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Originally published In Press as doi:10.1074/jbc.M700326200 on June 5, 2007

J. Biol. Chem., Vol. 282, Issue 31, 22525-22533, August 3, 2007
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Differential Regulation of ATP Binding Cassette Protein A1 Expression and ApoA-I Lipidation by Niemann-Pick Type C1 in Murine Hepatocytes and Macrophages*Formula

Ming-Dong Wang, Vivian Franklin, Meenakshi Sundaram, Robert S. Kiss, Kenneth Ho, Michel Gallant, and Yves L. Marcel1

From the Lipoprotein and Atherosclerosis Research Group, University of Ottawa Heart Institute, and Departments of Biochemistry, Microbiology, and Immunology, and Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Ontario K1Y 4W7, Canada

Received for publication, January 11, 2007 , and in revised form, May 11, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Niemann-Pick type C1 (Npc1) protein inactivation results in lipid accumulation in late endosomes and lysosomes, leading to a defect of ATP binding cassette protein A1 (Abca1)-mediated lipid efflux to apolipoprotein A-I (apoA-I) in macrophages and fibroblasts. However, the role of Npc1 in Abca1-mediated lipid efflux to apoA-I in hepatocytes, the major cells contributing to HDL formation, is still unknown. Here we show that, whereas lipid efflux to apoA-I in Npc1-null macrophages is impaired, the lipidation of endogenously synthesized apoA-I by low density lipoprotein-derived cholesterol or de novo synthesized cholesterol or phospholipids in Npc1-null hepatocytes is significantly increased by about 1-, 3-, and 8-fold, respectively. The increased cholesterol efflux reflects a major increase of Abca1 protein in Npc1-null hepatocytes, which contrasts with the decrease observed in Npc1-null macrophages. The increased Abca1 expression is largely post-transcriptional, because Abca1 mRNA is only slightly increased and Lxr{alpha} mRNA is not changed, and Lxr{alpha} target genes are reduced. This differs from the regulation of Abcg1 expression, which is up-regulated at both mRNA and protein levels in Npc1-null cells. Abca1 protein translation rate is higher in Npc1-null hepatocytes, compared with wild type hepatocytes as measured by [35S]methionine incorporation, whereas there is no difference for the degradation of newly synthesized Abca1 in these two types of hepatocytes. Cathepsin D, which we recently identified as a positive modulator of Abca1, is markedly increased at both mRNA and protein levels by Npc1 inactivation in hepatocytes but not in macrophages. Consistent with this, inhibition of cathepsin D with pepstatin A reduced the Abca1 protein level in both Npc1-inactivated and WT hepatocytes. Therefore, Abca1 expression is specifically regulated in hepatocytes, where Npc1 activity modulates cathepsin D expression and Abca1 protein translation rate.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Niemann-Pick type C (NPC)2 disease is an autosomal lipid storage disease resulting from mutations in either the Npc1 (95% of families) or Npc2 genes (1–3). It is characterized by progressive hepatosplenomegaly and neurodegeneration, leading to premature death (4, 5). The exact function of both proteins remains unknown, but there is much evidence that they facilitate the transport of lipids, primarily cholesterol, from late endosome to the Golgi apparatus, endoplasmic reticulum (ER), mitochondria, and plasma membrane (6, 7). Thus the storage involves the accumulation of unesterified cholesterol, sphingolipids, and other lipids within the endosomal/lysosomal compartments of cells in almost all body tissues (8). Impaired cholesterol traffic in NPC disease cells prevents the normal down-regulation of endogenous cholesterol synthesis and the low density lipoprotein (LDL) receptor (9, 10), through the interruption of normal cholesterol regulation by sterol regulatory element-binding protein at the ER and the interruption of generation of LDL cholesterol-derived oxysterols in the mitochondria (11).

Liver dysfunction in patients with NPC disease is more common than previously believed (12) and understanding the mechanism of cholesterol homeostasis in hepatocytes is most important for the treatment of this disease. In Npc1-null mice, biliary secretion is altered (13, 14) and hepatocyte plasma membrane and ER compartments accumulate large amounts of cholesterol (15, 16). The abnormal cholesterol homeostasis in Npc1-null hepatocytes leads to increased secretion of cholesterol-rich VLDL (17). However, one report indicated that NPC1-/- subjects do not exhibit elevated apoB lipoproteins but rather low HDL-C (18). Because the liver is a major expression site for both apoA-I and Abca1 (19, 20) and a main contributor to maintaining plasma level of HDL (21, 22), we hypothesized that HDL secretion might be impaired by Npc1 deficiency. Here we studied the effect of Npc1 ablation on hepatocyte secretion of lipidated apoA-I and compared lipid efflux to apoA-I in Npc1-/- hepatocytes and macrophages. We show that Npc1 deficiency strikingly increases efflux and HDL formation in hepatocytes, whereas it decreases efflux in macrophages. We have explored the regulatory mechanisms involved and demonstrate a post-transcriptional up-regulation of Abca1 in hepatocytes linked to increased Ctsd expression and Abca1 translation rate.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—[1,2-3H]Cholesterol, [5-3H]mevalono-lactoneRs, choline [methyl-3H]chloride, and [35S]methionine were obtained from PerkinElmer Life Sciences. Williams medium E, HepatoZYME-SFM, and antibiotic-antimycotic were purchased from Invitrogen. Rabbit polyclonal anti-human apoA-I antibody was purchased from Calbiochem. Monoclonal antibodies directed against human apoA-I (a combination of 4H1 (against the extreme N terminus) and 5F6 (against the central region)) were described previously (23) and biotinylated with sulfo-NHS-biotin from Pierce. Streptavidin-horseradish peroxidase conjugate and protein G-Sepharose were obtained from Amersham Biosciences.

Animals and Primary Hepatocyte Cultures—Breeder Npc1+/- mice were purchased from Jackson Laboratories and bred in our animal facility. All mice were maintained on a normal chow diet in a 12-h light/12-h dark schedule and used between the ages of 6 and 8 weeks. All experiments performed were in accordance with protocols approved by the University of Ottawa Animal Care Committee. Primary hepatocytes were isolated from these mice by liver collagenase perfusion according to established protocols (24). Briefly, the cells were plated in fibronectin-precoated (25 µg/well) 6-well plates at an initial density of 1.5 x 106 cells/well in Williams medium E containing penicillin (100 units/ml), streptomycin sulfate (100 units/ml), Fungizone (250 ng/ml), and 10% fetal bovine serum (25, 26).

Generation of Macrophages—Bone marrow-derived macrophages were flushed from mouse femurs. Macrophages were generated by incubating bone marrow cells (106 cells/ml) with DMEM of 10% FBS complemented with 15% L929 conditioned medium for 7 days (27).

Hepatocyte Labeling and ApoA-I Lipidation—The hepatocytes following a 5-h attachment period with 10% FBS (1 ml/well) were washed in serum-free Williams medium E (3 x 2 ml) and incubated with 2 ml of HepatoZYME-SFM containing an antibiotic/antimycotic mixture, 10 µCi/ml [3H]mevalonate, or 5 µCi/ml [3H]cholesterol delivered with LDL (50 µg/ml) or [3H]choline (5 µCi/ml) for 40 h. For exogenous apoA-I-mediated efflux, cells were washed once and incubated with 1 ml/well DMEM containing 10 µg/ml human recombinant apoA-I (28). For endogenous apoA-I-mediated efflux assay, 24 h after labeling, the medium was removed, and the cells in serum-free Williams medium E were infected for 1 h with either the recombinant adenovector expressing human apoA-I (Ad5-Ad AI) or as control, adeno-luciferase (Ad5-Ad Luc) at a multiplicity of infection of 75:1 plaque-forming units/cell (25, 26). After a 1-h infection, hepatocytes were incubated for an additional 24 h with the original labeling medium, then washed and incubated for the indicated period with efflux medium as described above.

Immunoprecipitation of ApoA-I-associated [3H]Cholesterol or [3H]Choline-Phospholipid—ApoA-I was immunoprecipitated either directly from the efflux medium or from lipoprotein fractions separated by fast protein liquid chromatography with a polyclonal anti-human apoA-I antiserum and pulled down with protein G-Sepharose. Control immunoprecipitations were carried out with equal volumes of an anti-human apoB antiserum from sheep, which does not cross-react with murine apoB, or with the time 0 efflux medium immunoprecipitated with anti-human apoA-I antiserum. The immunoprecipitates were collected as described previously (25). Radioactivity of cholesterol and phospholipid associated with human apoA-I was quantified by scintillation counting.

Lipid Efflux in Macrophages—Unless indicated in legends, labeling conditions are as follows. Macrophages were washed three times with plain DMEM and then labeled with LDL or acetylated LDL (50 µg of protein/ml) that had been preincubated with 5 µCi/ml [3H]cholesterol in 1% FBS of DMEM for 24 h. The cells were equilibrated with 2 mg/ml bovine serum albumin overnight. For labeling with [3H]mevalonate (10 µCi/ml) or [3H]choline (5 µCi/ml), the cells were incubated for 40 h in DMEM with 1% FBS. Efflux to apoA-I (10 µg in 2 mg/ml bovine serum albumin medium) was monitored for 3–5 h (29). Efflux to mbeta-CD (10 mM in 2 mg/ml bovine serum albumin DMEM) was carried out for 15 min at 37 or 4 °C (27).

Lipid Analysis—Cellular lipids were extracted (30), separated by thin layer chromatography (TLC) using hexane:diethyl ether:acetic acid (105:45:1.5) as running solvent on Sil-G TLC plates (EMD Chemicals, Darmstadt). Lipid bands were detected by exposure to iodine vapors, scraped off the TLC plate, and radioactivity measured with a scintillation counter. For total cholesterol determination, cells were washed with cold phosphate-buffered saline, cholesterol was extracted by isopropyl alcohol, and measured by colorimetric assay (Wako Chemicals, Richmond, VA).

RT-PCR—For transcription analysis in ex vivo hepatocytes or macrophages, the cells were transferred immediately after isolation into a tube with ice-cold DMEM containing 10% FBS and total RNA was isolated. In some experiments, hepatocytes also were cultured in media with 5% lipoprotein-deficient serum or 10% FBS or in HepatoZYME for 2 to 5 days. Total RNA from bone marrow-derived macrophages was isolated under the efflux conditions. DNA was removed by incubation with DNase and cDNA was reverse transcribed by addition of 1 µl of Moloney murine leukemia virus reverse transcriptase (Invitrogen) in the buffer with 2 µl of decamers, 1 µl of dNTP (25 mM), 5 µl of H2O plus 5 µl (0.5 µg) of mRNA and incubated for 1 h at 42°C. The products of 0.5 µg of target gene were amplified in a PCR tube with a specific set of primers (for one or more target genes) and one pair of internal control glyceraldehyde-3-phosphate dehydrogenase primers (see supplementary materials Table S1). All primers were designed based on same annealing temperature (60 °C) and the PCR product size for each gene was preset. The PCR was run for 30 cycles. Specificity of the primers of each target gene was tested by individual PCR before mixing with internal control primers or primers of other genes. The bands of final PCR products were analyzed with Quantity One software after separation by 1% agarose gel electrophoresis and staining with ethidium bromide.

Western Blotting—Cellular proteins were solubilized in RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 1% deoxycholic acid, 1% Triton X-100, mixture protease inhibitors (Roche)), electrophoresed on a 6% SDS-polyacrylamide gel, and transferred to nitrocellulose at 125 V for 4 h. Rabbit anti-Abca1 and Abcg1 antibodies (Novus Biologicals, 1:500 dilution) were used for detection, and an anti-rabbit antibody conjugated with horseradish peroxidase (Amersham Biosciences) was used as secondary antibody. The immunoblots were developed by incubating with SuperSignal chemiluminescent substrate (Pierce). The picture was taken with a mutiImageTM (AlphaInnotech, CA).

Measurement of Abca1 Synthesis—Newly isolated hepatocytes were plated onto fibronectin pre-coated 60-mm Petri dishes and incubated with 10% FBS DMEM for 5 h to allow the cells to attach. Before labeling with [35S]methionine/cystine (100µCi/ml), the cells were cultured with methionine/cystine-deficient medium for 1 h to increase the labeling efficiency. After labeling with various time intervals, cells were washed twice with cold phosphate-buffered saline and lysed with 0.5 ml of 1% SDS-RIPA buffer. The cell lysate was reconstituted with 4.5 ml of RIPA buffer. Total cellular Abca1 protein was immunoprecipitated with rabbit anti-mouse Abca1 polyclonal antibody. The precipitated Abca1 was solubilized and separated by SDS-PAGE. The intact and cleaved Abca1 bands were cut and counted.

Statistics—Significance was evaluated by Student's t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Npc1 Deficiency Increases the Lipidation of Newly Synthesized ApoA-I in Hepatocytes—Whereas Npc1 inactivation is well known to impair intracellular cholesterol traffic and decrease cholesterol efflux to apoA-I in macrophages or fibroblasts (6, 18, 31, 32), its effect on the lipidation of apoA-I in liver, the major site of HDL synthesis, is unknown. Here, we evaluated the effect of Npc1 inactivation on the lipidation of endogenous apoA-I in hepatocytes that were labeled with either endogenously synthesized lipids or exogenous lipids delivered by lipoproteins. Surprisingly, the lipidation of endogenously synthesized apoA-I with LDL-derived cholesterol or de novo synthesized cholesterol or de novo synthesized phospholipids was not impaired in Npc1-null hepatocytes; rather it was significantly increased by 68, 310, and 840%, respectively (Fig. 1, A–C).

We also compared the effect of Npc1 inactivation on the lipidation of exogenously added apoA-I in hepatocytes and macrophages, pre-labeled with exogenously added or de novo synthesized lipids. Compared with control, exogenous apoA-I lipidation by cholesterol was significantly increased by 2.6-, 3.2-, 4.8-, and 6.0-fold in NPC1-null hepatocytes labeled with [3H]cholesterol delivered by chylomicrons, VLDL, HDL, and LDL, respectively (Fig. 2A). Similarly, the lipidation of exogenous apoA-I by newly synthesized cholesterol and phospholipids was significantly increased by 5- and 6-fold, respectively, in Npc1-null hepatocytes compared with wild type (Fig. 2B). These results contrast with those in macrophages, where the lipidation of exogenous apoA-I was reduced by 40 and 80% in Npc1-null cells labeled with LDL and acetylated LDL, respectively (Fig. 2C). As well, exogenous apoA-I lipidation by newly synthesized cholesterol or phospholipids was decreased by 30 and 55%, respectively, in Npc1-null versus wild type macrophages (Fig. 2D). These observations indicate that lipid transport and homeostasis in hepatocytes and macrophages is differentially affected by Npc1 inactivation.


Figure 1
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  		FIGURE 1.
Endogenous apoA-I lipidation is increased in Npc1-null hepatocytes. Hepatocytes were isolated from Npc1-null or WT mouse and labeled with [3H]cholesterol-LDL (A), [3H]mevalonate (B), or [3H]choline (C) in hepatozyme (5 µCi/ml) for 40 h. The cells were transfected with adenovirus encoding human apoA-I or with an adenovector encoding luciferase as a control during the last 24 h of the labeling period. The cells were then washed with plain Williams medium twice; 1 ml of fresh hepatozyme was added to each well and the cells incubated for 4 h. ApoA-I lipidation was determined after immunoprecipitation with anti-human apoA-I antibody. Cellular cholesterol labeling was not affected by infection with adenovirus apoA-I compared with control virus. The results are presented as apoA-I-associated counts adjusted for cellular protein. This experiment was repeated three times with similar results.

 
Cholesterol Traffic from Intracellular Compartments to the Plasma Membrane Is Similarly Impaired in Npc1-null Hepatocytes and Macrophages—Npc1 regulates cellular cholesterol traffic from late endosomes to other compartments, including plasma membrane and ER (1, 6, 33). The decreased cholesterol efflux to apoA-I observed in macrophages, human fibroblasts, and Chinese hamster ovary cell lines expressing mutant Npc1 is commonly ascribed to impaired cholesterol traffic of late endosomes to plasma membrane (1, 6, 33), where a pool of Abca1 is situated and where apoA-I-mediated lipid efflux occurs (34, 35). To test if cholesterol traffic from intracellular compartments to plasma membrane is differentially impaired in Npc1-null hepatocytes versus macrophages, cells were labeled with [3H]cholesterol delivered by LDL (Fig. 3, A and C) or with [3H]mevalonate (Fig. 3, B and D). Cholesterol efflux to mbeta-CD at 4 °C, which measures labeled cholesterol associated with the plasma membrane (27), was significantly and similarly reduced by more than 30% in Npc1-null hepatocytes (Fig. 3A) and macrophages (Fig. 3C) labeled with LDL. Cholesterol efflux to mbeta-CD at 37 °C, which measures labeled cholesterol associated with both plasma membrane and recycling compartments (26, 27), was also reduced by about 40% in LDL-labeled Npc1-null hepatocytes and macrophages. Similar results were obtained with hepatocytes and macrophages metabolically labeled with [3H]mevalonate (Fig. 3, B and D), where cholesterol efflux to mbeta-CD at 4 °C and at 37 °C was proportionately lower. Therefore, the similar impairment of cholesterol traffic in Npc1-null hepatocytes and macrophages cannot account for the difference in cholesterol efflux between Npc1-null hepatocytes and macrophages.


Figure 2
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  		FIGURE 2.
Lipid efflux to exogenous apoA-I in hepatocytes and macrophages is differentially regulated by Npc1 inactivation. Primary hepatocytes (A and B) or bone marrow-derived macrophages (C and D) were isolated from WT or Npc1-null mice. The cells were labeled with [3H]cholesterol carried by various lipoproteins (A and C) or by preincubation with [3H]mevalonate or [3H]choline to label endogenous lipids (B and D). Exogenous apoA-I lipidation in hepatocytes was determined as described in the legend to Fig. 1. The experiments with hepatocytes were repeated three times and the data pooled. The -fold change was calculated from the ratio of efflux in Npc1-null versus WT hepatocytes (-fold change). The experiments with macrophages were also repeated 3 times and apoA-I lipidation expressed as the percent radioactivity of total cellular radioactivity associated with apoA-I and corrected for cellular protein.

 


Figure 3
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  		FIGURE 3.
Cholesterol traffic is similarly impaired by Npc1 inactivation in both mouse primary hepatocytes and mouse primary macrophages. Primary hepatocytes (A and B) or bone marrow-derived macrophages (C and D) were isolated from WT or Npc1-null mice. The hepatocytes were labeled with [3H]cholesterol carried by LDL (A) or with [3H]mevalonate (B) for 40 h. The macrophages were labeled with [3H]cholesterol carried by LDL (C) or [3H]mevalonate (D) for 24 h. Cholesterol efflux to mbeta-CD was carried out at 4 or 37 °C for 15 min. The efflux was calculated as a percentage of counts in the media divided by the total counts in cells and supernatant.

 
Abca1 and Abcg1 Expression Is Up-regulated by Npc1 Inactivation in Hepatocytes but Not in Macrophages—The similar defect in cholesterol traffic in hepatocytes and macrophages suggested that the difference observed for apoA-I lipidation between these cells could be related to the regulation of transporters. Under basal conditions in complete medium, Abca1 protein was greatly up-regulated (3–7-fold) in Npc1-null hepatocytes compared with control (Fig. 4), and addition of LDL further increased Abca1 expression independently of Npc1 expression (data not shown). This increase in Abca1 protein was independent of the hepatocyte culture conditions and was also observed in media with 10% FBS, 5% LPDS or synthetic medium (Hepatozyme; lacking cholesterol containing serum) and in culture for 1–3 days (data not shown). In contrast, Abca1 protein was decreased in Npc1-null macrophages compared with wild type (Fig. 4 and Table 1) and, while loading the cells with acetylated LDL increased Abca1 protein in wild type macrophages as expected (36), it had no effect on Abca1 in Npc1-null macrophages. Abcg1 is a transporter that mediates efflux to HDL and is highly expressed in both hepatocytes and macrophages (37). We evaluated whether its expression would be differentially regulated by Npc1 in theses cells. Like Abca1, Abcg1 protein was highly significantly increased in Npc1-null hepatocytes but not in macrophages (Fig. 4).


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  		TABLE 1
Effect of Npc1 inactivation on gene expression measured by RT-PCR in hepatocytes and macrophages (fold change ± S.D.) The expression of 80 lipid metabolism related genes was determined by RT-PCR. Here we present the results for selected genes, assayed in two experiments each in triplicate. A number >1.0 indicates up-regulation, and a number <1.0 indicates down-regulation. The numbers represent the -fold change in expression in Npc1-null versus control cells. Values are expressed in reference to an internal control (Gapdh).

 


Figure 4
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  		FIGURE 4.
Npc1 deficiency leads to increased Abca1 and Abcg1 expression in hepatocytes, but not in macrophages. Total cellular protein was isolated from hepatocytes (upper panel) or macrophages (lower panel) obtained from WT or Npc1-null mice. Western blots (WB) were performed using antibodies against Abca1 and Abcg1. Equal amounts of protein were loaded onto the gels for comparison of WT or Npc1-null cells. The results presented are representative of 10 different experiments. The mRNA levels of Abca1 and Abcg1 were determined by RT-PCR. The number below each band indicates the -fold change against control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 


Figure 5
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  		FIGURE 5.
Ctsd mRNA and protein are up-regulated by Npc1 deficiency in hepatocytes but not in macrophages. Ctsd mRNA was measured by RT-PCR with the mRNA isolated from WT and Npc1-null hepatocytes or macrophages (top panel). Ctsd protein was measured by Western blot of hepatocyte cell lysates (bottom panel). These results are representative of two separate experiments. The number below each band indicates the -fold change against control. WB, Western blot. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

 
To test if the regulation of these Abc transporters by Npc1 also occurred transcriptionally, mRNA levels of Abca1 and Abcg1 were determined by RT-PCR. Whereas Abcg1 mRNA was markedly and significantly increased in Npc1-null hepatocytes, Abca1 mRNA was only modestly but significantly increased (Fig. 4 and Table 1). In contrast, transcriptional expression of Abca1 and Abcg1 in macrophages was slightly reduced. These results suggested that unlike Abcg1 regulation, the up-regulation of Abca1 observed in Npc-null hepatocytes was mostly post-transcriptional.

The Increased Abca1 Transcription in Npc1-null Hepatocytes Is Independent of Regulation by Lxr{alpha}, Lxrbeta, and Retinoid X Receptor {alpha}—To elucidate the origin of the differential regulation of Abca1 and Abcg1 expression, the expression of their known transcriptional regulatory factors (38, 39) and of other major genes for lipid homeostasis were determined by RT-PCR. The expression of Lxr{alpha}, Lxrbeta, and retinoid X receptor {alpha} were not significantly altered in Npc1-null hepatocytes compared with wild type, and furthermore, the expression of Lxr{alpha} target genes, such as Abcg5/8, sterol regulatory element-binding protein-1c (Srebp), stearoyl-CoA desaturase 1 (Scd1), CYP7{alpha}, and CYP27{alpha}, were not up- but down-regulated (Table 1), again suggesting that Lxr{alpha} was not up-regulated and that the increased expression of Abca1 in Npc1-null hepatocytes was not linked to the transcriptional up-regulation by Lxr{alpha} activation.

The Differential Expression of Genes Regulating Lipid Transport and Homeostasis in Hepatocytes and Macrophages Indicates a Tissue-specific Regulation of Abca1 Expression—The observation that Abca1 mRNA is not as up-regulated as Abca1 protein indicates that post-transcriptional regulation of Abca1 may be involved. Recently, we identified a lysosomal protease, cathepsin D (Ctsd), whose expression positively regulated Abca1 protein expression (40). Here, Ctsd expression in Npc1-null hepatocytes and macrophages was measured by RT-PCR and Western blot (Fig. 5). Ctsd mRNA and protein levels were significantly up-regulated in Npc1-null hepatocytes compared with control. In contrast, Ctsd basal expression was high in control macrophages as reported earlier (41), and its expression in Npc1-null macrophages was decreased compared with control (Fig. 5). These results suggest that Ctsd can regulate the Abca1 protein level in a tissue-specific manner.

To confirm that Ctsd is positively involved in the up-regulation of Abca1 in Npc1-null hepatocytes, the Ctsd-specific inhibitor, pepstatin A, was used. The results show that pepstatin A inhibits Abca1 in WT and Npc1-null hepatocytes (Fig. 6) in a dose-dependent fashion. These results indicate that Ctsd is involved in Abca1 up-regulation. Interestingly, even at the highest dose of pepstatin A, the suppression of Abca1 protein expression in Npc1-null hepatocytes was not as effective as in WT hepatocytes, indicating other mechanisms may also be involved in the up-regulation.

Because the mRNA level of Abca1 is modestly increased in Npc1-null hepatocytes, unlike the protein level, which is increased several folds, the rate of Abca1 translation was measured by incubation of the hepatocytes with [35S]methionine/cystine. Surprisingly, the Abca1 translation rate was also significantly increased in Npc1-null hepatocytes (Fig. 7). The profile of radioactivity distribution in the full size of Abca1 band (Fig. 7A) and the cleaved Abca1 bands (Fig. 7, B–D) was similar in Npc1-null and WT, suggesting that Abca1 degradation rates were unaffected by Npc1 activity. This conclusion was further confirmed by a cycloheximide chase experiment, where Abca1 translation in Npc1-null or WT hepatocytes was blocked with cycloheximide. After 1 and 3 h, the residual Abca1 band was about 60 and 35% of the time 0 value in either WT or Npc1-null hepatocytes. These results suggest that the increased Abca1 expression in Npc1-null hepatocytes is contributed by increased translation rate and increased Ctsd activity, but not by an increased half-life of the Abca1.


Figure 6
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  		FIGURE 6.
CtsD-specific inhibitor pepstatin A decreases Abca1 expression in a dose-dependent manner. Hepatocytes were isolated from Npc1-null or WT mice. After attachment in 6-well plates, the cells were further cultured for 24 h in the presence of various doses of pepstatin A (0, 10, and 20 µg/ml). The cell protein was harvested and analyzed by Western blot. The number below each band indicates the -fold change against control.

 


Figure 7
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  		FIGURE 7.
Abca1 translation rate is increased in Npc1-null hepatocytes. Newly isolated hepatocytes were plated into fibronectin pre-coated 60-mm Petri dishes and labeled and processed as described under "Experimental Procedures." Total cellular Abca1 was immunoprecipitated with anti-mouse Abca1 polyclonal antibody. The precipitated Abca1 was solubilized and separated by SDS-PAGE. The full size and cleaved Abca1 bands were cut and counted. A, B, C, and D, represent the 220, 160, 110, and 90 kDa bands of Abca1, respectively.

 
We also screened by RT-PCR the expression of a number of lipid metabolism-related genes (Table 1 and data not shown). Many genes, measured in several replicate experiments, display a differential expression profile in Npc1-null hepatocytes compared with Npc1-null macrophages, and others are similarly up- or down-regulated, but with differing magnitude in the two cell types (Table 1). These data further support the conclusion that the regulation of Abca1 is tissue-specific and differentially responsive to the inactivation of Npc1 in hepatocytes and macrophages.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here, we have demonstrated that both endogenous apoA-I lipidation and exogenous apoA-I-mediated lipid efflux are markedly increased by Npc1 inactivation in hepatocytes, in contrast to macrophages where apoA-I-mediated efflux is decreased. The difference in apoA-I lipidation is independent of lipid accumulation, because cultured Npc1-null hepatocytes and bone marrow-derived macrophages similarly accumulated lipids, including free cholesterol and sphingomyelin (data not shown). However, cholesteryl esters accumulated in Npc1-null hepatocytes as reported by others (17), but decreased in Npc1-null macrophages compared with wild type. The increased apoA-I lipidation in Npc1-null hepatocytes is linked primarily to the increased Abca1 protein level, which is only partly explained by increased Abca1 mRNA expression. Effectively, the translation rate of Abca1 in Npc1-null hepatocytes is significantly increased (Fig. 7) and largely explains the increased Abca1 expression in Npc1-null hepatocytes. We have also shown that Npc1 inactivation increases Ctsd expression both transcriptionally and post-transcriptionally in Npc1-null hepatocytes, whereas no such effect is seen in macrophages. Ctsd, an aspartic protease involved in the degradation of proteins in the lysosome-late endosome compartment, has been recently identified by our laboratory as a positive enhancer of Abca1 protein expression (40). Suppression of Ctsd expression by a specific inhibitor, pepstatin A, or by specific short hairpin RNA reduces Abca1 expression and apoA-I-mediated cholesterol and phospholipid efflux in a dose-dependent manner. The mechanism of action of Ctsd includes the activation of prosaposins and its maturation into saposins, which regulates transport of glycosphingolipids and cholesterol through the late endosomes, which in turn regulates Abca1 expression and activity (40). Here we demonstrated that Ctsd activity was also responsible for the increased Abca1 observed in Npc1-null hepatocytes (Fig. 6). Whereas Ctsd activity can by itself suffice to increase Abca1 levels, we also observed that ablation of Npc1 elicits distinct transcriptome profiles in hepatocytes and macrophages (Table 1), which supports the specific regulation of Abca1 and Abcg1 in these two cell types. In addition to the increase of Ctsd and Abca1 protein levels, the down-regulation of stearoyl-CoA desaturase 1 in Npc1-null hepatocytes but not in macrophages may change the membrane cholesterol organization supporting Abca1-mediated cholesterol efflux. The enhanced neutral cholesterol hydrolase activity may contribute to the mobilization of cytoplasmic cholesteryl esters and increase the availability of free cholesterol for efflux via Abca1 (Table 1) (42, 43). Ctsd regulation by Npc1 is clearly cell specific: whereas its basal level is very low in control hepatocytes, it is markedly increased by both transcriptional and post-transcriptional mechanisms by Npc1 inactivation. In contrast, Ctsd is highly expressed in macrophages but unaffected by Npc1 expression (Fig. 5). Why Ctsd is activated in Npc1-null hepatocytes but not in macrophages is not clear. The ceramide produced by acid sphingomyelinase directly binds and activates Ctsd (44), which up-regulates Abca1, possibly contributing to its downstream effect on Abca1 presentation to plasma membrane (45). However, the reduction of acid sphingomyelinase mRNA expression measured by RT-PCR in both Npc1-null hepatocytes and macrophages does not favor such a pathway (data not shown). In further studies, it will be of interest to evaluate if the varied causes of late endosomal cholesterol accumulation, such as progesterone treatment (46, 47), Npc2 inactivation (6), Gulp expression (48), or Rab7 inactivation (49), equally stimulate Ctsd expression.

Protein kinases, notably in the PKA pathway, do not appear to be major contributors to the increased Abca1 in Npc1-null hepatocytes. The major inducer of these kinases is the interaction of apoA-I and Abca1 at the plasma membrane (50), but the increased Abca1 expression in Npc1-null hepatocytes observed in this study is independent of the addition of exogenous apoA-I, and the levels of endogenous apoA-I secretion or expression in hepatocytes in vitro and in vivo are similar in both Npc1-null and wild type mice (13). Furthermore, the potent PKA stimulator, cAMP, failed to increase apoA-I lipidation in hepatocytes (data not shown). Interestingly, recent evidence showed that intracellular adaptors containing PDZ domains, {alpha}1,beta1-syntrophins, modulate the stabilization of Abca1 proteins in hepatic cells, macrophages, and transfected cell lines via binding to the KESYV motif at the C terminus of Abca1 (51, 52). The mechanisms by which Abca1 interacts with these adaptor proteins and the subsequent reduction of Abca1 protein degradation are unknown. Further studies will be required to identify the factors that bridge the gap between the lysosome protease Ctsd up-regulation and the binding of those adaptor proteins to Abca1 in Npc1-null hepatocytes.

It is noteworthy and unexpected that Abcg1 and Abca1 regulation differs in Npc1-null hepatocytes compared with wild type cells (Table 1). The synthetic Lxr ligand T091713 stimulates the expression of both Abc transporters in these hepatocytes (data not shown) in agreement with other studies (53–56). The up-regulation of Abcg1 expression in Npc1-null hepatocytes is mainly transcriptional, whereas Abca1 expression is largely dependent on post-transcriptional mechanisms. A differential regulation was also observed in human macrophages (57), in which the advanced glycation end products of bovine serum albumin decreased Abcg1 expression and cholesterol efflux to HDL, but did not affect Abca1 expression and apoA-I-mediated efflux. We also observed that lipopolysaccharide treatment reduces Abcg1 mRNA expression, but not Abca1 mRNA in mouse hepatocytes, whereas TNF{alpha} treatment reduces the mRNA levels of both transporters.3 It is clear therefore that the transcriptional factors regulating expression of these transporters are not identical, particularly in the liver.

The LDL receptor pathway, through complex regulated steps, integrates the cellular homeostasis of exogenous and endogenous cholesterol (58). Free cholesterol normally released from the late endosome-lysosome is transported to the ER and down-regulates cholesterol synthesis, but this process is impaired in NPC disease. Cholesterol homeostasis in the macrophage as a function of Npc1 activity is consistent with this paradigm. However, in the Npc1-null hepatocyte, free cholesterol elicits a regulatory response that suggests leakage from the lysosome by an unknown mechanism. As reported by others (17), we observed that cholesterol secretion into the media of cultured Npc1-null hepatocytes was increased (data not shown). This increase reflects the increase in apoB synthesis and secretion of cholesterol-rich VLDL (17). Here, we showed that apoA-I lipidation and HDL secretion are also increased in Npc-1 hepatocytes, in keeping with the increased HDL cholesterol levels and formation of larger HDL particles determined by fast protein liquid chromatography in Npc1-null mice (8, 13). In contrast, human NPC homozygote subjects apparently exhibit a moderate decrease in plasma HDL levels (18). It is unclear whether this discrepancy reflects the effects of chronic illness, a species difference, or methodologies for lipoprotein quantification. In the latter study, HDL levels were measured not by fast protein liquid chromatography but by routine clinical laboratory methods. In the murine system, all evidence is consistent with increased HDL-C. Indeed, HDL-C levels and HDL size are further increased in Npc1-null mice fed a high fat diet (13) and in Npc1-null mice crossed with LDL receptor null mice (8). Amigo and colleagues (13) also showed that Abca1 expression was up-regulated in Npc1-null liver homogenates in agreement with our results in cultured hepatocytes. Because the secretion of cholesterol-rich VLDL by Npc1-null hepatocytes is accompanied by up-regulation of apoB synthesis (17), we considered that the increased lipidation of apoA-I might be associated with up-regulation of endogenous apoA-I and apoE. However, the mRNA levels of apoA-I and apoE are not altered in Npc1-null hepatocytes (data not shown), in agreement with a previous report (13). Thus, the major effect of Npc1 inactivation in hepatocytes on the HDL secretion is a stimulation of the apoA-I lipidation pathway.

Taken together, our study demonstrates that regulation of Abca1 expression is tissue specific. Inactivation of Npc1 in hepatocytes up-regulates Abca1 expression, largely by post-transcriptional mechanisms, including up-regulation of Ctsd, increasing Abca1 translation rate, and down-regulation of stearoyl-CoA desaturase 1, but independent of the Lxr{alpha}-related pathway. Because plasma HDL-C levels are predominantly regulated by the expression of hepatic apoA-I and Abca1, the observation that Npc1 activity regulates hepatic apoA-I lipidation and HDL secretion in a cell-specific manner is of major importance. The specific regulation of hepatic Ctsd is one such mechanism, but the association of Abca1 with syntrophins (51, 52), which can control Abca1 localization and transport in a cell-specific manner, might also play a role.


   FOOTNOTES
 
* This work was supported in part by Canadian Institutes of Health Research Grant 44359 (to Y. L. M.). 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. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1. Back

1 To whom correspondence should be addressed: 40 Ruskin St., Ottawa, Ontario K1Y 4W7, Canada. Tel.: 613-761-5254; Fax: 613-761-5281; E-mail: ylmarcel{at}ottawaheart.ca.

2 The abbreviations used are: NPC1, Niemann-Pick type C1; Abca1, ATP binding cassette protein A1; apoA-I, apolipoprotein A-I; Ctsd, cathepsin D; ER, endoplamic reticulum; HDL, high density lipoprotein; LDL, low density lipoprotein; Lxr, liver X receptor; mbeta-CD, methyl-beta-cyclodextrin; PKA, protein kinase A; VLDL, very low density lipoprotein; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; RT, reverse transcriptase; WT, wild type. Back

3 M.-D. Wang, V. Franklin, M. Sundaram, R. S. Kiss, K. Ho, M. Gallant, and Y. L. Marcel, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Zemin Yao for support with the methionine labeling experiments, Drs. Ruth McPherson and Ross Milne for critical reading of the manuscript, and Roch Brunet for technical assistance.



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