Fig. 6. Specific activities are calculated to be cpm/µg cholesterol. Final SA shown for esterified cholesterol (box) and free cholesterol (circle) are the mean and standard deviations of triplicate cultures. The asterisks indicate significant differences (t test) between FC SA and EC SA at p < 0.05 (*) and p < 0.005 (***).




Figure 8: Effect of mevinolin on the incorporation of exogenous radiolabeled cholesterol into esterified cholesterol. CHO 25-RA cells were plated at 1 times 10^5 cells/ml, 2 ml/35-mm dish in standard growth medium (Ham's F-12/10% FBS) containing 0.5 µCi/ml [1,2-^3H]cholesterol, mevinolin (20 µM), and mevalonate (0.5 mM). On each of 5 days after plating the cells, a set of cultures was analyzed for esterified lipid content as described under ``Experimental Procedures.'' Specific activities, expressed as cpm/µg cholesterol, for free (bullet) and esterified () cholesterol are the mean and standard deviations of triplicate cultures. The asterisks indicate significant differences (t test) between FC SA and EC SA at p < 0.05 (*), p < 0.01 (**), and p < 0.005 (***).



Because equilibration of radiolabeled cholesterol was not achieved in the 25-RA cell system unless cholesterol synthesis was inhibited, we examined two cell lines we have used extensively as foam cell models to determine rates of equilibration of the specific activities of free and esterified cholesterol. Cells, J774 mouse macrophages and Fu5AH rat hepatomas, were exposed to cholesterol-enriched media containing [^3H]cholesterol as described under ``Experimental Procedures'' to load the cells with cholesteryl ester. Equilibration was initiated by washing the monolayers and incubating the cells with 1% BSA for 24 h. Careful examination of the specific activities of the free and esterified cholesterol revealed statistically different values at the onset of the 24-h equilibration period (Fig. 9). The specific activities of free and esterified cholesterol move toward equilibration. However, with the exception of two analyses of J774 cells, the specific activities remain significantly distinct at every time point, suggesting that these cells also sequester cholesterol in the cholesteryl ester cycle. We conclude that the compartmentalization of cholesterol derived from exogenous and endogenous sources is a general cellular phenomenon that is exaggerated in the CHO 25-RA cells because of their lack of regulation of cholesterol synthesis and uptake.


Figure 9: Specific activity determination in loaded Fu5AH hepatoma cells and J774 macrophages after 1% BSA equilibration. Fu5AH cells and J774 mouse macrophages were plated and loaded with free cholesterol-rich lipid dispersions as described under ``Experimental Procedures.'' Equilibration was initiated after the cultures were rinsed three times with phosphate-buffered saline, and medium containing 1% BSA was added to the wells. Fu5AH FC SA (bullet), EC SA (), J774 FC SA (circle), and EC SA (box) are expressed as cpm/µg cholesterol and are the mean and standard deviations of triplicate cultures. The asterisks indicate significant differences (t test) between FC SA and EC SA within the same cell type at p < 0.05 (*), p < 0.01 (**), and p < 0.005 (***).




DISCUSSION

The formation of foam cells, a key event of the atherosclerotic process, rests on the continual presence of excess cholesterol available for storage within the cell, which is stored as cholesteryl ester in cytoplasmic droplets. Using mouse peritoneal macrophages, it has been demonstrated that this cholesteryl ester is in a dynamic state, undergoing continuous hydrolysis and re-esterification unless an extracellular acceptor of free cholesterol, such as high density lipoprotein, is present(35, 36) . In that case, there is a net efflux of free cholesterol, the only form of cholesterol that can leave the cell, that effectively diverts free cholesterol from the cholesteryl ester cycle. The morphological studies of McGookey and Anderson (37) documented that in the presence of extracellular acceptors, the size of the cholesteryl ester droplets decreases, but the number of droplets was not changed. When progesterone was used to inhibit re-esterification by ACAT in the absence of an extracellular acceptor, the free cholesterol was seen in multilamellar whorls. These experiments suggested that the free cholesterol derived from cholesteryl ester hydrolysis could move freely from the droplets to other locations in the cell and could fully equilibrate with the bulk cellular free cholesterol. The potential for clearance of cholesteryl esters from foam cells thus rests on hydrolysis and subsequent diversion of the free cholesterol away from ACAT.

In contrast to the view that free cholesterol can move freely between intracellular locations are several pieces of inferential evidence that cholesterol derived from different sources is preferentially moved to different locations. These include the studies of Xu and Tabas (11) from which they concluded that lipoprotein-derived cholesterol moves preferentially to the plasma membrane prior to becoming available for esterification by ACAT, and the conclusions of Liscum and Faust (8) that ACAT preferentially esterifies endogenously synthesized cholesterol. In contrast, the studies of Lange et al.(38) showed that only a small fraction of endogenously synthesized cholesterol was esterified, whereas the remaining was rapidly moved to the plasma membrane. To definitively demonstrate this selective trafficking has been difficult because the intact regulatory mechanisms found in normal cells minimize de novo synthesis of cholesterol and make studies tracing the fate of synthesized cholesterol very difficult. The CHO 25-RA system, which fails to down-regulate cholesterol biosynthesis or uptake of exogenous cholesterol, has proved to be an informative model in which to demonstrate the validity of the assumptions drawn from other systems. These cells have large stores of unlabeled cholesteryl ester that turn over rapidly (t = 7.5 h). If there were no selective handling of cholesterol within the cell, provision of exogenous radiolabeled free cholesterol would lead very quickly to the uniform labeling of both free and esterified cholesterol pools. As demonstrated here, this is not the case. As seen in Fig. 5, the specific activity of the free cholesterol remains far greater than the specific activity of the esterified cholesterol, even when the radiolabeled precursor ([^3H]cholesterol) is removed from the medium. These data support the idea that cholesteryl ester is derived largely from de novo synthesis. To demonstrate this, the converse experiments, using radiolabeled acetate to track endogenous cholesterol, were done. Again, there were large differences in the specific activities of the free and esterified cholesterol. However, with endogenous labeling the specific activity of the esterified cholesterol exceeded that of the free cholesterol, as would be expected if the cell preferentially esterifies endogenously synthesized cholesterol. An interesting result is the maintenance of the higher SA found in the esterified cholesterol pool, which was seen when the radiolabeled precursor was removed from the experimental system. These data imply a recycling and recapturing of the free cholesterol generated during hydrolysis in the cholesteryl ester cycle, suggesting that there is a physical compartmentalization of cholesterol that is part of the cycle from the other pools of free cholesterol within the cell.

The results presented here demonstrate that in CHO 25-RA foam cells, there is a specific compartmentalization of cholesterol within the cholesteryl ester cycle. This conclusion is based on the observation that equilibration of the specific activity of cholesterol in free and esterified cholesterol is not attained regardless of whether the radiolabeled precursor is exogenous cholesterol or is synthesized from radiolabeled acetate. In addition, the differences in specific activity are maintained after the radiolabeled precursors are removed. Fig. 10summarizes our findings in light of current thinking about intracellular cholesterol trafficking. There are two distinct pathways of cholesterol transport. First, endogenous cholesterol synthesized at the ER is preferentially used as a substrate for ACAT and stored in cholesteryl ester droplets, a reasonable hypothesis because enzymes of cholesterol synthesis and ACAT both appear to reside in ER membranes(13, 14) . In our experiments tracing the fate of endogenously synthesized cholesterol, a higher specific activity was consistently seen in the product (esterified cholesterol) rather than in the precursor (free cholesterol), implying that the cells were preferentially esterifying endogenously synthesized cholesterol. Our data also suggest that the cholesteryl ester cycle is isolated. The droplets turn over, generating free cholesterol, which apparently does not fully equilibrate with cellular cholesterol prior to re-esterification. Removal of the radiolabeled precursor ([^3H]acetate) did not change the distribution of radiolabel between free and esterified cholesterol. If there were complete mixing of cholesterol, i.e. that which is part of the cycle leaving and becoming part of bulk cellular cholesterol pools, the specific activities would have moved toward equilibrium. Instead, the product (esterified cholesterol) remained significantly higher. One possible explanation for this compartmentalization is the following. As the build-up of cholesterol occurs in the ER, ACAT rapidly esterifies the excess cholesterol. It has been proposed that the esterification of excess cholesterol in the membrane by ACAT may in fact be the cellular means of detoxifying the ER membrane, because abundance of cholesterol may alter protein function of membranes(39) . When ACAT forms cholesteryl esters, vesicles bud off and form droplets that contain ER membrane constituents and cholesteryl esters. Thus, a lipid droplet may contain all the enzymes of esterification and hydrolysis, explaining the dynamic nature of the cholesteryl ester cycle and compartmentalization of cholesterol seen in model foam cells. With the recent cloning of the cDNA encoding human ACAT(40) , experimental means documenting the localization of ACAT and whether the intracellular cholesteryl ester droplets contain this enzyme should be possible in the near future. A more likely possibility for the compartmentalization is that cholesterol generated by cholesteryl ester hydrolysis is selectively moved to the ER, where it again becomes a substrate for ACAT. A recent publication by Mazzone et al.(41) suggests a similar trafficking pattern.


Figure 10: Hypothesis. For an explanation for lack of equilibration of cholesterol pools see ``Discussion.''



The second major pathway (see Fig. 10) involves lipoprotein-derived cholesterol, which appears to be separately regulated within the cell, as evidenced by specific disorders of cholesterol trafficking(42, 43, 44, 45) , e.g. Niemann Pick type C disease. Cholesteryl esters in exogenous lipoprotein, taken in by receptor mediated endocytosis and degraded to free cholesterol in the lysosomal compartment(34) , appear to preferentially provide cholesterol for new membrane synthesis prior to entering the ACAT substrate pool(10, 11, 18, 46) . These two pathways, designated here as the endogenous and exogenous pathways, although distinct, are not mutually exclusive of one another. As evidenced by all studies presented herein using radiolabel tracers, as well as the work of other investigators, lipoprotein-derived cholesterol is esterified by ACAT, and endogenously synthesized cholesterol is incorporated into the plasma membrane.

Taken together, the data presented here and suggestions made by several other investigators have many implications for foam cell metabolism. First, with regard to cholesterol trafficking, the data presented confirm conclusions of other investigators. The discrepancy in specific activities of the free and esterified cholesterol, seen whether the 25-RA cells were endogenously or exogenously radiolabeled, demonstrate that endogenously synthesized or lipoprotein-derived cholesterol undergoes directed transport along different pathways. Secondly, even if synthesis is minimal in cells with intact regulatory mechanisms, these findings hold significance for the maintenance of foam cells. Unless there is a concerted movement of free cholesterol from the cholesteryl ester cycle to the plasma membrane for efflux, the cholesterol may remain isolated in the cycle, as demonstrated here.


APPENDIX

The fundamentals of first-order kinetics can be used to predict how rapidly two cholesterol pools should equilibrate. Assuming the limiting case of a closed system where the entire cellular pool of free cholesterol constitutes that which participates in the cholesteryl ester cycle, a two compartment system can be described by the following general scheme:

where A (substrate) and B (product) represent the free and esterified cholesterol pools, respectively, and k(1) and k are the rate constants describing esterification and hydrolysis, respectively. The data from the experiment shown in Fig. 3, in which esterification is inhibited with 58035, demonstrate a half-time for hydrolysis of approximately 7.5 h. Using the following equation which describes the relationship between rate constant and half-time,

the rate constant for hydrolysis (k) was determined to be 0.09 h. When a system is at equilibrium, the forward and reverse reactions are equal, and an equilibrium constant K, can be defined as follows:

For the experiment shown in Fig. 5, the esterified cholesterol mass [B] was 3.3 times greater than the mass of free cholesterol [A]. Using Equation 3, the rate constant for esterification (k(1)) is predicted to be 0.30 h. For two-pool compartment analysis, the half-time to reach equilibrium (t) depends on the sum of the two rates within the system (47) and is defined as follows:

Using the measured rate constant for hydrolysis and the predicted rate constant for esterification, the t is predicted to be 1.75 h. This would lead to essentially complete equilibration in 12 h (approximately 7 half-times).


FOOTNOTES

*
This work was supported in part by National Institutes of Health Program Project Grant 22633. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by Training Grant HL07443 from the National Institutes of Health and a pre-doctoral fellowship from the American Heart Association, Southeastern Pennsylvania Affiliate. This work was performed in partial fulfillment of the requirements for the degree of Doctor of Philosophy from the Medical College of Pennsylvania.

To whom correspondence should be addressed: Inst. for Human Gene Therapy, University of Pennsylvania Medical Center, 3400 Spruce St., Maloney 601, Philadelphia, PA 19104-4283. Tel.: 215-662-6245; Fax: 215-349-8623.

(^1)
The abbreviations used are: CHO, Chinese hamster ovary; ACAT, acyl CoA:cholesterol acyltransferase; BSA, bovine serum albumin; EC, esterified cholesterol; ER, endoplasmic reticulum; FBS, fetal bovine serum; FC, free cholesterol; LDL, low density lipoprotein; SA, specific activity.


ACKNOWLEDGEMENTS

We gratefully acknowledge the advice and support of Drs. George H. Rothblat and Diane W. Morel.


REFERENCES

  1. Hussain, M. M., Glick, J. M., and Rothblat, G. H. (1992) Curr. Opin. Lipidol. 3, 173-178
  2. Leonard, S., and Sinensky, M. (1988) Biochim. Biophys. Acta 947, 101-112 [Medline] [Order article via Infotrieve]
  3. Gottesman, M. M. (1987) Methods Enzymol. 151, 3-8, 113-121 [Medline] [Order article via Infotrieve]
  4. Chang, T. Y., and Limanek, J. S. (1980) J. Biol. Chem. 255, 7787-7795 [Free Full Text]
  5. Chang, T. Y., and Chang, C. C. Y. (1982) Biochemistry 21, 5316-5323 [Medline] [Order article via Infotrieve]
  6. Yang, J., Sato, R., Goldstein, J. L., and Brown, M. S. (1994) Genes & Dev. 8, 1910-1919
  7. Liscum, L., and Dahl, N. K. (1992) J. Lipid Res. 33, 1239-1254 [Abstract]
  8. Liscum, L., and Faust, J. R. (1994) Curr. Opin. Lipidol. 5, 221-226 [Medline] [Order article via Infotrieve]
  9. Reinhart, M. P. (1990) Experientia 46, 569-611 [Medline] [Order article via Infotrieve]
  10. Brown, M. S., and Goldstein, J. L. (1986) Science 232, 34-47 [Medline] [Order article via Infotrieve]
  11. Xu, X.-X., and Tabas, I. (1991) J. Biol. Chem. 266, 17040-17048 [Abstract/Free Full Text]
  12. Lange, Y. (1994) J. Biol. Chem. 269, 3411-3414 [Abstract/Free Full Text]
  13. Chesterton, C. J. (1968) J. Biol. Chem. 243, 1147-1151 [Abstract/Free Full Text]
  14. Reinhart, M. P., Billheimer, J. T., Faust, J. R., and Gaylor, J. L. (1987) J. Biol. Chem. 262, 9649-9655 [Abstract/Free Full Text]
  15. Lange, Y., and Ramos, B. V. (1983) J. Biol. Chem. 258, 15130-15134 [Abstract/Free Full Text]
  16. Hatch, F. T., and Lees, R. S. (1968) Adv. Lipid Res. 6, 1-68 [Medline] [Order article via Infotrieve]
  17. Basu, S. K., Goldstein, J. L., Anderson, R. G. W., and Brown, M. S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3178-3182 [Abstract]
  18. Johnson, W. J., Chacko, G. K., Phillips, M. C., and Rothblat, G. H. (1990) J. Biol. Chem. 265, 5546-5553 [Abstract/Free Full Text]
  19. Yancey, P. G., and St. Clair, R. W. (1992) Arterioscler. Thromb. 12, 1291-1304 [Abstract]
  20. Sokoloff, L., and Rothblat, G. H. (1974) Proc. Soc. Exp. Biol. Med. 146, 1166-1172
  21. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 [Free Full Text]
  22. Markwell, M. A. K., Haas, S. M., Bieber, L. L., and Tolbert, N. E. (1978) Anal. Biochem. 87, 206-210 [Medline] [Order article via Infotrieve]
  23. Arbogast, L. Y., Rothblat, G. H., Leslie, M. H., and Cooper, R. A. (1976) Proc. Natl. Acad. Sci. U. S. A. 7, 3680-3684
  24. Ham, R. G. (1965) Proc. Natl. Acad. Sci. U. S. A. 53, 288-293 [Medline] [Order article via Infotrieve]
  25. Klansek, J. J., Yancey, P., St. Clair, R. W., Fischer, R. T., Johnson, W. J., and Glick, J. M. (1995) J. Lipid Res. 36, 2261-2266 [Abstract]
  26. Kilsdonk, E. P. C., Morel, D. W., Johnson, W. J., and Rothblat, G. H. (1995) J. Lipid Res. 36, 505-516 [Abstract]
  27. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. 37, 911-917
  28. Folch, J., Lees, M., and Sloane-Stanley, G. H. (1957) J. Biol. Chem. 226, 497-509 [Free Full Text]
  29. Bamberger, M., Lund-Katz, S., Phillips, M. C., and Rothblat, G. H. (1985) Biochemistry 24, 3693-3701 [Medline] [Order article via Infotrieve]
  30. Ross, A. C., Go, K. J., Heider, J. G., and Rothblat, G. H. (1984) J. Biol. Chem. 259, 815-819 [Abstract/Free Full Text]
  31. Glick, J. M., Adelman, S. J., and Rothblat, G. H. (1987) Atherosclerosis 64, 223-230 [Medline] [Order article via Infotrieve]
  32. Brown, M. S., Goldstein, J. L., Krieger, M., Ho, Y. K., and Anderson, R. G. W. (1979) J. Cell Biol. 82, 597-613 [Abstract]
  33. Hosie, L., Sutton, L. D., and Quinn, D. M. (1987) J. Biol. Chem. 262, 260-264 [Abstract/Free Full Text]
  34. Harrison, E. H., Bernard, D. W., Scholm, P., Quinn, D. M., Rothblat, G. H., and Glick, J. M. (1990) J. Lipid Res. 31, 2187-2193 [Abstract]
  35. Brown, M. S., Dana, S. E., and Goldstein, J. L. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 2925-2929 [Abstract]
  36. Brown, M. S., Ho, Y. K., and Goldstein, J. L. (1980) J. Biol. Chem. 255, 9344-9352 [Free Full Text]
  37. McGookey, D. J., and Anderson, R. G. W. (1985) J. Cell Biol. 97, 1156-1168 [Abstract]
  38. Lange, Y., Strebel, F., and Steck, T. L. (1993) J. Biol. Chem. 268, 13838-13843 [Abstract/Free Full Text]
  39. Spector, A. A., and Yorek, M. A. (1985) J. Lipid Res. 26, 1015-1035 [Abstract]
  40. Chang, C. C. Y., Huh, H. Y., Cadigan, K. M., and Chang, T. Y. (1994) J. Biol. Chem. 268, 20747-20755 [Abstract/Free Full Text]
  41. Mazzone, T., Krishna, M., and Lange, Y. (1995) J. Lipid Res. 36, 544-551 [Abstract]
  42. Pentchev, P. G., Comly, M. E., Kruth, H. S., Vanier, M. T., Wenger, D. A., Patel, S., and Brady, R. O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8247-8251 [Abstract]
  43. Pentchev, P. G., Kruth, H. S., Comly, M. E., Butler, J. D., Vanier, M. T., Wenger, D. A., and Patel, S. (1986) J. Biol. Chem. 261, 16775-16780 [Abstract/Free Full Text]
  44. Pentchev, P. G., Comly, M. E., Kruth, H. S., Tokoro, T., Butler, J., Solkol, J., Filling-Katz, M., Quirk, J. M., Marshall, D. C., Patel, S., Vanier, M. T., and Brady, R. O. (1987) FASEB J. 1, 40-45 [Abstract/Free Full Text]
  45. Liscum, L., Ruggiero, R. M., and Faust, J. R. (1989) J. Cell Biol. 108, 1625-1636 [Abstract]
  46. Tabas, I., Weiland, D. A., and Tall, A. R. (1986) J. Biol. Chem. 261, 3147-3155 [Abstract/Free Full Text]
  47. Shipley, R. A., and Clark, R. E. (1972) Tracer Methods for in Vivo Kinetics , pp. 1-42, Academic Press, New York

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