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(Received for publication, May 9, 1996, and in revised form, June 5, 1996)
From the ABSTRACT The sterol binding agent
2-hydroxypropyl- INTRODUCTION Cholesterol is an integral membrane component required for normal
cellular function (1). In mammalian cells this sterol is derived either
from low density lipoprotein (LDL)1
following receptor-mediated endocytosis and subsequent hydrolysis in
lysosomes (2) or via de novo biosynthesis in the endoplasmic
reticulum (ER) (3). The mechanism for the ensuing movement of
cholesterol from these intracellular sites to their ultimate cellular
destinations is an unresolved question of fundamental importance to
cell biology and medicine. Defects in these transport pathways can
alter cellular cholesterol metabolism resulting in pathological states.
Niemann-Pick C (NP-C) disease is characterized by sequestration of
LDL-derived cholesterol in lysosomes that is linked to delays in the
induction of cholesterol-mediated homeostatic responses (4, 5).
Development of atherosclerotic lesions involves in part a failure to
maintain adequate transfer of cholesterol from intracellular sites of
accumulation to the cell surface for removal by extracellular acceptors
(6). Understanding pathways of intracellular cholesterol transport is a
critical step toward the correction of cellular cholesterol
lipidoses.
The intracellular distribution of cholesterol generated by these
transport pathways is not uniform, with a major fraction present in the
plasma membrane (PM) (7). It has been suggested that this high
concentration of cholesterol at the cell surface results in part from
its close association with sphingomyelin (SM) (8). Enzymatic digestion
of SM by exogenously added sphingomyelinase (SMase) mobilizes PM
cholesterol for esterification in the ER (9, 10, 11, 12).
Several methods have been employed to monitor intracellular movement of
cholesterol. Arrival of cholesterol at the ER is accompanied by
esterification catalyzed by the resident enzyme acetyl-coenzyme
A:cholesterol acyltransferase. Transfer of cholesterol to the PM has
been studied by subcellular fractionation (13), chemical modification
by exogenously added cholesterol oxidase (14), or via removal from the
cell surface to extracellular cholesterol acceptors (15, 16). This
latter approach has been limited by the lack of an acceptor that
efficiently removes cholesterol. The effectiveness of cholesterol
depletion from the PM by HDL varies with cell type and is neither an
extensive nor a rapid process (16, 17).
In the present study we demonstrate the utility of
2-hydroxypropyl- EXPERIMENTAL PROCEDURES Fetal bovine serum was obtained from HyClone
Laboratories, Inc., Logan, UT. Lipoprotein-deficient bovine serum
(LPDS) and human low density lipoprotein (LDL) were prepared by
Advanced Bioscience Laboratories, Rockville, MD. Glass and plastic
microscope culture wells (Lab-Tek) were purchased from Thomas
Scientific. [9,10-3H]Oleic acid (10-14 Ci/mmol),
[3H]acetate (135 mCi/mmol),
[1,2,6,7-3H]cholesteryl oleate (98.5 Ci/mmol),
[1,2,6,7-3H]cholesteryl linoleate (87 Ci/mmol), and
[5-3H]mevalonate (33 Ci/mmol) were purchased from DuPont
NEN. Filipin was purchased from Polysciences, Warrington, PA.
2-Hydroxypropyl- Normal and NP-C fibroblasts were derived
from volunteers and confirmed patients of the Developmental and
Metabolic Neurology Branch under the guidelines approved by clinical
and research committees of the National Institutes of Health.
Fibroblasts (3-15 passages) were cultured in Eagle's minimal
essential medium supplemented with 10% fetal bovine serum, 1%
nonessential amino acids, 2 m glutamine, and 100 units of
penicillin/streptomycin/ml in humidified 95% air and 5%
CO2 at 37 °C. For biochemical analyses, fibroblasts were
seeded at a density of 40,000 cells/well (``subconfluent'') in
plastic 6-well dishes (Costar, Cambridge, MA) in McCoy's medium
supplemented with 5% LPDS, 2 m glutamine, and 100 units
of penicillin/streptomycin/ml (McCoy's/5% LPDS medium) in humidified
95% air and 5% CO2 at 37 °C for 4 days. For
cytochemical analyses, fibroblasts were seeded at a density of 20,000 cells/well in McCoy's/5% LPDS medium in 9.5-cm glass microscope wells
(Nunc, Inc., Naperville, IL) coated with human fibronectin. For
cholesterol depletion studies, 2-hydroxy-propyl- Human LDL was
reconstituted with [3H]cholesteryl linoleate or
[3H]cholesteryl oleate as described by Krieger (23). A
modification of the procedure described by Rome et al. (24)
for the fractionation of human fibroblasts was employed. Cells were
harvested by scraping with a razor blade and disrupted by
N2 cavitation. A combined postnuclear supernatant fraction
(600 × g, 10 min) was obtained from 1 to 2 confluent
75-cm2 flasks that generally contained 0.5-1.0 mg of
protein in 2.0 ml of 0.25 sucrose and 1.0 m
EDTA, pH 7.0. Stock Percoll was diluted to 10% (v/v) with 0.25 sucrose and 1.0 m EDTA, pH 7.0. The
postnuclear supernatant (2.0 ml) was layered on top of 27 ml of 10%
Percoll and a 4.0-ml cushion of 2.5 sucrose in Sorvall
polyallomer tubes (1 × 3.5 cm). Centrifugation was carried out in
a Sorvall SV288 vertical rotor at 28,000 × g for
1 h at 4 °C in a Sorvall RC2C Plus centrifuge equipped with a
rate controller. Fractions (2.0 ml) were collected from the bottom of
the gradients with the aid of a Beckman Fraction Recovery System. A
light density membrane fraction as well as a heavy density membrane
fraction was found in both normal and NP-C human fibroblast
preparations. Alkaline phosphatase assayed by the method of Touster
et al. (25) sedimented with the light density protein
peak.
Cell monolayers were washed directly in
their plastic 35-mm wells twice with chilled phosphate-buffered saline
(PBS) and then extracted with 0.5 ml of isopropyl alcohol by gentle
rocking for 30 min at room temperature in sealed plates. The isopropyl
alcohol-extracted lipids were stored at Cells in glass slide chambers were
washed with PBS, fixed in 3% paraformaldehyde for 30 min at room
temperature, washed with PBS, and then incubated overnight with 0.05%
filipin in PBS at room temperature. Cells were washed with PBS, mounted
in p-phenylenediamine glycerol, and viewed with a Leitz
fluorescence microscope by using excitation filter BP-350-410 for
filipin.
RESULTS 2-Hydroxypropyl-
Cholesterol mass in normal human fibroblasts after incubation with
various concentrations of 2-hydroxypropyl-
Exposure of normal and NP-C cultures to neutral sphingomyelinase
(SMase) for 1 h resulted in the digestion of 80% of
[3H]sphingomyelin formed by de novo
biosynthesis (data not shown). SMase treatment dramatically enhances
both the rate and extent of removal of cellular
[3H]sterol by CD (Fig. 2). Treatment with
CD following SM digestion resulted in the removal of cellular sterol in
a biphasic manner with an initial rapid rate of depletion (70% within
15 min) followed by a subsequent slower rate of depletion
(t1/2 = 5-6 h). Similar results were obtained when
cells were treated with CD and SMase concurrently (data not shown).
There was no detectable release of lactic acid dehydrogenase to the
medium nor altered triglyceride synthesis for up to 2 h of this
combined treatment (data not shown).
We employed fluorescence microscopy using filipin, a fluorescent marker
specific for unesterified cholesterol (30), to demonstrate that CD
primarily targets the PM for removal of cellular cholesterol. In normal
cells, we have confirmed (31) that filipin-cholesterol fluorescence is
associated with both the PM and intracellular structures (Fig.
3A). Treatment of these cells with CD alone
greatly diminishes PM fluorescence without substantially affecting
intracellular staining (Fig. 3B). Filipin-cholesterol
fluorescence associated with the PM is essentially eliminated after
treatment with CD/SMase (Fig. 3C). In NP-C fibroblasts
cultured with LDL, we also confirmed (31) that considerable
filipin-cholesterol fluorescence is seen at the PM as well as intense
fluorescence in perinuclear lysosomes resulting from defective
intracellular mobilization of LDL-derived cholesterol in these cells
(Fig. 4A). Treatment of these NP-C cells with
CD/SMase resulted in complete loss of filipin-cholesterol fluorescence
from the PM with no substantial loss of fluorescence from intracellular
sites (Fig. 4B).
CD preferentially removes LDL-derived cholesterol from PM as determined
by subcellular fractionation. Following incubation of both normal and
NP-C fibroblast cultures with LDL [3H]cholesteryl
linoleate for 24 h, a portion of the cultures was subsequently
treated with CD/SMase. Subcellular fractionation of cell extracts on
10% Percoll gradients reveals two peaks of
[3H]cholesterol representing heavy density (fractions
1-5) and light density (fractions 10-15) membranes (Fig.
5). A lysosomal marker (
CD can also prevent internalization of PM cholesterol to intracellular
compartments. Fibroblasts were treated with SMase to induce movement of
PM cholesterol to the ER (10). This incubation, performed in the
presence of [3H]oleate to follow arrival of cholesterol
at the ER by esterification, was carried out in the absence or presence
of CD. Treatment of fibroblasts with SMase stimulated cholesterol
esterification (Fig. 6). This SMase-stimulated
esterification of cholesterol is markedly suppressed by CD.
Fibroblasts were
incubated with [3H]mevalonate in the presence or absence
of CD to follow incorporation of nascent [3H]sterol into
the PM (Fig. 7). CD substantially reduces this
cell-associated [3H]sterol (Fig. 7A) without
altering total sterol synthesis. A major fraction (80%) of the newly
synthesized [3H]sterol was transferred to the medium at
24 h in the presence of CD (Fig. 7B). The relatively
small proportion of newly synthesized [3H]sterol (20%)
that remained associated with cells may represent intracellular pools
or a more highly resistant PM sterol pool.
The arrival of lysosomal cholesterol at the PM can be detected with CD.
Normal and NP-C fibroblasts were incubated with
LDL[3H]cholesteryl oleate in the presence of progesterone
in order to sequester [3H]cholesterol in lysosomes (32).
Progesterone was washed from cultures (to initiate
[3H]cholesterol transport out of lysosomes) in the
presence of CD. Reduction in this cell-associated
[3H]cholesterol in the presence of CD corresponded to its
appearance in the medium (Fig. 8). Lysosomally
derived [3H]cholesterol was lost from normal cells with a
half-life of 4 h and from NP-C cells with a half-life of 8.5 h, reflecting the documented lesion in transfer of cholesterol from
lysosomes to the PM (15).
Arrival of lysosomal cholesterol at the PM can also be indirectly
monitored by assessing the effect of CD on the level of cholesterol
esterified during its release from lysosomes. LDL-derived lysosomal
cholesterol has been shown to be transported to the PM (15, 33, 34, 35)
where it enriches a precursor pool of cholesterol destined to be
esterified in the ER (35, 36, 37, 38). One would predict that CD can retard
cellular cholesterol esterification by removing substrate from the PM.
This was tested by loading the lysosomes of cultured fibroblasts with
LDL-derived cholesterol using progesterone. During a subsequent period
of progesterone washout, cells were incubated with
[3H]oleate (to assay esterification) in the absence or
presence of 2% CD (to remove PM cholesterol). CD strongly
inhibited the esterification normally seen during progesterone washout
(Fig. 9A) consistent with the relocation of a
large portion of lysosomal cholesterol to the PM prior to its
esterification in the ER. The metabolic suppression of
[3H]oleate incorporation is specific for cholesterol
esterification since CD did not alter triglyceride synthesis (data not
shown). The level of esterification induced in cholesterol-loaded NP-C
cells during progesterone washout was approximately 20-fold less than
that observed in normal cells (Fig. 9B). CD did not suppress
this attenuated level of esterification (Fig. 9B),
consistent with a disrupted movement of lysosomal cholesterol to the PM
in the mutant cells. The residual esterification in NP-C cells may
represent intracellular routing of lysosomal cholesterol to the ER via
a pathway that bypasses the PM.
Removal of PM sphingomyelin can affect
the intracellular transport path of lysosomal cholesterol. Our
experimental protocol was to load the lysosomes of cultured normal
fibroblasts with LDL-cholesterol using progesterone. These cells were
then briefly treated with SMase to deplete PM SM, followed by washing
the progesterone from the cultures to establish cholesterol transport
out of lysosomes. During progesterone washout cells were incubated with
[3H]oleate in the absence or presence of CD. As
established above (Fig. 9), CD alone can effectively suppress
esterification of cholesterol in untreated cells (Fig.
10A). Depletion of PM SM alone does not
alter the level of esterification of lysosomal cholesterol
(cf. Fig. 10, A and B). However, when
cells were exposed to SMase, CD no longer blocked cholesterol
esterification (Fig. 10B). This loss in the sensitivity of
esterification to CD modulation occurs under conditions that render
cells particularly sensitive to removal of PM sterol (Fig. 2). These
data suggest that depletion of PM SM diverts the trafficking of
lysosomal cholesterol away from the PM without diminishing its
accessibility to the ER.
The current studies with CD provide evidence
that the Golgi plays a key role in the movement of LDL-derived
cholesterol from lysosomes to the PM. Lysosomes of normal and NP-C
fibroblasts were loaded with LDL-cholesterol using progesterone.
Lysosomal cholesterol transport was re-established with progesterone
washout, and cells were incubated with [3H]oleate in the
presence or absence of BFA and/or CD. The effective suppression of
cholesterol esterification by CD during progesterone washout in normal
fibroblasts (Fig. 11A) was largely lost when
BFA was added (Fig. 11B). This finding does not result from
a loss in the ability of CD to remove [3H]sterol from the
PM of BFA-treated cells labeled to equilibrium with
[3H]acetate (data not shown). The loss of CD-mediated
suppression of esterification in the presence of BFA suggests that
disruption of the Golgi complex (39) reduces the intracellular
trafficking of lysosomal cholesterol to the PM without substantially
diminishing its accessibility to the ER. In NP-C cells, CD does not
suppress the attenuated cholesterol esterification either in the
presence (Fig. 11C) or absence (Fig. 11D) of BFA.
In these mutant cells, BFA stimulates cholesterol esterification 4-fold
(Fig. 11D). The BFA-induced increase in cholesterol
esterification is consistent with previous observations (40, 41, 42)
and suggests that Golgi cholesterol can be absorbed into the ER along
with other Golgi components.
The Golgi also appears to play a role in the movement of PM cholesterol
to the ER in normal and NP-C cells. Such movement can be induced by
treating cells with SMase and assessed by measuring esterification
(10). When cells are treated with either BFA or SMase alone there is a
relatively weak stimulation in esterification of PM sterol (Fig.
12). However, when BFA and SMase are both present,
there is a dramatic increase in the esterification of PM sterol in
normal and mutant cell cultures (Fig. 12). In NP-C cells, stimulation
of esterification by co-treatment with BFA and SMase is 50% of that
seen in normal cells. It appears that BFA-mediated perturbations of the
Golgi complex enhance access of SMase-mobilized PM sterol to the ER for
esterification.
DISCUSSION We have demonstrated that CD provides a simple and effective
method for monitoring the flow of cellular cholesterol pools to and
from the plasma membrane of living cells. Using this approach we have
followed the movement of cholesterol derived from LDL or de
novo biosynthesis. We have found that the bulk of LDL-derived
cholesterol destined for esterification in the ER is first delivered to
the PM. In addition we provide evidence that transport of cholesterol
from lysosomes via this pathway can be dramatically affected by the
integrity of the Golgi complex and the SM content of the PM.
Our experimental approach depends on the effective removal of
unesterified cholesterol by CD from viable cells. CD depletes up to
80% of cellular cholesterol from intact, viable cells in a
concentration (Table I) and time (Fig. 1) -dependent
manner. The kinetics of sterol efflux from human fibroblasts to CD are
similar to those recently reported for mouse L-cells (22). The efflux
process likely involves cholesterol desorption from cells followed by
diffusion to an extracellular acceptor (6, 43). An excess pool of CD,
routinely used in our present studies, serves as a sink for cellular
cholesterol. Biochemical and cytochemical analyses strongly suggest
that CD specifically targets removal of cholesterol from the cell
surface. Depletion of cellular cholesterol with CD corresponds to a
marked loss of filipin-cholesterol fluorescence from the PM but not
from intracellular pools. Subcellular fractionation of normal and
Niemann-Pick C cells reveals that CD preferentially removes LDL-derived
[3H]cholesterol primarily from light membranes (PM). The
kinetics and extent of cholesterol removal from the PM of normal or
NP-C fibroblasts by CD are indistinguishable (Fig. 1). This suggests
that the defective intracellular transport of cholesterol in NP-C cells
(4, 15) is not an intrinsic property of the PM.
The large fraction of cellular cholesterol associated with the PM has
often been attributed to its interaction with SM at this site (38).
This hypothesis is based in part on observations that cells treated
with SMase display a burst of esterification at the ER of cholesterol
derived from the PM (9, 10, 11). These studies suggested that a large
portion of PM cholesterol relocated to intracellular sites that
appeared to be inaccessible to extracellular lipid acceptors such as
HDL3 (40, 44). In the present study we find, however, that
removal of cellular cholesterol by CD is, in fact, significantly
enhanced after enzymatic depletion of PM SM (Fig. 2). Our data are
consistent with the recent report that the majority of cellular
cholesterol remains at the PM after SM depletion (45). The observed
level of enhancement of CD-mediated cholesterol removal after SM
depletion suggests that a considerable portion of PM cholesterol
(~50%) may be associated with SM. We also show that CD, in contrast
to HDL (40, 44), can divert PM cholesterol from intracellular transport
pathways, such as movement to the ER for esterification (Fig. 6). This
difference most likely reflects the relative effectiveness of CD as a
cholesterol acceptor (22).
Because of its effective removal of surface cholesterol, CD provides a
facile means for monitoring sterol flux through the PM. CD removes a
significant fraction of de novo cellular sterol (Fig. 7). It
has been estimated that the half-time for delivery of cholesterol from
its site of synthesis in the ER to the cell surface is 10-30 min in
several cell types (46, 47, 48). The half-times reported for fibroblasts
appear to depend on the methods employed, ranging from 10 min (13) to
1-2 h (49). The reason for these discrepancies remains unclear. Our
data (Fig. 7B) show a marked increase in the rate of removal
of de novo synthesized sterol to CD after a few hours of
incubation with the [3H]mevalonate precursor, consistent
with the extended time of transfer in fibroblasts (49). Further studies
will enable us to provide a more precise measurement for the rate of
transfer.
Transport of LDL-derived cholesterol from lysosomes to the PM can also
be followed with CD. We have previously shown that progesterone
treatment of normal cells produces an accumulation of LDL-derived
cholesterol in lysosomes, as seen with the NP-C mutation (32). After
progesterone removal, this cholesterol pool leaves lysosomes. The
reversibility of the progesterone-induced block provides a direct
method to study egress of cholesterol from lysosomes. We find that
removal of LDL-derived cholesterol by CD during progesterone washout
occurs with half-times of 4 h in normal cells and 8 h in NP-C
cells (Fig. 8). This difference appears to reflect the lesion in
lysosomal cholesterol trafficking to the PM reported in the mutant
cells (15), since the rates of removal of resident PM sterol itself by
CD are identical in the two cell types. Our measurements of removal of
lysosomal cholesterol from cells by CD during progesterone washout are
considerably longer than those previously reported for the movement of
pulse-labeled lysosomal cholesterol to the PM with values of 1-2 min
(33), 40-50 min (34), and 1-2 h (15). Differences among these data
may be attributable to variability in the cell type and the methods
employed. Our measurements may also reflect extensive loading of
lysosomes with cholesterol that occurs when cultures are exposed to
progesterone during LDL uptake (32). The slower rates of clearance that
we report may therefore reflect saturation of the transport pathways
that mediate movement of lysosomal cholesterol to the PM as well as the
rate at which CD can remove cholesterol from the PM. Nonetheless, it is
important to note that we detect a slower rate in the movement of
LDL-derived cholesterol to the PM in NP-C cells compared with normal
cells.
Movement of cholesterol from lysosomes during progesterone washout can
also be monitored through its subsequent esterification (32). This
pathway appears to involve an initial transfer of lysosomal cholesterol
to the PM since PM cholesterol is considered to provide the principal
source of substrate for acetyl-coenzyme A:cholesterol acyltransferase
located in the ER (35, 36, 37, 38). It has, however, been speculated that a
portion of LDL-derived cholesterol destined for esterification may be
directly transferred from lysosomes to the ER (36). We find that CD in
normal cells can block esterification by as much as 70% (Fig. 9) and
conclude that this fraction of endocytosed cholesterol, mobilized from
lysosomes, is translocated to the ER through a PM-mediated pathway. The
remaining sterol fraction presumably moves to the ER by a
PM-independent pathway. In NP-C fibroblasts, the attenuated cholesterol
esterification associated with the defective lysosomal cholesterol
transport pathway is insensitive to suppression by CD (Fig. 9) and thus
appears to bypass the PM.
The current studies show that cell surface SM affects the trafficking
of lysosomal cholesterol to the PM. In SMase-treated cells, CD no
longer suppresses the esterification of lysosomal cholesterol during
progesterone washout (Fig. 10). Thus, removal of SM from the PM appears
to alter, without apparently attenuating, intracellular trafficking of
lysosomal cholesterol. Since esterification of LDL-derived cholesterol
in SM-depleted cells is no longer affected by CD, we conclude that it
has been re-routed to the ER by a PM-independent pathway. It is
conceivable that SM depletion renders the PM incapable of accepting
cholesterol delivered from lysosomes. Further investigation is required
to evaluate this unexpected finding.
Several studies have provided a link between the Golgi complex and the
processing of cellular cholesterol. It has been postulated that the
distribution of cholesterol within the complex, increasing in an
anterograde cis to trans direction (50), plays a role in the sorting of
membrane proteins (51) and lipids (52). We have previously shown that
the processing of LDL-derived cholesterol in normal cells is
accompanied by an enrichment of the cholesterol content of the Golgi
complex in an anterograde fashion (31, 53, 54). By contrast LDL
processing in NP-C fibroblasts leads to a premature (31) and abnormal
(53) enrichment of cholesterol in Golgi cisternae. These studies were
the first to implicate the Golgi with the intracellular trafficking of
LDL-derived cholesterol.
We have now obtained further evidence for a role of the Golgi complex
in the transport of LDL-derived cholesterol. Treatment of normal cells
with BFA renders esterification of lysosomal cholesterol insensitive to
exogenous CD (Fig. 11). This finding implies that cholesterol is
normally transferred from lysosomes to the PM via the Golgi and that
BFA disruption of this organelle results in re-routing of cholesterol
to the ER via a PM-independent pathway. Our present studies also
provide further insights to suggest that defective transport of
lysosomal cholesterol in NP-C cells may be linked to disruption of its
passage through the Golgi. As shown above, in these mutant cells,
lysosomal cholesterol appears to be translocated to the ER via a
PM-independent pathway, which is both attenuated and insensitive to
exogenous CD. A curtailment in cholesterol transfer from lysosomes to
the PM in NP-C cells due to a defect in movement through the Golgi
complex is suggested by the finding that BFA curtails this transfer in
normal cells. The observed 4-fold stimulation of cholesterol
esterification in NP-C cells during disruption of the Golgi with BFA is
consistent with the notion that abnormal cholesterol accumulation in
the Golgi complex leads to an increase in the supply of substrate for
acetyl-coenzyme A:cholesterol acyltransferase when Golgi components
fuse with the ER.
The Golgi complex also appears to play a role in shuttling cholesterol
between the PM and other organelles. Transfer of cholesterol between
the Golgi and PM is thought to be involved in the formation of
cholesterol-rich PM caveolae (55). The caveolar-specific cholesterol
binding protein, caveolin (56), cycles constitutively between the PM
and intracellular sites including the Golgi complex (57). Our present
studies support a retrograde movement of cholesterol from the PM to the
ER that may be modulated by the Golgi complex. Mobilization of
cholesterol from the PM following SMase treatment results in an
enrichment of the cholesterol content in the Golgi complex (data not
shown), as monitored by the stabilized fluorescence of
C6-NBD-ceramide (54). The movement of cholesterol between
the PM and the Golgi may in turn be linked to its appearance in the ER.
This is supported by our observation that BFA-induced disruption of the
Golgi complex greatly enhances esterification of PM cholesterol
mobilized by SM depletion (Fig. 12). This marked enhancement of
cholesterol esterification by BFA suggests a direct role for the Golgi
in regulating trafficking of PM-derived cholesterol to the ER.
Mobilization of PM cholesterol to the ER remains deficient in NP-C
cells, even with BFA treatment, suggesting an inherent defect in the
processing of cholesterol by the Golgi in these mutant cells. Golgi
processing of cholesterol derived from LDL and the PM may play an
essential role in maintaining the appropriate distribution of
cholesterol within the cell.
FOOTNOTES REFERENCES
Volume 271, Number 35,
Issue of August 30, 1996
pp. 21604-21613
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
§,
,,
Lipid Cell Biology Section, Laboratory of
Cell Biochemistry and Biology, NIDDK, the ¶ Developmental and
Metabolic Neurology Branch, NINDS, National Institutes of Health,
Bethesda, Maryland 20892, the 
Gerontology Research Center,
NIA, National Institutes of Health, Baltimore, Maryland 21224, and
the '' Marine Biological Laboratory, Woods Hole,
Massachusetts 02543
-cyclodextrin is shown to be a convenient and useful
experimental tool to probe intracellular pathways of cholesterol
transport. Biochemical and cytochemical studies reveal that
cyclodextrin specifically removes plasma membrane cholesterol.
Depletion of plasma membrane sphingomyelin greatly accelerated
cyclodextrin-mediated cholesterol removal. Cholesterol arriving at the
plasma membrane from lysosomes and the endoplasmic reticulum was also
removed by cyclodextrin. Cellular cholesterol esterification linked to
the mobilization of cholesterol from lysosomes was strongly attenuated
by cyclodextrin, suggesting that the major portion of endocytosed
cholesterol is delivered from lysosomes to the endoplasmic reticulum
via the plasma membrane. Evidence for translocation of lysosomal
cholesterol to the endoplasmic reticulum by a plasma
membrane-independent pathway is provided by the finding that
cyclodextrin loses its ability to suppress esterification when plasma
membrane sphingomyelin is depleted. The Golgi apparatus appears to play
an active role in directing the relocation of lysosomal cholesterol to
the plasma membrane since brefeldin A also abrogated
cyclodextrin-mediated suppression of cholesterol esterification. Using
cyclodextrin we further show that attenuated esterification of
lysosomal cholesterol in Niemann-Pick C cells reflects defective
translocation of cholesterol to the plasma membrane that may be linked
to abnormal Golgi trafficking.
-cyclodextrin (CD), as an effective extracellular
cholesterol acceptor, to monitor the flux of cholesterol through the PM
of living cells. Cyclodextrins are cyclic oligomers of glucose that
have the capacity to sequester lipophiles in their hydrophobic core
(18). The water-soluble cyclodextrins preferentially form inclusion
complexes with sterols thereby greatly enhancing their solubility in
aqueous solution (19, 20, 21). It has recently been shown that CD is
capable of stimulating cholesterol efflux from cultured cells with very
high efficiency (22). We now report our findings on the use of CD to
follow movement of de novo synthesized cholesterol to and
from the PM and the egress of LDL-derived cholesterol from lysosomes.
We further used CD to reveal that cell surface SM and the Golgi
apparatus regulate intracellular transport of cholesterol.
-cyclodextrin was purchased from Research Plus,
Inc., Bayonne, NJ. Neutral sphingomyelinase (from
Staphylococcus aureus) was purchased from
Sigma. Brefeldin A was purchased from Epicentre
Technologies (Madison, WI) and stored as a 1 mg/ml stock solution in
ethanol at 
20 °C.
-cyclodextrin was
routinely added at 2 g/100 ml to McCoy's/5% LPDS medium
supplemented with 25 m HEPES, pH 7.0, to adjust pH to
7.2.
20 °C in sealed tubes
until analyzed. The lipid-free cell residues remaining in the culture
wells were taken up in 0.5 ml of 0.5 NaOH and protein
measured by the method of Lowry et al. (26). Unesterified
and esterified cholesterol levels were measured in aliquots of the
isopropyl alcohol extract corresponding to approximately 10 µg of
protein with a fluorescently linked assay employing cholesteryl oxidase
and cholesteryl esterase as described previously (27). Cellular
cholesterol formed by de novo synthesis from
[3H]acetate or [3H]mevalonate was measured
in a [3H]sterol fraction separated by thin layer
chromatography (28). Cellular cholesterol esterification was measured
by the incorporation of [3H]oleate into newly synthesized
sterol esters as described previously (28) utilizing thin layer
chromatography to separate the formed
cholesteryl-[3H]oleate. [3H]Cholesterol in
medium was extracted by the Folch method (29).
-cyclodextrin (CD) is an effective
sterol acceptor that removes the unesterified cholesterol mass from
viable cells in a saturable, concentration-dependent manner
(Table I). Approximately 70% of cellular cholesterol
can be removed after 4 h treatment with 2% CD at 37 °C. The
kinetics of cholesterol removal from both normal and NP-C fibroblasts
are similar with a biphasic nature (Fig. 1). In cells
uniformly labeled with [3H]acetate, approximately
40-50% of the [3H] sterol is depleted within 60 min
followed by a slower rate of depletion (t1/2 = 5-6
h). It appears that esterified cholesterol cannot be mobilized to an
appreciable extent by CD (Table I). The small amount of cholesterol
ester removed does not appear to depend on the concentration of CD.
-cyclodextrin for 4 h
Concentration
CD
Cellular cholesterol
Freea
Esterifieda
g/100 ml
0
81
± 4 (100)
16 ± 4 (100)
0.05
43 ± 2 (53)
7
± 1 (44)
1
38 ± 2 (47)
10 ± 1 (62)
2
24
± 1 (30)
8 ± 2 (50)
4
23 ± 1 (28)
10
± 1 (62)
8
18 ± 1 (22)
12 ± 3 (75)
a
Sterol mass is expressed as nmol per mg cell protein
of six wells (mean ± S.D.). Values in parentheses are expressed
as % of initial cell sterol remaining.
Fig. 1.
Kinetics of efflux of de novo
synthesized [3H]sterol from normal and NP-C fibroblasts
to CD. Subconfluent normal and NP-C human fibroblast cultures were
incubated in McCoy's/5% LPDS medium at 37 °C for 4 days. Cell
monolayers were then incubated with the same medium containing 8.9 µ [3H]acetate (specific activity = 1000 dpm/pmol) at 37 °C for 24 h to label PM pools of
cholesterol. A portion of the cultures were washed in PBS, and then
lipids and protein were extracted in the dishes as described. The
remaining cultures were incubated at 37 °C in McCoy's/5% LPDS
medium containing 2% CD for the times indicated, washed, and then
extracted. Levels of cell-associated [3H]sterol were
determined by thin layer chromatography as described. Total levels of
cell-associated [3H]sterol formed from
[3H]acetate remained constant in the absence of CD.
Values are mean ± S.D. of six wells.
Fig. 2.
The effect of SMase on the efflux of de
novo synthesized [3H]sterol to CD.
Subconfluent normal human fibroblast cultures were incubated in
McCoy's/5% LPDS medium at 37 °C for 4 days. Cell monolayers were
then incubated with the same medium containing 8.9 µ
[3H]acetate (specific activity = 1000 dpm/pmol) at
37 °C for 24 h to label PM pools of cholesterol. Cultures were
washed in PBS, chased in McCoy's/5% LPDS medium for 30 min at
37 °C, and then incubated in McCoy's/5% LPDS medium in the absence
or presence of 0.12 units/ml SMase for 30 min at 37 °C. A portion of
cultures were washed, and then lipids and protein were extracted in the
dishes as described. The remaining cultures were washed and then
incubated in 2% CD for the times indicated, washed, and then
extracted. Levels of cell-associated [3H]sterol were
determined by thin layer chromatography as described. Total levels of
cell-associated [3H]sterol formed from
[3H]acetate remained constant in the absence of CD.
Values are mean ± S.D. of six wells.
Fig. 3.
Cytochemical assessment of the effect of CD
on the distribution of cholesterol in membranes of normal
fibroblasts. Normal human fibroblasts were incubated in
McCoy's/5% LPDS medium at 37 °C for 4 days. A portion of the
cultures were washed in PBS and then incubated in McCoy's/5% LPDS
medium alone (A) or in McCoy's/5% LPDS medium
containing 2% CD (B) for 6 h at 37 °C. The
remaining cultures were incubated in McCoy's/5% LPDS medium
containing 2% CD and 0.12 units/ml SMase for 1 h at 37 °C
(C). The PM of control fibroblasts revealed substantial
filipin-cholesterol fluorescence (A, arrowheads).
Note the diminution (B, arrowheads) and
elimination (C, arrowheads) of PM
filipin-cholesterol fluorescence following treatment with CD and
CD/SMase, respectively. All photographic exposures and processing were
performed identically. (A-C, × 370).
Fig. 4.
Cytochemical assessment of the effect of CD
on the distribution of cholesterol in membranes of NP-C fibroblasts
cultured with LDL. NP-C human fibroblasts were incubated in
McCoy's/5% LPDS medium at 37 °C for 4 days. Cells were then
incubated in fresh medium containing LDL (50 µg/ml) for 24 h. A
portion of the cultures were washed in PBS and then incubated in
McCoy's/5% LPDS medium alone for 1 h at 37 °C (A).
The remaining cultures were incubated in McCoy's/5% LPDS medium
containing 2% CD and 0.12 units/ml SMase for 1 h at 37 °C
(B). The PM of NP-C fibroblasts demonstrates considerable
filipin fluorescence (A, arrowheads). Note that
CD/SMase treatment eliminated filipin-cholesterol fluorescence at the
PM (B, arrowheads) but did not appreciably alter
perinuclear lysosomal filipin-cholesterol fluorescence. All
photographic exposures and processing were performed identically.
(A and B, × 150).
-hexosaminidase) was found
predominantly in the heavy membrane fraction (HM), whereas markers for
the PM, Golgi, and ER were found in the light membrane fraction (LM)
(data not shown). In normal cells, 60-70% of the total recovered
radioactivity was distributed in the LM while 18% was associated with
the HM (Fig. 5A). Treatment of these cells with CD/SMase
reduced the level of [3H]cholesterol in LM and HM by 64 and 50%, respectively. The loss of radioactivity from LM is consistent
with the removal of [3H]cholesterol from the PM.
[3H]Cholesterol remaining in LM after CD/SMase treatment
may in part represent [3H]cholesterol associated with the
Golgi complex and/or ER. The loss of [3H]cholesterol from
HM is consistent with a partial transfer of
[3H]cholesterol from lysosomes to the PM during depletion
with CD. In NP-C cells, the subcellular distribution of LDL-derived
[3H]cholesterol was higher in HM (30%) and lower in LM
(58%) when compared with normal cells (Fig. 5B), reflecting
the defective intracellular distribution of lysosomal cholesterol (15).
Unlike normal fibroblasts, CD/SMase treatment did not substantially
alter the LDL-derived [3H]cholesterol distributed in HM
of NP-C fibroblasts. This treatment did, however, produce a decrease by
2/3 in the percentage of the total [3H]cholesterol
distributed in LM. Although less lysosomal cholesterol appears at the
PM of NP-C cells, a comparable proportion of LDL-derived cholesterol
was removed by CD/SMase from this membrane in both cell types.
Fig. 5.
Effect of combined treatment with CD and
SMase on the distribution of [3H]cholesterol derived
from LDL[3H]cholesteryl linoleate in Percoll
fractions of normal and NP-C fibroblasts. Stock cultures of normal
and NP-C fibroblasts were harvested, and 1.0-1.5 × 106 cells were seeded in 75-cm2 flasks in
McCoy's/5% LPDS medium at 37 °C for 4 days. Cultures were then
incubated with fresh medium containing 15 µg/ml LDL[3H]cholesteryl
linoleate (specific activity = 3.94 × 107 dpm/mg
protein) for 24 h, washed, and then incubated in McCoy's/5% LPDS
medium in the absence or presence of 2% CD and 0.12 units/ml SMase at
37 °C for 1 h. Cultures were washed and then fractionated in
Percoll gradients, as described. 0.5-ml aliquots of Percoll fractions
were added to scintillant mixture, and [3H] radioactivity
was counted. Total radioactivity associated with NP-C fibroblasts was
normalized to the level observed in normal fibroblasts.
Fig. 6.
Effect of CD on ability of SMase to stimulate
esterification of PM cholesterol in normal human fibroblasts.
Subconfluent normal human fibroblast cultures were incubated in
McCoy's/5% LPDS medium at 37 °C for 4 days. Cultures were then
washed in PBS and incubated in McCoy's/5% LPDS medium containing LDL
(50 µg/ml) for 48 h to enrich PM pools of cholesterol. A portion
of the cultures were then washed in PBS and incubated in McCoy's/5%
LPDS medium containing 12.5 m [3H]oleate
(specific activity = 200 dpm/pmol) in the absence or presence of
2% CD at 37 °C for the times indicated. The remaining cultures were
washed in PBS and incubated in McCoy's/5% LPDS medium containing 0.12 units/ml SMase and 12.5 m [3H]oleate
(specific activity = 200 dpm/pmol) in the absence or presence of
2% CD at 37 °C for the times indicated. Cell monolayers were
subsequently washed, and lipids and protein were extracted in the
dishes as described. Levels of cholesteryl [3H]oleate
formed in vitro were determined in lipid extracts of cells
by thin layer chromatography as described. Values are mean ± S.D.
of six wells.
Fig. 7.
Effect of CD on cellular retention of
[3H]sterol synthesized from exogenously added
[3H]mevalonate. Subconfluent normal human fibroblast
cultures were incubated in McCoy's/5% LPDS medium at 37 °C for 4 days. Cell monolayers were then incubated with the same medium
containing 0.5 m [3H]mevalonate (specific
activity = 22 dpm/pmol) at 37 °C in the absence or presence of
2% CD for the time intervals indicated. At the end of the indicated
periods, 1-ml aliquots of medium were removed and lipids were extracted
as described. Cell monolayers were washed and then cellular lipids and
protein were extracted. Levels of [3H]sterol formed
in vitro were determined in lipid extracts of cells
(A) and medium (B) by thin layer chromatography
as described. Values are mean ± S.D. of triplicate wells.
Fig. 8.
Effect of CD on cellular clearance of
lysosomally derived [3H]cholesterol. Subconfluent
normal and NP-C human fibroblast cultures were incubated in
McCoy's/5% LPDS medium at 37 °C for 4 days. Cultures were then
incubated with fresh medium containing 10 µg/ml LDL [3H]cholesteryl
oleate (specific activity = 4.45 × 107 dpm/mg
protein) and 20 µg/ml progesterone at 37 °C for 24 h.
Cultures were then washed in PBS and incubated in McCoy's/5% LPDS
medium in the absence or presence of 2% CD at 37 °C for the
indicated times. At the end of the indicated time periods, 1-ml
aliquots of medium were removed. Cell monolayers were subsequently
washed, and lipids and proteins were extracted in the dishes. Levels of
[3H]cholesterol formed in vitro were
determined in Folch extracts of medium and in isopropyl alcohol
extracts of cells by thin layer chromatography as described. % endocytosed [3H]cholesterol retained in cells = ((cell-associated [3H]cholesterol)/(cell-associated
[3H]cholesterol + medium [3H]cholesterol)) × 100. Values are mean ± S.D. of triplicate wells.
Fig. 9.
Comparison of the ability of CD to block
esterification of lysosomally derived cholesterol in normal and NP-C
human fibroblasts. Subconfluent normal (A) and NP-C
(B) human fibroblast cultures were incubated in McCoy's/5%
LPDS medium at 37 °C for 4 days. Cultures were washed in PBS and
then incubated in McCoy's/5% LPDS medium containing LDL (50 µg/ml)
and progesterone (10 µg/ml) for 24 h at 37 °C. Cultures were
washed in PBS and then incubated in McCoy's/5% LPDS medium containing
12.5 m [3H]oleate (specific activity = 200 dpm/pmol) in the absence or presence of 2% CD at 37 °C for the
times indicated. Cultures were washed, and then lipids and protein were
extracted in the dishes as described. Levels of cholesteryl
[3H]oleate formed in vitro were determined by
thin layer chromatography. Values are mean ± S.D. of triplicate
wells.
Fig. 10.
Effect of SMase on the CD-induced block of
esterification of lysosomally derived cholesterol. Subconfluent
normal human fibroblast cultures were incubated in McCoy's/5% LPDS
medium at 37 °C for 4 days. Cultures were then washed in PBS and
incubated in McCoy's/5% LPDS medium containing LDL (50 µg/ml) and
progesterone (10 µg/ml) for 24 h at 37 °C. A portion of the
cultures was then washed in PBS and incubated in McCoy's/5% LPDS
medium for 30 min at 37 °C, washed, and incubated McCoy's/5% LPDS
medium containing 12.5 m [3H]oleate
(specific activity = 200 dpm/pmol) in the absence or presence of
2% CD at 37 °C for the times indicated (A). The
remaining cultures were washed in PBS and then incubated in
McCoy's/5% LPDS medium containing 0.12 units/ml SMase for 30 min at
37 °C, washed, and then incubated in McCoy's/5% LPDS medium
containing 12.5 m [3H]oleate (specific
activity = 200 dpm/pmol) in the absence or presence of 2% CD at
37 °C for the times indicated (B). Cell monolayers were
subsequently washed, and lipids and protein were extracted in the
dishes. Levels of cholesteryl [3H]oleate formed in
vitro were determined as described. Values are mean ± S.D.
of six wells.
Fig. 11.
Effect of BFA on the CD-induced block of
esterification of lysosomally derived cholesterol. Subconfluent
normal (A, B) and NP-C (C,
D) human fibroblast cultures were incubated in McCoy's/5%
LPDS medium at 37 °C for 4 days. Cultures were then washed in PBS
and incubated in McCoy's/5% LPDS medium containing LDL (50 µg/ml)
and progesterone (10 µg/ml) for 24 h at 37 °C. A portion of
the cultures were then washed in PBS and incubated in McCoy's/5% LPDS
medium containing 12.5 m [3H]oleate
(specific activity = 200 dpm/pmol) in the absence or presence of
2% CD at 37 °C for the times indicated (A,
C). The remaining cultures were washed in PBS, incubated in
McCoy's/5% LPDS medium containing 2 µg/ml BFA and 12.5 m [3H]oleate (specific activity = 200 dpm/pmol) in the absence or presence of 2% CD at 37 °C for the
times indicated (B, D). Cell monolayers were
subsequently washed and then lipids and protein were extracted in the
dishes. Levels of cholesteryl [3H]oleate formed in
vitro were determined.
Fig. 12.
Effect of BFA on SMase-stimulated
esterification of PM sterol. Subconfluent normal and NP-C human
fibroblast cultures were incubated in McCoy's/5% LPDS medium at
37 °C for 4 days. Cell monolayers were then incubated with the same
medium containing 8.9 µ [3H]acetate
(specific activity = 1000 dpm/pmol) at 37 °C for 24 h to
label PM pools of cholesterol. Cultures were washed in PBS and then
chased in McCoy's/5% LPDS medium for 24 h at 37 °C. A portion
of the cultures was washed in PBS and then incubated in McCoy's/5%
LPDS medium alone or in McCoy's/5% LPDS medium containing 2 µg/ml
BFA for 6 h at 37 °C. The remaining cultures were incubated in
McCoy's/5% LPDS medium containing 0.12 units/ml SMase in the absence
or presence of 2 µg/ml BFA for 6 h at 37 °C. Cell monolayers
were subsequently washed and then lipids and proteins were extracted in
the dishes. Levels of cell-associated [3H]sterol and
[3H]steryl ester formed in vitro were
determined in lipid extracts by thin layer chromatography as described.
Values are mean ± S.D. of six wells.
§
To whom correspondence should be addressed: Lipid Cell Biology
Section, Laboratory of Cell Biochemistry and Biology, NIDDK, National
Institutes of Health, Bldg. 8, Rm. 427, 8 Center Dr., MSC 0850, Bethesda, MD 20892. Tel.: 301-496-2050; Fax: 301-402-0723.
1
The abbreviations used are: LDL, low density
lipoprotein; BFA, brefeldin A; CD, 2-hydroxypropyl--cyclodextrin;
ER, endoplasmic reticulum; HDL, high density lipoprotein; HM, heavy
membrane fraction; LM, light membrane fraction; LPDS,
lipoprotein-deficient serum; NP-C, Niemann-Pick type C; PM, plasma
membrane; SM, sphingomyelin; SMase, neutral sphingomyelinase; PBS,
phosphate-buffered saline.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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 S. B. Sieczkarski and G. R. Whittaker Dissecting virus entry via endocytosis J. Gen. Virol., June 1, 2002; 83(7): 1535 - 1545. [Abstract] [Full Text] [PDF]
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A. A. Wolf, Y. Fujinaga, and W. I. Lencer Uncoupling of the Cholera Toxin-GM1 Ganglioside Receptor Complex from Endocytosis, Retrograde Golgi Trafficking, and Downstream Signal Transduction by Depletion of Membrane Cholesterol J. Biol. Chem., May 3, 2002; 277(18): 16249 - 16256. [Abstract] [Full Text] [PDF]
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W. S. Garver, K. Krishnan, J. R. Gallagos, M. Michikawa, G. A. Francis, and R. A. Heidenreich Niemann-Pick C1 protein regulates cholesterol transport to the trans-Golgi network and plasma membrane caveolae J. Lipid Res., April 1, 2002; 43(4): 579 - 589. [Abstract] [Full Text] [PDF]
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M. Alfalah, R. Jacob, and H. Y. Naim Intestinal Dipeptidyl Peptidase IV Is Efficiently Sorted to the Apical Membrane through the Concerted Action of N- and O-Glycans as Well as Association with Lipid Microdomains J. Biol. Chem., March 15, 2002; 277(12): 10683 - 10690. [Abstract] [Full Text] [PDF]
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O. Kovbasnjuk, M. Edidin, and M. Donowitz Role of lipid rafts in Shiga toxin 1 interaction with the apical surface of Caco-2 cells J. Cell Sci., March 13, 2002; 114(22): 4025 - 4031. [Abstract] [Full Text] [PDF]
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 C. Gajate and F. Mollinedo The antitumor ether lipid ET-18-OCH3 induces apoptosis through translocation and capping of Fas/CD95 into membrane rafts in human leukemic cells Blood, December 15, 2001; 98(13): 3860 - 3863. [Abstract] [Full Text] [PDF]
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A. Frolov, K. Srivastava, D. Daphna-Iken, L. M. Traub, J. E. Schaffer, and D. S. Ory Cholesterol Overload Promotes Morphogenesis of a Niemann-Pick C (NPC)-like Compartment Independent of Inhibition of NPC1 or HE1/NPC2 Function J. Biol. Chem., November 30, 2001; 276(49): 46414 - 46421. [Abstract] [Full Text] [PDF]
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W. Chen, Y. Sun, C. Welch, A. Gorelik, A. R. Leventhal, I. Tabas, and A. R. Tall Preferential ATP-binding Cassette Transporter A1-mediated Cholesterol Efflux from Late Endosomes/Lysosomes J. Biol. Chem., November 16, 2001; 276(47): 43564 - 43569. [Abstract] [Full Text] [PDF]
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S. S.-S. Wang, D. L. Rymer, and T. A. Good Reduction in Cholesterol and Sialic Acid Content Protects Cells from the Toxic Effects of beta -Amyloid Peptides J. Biol. Chem., November 2, 2001; 276(45): 42027 - 42034. [Abstract] [Full Text] [PDF]
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 F. Schroeder, A. M. Gallegos, B. P. Atshaves, S. M. Storey, A. L. McIntosh, A. D. Petrescu, H. Huang, O. Starodub, H. Chao, H. Yang, et al. Recent Advances in Membrane Microdomains: Rafts, Caveolae, and Intracellular Cholesterol Trafficking Experimental Biology and Medicine, November 1, 2001; 226(10): 873 - 890. [Abstract] [Full Text] [PDF]
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 J. Herreros, T. Ng, and G. Schiavo Lipid Rafts Act as Specialized Domains for Tetanus Toxin Binding and Internalization into Neurons Mol. Biol. Cell, October 1, 2001; 12(10): 2947 - 2960. [Abstract] [Full Text] [PDF]
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H. S. Kruth, I. Ifrim, J. Chang, L. Addadi, D. Perl-Treves, and W.-Y. Zhang Monoclonal antibody detection of plasma membrane cholesterol microdomains responsive to cholesterol trafficking J. Lipid Res., September 1, 2001; 42(9): 1492 - 1500. [Abstract] [Full Text] [PDF]
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A. Mohammadi, R. J. Perry, M. K. Storey, H. W. Cook, D. M. Byers, and N. D. Ridgway Golgi localization and phosphorylation of oxysterol binding protein in Niemann-Pick C and U18666A-treated cells J. Lipid Res., July 1, 2001; 42(7): 1062 - 1071. [Abstract] [Full Text] [PDF]
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 B. Darblade, D. Caillaud, M. Poirot, M.-J. Fouque, J.-C. Thiers, J. Rami, F. Bayard, and J.-F. Arnal Alteration of plasmalemmal caveolae mimics endothelial dysfunction observed in atheromatous rabbit aorta Cardiovasc Res, June 1, 2001; 50(3): 566 - 576. [Abstract] [Full Text] [PDF]
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K. Fassbender, M. Simons, C. Bergmann, M. Stroick, D. Lütjohann, P. Keller, H. Runz, S. Kühl, T. Bertsch, K. von Bergmann, et al. Simvastatin strongly reduces levels of Alzheimer's disease beta -amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo PNAS, April 5, 2001; (2001) 81620098. [Abstract] [Full Text]
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P. G. Yancey and W. G. Jerome Lysosomal cholesterol derived from mildly oxidized low density lipoprotein is resistant to efflux J. Lipid Res., March 1, 2001; 42(3): 317 - 327. [Abstract] [Full Text]
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D. C. Ko, M. D. Gordon, J. Y. Jin, and M. P. Scott Dynamic Movements of Organelles Containing Niemann-Pick C1 Protein: NPC1 Involvement in Late Endocytic Events Mol. Biol. Cell, March 1, 2001; 12(3): 601 - 614. [Abstract] [Full Text]
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K. Simons and E. Ikonen How Cells Handle Cholesterol Science, December 1, 2000; 290(5497): 1721 - 1726. [Abstract] [Full Text]
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 N. L. Cross Sphingomyelin Modulates Capacitation of Human Sperm In Vitro Biol Reprod, October 1, 2000; 63(4): 1129 - 1134. [Abstract] [Full Text]
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 A. D. Tepper, P. Ruurs, T. Wiedmer, P. J. Sims, J. Borst, and W. J. van Blitterswijk Sphingomyelin Hydrolysis to Ceramide during the Execution Phase of Apoptosis Results from Phospholipid Scrambling and Alters Cell-surface Morphology J. Cell Biol., July 11, 2000; 150(1): 155 - 164. [Abstract] [Full Text] [PDF]
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S. Heino, S. Lusa, P. Somerharju, C. Ehnholm, V. M. Olkkonen, and E. Ikonen Dissecting the role of the Golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface PNAS, July 5, 2000; (2000) 140218797. [Abstract] [Full Text]
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W. S. Garver, R. A. Heidenreich, R. P. Erickson, M. A. Thomas, and J. M. Wilson Localization of the murine Niemann-Pick C1 protein to two distinct intracellular compartments J. Lipid Res., May 1, 2000; 41(5): 673 - 687. [Abstract] [Full Text]
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I. Coppens, A. P. Sinai, and K. A. Joiner Toxoplasma gondii Exploits Host Low-Density Lipoprotein Receptor-mediated Endocytosis for Cholesterol Acquisition J. Cell Biol., April 3, 2000; 149(1): 167 - 180. [Abstract] [Full Text] [PDF]
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G. H. Hansen, L.-L. Niels-Christiansen, E. Thorsen, L. Immerdal, and E. M. Danielsen Cholesterol Depletion of Enterocytes. EFFECT ON THE GOLGI COMPLEX AND APICAL MEMBRANE TRAFFICKING J. Biol. Chem., February 18, 2000; 275(7): 5136 - 5142. [Abstract] [Full Text] [PDF]
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J. C. Cruz, S. Sugii, C. Yu, and T.-Y. Chang Role of Niemann-Pick Type C1 Protein in Intracellular Trafficking of Low Density Lipoprotein-derived Cholesterol J. Biol. Chem., February 11, 2000; 275(6): 4013 - 4021. [Abstract] [Full Text] [PDF]
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Y. Lange, J. Ye, M. Rigney, and T. L. Steck Regulation of endoplasmic reticulum cholesterol by plasma membrane cholesterol J. Lipid Res., December 1, 1999; 40(12): 2264 - 2270. [Abstract] [Full Text] [PDF]
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A. G. Ostermeyer, B. T. Beckrich, K. A. Ivarson, K. E. Grove, and D. A. Brown Glycosphingolipids Are Not Essential for Formation of Detergent-resistant Membrane Rafts in Melanoma Cells. METHYL-beta -CYCLODEXTRIN DOES NOT AFFECT CELL SURFACE TRANSPORT OF A GPI-ANCHORED PROTEIN J. Biol. Chem., November 26, 1999; 274(48): 34459 - 34466. [Abstract] [Full Text] [PDF]
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L. Abrami and F. G. van der Goot Plasma Membrane Microdomains Act as Concentration Platforms to Facilitate Intoxication by Aerolysin J. Cell Biol., October 4, 1999; 147(1): 175 - 184. [Abstract] [Full Text] [PDF]
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R. B. Rawson, R. DeBose-Boyd, J. L. Goldstein, and M. S. Brown Failure to Cleave Sterol Regulatory Element-binding Proteins (SREBPs) Causes Cholesterol Auxotrophy in Chinese Hamster Ovary Cells with Genetic Absence of SREBP Cleavage-activating Protein J. Biol. Chem., October 1, 1999; 274(40): 28549 - 28556. [Abstract] [Full Text] [PDF]
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A. E. Christian, H.-S. Byun, N. Zhong, M. Wanunu, T. Marti, A. Fürer, F. Diederich, R. Bittman, and G. H. Rothblat Comparison of the capacity of {beta}-cyclodextrin derivatives and cyclophanes to shuttle cholesterol between cells and serum lipoproteins J. Lipid Res., August 1, 1999; 40(8): 1475 - 1482. [Abstract] [Full Text]
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N. Naslavsky, H. Shmeeda, G. Friedlander, A. Yanai, A. H. Futerman, Y. Barenholz, and A. Taraboulos Sphingolipid Depletion Increases Formation of the Scrapie Prion Protein in Neuroblastoma Cells Infected with Prions J. Biol. Chem., July 23, 1999; 274(30): 20763 - 20771. [Abstract] [Full Text] [PDF]
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S. Maekawa, C. Sato, K. Kitajima, N. Funatsu, H. Kumanogoh, and Y. Sokawa Cholesterol-dependent Localization of NAP-22 on a Neuronal Membrane Microdomain (Raft) J. Biol. Chem., July 23, 1999; 274(30): 21369 - 21374. [Abstract] [Full Text] [PDF]
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A. Subtil, I. Gaidarov, K. Kobylarz, M. A. Lampson, J. H. Keen, and T. E. McGraw Acute cholesterol depletion inhibits clathrin-coated pit budding PNAS, June 8, 1999; 96(12): 6775 - 6780. [Abstract] [Full Text] [PDF]
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E. B. Neufeld, M. Wastney, S. Patel, S. Suresh, A. M. Cooney, N. K. Dwyer, C. F. Roff, K. Ohno, J. A. Morris, E. D. Carstea, et al. The Niemann-Pick C1 Protein Resides in a Vesicular Compartment Linked to Retrograde Transport of Multiple Lysosomal Cargo J. Biol. Chem., April 2, 1999; 274(14): 9627 - 9635. [Abstract] [Full Text] [PDF]
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S. K. Rodal, G. Skretting, O. Garred, F. Vilhardt, B. van Deurs, and K. Sandvig Extraction of Cholesterol with Methyl-beta -Cyclodextrin Perturbs Formation of Clathrin-coated Endocytic Vesicles Mol. Biol. Cell, April 1, 1999; 10(4): 961 - 974. [Abstract] [Full Text]
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T. A. Lagace, D. M. Byers, H. W. Cook, and N. D. Ridgway Chinese hamster ovary cells overexpressing the oxysterol binding protein (OSBP) display enhanced synthesis of sphingomyelin in response to 25-hydroxycholesterol J. Lipid Res., January 1, 1999; 40(1): 109 - 116. [Abstract] [Full Text]
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Y.-H. Choi and Y. Toyoda Cyclodextrin Removes Cholesterol from Mouse Sperm and Induces Capacitation in a Protein-Free Medium Biol Reprod, December 1, 1998; 59(6): 1328 - 1333. [Abstract] [Full Text]
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Y. Liu, D. A. Peterson, and D. Schubert Amyloid beta peptide alters intracellular vesicle trafficking and cholesterol homeostasis PNAS, October 27, 1998; 95(22): 13266 - 13271. [Abstract] [Full Text] [PDF]
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T. Furuchi and R. G. W. Anderson Cholesterol Depletion of Caveolae Causes Hyperactivation of Extracellular Signal-related Kinase (ERK) J. Biol. Chem., August 14, 1998; 273(33): 21099 - 21104. [Abstract] [Full Text] [PDF]
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Y. Lange, J. Ye, and T. L. Steck Circulation of Cholesterol between Lysosomes and the Plasma Membrane J. Biol. Chem., July 24, 1998; 273(30): 18915 - 18922. [Abstract] [Full Text] [PDF]
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S. Bose, S. J. Chapin, S. Seetharam, J. Feix, K. E. Mostov, and B. Seetharam Brefeldin A (BFA) Inhibits Basolateral Membrane (BLM) Delivery and Dimerization of Transcobalamin II Receptor in Human Intestinal Epithelial Caco-2 Cells. BFA EFFECTS ON BLM CHOLESTEROL CONTENT J. Biol. Chem., June 26, 1998; 273(26): 16163 - 16169. [Abstract] [Full Text] [PDF]
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M. Simons, P. Keller, B. De Strooper, K. Beyreuther, C. G. Dotti, and K. Simons Cholesterol depletion inhibits the generation of beta -amyloid in hippocampal neurons PNAS, May 26, 1998; 95(11): 6460 - 6464. [Abstract] [Full Text] [PDF]
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P. A. Orlandi and P. H. Fishman Filipin-dependent Inhibition of Cholera Toxin: Evidence for Toxin Internalization and Activation through Caveolae-like Domains J. Cell Biol., May 18, 1998; 141(4): 905 - 915. [Abstract] [Full Text] [PDF]
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N. Khelef, X. Buton, N. Beatini, H. Wang, V. Meiner, T.-Y. Chang, R. V. Farese Jr., F. R. Maxfield, and I. Tabas Immunolocalization of Acyl-Coenzyme A:Cholesterol O-Acyltransferase in Macrophages J. Biol. Chem., May 1, 1998; 273(18): 11218 - 11224. [Abstract] [Full Text] [PDF]
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M. D. Ledesma, K. Simons, and C. G. Dotti Neuronal polarity: Essential role of protein-lipid complexes in axonal sorting PNAS, March 31, 1998; 95(7): 3966 - 3971. [Abstract] [Full Text] [PDF]
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P. Keller and K. Simons Cholesterol Is Required for Surface Transport of Influenza Virus Hemagglutinin J. Cell Biol., March 23, 1998; 140(6): 1357 - 1367. [Abstract] [Full Text] [PDF]
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K. W. Underwood, N. L. Jacobs, A. Howley, and L. Liscum Evidence for a Cholesterol Transport Pathway from Lysosomes to Endoplasmic Reticulum That Is Independent of the Plasma Membrane J. Biol. Chem., February 13, 1998; 273(7): 4266 - 4274. [Abstract] [Full Text] [PDF]
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A.T. Remaley, U.K. Schumacher, J.A. Stonik, B.D. Farsi, H. Nazih, and H.B. Brewer Decreased Reverse Cholesterol Transport from Tangier Disease Fibroblasts : Acceptor Specificity and Effect of Brefeldin on Lipid Efflux Arterioscler. Thromb. Vasc. Biol., September 1, 1997; 17(9): 1813 - 1821. [Abstract] [Full Text]
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S. K. Loftus, J. A. Morris, E. D. Carstea, J. Z. Gu, C. Cummings, A. Brown, J. Ellison, K. Ohno, M. A. Rosenfeld, D. A. Tagle, et al. Murine Model of Niemann-Pick C Disease: Mutation in a Cholesterol Homeostasis Gene Science, July 11, 1997; 277(5323): 232 - 235. [Abstract] [Full Text]
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Y. Lange, J. Ye, and J. Chin The Fate of Cholesterol Exiting Lysosomes J. Biol. Chem., July 4, 1997; 272(27): 17018 - 17022. [Abstract] [Full Text] [PDF]
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S. Peiro, J. X. Comella, C. Enrich, D. Martin-Zanca, and N. Rocamora PC12 Cells Have Caveolae That Contain TrkA. CAVEOLAE-DISRUPTING DRUGS INHIBIT NERVE GROWTH FACTOR-INDUCED, BUT NOT EPIDERMAL GROWTH FACTOR-INDUCED, MAPK PHOSPHORYLATION J. Biol. Chem., November 22, 2000; 275(48): 37846 - 37852. [Abstract] [Full Text] [PDF]
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E. E. Millard, K. Srivastava, L. M. Traub, J. E. Schaffer, and D. S. Ory Niemann-Pick Type C1 (NPC1) Overexpression Alters Cellular Cholesterol Homeostasis J. Biol. Chem., December 1, 2000; 275(49): 38445 - 38451. [Abstract] [Full Text] [PDF]
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M. Fukasawa, M. Nishijima, H. Itabe, T. Takano, and K. Hanada Reduction of Sphingomyelin Level without Accumulation of Ceramide in Chinese Hamster Ovary Cells Affects Detergent-resistant Membrane Domains and Enhances Cellular Cholesterol Efflux to Methyl-beta -cyclodextrin J. Biol. Chem., October 27, 2000; 275(44): 34028 - 34034. [Abstract] [Full Text] [PDF]
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S. Dhanvantari and Y. P. Loh Lipid Raft Association of Carboxypeptidase E Is Necessary for Its Function as a Regulated Secretory Pathway Sorting Receptor J. Biol. Chem., September 15, 2000; 275(38): 29887 - 29893. [Abstract] [Full Text] [PDF]
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K. Schmidt, M. Schrader, H.-F. Kern, and R. Kleene Regulated Apical Secretion of Zymogens in Rat Pancreas. INVOLVEMENT OF THE GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED GLYCOPROTEIN GP-2, THE LECTIN ZG16p, AND CHOLESTEROL-GLYCOSPHINGOLIPID-ENRICHED MICRODOMAINS J. Biol. Chem., April 20, 2001; 276(17): 14315 - 14323. [Abstract] [Full Text] [PDF]
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J. C. Cruz and T.-Y. Chang Fate of Endogenously Synthesized Cholesterol in Niemann-Pick Type C1 Cells J. Biol. Chem., December 22, 2000; 275(52): 41309 - 41316. [Abstract] [Full Text] [PDF]
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H. Shogomori and A. H. Futerman Cholera Toxin Is Found in Detergent-insoluble Rafts/Domains at the Cell Surface of Hippocampal Neurons but Is Internalized via a Raft-independent Mechanism J. Biol. Chem., March 16, 2001; 276(12): 9182 - 9188. [Abstract] [Full Text] [PDF]
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X. Chen and M. D. Resh Activation of Mitogen-activated Protein Kinase by Membrane-targeted Raf Chimeras Is Independent of Raft Localization J. Biol. Chem., September 7, 2001; 276(37): 34617 - 34623. [Abstract] [Full Text] [PDF]
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M. Hao, S. X. Lin, O. J. Karylowski, D. Wustner, T. E. McGraw, and F. R. Maxfield Vesicular and Non-vesicular Sterol Transport in Living Cells. THE ENDOCYTIC RECYCLING COMPARTMENT IS A MAJOR STEROL STORAGE ORGANELLE J. Biol. Chem., January 4, 2002; 277(1): 609 - 617. [Abstract] [Full Text] [PDF]
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K. Fassbender, M. Simons, C. Bergmann, M. Stroick, D. Lutjohann, P. Keller, H. Runz, S. Kuhl, T. Bertsch, K. von Bergmann, et al. Simvastatin strongly reduces levels of Alzheimer's disease beta -amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo PNAS, May 8, 2001; 98(10): 5856 - 5861. [Abstract] [Full Text] [PDF]
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S. Heino, S. Lusa, P. Somerharju, C. Ehnholm, V. M. Olkkonen, and E. Ikonen Dissecting the role of the Golgi complex and lipid rafts in biosynthetic transport of cholesterol to the cell surface PNAS, July 18, 2000; 97(15): 8375 - 8380. [Abstract] [Full Text] [PDF]
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 H. Li, A. Lewis, S. Brodsky, R. Rieger, C. Iden, and M. S. Goligorsky Homocysteine Induces 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase in Vascular Endothelial Cells: A Mechanism for Development of Atherosclerosis? Circulation, March 5, 2002; 105(9): 1037 - 1043. [Abstract] [Full Text] [PDF]
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