NPC2 Is Expressed in Human and Murine Liver
and Secreted Into Bile: Potential Implications for
Body Cholesterol Homeostasis
Andrés Klein, Ludwig Amigo, Marı́a José Retamal, Marı́a Gabriela Morales, Juan Francisco Miquel,
Attilio Rigotti, and Silvana Zanlungo
The liver plays a critical role in the metabolism of lipoprotein cholesterol and in controlling
its elimination through the bile. Niemann-Pick type C 2 (NPC2), a cholesterol-binding
protein, is key for normal intracellular trafficking of lipoprotein cholesterol, allowing its exit
from the endolysosomal pathway into the metabolically active pool of the cell. In addition,
NPC2 is a secretory protein from astrocytes and epididymal cells. Although NPC2 mRNA is
detected in the liver, plasma and biliary NPC2 protein levels and function have not been
reported. This study demonstrates that NPC2 is present in murine and human plasma and
bile. In addition, hepatic NPC2 protein expression was dramatically increased in NPC1deficient mice but not regulated by cholesterol feeding or pharmacological modulation of
various nuclear receptors involved in cholesterol and bile acid metabolism. Interestingly,
biliary NPC2 levels were 3-fold increased in gallstone-susceptible C57BL6/J versus gallstone-resistant BALB/c mice. Furthermore, NPC2 was exclusively found in the cholesterol
pro-nucleating ConA-binding fraction of human bile. In conclusion, NPC2 is secreted from
the liver into bile and plasma, where it may have a functional role in cholesterol transport in
normal and disease conditions. (HEPATOLOGY 2006;43:126-133.)
N
iemann Pick type C (NPC) disease is a fatal,
autosomal recessive lipid storage disorder characterized by cholesterol accumulation in the
liver, spleen, and central nervous system.1,2 Mutations in
NPC1 and NPC2 genes are responsible for this disease
condition.3,4 NPC1 encodes an intracellular transmembrane protein with a putative sterol sensing domain similar to those found in hydroxymethylglutaryl-coenzyme A
reductase and sterol regulatory element binding protein
cleavage-activating protein,3,5 which are known to play
Abbreviations: NPC, Niemann-Pick type C; NPC1, Niemann-Pick C1 protein;
NPC2, Niemann-Pick C2 protein; LDL, low-density lipoprotein; NPC1 (⫺/⫺)
mice, NPC1-deficient BALB/c mice; wild-type mice,control BALB/c mice; Con-A,
concanavalin–A; PNGaseF, peptide N-glycosidase F.
From the Departamento de Gastroenterologı́a, Facultad de Medicina, Pontificia
Universidad Católica, Santiago, Chile.
Received May 11, 2005; accepted October 9, 2005.
Supported by Fondo Nacional de Desarrollo Cientı́fico y Tecnológico (FONDECYT, Grant #1030415 to SZ) and Dirección de Investigación, Facultad de
Medicina, PUC (Programa Inmersión de Verano, Grant #02/05 to MJR).
Address reprint requests to: Dr. Silvana Zanlungo, Departamento de Gastroenterologı́a, Pontificia Universidad Católica de Chile, Marcoleta 367, Casilla 114-D,
Santiago, Chile. E-mail: silvana@med.puc.cl; fax: (56) 2-639 7780.
Copyright © 2005 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/hep.20985
Potential conflict of interest: Nothing to report.
126
important roles in maintaining cellular sterol balance.
Conversely, NPC2 encodes a small soluble lysosomal cholesterol-binding protein.4,6-8 Thus, the pathogenesis of
NPC disease is closely related to abnormal cellular cholesterol transport and deposition.
NPC proteins appear to play important roles in intracellular traffic of both endogenously synthesized cholesterol and low-density lipoprotein (LDL)– derived
cholesterol acquired by endocytosis.1,2,9 NPC1 overexpression in CHO cells increased cholesterol synthesis and
total cellular cholesterol concentration.10 Cultured cells
harboring mutations in NPC1 or NPC2 genes accumulate endocytosed lipoprotein cholesterol within lysosomes
and exhibit delayed sterol-regulated gene expression.1,2,11,12 More recently, NPC1 and NPC2 were found
to regulate cellular cholesterol homeostasis through generation of LDL cholesterol– derived oxysterols.12 Taken
together, these studies demonstrate that NPC1 and
NPC2 play critical roles in regulating intracellular cholesterol transport and homeostasis.
Interestingly, NPC proteins also may modulate overall
body cholesterol metabolism. We recently found that
NPC1 plays a key role in controlling the transhepatic
trafficking of cholesterol from plasma into the bile in
mice.13 Also, the studies of Xie et al.14-16 have shown that
HEPATOLOGY, Vol. 43, No. 1, 2006
cholesterol carried in LDL and chylomicrons was sequestered in an intracellular pool of a variety of tissues in
NPC1-deficient mice [NPC1(⫺/⫺ ) mice].14-16 In contrast, the significance of NPC2 for in vivo cholesterol
homeostasis has not been fully established. NPC2 is a
major protein of epididymal fluid, where it may modulate
sperm formation.6,17 However, NPC2 expression has
been detected in several tissues, including neurons and
astrocytes,18,19 suggesting that it could have a more global
function in lipid homeostasis.4,17
In mammals, the liver plays a critical role in lipoprotein
cholesterol metabolism and is a key organ for body cholesterol removal into the bile.20,21 Hepatocytes acquire
cholesterol by 3 metabolic pathways: (1) endogenous biosynthesis from acetate, (2) receptor-mediated endocytosis
of chylomicrons, very low-density lipoproteins and LDL,
and (3) selective cholesterol uptake from high-density lipoproteins via the scavenger receptor class B, type I.21 The
receptor-mediated endocytic pathway is one of the major
mechanisms for uptake of lipoprotein cholesterol into the
liver. In fact, more than 80% of circulating plasma LDL
cholesterol is cleared by endocytosis in this organ.20 Furthermore, the hepatic endocytic pathway is also responsible for metabolism of lipoprotein remnants. Finally,
hepatocytes efficiently eliminate sterols through the bile
as unesterified cholesterol and by its catabolism and biliary secretion as bile acids.21 Biliary cholesterol disposal is
critical not only for normal body cholesterol homeostasis,
but also for the pathogenesis of cholesterol gallstone disease, a highly prevalent and costly condition in Western
countries.22-24
We have established that hepatic NPC1 is critical in
controlling plasma cholesterol and biliary lipid secretion
in the murine liver.13 However, the expression and functional relevance of NPC2 in the hepatocytes are not fully
understood. We evaluated the expression and regulation
of NPC2 in the murine liver as well as the consequence of
adenovirus-mediated hepatic NPC2 overexpression in
mice. We also analyzed the presence of NPC2 protein in
plasma and bile of human and 2 murine strains with different susceptibility to diet-induced gallstone formation.
Materials and Methods
Animals and Diets. The C57BL/6J murine strain
was originally purchased from Jackson Laboratory (Bar
Harbor, ME) and bred to generate our own colony.
BALB/c mice carrying a heterozygous mutation in the
NPC1 gene5 were donated by Dr. Peter Pentchev from
the National Institutes of Health (NIH; Bethesda, MD)
and were used to generate animals that were wild-type
[NPC1 (⫹/⫹)] controls and homozygous NPC1(⫺/⫺)
KLEIN ET AL.
127
mutants. The genotype of offsprings from NPC1 (⫹/⫺)
crosses was identified using a polymerase chain reaction–
based screening as previously described.5 All mice were
maintained with free access to water and a chow diet
(⬍0.02% cholesterol; Prolab RMH 3000, PMI Feeds
Inc., St. Louis, MO).
Wild-type male C57BL/6J and BALB/c mice (2-3
months old) were used in experimental protocols. In some
experiments, C57BL/6J mice (2-3 months old) were
switched from chow diet to diets containing 2% cholesterol (Sigma, St. Louis, MO) or 0.2% ciprofibrate
(Sanofi, Gentilly, France) for 7 days, or 0.5% chenodeoxycholate (CDCA) (Sigma) for 48 hours. In other experiments, C57BL6/J mice were treated through oral gavage
with either vehicle (0.5% methyl cellulose, Sigma) or 2.5
mg/mouse guggulsterone (Steraloids Inc., Newport, RI)
or 1 mg/25 g mouse T0901317 (Amgen Inc., South San
Francisco, CA) daily for 7 days. In one experiment, male
BALB/c NPC1(⫺/⫺) mice of 6 weeks of age as well as
age- and sex-matched BALB/c controls were used to compare hepatic NPC2 expression levels.
Protocols were performed according to accepted criteria for humane care of experimental animals and approved
by the review board for animal studies of our institution.
Recombinant Adenoviruses Preparation and Administration. A recombinant adenovirus containing the
murine NPC2 cDNA (Ad.NPC2) under control of the
cytomegalovirus promoter was generated by homologous
recombination in bacterial cells using the AdEasy system
generously provided by Dr. Bert Vogelstein (Johns Hopkins University, Baltimore, MD).25 The control adenovirus Ad.E1⌬ without transgene was kindly donated by Dr.
Karen Kozarsky (SmithKline Beecham Pharmaceuticals,
King of Prussia, PA). Large-scale production of recombinant adenoviruses was performed in HEK293 cells as previously described.13
For in vivo administration of viruses, mice were anesthetized by ether inhalation, the femoral vein was exposed, and 1 ⫻ 1011 viral particles (in 0.1 mL isotonic
saline buffer) of control or recombinant adenoviruses
were injected intravenously. An additional control group
received 0.1 mL saline buffer only. Mice were euthanized
for analysis 7 days after adenoviral infection.
Murine Bile, Blood, Epididymal Fluid, and Liver
Sampling. Mice were anesthetized by intraperitoneal
injection of sodium pentobarbital. The cyst duct was ligated and a common bile duct fistula was performed using
a polyethylene catheter. Hepatic bile specimens followed
by plasma and liver samples were obtained as described
previously.26,27 Mouse epididymis fluid was prepared as
previously described.28
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KLEIN ET AL.
Human Plasma and Bile Sampling and Processing.
Human plasma was obtained from healthy volunteers.
Hepatic bile samples were obtained from patients with
indwelling T-tubes 3 to 5 days after cholecystectomy for
gallstones and immediately delipidated as previously described.29,30 Gallbladder bile was obtained during laparoscopic cholecystectomy. Biliary glycoproteins were
extracted with Concanavalin–A (Con-A) sepharose beads
using a method previously described.31
Plasma Biochemical Analyses. Serum alkaline phosphatase and alanine aminotransferase were measured by
routine automated methods in the clinical facilities of our
institution.
Glycosidase Digestion and Immunoblotting Analysis. For glycosidase digestion, liver homogenates were
prepared as previously described.26 Sixty micrograms total
protein were incubated with or without peptide N-glycosidase F (PNGaseF) (New England Biolabs, Ipswich,
MA) according to manufacturer’s instructions.
For hepatic NPC2 protein analysis, proteins (40-60 ␮g
protein/sample from liver extracts with or without PNGaseF treatment; 10 ␮g protein/sample from epididymal
fluid; 5 or 10 ␮L murine hepatic bile; 40 ␮g protein/
sample of delipidated human hepatic or gallbladder bile;
2-3 ␮L plasma) were separated by 12% to 15% sodium
dodecyl sulfate polyacrylamide gel electrophoresis and
immunoblotted using an anti-NPC2 antiserum donated
by Dr. Peter Lobel (Robert Wood Johnson Medical
School, Piscataway, NJ). An anti-albumin antibody was
used as protein loading control for immunoblotting. For
biliary cathepsin D and ␤-galactosidase protein analysis,
10 ␮L murine hepatic bile were separated by 10% sodium
dodecyl sulfate polyacrilamide gel electorphoresis and immunoblotted using anti-Cathepsin D (Santa Cruz Biotechnology, Santa Cruz, CA) and anti–␤-galactosidase
(Promega, Madison, WI) antibodies. Antibody binding
to protein samples was visualized using enhanced chemiluminescence and measured using the GS-525 Molecular
Image System (Bio-Rad, Hercules, CA).
Hepatic Immunofluorescence. Fresh-frozen livers
were sectioned, and cryosections (4-5 ␮m) were fixed in
acetone, rinsed 3 times in phosphate-buffered saline, permeabilized with 0.1% Triton X-100 for 15 minutes,
blocked overnight in 10% goat serum in phosphate-buffered saline, and incubated for 2 hours at 37°C with the
polyclonal antibody against NPC2 (1:100). As secondary
antibody, fluorescein isothiocyanate– conjugated goat
anti-rabbit IgG (dilution 1:150) was used. After washing
in phosphate-buffered saline, samples were mounted with
coverslips using Fluoromont-G (EMS, Fort Washington,
MD). Labeled sections were examined by immunofluorescence microscopy.
HEPATOLOGY, January 2006
Quantitative Northern Blot Analysis. cDNA
probes for murine NPC2 and 18S rRNA were prepared
from total liver RNA by a standard reverse transcription
polymerase chain reaction procedure using primers based
on mouse cDNA sequences available through GeneBank
databases.
For Northern blotting, total RNA was prepared from
murine livers using the acid guanidinium thiocyanatephenol-chloroform method.32 Equal amounts of total
RNA (30 ␮g per individual mouse from each experimental group) were size-fractionated by agarose-formaldehyde
gel electrophoresis and transferred to nylon membranes.
Probes were labeled by the random primer method (Promega, Madison, WI) and used for hybridization as previously described.13 Radioactive bands were quantified by
phosphorimaging using the GS-525 Molecular Image
System (Bio-Rad). Results were normalized to the signal
generated from hybridization of a [32P]-labeled mouse
18S rRNA probe on the same filter.
Statistics. The statistical significance of the differences between the means of the experimental groups was
evaluated using the Student t test for unpaired data. A
difference was considered statistically significant at a P
value ⬍ .05.
Results
NPC2 Protein Expression and Regulation in the
Murine Liver. Using immunoblot analysis, NPC2 was
detected in total liver extracts as protein bands of approximately 16 to 22 kd in BALB/c and C57BL6/J mice (Fig.
1A). This signal matches the molecular size of NPC2
protein present in murine epididymal fluid, which was
used as a positive control for immunoblotting (Fig. 1A).
We did not observe differences in the constitutive expression levels of NPC2 in the liver samples of these two
mouse strains. NPC2 protein heterogeneity seems to be
attributable to differences in posttranslational glycosylation. After glycosidase F treatment, NPC2 was visualized
as a single immunoreactive band of faster mobility approximately 16 kd in the epididymal fluid and liver extracts from both BALB/c and C57BL6/J mice (Fig. 1B).
To explore the regulation of hepatic NPC2 protein in
vivo, we next tested the effect of a high-cholesterol diet as
well as different agonists for nuclear receptors involved in
the regulation of cholesterol and bile salt metabolism in
the liver. C57BL6/J mice were fed with diets containing
2% cholesterol or the PPAR␣ agonist cipofibrate, for 7
days, or the primary bile acid and FXR agonist chenodeoxycholic acid for 2 days. Also, mice were treated by gavage with the FXR antagonist guggulsterone or the LXR
agonist T0901317 for 7 days. These treatments did not
HEPATOLOGY, Vol. 43, No. 1, 2006
Fig. 1. Hepatic NPC2 protein in BALB/c and C57BL6/J mice. (A) For
NPC2 expression analysis, proteins from liver homogenates were size
fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotted with anti-NPC2 antibodies, and subjected to densitometric analysis. Sixty micrograms liver homogenate proteins were
analyzed, and 10 ␮g epididymal fluid proteins were used as NPC2
positive control (C⫹). Relative NPC2 expression levels are shown after
normalization for pre-albumin as a protein loading control. (B) For
endoglycanase digestion, homogenates from epididymal fluid (10 ␮g
protein/sample) and livers (60 ␮g protein/sample) from BALB/c and
C57BL6/J mice were incubated with peptide N-glycosidase F (PNGaseF),
followed by Western blotting using anti-NPC2 antibodies.
change hepatic NPC2 protein expression significantly,
suggesting that this protein is not under the control of
transcriptional factors that are critical for the regulation of
cholesterol and bile acid synthesis and transport in the
liver (Fig. 2).
NPC2 has been described as being overexpressed in
fibroblasts of NPC1 patients.33 To evaluate whether this
regulation also occurs in hepatocytes in vivo, we analyzed
NPC2 mRNA and protein levels in livers of NPC1-deficient mice. As shown in Fig. 3A, hepatic NPC2 protein
levels were increased by 5-fold in the absence of a functional NPC1 protein in mice. This finding was paralleled
with a 70% increase in NPC2 mRNA levels in
NPC1(⫺/⫺) livers as shown by Northern blot analysis
(Fig. 3B). These results suggest that the hepatic deficiency
of NPC1 protein is sensed in liver cells by upregulating
NPC2 gene expression and protein levels in vivo.
Adenovirus-Mediated Hepatic NPC2 Overexpression in Mice. Infection of C57BL6/J mice with recombinant adenovirus encoding NPC2 markedly increased
NPC2 protein levels in the liver (Fig. 4A). The expression
of NPC2 in liver was also characterized by immunofluorescence microscopy (Fig. 4B). As shown in Fig. 4B,
NPC2 staining displayed a punctuate cytoplasmic pattern
consistent with NPC2 protein localization in intracellular
vesicular compartments as previously shown.33 Unfortunately, adenovirus-mediated NPC2 overexpression in-
KLEIN ET AL.
129
Fig. 2. Hepatic NPC2 protein levels after cholesterol feeding and
pharmacological modulation of nuclear receptors in C57BL6/J mice.
Hepatic NPC2 protein levels were analyzed in C57BL6/J mice fed a 2%
cholesterol-, 0.2% fibrate-, or 0.5% chenodeoxycholic acid– containing
diets or treated by gavage with guggulsterone (2.5 mg/day) or
T0901317 (1 mg/day). Liver samples were removed, and total homogenates were prepared. Proteins (40 ␮g per lane) were fractionated by
sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to
nitrocellulose, and immunoblotted with anti-NPC2 and anti-prealbumin
antibodies. NPC2 protein expression was normalized to the signal generated by the anti-prealbumin antibody on the same membrane. Results
are representative of three independent experiments.
Fig. 3. Hepatic NPC2 expression analysis by immuno- and northern
blotting in wild-type and NPC1(⫺/⫺) mice. (A) Total homogenate
proteins (60 ␮g per lane) were fractionated by SDS-PAGE, transferred to
nitrocellulose, and immunoblotted with anti-NPC2 and anti-prealbumin
antibodies. NPC2 protein expression was normalized to the signal generated by the anti-prealbumin antibody on the same membrane. (B) Total
hepatic RNA was prepared, electrophoresed, and transferred to nylon
membranes. Gene expression was evaluated by RNA blot hybridization
with 32P-labeled cDNA probes. The mRNA expression data for npc2 gene
is shown after normalization against 18S rRNA. The results shown in this
figure are representative of 2 or 3 independent expression analyses.
NPC1 (⫹/⫹): wild-type mice; NPC1 (⫺/⫺): NPC1-deficient mice.
*indicates a significant difference (P ⬍ .05).
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KLEIN ET AL.
Fig. 4. Hepatic NPC2 expression in mice after infection with an NPC2
recombinant adenovirus. C57BL/6 mice were infected with murine npc2
recombinant adenovirus (Ad.NPC2) or a control adenovirus (Ad.E1⌬).
Seven days after adenoviral infection, liver samples were collected for
NPC2 immunoblotting (A) and immunofluorescence (B) analysis.
duced a strong hepatic inflammatory response as
evidenced by a dramatic increase on liver enzyme levels in
plasma of Ad.NPC2-infected mice compared with animals infected with the control adenovirus (data not
shown). For this reason, we did not further characterize
the effect of hepatic NPC2 overexpression on plasma,
liver, and biliary parameters related to cholesterol metabolism in vivo.
NPC2 Protein Levels in Murine and Human
Plasma, Liver, and Bile. Because NPC2 has been
found as a major secretory product in the epididymal
fluid, we analyzed whether NPC2 could be detected in
plasma and bile. As shown in Fig. 5A, plasma from
BALB/c and C57BL6/J mice contained polypeptides that
were immunoreactive with the NPC2 antibody. Furthermore, NPC2 protein was also detected in bile obtained
from these 2 murine strains (Fig. 5B). Noteworthy, biliary
NPC2 levels were 3-fold higher in gallstone-susceptible
C57BL6/J mice compared with the gallstone-resistant
BALB/c strain. To analyze whether NPC2 secretion into
bile was specific or correlated with the secretion of common lysosomal markers, we also performed immunoblot
analysis of cathepsin D and ␤-galactosidase in bile. We
found that both lysosomal markers were also increased in
murine bile from C57BL/6 compared with BALB/c (Fig.
5B), suggesting that biliary secretion of NPC2 in mice is
not a specific event and it occurs in conjunction with the
other lysosomal hydrolases. In any case, these results indicate that NPC2 is a secretory protein present in murine
plasma and bile. These findings were further supported by
increased levels of plasma and biliary NPC2 protein levels
HEPATOLOGY, January 2006
Fig. 5. NPC2 plasma and biliary levels in mice. For NPC2 expression
analysis, proteins were size fractionated by SDS-PAGE and immunoblotted with anti-NPC2 antibodies. (A) Two microliters of murine plasma were
analyzed. Two micrograms of epididymal fluid proteins were used as
NPC2 positive control (C⫹). (B) Ten microliters murine hepatic bile were
analyzed with antibodies against NPC2, pre-albumin, cathepsin-D, and
␤-galactosidase. (C) Two microliters plasma and 5 ␮L of bile from
Ad.E1⌬ and Ad.NPC2-infected mice were analyzed.
found in mice with adenovirus-mediated hepatic NPC2
overexpression (Fig. 5C).
Finally, we tested whether NPC2 was also present in
human plasma and bile. The rabbit polyclonal antisera
raised against recombinant NPC2/HE14 detected immunoreactive bands of approximately 25 to 27 kd in human
plasma as well as hepatic and gallbladder bile (Fig. 6, lanes
3, 7, 8). Next, extraction of biliary glycoproteins was performed by standard Con A-Sepharose affinity purification
as previously described.31 As shown in Fig. 6, lane 9,
Fig. 6. NPC2 plasma and biliary levels in humans. For NPC2 expression analysis, proteins were size fractionated by sodium dodecyl sulfate
polyacrylamide gel electrophoresis, and immunoblotted with anti-NPC2
antibodies. Lane 1, 10 ␮g mouse epididymal fluid; lane 2, 3 ␮L mouse
plasma; lane 3, 3 ␮L of human plasma; lane 4; 40 ␮g mouse liver
homogenate; lane 5, 40 ␮g human liver homogenate; lane 6, 10 ␮L
mouse hepatic bile; lane 7, 40 ␮g human hepatic bile after delipidation;
lane 8, 40 ␮g of human gallbladder bile after delipidation; lane 9, Con-A
bound fraction (Con-ABF) from a human gallbladder bile sample; lane
10, Con-A unbound fraction (Con-AUBF) from a human gallbladder bile
sample.
HEPATOLOGY, Vol. 43, No. 1, 2006
human biliary NPC2 was completely recovered in the
Con A– binding fraction as expected for glycoprotein features of NPC2.34 The antigens detected with the NPC2
antibody in human plasma and bile had a molecular size
range similar to that observed for NPC2 immunoreactivity in human liver samples (Fig. 6, lane 5). Interestingly,
human NPC2 (Fig. 6, lanes 3, 5, 7, and 8) exhibited a
molecular size bigger than that found in murine epididymal fluid, plasma, liver, and bile (Fig. 6, lanes 1, 2, 4, 6).
These murine versus human differences in the NPC2 protein size have been previously reported19 and are fully
consistent with an extra consensus site for N-glycosylation present in human NPC2 compared with its murine
ortholog.17,34
Discussion
This study demonstrates that NPC2 is present in murine and human plasma and bile. In addition, NPC2 was
isolated in the cholesterol pro-nucleating Con A– binding
fraction of human bile. Furthermore, biliary NPC2 levels
were 3-fold increased in gallstone-susceptible C57BL6/J
mice compared with the gallstone-resistant BALB/c
strain. Conversely, hepatic expression was dramatically
increased in NPC1-deficient mice, but not regulated by
cholesterol feeding or various nuclear receptor ligands involved in cholesterol and bile acid metabolism in vivo.
One significant finding of this study was the dramatic
increase on NPC2 levels observed in livers of NPC1 mutant mice. Our results extend previous data obtained in
fibroblast cells33 and astrocytes19 and murine cerebellum,35 indicating that NPC1 deficiency is also sensed in
vivo in liver cells by upregulating NPC2 expression. This
reciprocal co-regulation suggests that NPC1 and NPC2
function by complementation in a common pathway.
This idea is also supported by recent findings studying
NPC2 hypomorphic mice (0%-4% of residual protein
expressed in different tissues) and NPC1/NPC2 double
mutant animals.36 In this latter study, single NPC1 or
NPC2 mutants as well as double mutants were similar in
disease onset and progression, biochemical patterns of
lipid accumulation, and pathology.
To explore the regulation of hepatic NPC2 protein in
vivo, we tested the effect of a high-cholesterol diet as well
as different agonists for the nuclear receptors FXR, LXR,
and PPAR␣ involved in the regulation of cholesterol and
bile salt metabolism in the liver. Neither of these treatments changed significantly hepatic NPC2 expression at
the protein level, suggesting that intrahepatic cholesterol
content does not play a dominant role in controlling hepatic NPC2 expression in mice and that this protein is not
under the control of transcriptional factors that are critical
KLEIN ET AL.
131
for the regulation of cholesterol and bile acid synthesis
and transport in the liver.
NPC2, a protein with lysosomal localization when
found within cells,33 binds cholesterol with high affinity.7,8 The phenotype of NPC2 mutant cells and the
knowledge of its structure and intracellular location has
supported a working model in which NPC2 binds unesterified cholesterol released within the lysosomes after lipoprotein endocytosis and degradation, facilitating
cholesterol export from this organelle through NPC1 or
other proteins to various subcellular compartments.37 Remarkably, NPC2 also has been reported as an extracellular
secretory product. In fact, NPC2 is particularly abundant
in the epididymal fluid, where it has been suggested that
this protein plays an important role in sperm formation
by reducing cholesterol content in male spermatozoidal
membranes,6 which seems critical for sperm capacitation.38 Furthermore, bovine NPC2 (also known as
EPV20) is mainly present in the milk.39 More recently,
NPC2 has been reported to also be secreted from primary
astrocytes independently from the secretion of astrocytederived sterols.19
Our major finding is the demonstration that NPC2 is
also found in plasma and bile. The principal sources and
the functional role of plasma and biliary NPC2 and its
relation with body cholesterol trafficking and homeostasis
require further study. We were able to demonstrate that
NPC2 hepatic overexpression in the murine liver correlated with a significant increment in plasma and biliary
NPC2 protein levels, suggesting that hepatocytes actively
secrete NPC2 through the basolateral and canalicular domains. The secretion of NPC2 is rather unique and not
just a secondary effect of the overexpression induced by
the recombinant adenoviral vector. In fact, we have not
detected plasma or biliary secretion of other lipid transport proteins (e.g., sterol carrier protein-2, caveolin-1, or
caveolin-2) when overexpressed in mice26,40 (also unpublished personal observations). Based on our findings, the
liver may represent a main source of plasma and biliary
NPC2, even though we cannot exclude significant contribution of nonhepatic tissues to plasma NPC2 levels or
bile duct epithelial cells to the biliary content of this protein. The detailed mechanism by which NPC2 is secreted
into plasma and bile is not known. Our results indicated
that NPC2 secretion into bile correlated with biliary levels
of soluble lysosome proteins, suggesting that it may share
the mechanism underlying lysosomal hydrolases secretion. Biliary secretion of these enzymes into bile occurs
through a microtubule-dependent mechanism, suggesting that lysosomes follow an exocytotic pathway in which
the luminal content is discharged into plasma or bile after
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KLEIN ET AL.
fusion of the lysosomal membrane with the sinusoidal or
canalicular membrane.41-43
Physiologically, NPC2 synthesis and secretion from
the liver may play a role in cholesterol secretion from the
liver into plasma and bile and, if so, in the maintenance of
normal cholesterol metabolism. However, recent studies
have indicated that NPC2 is not co-secreted with cellular
sterols, and its overexpression cannot drive an increase in
cellular cholesterol efflux from cultured astrocytes.19
Whether these latter findings are also valid to liver cells
remains to be studied. Unfortunately, adenovirus-mediated hepatic NPC2 overexpression induced a strong hepatic inflammatory response in C57BL6/J mice. Infection
of this murine strain with adenoviruses carrying a transgene usually causes some inflammation, which is comparable to that produced with the control adenovirus
(Ad.E1⌬). Currently, we do not have an apparent explanation for the major hepatic inflammation observed in
NPC2-overexpressing mice. Because severe liver inflammation leads to significant modifications in cholesterol
metabolism,44 evaluation of the specific role of hepatic
NPC2 expression in cholesterol traffic and homeostasis in
this animal model of NPC2 overexpression was not possible.
That the primary pathogenic event for gallstone formation is biliary cholesterol hypersecretion followed by cholesterol crystallization and crystal growth in bile21,45 has
been established. A variety of biliary proteins can influence these early and critical steps during gallstone formation.46-48 Considering its cholesterol-binding activity,
NPC2 secretion into bile led us to speculate that it may
modulate biliary cholesterol precipitation. Interestingly,
we found that biliary, but not hepatic, NPC2 content was
significantly increased in C57BL/6 mice susceptible to
diet-induced gallstone formation compared with BALB/c
mice. Because NPC2 is secreted into bile, steady-state
protein levels in the liver could not fully reflect NPC2
production rate. Further pulse-chase studies and mRNA
expression analysis are required to elucidate whether hepatic NPC2 biosynthesis is different in these 2 mouse
strains. Whether the strain-dependent difference in biliary NPC2 plays a role in the different susceptibility to
gallstone disease also needs additional study.
Furthermore, we demonstrated that NPC2 present in
human bile was fully recovered in a Con A– binding fraction, which has been characterized as a potent promoter of
cholesterol crystallization in vitro.31,49 The cholesterol
pronucleating activity of this protein mixture has been
well established, and a variety of proteins of this fraction
have been partially identified.31,49,50 Exactly which of
these biliary proteins is more relevant in cholesterol crystallization and how these glycoproteins facilitate choles-
HEPATOLOGY, January 2006
terol nucleation and crystallization remain unknown.50
Some of these glycoproteins may facilitate the proximity
and fusion of cholesterol molecules carried in vesicular
transporters enriched in cholesterol. NPC2 is a novel protein found in this biliary cholesterol pronucleating Con
A– binding fraction and the first that specifically binds
cholesterol. Interestingly, secreted NPC2 seems to be
functional, retaining its cholesterol-binding activity.6
Therefore, biliary NPC2 may play a relevant role in the
cholesterol crystallization defect present in gallstone patients.
In conclusion, we have described that NPC2 is present
in plasma and bile, suggesting a potential role for this
protein in cholesterol transport and gallstone disease in
mammals. Further studies are required to elucidate the
physiological and pathological relevance of our findings.
Acknowledgment: The authors thank Flavio Nervi
for constant support and helpful discussions. They also
thank Margrit Schwarz and Ricardo Moreno for help in
obtaining T0901317 and mice epididymal fluid, respectively.
References
1. Sturley SL, Patterson MC, Balch W, Liscum L. The pathophysiology and
mechanisms of NP-C disease. Biochim Biophys Acta 2004;1685:83-87.
2. Mukherjee S, Maxfiled FR. Lipid and cholesterol trafficking in NPC.
Biochim Biophys Acta 2004;1685:28-37.
3. Cartsea ED, Morris JA, Coleman JK, Loftus SK, Zhang D, Cummings C,
et al. Niemann-Pick C1 disease gene: homology to mediators to cholesterol
homeostasis. Science 1997;277:228-231.
4. Naureckiene S, Sleat DE, Lackland H, Fensom A, Vanier MT, Wattiaux
R, et al. Identification of HE1 as the second gene of Niemann-PicK C
disease. Science 2000;290:2298-2301.
5. Loftus SK, Morris JA, Cartsea ED, Gu JZ, Cummings C, Brown A, et al.
Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene. Science 1997;277:232-235.
6. Okamura N, Kiuchi S, Tamba M, Kashima T, Hiramoto S, Baba T, et al.
A porcine homolog of the major secretory protein of human epididymis
HE1, specifically binds cholesterol. Biochim Biophys Acta 1999;1438:
377-387.
7. Friedland N, Liou HL, Lobel P, Stock AM. Structure of a cholesterolbinding protein deficient in Niemann-Pick type C2 disease. Proc Natl
Acad Sci U S A 2003;100:2512-2517.
8. Ko DC, Binkley J, Sidow A, Scott MP. The integrity of a cholesterolbinding pocket in Niemann–Pick C2 protein is necessary to control lysosome cholesterol levels. Proc Natl Acad Sci U S A 2003;100:2518-2525.
9. Neufeld EB, Wastney M, Patel S, Suresh S, Cooney AM, Dwyer NK, et al.
The Niemann-Pick C1 protein resides in a vesicular compartment linked
to retrograde transport for multiple lysosomal cargo. J Biol Chem 1999;
274:9627-9635.
10. Millard EE, Srivastava K, Traub LM, Schaffer JE, Ory DS. NPC1 overexpression alters cellular cholesterol homeostasis. J Biol Chem 2000;275:
38445-38451.
11. Liscum L, Faust JR. Low density lipoprotein (LDL)-mediated suppression
of cholesterol synthesis and LDL uptake is defective in Niemann-Pick type
C fibroblasts. J Biol Chem 1987;262:17002-17008.
12. Frolov A, Zielinski SE, Crowley JR, Dudley-Rucker N, Schaffer JE, Ory
DS. NPC1 and NPC2 regulate cellular cholesterol homeostasis through
generation of low density lipoprotein cholesterol-derived oxysterols. J Biol
Chem 2003;278:25517-25525.
HEPATOLOGY, Vol. 43, No. 1, 2006
13. Amigo L, Mendoza H, Castro J, Quiñones V, Miquel JF, Zanlungo S.
Relevance of Niemann-Pick Type C1 protein expression in controlling
plasma cholesterol and biliary lipid secretion in mice. HEPATOLOGY 2002;
36:819-828.
14. Xie C, Turley SD, Pentchev PG, Dietschy JM. Cholesterol balance and
metabolism in mice with loss of function of Niemann-Pick C protein.
Am J Physiol (Endocrinol Metabol 39) 1999;276:E336-E334.
15. Xie C, Turley SD, Dietschy JM. Cholesterol accumulation in tissues of the
Niemann-Pick type C mouse is determined by the rate of lipoproteincholesterol uptake through the coated-pit pathway in each organ. Proc
Natl Acad Sci U S A 1999;96:11992-11997.
16. Xie C, Turley SD, Dietschy JM. Centripetal cholesterol flow from the
extrahepatic organs through the liver is normal in mice with mutated
Niemann-Pick type C protein. J Lipid Res 2000;41:1278-1289.
17. Vanier MT, Millat G. Structure and function of the NPC2 protein. Biochim Biophys Acta 2004;1685:14-21.
18. Ong WY, Sundaram RK, Huang E, Ghoshal S, Kumar U, Pentchev PG, et
al. Neuronal localization and association of Niemann Pick C2 protein
(HE1/NPC2) with the postsynaptic density. Neuroscience 2004;128:561570.
19. Mutka AL, Lusa S, Linder MD, Jokitalo E, Kopra O, Jauhiainen M, et al.
Secretion of sterols and the NPC2 protein from primary astrocytes. J Biol
Chem 2004;279:48654-48662.
20. Dietschy JM, Turley SD, Spady DK. Role of the liver in the maintenance
of cholesterol and low density lipoprotein homeostasis in different animal
species, including humans. J Lipid Res 1993;34:1637-1659.
21. Zanlungo S, Rigotti A, Nervi F. Hepatic cholesterol transport from plasma
into bile: implications for gallstone disease. Curr Opin Lipidol 2004;15:
279-286.
22. Miquel JF, Covarrubias C, Villarroel L, Mingrone G, Greco AV, Puglielli L, et
al. Genetic epidemiology of cholesterol cholelithiasis among Chilean Hispanics, Amerindians, and Maoris. Gastroenterology 1998;115:937-946.
23. Everhart JE, Khare M, Hill M, Maurer KR. Prevalence and ethnic differences in gallbladder disease in the United States. Gastroenterology 1999;
117:632-639.
24. Amigo L, Zanlungo S, Mendoza H, Miquel JF, Nervi F. Risk factors and
pathogenesis of cholesterol gallstones: state of the art. Eur Rev Med Pharmacol Sci 1999;3:241-246.
25. He T-G, Zhou S, Da Costa LT, Yu J, Kinzler KW, Vogelstein B. A
simplified system for generating recombinant adenoviruses. Proc Natl
Acad Sci U S A 1998;95:2509-2514.
26. Zanlungo S, Amigo L, Mendoza H, Miquel JF, Vio C, Glick JM, et al.
Overexpression of sterol carrier protein-2 leads to altered lipid metabolism
and enhanced enterohepatic sterol circulation in mice. Gastroenterology
2000;119:1708-1719.
27. Mardones P, Quiñones V, Amigo L, Moreno M, Miquel JF, Schwarz M, et
al. Hepatic cholesterol and bile acid metabolism and intestinal cholesterol
absorption in scavenger receptor class B type I (SR-BI)-deficient mice. J
Lipid Res 2001;42:170-180.
28. Fouchecourt S, Metayer S, Locatelli A, Dacheux F, Dacheux JL. Stallion
epididymal fluid proteome: qualitative and quantitative characterization;
secretion and dynamic changes of major proteins. Biol Reprod 2000;62:
1790-1803.
29. Wesel D, Flügge UL. A method for the quantitative recovery of protein in
dilute solution in the presence of detergents and lipids. Anal Biochem
1984;38:141-143.
30. Miquel JF, Nunez L, Amigo L, González S, Raddatz A, Rigotti A, et al.
Cholesterol saturation, not proteins or cholecystitis, is critical for crystal
formation in human gallbladder bile. Gastroenterology 1998;114:10161023.
31. Miquel JF, Van der Putten J, Pimentel F, Mok KS, Groen AK. Increased
activity in the biliary Con A-binding fraction accounts for the difference in
KLEIN ET AL.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
133
crystallization behavior in bile from Chilean gallstone patients compared
with Dutch gallstone patients. HEPATOLOGY 2001;33:328-332.
Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid
guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem
1987;162:156-159.
Blom TS, Linder MD, Snow K, Pihko H, Hess MW, Jokitalo E, et al.
Defective endocytic trafficking of NPC1 and NPC2 underlying infantile
Niemann–Pick type C disease. Hum Mol Genet 2003;12:257-272.
Kirchoff C, Osterhoff C, Young L. Molecular cloning and characterization
of HE1, a major secretory protein of the human epididymis. Biol Reprod
1996;54:847-856.
Li H, Repa JJ, Valasek MA, Beltroy EP, Turley SD, German DC, et al.
Molecular, anatomical, and biochemical events associated with neurodegeneration in mice with Niemann-Pick type C disease. J Neuropathol Exp
Neurol 2005;64:323-333.
Sleat DE, Wiseman JA, El-Banna ME, Price SM, Verot L, Shen MM, et al.
Genetic evidence for non-redundant functional cooperativity between
NPC1 and NPC2 in lipid transport. Proc Natl Acad Sci U S A 2004;101:
5886-5891.
Ioannou YA. Multidrug permeases and subcellular cholesterol transport.
Nature Rev Mol Cell Biol 2001;2:657-668.
Cross NL. Role of cholesterol in sperm capacitation. Biol Reprod 1998;
59:7-11.
Larsen LB, Ravn P, Boisen A, Berglund L, Petersen TE. Primary structure
of EPV20, a secretory glycoprotein containing a previously uncharacterized type of domain. Eur J Biochem 1997;243:437-441.
Moreno M, Molina H, Amigo L, Zanlungo S, Arrese M, Rigotti A, et al.
Hepatic overexpression of caveolins increases bile salt secretion in mice.
HEPATOLOGY 2003;38:1477-1488.
Godfrey PP, Lembra L, Coleman R. Effects of colchicine and vinblastine
on output of proteins into bile. Biochem J 1982;208:153-157.
Barnwell SG, Coleman R. Abnormal secretion of proteins into bile from
colchicine treated isolated perfused rat livers. Biochem J 1983;216:409414.
Rahman K, Coleman R. Effect of chloroquine on biliary lipid and lysosomal enzyme output in the isolated perfused rat liver at low bile salt output
rates. Biochim Biophys Acta 1987;922:395-397.
Khovidhunkit W, Kim MS, Memon RA, Shigenaga JK, Moser AH, Feingold KR, et al. Effects of infection and inflammation on lipid and lipoprotein metabolism: mechanisms and consequences to the host. J Lipid Res
2004;45:1169-1196.
Zanlungo S, Nervi F. The molecular and metabolic basis of biliary cholesterol secretion and gallstone disease. Front Biosci.2003;8:S1166-S1174.
Groen AK, Stout JPJ, Drapers JAG, Hoek FJ, Grijm R, Tytgat GNJ. Cholesterol nucleation-influencing activity in T-tube bile. HEPATOLOGY 1988;
8:347-352.
Miquel JF, Nuñez L, Rigotti A, Amigo L, Brandan E, Nervi F. Isolation
and partial characterization of cholesterol pronucleating hydrophobic glycoproteins associated to native biliary vesicles. FEBS Lett 1993;318:45-49.
Secknus R, Darby GH, Chernosky A, Juvonen T, Moore EW, Holzbach
RT. Apolipoprotein A-I in bile inhibits cholesterol crystallization and
modifies transcellular lipid transfer through cultured human gall-bladder
epithelial cells. J Gastroenterol Hepatol 1999;14:446-456.
Keulemans YC, Mok KS, Slors JF, Brink MA, Gouma DJ, Tytgat GN, et
al. Concanavalin A-binding cholesterol crystallization inhibiting and promoting activity in bile from patients with Crohn’s disease compared to
patients with ulcerative colitis. J Hepatol 1999;31:685-691.
Jirsa M, Groen AK. Role of biliary proteins and non-protein factors in
kinetics of cholesterol crystallisation and gallstone growth. Front Biosci
2001;6:E154-E167.