From the Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111
Received for publication, January 16, 2003, and in revised form, February 13, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Niemann-Pick disease type C (NPC) is
characterized by lysosomal storage of cholesterol and gangliosides,
which results from defects in intracellular lipid trafficking. Most
studies of NPC1 have focused on its role in intracellular cholesterol
movement. Our hypothesis is that NPC1 facilitates the egress of
cholesterol from late endosomes, which are where active NPC1 is
located. When NPC1 is defective, cholesterol does not exit late
endosomes; instead, it is carried along to lysosomal storage bodies,
where it accumulates. In this study, we addressed whether
cholesterol is transported from endosomes to the plasma membrane before
reaching NPC1-containing late endosomes. Our study was conducted in
Chinese hamster ovary cell lines that display the classical NPC
biochemical phenotype and belong to the NPC1 complementation group. We
used three approaches to test whether low density lipoprotein
(LDL)-derived cholesterol en route to NPC1-containing organelles
passes through the plasma membrane. First, we used cyclodextrins to
measure the arrival of LDL cholesterol at the plasma membrane and found
that the arrival of LDL cholesterol in a cyclodextrin-accessible pool
was significantly delayed in NPC1 cells. Second, the movement of LDL
cholesterol to NPC1-containing late endosomes was assessed and found to
be normal in Chinese hamster ovary mutant 3-6, which exhibits
defective movement of plasma membrane cholesterol to intracellular
membranes. Third, we examined the movement of plasma membrane
cholesterol to the endoplasmic reticulum and found that this pathway is
intact in NPC1 cells, i.e. it does not pass through
NPC1-containing late endosomes. Our data suggest that in NPC1 cells LDL
cholesterol traffics directly through endosomes to lysosomes, bypassing
the plasma membrane, and is trapped there because of dysfunctional NPC1.
Niemann-Pick disease type C
(NPC)1 is an autosomal
recessive lysosomal storage disease that is caused by mutations in
either the NPC1 or NPC2 genes and leads to
progressive neurodegeneration and premature death (1). Fibroblasts from
NPC1 patients exhibit massive storage of unesterified cholesterol and
gangliosides in lysosomes, which results from defects in intracellular
lipid trafficking (reviewed in Refs. 2 and 3). Most studies of NPC1
have focused on its role in intracellular cholesterol movement. In
normal cells, low density lipoproteins (LDL) are internalized and
transported through the endocytic pathway where the cholesteryl esters
are hydrolyzed. The cholesterol that is released is transported to the
plasma membrane and endoplasmic reticulum (4). Our working hypothesis
has been that NPC1 facilitates the egress of free cholesterol from late
endosomes, which are the location of functional NPC1 (5, 6). When NPC1
is defective, the free cholesterol does not exit the late endosomes;
instead, it is carried along the endocytic pathway to lysosomal storage
bodies where it accumulates along with mutant NPC1 protein.
In this study, we addressed whether cholesterol released by hydrolysis
of LDL-derived cholesteryl linoleate (CL) is transported to the plasma
membrane in its transit from early endosomes to NPC1-containing late
endosomes and lysosomes. Our work was prompted by two recent studies.
First, Cruz et al. (7) reported that LDL cholesterol is
transported to the plasma membrane independently of NPC1. On reaching
the plasma membrane, LDL-derived cholesterol is proposed to be
specifically reinternalized and delivered to a compartment that is
functionally distinct from lysosomes and contains NPC1. Cholesterol is
then mobilized to other cellular destinations such as the plasma
membrane and endoplasmic reticulum. It is the movement of cholesterol
from these storage vesicles that is proposed to be defective in NPC1
cells (7-9). Second, Lange et al. (9, 10) have reported
that the rate of cholesterol movement from lysosomes to the plasma
membrane in NPC cells is equal to or greater than that in normal cells.
Our study was conducted in Chinese hamster ovary (CHO) cell lines that
display the classical NPC biochemical phenotype (11) and belong to the
same complementation group as NPC1 fibroblasts and CT60, a CHO cell
mutant isolated previously (12, 13). Mutations in the NPC1
gene were defined in two mutants from this complementation class.
We used three approaches to test our hypothesis that LDL-derived
cholesterol en route to NPC1-containing late endosomes does not pass
through the plasma membrane. First, we used cyclodextrins (CDs) to
measure the arrival of LDL cholesterol at the plasma membrane and found
that the arrival of LDL cholesterol in a CD-accessible pool was
significantly delayed in NPC1 cells. Second, the movement of LDL
cholesterol to late endosomes and lysosomes was assessed and found to
be normal in CHO mutant 3-6, which exhibits defective movement of
plasma membrane cholesterol to intracellular membranes (14). Third, we
examined the movement of plasma membrane cholesterol to the endoplasmic
reticulum and found that this pathway is intact in NPC1 cells,
i.e. passage through NPC1-containing organelles is not
obligatory for this pathway. Together our data suggest that in NPC1
mutant cells LDL cholesterol traffics directly through endosomes to
lysosomes, bypassing the plasma membrane, and becomes trapped there
because of dysfunctional NPC1.
Materials--
[1,2,6,7-3H]Cholesteryl linoleate
(86 Ci/mmol), [1,2-3H]cholesterol (45 Ci/mmol),
cholesteryl [1-14C]oleate (59.5 mCi/mmol), and
[
2-Hydroxy-propyl- Cultured Cells, Preparation of LDL, Lipoprotein-deficient Serum,
Media, and Buffers--
LDL was prepared by ultracentrifugation (16).
LDL labeled with [3H]CL ([3H]CL-LDL) was
prepared with an average specific activity of 50,000 dpm/nmol total
cholesteryl linoleate (17). Lipoprotein-deficient serum was prepared as
described, omitting the thrombin incubation (16). The following media
were prepared: Ham's F-12 medium containing 5% (v/v) newborn calf
serum, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (H-5% NCS); and H-5% NCS in which 5% (v/v)
newborn calf serum was replaced with 5% or 10% (v/v)
lipoprotein-deficient newborn calf serum (H-5% LPDS and H-10%
LPDS).
The following buffers were prepared: TBS (150 mM NaCl, 50 mM Tris-chloride, pH 7.4), TBS-bovine serum albumin (TBS
containing 2 mg/ml bovine serum albumin) and phosphate-buffered saline
(1.5 mM KH2PO4, 2.8 mM
KCl, and 137 mM NaCl, pH 7.3).
Cholesterol transport-defective CHO-K1 mutants 2-2, 4-4, 10-3, and 1-2 were identified in a screen to isolate NPC1-defective CHO cells (11).
They belong to the same complementation group as CT60 (12, 13), which
was isolated by Chang and colleagues and displays the classical
NPC phenotype. The CHO mutant 3-6 belongs to a second complementation
class and exhibits impaired cholesterol movement from the plasma
membrane to the endoplasmic reticulum (14). All cells were grown in a
monolayer in a humidified incubator (5% CO2) at 37 °C.
Stocks were cultured in H-5% NCS.
Efflux of LDL-derived [3H]Cholesterol--
On day
0, CHO cells were seeded into six-well plates (25,000 cells/well) in
H-5% NCS. On day 1, cells were washed in balanced Hanks' salt
solution and refed H-5% LPDS. On day 3 or 4, cells were refed H-5%
LPDS and incubated with [3H]CL-LDL for various times at
37 °C. Cells were washed twice with phosphate-buffered saline and
incubated in a medium containing no addition, 12.5 mM CD, or 25 mM CD/chol for 10-120 min. Cells were then washed with TBS, and lipids were extracted with
hexane/isopropyl alcohol (3:2). The media were centrifuged to remove
cellular debris, and lipids were extracted with petroleum ether. A
chromatography standard was added (50 µg of cholesterol, 25 µg of
cholesteryl oleate, and 1500 dpm of [14C]cholesterol) to
cells and media. Labeled cellular lipids were analyzed by TLC with
toluene/ethyl acetate (2:1). Radioactivity was measured with liquid
scintillation counting in ReadySafe. After lipid extraction, monolayers
were incubated with 0.1 N NaOH, and aliquots were taken for
protein determination (18).
Filipin Fluorescence Microscopy--
On day 0, cells were seeded
into chamber slides in H-5% NCS. On day 3 cells were refed with H-10%
LPDS. On day 4 cells were refed with H-10% LPDS, and the following
additions were made: no addition, 50 µg/ml LDL, or 50 µg/ml LDL
plus 20 µg/ml progesterone. Cells were incubated for 4 h at
37 °C, and filipin fluorescence microscopy was performed as
described previously (14).
Basal Esterification of Plasma Membrane Cholesterol--
On day
0, cells were seeded in 12-well plates (10,000 cells/well) in H-5%
NCS. On day 2, cells were refed H-5% LPDS. On day 3, cells were refed
1 ml of H-5% LPDS. Additions of 0.5 µCi of [3H]cholesterol in ethanol were made at staggered times.
Cells were washed with TBS after which lipids were extracted,
separated, and quantified as described previously (4).
Cloning of Hamster NPC1 cDNAs--
Total RNA was isolated
from parental CHO cells and NPC1 mutants by using the RNeasy Mini Kit
(Qiagen). First strand cDNA was synthesized by using SuperScript II
reverse transcriptase and Oligo(dT)12-18 (Invitrogen).
Primers were designed to amplify the hamster NPC1 cDNA
on the basis of the mouse NPC1 cDNA sequence (19), and
several overlapping products were generated by PCR. Primers to amplify
the 5'- and 3'-coding region of NPC1 were designed on the
basis of the hamster NPC1 cDNA sequence, generously
supplied by Chang (7). Using the primer set (forward primer:
5'-ATGAGCGCGCGCCACCCGGCCCTG-3'; reverse primer:
5'-CTAAAAATTGAGGAGCCGTTCTCGCTC-3'), a full-length NPC1 product
was generated by PCR and sequenced by the Tufts University Core
Facility. Sequences were assembled by using the AssemblyLIGN and
MacVector programs. Mutations were identified by using BLAST 2.
Northern Blot Analysis--
mRNA was isolated with the
Oligotex Direct mRNA Minikit (Qiagen). Four µg of mRNA was
loaded onto a formamide gel and transferred to nylon membrane (Ambion).
A 32P-labeled 800-nucleotide NPC1 cDNA probe
was generated by PCR amplification by using the primer set (forward
primer: 5'-CATTTCCTTCATTGCTGAGAG-3'; reverse primer:
5'-GGCATTGAAAGAGACTGATCC-3') and the Prime-It Random Labeling kit
(Stratagene). The membrane was then stripped (2 h in 10 mM
Tris-Cl, pH 7.5, 0.2% SDS at 70 °C) and reprobed with a
32P-labeled Transfections--
Cells were seeded into Falcon chamber slides
in H-5% NCS. Cells were transiently transfected at 60-70% confluency
by using LipofectAMINE PLUS reagent (Invitrogen) with a total of 0.7 µg DNA/chamber of human NPC1 (generously supplied by Jill
Morris and Peter Pentchev, National Institutes of Health) or no DNA, along with pEGFP (Clontech). After 3 h
cells were refed H-5% LPDS and incubated for another 48 h.
Additions of 50 µg/ml LDL were made, and cells were incubated for an
additional 16-24 h. Filipin fluorescence microscopy was performed as
described previously (14).
In normal mammalian cells, LDL is taken up and the cholesteryl
ester core is hydrolyzed in endocytic vesicles. The cholesterol that is
released eventually reaches NPC1-containing organelles. What itinerary
does cholesterol follow? Previous studies have concluded that
LDL-derived cholesterol is transported to multivesicular late endosomes
where active NPC1 resides (5) (Fig
1A). NPC1-containing vesicles
bud off of late endosomes, presumably carrying cholesterol and other
cargo (6), move to the cell interior and cell periphery, and then
return (20). In NPC1 cells, the movement of cholesterol out of late
endosomes is greatly delayed (11, 21, 22). Thus, cholesterol and other
lipids accumulate in lysosomes along with dysfunctional NPC1 protein
(23). Some of the previous cholesterol transport studies relied on
inefficient methods to assess the transit of LDL-derived cholesterol to
the plasma membrane, such as spontaneous desorption of
[3H]cholesterol from the plasma membrane and its
entrapment in small unilamellar vesicles in the medium (11, 22). Other
studies examined LDL cholesterol sequestration in lysosomes
versus transport to the plasma membrane after long
incubation periods with [3H]CL-LDL and subcellular
fractionation (11, 21, 22). All of these studies found delayed arrival
of LDL cholesterol in the plasma membrane. Most recently, cholesterol
movement to the plasma membrane has been quantified by using CD, which
can stimulate cholesterol efflux from cells (24) and act as cholesterol
shuttles (15, 25). By using CD that are complexed with varying levels of cholesterol, it is possible to manipulate the cholesterol content of
cells ranging from net cholesterol enrichment to depletion (15, 26).
Cruz et al. (7) assessed LDL cholesterol movement in NPC1
cells using short incubation times and CD to measure the arrival of
[3H]cholesterol in the plasma membrane. Their results
indicated that the pathway of LDL cholesterol transport was more
complex than suspected previously, involving LDL cholesterol movement to the plasma membrane before arrival in NPC1-containing organelles (Fig. 1B).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP (3000 Ci/mmol) were obtained from
PerkinElmer Life Sciences. Tissue culture reagents were from
Mediatech Cellgro or Fisher. Other chemicals were from Sigma unless
otherwise indicated. Falcon CultureSlide tissue culture-treated glass
chamber slides were obtained from BD Biosciences.
-cyclodextrin (Mr
1576) was obtained from Sigma, and a 100 mM stock was
prepared in Ham's F-12 medium. A 12.5 mM CD was prepared
by diluting the stock in medium. A 25 mM CD that was 50%
saturated with cholesterol (CD/chol) was prepared by modification of a
method described previously (15). Sixteen mg of recrystallized
cholesterol was added to 10 ml of a 25-mM CD in medium and
incubated overnight at 37 °C in a shaking water bath. The solution
was filtered through a 0.45-µm syringe filter to remove excess
cholesterol crystals, and the cholesterol-saturated cyclodextrin
solution was diluted with an equal volume of 25 mM CD.
-actin cDNA (Ambion).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (89K):
[in a new window]
Fig. 1.
Alternative models of the role of NPC1 in
cholesterol transport. A, vesicles carry plasma membrane
lipids to and from the endocytic recycling compartment
(ERC). Membrane-bound receptors deliver LDL to the ERC,
where the LDL is sorted into endosomes. Vesicles carry membrane lipids
and LDL to multivesicular late endosomes. LDL-cholesteryl esters are
hydrolyzed during this transit. NPC1-containing vesicles bud from late
endosomes and carry cholesterol and other cargo to perinuclear
(e.g. endoplasmic reticulum (ER)) and peripheral
(e.g. ERC) compartments. When NPC1 is
dysfunctional, cholesterol accumulates in lysosomes. Plasma membrane
(PM) cholesterol can reach the endoplasmic reticulum by
NPC1-dependent and NPC1-independent pathways. L,
lysosomes. B, after LDL uptake and cholesteryl
ester hydrolysis, LDL cholesterol is transported to the plasma membrane
and then quantitatively internalized to an NPC1-containing
cholesterol-sorting compartment. NPC1 is involved in transporting
cholesterol from this compartment to the plasma membrane and
endoplasmic reticulum. ACAT, acyl-CoA/cholesterol
acyltransferase.
Here we have evaluated this itinerary in CHO mutants 2-2 and CT60. Isolated in our laboratory and Chang's laboratory, respectively, these NPC1-defective mutants display the classical NPC disease phenotype (11, 13). We compared the use of CD with CD/chol, which is 50% saturated with cholesterol, i.e. a shuttle system that samples the amount of cholesterol at the plasma membrane, presumably without disrupting the normal cholesterol trafficking pattern (15, 26).
Analysis of LDL Cholesterol Movement to the Plasma Membrane Using a
Continuous Label Protocol--
Wild-type CHO cells and mutants 2-2 and
CT60 were incubated for 10-120 min at 37 °C with
[3H]CL-LDL. During the incubation, the
[3H]CL-LDL will be internalized and hydrolyzed and
[3H]cholesterol will be released. To measure
[3H]cholesterol arrival in the plasma membrane, cells
were briefly exposed to media containing CD/chol, which acts as a
shuttle to sample cholesterol as it arrives at the plasma membrane (15, 26). Cell-associated [3H]cholesteryl linoleate and
[3H]cholesterol and [3H]cholesterol that
had desorbed to CD/chol were quantified (Fig. 2). We found that
[3H]CL-LDL uptake was equivalent in all cells (Fig.
2A), and similar amounts of the
[3H]cholesteryl linoleate were hydrolyzed at each time
point (data not shown). No [3H]cholesterol appeared in
media lacking CD/chol (data not shown). When cells were incubated for
10 min with [3H]CL-LDL and then exposed to media
containing CD/chol for 10 min, we found no
LDL-[3H]cholesterol in CD/chol-accessible pools. By 30 min of [3H]CL-LDL incubation, discernible LDL-derived
[3H]cholesterol was found in the plasma membrane of
wild-type cells but none in mutant 2-2 or CT60 cells. After 30 min, we
observed a time-dependent increase in CD/chol- accessible
[3H]cholesterol in wild-type CHO cells; however, very
little LDL-derived [3H]cholesterol became accessible to
CD/chol in mutant 2-2 or CT60 cells (Fig. 2, B and
C). These data suggest that either LDL cholesterol does not
move through the plasma membrane in NPC1 cells or that its appearance
there is transient. Therefore, we next evaluated LDL-[3H]cholesterol transport following the protocol used
by Cruz et al. (7).
|
Analysis of LDL Cholesterol Movement to the Plasma Membrane Using a
Pulse-Chase Protocol--
In this experiment, wild-type and mutant
cells were pulsed for 1 h at 37 °C with
[3H]CL-LDL and then incubated with either 25 mM CD/chol (as described above) or 12.5 mM CD
alone (as used by Cruz et al. (7)). Cell-associated [3H]cholesteryl linoleate and
[3H]cholesterol and [3H]cholesterol that
had desorbed to CD/chol or CD were quantified (Fig.
3). All cells internalized equivalent
amounts of [3H]CL-LDL during the 1-h pulse (data not
shown). During the chase incubations, the [3H]cholesteryl
linoleate was hydrolyzed to release free [3H]cholesterol
(Fig. 3, A and C). In this experiment less
[3H]cholesteryl linoleate hydrolysis occurred in CT60
than in CHO or 2-2 cells. However, the amount of LDL-derived
[3H]cholesterol accessible to CD was calculated as a
percentage of the available [3H]cholesterol. Under all
conditions we found that LDL-derived [3H]cholesterol
movement to the CD-accessible compartment was delayed by ~60 min in
mutant 2-2 or CT60 cells as compared with wild-type CHO cells (Fig. 3,
B and D). After 60 min the rate at which LDL [3H]cholesterol appeared in a CD-accessible pool was
equivalent in mutant and wild-type cells.
|
Analysis of LDL Cholesterol Movement in Mutant 3-6 Cells-- A second strategy to test whether LDL cholesterol traffics through the plasma membrane en route to NPC1-containing organelles used a somatic cell mutant with impaired transport of plasma membrane cholesterol into the cell interior combined with pharmacological inhibition of NPC1. In such a cell model, LDL-derived cholesterol should not reach the NPC1-containing late endosomes if its transport pathway takes it through the plasma membrane, but the LDL cholesterol should accumulate internally if it passes through late endosomes on its way to the plasma membrane. We created such a cell model by treating mutant 3-6 with either progesterone or hydrophobic amines. Mutant 3-6 is a CHO cell line that exhibits defective transport of cholesterol from the plasma membrane to the cell interior (14). Progesterone and hydrophobic amines such as U18666A and imipramine inhibit LDL cholesterol egress from lysosomes, causing an NPC phenotype in cultured cells (27-29).
Parental CHO and mutant 3-6 cells were incubated for 4 h in H-10%
LPDS containing no addition, 50 µg/ml LDL, or 50 µg/ml LDL plus 20 µg/ml progesterone (or 100 µM imipramine). Cells were then stained with filipin and inspected by fluorescence microscopy (Fig. 4). Parental CHO cells showed
distinct plasma membrane fluorescence when given no addition and some
additional punctate staining when given LDL. As expected, CHO cells
given LDL plus progesterone displayed intense punctate perinuclear
fluorescence indicative of lysosomal cholesterol storage. Mutant 3-6 cells were indistinguishable from parental CHO cells. They displayed
distinct plasma membrane fluorescence when given LDL and punctate
fluorescence when given LDL plus progesterone. CHO and mutant 3-6 cells
treated with imipramine were identical to those treated with
progesterone (data not shown). These results provide further support to
our hypothesis that LDL cholesterol traffics through NPC1 vesicles
before arriving at the plasma membrane.
|
Basal Esterification of Plasma Membrane Cholesterol--
A third
strategy to test this hypothesis is to determine whether plasma
membrane cholesterol is transported through NPC1-containing organelles
on the way to the endoplasmic reticulum. Cultured cells incubated with
[3H]cholesterol adsorb the [3H]cholesterol
and distribute it according to their cellular cholesterol pools. With
time, [3H]cholesterol reaches acyl-CoA/cholesterol
acyltransferase in the endoplasmic reticulum and is esterified. We
incubated parental CHO cells and four mutant cell lines from the NPC1
complementation group with [3H]cholesterol for various
times. Fig. 5 shows a
time-dependent incorporation of
[3H]cholesterol into [3H]cholesteryl esters
in parental CHO cells and equivalent [3H]cholesterol
ester formation in each of the mutant cell lines. This result is not
consistent with plasma membrane cholesterol being transported through
NPC1-containing organelles to the cell interior.
|
Sequence Analysis of Hamster NPC1 Mutants--
To confirm genetic
evidence that our cholesterol transport-defective CHO cells are NPC1
mutants, we cloned and sequenced the NPC1 cDNA from our
parental CHO-K1 cells and four independently isolated cell lines,
mutants 2-2, 4-4, 10-3, and 1-2. The nucleotide and deduced amino acid
sequence of NPC1 from our CHO-K1 cells was identical to that
reported previously (7). The NPC1 mutations in the mutant
cell lines were located by using a BLAST2 search after initial sequence
analysis with AssemblyLIGN and MacVector programs. Mutant 2-2 contains
a base-pair insertion of A at nucleotide 1335, resulting in a
frameshift and premature translational termination after 450 amino
acids (Fig. 6). Mutant 4-4 contains a
base insertion of C at nucleotide 1946, which results in a frameshift
and premature translational termination after 652 amino acids (Fig. 6).
Mutant 1-2 contains no apparent mutations in the NPC1 coding
region. It is possible that mutations exist in the 3'- or
5'-untranslated regions. We were unable to obtain the complete sequence
for mutant 10-3.
|
Northern blot analysis showed that parental and mutant cells express an
NPC1 mRNA of comparable size. However, mRNA levels are reduced in mutant 2-2 and practically absent in mutant 10-3 (Fig.
7). The lack of NPC1 mRNA
in mutant 10-3 may explain our difficulty in sequencing a cDNA.
|
Transient Expression of Human NPC1--
A hallmark of NPC1 cells
is the punctate filipin fluorescence, which denotes lysosomal
cholesterol storage. To determine whether expression of the human
NPC1 cDNA (phuNPC1) was able to correct the
filipin-staining pattern in our mutants, we performed co-transfection experiments with phuNPC1 and pEGFP, which expresses the
green fluorescent protein (GFP). Fig. 8
shows that filipin-stained parental CHO cells have diffuse
fluorescence, whether they are untransfected, expressing pEGFP or
phuNPC1 plus pEGFP. The expression of pEGFP alone in mutant
cells had no effect on filipin fluorescence; however, when mutant cells
were transfected with pEGFP plus phuNPC1, the GFP-positive
cells showed correction of the filipin staining. These results support
the complementation and sequence analyses of our NPC1-deficient
mutants.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we have evaluated the fate of cholesterol released by LDL-cholesteryl ester hydrolysis in the endocytic pathway. It is generally accepted that in normal cells LDL cholesterol is rapidly transported to the plasma membrane and other cellular membranes, whereas in NPC cells LDL cholesterol ends up sequestered in terminal storage compartments along with glycosphingolipids. What is disputed is whether NPC1 facilitates the initial transport of LDL cholesterol to the plasma membrane, or whether LDL cholesterol is transported by other means to the plasma membrane and is then internalized to NPC1-containing organelles for further disposition (Fig. 1). The latter model calls for all plasma membrane cholesterol to be routed through the NPC1-containing organelles before arrival in other intracellular membranes (7).
We used three experimental approaches to evaluate these models in wild-type and NPC1-deficient CHO cell lines. First, CD/chol complexes were used to detect the arrival of LDL-derived [3H]cholesterol in the plasma membrane. We found that under all experimental conditions NPC1 mutants exhibited a distinct lag in the movement of LDL-derived [3H]cholesterol to the CD-accessible plasma membrane. We were unable to demonstrate any early transient appearance of LDL-[3H]cholesterol at the plasma membrane.
We found the same qualitative results using either CD or CD/chol as cholesterol acceptors in the media, although CD/chol consistently promoted more [3H]cholesterol efflux from cells than an equal concentration of CD alone. This finding is consistent with the notion of CD/chol as a effective cholesterol shuttle and suggests that cholesterol transfer to the plasma membrane stimulates the reciprocal cholesterol movement.
In the second approach, we tested specifically whether LDL-[3H]cholesterol passes through the plasma membrane before its arrival in the NPC1-containing late endosomes. We used mutant 3-6, which exhibits defective transport of plasma membrane cholesterol to internal membranes (14). A classical genetic approach to this problem would be to perform epistasis analysis, in which an NPC1/3-6 double mutant would be evaluated. If LDL cholesterol passed through NPC1-containing organelles before arrival in the plasma membrane, then an NPC1/3-6 mutant would show lysosomal accumulation of cholesterol. However, if LDL cholesterol were transported to the plasma membrane and then to the NPC1-containing organelles, then an NPC1/3-6 double mutant would show no intracellular storage of cholesterol. An NPC1/3-6 double mutant is not available; however, we created an equivalent cell model using pharmacological agents that cause an NPC1 phenotype. We found that when 3-6 cells were treated with either progesterone or imipramine, incubation with LDL led to punctate filipin fluorescence that was indistinguishable from the classical NPC phenotype. This result is consistent with LDL cholesterol reaching the NPC1-containing late endosomes before reaching the plasma membrane.
In the third approach, we evaluated whether plasma membrane cholesterol passes through NPC1-containing late endosomes en route to the endoplasmic reticulum. Lange et al. (30) have estimated that in cultured cells the entire plasma membrane cholesterol pool cycles between the plasma membrane and endoplasmic reticulum with a half-time of 40 min; thus, delayed kinetics in cholesterol exiting the NPC1 organelles should be readily apparent. We found identical basal [3H]cholesterol esterification in wild-type and NPC1-deficient cells. These results are consistent with our previous finding that imipramine has no effect on plasma membrane to endoplasmic reticulum cholesterol movement at concentrations that cause the NPC1 phenotype (4). Together they indicate that the plasma membrane to endoplasmic reticulum cholesterol transport pathway is intact and that the NPC1-containing late endosomes are not an obligate part of that pathway.
An intact plasma membrane to endoplasmic reticulum cholesterol transport pathway was also indicated in studies of cholesterol balance in the NPCnih mouse model of NPC disease. Xie et al. (31) found that cholesterol delivered via HDL to the adrenal was used as normal for cholesterol esterification and steroid hormone production, and that HDL-cholesterol delivered to the liver could apparently be secreted into the bile or converted to bile acids as normal in NPCnih mice. This activity of cholesterol delivered via HDL contrasted with cholesterol delivered via LDL or chylomicrons, which was sequestered within a metabolically inactive pool (31).
We can think of no apparent reason for LDL cholesterol to be released by non-NPC1-containing late endosomes/lysosomes and rapidly transported to the plasma membrane followed by quick retrieval and delivery to NPC1-containing organelles, as proposed by Cruz et al. (7) (Fig. 1B). If this rapid transit does take place, it is not clear how those cholesterol molecules would be quantitatively retrieved. Does the retrieval mechanism distinguish between the newly delivered cholesterol molecules and the other plasma membrane pool(s), or are they delivered to a plasma membrane domain that does not diffuse laterally? One possibility is that the cholesterol detected by Cruz et al. (7) is actually an internal pool that is capable of being diverted to the plasma membrane by CD depletion of plasma membrane cholesterol content.
Our hypothesis is that membrane lipid and receptor-bound cargo such as
LDL is internalized and delivered to the endocytic recycling
compartment (32) (Fig. 1A). LDL is released from its receptor and sorted into early endosomes. LDL-cholesteryl ester hydrolysis likely begins when the LDL particle is in transit through the endocytic pathway. In multivesicular late endosomes, NPC1 facilitates the rapid transport of lipids, whether they arrived as
constituents of the lipoprotein particles or of the vesicle membranes.
NPC1-containing vesicles have been observed to bud from late endosomes,
presumably carrying cholesterol and other cargo, and fuse with
perinuclear and peripheral membranous compartments, although not the
plasma membrane (20). Cholesterol may be delivered via NPC1 to the
endocytic recycling compartment (32) from which it recycles to the
plasma membrane. When NPC1 is dysfunctional, cholesterol accumulates in
terminal organelles because its acquisition by vesicular transport and
lipoprotein uptake is greater than its dispersal by vesicular
trafficking alone. We conclude that plasma membrane cholesterol can
reach the endoplasmic reticulum by NPC1-dependent and
NPC1-independent pathways. The NPC1-dependent pathway
involves the movement of membrane lipids by vesicle trafficking through
endosomes to the NPC1-containing late endosomes, where it is dispersed
by NPC1. The NPC1-independent pathway is responsible for rapid cycling
of cholesterol to the cell interior and back and represents the pathway
that is defective in mutant 3-6.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Peter Pentchev for very helpful discussions, Jill Morris and Peter Pentchev for providing the human NPC1 cDNA, and T. Y. Chang for generously supplying the hamster NPC1 cDNA sequence. Emily Arnio provided excellent assistance with filipin fluorescence microscopy.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant DK49564, National Institutes of Health Training Grant DK07704, and by the Center for Gastroenterology Research on Absorptive and Secretory Processes, Molecular Biology and Image Analysis Core (NIDDK P30 DK34928).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Physiology,
Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA
02111. Tel.: 617-636-6945; Fax: 617-636-0445; E-mail: Laura.Liscum@Tufts.Edu.
Published, JBC Papers in Press, February 18, 2003, DOI 10.1074/jbc.M300488200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NPC, Niemann-Pick disease type C;
CD, 2-hydroxy-propyl--cyclodextrin;
CD/chol, CD that is 50% saturated with cholesterol;
CHO, Chinese
hamster ovary;
CL, cholesteryl linoleate;
LDL, low density
lipoprotein;
LPDS, lipoprotein-deficient serum;
NCS, newborn calf
serum;
GFP, green fluorescent protein;
TBS, Tris-buffered saline;
pEGFP, plasmid encoding green fluorescent protein.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Patterson, M. C., Vanier, M. T., Suzuki, K., Morris, J. A., Carstea, E., Neufeld, E. B., Blanchette-Mackie, J. E., and Pentchev, P. G. (2001) in The Metabolic and Molecular Bases of Inherited Disease (Scriver, C. R. , Beaudet, A. L. , Sly, W. S. , and Valle, D., eds), 8th Ed., Vol. III , pp. 3611-3633, McGraw-Hill, New York |
2. | Liscum, L. (2000) Traffic 1, 218-225[CrossRef][Medline] [Order article via Infotrieve] |
3. | Ory, D. S. (2000) Biochim. Biophys. Acta 1529, 331-339[Medline] [Order article via Infotrieve] |
4. |
Underwood, K. W.,
Jacobs, N. L.,
Howley, A.,
and Liscum, L.
(1998)
J. Biol. Chem.
273,
4266-4274 |
5. | Higgins, M. E., Davies, J. P., Chen, F. W., and Ioannou, Y. A. (1999) Mol. Genet. Metab. 68, 1-13[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Neufeld, E. B.,
Wastney, M.,
Patel, S.,
Suresh, S.,
Cooney, A. M.,
Dwyer, N. K.,
Roff, C. F.,
Ohno, K.,
Morris, J. A.,
Carstea, E. D.,
Incardona, J. P.,
Strauss, J. F., III,
Vanier, M. T.,
Patterson, M. C.,
Brady, R. O.,
Pentchev, P. G.,
and Blanchette-Mackie, E. J.
(1999)
J. Biol. Chem.
274,
9627-9635 |
7. |
Cruz, J. C.,
Sugii, S., Yu, C.,
and Chang, T. Y.
(2000)
J. Biol. Chem.
275,
4013-4021 |
8. |
Cruz, J. C.,
and Chang, T. Y.
(2000)
J. Biol. Chem.
275,
41309-41316 |
9. |
Lange, Y.,
Ye, J.,
Rigney, M.,
and Steck, T.
(2000)
J. Biol. Chem.
275,
17468-17475 |
10. |
Lange, Y.,
Ye, J.,
Rigney, M.,
and Steck, T. L.
(2002)
J. Lipid Res.
43,
198-204 |
11. |
Dahl, N. K.,
Reed, K. L.,
Daunais, M. A.,
Faust, J. R.,
and Liscum, L.
(1992)
J. Biol. Chem.
267,
4889-4896 |
12. | Dahl, N. K., Daunais, M. A., and Liscum, L. (1994) J. Lipid Res. 35, 1839-1849[Abstract] |
13. | Cadigan, K. M., Spillane, D. M., and Chang, T.-Y. (1990) J. Cell Biol. 110, 295-308[Abstract] |
14. | Jacobs, N. L., Andemariam, B., Underwood, K. W., Panchalingam, K., Sternberg, D., Kielian, M., and Liscum, L. (1997) J. Lipid Res. 38, 1973-1987[Abstract] |
15. | Christian, A. E., Haynes, M. P., Phillips, M. C., and Rothblat, G. H. (1997) J. Lipid Res. 38, 2264-2272[Abstract] |
16. | Goldstein, J. L., Basu, S. K., and Brown, M. S. (1983) Methods Enzymol. 98, 241-260[Medline] [Order article via Infotrieve] |
17. | Faust, J. R., Goldstein, J. L., and Brown, M. S. (1977) J. Biol. Chem. 252, 4861-4871[Medline] [Order article via Infotrieve] |
18. |
Lowry, O. H.,
Rosebrough, N. J.,
Farr, A. L.,
and Randall, R. J.
(1951)
J. Biol. Chem.
193,
265-275 |
19. |
Carstea, E. D.,
Morris, J. A.,
Coleman, K. G.,
Loftus, S. K.,
Zhang, D.,
Cummings, C.,
Gu, J.,
Rosenfeld, M. A.,
Pavan, W. J.,
Krizman, D. B.,
Nagle, J.,
Polymeropoulos, M. H.,
Sturley, S. L.,
Ioannou, Y. A.,
Higgins, M. E.,
Comly, M.,
Cooney, A.,
Brown, A.,
Kaneski, C. R.,
Blanchette-Mackie, E. J.,
Dwyer, N. K.,
Neufeld, E. B.,
Chang, T.-Y.,
Liscum, L.,
Strauss, J. F.,
Ohno, K.,
Zigler, M.,
Carmi, R.,
Sokol, J.,
Markie, D.,
O'Neill, R. R.,
Diggelen, O. P. v.,
Elleder, M.,
Patterson, M. C.,
Brady, R. O.,
Vanier, M. T.,
Pentchev, P. G.,
and Tagle, D. A.
(1997)
Science
277,
228-231 |
20. |
Ko, D. C.,
Gordon, M. D.,
Jin, J. Y.,
and Scott, M. P.
(2001)
Mol. Biol. Cell
12,
601-614 |
21. |
Pentchev, P. G.,
Comly, M. E.,
Kruth, H. S.,
Tokoro, T.,
Butler, J.,
Sokol, J.,
Filling-Katz, M.,
Quirk, J. M.,
Marshall, D. C.,
Patel, S.,
Vanier, M. T.,
and Brady, R. O.
(1987)
FASEB J.
1,
40-45 |
22. | Liscum, L., Ruggiero, R. M., and Faust, J. R. (1989) J. Cell Biol. 108, 1625-1636[Abstract] |
23. |
Watari, H.,
Blanchette-Mackie, E. J.,
Dwyer, N. K.,
Watari, M.,
Neufeld, E. B.,
Patel, S.,
Pentchev, P. G.,
and Strauss, J. F., III.
(1999)
J. Biol. Chem.
274,
21861-21866 |
24. |
Kilsdonk, E. P. C.,
Yancey, P. G.,
Stoudt, G. W.,
Bangerter, F. W.,
Johnson, W. J.,
Phillips, M. C.,
and Rothblat, G. H.
(1995)
J. Biol. Chem.
270,
17250-17256 |
25. |
Christian, A. E.,
Byun, H. S.,
Zhong, N.,
Wanunu, M.,
Marti, T.,
Furer, A.,
Diederich, F.,
Bittman, R.,
and Rothblat, G. H.
(1999)
J. Lipid Res.
40,
1475-1482 |
26. |
Yancey, P. G.,
Rodrigueza, W. V.,
Kilsdonk, E. P.,
Stoudt, G. W.,
Johnson, W. J.,
Phillips, M. C.,
and Rothblat, G. H.
(1996)
J. Biol. Chem.
271,
16026-16034 |
27. |
Butler, J. D.,
Blanchette-Mackie, J.,
Goldin, E.,
O'Neill, R. R.,
Carstea, G.,
Roff, C. F.,
Patterson, M. C.,
Patel, S.,
Comly, M. E.,
Cooney, A.,
Vanier, M. T.,
Brady, R. O.,
and Pentchev, P. G.
(1992)
J. Biol. Chem.
267,
23797-23805 |
28. |
Liscum, L.,
and Faust, J. R.
(1989)
J. Biol. Chem.
264,
11796-11806 |
29. | Rodriguez-Lafrasse, C., Rousson, R., Bonnet, J., Pentchev, P. G., Louisot, P., and Vanier, M. T. (1990) Biochim. Biophys. Acta 1043, 123-128[Medline] [Order article via Infotrieve] |
30. |
Lange, Y.,
Strebel, F.,
and Steck, T. L.
(1993)
J. Biol. Chem.
268,
13838-13843 |
31. |
Xie, C.,
Turley, S. D.,
and Dietschy, J. M.
(2000)
J. Lipid Res.
41,
1278-1289 |
32. |
Hao, M.,
Lin, S. X.,
Karylowski, O. J.,
Wustner, D.,
McGraw, T. E.,
and Maxfield, F. R.
(2002)
J. Biol. Chem.
277,
609-617 |