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J. Biol. Chem., Vol. 278, Issue 28, 25517-25525, July 11, 2003
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NPC1 and NPC2 Regulate Cellular Cholesterol Homeostasis through Generation of Low Density Lipoprotein Cholesterol-derived Oxysterols*
**
From the
Center for Cardiovascular Research,
Department of Internal Medicine, and the ¶Mass
Spectrometry Facility, Department of Internal Medicine,
**Department of Molecular Biology and Pharmacology,
and the Department of Cell Biology and
Physiology, Washington University School of Medicine, St. Louis, Missouri
63110-1010
Received for publication, March 13, 2003 , and in revised form, April 23, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Mutations in the Niemann-Pick disease genes cause lysosomal cholesterol accumulation and impaired low density lipoprotein (LDL) cholesterol esterification. These findings have been attributed to a block in cholesterol movement from lysosomes to the site of the sterol regulatory machinery. In this study we show that Niemann-Pick type C1 (NPC1) and Niemann-Pick type C2 (NPC2) mutants have increased cellular cholesterol, yet they are unable to suppress LDL receptor activity and cholesterol biosynthesis. Cholesterol overload in both NPC1 and NPC2 mutants results from the failure of LDL cholesterol tobothsuppresssterolregulatoryelement-bindingprotein-dependent gene expression and promote liver X receptor-mediated responses. However, the severity of the defect in regulation of sterol homeostasis does not correlate with endoplasmic reticulum cholesterol levels, but rather with the degree to which NPC mutant fibroblasts fail to appropriately generate 25-hydroxycholesterol and 27-hydroxycholesterol in response to LDL cholesterol. Moreover, we demonstrate that treatment with oxysterols reduces cholesterol in NPC mutants and is able to correct the NPC1I1061T phenotype, the most prevalent NPC1 disease genotype. Our findings support a role for NPC1 and NPC2 in the regulation of sterol homeostasis through generation of LDL cholesterol-derived oxysterols and have important implications for the treatment of NPC disease.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Niemann-Pick type C (NPC)1 disease is a fatal, autosomal recessive lipid storage disorder characterized by cholesterol accumulation in the liver, spleen, and central nervous system (1). Mutations in two independent genes result in the clinical and biochemical NPC phenotype (2, 3). The NPC1 disease gene encodes a late endosomal protein that has 13 membrane-spanning domains, five of which share sequence homology with the sterol-sensing domains of hydroxymethylglutaryl-CoA reductase, sterol regulatory element-binding protein (SREBP), cleavage-activating protein (SCAP), and Patched (4, 5). The minor disease locus, the NPC2 gene, encodes a 132-amino acid soluble lysosomal protein that has been shown to specifically bind cholesterol with a 1:1 stoichiometry and submicromolar affinity (6–9).
The NPC disease genes are key participants in the intracellular trafficking of cholesterol. Cells with mutations in NPC1 and NPC2 accumulate unesterified cholesterol in an aberrant late endosomal/lysosomal organelle and have markedly impaired rates of esterification of LDL cholesterol (2, 10). Fibroblasts from NPC1 patients also exhibit a defect in mobilization of endosomal cholesterol to the plasma membrane (11–13) and delayed down-regulation of the LDL receptor and de novo cholesterol biosynthesis (14, 15). Similarly, in the NPC1–/– mice, which phenocopy human NPC disease, cholesterol synthesis in the whole animal is elevated, and there is increased accumulation of unesterified cholesterol (16). These sterol regulatory defects have been attributed to impaired delivery of LDL-derived free cholesterol to the endoplasmic reticulum (ER). Decreased ER cholesterol promotes SCAP-mediated trafficking of SREBP to the Golgi, enabling SREBP proteolysis and up-regulation of genes involved in cholesterol synthesis and uptake (17).
The goal of the present study is to characterize the relationship between the NPC proteins and the sterol regulatory machinery. Thus, we examined the consequence of NPC1and NPC2 loss of function on the key cellular pathways that govern cholesterol homeostasis. We find that cholesterol overload in NPC1 and NPC2 mutants results from the failure of LDL cholesterol to both suppress SREBP-dependent gene expression and promote liver X receptor (LXR)-mediated responses. We show that the sterol regulatory defects in the NPC mutants correlate with the failure to generate oxysterols in response to LDL cholesterol loading, rather than with ER cholesterol levels. Our findings indicate that the NPC proteins regulate sterol homeostasis through production of LDL cholesterol-derived oxysterols and have implications for the treatment of NPC disease.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Materials—Dulbecco's modified Eagle's medium (DMEM), Ham's F-12 medium, glutamine, and penicillin/streptomycin were obtained from Invitrogen. Lipoprotein-deficient fetal calf serum (LPDS) was obtained from Cocalico Laboratories. Fetal bovine serum (FBS), filipin complex and human LDL were obtained from Sigma. DiI-LDL was obtained from Molecular Probes. Oleic acid, triolein, and cholesteryl oleate were obtained from Nu-Check Prep. [9,10-3H]oleic acid (5 Ci/mmol), [cholesteryl-1,2,6,7-3H]linoleate (84 Ci/mmol), [3H]acetic acid (5.1 Ci/mmol), and [oleate-1-14C]cholesteryl oleate (59.5 mCi/mmol) were obtained from PerkinElmer Life Sciences. 27-Hydroxycholesterol was obtained from Research Plus, Inc. Cholesterol, 24(S)-hydroxycholesterol (24-HC), and 25-hydroxycholesterol (25-HC) were obtained from Steraloids, Inc.
Plasmids—The LDLp-588luc construct containing the human LDL receptor (LDLr) promoter linked to a luciferase reporter was a gift of M. Issandou (18). The TK-LXRE3-luc construct containing three copies of the LXR response element upstream of a luciferase reporter was a gift of D. Mangelsdorf (19). The TK promoter-Renilla luciferase construct, pRL-TK, was from Promega.
Cell Lines—Normal skin fibroblasts (CRL-1474) were obtained
from ATCC. The NPC1 mutant human skin fibroblast cell lines with the
NPC1I1061T (GM3123) and NPC11628delC (NIH 98.016) were
provided by P. Pentchev (National Institutes of Health)
(20). The NPC2 mutant human
skin fibroblast cell lines, NPC2G58T (NIH 99.04) and
NPC2IVS2 + 5G
A
(NIH 94.85), were provided by A. Fensom (United Medical & Dental Schools
of Guy's & St. Thomas's Hospitals, London, UK) and M. Vanier (U189
INSERM/Fondation Gillet-Mérieux, Lyon-Sud, France), respectively
(2,
3). All fibroblasts cell lines
were passaged in medium containing DMEM, 10% FBS.
Cholesterol Esterification Assays—Cholesterol esterification assays were performed as described previously (12). For measurement of 25-HC-stimulated cholesterol esterification, cells were fed medium containing 0–5 µM 25-HC instead of LDL, pulsed with [3H]oleate for 2 h, and lipids extracted as described. A chromatography recovery standard was added (30 µg of cholesteryl oleate, 30 µg of triolein, 0.0005 µCi of [14C]cholesteryl oleate), and samples were dried under nitrogen. The lipids were separated by thin-layer chromatography (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 performed using the MicroBCA assay (Pierce).
Measurement of Cellular Cholesterol—Total cellular cholesterol was determined by an enzymatic method using the Cholesterol CII Kit (Wako) as described previously (21).
LDL Receptor Activity Assay—Cells were grown for 48 h in DMEM, 10% FBS or DMEM, 10% LPDS, incubated with DiI-LDL (6 µg/ml) for 1 h, and the mean fluorescence of 10,000 cells was measured by flow cytometetry using a FACScan (BD Biosciences). For studies involving oxysterol treatment, cells were lipoprotein-starved for 48 h and then incubated with LDL (25 µg/ml), with LDL plus 27-HC (0.25 µM), with LDL plus 25-HC (2.5 µM), and with LDL plus both 27-HC and 25-HC. Cells were then incubated with DiI-LDL (6 µg/ml) for 1 h and examined by flow cytometetry as described above.
Measurement of de Novo Cholesterol Synthesis—Metabolic labeling of de novo synthesized cholesterol was performed as described previously (12).
Luciferase Reporter Assays—For quantification of SREBP-dependent gene expression, fibroblasts were co-transfected using nucleofection (Amaxa) with 2 µg of LDLp-588luc and 0.2 µg of pRL-TK. Using the nucleofection method, transfection efficiencies of >90% were achieved in the human fibroblasts. On day 1, cells were re-fed lipoprotein-deficient medium. On day 2 cells were pulsed with LDL (0–80 µg/ml) for 24 h, followed by harvest of cell lysates and determination of luciferase and Renilla activity (Promega). Normalization of luciferase activity to Renilla activity controlled for transfection efficiency. For determination of LXR-activated gene expression, fibroblasts were co-transfected with 2 µg of TK-LXRE3-luc and 0.2 µg of pRL-TK. On day 1, cells were re-fed lipoprotein-deficient medium. On day 2 cells were pulsed with LDL (0–80 µg/ml) for 24 h, followed by harvest of cell lysates and determination of luciferase and Renilla activity.
In Vitro Cholesterol Esterification Assay—Fibroblasts were grown for 48 h in DMEM, 10% FBS, or for 24 h in DMEM, 10% LPDS followed by re-feeding with DMEM, 10% LPDS in the presence and absence of 50 µg/ml LDL or 2.5 µM 25-HC. The in vitro esterification assay was performed as described by Lange and Steck (22). Cells were trypsinized, pelleted, and washed in 0.25 M sucrose, 5 mM sodium phosphate, pH 7.5. Pelleted cells were resuspended in 0.1 M sucrose, 5 mM sodium phosphate, pH 7.5, and swelled on ice for 10 min. The cells were homogenized with 10 strokes using a ball bearing homogenizer with an 11-µm clearance, centrifuged to remove large particles, and adjusted to 1 mM dithiothreitol and 1 mg/ml bovine serum albumin. Esterification reactions were started by addition of 25 µM [14C]oleoyl-CoA and incubated for 2 h at 37 °C. After extraction with CH3Cl:methanol (2:1) and addition of a recovery standard (40 µg of cholesterol, 30 µg of cholesteryl oleate, 0.002 µCi of [3H]cholesterol), lipids were dried under nitrogen. Cholesteryl oleate was recovered by TLC and quantified as described under "Cholesterol Esterification Assays." Protein determinations were performed using the BCA assay.
Gas Chromatography/Mass Spectrometry (GC/MS) Determinations— For oxysterol measurements, fibroblasts were grown in DMEM, 10% LPDS for 48 h and then re-fed for 24 h DMEM, 10% LPDS containing 50 µg/ml LDL. Oxysterols were extracted from the cells and media as described (23, 24). Since 24-HC was not detected in the fibroblasts, we used 200 pmol of 24-HC as an internal standard during oxysterol isolation. Oxysterols were derivatized to trimethylsilyl ethers by treatment with Sigma Sil-A for 1 h at 60 °C. Derivatized samples were analyzed on a Varian 3400 gas chromatograph interfaced to a Finnigan SSQ 7000 mass spectrometer. The GC column used for the study was a DB-1 (12.5 m, 0.2 mm inner diameter, 0.33-µm film coating, P. J. Cobert, St. Louis, MO). A gradient was run as follows. The initial temperature of 180° was held for 1 min and increased to 250 at 20°/min. The temperature was increased from 250 to 300° at 5°/min and held for 10 min. The mass spectrometer was operated in the electron ionization mode, and the source temperature, electron energy, and emission current were 200°, 100 eV, and 300 µA, respectively. The injector and transfer line temperatures were 250°. The presence of 27-HC and 25-HC was monitored with ions at m/z 456 at 16.3 min and m/z 131 at 15.6 min, respectively. Quantitative GC/MS determinations for 25-HC and 27-HC were calculated from triplicate injections. Total oxysterol production was determined as the sum of oxysterols in the cells and secreted into the medium.
Filipin Staining—Cells were grown in DMEM, 10% FBS for 48 h in the presence and absence of 0.25 µM 25-HC or 2.5 µM 25-HC. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline for 30 min, washed twice with phosphate-buffered saline, and stained with 50 µg/ml filipin in phosphate-buffered saline for 30 min. Filipin fluorescence was detected by fluorescence microscopy on a Zeiss Axiovert epifluorescence microscope using 360/40 nm excitation and 460/50 emission filters (Chroma). For each condition a minimum of 70 cells were scored on each of two coverslips, and only filipin-negative cells (i.e. fluorescence intensity equivalent to normal fibroblasts) were scored as fully corrected cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Regulation of Sterol Homeostatic Responses Is Defective in NPC Mutants—To gain insight into the molecular function of the NPC1 and NPC2 proteins, we examined the consequence of NPC mutations on regulation of cellular cholesterol homeostasis. We first compared the rates of LDL-stimulated cholesterol esterification in NPC1 and NPC2 mutants. In these experiments we examined NPC1 fibroblasts harboring NPC1I1061T or NPC11628delC mutations and NPC2 cells with NPC2IVS2 + 5G
A or NPC2G58T
mutations (20,
25). The rate of cholesterol
esterification in these NPC genotypes is reduced to less than 8% of normal
fibroblasts (Fig. 1A).
Since both NPC1 and NPC2 mutants exhibit normal acyl-coenzyme A:cholesterol
acyltransferase (ACAT) activity in response to stimulation by 25-HC
(Fig. 1B), the absence
of LDL cholesterol esterification is not due to defective ACAT activity in
these cells. These results are consistent with current models for NPC1 and
NPC2 function in trafficking of LDL cholesterol out of the lysosomal
compartment.
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We next examined the effect of the NPC mutations on accumulation of cholesterol. For these experiments, we compared NPC1I1061T and NPC2G58T cells to normal fibroblasts. In lipoprotein-fed NPC1I1061T and NPC2G58T mutant fibroblasts, we find that total cellular cholesterol is elevated 1.5- and 2-fold, respectively, over normal fibroblasts (Fig. 2A). To determine whether the increased cellular cholesterol in the NPC mutants was due to defective regulation of LDL cholesterol uptake and/or de novo cholesterol synthesis, we performed the following studies. LDLr activity in the fibroblasts was measured using an established, quantitative assay that monitors the uptake of DiI-LDL, a fluorescent-tagged LDL, by flow cytometry (26, 27). With this method, uptake of DiI-LDL was abolished in the presence of 80-fold excess of unlabeled LDL, demonstrating that the DiI-LDL uptake is a specific measure of LDLr activity (data not shown). Despite the marked cholesterol overload in NPC fibroblasts, LDLr activity in the NPC mutants fails to suppress in response to LDL feeding (Fig. 2B). In comparison with normal fibroblasts, LDLr is increased 1.6- and 2-fold in the NPC1I1061T and NPC2G58T, respectively. Similar results were obtained using an 125I-LDL receptor binding assay (data not shown). These findings mirror the inability of LDL cholesterol to suppress de novo cholesterol synthesis in NPC1I1061T and NPC2G58T mutants (elevated 1.5- and 3-fold, respectively) (Fig. 2C). In response to lipoprotein starvation, the NPC2G58T mutant up-regulates LDLr activity and de novo cholesterol synthesis, responses that are inappropriate for cholesterol overloaded cells. On the other hand, the NPC1I1061T mutant only partially up-regulates LDLr activity and de novo cholesterol synthesis. This finding suggests that in the NPC1I1061T mutant, accumulated lysosomal cholesterol is partially available for regulation of cholesterol homeostatic responses.
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NPC Mutants Fail to Suppress SREBP-dependent Gene Expression—It has been proposed that the local ER cholesterol pool accessible to ACAT serves as the cholesterol pool that controls cellular cholesterol content through regulated proteolysis of SREBPs (28). To determine whether the altered LDLr activity and de novo cholesterol synthesis in NPC mutants are attributable to a failure to suppress SREBP proteolysis, we examined the effect of NPC mutations on SREBP-dependent gene transcription using an sterol regulatory element-containing reporter construct as an indicator of the status of SREBP maturation (18). For these and all subsequent experiments, we compared NPC mutants with moderate (NPC1I1061T) and severe (NPC2G58T) defects in sterol regulatory activity to normal fibroblasts. Constructs were introduced into these primary fibroblasts by nucleofection, a method that routinely affords >90% transfection efficiency in our hands (data not shown). Compared with normal fibroblasts, the NPC1I1061T mutant is resistant to suppression of SREBP-dependent activity at low concentrations of LDL (20 µg/ml), but is partially suppressed at high concentrations of LDL (60–80 µg/ml). In contrast, the NPC2G58T mutant shows no suppression of SREBP-dependent gene expression by LDL cholesterol over the range of concentrations examined (Fig. 3A). Failure to appropriately suppress SREBP-mediated gene suppression is not unexpected in NPC cells given their known cholesterol trafficking defects.
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Defects in Sterol Regulation in NPC Mutants Do Not Correlate with ER Cholesterol Levels—We hypothesized that if failure to suppress SREBP processing in NPC cells results from failure to deliver cholesterol to the ER, then ER cholesterol levels should correlate with the severity of the biochemical phenotype. We measured cholesterol levels in the ER using an in vitro esterification assay (22). In normal fibroblasts, cholesterol in the ACAT-accessible pool is reduced under conditions of lipoprotein starvation, and is elevated following treatment with either LDL cholesterol or 25-HC, consistent with the expected changes in the ER regulatory pool (Fig. 3B). In lipoprotein-fed NPC1I1061T and NPC2G58T mutant fibroblasts, the size of the ER cholesterol pool is identical to that of normal fibroblasts. Nonetheless, these cells show persistent LDLr activity and de novo cholesterol synthesis in the mutants. Similarly, in lipoprotein-starved NPC mutants, ER cholesterol content is comparable with that of normal fibroblasts. Upon LDL stimulation, the ER cholesterol levels achieved are lower in both the NPC1I1061T and NPC2G58T mutants compared with normal fibroblasts, consistent with impaired trafficking of LDL cholesterol. However, the failure to increase ER cholesterol in response to LDL is more pronounced in the NPC1I1061T than the NPC2G58T mutant (0.86 ± 0.06 versus 1.23 ± 0.13 pmol/ mg/h), whereas the defect in regulation of sterol homeostatic responses is more severe in the NPC2G58T mutant. Thus, the relative biochemical defects between the NPC mutants do not correlate with differences in steady-state ER cholesterol levels.
LXR-activated Gene Expression Is Attenuated in NPC Mutants—The disparity between ER cholesterol levels and SREBP-mediated transcriptional responses suggests that regulatory molecules other than free ER cholesterol may be important for genesis of the NPC phenotype. Recent studies demonstrate that oxysterol cholesterol metabolites are ligands for LXRs, which regulate cellular cholesterol balance by transactivation of genes that promote catabolism and elimination of excess free cholesterol (29). Treatment with LXR agonists has been shown to induce expression of sterol transporters, such as ABCA1 and ABCG5/ABCG8, as well as a number of target genes that function in the reverse cholesterol transport pathway (30, 31). To determine the effect of NPC mutations on LXR-mediated gene expression, we examined in NPC mutant fibroblasts the expression of a reporter construct driven by an LXR response element (19). In normal fibroblasts LXR activity increases with increasing concentrations of LDL cholesterol (Fig. 4). In NPC fibroblasts, on the other hand, stimulation of LXR activity in the NPC1I1061T mutant occurs only at high concentrations of LDL (60–80 µg/ml), and no stimulation of LXR activity is observed in the NPC2G58T mutant. Thus, the relative impairment of LXR-mediated gene regulation correlates with the severity of the biochemical phenotype in NPC mutants.
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Production of LDL Cholesterol-derived Oxysterols Is Defective in NPC Mutants—Taken together, the failure to suppress SREBP-dependent gene expression and the impaired LXR-mediated activity in the NPC mutants suggested a possible defect in the generation of endogenous sterol ligands (e.g. LDL-derived cholesterol oxidation products) or in the trafficking of such ligands to their site of action. To test whether oxysterol production is deficient in NPC fibroblasts, we measured the concentration of 25-HC and 27-HC in conditioned medium and cell lipid extracts from LDL fed normal fibroblasts, and NPC1I1061T and NPC2G58T mutants using GC/MS (Fig. 5A). We specifically examined the production of these cholesterol metabolites, since they are the predominant oxysterols produced by skin fibroblasts (32), they are potent suppressors of hydroxymethylglutaryl-CoA reductase (33–35), and they are known ligands for LXR (24, 36). In NPC1I1061T and NPC2G58T mutants, LDL-stimulated production of 25-HC is decreased by 34 and 77%, respectively, as compared with normal fibroblasts (Fig. 5B). LDL-stimulated production of 27-HC is decreased by 66 and 86% in the NPC1I1061T and NPC2G58T mutants, respectively. Because 25-HC and 27-HC, in contrast to 5,6- and 7-oxygenated sterols, are not major auto-oxidation products, it is unlikely that the oxysterols detected by GC/MS analysis were generated non-enzymatically during the oxysterol isolation procedure (37). Additionally, to exclude auto-oxidation in our experimental system, we performed control studies in which deuterated free cholesterol was added to the cells and media at the time of lipid extraction, and incorporation of the deuterated label into 25-HC and 27-HC was monitored by GC/MS. We were unable to detect any of the deuterated precursor in either of the oxysterol ion peaks (data not shown), indicating that our oxysterol measurements were not confounded by auto-oxidation products. Thus, the observed defects in oxysterol generation correlate with the relative decrease in LDL-stimulated esterification and the relative increase in total cholesterol in these NPC mutants.
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Exogenous Oxysterols Normalizes LDLr Activity in NPC Mutants—In light of the defect in oxysterol generation in NPC mutants, we examined whether supplementation with exogenous oxysterols is able to restore appropriate regulation of cholesterol homeostasis in these cells. We measured LDLr activity in NPC1I1061T and NPC2G58T mutants in response to LDL feeding, in the presence and absence of 25-HC, 27-HC, or both compounds. In normal fibroblasts exposure to LDL appropriately suppresses LDLr activity, and no further reduction is observed in the presence of 27-HC (Fig. 6). In contrast, LDLr activity remains elevated in LDL-fed NPC1I1061T fibroblasts (increased 2.7-fold as compared with normal fibroblasts), but in the presence of 27-HC LDLr activity is suppressed to a level comparable with that of normal fibroblasts. NPC2G58T fibroblasts are even more resistant than NPC1I1061T fibroblasts to LDL suppression (LDLr activity increased 5.2-fold). LDLr activity in these cells is only partially suppressed in the presence of 27-HC (increased 2.1-fold). Treatment with either 25-HC alone or in combination with 27-HC normalizes LDLr activity in both mutant cell lines. The underlying deficiency in production of these oxysterols, combined with the ability of exogenously supplied oxysterols to suppress LDLr activity, suggest that NPC1 and NPC2 play critical roles in generation of oxysterols in response to LDL. Furthermore, failure to generate these oxysterols appears to be central to the genesis of the defect in sterol homeostasis in NPC mutants.
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Treatment with Exogenous Oxysterols Reduces Cholesterol Accumulation in NPC Mutants—Because the addition of oxysterols is able to normalize LDLr activity in the NPC fibroblasts, we investigated whether treatment with exogenous 25-HC and 27-HC would reduce the cellular cholesterol burden, and thereby mitigate the NPC phenotype. We measured total cellular cholesterol and performed filipin staining in NPC1I1061T and NPC2G58T fibroblasts that were cultured in lipoprotein-containing medium supplemented with either 25-HC or 27-HC. After 2 days of oxysterol treatment, cholesterol accumulation is reduced in the NPC1I1061T fibroblasts to levels found in lipoprotein-fed normal fibroblasts (Fig. 7). While therapy with 25-HC and 27-HC reduces total cholesterol in the NPC2G58T mutant by 25 and 20%, respectively, total cholesterol remains elevated 1.5-fold above that of normal fibroblasts. Treatment with 25-HC and 27-HC normalizes the filipin-staining pattern in 71 and 79% of NPC1I1061T mutant cells, respectively (Fig. 8). Treatment of the NPC2G58T mutant with 25-HC, but not 27-HC, reduces filipin fluorescence, but to a lesser extent than is observed with the NPC1I1061T mutant and is unable to fully correct the filipin staining pattern. These results show that exogenous oxysterols are capable of effecting appropriate cholesterol homeostatic responses despite the sterol trafficking defect in the NPC1I1061T mutant.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In the present study we provide evidence that NPC1 and NPC2 function in the regulation of cellular cholesterol homeostasis through generation of LDL cholesterol-derived oxysterols. Our results confirm and extend the findings of Liscum et al. (15). First, we demonstrate that in response to LDL cholesterol NPC1 and NPC2, loss of function mutants fail to appropriately suppress cholesterol synthesis and uptake due to persistent activation of SREBP-dependent gene expression. Additionally, we show that LXR-mediated gene expression is attenuated in NPC mutants. Second, we measure the ER cholesterol in the ACAT-accessible pool and demonstrate that ER cholesterol levels fail to account for marked impairment in rate of cholesterol esterification and the relative biochemical defects in the NPC mutants. Third, we find that LDL cholesterol-stimulated production of 25-HC and 27-HC is deficient in both NPC1 and NPC2 mutants. Moreover, in the NPC mutants the degree of oxysterol deficiency correlates with the severity of the sterol regulatory defects. Finally, we show that treatment with exogenous oxysterols reduces cholesterol overload in both NPC1 and NPC2 mutants, and is able to correct the NPC1I1061T phenotype, the most prevalent NPC1 disease genotype (20). While it is not unexpected that a block in the processing of LDL cholesterol in NPC mutants would lead to lower cellular levels of oxysterols, our findings provide the first evidence that deficiency in oxysterol production is linked to the pathogenesis of NPC disease.
Previous studies have shown that both NPC1 and NPC2 mutants accumulate free cholesterol in lysosomes and exhibit similar cytochemical phenotypes. We now show that cholesterol overload in both NPC1 and NPC2 mutants results from the inability of LDL cholesterol to both suppress SREBP-dependent gene expression and promote LXR-mediated responses. Based on these measures, the NPC2G58T mutant has a more severe phenotype than the NPC1I1061T. To determine whether these observations reflect a general difference between NPC1 and NPC2 genotypes, it will be necessary in future studies to examine fibroblast cell lines derived from multiple different NPC1 and NPC2 genotypes. At present the few NPC2 fibroblast lines available are limiting.
How do NPC1 and NPC2 contribute to the generation of LDL cholesterol-derived oxysterols? We hypothesize that the NPC proteins channel excess LDL cholesterol to intracellular sites of oxysterol synthesis. Our findings suggest that in fibroblasts NPC1 and NPC2 function in transfer of free cholesterol substrate to both the mitochondrial sterol 27-hydroxylase and the ER/Golgi-localized cholesterol 25-hydroxylase (38). Thus, in NPC disease, the failure to generate appropriate levels of 25-HC and 27-HC prevents feedback inhibition of SREBP-dependent gene expression and prevents activation of LXR-regulated pathways. Persistent activation of LDL uptake and de novo cholesterol synthesis, coupled with impaired ABCA1-mediated cholesterol efflux, results in a net accumulation of free cholesterol. Exposure of NPC1 mutants to 7-ketocholesterol and 25-HC has been shown previously to reduce total cellular cholesterol (39). We now show that treatment with oxysterols corrects the NPC1I1061T phenotype and provide a mechanistic basis for these findings. Our findings suggest that exogenously supplied oxysterols prevent induction of the NPC phenotype by limiting cholesterol synthesis and uptake, although we cannot exclude a role for oxysterols in mobilization of free cholesterol from the NPC compartment.
The mechanism by which oxysterols suppress sterol synthesis and LDL cholesterol uptake is not well understood. It has been proposed that oxysterols, such as 25-HC, promote translocation of free cholesterol from the plasma membrane to the ER (40). Increased free cholesterol in the ER regulatory pool prevents SCAP-mediated trafficking of SREBPs to the Golgi, thereby suppressing SREBP proteolysis (17). The recent demonstration that free cholesterol, but not 25-HC, can change the conformation of SCAP in vitro lends support to this hypothesis, although the mechanism by which this conformational change contributes to the regulation of sterol homeostasis is not yet known (41). In the present study we use an in vitro esterification assay to quantify the level of cholesterol in the ACAT-accessible pool (22). Previous studies have shown that the size of the ACAT-accessible pool measured by this assay correlates well with ER membrane cholesterol in vivo (22, 39). Our finding that the ER cholesterol pool in lipoprotein-fed NPC mutants is essentially normal is in agreement with an earlier study (39). Why does the ER cholesterol pool in NPC mutants fail to sense total cellular cholesterol overload? Lange and colleagues have proposed that the size of the ER cholesterol pool is set by the needs of the plasma membrane (39). While in NPC mutants the plasma membrane cholesterol pool is normal, the flow of cholesterol between the cell surface and interior membrane compartments is impaired (39). It is possible that endogenous production of LDL cholesterol-derived oxysterols, which is defective in NPC mutants, may normally signal the needs of the plasma membrane by modulating the brisk flow of cholesterol to the ER. Alternatively, oxysterols may exert their effects on the sterol regulatory machinery through a more direct mechanism. These hypotheses will need to be tested directly in future studies.
Oxysterols are known to be potent suppressors of sterol synthesis both in cultured cells and in vivo (33–35) and are capable of activation of LXR-mediated pathways (19, 24). Previous studies have found altered sterol regulatory responses in fibroblasts from patients with cerebrotendinous xanthomatosis, a disorder caused by deficiency in sterol 27-hydroxylase (CYP27) and characterized by absence of 27-HC (32). The physiologic role of 27-HC as a regulator of whole body sterol homeostasis, though, is less clear. While in vivo studies of Cyp27–/– mice demonstrated a 2.5-fold increase in whole body cholesterol synthesis, the increase in sterol synthesis was not distributed uniformly across all tissues, suggesting that mechanisms other than production of 27-HC contribute to the regulation of sterol synthesis in these tissues (42). NPC mutants, on the other hand, fail to appropriately generate both 25-HC and 27-HC, indicative of a more widespread defect in oxysterol production that underlies the cholesterol overload phenotype. Consistent with these findings, studies in NPC1–/– mice show increased cholesterol synthesis in nearly all tissues (16).
Oxysterols not only regulate lipid and cholesterol biosynthesis, but in some tissues the synthesis and secretion of oxysterols represents a form of reverse cholesterol transport to return excess sterol to the liver to maintain tissue lipid homeostasis (38, 43). In the brain excess cholesterol is converted into 24-HC, which freely crosses the blood-brain barrier down a concentration gradient, contributing to maintenance of cholesterol homeostasis in this organ (44). In NPC disease, failure to generate oxysterols may not only prevent elimination of excess cellular cholesterol, but at the same time prevent suppression of cholesterol synthesis and lipoprotein uptake, leading to potentially toxic accumulation of free cholesterol. Purkinje cells in the cerebellum, which express high levels of both NPC1 and 24(S)-cholesterol hydroxylase (43, 45), may require higher rates of oxysterol secretion to maintain cellular homeostasis. Such a mechanism offers a possible explanation for the selective loss of this neuronal population in NPC disease (46–48). Our finding that treatment with oxysterols corrects the cellular NPC1I1061T phenotype raise the possibility such therapies targeted to the central nervous system could be used to treat NPC disease.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by an American Heart Association Scientist Development Grant.
|| Supported by National Institutes of Health Grants DK56341 and
P41-RR-00954.
Supported by grants from the National Niemann-Pick Disease Foundation, the Ara
Parseghian Medical Research Foundation, and by National Institutes of Health
Grants HL04482 and HL67773. To whom correspondence should be addressed: Center
for Cardiovascular Research, Washington University School of Medicine, Box
8086, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-8737; Fax:
314-362-0186; E-mail:
dory{at}im.wustl.edu.
1 The abbreviations used are: NPC, Niemann-Pick type C; ACAT,
acyl-CoA:cholesterol acyltransferase; DMEM, Dulbecco's modified Eagle's
medium; ER, endoplasmic reticulum; FBS, fetal bovine serum; GC/MS, gas
chromatography/mass spectrometry; 24-HC, 24(S)-hydroxycholesterol;
25-HC, 25-hydroxycholesterol; 27-HC, 27-hydroxycholesterol; LDL, low density
lipoprotein; LDLr, LDL receptor activity; LXR, liver X receptor; LPDS,
lipoprotein-deficient serum; NPC1, Niemann-Pick type C1; NPC2, Niemann-Pick
type C2; SREBP, sterol regulatory element-binding protein; SCAP, SREBP
cleavage-activating protein; WT, wild type.
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ACKNOWLEDGMENTS |
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We thank A. Fensom and M. Vanier for providing NPC mutant cell lines. We are grateful for P. Pentchev for critical review of this manuscript.
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REFERENCES |
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