Effects of 25-hydroxycholesterol on cholesterol esterification and sterol regulatory element-binding protein processing are dissociable: implications for cholesterol movement to the regulatory pool in the endoplasmic reticulum.

The regulatory pool of cholesterol is located in the endoplasmic reticulum (ER) and is key to how mammalian cells sense and respond to changes in cellular cholesterol levels. The extent of cholesterol esterification by the ER-resident protein, acyl-coenzyme A:cholesterol acyl-transferase (ACAT), has become the standard method for monitoring cholesterol transport to the ER and is assumed to reflect the regulatory pool of ER cholesterol. The oxysterol, 25-hydroxycholesterol (25HC), is thought to trigger intracellular cholesterol transport to the ER. In support of this contention, we confirmed previous reports that 25HC activates cholesterol esterification and is a potent suppressor of the sterol regulatory element-binding protein (SREBP) pathway. Processing of the ER membrane-bound SREBP into a soluble transcription factor is controlled by cholesterol levels in the ER. In this study, we addressed whether or not cholesterol esterification necessarily reflects cholesterol movement to the cholesterol homeostatic machinery in the ER as determined by SREBP processing. We found that three agents that inhibited the ability of 25HC to induce cholesterol esterification (progesterone, nigericin, and monensin) did not have a corresponding effect on 25HC suppression of SREBP processing. Moreover, ACAT inhibition did not alter the sensitivity of SREBP processing to 25HC. Therefore, cholesterol esterification by the ER-resident protein ACAT is dissociable from cholesterol transport to the cholesterol homeostatic machinery in the ER. In light of our results, we question the security of previous work that has inferred cholesterol transport to the ER regulatory pool based solely on cholesterol esterification.

SREBP processing when added to intact cells than cholesterol (15,16) and suppress cholesterol synthesis when their endogenous levels have been increased through pharmacologic or genetic manipulation (16 -18). Interestingly, these oxysterols are not sensed directly by SCAP as SCAP undergoes a conformational change in response to cholesterol but not these sidechain oxygenated sterols (7). Consistent with these observations, we proposed that these oxysterols may inhibit SREBP processing by causing intracellular cholesterol transport to the ER, where cholesterol produces a conformational change in SCAP (7). The idea that oxysterols like 25HC trigger movement of cholesterol to the ER is based on the well-documented ability of 25HC to increase cholesterol esterification (13, 19 -21).
In this study, we addressed whether or not cholesterol esterification by the ER-resident protein ACAT necessarily reflects cholesterol movement to the cholesterol homeostatic machinery in the ER as determined by SREBP processing. We employed 25HC, a commonly used model oxysterol, to elicit cholesterol transport, and a number of pharmacologic agents that inhibit 25HC-induced cholesterol esterification.
Cell Culture-Chinese hamster ovary cells K1 (CHO-K1) were grown in monolayer in a humidified incubator at 37°C with 5% CO 2 atmosphere. The cells were grown in 1:1 (v/v) Dulbecco's modified Eagle's medium:Ham's F-12 containing penicillin (100 units/ml), streptomycin (100 g/ml), and L-glutamine (2 mM), supplemented with various sera. The different media used were supplemented as follows: Medium A, 10% (v/v) NBS; Medium B, 5% NBS; Medium C, 5% lipoprotein deficient NBS; Medium D, 5% lipoprotein-deficient NBS plus 5 M compactin and 50 M mevalonate. Compactin, mevalonate, sterols, and other test agents were added in ethanol. Within an experiment, the final ethanol concentration was kept constant between conditions and did not exceed Cholesterol Esterification-The effect of different compounds on cholesterol esterification was tested using a method described by Lange and colleagues with minor modifications (10). On day 0, CHO-K1 cells were set up (in triplicate) at a density of 4 ϫ 10 5 cells/60-mm dish in medium A. On day 1, cells were switched to medium C and incubated for 16 h. On day 2, cells were pulse-labeled with [ 3 H]cholesterol complexed to 2-hydroxypropyl-␤-cyclodextrin (10 min, 4°C). After washing with a 0.5 mg/ml solution of bovine serum albumin in phosphatebuffered saline (PBS) (3 ϫ 1 ml, 4°C), cells were incubated with medium C plus test agents (4 h, 37°C). Cells were washed twice with PBS and lysed with 0.1 M NaOH (1 ml). Cell lysate was neutralized by 0.1 M HCl. Methanol was added to cell lysate, and lipids were extracted with hexane. Hexane was evaporated, and samples were redissolved in 60 l of hexane for thin layer chromatography (TLC). [  , and the indicated concentrations of 25HC in the presence or absence of the ACAT inhibitor, Sandoz 58-035 (0.5 g/ml). Cells were washed and harvested, lipids were extracted, and the extent of cholesterol esterification was determined as described above.
ACAT Enzyme Activity-On day 0, CHO-K1 cells were plated at a density of 10 6 cells/100-mm dish in medium A (7 dishes per condition). On day 1, cells were switched to medium C and incubated for 16 h. On day 2, cells were incubated in medium C in the absence and presence of progesterone (10 M) for 4 h. Cells were washed with PBS, harvested, and resuspended in 1 ml of Buffer A (10 mM HEPES-KOH at pH 7.4, 10 mM KCl, 1.5 mM MgCl 2 , 5 mM sodium EDTA, 5 mM sodium EGTA, and 250 mM sucrose). Cells were lysed by passing through a syringe needle (22 gauge, 30 times), and then centrifuged (1,000 ϫ g, 5 min, 4°C). The post-nuclear supernatant was centrifuged (20,800 ϫ g, 20 min, 4°C). The pellet was resuspended in Buffer B (0.1 M Tris-HCl at pH 7.5, 250 mM sucrose containing 1 mg/ml bovine serum albumin). Microsomal protein was determined using the bicinchoninic acid protein assay kit (Pierce). ACAT activity in microsomal fractions was determined by the method of Billheimer and colleagues (27)  The inset shows that 25HC stimulates cholesterol esterification within the first hour. B, CHO-K1 cells were incubated with medium D containing 25HC (1 g/ml) and then harvested for preparation of cell fractions. Aliquots of membranes (25 g) and nuclear extracts (40 g) were subjected to 8% SDS-PAGE and Western blotting with IgG-7D4 for SREBP-2 (membranes and nuclear extracts) and IgG-R139 for SCAP (membranes only). P and N denote the precursor and nuclear (cleaved) forms of SREBP-2, respectively. Nitrocellulose membranes were exposed to film for 30 s. Analysis of SREBP Processing by Western Blotting-On day 0, CHO-K1 cells were set up at 8 ϫ 10 5 cells/100-mm dish (in duplicate) in medium B. On day 2, cells were switched to medium D and incubated for 16 h. On day 3, cells were switched to fresh medium D and incubated with various additions (0 -1 g/ml 25HC with or without 10 M progesterone) for 0 -4 h. Cells were harvested, and membrane and nuclear extract fractions were prepared and analyzed by 8% SDS-PAGE and Western blotting as described previously (7,28).
PLAP-BP2 Cleavage-On day 0, CHO-K1 cells were set up at 1.2 ϫ 10 5 cells/well in 12-well plate in medium A. On day 1, triplicate wells of cells were refed fresh medium A and then transfected with the indicated plasmids using FuGENE 6 transfection reagent according to the manufacturer's instructions (Roche Diagnostics). A ratio of 1:3 plasmid DNA to FuGENE 6 reagent was used. After incubation for 5-6 h, the cells were rinsed with PBS and incubated with medium D in the absence or presence of test agents as indicated in the figure legends. After incubation for 16 h, the medium was removed and centrifuged (20,800 ϫ g, 20 min, 4°C). An aliquot of supernatant (50 l) was diluted, heattreated (30 min, 65°C) to inactivate non-placental alkaline phosphatase, and assayed for secreted alkaline phosphatase activity using the SEAP gene reporter assay kit (Roche Molecular Biochemicals) according to the manufacturer's instructions. After 10 -20 min, chemiluminescence was quantified on a Wallac Microbeta luminometer. Cells were lysed with 0.1 M NaOH (0.25 ml), and proteins were determined using the bicinchoninic acid protein assay kit (Pierce) to normalize secreted alkaline phosphatase activity (expressed as relative light units).

RESULTS
The regulatory pool of cholesterol is located in the ER and is key to how mammalian cells sense and respond to changes in cellular cholesterol levels. The aim of this study was to gain greater insight into how cellular cholesterol levels are regulated via transport of cholesterol to the cholesterol homeostatic machinery in the ER. We have focused on cholesterol transport induced by the oxysterol, 25HC. Although the precise mechanism remains obscure, the current view is that oxysterols like 25HC trigger intracellular cholesterol transport to the ER (29). In support of this contention, others have shown that 25HC activates cholesterol esterification (e.g. (13, 19 -21)) and is a potent suppressor of SREBP processing (e.g. Refs. 5-7 and 15). Our results confirm these previous reports. When the plasma membrane of CHO-K1 cells was pulsed with [ 3 H]cholesterol, 25HC stimulated esterification of the labeled cholesterol within an hour (Fig. 1A). 25HC suppressed SREBP processing to the mature nuclear form within a similar time-frame (Fig. 1B). SREBP processing is determined under conditions of sterol deprivation, i.e. in lipoprotein-deficient medium in the presence of a cholesterol synthesis inhibitor (compactin). Therefore, suppression of SREBP processing by 25HC is consistent with the idea that 25HC shifts an existing pool of cellular cholesterol to the cholesterol homeostatic machinery in the ER.
To enable higher sample throughput and allow ready quantification, we adopted a reporter assay devised by Sakai et al. (25) to measure SREBP processing ( Fig. 2A). In agreement with previous work (25,30), more PLAP was secreted when cells were co-transfected with a plasmid encoding the SREBP escort protein, SCAP (data not shown), whereas less PLAP was secreted when cells were transfected with a plasmid encoding the SREBP⅐SCAP retention protein, INSIG-1 (Fig. 2B). As predicted, more PLAP was secreted in the absence of sterols (active SREBP pathway) than in the presence of sterols (inactive SREBP pathway) (Fig. 2B). It was previously noted that 25HC was far more potent in suppressing SREBP processing than cholesterol added in ethanol (15). In support of that work, we found that the suppression of PLAP-BP2 processing was due to the addition of 25HC rather than cholesterol (Fig. 2C).
The effect of various inhibitors of intracellular cholesterol traffic, especially transport to the ER, is gauged on the ability of these compounds to inhibit cholesterol esterification induced by LDL or 25HC. One of the commonly used compounds to disrupt intracellular traffic, particularly cholesterol movement from plasma membrane to ER, is the steroid hormone, proges-terone (31,32). Previously (21,33,34), progesterone was found to inhibit 25HC-induced cholesterol esterification, indicating that progesterone inhibits cholesterol delivery to the ER. The question is whether or not progesterone also inhibits the suppressive effect of 25HC on SREBP processing (Fig. 3A). If progesterone were to inhibit the expansion of 25HC of the ER regulatory pool of cholesterol, it would also be predicted to relieve 25HC-mediated suppression of SREBP-2 processing, causing increased nuclear SREBP-2. In accordance with previous reports (21,33,34), we found that progesterone inhibited 25HC-induced cholesterol esterification (Fig. 3B). However, contrary to expectations, progesterone treatment tended to decrease the nuclear form of SREBP-2 (Fig. 3C). Moreover, there was no indication that the response of SREBP processing to 25HC was blunted (Fig. 3C). This result was confirmed using the PLAP-BP2 reporter assay, i.e. 25HC still inhibited PLAP-BP2 processing in the presence of progesterone (Fig. 3D).
In further experiments, varying concentrations of progesterone and two other agents (nigericin and monensin) were tested for their ability to block the effect of 25HC on cholesterol esterification and suppression of PLAP-BP2 processing. Like progesterone, nigericin, a potassium ionophore, and monensin, a sodium ionophore, were shown previously to inhibit the ability of 25HC to stimulate cholesterol esterification (35,36). Results in Fig. 4 are presented relative to the effect induced by 1 g/ml 25HC, so that 0% signifies that this effect was completely blocked, whereas 100% signifies that the effect was unaltered. In agreement with previous studies (21, 33-36), all FIG. 3. Does progesterone treatment of CHO-K1 cells block 25HC suppression of SREBP processing? A, the schematic shows that progesterone blocks the ability of 25HC to stimulate esterification of free cholesterol (FC) to cholesteryl esters (CE) by ACAT and questions whether or not 25HC suppression of the SREBP⅐SCAP system is also blocked by progesterone. The SCAP⅐ SREBP system includes INSIG proteins that can bind to both SCAP (5) and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (50). B, triplicate 60-mm dishes of CHO-K1 cells were incubated with medium C for 16 h. and nuclear extracts (40 g) were subjected to 8% SDS-PAGE and Western blotting with IgG-7D4 for SREBP-2 (membranes and nuclear extracts) and IgG-R139 for SCAP (membranes only). P and N denote the precursor and nuclear (cleaved) forms of SREBP-2, respectively. Nitrocellulose membranes were exposed to film for 30 s. D, triplicate wells of CHO-K1 cells in a 12-well plate were transfected with pCMV-PLAP-BP2(513-1141) and pGFP-SCAP as described in Fig. 2C. Cells were incubated in medium D containing 25HC (0 -1 g/ml) in the absence or presence of progesterone (10 M) for 16 h, and medium was assayed for PLAP secretion.

25HC, Cholesterol Esterification, and SREBP Processing
three agents decreased the ability of 25HC to stimulate cholesterol esterification in a dose-dependent fashion (Fig. 4, A, C,  and E). Importantly, these agents did not inhibit the ability of 25HC to suppress PLAP-BP2 processing (Fig. 4, B, D, and F), indicating that cholesterol esterification does not necessarily reflect cholesterol transport to the regulatory pool in the ER.
To validate our approach using pharmacologic agents, we performed positive control experiments. To date, the hypocholesterolemic agent, LY295427, is the only compound shown to block the effect of 25HC both in increasing cholesterol esterification and suppressing SREBP processing (37). LY295427 is a 3␣-hydroxysteroid (4␣-allylcholestan-3␣-ol) and was identified for its ability to activate the LDL-receptor promoter in the presence of 25HC (38). Its 3␤-isomer (LY306039) is inactive (37) and was used as a control. We found that LY295427 (but not LY306039) inhibited 25HC-induced effects on cholesterol esterification at relatively high concentrations (Fig. 5A). In agreement with previous work (30,37), we found that the active isomer, LY295427, also inhibited the effect of 25HC of suppressing SREBP (PLAP-BP2) processing and that this effect was maximal at 20 M (Fig. 5B). Therefore, our experiments with LY295427 confirm previous re-ports, and indicate the soundness of our pharmacologic approach to dissect the effects of 25HC on cholesterol esterification and SREBP processing.
A trivial explanation of why a pharmacologic agent could have a different effect on cholesterol esterification by ACAT and on the cholesterol homeostatic machinery would be that the agent inhibits ACAT activity, and therefore may not directly influence cholesterol transport to the ER. Others have reported that nigericin and monensin do not inhibit ACAT activity (39). However, under some (40,41) but not all circumstances (31,39), progesterone can inhibit ACAT activity. Therefore, we determined ACAT activity in membrane microsomes prepared from CHO-K1 cells treated with 10 M progesterone for 4 h. We found no effect of progesterone (mean Ϯ S.E.: 454 Ϯ 17 versus 430 Ϯ 22 pmol/min/mg of protein for ethanol control versus progesterone, respectively). This in vitro assay was performed under conditions of cholesterol excess, meaning that substrate delivery to the enzyme was not limiting. Therefore, the ability of progesterone to block 25HC stimulation of cholesterol esterification in intact cells is not due to a direct inhibitory effect on ACAT activation, supporting the view that progesterone inhibits intracellular cholesterol transport.
ACAT is proposed to maintain the regulatory pool of ER cholesterol through esterification. Inhibition of ACAT might then be predicted to expand the regulatory cholesterol pool and hence to suppress SREBP processing. Under conditions employed in the PLAP-BP2 cleavage assay, 25HC induces cholesterol transport to the ER from an existing cellular pool, because the assay is performed in lipoprotein-deficient serum with cholesterol synthesis inhibited. Therefore, inhibition of ACAT might also be predicted to increase the sensitivity of the SREBP pathway to 25HC. The sensitivity of PLAP-BP2 processing to a range of 25HC concentrations was assessed in the presence and absence of the ACAT inhibitor, S58-035 (0.5 g/ ml). The ACAT inhibitor ablated the formation of cholesteryl esters during the 16-h incubation (Fig. 6A). However, ACAT inhibition did not significantly alter the sensitivity of PLAP-BP2 processing to 25HC (Fig. 6B), further indicating that ACAT is distinct from the SREBP⅐SCAP system in the ER. DISCUSSION Mechanisms by which cholesterol is transported to the ER remain largely undefined. By questioning our current knowledge based on cholesterol esterification, we have made an initial and necessary step toward re-evaluating cholesterol transport to the regulatory pool in the ER. We present two lines of Results are presented as the relative effect induced by 25HC (1 g/ml) so that 0% signifies that this effect is completely blocked, whereas 100% signifies that the effect was unaltered. Cholesterol esterification and PLAP-BP2 cleavage were determined as outlined in Figs. 3A and 2C, respectively, for the indicated concentrations of progesterone (A and B), nigericin (C and D), and monensin (E and F). Values are means Ϯ S.E. for triplicate dishes (A, C, and E) or wells (B) or three separate experiments (D and F). The enhanced effect of 10 M progesterone in addition to 25HC was consistent between experiments (mean Ϯ S.E. from four separate experiments was 145 Ϯ 6% relative to the effect of 25HC alone). evidence that cholesterol esterification by the ER-resident protein, ACAT, is dissociable from cholesterol transport to the cholesterol homeostatic machinery in the ER. First, three agents that inhibited the ability of 25HC to induce cholesterol esterification did not have a corresponding effect on 25HC suppression of SREBP processing. We demonstrated that our approach is valid, because LY295427, shown previously to block the effect of 25HC on cholesterol esterification and SREBP processing (30,37), did both in our hands. Second, inhibition of ACAT, which should in theory expand the cholesterol content of the ER regulatory pool, did not alter SREBP processing or its response to 25HC. Together, these data indicate that cholesterol transport to ACAT in the ER may be different from cholesterol transport to the homeostatic machinery where SREBP/SCAP resides. Therefore, cholesterol esterification by ACAT may not necessarily reflect cholesterol transport to the ER regulatory pool.
Holtta-Vuori and colleagues (42) arrived at a similar conclusion using a genetic approach. They examined cholesterol metabolism in cells overexpressing the small GTPase, Rab11, and compared it with reports of cholesterol metabolism in cells lacking a functional NPC1 gene. In NPC1-deficient cells, LDLstimulated cholesterol esterification is impaired while cholesterol synthesis is increased (43), consistent with a role for NPC1 in delivering LDL-cholesterol to the regulatory pool of ER. In Rab11 cells, cholesterol esterification was also impaired, but there was no corresponding change in cholesterol synthesis, which would be expected if cholesterol esterification reflects cholesterol homeostatic mechanisms. The authors con-cluded that several membrane-trafficking pathways may feed ACAT with cholesterol (42).
Another possibility is that there may be more than one pool of ACAT. Subcellular fractionation studies of rat liver concluded that ACAT mostly resides in the rough ER (44,45). Similarly, Chang et al. (46) showed, by indirect immunofluorescent microscopy, that ACAT in melanoma cells was distributed in a typical ER-like pattern. However, more recently, Khelef et al. (47,48) observed in murine macrophages, that a significant fraction of ACAT was in a distinct undefined subcompartment of the ER, close to the trans-Golgi network and endocytic recycling compartment. Further work is required to determine if the dissociation between the effect of 25HC on cholesterol esterification and SREBP processing represents different cholesterol trafficking pathways and/or trafficking to a distinct pool of ACAT.
How the ER gains access to the regulatory pool of cholesterol is an important unresolved issue in our understanding of cholesterol homeostasis. The recent finding that the ER is the site of cholesterol-induced cytotoxicity in macrophages (49) further reinforces the importance of increasing our understanding of cholesterol traffic to and from the ER. In light of our results, we question the security of previous work that inferred cholesterol transport to the ER regulatory pool based solely on cholesterol esterification. We suggest that our current understanding of cholesterol transport to the ER, largely based on such studies, may need reassessing.