© 1997 American Heart Association, Inc.
Articles |
Glucocorticoids Stimulate Cholesteryl Ester Formation in Human Smooth Muscle Cells
From the Department of Biochemistry, Laboratory of Risk Factors Biochemistry, National Research Center for Preventive Medicine, Moscow (I.E.P., G.V.K., Y.A.S.), and Unite de Recherches de Cardiologie, U-8 INSERM, Pessac, France (D.D., J.L.).
Correspondence to Igor E. Petrichenko, Laboratory of Risk Factors Biochemistry, National Research Center for Preventive Medicine, 10, Petroverigsky str, Moscow 101953, Russia.
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Abstract The aim of the present study was to investigate the effect of synthetic glucocorticoid dexamethasone (Dex) on cholesterol esterification in cultured human smooth muscle cells (SMC). In labeled SMC, Dex stimulated the esterification of [3H]cholesterol in a dose-dependent manner. This effect was specific for glucocorticoid hormones and could be inhibited by cycloheximide (3 ng/mL), actinomycin D (10-5 mol/L), and the specific glucocorticoid antagonist RU 486 (10-8 mol/L). When plasma membrane was selectively labeled with trace quantities of [3H]cholesterol (0.25 µCi/mL, 1 hour, 10°C), Dex (10-8 mol/L) caused a net flux of free [3H]cholesterol into the cells. Moreover, Dex (10-8 mol/L, 24 hours) stimulated the esterification of sterols, newly synthesized from [14C]mevalonate (10 µCi/mL, 4 hours) and lowered the amount of [14C]sterols susceptible for cholesterol oxidase. The incorporation of [14C]oleic acid into cholesteryl esters was markedly higher in Dex-pretreated SMC than in the control cells (2.1±0.07 and 1.4±0.1 pmol/h/µg protein, respectively, P<.01). At the time, cholesteryl ester hydrolysis in Dex-treated cells was reduced (72±8 pmol cholesteryl esters/h per milligram versus 130±10 in the control cells). HDL3-mediated [3H]cholesterol efflux was also inhibited in Dex-treated cells; moreover, HDL3 (40 µg/mL, 24 hours) had practically no effect on [3H]cholesteryl ester content in Dex-treated SMC but caused a 50% reduction of [3H]cholesteryl esters in the control cells. Thus, in human SMC glucocorticoids alter the redistribution of cholesterol between the pools of free and esterified cholesterol, paralleled by the change in acyl coenzyme A:cholesteryl acyltransferase and neutral cholesteryl ester hydrolase activities, leading to the impaired HDL3-mediated cholesterol efflux.
Key Words: smooth muscle cells • glucocorticoids • cholesteryl esters • HDL3 • cholesterol efflux
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Introduction |
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Glucocorticoids have been proposed as a factor involved in lipid metabolism and body fat distribution.1 It is well known that glucocorticoid treatment is associated with an accumulation of abdominal fat and metabolic and physiological complications such as insulin resistance, arterial hypertension, and hyperlipidemia. Furthermore, retrospective studies including pathomorphological data obtained at autopsy suggested that in humans, glucocorticoids adversely affected atherogenesis.2
Glucocorticoids stimulate hepatic VLDL secretion, decrease the binding and degradation of native LDL by rat hepatocytes, human macrophages, SMC, and fibroblasts and, at the same time, increase the binding of acetylated LDL to macro-phages.3 4 Glucocorticoids are well-known inhibitors of protein and cholesterol synthesis in various cell types and target tissues, including SMC and macrophages.5 All these effects are thought to be mediated through specific glucocorticoid receptors and required synthesis of regulatory proteins.5 On the other hand, despite the long-term clinical observations and abundant experimental data, the mechanism of atherogenic action of glucocorticoids in humans remains to be elucidated.
The characteristic feature of the atherosclerotic lesion is the presence of cells (SMC, macrophages) heavily loaded with cytoplasmic droplets of cholesteryl esters, which are not inert but undergo continual hydrolysis by a cytoplasmic enzyme, NCEH, and reesterification by ACAT.6 Among the factors regulating the cholesteryl ester cycle, the inhibition of cholesterol7 or protein8 synthesis has been shown to cause a change in intracellular cholesterol trafficking and burst of cholesterol esterification in cultured cells. Moreover, a coordinate, reciprocal regulation of HMG CoA reductase, the rate-controlling enzyme in cholesterol biosynthesis, and ACAT through a phosphorylation/dephosphorylation mechanism has been proposed.9 Although glucocorticoids markedly inhibit cholesterol synthesis in extrahepatic cells, not much is known about their effect on the processes involved in intracellular cholesterol trafficking and esterification.
The purpose of the present study was to determine whether intracellular cholesterol trafficking and esterification in human SMC can be modulated by glucocorticoids. We have demonstrated that glucocorticoids, apart from their well-known inhibitory action on sterol synthesis in peripheral cells, may specifically, through receptor-mediated mechanisms, regulate the activity of ACAT and NCEH, stimulate the esterification of cholesterol, and decrease the HDL3-mediated cholesterol efflux from human SMC.
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Materials
All steroid hormones were research grade and purchased from Serva. Fatty acid–free bovine serum albumin, cycloheximide, and actinomycin D were from Sigma. Cholesterol oxidase (Streptomyces) was from Calbiochem. [7(n)-3H]cholesterol (50 Ci/mmol), [1-14C]oleic acid (52 mCi/mmol), [1,2(n)-3H]cholesteryl oleate (50 Ci/mmol), [2-14C]mevalonic acid (DBED salt, 70 mCi/mmol), and [2-14C]acetic acid (sodium salt, 54 mCi/mmol) were purchased from Amersham. Fetal calf serum, Ham's F-10 medium, penicillin, and streptomycin were from Flow. All other chemicals were research grade and were obtained from Merck.
Cell Culture
SMC were obtained by dissociation of the tissue from human abdominal aorta (26-year-old man, autopsy material) with 0.05% collagenase and 0.1% trypsin in Ham's F-10 medium.10 During the first week, cells were cultured at 37°C, 5% CO2, 80% humidity in Ham's F-10 medium supplemented with 20% fetal calf serum. When a monolayer was reached, the cells were removed by trypsin-EDTA (0.05% to 0.02%) and cultured in 25-cm2 flasks in Ham's F-10 medium, supplemented with 10% fetal calf serum, 1% penicillin-streptomycin at 37°C in 5% CO2 in air. Cultured SMC grew in atypical "hill and valley" formation. For experiments, cells were seeded in 24-well plates (at about 20 000 cells/well) or 25-cm2 flasks (300 000 cells/flask) and were grown to confluence (7 days). All experiments were performed on cells between passages 9 and 11.
Lipoproteins
Lipoproteins were isolated by standard preparative ultracentrifugation techniques from the pooled plasma of healthy male volunteers according to density as follows: LDL, d=1.019 to 1.063 g/L; HDL3, d=1.125 to 1.21 g/L and stored under N2 in the dark at 4°C until used within 2 weeks of preparation. Before being added to the cell culture, lipoprotein was dialyzed extensively against 0.15 mol/L NaCl, 1 mmol/L EDTA and filtered through a sterile Millipore filter (0.45 µm). HDL was iodinated by the monochloride procedure.11 Specific activity ranged from 300 to 500 cpm/ng protein.
Cell Labeling With [3H]Cholesterol
Cells in 24-well plates were incubated for 48 hours in growing medium with 10% fetal calf serum containing 1 µCi/mL [3H]cholesterol. During the first 24 hours of incubation, 45% to 55% of label was incorporated into the cells and this value did not increase significantly when incubation was continued for an additional 24 hours. At the end of the labeling period, cells were washed four times with PBS (pH 7.4) and incubated for 3 hours in the Ham's F-10 medium without serum.12
To label the cell plasma membrane with [3H]cholesterol, confluent SMC growing in 25-cm2 flasks were washed four times with PBS and incubated for 1 hour at 10°C in the medium without serum containing 0.25 µCi/mL [3H]cholesterol added in ethanol.13 At the end of incubation, cells were chilled on ice, washed four times with ice-cold PBS, and used in experiments.
To label newly synthesized sterols, human SMC were incubated with [14C]-mevalonate (10 µCi/mL, 0.4 mmol/L) for 4 hours at 37°C. With this labeling procedure the rate of translocation of newly synthesized sterols to the plasma membrane is rapid, with a half-time of 
1 hour.14
Neutral lipid synthesis was also measured by the incorporation of Na-[14C]acetate (1 µCi/mL) into cellular sterols, triglycerides, and phospholipids during a 2-hour incubation at 37°C.
Treatment of Cells With Cholesterol Oxidase
Human SMC were washed four times with ice-cold PBS followed by addition of 2 mL of 1% glutaraldehyde in PBS, containing 350 mmol/L sucrose (10 minutes, 0°C). After fixation the cells were washed four times, transferred to a 37°C water bath, and incubated for 30 minutes with 2 U of cholesterol oxidase (CO) in PBS, 350 mmol/L sucrose. Then, cells were chilled on ice, washed with PBS, and analyzed for radioactive sterols. The ratio of label cholesterol and cholestenone was determined from the neutral lipid extract. Cellular labeled cholesterol resistant to conversion to cholestenone by CO was assumed to reside in an intracellular pool.15 Results are expressed as counts per minute (cpm) per well because after glutaraldehyde fixation it was not possible to determine protein concentration.
Whole-Cell ACAT Assay
The relative activity of ACAT was assayed by pulse-labeling the cells with [14C]oleate-albumin complex as described.14 Briefly, 1 hour before the end of experiments [14C]oleate (10 µCi/mL, 0.2 mmol/L) in the presence of albumin (0.07 mmol/L) was added directly into the incubation medium. Cells were washed 4 times with ice-cold PBS, and incorporation of label into cellular neutral lipids was studied.
Assay of Acid and Neutral Cholesteryl Ester Hydrolase
The activity of ACEH and NCEH was determined by the release of [3H]cholesterol from [3H]cholesteryl oleate at the different pH values.16 17 Cells cultured in 25-cm2 flasks were washed 4 times with PBS and scraped into 10 mmol/L Tris-HCl buffer, pH 7.4, containing 250 mmol/L sucrose and 0.1 mmol/L EDTA. Homogenate was prepared by sonication of the cells with a Branson sonifier three times for 8 seconds in ice-water at an intensity of 30 W. Substrate for NCEH contained 85 mmol/L phosphate buffer (pH 7.0), 12.5 mmol/L Na-taurocholate, 6 µmol/L [3H]cholesteryl oleate (specific activity, 150 mCi/mmol), and 0.04% albumin. Substrate for ACEH was 50 mmol/L Na-acetate buffer (pH 3.9), 2 mmol/L Na-taurocholate, 13 µmol/L [3H]cholesteryl oleate (specific activity 75 mCi/mmol), 1.3 mmol/L lecithin, and 0.005% digitonin. The reaction was initiated by the addition of 100 µL of cell homogenate (60 µg protein) to 100 µL substrate solution. The incubation was continued for 1 hour at 37°C and stopped by cooling the tubes in ice water. Substrate blanks were run under identical conditions with sucrose buffer in place of enzyme. Neutral lipids were extracted with 3 mL of chloroform/methanol (1:1 vol:vol), followed by the addition of 0.1 mL of 1 mol/L NaOH. The mixture was then agitated and centrifuged to separate the phase. The chloroform phase was collected, and neutral lipids were separated by TLC.
Binding of [125I]HDL3 to Cultured SMC
Cells were washed and incubated in the medium supplemented with albumin (1 mg/mL) and various concentrations of [125I]HDL3 (from 1 to 10 µg/mL) in the presence or absence of a 100-fold excess of unlabeled HDL3 for 1 hour, at 37°C. Then cells were chilled on ice, washed, incubated twice for 10 minutes in the medium containing albumin, and finally washed in the medium without albumin. Cells were digested in 0.1N NaOH and aliquots were assayed for radioactivity and protein content.14
Assay Procedures
Washed cell monolayers were extracted with hexane/isopropanol (3:2 vol:vol) for 1 hour at room temperature.15 Labeled neutral lipids from cell extract were isolated on normal phase TLC (Whatman silica gel G plates) in hexane/diethyl ether/acetic acid (130:30:1.5 vol:vol:vol); the radioactivity of labeled neutral lipids was either scanned and quantified directly on the plates with a Berthold Automatic TLC Linear Analyzer LB 2832 or appropriate spots were detected by I2 staining, scraped into scintillation vials, and counted in a Mark III liquid scintillation counter. Spots for labeled lipids were identified from standards run in parallel.
To determine sterol mass, neutral lipids were extracted with hexane/isopropanol (3:2 vol:vol) for 1 hour at room temperature and separated on normal-phase TLC in hexane/diethyl ether/acetic acid (130:30:1.5 vol:vol:vol); free and esterified cholesterol spots were scraped, extracted, and assayed by the cholesterol oxidase procedure.14
Cell proteins after lipid extraction were digested into 1 mL of 0.1 mol/L NaOH (1 hour, 37°C), and the protein concentration in an aliquot of the hydrolysate was determined according to Lowry using bovine serum albumin as the standard.18
Statistical Analysis
All data are expressed as the mean of three or more experiments done in triplicate±SEM. The coefficient of variation was less than 10% for each experiment. Statistical significance for the experimental data and their respective controls was evaluated by Student's t test.
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Effect of Glucocorticoids on [3H]Cholesterol Redistribution in SMC Pools
In our experiments treatment of human SMC with a synthetic analogue of glucocorticoids, Dex (10-8 mol/L, 24 hours), inhibited the incorporation of Na-[14C]acetate into total lipids, phospholipids, C27-sterols, and triglycerides, reflecting the general inhibitory effect of glucocorticoids on sterol synthesis in these cells (Table 1
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However, concurrent with a decrease of sterol synthesis, glucocorticoid treatment significantly stimulated the esterification of exogenously derived cholesterol. In human SMC incubated for 48 hours in growth medium containing 10% fetal calf serum and [3H]cholesterol (1 µCi/mL), <8% (44.7±1.3 cpm/µg) of total cellular [3H]cholesterol was esterified at the end of the incubation. Treatment of cells with Dex during the last 24 hours of incubation stimulated the formation of [3H]cholesteryl esters in a dose-dependent manner, with the total amount of label incorporated being unaffected by hormone (Fig 1A). This effect was specific for glucocorticoids: progesterone, estradiol, testosterone, and aldosterone at a concentration of 10-8 mol/L had no effect on [3H]cholesterol esterification in cultured SMC (Fig 1B). Among glucocorticoids their ability to stimulate the formation of [3H]cholesteryl esters decreased in the order: triamcinolone acetonide (TA)
Dex>cortisol (F) (Fig 1C). Although the addition of glucocorticoids caused a change in cell morphology, with the cells appearing larger and flatter, a general toxic effect of glucocorticoids did not appear to account for these observations, since Dex up to the concentration of 10-7 mol/L, during 24 hours of incubation had no significant effect on the total protein content of the cultured cells (105±3 µg/well versus 110±4 µg/well in the control). Trypan blue dye exclusion analysis of human SMC treated with Dex (10-7 mol/L) for 24 hours showed >95% viability, similar to that of the control cells.
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Given the fact that Dex-induced cholesterol esterification was specific for glucocorticoids and could be done with relatively low concentrations (10-8 mol/L) of hormone, this effect was presumed to be a classic steroid receptor-mediated event, which involves the binding of activated hormone-receptor complexes with DNA, expression of certain gene(s), and modulation of RNA and regulatory protein synthesis. Indeed, washout of Dex from the incubation medium did not diminish the glucocorticoid-induced cholesterol esterification. In our experiments, SMC treated with Dex for 24 hours were carefully washed, a new portion of Ham's F-10 medium supplemented with 1% albumin was added, and the amount of [3H]cholesteryl esters 24 hours after hormone removal was measured. While the incubation of human SMC in the medium without fetal calf serum stimulates by itself the [3H]cholesterol esterification (12.1±0.9% of total cellular [3H]sterols, 67.2±3.7 cpm/µg), Dex-treated cells retained twice as much [3H]cholesteryl ester (21.8±1.8%, 123±7 cpm/µg) as the control cells. After 24 hours of incubation, the amount of [3H]cholesterol that appeared in the medium supplemented with 1% albumin was fairly high in the control cells (3.2±0.4% of total label, 18.3±0.2 cpm/µg), and two times as low as in Dex-treated cells (1.4±0.1%, 8.1±0.1 cpm/µg). Moreover, the addition of cycloheximide (3 ng/mL), actinomycin D (10-5 mol/L) and glucocorticoid antagonist RU 486 (10-8 mol/L) completely inhibited Dex-induced [3H]cholesteryl ester formation (Fig 2). At these concentrations, cycloheximide and actinomycin D by themselves had no effect on cholesterol esterification and did not change viability of cultured SMC compared with the control cells (>95%).
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Effect of Dexamethasone on Cholesterol Mass in Cultured Smooth Muscle Cells
To further characterize the effect of Dex on cholesterol redistribution in human SMC, free and esterified cholesterol mass in control and Dex-treated cells was determined. When not loaded with exogenous cholesterol, SMC were treated with 10-8 mol/L Dex for 24 hours, and there was a slight shift between intracellular cholesterol pools toward an enlargement of esterified cholesterol, without any changes in the total cholesterol mass (Table 2, A). The same, but more pronounced, tendency was observed when SMC were loaded with LDL cholesterol prior to Dex treatment (Table 2, B).
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Dexamethasone-Mediated Translocation of Cholesterol Between Cellular Pools
The cholesterol in the plasma membrane pool(s) is thought to be a preferred substrate for the ACAT-catalyzed reaction. To verify if Dex treatment could lead to a net flux of the plasma membrane–located cholesterol into the cells, plasma membrane was selectively labeled with a trace amount of [3H]cholesterol; then cells were washed and incubated in the medium supplemented with Dex. When control cells were placed at 37°C there was a time-dependent decrease in the [3H]cholesterol fraction susceptible to cholesterol oxidase (CO) treatment, concomitant with a progressive increase of label in the CO-resistant pool. The addition of Dex caused, however, a more pronounced shift of [3H]cholesterol from a CO-susceptible to the CO-resistant pool, with the hormone effect became detectable by 4 hours of incubation (Fig 3). The amount of label that appeared in the medium was negligible in both the control and Dex-treated cells.
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, Control cells, 
, Dex-treated SMC.It is known that in the cells not loaded with cholesterol, newly synthesized sterols rapidly transfer to the plasma membrane.14 To enrich the plasma membrane with label sterols and bypass the inhibitory action of glucocorticoids on sterol synthesis, human SMC were pulse labeled with the biosynthetic precursor of cholesterol, [14C]mevalonate. When confluent SMC were grown in the standard culture medium and pulsed with 10 µCi/mL [14C]mevalonate for 4 hours at 37°C, most of the biosynthetically labeled C27-sterols (95%) appeared in CO-susceptible pools, with only about 4% of [14C]sterols being esterified (1237±41 and 48±1 cpm/well, respectively). Pretreatment of the cells with Dex (10-8 mol/L) for 24 hours led to a decrease in the relative proportion of unesterified [14C]sterols (which were practically all oxidized by CO and comigrated with cholestenone) and to a fivefold increase in the esterification of newly synthesized C27-sterols (910±17 and 290±12 cpm/well, respectively; P<.001 compared with the control cells).
Effect of Dexamethasone on ACAT and NCEH Activities
A tight relationship is thought to exist between the rate of cholesterol biosynthesis and activities of the enzymes that regulate the intracellular cholesteryl ester cycle, ACAT and NCEH.9 To verify if glucocorticoid treatment can alter the relative activity of ACAT, human SMC were incubated with 10-8 mol/L Dex for 24 hours and pulsed with [14C]oleate-albumin complex during the last hour of incubation. Dex markedly stimulated the formation of cholesteryl-[14C]oleate (2.1±0.07 versus 1.4±0.1 pmol/h/µg protein in the control cells, P<.01) that seems to reflect the upregulation of the apparent ACAT activity.
However, glucocorticoids not only stimulate the incorporation of label oleate into cholesteryl ester but also modify the redistribution of fatty acids between phospholipids and neutral lipids (Table 3). Although the total incorporation of [14C]oleate was reduced in Dex-treated cells, there was a threefold increase in [14C]triglycerides, accompanied by a significant (up to 50%) decrease of label incorporated into cellular phospholipids. Thus, the stimulation of apparent ACAT activity by Dex may be just a part of the combined action of glucocorticoids on fatty acid turnover in human SMC.
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On the other hand, glucocorticoids seem to strongly inhibit the hydrolytic part of cholesteryl ester cycle. Both neutral and acid cholesteryl ester hydrolase activities were decreased in the homogenates from Dex-treated SMC (Table 4), with a more notable effect on ACEH activity.
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When Dex (10-8 mol/L, 24 hours) was removed and cells were further incubated in the medium without serum (plus 1 mg/mL albumin) for an additional 24 hours, the hydrolytic activities (both NCEH and ACEH) in Dex-pretreated cells stilled half as much as in the control SMC (data not shown).
Effect of Dexamethasone on HDL3-Mediated Cholesterol Efflux
Inasmuch as glucocorticoids stimulated the translocation of plasma membrane–located cholesterol into the cells and promoted the cholesterol esterification, it was likely that HDL3-mediated [3H]cholesterol efflux could be impaired in Dex-treated cells. The efflux of [3H]cholesterol to HDL3 in cultured human SMC was relatively weak, as consistent with the data19 showing the slow rate of cholesterol desorption from the plasma membrane of SMC. Pretreatment of SMC with Dex (10-8 mol/L) during 24 hours decreased the rate of appearance of [3H]cholesterol in the medium either in the presence or absence of HDL3 (Fig 4) and even high concentrations of HDL3 (up to 200 µg/mL) was unable to overcome this inhibitory action. When control SMC were exposed to HDL3 (40 µg/mL, 24 hours) both free and esterified [3H]cholesterol content were reduced, in contrast to Dex-treated cells, where the pools of [3H]cholesterol were significantly less altered (Table 5). In alternative experiments cultured SMC were labeled with [14C]mevalonate (10 µCi/mL) for 6 hours at 37°C, chased for 18 hours with serum-free medium at 37°C, and treated with Dex (10-8 mol/L, 24 hours). HDL3-mediated [14C]sterol efflux (40 µg/mL, 24 hours) was also reduced in Dex-treated cells in comparison with the control cells (6.1±0.2 versus 10.1±0.1% of cellular [14C]sterols, n=3, P<.001). It should be mentioned that Dex treatment did not change the parameters of HDL3 binding to cultured SMC (Table 6), suggesting that the effect of Dex can be ascribed to alteration in intracellular cholesterol trafficking rather then specific glucocorticoid action on the HDL receptor.
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Thus, in cultured human SMC, glucocorticoids dramatically alter the redistribution of cholesterol between the pools of free and esterified cholesterol, paralleled by the change in ACAT and NCEH activities, leading to the impaired HDL3-mediated cholesterol efflux.
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Discussion |
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The mechanisms of glucocorticoid action on atherogenesis are poorly understood. When applied as anti-inflammatory drugs at high pharmacological doses, glucocorticoids seem to suppress the development of atherosclerosis in experimental animals, despite enhancement of hypertriglyceridemia and hypercholesterolemia.20 21 Glucocorticoids were shown to inhibit leukocyte accumulation in the rabbit carotid artery and intimal thickening induced by cuff sheathing22 and suppress the development of atherosclerosis in the aorta of rabbits by inhibiting recruitment and proliferation of macrophages and formation of foam cells in plaques.23 On the other hand glucocorticoids are able to decrease the expression of hepatic LDL receptors,24 stimulate the net synthesis of apoB-100 and apoB-48 and also decrease their intracellular degradation.25 These changes are potentially atherogenic, and the involvement of glucocorticoids in atherogenesis is supported by the strong correlation between increased serum cortisol in human beings and the extent of coronary artery disease.2 26 27 28 29 Data presented in this article indicate that intracellular cholesterol movement in human SMC is also under crucial glucocorticoid control, manifested as an increase in the flux of plasma membrane–located cholesterol into cells, promotion of cholesterol esterification (both [3H]cholesterol and cholesteryl mass in LDL-loaded SMC), and reduction of HDL3-mediated cholesterol efflux.
It is well established that glucocorticoids exert most of their effects through the binding with high-affinity cytoplasmic receptors. Glucocorticoid receptors have been reported in aortic SMC30 31 and, earlier, we have presented the characteristics of high-affinity binding of Dex in cultured human and rat SMC.10 Apparently, the ability of glucocorticoids to modulate the redistribution of cholesterol and stimulate cholesteryl ester formation in SMC represents a classic receptor-mediated event. Indeed, glucocorticoid-induced cholesterol esterification was blocked by actinomycin D, cycloheximide, and glucocorticoid antagonist RU 486; it was specific for glucocorticoids and the magnitude of glucocorticoid action ranged according to their binding affinity with glucocorticoid receptor (triamcinolone acetonidedexamethasone>cortisol). The effect could be reached at low concentrations of Dex and did not fade instantly under Dex removal.
Several groups of steroids were previously described that modulate cholesterol movement in cultured cells7 32 : cholesterol, 25-hydroxycholesterol, 7-ketocholesterol, and 6-ketocholestanol (suppress HMG CoA reductase and raise the activity of ACAT); synthetic analogues of 7-ketocholesterol (suppress HMG CoA reductase and directly inhibit ACAT); and progesterone and 20
-dihydroprogesterone (directly inhibit the ACAT). However, all these actions clearly do not represent steroid hormone receptor–mediated events. In fact, the inhibition of cholesterol synthesis by 25-hydroxycholesterol was no longer visible at 10-7 mol/L, increasing abruptly with increasing concentrations of drug up to 10-5 mol/L; did not appear to require RNA synthesis; and could not be blocked by cycloheximide or actinomycin D.33 34 Furthermore, those steroids that inhibit ACAT activity were shown to have a direct effect on the enzyme in a cell-free extract.7 The progesterone effect was detectable at a very high concentration (10-5 mol/L) and readily reversible through steroid washout.32
In our experiments, Dex stimulated the esterification of exogenously derived [3H]cholesterol concurrent with an inhibition of sterol synthesis in SMC, as judged by the rate of [14C]acetate incorporation into neutral lipids. Glucocorticoid treatment has been reported to markedly inhibit cholesterol synthesis in various tissues (cultured human fibroblasts, HeLa cells, lymphocytes), presumably through the inhibition of both HMG CoA reductase and synthase activities.35 36 37 38 39 The rate of sterol synthesis and proliferating stage of the cells are thought to regulate the cholesterol esterification in human cells.9 SMC become quiescent with serum deprivation and stop at G0/G1 phase.40 Glucocorticoids, in their turn, notably inhibit the proliferation of SMC growing in culture.41 On the other hand, our data indicate that human SMC, labeled with [3H]cholesterol in the presence of fetal calf serum for 48 hours, start to accumulate [3H]cholesteryl esters over the initial value, having been incubated for an additional 24 hours in the medium without serum (44.7 cpm/µg of [3H]cholesteryl esters at the end of the labeling period versus 67.2±3.7 cpm/µg after additional incubation). Dex pretreatment significantly potentates this effect (123±7 cpm/µg). Additionally, in the control cells a notable portion of [3H]cholesterol (3.2±0.4% of total labeled sterols) was detectable in the medium, while Dex-treated SMC released labeled cholesterol to a considerably lesser extent (1.4±0.1%). Thus, it can be proposed that the inhibition of sterol synthesis (and possibly reduction of proliferating activity), induced by glucocorticoids, reverses the direction of cholesterol movement in human SMC and makes a net flux of cholesterol into the cells prevalent, which, in turn, enlarges the substrate pool of ACAT, leading to an increase in cholesterol esterification. This assumption can also be supported by the fact that glucocorticoids cause SMC hypertrophy42 and increased cholesteryl esters and free cholesterol deposition may be required of an enlarged cell area.
The ability of glucocorticoids to increase the flux of cholesterol within the cells can be strongly supported by the fact that glucocorticoids stimulate the translocation of cholesterol from CO-susceptible (cholestenone) to CO-resistant pools. Our results (Fig 3
) clearly demonstrate that Dex, after a 4-hour lag period, stimulates the translocation of [3H]cholesterol from the plasma membrane into human SMC. Tabas et al43 suggested that it is precisely cholesterol residing in the plasma membrane that is translocated to ACAT in the endoplasmic reticulum and preferably esterified inside the cells. In our experiments, when cellular sterols were biosynthetically labeled with [14C]mevalonate, Dex treatment decreased the amount of C27-sterols in CO-susceptible pools and stimulated the appearance of the label in the cholesteryl ester moiety. Newly synthesized sterols rapidly translocated to the plasma membrane can be oxidized by CO and comigrate with cholestenone on normal-phase chromatography.44 The above results suggest that glucocorticoids can either trap newly synthesized sterols within intracellular pools, that seem to be in rapid equilibrium with the substrate pool of ACAT44 or increase their retranslocation from the plasma membrane to the endoplasmic reticulum.
The addition of glucocorticoids to the cultured SMC notably alters the activities of the enzymes, which catalyze the cholesteryl ester cycle reactions—ACAT and NCEH. The stimulation of cholesteryl ester formation in Dex-treated SMC was accompanied by a 1.5-fold increase in the relative activity of ACAT. However, it is not clear to what extent these data can represent the true activation of ACAT, since Dex dramatically changes the redistribution of fatty acids between cellular phospholipids and neutral lipids (Table 3). The formation of [14C]triglycerides after Dex treatment was most pronounced and attended with the decrease of the label incorporated into cellular phospholipids. In certain tissues (eg, macrophages and adipose tissue), the formation of cholesteryl esters and triglycerides proceeds in a similar manner: in both cases there are continual hydrolysis and resynthesis in a series of transesterification reactions that use fatty acyl-CoA derivatives.45 The triglyceride formation has been shown to be regulated by glucocorticoids in various tissues,46 47 and though our data cannot unambiguously indicate that glucocorticoids stimulate triglyceride synthesis in human SMC, it is clear that Dex treatment increases the availability of fatty acyl-CoA derivatives to the esterification reactions and decreases the rate of fatty acid exchange between neutral lipids and phospholipids.
Unlike ACAT, the activities of NCEH and ACEH were inhibited in Dex-treated human SMC (Table 4). Previously, it has been shown that glucocorticoids can inhibit both ACAT and cholesteryl ester hydrolase activity in the outer adrenal cortex,48 the effect which was also accompanied by the reduction of HMG CoA reductase activity.49 In some tissues cholesteryl ester hydrolase activity is thought to be regulated by cAMP-mediated mechanisms. Thus, the neutral hydrolytic activity and cholesterol efflux to HDL3 are stimulated by cAMP or its analogues in the SMC.50 51 52 It was suggested that neutral hydrolytic activity in SMC can be catalyzed by a cAMP-dependent enzyme similar to hormone-sensitive lipase (HSL, EC 3.1.1.3) that catalyzes the rate-limiting step in adipose tissue triglyceride lipolysis and has the same catalytic activity toward both cholesteryl esters and triglycerides.50 53 54 Glucocorticoid hormones, in turn, not only have been shown to inhibit cAMP production in rat SMC55 but also directly regulate the HSL mRNA level in adipose tissue.56 Since glucocorticoid action not only stimulates the formation of cholesteryl esters but also increases the incorporation of fatty acids into the triglyceride fraction (Table 3), it is conceivable that in human SMC, glucocorticoids can inhibit the activity or reduce the expression of the enzyme that closely resembles HSL.
In cultured human SMC glucocorticoids significantly inhibited HDL3-mediated cholesterol efflux (Table 5 and Fig 4). The long-term labeling (48 hours) of cells with [3H]cholesterol in serum allowed to achieve uniform distribution of the label among the cholesterol compartments of SMC, and therefore the possible receptor-mediated responses of HDL were not largely involved in the study.12 57 Moreover, despite earlier published data showed that glucocorticoids can upregulate HDL-specific binding in rat hepatocytes,58 in our experiments Dex had no effect on specific binding of HDL3 to cultured SMC (Table 6). In the control SMC, HDL3 caused a notable decrease in both free cholesterol and cholesteryl ester content (Table 5), the effect that is thought to be connected to a decrease in ACAT activity; continued hydrolysis of cholesteryl esters; and cholesterol efflux to the medium.59 In contrast, HDL3 failed to clear Dex-treated SMC from cholesteryl esters and did not change significantly the amount of free cholesterol. These results indicate that glucocorticoid treatment not only inhibits the cholesteryl ester hydrolysis but catches free cholesterol inside the cells as well, making this pool insensitive to HDL-induced cholesterol translocation from the intracellular compartment to the plasma membrane.
The effect of glucocorticoids on cholesterol esterification in human SMC is not unique. Recently, dexamethasone has been reported to enhance accumulation of cholesteryl esters by human macrophages.60 Although glucocorticoid action is known to be mediated in several ways, including stimulation of lipocortin I (annexin I) synthesis,61 inhibition of cAMP production,55 or direct action on enzyme expression,5 further investigations are required to elucidate the mechanism of glucocorticoid action on cholesterol esterification.
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Acknowledgments |
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The study was performed thanks to the Russia-France Intergovernmental Agreement for Cooperation in Public Health and partly supported by a grant from INSERM, France. We gratefully acknowledge Drs N. Perova, V. Metelskaya, and A. Sigalov for helpful discussion.
Received August 31, 1996; accepted October 17, 1996.
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