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Journal of Lipid Research, Vol. 45, 223-231, February 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

The steroidal analog GW707 activates the SREBP pathway through disruption of intracellular cholesterol trafficking

Jessie Zhang^*, Nicole Dudley-Rucker^*, Jan R. Crowley, Elvira Lopez-Perez, Marc Issandou, Jean E. Schaffer^*,** and Daniel S. Ory^*,

^* Center for Cardiovascular Research, 91951 Les Ulis cedex, France
Department of Internal Medicine, and Mass Spectrometry Facility, 91951 Les Ulis cedex, France
Department of Internal Medicine, Laboratoires GlaxoSmithKline, 91951 Les Ulis cedex, France
^** Departments of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110-1010
Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110-1010

Published, JLR Papers in Press, November 16, 2003. DOI 10.1194/jlr.M300409-JLR200

To whom correspondence should be addressed. e-mail: dory{at}wustl.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Recently, a new class of lipid-lowering agents has been described that upregulate LDL receptor (LDLr) activity. These agents are proposed to activate sterol-regulated gene expression through binding to the sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP). Here, we show that the steroidal LDLr upregulator, GW707, induces accumulation of lysosomal free cholesterol and inhibits LDL-stimulated cholesterol esterification, similar to that observed in U18666A-treated cells and in Niemann-Pick type C1 (NPC1) mutants. Moreover, we demonstrate that induction of the NPC-like phenotype by GW707 is independent of SCAP function. We find that treatment with GW707 does not increase SREBP-dependent gene expression above that observed in lipoprotein-starved cells. Rather, we show that the apparent increase in SREBP-dependent activity in GW707-treated cells is attributable to a failure to appropriately suppress sterol-regulated gene expression, as has been shown previously for U18666A-treated cells and NPC mutant fibroblasts. We further demonstrate that cells treated with either GW707 or U18666A fail to appropriately generate 27-hydroxycholesterol in response to LDL cholesterol.

Taken together, these findings support a mechanism in which GW707 exerts its hypolipidemic effects through disruption of late endosomal/lysosomal sterol trafficking and subsequent stimulation of LDLr activity.

Abbreviations: ER, endoplasmic reticulum; 25-HC, 25-hydroxycholesterol; 27-HC, 27-hydroxycholesterol; LDLr, LDL receptor; LPDS, lipoprotein-deficient serum; LXR, liver X receptor; NPC, Niemann-Pick type C; SRE, sterol regulatory element; SREBP, sterol regulatory element binding protein

Supplementary key words sterol regulatory element binding protein • cleavage-activating protein ligands • Niemann-Pick type C • sterol-sensing domain • 27-hydroxycholesterol

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Hypercholesterolemia is a major risk factor for the development of coronary artery and cerebral vascular disease. Increased plasma levels of LDL lead to deposition of excess cholesterol in arteries, initiating atherosclerosis. Treatment with 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors (statins) lowers plasma LDL and decreases cardiovascular mortality. The beneficial effects of statin therapy primarily result from the depletion of cellular cholesterol and the increased expression of the LDL receptor (LDLr), thereby promoting clearance of LDL particles and reducing plasma LDL (1, 2). Recently, a novel class of potent cholesterol-lowering agents has been described that upregulates LDLr activity, leading to marked reduction of both LDL cholesterol and triglycerides in vivo (3). These cholesterol-lowering agents, initially designated SCAP ligands and referred to here as LDLr upregulators, were identified in a screen for transcriptional activators of the human LDLr promoter and are proposed to bind to the sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) through direct interaction with the sterol-sensing domain of SCAP.

The steroidal LDLr upregulator GW707 is structurally similar to the amphiphilic compound 3-ß-[2-(diethylamino) methoxy]androst-5-en-17-one (U18666A) (Fig. 1) , which is known to interfere with intracellular sterol trafficking (4). Both compounds consist of a planar sterol nucleus with an ether linkage at the C-3 position and an alkyl side chain that contains a tertiary amine. U18666A differs from GW707 in that the sterol side chain is absent from the C-17 position. Cells treated with U18666A accumulate lysosomal free cholesterol and demonstrate impaired movement of cholesterol both to and from the plasma membrane (4–8). Treatment of cells with U18666A also inhibits LDL-stimulated cholesterol esterification and prevents the suppression of sterol-regulated gene expression (4).

View larger version (8K): [in this window] [in a new window]   Fig. 1. Structures of the GW707 and U18666A steroidal analogs. Both GW707 and U18666A possess an ether linkage at the C-3 position and alkyl side chain attached to the steroid A-ring that contains a tertiary amine. GW707 has two heterocyclic spiro rings at the C-17 position, whereas in U18666A the sterol side chain is absent.

Although the mechanism of action of U18666A is not well understood, the cellular phenotype induced by this compound suggests that it disrupts trafficking of LDL cholesterol through the NPC pathway (4, 6). We hypothesized that the steroidal compound GW707 similarly may act through inhibition of either NPC1 or NPC2 function. Therefore, the lipid-lowering effects of LDLr upregulators may result from disruption of intracellular sterol-trafficking pathways and downstream sterol-regulated gene expression, rather than through the direct activation of SCAP. In this study, we show that GW707, like the amphiphilic compound U18666A, induces an NPC-like phenotype in Chinese hamster ovary (CHO) cells and that induction of this phenotype is independent of SCAP function. Rather than upregulating SREBP-dependent gene expression, we show that the increased sterol-regulated activity in GW707-treated cells is attributable to a failure to appropriately suppress SREBP-dependent gene expression, similar to that observed in NPC mutant fibroblasts. Our findings support a mechanism in which GW707 exerts its lipid-lowering effects through inhibition of the late endosomal/lysosomal sterol-trafficking pathway. Moreover, GW707 may serve as a useful tool to probe pathways of intracellular cholesterol trafficking.

	MATERIALS AND METHODS

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Materials
Dulbecco's modified Eagle's medium (DMEM), Ham's F12 medium, glutamine, and penicillin/streptomycin were obtained from Invitrogen. Lipoprotein-deficient serum (LPDS) was obtained from Cocalico Labs. U18666A was obtained from Biomol. Steroidal compound GW707 was provided by GSK. Oleic acid and cholesteryl oleate were obtained from Nu-Check Prep. [³H]Cholesterol (75 Ci/mmol), [9,10-³H]oleic acid (5 Ci/mmol), and [oleate-1-¹⁴C]cholesteryl oleate (59.5 mCi/mmol) were obtained from Perkin-Elmer Life Sciences Products. 27-Hydroxycholesterol (27-HC) was obtained from Research Plus, Inc. Compactin, fetal bovine serum (FBS), filipin complex, human LDL, and mevalonic acid were obtained from Sigma. Cholesterol, 24(S)-hydroxycholesterol (24-HC), and 25-hydroxycholesterol (25-HC) were obtained from Steraloids, Inc.

Plasmids
The LDLp-588luc construct used in the reporter assays contains the human LDLr promoter linked to a luciferase reporter (3). The thymidine kinase (TK) promoter-Renilla luciferase construct, pRL-TK, was from Promega.

Cell lines
CHO-K1 cells were obtained from the American Type Culture Collection (ATCC; CRL-9618). M12 cells are a mutant CHO-K1 cell line with a deletion of the NPC1 locus (7). The CHO/NPC1-28 cell line expressing human NPC1 was generated as previously described (7). 25-RA cells were provided by T. Y. Chang (Dartmouth College) (21). SRD-13A cells were a gift from M. Brown (22). Normal skin fibroblasts (CRL-1474) were obtained from ATCC. The NPC1-null mutant human skin fibroblast cell line NPC1^1628delC (NIH 98.016) was provided by P. Pentchev (National Institutes of Health) (23). The NPC2-null mutant human skin fibroblast cell line NPC2^G20x (NIH 99.04) was provided by A. Fensom (United Medical and Dental Schools of Guy's and St. Thomas's Hospitals, London, UK) (24). The use of human fibroblast cell lines was approved by the Human Studies Committee at the Washington University Medical Center.

Cell culture
Cells were maintained in monolayer culture at 37°C with 5% CO₂. CHO cell lines were maintained in medium containing 1:1 DMEM-Ham's F12, 5% (v/v) FBS, 2 mM glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. Fibroblasts cell lines were grown in DMEM with 10% (v/v) FBS, 2 mM glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin.

Immunocytochemistry
On day 0, CHO cell lines were plated at 1.4 x 10⁴ cells/ml on gelatin-coated 12 mm glass coverslips in 24-well dishes in lipoprotein-containing medium. On day 1, the cells were refed medium containing LPDS. On day 2, the medium was replaced with LPDS medium supplemented with 50 µg/ml LDL in the presence or absence of 1 µM GW707 or 0.5 µM U18666A. Conditions for fixation and antibody and filipin staining have been described previously (25). Cells were examined by fluorescence microscopy on a Zeiss Axiovert epifluorescence microscope. The following filter sets (Chroma) were used: for filipin, excitation filter 360/40 nm, beamsplitter 400 nm, emission filter 460/50 nm; for LysoSensor, excitation filter 470/40 nm, beamsplitter 500 nm, emission filter 535/40 nm; and for Cy3, excitation filter 535/50 nm, beamsplitter 565 nm, emission filter 590 nm.

Cholesterol esterification assays
Cholesterol esterification assays were performed as previously described (7). After lipid extraction, a chromatography recovery standard was added (30 µg of cholesteryl oleate and 0.0005 µCi of [¹⁴C]cholesteryl oleate) and samples were dried under nitrogen. The lipids were separated by TLC (PE SIL G plates from Whatman) using heptane-ethyl ether-acetic acid (90:30:1) and visualized with iodine. [³H]Cholesteryl oleate was quantified by liquid scintillation counting. After lipid extraction, monolayers were incubated with 0.1 N NaOH and protein determination was performed using the MicroBCA assay (Pierce).

Luciferase reporter assays
For quantification of SREBP-dependent gene expression, CHO and M12 cells (1 x 10⁶ cells) were electroporated using nucleofection (Amaxa) with 2 µg of LDLp-588luc and 0.5 µg of pRL-TK and then plated at 1 x 10⁵ cells/35 mm well. For experiments shown in Fig. 5A and D, cells were fed LPDS medium on day 2, and on day 3 they were refed LPDS medium in the presence or absence of GW707 (1 µM) and U18666A (0.5 µM). After 24 h, cells were incubated with LDL (50 µg/ml) in the presence or absence of the compounds for an additional 6 h. For the experiment shown in Fig. 5B, cells were fed LPDS medium on day 2, and on day 3 they were refed LPDS medium in the presence of LDL (50 µg/ml) (lipoprotein-fed) or 1 µM GW707 or 20 µM compactin and 50 µM mevalonate (lipoprotein-starved). For the experiment shown in Fig. 5C, cells were fed LPDS medium on day 2, and on day 3 they were refed LPDS medium supplemented with LDL (0–80 µg/ml) in the presence or absence of GW707 (1 µM) and U18666A (0.5 µM). For all experiments, cell lysates were harvested on day 4 and luciferase and Renilla activity (Promega) were determined. Normalization of luciferase activity to Renilla activity controlled for transfection efficiency.

View larger version (34K): [in this window] [in a new window]   Fig. 5. GW707-treated cells fail to suppress sterol regulatory element (SRE)-dependent gene expression. Cells were cotransfected with a luciferase reporter driven by the human LDL receptor promoter and with a thymidine kinase (TK)-Renilla transfection control. A: Lipoprotein-fed conditions. CHO (WT) and NPC1-null cells were grown in LPDS medium for 24 h in the presence or absence of 1 µM GW707 or 0.5 µM U18666A and then incubated with LDL for an additional 6 h in the presence or absence of the compounds. Lysates were harvested, and luciferase and Renilla activity was determined. Data are presented as relative light units. Luciferase activity is normalized to Renilla activity. Values are means ± SEM and are representative of two independent experiments. * P < 0.005 versus untreated WT CHO cells; ** P < 0.001 versus untreated WT CHO cells. B: Lipoprotein-starved conditions. CHO cells were cultured in LPDS medium in the absence (LPDS) or presence of LDL (LPDS + LDL), 1 µM GW707 (LPDS + GW707), or 20 µM compactin and 50 µM mevalonate (LPDS + compactin/mev). Luciferase activity was determined as described above. Values are means ± SEM and are representative of two independent experiments. * P < 0.025 versus LDL-fed cells; ** P < 0.002 versus LDL-fed cells. C: Lipoprotein-suppression conditions. Cells were grown in LPDS medium supplemented with LDL (0–80 µg/ml) in the presence or absence of GW707 (1 µM) and U18666A (0.5 µM). Lysates were harvested and luciferase activity determined as described above. Values are means ± SEM and are representative of four independent experiments. * P < 0.02 versus WT CHO cells; ** P < 0.002 versus WT CHO cells. D: CHO and NPC1-null cells were grown in LPDS medium for 24 h in the presence or absence of 1 µM GW707 and then incubated with LDL for an additional 6 h in the presence or absence of the compounds. Luciferase activity was determined as described above. Values are means ± SEM and are representative of two independent experiments. * P < 0.005 versus WT cells.

Statistics
All results are expressed as means ± SEM. The statistical significance of differences in mean values was determined by one-way ANOVA. Data shown are representative of at least two similar experiments.

	RESULTS

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Treatment with the steroidal analog GW707 disrupts trafficking of LDL cholesterol
To explore the cellular mechanism of action of the GW707 compound, we examined the effect of treatment with GW707 on intracellular cholesterol trafficking. Wild-type CHO, M12 (NPC1-null), and human NPC1-expressing CHO (CHO/NPC1) cells treated with 1 µM GW707 or 0.5 µM U18666A were costained with filipin, a specific fluorescent marker of unesterified cholesterol, and antisera to human NPC1 and LAMP-2, a late endosomal/lysosomal marker. Using immunofluorescence microscopy, we found that CHO/NPC1 cells incubated with GW707 accumulate free cholesterol in aberrant LAMP-2-positive, NPC1-positive organelles, similar to the cholesterol-laden compartment present in NPC1-null cells and in cells incubated with U18666A (Fig. 2) . To further demonstrate that GW707 disrupts NPC1-mediated sterol trafficking, we examined the effect of the compound on the rate of LDL-stimulated cholesterol esterification. Cells treated with GW707 show dose-dependent inhibition of LDL cholesterol esterification, similar to that observed in U18666A-treated cells (Fig. 3) . Moreover, at the highest concentrations examined, both compounds result in severe impairment of LDL cholesterol esterification, characteristic of NPC1-null cells. Although there is a steeper dose response in the U18666A-treated cells, these findings suggest that GW707 impairs mobilization of lysosomal cholesterol in an amphiphile-like manner.

View larger version (52K): [in this window] [in a new window]   Fig. 2. GW707 induces free cholesterol accumulation in a lysosomal compartment. A: CHO/NPC1 cells (a–c) and NPC1-null cells (d–f) were cultured in lipoprotein-deficient serum (LPDS) medium, pulsed with LDL (50 µg/ml), and costained for cholesterol (a and d), NPC1 (b and e), and LAMP-2 (c and f). Cells were examined by fluorescence microscopy. Note the absence of NPC1 staining in the NPC1-null cells (e). Arrowheads identify LAMP-2 staining at the limiting membrane of cholesterol-laden lysosomes in NPC1-null cells. For each set of panels (a–c and d–f), the images are from a single field. Bar = 10 µm. B: CHO/NPC1 cells were cultured in LPDS medium containing 1 µM GW707 (a–c) or 0.5 µM U18666A (d–f), pulsed with LDL (50 µg/ml), and costained for cholesterol (a and d), NPC1 (b and e), and LAMP-2 (c and f). Cells were examined by fluorescence microscopy. Arrowheads identify colocalization of NPC1 and LAMP-2 in cholesterol-laden lysosomes. For each set of panels (a–c and d–f), the images are from a single field. Bar = 10 µm.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Inhibition of LDL cholesterol esterification by GW707 and U18666A. CHO and NPC1-null cells were plated in triplicate (2.5 x 104 cells/well) and then grown in LPDS medium for 48 h. CHO cells were fed medium supplemented with LDL in the presence or absence of GW707 (A) and U18666A (B) for 16 h and pulsed for 2 h with [3H]oleate, and the rate of incorporation of [3H]oleate into cholesteryl-[3H]oleate was determined. Esterification rates (picomoles per minute per milligram) are normalized to untreated CHO cells. Values are means ± SEM and are representative of two independent experiments. * P < 0.005 versus wild-type (WT) CHO cells; ** P < 0.001 versus WT CHO cells.

View larger version (116K): [in this window] [in a new window]   Fig. 4. Free cholesterol accumulation in GW707-treated cells is sterol regulatory element binding protein cleavage-activating protein (SCAP)-independent. 25-RA cells (D443N SCAP mutation) (A and B) and SRD-13A (SCAP-null) cells (C and D) were cultured in LPDS medium plus LDL (50 µg/ml) in the absence (A and C) or presence (B and D) of 1 µM GW707 and stained for cholesterol. Bar = 10 µm.

In light of the sterol-trafficking defect in the compound-treated and NPC1 mutant cells, we hypothesized that the increase in SRE reporter activity in the LDL-fed cells was not attributable to additional activation of SRE-dependent gene transcription but rather to a failure to appropriately suppress SRE-dependent activity in response to LDL cholesterol. To test this hypothesis, we examined the effect of GW707 on SRE reporter activity under conditions of lipoprotein starvation (Fig. 5B). CHO cells transfected with the SRE reporter were incubated in lipoprotein-deficient medium in the presence or absence of GW707, and SRE reporter activity was determined. In the absence of LDL suppression, no increase in SRE activity was observed in the GW707-treated cells compared with untreated CHO cells. Note that additional activation of SRE-dependent activity was achieved by complete sterol starvation (cells incubated in lipoprotein-deficient medium plus compactin and mevalonate). To demonstrate that GW707 prevents the suppression of sterol-regulated gene expression by LDL cholesterol, we next measured SRE reporter activity in CHO and NPC1-null cells that were incubated in lipoprotein-deficient medium in the presence or absence of GW707 and U18666A, followed by LDL feeding (Fig. 5C). Compared with wild-type CHO cells, GW707- and U18666A-treated cells only partially suppress SRE-dependent activity in response to LDL cholesterol, and NPC1-null cells are resistant to suppression of SRE-dependent activity by LDL cholesterol over the range of concentrations examined. Treatment of NPC1-null cells with GW707 did not result in a further increase in SRE reporter activity (Fig. 5D; P = NS for treated versus untreated NPC1-null cells). These findings imply that induction of an NPC-like sterol-trafficking defect in GW707-treated cells is sufficient to explain the apparent activation of SREBP-dependent gene expression.

GW707 inhibits the production of LDL cholesterol-derived oxysterols
Recently, we have shown that cells with NPC1 or NPC2 loss of function are deficient in the generation of oxysterols in response to LDL cholesterol (20). To determine whether treatment with GW707 similarly interferes with the delivery of free cholesterol substrate to sites of cellular oxysterol synthesis, we measured production of 25-HC and 27-HC in wild-type human fibroblasts in the presence and absence of GW707 and U18666A and in NPC1-null and NPC2-null fibroblasts. The rate of 25-HC secretion into the media was decreased by 54% and 70% in the NPC1 and NPC2 mutants, respectively (Fig. 6) . A similar reduction was observed in U18666A-treated cells (40% decrease), but not in cells exposed to GW707 (5% decrease; P = NS). In contrast, the rate of 27-HC secretion was markedly reduced in both GW707- and U18666A-treated cells (87% and 96% decrease, respectively), comparable to the decrease observed in the NPC1 and NPC2 mutants. Therefore, although both GW707 and U18666A disrupt the delivery of free cholesterol to the mitochondrial sterol 27-hydroxylase, only U18666A appears to interfere with cholesterol delivery to the endoplasmic reticulum (ER)/Golgi-localized cholesterol 25-hydroxylase (28).

View larger version (17K): [in this window] [in a new window]   Fig. 6. Determination of oxysterol production in fibroblasts treated with GW707 and U18666A in response to LDL cholesterol. WT, NPC1-null, and NPC2-null fibroblasts were lipoprotein starved for 24 h and refed LPDS medium supplemented with LDL (50 µg/ml) in the presence or absence of 1 µM GW707 or 0.5 µM U18666A. Quantitative gas chromatography-mass spectrometry determinations for 25-hydroxycholesterol (25-HC) and 27-hydroxycholesterol (27-HC) were calculated from triplicate injections and represent total oxysterols in cells and secreted into media. A: Rate of 25-HC production in cells and secretion into the media for WT, NPC1-null, NPC2-null, GW707-treated, and U18666A-treated fibroblasts. * P < 0.01 versus WT; ** P < 0.03 versus WT. B: Rate of 27-HC production in cells and secretion into the media for WT, NPC1-null, NPC2-null, GW707-treated, and U18666A-treated fibroblasts. * P < 0.0001 versus WT. All values are means ± SEM and are representative of two independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

GW707 was identified through a screen for compounds that activate expression from the human LDLr promoter (3). It has been proposed that these compounds upregulate LDLr activity based on their ability to bind SCAP and to promote SREBP maturation (3). Several lines of evidence, however, suggest that the GW707 compound modulates sterol homeostasis through an alternative mechanism. First, the screen that identified the LDLr upregulators was designed to detect transcriptional activators of the LDLr. Because the assay was performed under sterol-loading conditions, it is likely that the screen would have also identified classes of compounds that interfere with the endocytic processing of LDL cholesterol and block the delivery of cholesterol to the ER, thereby preventing downregulation of SREBP-dependent gene expression. Our studies support such as mechanism of action for GW707. Second, treatment with GW707 leads to the accumulation of free cholesterol in lysosomes, even in the absence of a functional SCAP. Therefore, induction of the sterol-trafficking defect by GW707 is sufficient to explain the upregulation of LDLr activity without the need to consider direct effects of the compound on SCAP. The finding that treatment with GW707 does not further augment sterol-regulated activity in NPC1-null cells supports this conclusion. Third, in GW707-treated cells, the profound deficiency in 27-HC synthesis suggests that this compound may exert its effects by preventing oxysterol-mediated suppression of sterol-regulated expression, similar to the mechanism proposed for NPC mutants (20).

Treatment with LDLr upregulators in vivo results in a marked reduction in the level of VLDL and LDL (3). In the treated animals, this hypolipidemic response is accompanied by an 3- to 3.5-fold increase in hepatic LDLr and HMG-CoA reductase mRNA levels. Although these observations imply an important role for the LDLr pathway in the clearance of apolipoprotein B-containing lipoproteins in the SCAP ligand-treated animals, the relative contribution of rates of lipoprotein clearance versus production has not been measured directly. In the liver tissue of treated animals, no increase in cholesteryl esters was observed, although the effect of the compound on free cholesterol levels in this setting is not known. Nonetheless, the effect of LDLr upregulators on lipoprotein metabolism in vivo could be attributed to a defect in the trafficking of LDL cholesterol and is consistent with the cellular mechanism of action we have proposed for GW707.

The steroid analog GW707 exhibits properties consistent with class II amphiphiles, a class of agents that includes steroids and hydrophobic amines (29). Although there is structural diversity among the amphiphiles, GW707 and U18666A, a prototypic class II agent, share common features. Both compounds possess an ether linkage at the C-3 position and alkyl side chain attached to the steroid A-ring that contains a tertiary amine. The compounds differ in the treatment of the sterol side chain: GW707 has two heterocyclic spiro rings at the C-17 position, whereas in U18666A the side chain is absent, indicating that the identity of the C-17 substituent is relatively unimportant compared with the steroid A-ring modifications. The observation that GW706, an inactive analog of GW707 that lacks the alkyl side chain and tertiary amine, neither induces cholesterol accumulation nor inhibits cholesterol esterification supports this conclusion (J. Zhang and D. S. Ory, unpublished observations) (3). Functionally, GW707 and U18666A both disrupt late endosomal/lysosomal trafficking of LDL cholesterol, inhibiting the egress of free cholesterol from lysosomes and bulk cholesterol esterification. Furthermore, both agents prevent the suppression by LDL cholesterol of sterol-regulated gene expression, as indicated by the failure to downregulate LDLr and HMG-CoA reductase activity in compound-treated cells (3, 4). These findings correlate with the ability of both agents to profoundly inhibit the production of 27-HC in response to LDL cholesterol.

On the other hand, GW707 differs from U18666A in several respects. In CHO cells, GW707 is approximately 10-fold less potent than U18666A with respect to inhibition of LDL-stimulated cholesterol esterification, whereas in HepG2 cells, GW707 is 2- to 3-fold more potent than U18666A in inhibition of cholesteryl ester synthesis (E. Lopez-Perez and M. Issandou, unpublished data). In addition, U18666A, unlike GW707, impairs cholesterol biosynthesis through the inhibition of 2,3-oxidosqualene cyclase (30). In the present study, we found that GW707 does not significantly affect the synthesis of 25-HC in response to LDL cholesterol, whereas U18666A-treated cells exhibit a 40% reduction in the rate of secretion of 25-HC. There are several possible explanations for the divergent effects of the compounds on 25-HC synthesis. GW707 may selectively inhibit trafficking of LDL cholesterol to the mitochondrial sterol 27-hydroxylase, whereas treatment with U18666A may interfere more broadly with the delivery of free cholesterol to sites of cellular oxysterol synthesis. Alternatively, the compounds may differentially affect the trafficking or presentation of the cholesterol substrate to sterol 27-hydroxylase. Given the ability of the enzyme to catalyze both 25- and 27-hydroxylation (31), it is possible that U18666A, but not GW707, affects the relative rate of 25-hydroxylation. At present, however, little is known of the mechanisms involved in the regulation of oxysterol synthesis. In future studies, GW707 may be used to probe specific limbs of the sterol-trafficking pathway or to explore the mechanism of the sterol 27-hydroxylase.

The molecular mechanism through which the amphiphilic compounds exert their effects is not known. The observation that cells overexpressing the NPC1 protein are resistant to the effects of U18666A implies that NPC1 or a protein in the same pathway may be the molecular target of this class of compounds (8). The finding that LDLr upregulators can bind to the sterol-sensing domain of SCAP raises the possibility that amphiphilic compounds likewise may interact with the sterol-sensing domain of NPC1, which is 28% identical and 54% similar to the homologous region of SCAP. Alternatively, amphiphiles may target the NPC2 protein, which has been shown to bind free cholesterol with high affinity (14, 15). Future studies examining the mechanism of action of these compounds may identify specific inhibitors of NPC1 or NPC2 function and will further our understanding of the role of the NPC proteins in the regulation of sterol trafficking.

	ACKNOWLEDGMENTS

 
The authors are grateful to D. Covey for helpful discussions. D.S.O. is supported by Grants HL-04482 and HL-67773 from the National Institutes of Health, and J.R.C. is supported by Grants DK-6341 and P-41-RR-00954 from the National Institutes of Health.

Manuscript received September 26, 2003 and in revised form October 31, 2003.

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