Multidrug Resistance (MDR1)
P-glycoprotein Enhances Esterification of Plasma Membrane
Cholesterol*
Gary D.
Luker
§,
Kent R.
Nilsson§,
Douglas F.
Covey§, and
David
Piwnica-Worms
§¶
From the
Laboratory of Molecular Radiopharmacology,
Mallinckrodt Institute of Radiology and the § Department of
Molecular Biology and Pharmacology, Washington University Medical
School, St. Louis, Missouri 63110
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ABSTRACT |
Class I P-glycoproteins (Pgp) confer multidrug
resistance in tumors, but the physiologic function of Pgp in normal
tissues remains uncertain. In cells derived from tissues that normally express Pgp, recent data suggest a possible role for Pgp in cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. We
investigated the esterification of plasma membrane cholesterol under
basal conditions and in response to sphingomyelinase treatment in
transfected and drug-selected cell lines expressing differing amounts
of functional class I Pgp. Compared with parental NIH 3T3 fibroblasts,
cells transfected with human multidrug resistance (MDR1)
Pgp esterified more cholesterol both without and with sphingomyelinase. Esterification also was greater in drug-selected Dox 6 myeloma cells
than parental 8226 cells, which express low and non-immunodetectable amounts of Pgp, respectively. However, no differences in total plasma
membrane cholesterol were detected. Transfection of fibroblasts with
the multidrug resistance-associated protein (MRP) did not alter esterification, showing that cholesterol trafficking was not
generally affected by ATP-binding cassette transporters. Steroidal (progesterone, dehydroepiandrosterone) and non-steroidal antagonists (verapamil, PSC 833, LY335979, and GF120918) were evaluated for effects
on both cholesterol trafficking and the net content of 99mTc-Sestamibi, a reporter of drug transport activity
mediated by Pgp. In Pgp-expressing cells treated with nonselective and
selective inhibitors, both the kinetics and efficacy of inhibition of
cholesterol esterification differed from the antagonism of drug
transport mediated by Pgp. Thus, although the data show that greater
expression of class I Pgp within a given cell type is associated with
enhanced esterification of plasma membrane cholesterol in support of a physiologic function for Pgp in facilitating cholesterol trafficking, the molecular mechanism is dissociated from the conventional drug transport activity of Pgp.
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INTRODUCTION |
P-glycoproteins (Pgp)1
are 140-180-kDa plasma membrane glycoproteins that initially were
identified because some members of this family confer multidrug
resistance (MDR) in tumor cells (1, 2). Mammalian Pgp are encoded by a
small gene family, consisting, for example, of two members in humans
(MDR1 and MDR2) and three members in mice
(mdr1a, mdr1b, and mdr2). Class I Pgp
(MDR1, mdr1a, and mdr1b) decrease
intracellular concentrations of a wide variety of structurally diverse
chemotherapeutic agents, resulting in MDR, whereas the closely related
class II Pgp (MDR2, mdr2) are not associated with
resistance to drugs (3).
Although class I Pgp are expressed normally in many different tissues
(4, 5), the physiologic function(s) and molecular mechanisms of the
protein remain under active investigation. Based on expression in
epithelia of the intestine, kidney, liver, and endothelial cells of the
blood-brain barrier, a role for Pgp in protection from xenobiotics has
been proposed. Such a function is supported by studies with mice that
have had homozygous disruption of both class I Pgp (6). These mice are
phenotypically normal but have increased penetration of drugs into the
brain and reduced elimination of these compounds. However, the
hypothesis that Pgp functions only to exclude xenobiotics does not
readily explain the high levels of the protein in adrenal gland (5) or
apical localization of Pgp in epithelial cells of the choroid plexus (7). Other studies suggest that Pgp functions as a flippase for
phospholipids. Recently, class I Pgp were shown to translocate a wide
variety of short chain analogs of phospholipids from the inner to outer
leaflet of the plasma membrane, and Pgp also may function as a flippase
for native phosphatidylcholine and sphingomyelin (8-10). Class II Pgp
also function as lipid flippases, although these proteins are specific
for phosphatidylcholine (11). Mice that are deficient in
mdr2 have severe liver disease caused by greatly reduced
excretion of phosphatidylcholine and cholesterol into bile (12).
In addition to these possible roles for Pgp in outward translocation of
substrates, several recent studies suggest a function for class I Pgp
in the trafficking of sterols within cells. Lange and Steck (13) have
shown that esterification of plasma membrane cholesterol in rat
hepatoma cells is inhibited rapidly by treatment with a wide variety of
amphiphilic compounds. Most of the compounds tested in these studies
were nonspecific inhibitors of class I Pgp. Likewise, in CaCo-2 cells
(14) and in a human hepatoma cell line (15), compounds known to inhibit
MDR1 Pgp nonspecifically also inhibited esterification of
plasma membrane cholesterol. In CaCo-2 cells, esterification was
reduced at concentrations of drugs which did not significantly inhibit
acyl-CoA:cholesterol acyltransferase (ACAT) in isolated microsomes,
suggesting that these nonspecific inhibitors of Pgp affected
cholesterol movement from the plasma membrane to the endoplasmic
reticulum (ER). Using a series of steroid hormones, Debry et
al. (16) found that inhibition of cholesterol trafficking from the
plasma membrane to the ER correlated directly with the hydrophobicity
of each steroid and its potency in reversing the effect of Pgp on drug
accumulation. These authors also demonstrated that esterification was
decreased by non-steroidal inhibitors of class I Pgp. Although these
data support a role for Pgp in cholesterol transport from the plasma membrane to the ER, this conclusion is based on results with
amphiphiles and inhibitors that also are known to affect targets other
than Pgp. Furthermore, cholesterol trafficking has not been analyzed in
cells with documented differences in expression and function of class I
Pgp. Thus, the correlation of Pgp with esterification of plasma
membrane cholesterol remains indirect.
To define better the role of Pgp in cholesterol trafficking, we
determined the esterification of plasma membrane cholesterol in cells
that express differing amounts of functional class I Pgp. Both
drug-selected cells expressing increased levels of Pgp and cells
transfected with MDR1 Pgp or other ATP-binding cassette transporters (ABC) were examined. In addition to evaluating selected, nonspecific steroidal and non-steroidal modulators of Pgp which have
been tested previously, we also investigated the effects of several
potent antagonists specifically targeted to Pgp on both cholesterol
esterification and intracellular accumulation of a known substrate for
Pgp. Results showed that within a given cell type, greater expression
of Pgp was correlated with increased esterification of plasma membrane
cholesterol. Specific modulators all increased the cellular content of
a substrate for Pgp but differed with respect to the effects on
cholesterol esterification, suggesting dissociation between the
functions of Pgp in cholesterol trafficking and MDR. Overall, the data
support an additional physiologic function for Pgp in cholesterol
trafficking within cells.
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EXPERIMENTAL PROCEDURES |
Materials--
[1
,2
-3H]Cholesterol (50 Ci/mmol), [4-14C]cholesterol (51.3 mCi/mmol), and
[1-14C]oleoyl coenzyme A (55 mCi/mmol) were purchased
from Sigma, NEN Life Science Products, and American Radiolabeled
Chemicals, Inc., respectively. Stock solutions of verapamil,
progesterone, (+)-dehydroepiandrosterone (DHEA) (Sigma), GF120918 (gift
of Glaxo-Wellcome), and LY335979 (gift of Eli Lilly and Co.) were
prepared in dimethyl sulfoxide. Solutions of PSC 833 (gift of
Mallinckrodt Medical, Inc.) and cholesterol were prepared in ethanol.
Sphingomyelinase (Bacillus cereus) and cholesterol oxidase
(Brevibacterium spp.) were obtained from Sigma.
99mTc-Sestamibi was prepared with a one-step kit
formulation (Cardiolite, gift of DuPont Medical Products Division).
Synthesis of the (
)-enantiomer of DHEA was performed as described
previously (17), and a stock solution was prepared in dimethyl
sulfoxide. All other reagents were from Sigma.
Cell Culture and Buffers--
NIH 3T3 fibroblasts were obtained
from the ATCC and grown in Dulbecco's modified Eagle's medium, 10%
heat-inactivated calf serum, and 0.1% penicillin/streptomycin. NIH 3T3
cells stably transfected with human MDR1 (obtained from
Michael Gottesman, NIH) or human multidrug-resistance associated
protein (MRP) (gift of Gary Kruh, Fox Chase Cancer Center)
were maintained in 60 ng/ml colchicine or passaged once/month in 750 µg/ml G418, respectively. Experiments with the MRP
transfectants were performed within 1 week of culture in the selection
drug. 8226 myeloma cells and the doxorubicin-selected derivative Dox 6 cell line (gift of William Dalton, Moffitt Cancer Center) were grown in
suspension culture in RPMI with 5% heat-inactivated fetal bovine
serum, 0.1% penicillin/streptomycin, and 1% L-glutamine
(18). All cells were cultured in a 5% C02 incubator at
37 °C. Control buffer for all assays was a modified Earle's
balanced salt solution (MEBSS) containing (mM): 145 Na+, 5.4 K+, 1.2 Ca2+, 0.8 Mg2+, 152 Cl
, 0.8 H2PO4
, 0.8 SO42
, 5.6 dextrose, 4.0 HEPES, and 0.1%
fatty acid-deficient bovine serum albumin (BSA) (w/v), pH 7.4. For
cellular accumulation of 99mTc-Sestamibi, 1% serum was
used instead of BSA (MEBSS-serum). Assays of ACAT activity in
homogenates of cells were performed in 0.1 M Tris-HCl, 0.25 M sucrose, 1 mg/ml BSA, 1 mM dithiothreitol, pH
7.5 (ACAT buffer) (19). TBS-BSA (150 mM NaCl, 50 mM Tris-Cl, 2 mg/ml BSA, pH 7.4) was used as a wash buffer
in cholesterol oxidase assays (20).
Esterification of Plasma Membrane Cholesterol--
NIH 3T3 cells
were seeded in 6- or 24-well plates at a density of 3-4 × 105 cells/well either 3 days (6-well plates) or 2 days
(24-well plates) prior to experiments and reached 70-90% confluence
at the beginning of all assays. 8226 cell lines were grown at an
initial density of 1 × 106 cells/10 ml of medium 3 days before each assay, and 2-2.5 × 106 cells were
suspended in 0.5 ml of buffer for each data point. Drugs used for
selection were omitted from the culture medium of all cells in the 2-3
days prior to each experiment. Cells were grown in culture medium
supplemented with serum to stimulate cholesterol esterification
maximally, as described previously (19). For selected experiments,
drugs were added to the culture medium for the indicated times prior to
transfer to MEBSS, and control cells received vehicle alone. For
equilibrium labeling of cellular cholesterol pools, cells were cultured
with [3H]cholesterol (5 µCi/well for fibroblasts or 1 µCi/ml for 8226 cells) for 72 h prior to each assay. For pulse
labeling of plasma membranes, cells were washed with PBS, equilibrated
in MEBSS at 15 °C for 30 min, and then incubated with 2 µCi/ml
[3H]cholesterol at 15 °C for 20 min as described by
Lange (21). Under these conditions, [3H]cholesteryl
oleate could not be detected above background activity. After labeling,
cells were washed with PBS and incubated at 37 °C for the indicated
times in MEBSS containing 10 µM cold cholesterol and the
appropriate drug and/or sphingomyelinase. For all experiments, the
concentration of solvent in MEBSS was
0.1% (v/v). Upon
termination of each experiment, cells were washed with ice-cold PBS,
and lipids were extracted and quantified as described previously (22). Counts from parallel incubations without cells were subtracted to
account for background activity. Data for cholesterol esterification are reported as the percent of [3H]cholesteryl oleate
from total cellular [3H]cholesterol.
ACAT Activity in Cell Homogenates--
Activity of ACAT in
homogenates of NIH 3T3 cells was determined essentially as published
previously by Lange et al. (19). Counts from parallel
incubations with no protein were subtracted to account for background
activity. Data for cholesterol esterification in the presence of
inhibitors are expressed as the percent of vehicle control.
Cholesterol Oxidase Treatment of Cells--
NIH 3T3 fibroblasts
in six-well plates and 8226 cell lines were labeled with
[3H]cholesterol for 72 h as described above. Cells
were washed with PBS (37 °C) to remove extracellular
[3H]cholesterol, preincubated in MEBSS at 37 °C for
1 h, and then washed three times with ice-cold TBS-BSA for 5 min
and twice rapidly with PBS (20). Cholesterol oxidase treatment was
performed according to the method of either Porn and Slotte (23) or
Smart et al. (24). The percent
[3H]cholestenone was determined from
[3H]cholestenone dpm divided by
[3H]cholestenone plus [3H]cholesterol dpm.
Western Blots--
Pgp was detected in enriched membrane
preparations using Western blotting with monoclonal antibody C219
(Signet Corp.) as described previously (25).
Cellular Accumulation of Tc-Sestamibi--
Transport function
and modulation of class I Pgp were assayed with 99
mTc-Sestamibi as described previously (26, 27). Data are
reported as fmol of Tc-Sestamibi (mg of protein)
1
(nMO)
1, where
(nMO)
1 represents the total
concentration of Tc-Sestamibi in the extracellular buffer.
Data Analysis--
Data are reported as mean values ± S.E.
using the number of replicates for each point as described in figure
legends. Pairs were compared by Student's t test.
Values of p
0.05 were considered significant.
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RESULTS |
Characterization of Pgp in Fibroblast and Myeloma Cell
Lines--
To determine relative expression of Pgp, we prepared
enriched membrane fractions from the fibroblast and myeloma cell lines and immunoblotted with a monoclonal antibody to Pgp (Fig.
1A). NIH 3T3 cells express a
low amount of murine Pgp, whereas immunodetectable Pgp is increased in
fibroblasts transfected with human MDR1. In agreement with
previous reports (18), parental 8226 cells do not express
immunodetectable levels of Pgp, whereas the Dox 6 derivative cell line
has a low level of class I Pgp.

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Fig. 1.
Detection of class I Pgp. Panel
A, expression of Pgp in fibroblast and myeloma cells was detected
in 50 µg of enriched membrane fractions using monoclonal C219 as
described under "Experimental Procedures." Lane 1, NIH
3T3 MDR1; lane 2, NIH 3T3; lane 3,
8226; lane 4, Dox 6. The position of 170 kDa is indicated.
Functional class I Pgp in NIH 3T3 (panel B) and myeloma cell
lines (panel C) was identified by incubating cells with
0.1-0.6 nM 99mTc-Sestamibi in MEBSS-serum
containing vehicle alone ( ) or 300 nM GF120918 ( ) for
30 min at 37 °C. The net cell content of Tc-Sestamibi was quantified
as described under "Experimental Procedures." Columns
represent the mean of four determinations; bars
represent ± S.E. when large enough to be seen.
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Because expression and function of Pgp are not always correlated
directly (28), we also characterized the cells functionally based on
accumulation of Tc-Sestamibi, a known substrate for class I Pgp (29).
The net cell content of Tc-Sestamibi is inverse to expression of
functional class I Pgp, and this effect is reversed by GF120918, a
specific modulator of the protein (25, 26, 30). Cells were incubated
with Tc-Sestamibi for 30 min, a time sufficient for the radiotracer
to reach a steady-state accumulation within cells, in the presence of
vehicle or a saturating concentration of GF120918. In parental NIH 3T3
cells, Tc-Sestamibi accumulation under base-line conditions was
13.9 ± 1.5 fmol (mg of protein)
1
(nMO)
1 and increased
approximately 6-fold to 81.1 ± 9.1 fmol (mg of protein)
1 (nMO)
1 in
response to treatment with 300 nm GF120918 (Fig. 1B).
The base-line uptake of radiotracer was reduced to 1.5 ± 0.4 fmol (mg of protein)
1
(nMO)
1 in fibroblasts transfected
with human MDR1, and modulation of Pgp by GF120918 increased
the content of Tc-Sestamibi by approximately 43-fold to 64.2 ± 2.2 fmol (mg of protein)
1
(nMO)
1. These data show that
parental NIH 3T3 cells express a low level of class I Pgp
(mdr1a or mdr1b) and that transfection of human MDR1 into these cells increases the total amount of
functional class I Pgp. In 8226 myeloma cells, the net content of
Tc-Sestamibi was 213.3 ± 14.1 fmol (mg of protein)
1
(nMO)
1 and did not increase in
the presence of 300 nm GF120918 (Fig. 1C).
Accumulation of radiotracer in Dox 6 cells was reduced to 11.9 ± 3.0 fmol (mg of protein)
1
(nMO)
1 in control buffer and
increased approximately 18-fold to 215.9 ± 10.0 fmol (mg of
protein)
1 (nMO)
1
when GF120918 was added. Thus, based on the data for Tc-Sestamibi accumulation, 8226 cells do not express functional Pgp, whereas Dox 6 cells have a modest amount of the protein. Overall, for these cells,
the data reflecting transport function of class I Pgp correspond
directly with expression levels of the protein determined by Western blotting.
Esterification of Plasma Membrane Cholesterol--
Cholesterol is
located predominantly in the plasma membranes of cells, separated from
ACAT, which resides primarily, if not exclusively, in the ER (31-33).
To investigate the function of Pgp in cholesterol trafficking, we used
esterification of plasma membrane cholesterol as a marker of
cholesterol movement from the cell surface to the ER. Plasma membranes
were pulse labeled with [3H]cholesterol at 15 °C for
20 min and then chased at 37 °C for up to 4 h (21). Under basal
conditions, the esterification of plasma membrane cholesterol was
greater in fibroblasts transfected with MDR1 Pgp than
in the parental cells (Fig.
2A). At all time points, the
percent cholesteryl oleate in the MDR1 transfectants was
increased by approximately 1.3-1.9-fold over the parental cells. To
verify that the correlation of Pgp with cholesterol esterification was
not unique to fibroblasts and to compare Pgp-negative and -expressing
cells directly, we also measured the time course of esterification in
8226 and Dox 6 myeloma cells. Similar to the fibroblasts, more plasma
membrane cholesterol was esterified in the Pgp-expressing Dox 6 cells
than the 8226 cells during the 4-h time course (Fig. 2B).
The percent cholesteryl oleate was approximately 1.2-fold greater in
the Dox 6 cells after 30 min and increased to a 3-fold difference by
4 h. The relative differences between cell lines were maintained
in all experiments with the fibroblasts and myeloma cells, although the
absolute percent cholesteryl oleate varied (see subsequent figures).
Similar variations in esterification among experiments have been noted
previously in human fibroblasts (34). Pilot data also indicate that the
kinetics of cholesterol esterification are enhanced in Pgp-expressing
KB 8-5 epidermoid carcinoma cells compared with the Pgp-negative KB 3-1 parental cell line (data not shown). Although Pgp is not essential for
cholesterol trafficking from the plasma membrane to the ER under basal
conditions in all cells, these data show that cholesterol
esterification within a given cell type increases in direct relation to
expression of functional Pgp.

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Fig. 2.
Time course of plasma membrane cholesterol
esterification. NIH 3T3 ( ) and NIH 3T3 MDR1 ( )
(panel A) or 8226 ( ) and Dox 6 ( ) (panel B)
cell lines were cultured as described under "Experimental
Procedures." Cells were equilibrated for 30 min at 15 °C in MEBSS
followed by labeling of plasma membranes with
[3H]cholesterol in MEBSS for an additional 20 min at
15 °C. After washing with PBS, cells were incubated at 37 °C in
MEBSS with 10 µM cholesterol for the indicated times.
Cells were harvested and assayed for incorporation of
[3H]cholesterol into [3H]cholesteryl oleate
as described under "Experimental Procedures." Each data point
represents the mean of three (fibroblasts) or four (myeloma cells)
determinations, and bars represent ± S.E. when larger
than the symbol. The data are representative of at least two
independent experiments for each cell line.
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Treatment of cells with sphingomyelinase stimulates esterification of
plasma membrane cholesterol without expanding total intracellular pools
of cholesterol (35). Although the mechanism for this effect is not
known, the pathway may involve an energy-independent mechanism of
vesicle formation which differs from esterification under basal
conditions (36). Nevertheless, modulators of Pgp have been shown to
inhibit cholesterol esterification induced by sphingomyelinase,
suggesting that this pathway also is affected by Pgp (16). To determine
if levels of functional Pgp also correlate with esterification
stimulated by sphingomyelinase, we pulse labeled the plasma membrane
with [3H]cholesterol and then chased cells in the medium
containing sphingomyelinase for various periods of time. Throughout the
4-h chase, more cholesterol was esterified in the MDR1
transfectants than parental NIH 3T3 cells (Fig.
3A). Differences between these
two cell lines were greatest at the earlier time points, with 6-fold
more cholesteryl oleate in the MDR1 cells at 1 h. After
4 h of chase, cholesteryl oleate was approximately 1.4-fold
greater in the MDR1 transfectants compared with the parental
fibroblasts. Relative to basal conditions, stimulation of
esterification by sphingomyelinase was greatest at the 30-min time
point, with approximately 11- and 6-fold increases in cholesteryl
oleate in the MDR1 transfectants and NIH 3T3 cells, respectively (Fig. 3 and compare with Fig. 2). Esterification was
approximately 2-fold greater than under basal conditions in both
fibroblast cell lines after 4 h of chase. Similarly, in the myeloma cells, esterification induced by sphingomyelinase correlated with expression of Pgp. Synthesis of cholesteryl oleate was
approximately 1.2-1.9-fold greater in Dox 6 cells compared with the
Pgp-negative 8226 cells (Fig. 3B). The percent cholesteryl
oleate in both cell lines was 2-3-fold greater than comparable time
points without sphingomyelinase treatment.

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Fig. 3.
Time course of plasma membrane cholesterol
esterification in response to sphingomyelinase. The plasma
membranes of NIH 3T3 ( )and NIH 3T3 MDR1 ( )
(panel A) or 8226 ( ) and Dox 6 ( ) (panel B)
cell lines were labeled with [3H]cholesterol as described
in the legend to Fig. 2. Cells were washed with PBS and then incubated
at 37 °C in MEBSS containing 10 µM cholesterol and 50 milliunits/ml sphingomyelinase for the indicated times. Cells were
extracted for [3H]cholesterol and
[3H]cholesteryl oleate as described under "Experimental
Procedures." Each data point is the mean of three (fibroblasts) or
four (myeloma cells) determinations. Bars represent ± S.E. when larger than the symbol.
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To confirm that these observations were not an artifact of partial
labeling of a cholesterol pool by the pulse-chase protocol, cholesterol
esterification in response to sphingomyelinase also was determined in
cells that were equilibrium labeled with [3H]cholesterol
for 72 h. After 1 h of incubation with sphingomyelinase, cholesterol esterification was significantly greater in
MDR1-transfected fibroblasts and Dox 6 cells compared with
the respective parental cell lines. Cholesteryl oleate increased by
4.8- and 2.8-fold for the MDR1-transfected fibroblasts and
parental NIH 3T3 cells (p < 0.02), respectively,
whereas the increases were 1.6-fold in Dox 6 and 1.1-fold in 8226 cells
(p < 0.01). Thus, using two different methods of
labeling with [3H]cholesterol, these data show that
esterification of plasma membrane cholesterol in response to
sphingomyelinase correlates with expression of Pgp.
To determine if another ABC transporter could increase cholesterol
esterification, the effects of MRP on trafficking of plasma membrane cholesterol were investigated. Expression of MRP
decreases intracellular accumulation of structurally diverse drugs,
some of which overlap with compounds included in the MDR phenotype mediated by class I Pgp (37), including Tc-Sestamibi (38, 39). NIH 3T3
cells stably transfected with human MRP or empty vector were
pulse labeled with [3H]cholesterol and chased for 1 h in the absence or presence of sphingomyelinase.
MDR1-transfected fibroblasts were included in these same
experiments to allow direct comparison of both ABC transporters. Under
both basal conditions (Fig.
4A) and in response to
sphingomyelinase (Fig. 4B), esterification of plasma
membrane cholesterol was not increased by expression of MRP
compared with vector alone. Relative to the cells expressing
MRP, esterification was increased by approximately 3-fold in
MDR1-transfected fibroblasts. These data indicate that the
function of Pgp in cholesterol trafficking is not a general effect of
ABC transporters.

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Fig. 4.
Effect of MRP and
MDR1 Pgp on plasma membrane cholesterol
esterification. NIH 3T3 fibroblasts transfected with vector alone,
MRP, or MDR1 were plated and cultured as described under
"Experimental Procedures." The plasma membranes of cells were
labeled with [3H]cholesterol as described in Fig. 2 and
then incubated for 1 h at 37 °C in MEBSS with 10 µM cholesterol (panel A) and 50 milliunits/ml
sphingomyelinase (panel B). Conversion of
[3H]cholesterol to [3H]cholesteryl oleate
was determined as described under "Experimental Procedures." Each
column is the mean of four determinations; bars
represent ± S.E. Data are representative of two independent
experiments.
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Effects of Modulators of Pgp--
Previous investigations of the
role of Pgp in cholesterol trafficking have used nonspecific modulators
such as verapamil and progesterone. Although these compounds are
reported to inhibit the effect of Pgp on drug accumulation, they are
not specific for this protein. We used concentrations of these
compounds which are comparable to concentrations shown previously to
inhibit cholesterol esterification in other cell types and tested these
drugs for effects on both accumulation of Tc-Sestamibi and cholesterol
trafficking (14, 16).
First, in the presence of 100 µM verapamil, the net
content of Tc-Sestamibi increased by approximately 18-fold in
MDR1-transfected fibroblasts (Fig.
5A) and 35-fold in Dox 6 cells
(Fig. 5B), but the amount of Tc-Sestamibi in the 8226 cells
was not significantly changed. Accumulation of Tc-Sestamibi in Dox 6 cells increased to the same absolute amount as in 8226 cells,
indicating complete inhibition of Pgp in Dox 6 cells. Conversely,
progesterone was an ineffective reversal agent of transport of
Tc-Sestamibi mediated by Pgp. In MDR1-transfected
fibroblasts, accumulation of Tc-Sestamibi was not increased
significantly by 20 µM progesterone (Fig. 5A), whereas in Dox 6 cells, the radiotracer content was only 2-fold greater
(Fig. 5C). The Tc-Sestamibi content in 8226 cells was not
affected by progesterone.

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Fig. 5.
Effects of nonspecific modulators on plasma
membrane cholesterol esterification and Tc-Sestamibi accumulation.
The plasma membranes of NIH 3T3 MDR1 (panel A),
or 8226 ( ) and Dox 6 ( ) cells (panels B and
C) were labeled with [3H]cholesterol as
described in Fig. 2 and then incubated for 1 h at 37 °C in
MEBSS containing 10 µM cholesterol and vehicle
(veh), 100 µM verapamil (verap), or
20 µM progesterone (prog). In panel
B, myeloma cells were pretreated with vehicle or verapamil for 24 or 48 h before labeling with [3H]cholesterol. Cells
that were pretreated with verapamil also were incubated with the
modulator during the 1-h chase period. [3H]Cholesterol
and [3H]cholesteryl oleate in cells were determined as
described under "Experimental Procedures." Each column
is the mean of three (fibroblasts) or four (myeloma cells)
determinations; bars represent ± S.E. Data for the
fibroblasts are representative of two independent experiments; data for
the myeloma cells are from single experiments for each modulator. The
inset to all panels shows cell accumulation of
Tc-Sestamibi during a 30-min assay in MEBSS-serum containing vehicle or
the indicated modulator. Each column is the mean of four
determinations for all cell lines; bars represent ± S.E. when larger than the column.
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We then tested these modulators for effects on cholesterol
esterification, using pulse labeling of plasma membrane cholesterol followed by a 1-h chase period. Verapamil inhibited esterification in
MDR1-transfected fibroblasts, reducing cholesteryl ester to 31% of control when the modulator was added only to the chase buffer
(Fig. 5A). Adding verapamil only to the chase medium also had a significant inhibitory effect on esterification in the
Pgp-expressing Dox 6 cells, reducing esterification to 53% of cells
treated with vehicle alone; the cholesteryl oleate was decreased only
to 75% of vehicle control in 8226 cells (Fig. 5B).
Incubating Dox 6 cells with verapamil for 24 h did not inhibit
esterification to a greater percentage than only including the drug in
the chase medium. However, treatment with verapamil for 48 h
further decreased esterification to 20% of control values for these
cells, but effects caused by cell toxicity could not be excluded over
this length of time. Cell growth was diminished markedly under this
condition, and cells were not viable when incubated with verapamil for
72 h. Compared with including verapamil only in the chase medium,
prolonged incubation of 8226 cells with verapamil produced a minimal
additional reduction in synthesis of cholesteryl oleate.
Progesterone was an effective inhibitor of cholesterol esterification
in MDR1-transfected fibroblasts, unlike the effects of this
steroid on the accumulation of Tc-Sestamibi. Esterification decreased
to 24% of control when these cells were treated with 20 µM progesterone (Fig. 5A). Similarly,
progesterone inhibited cholesterol esterification in both myeloma cell
lines, with a greater effect on Pgp-expressing Dox 6 cells. Using
progesterone in the chase buffer, the cholesteryl oleate was 77 and
64% of control in 8226 and Dox 6 cells, respectively (Fig.
5C).
Because these first-generation modulators are known to interact with
cellular targets other than Pgp, it was of interest to study compounds
developed specifically to inhibit Pgp in MDR. We used three such
compounds (PSC 833, GF120918, and LY335979) at concentrations that
maximally antagonize the effects of class I Pgp on drug accumulation
(25, 30, 40, 41) to investigate further the relationships of Pgp to the
intracellular content of drugs and cholesterol trafficking. Using 2 µM PSC 833, 300 nM GF120918, or 1 µM LY335979, the net cell content of Tc-Sestamibi increased by approximately 40-fold in both MDR1-transfected
fibroblasts and Dox 6 cells (see Figs. 6-8). At the concentrations
tested, none of the compounds altered the accumulation of Tc-Sestamibi
in Pgp-negative 8226 cells. In Dox 6 cells, the accumulation of
Tc-Sestamibi increased to the amount present in the parental myeloma
cells. The antagonism was seen without preincubation of cells in the
modulator prior to adding the radiotracer substrate, showing that these
compounds rapidly inhibit Pgp-mediated effects on drug accumulation
during short term assays.
We then determined the effects of these same concentrations of
modulators on cholesterol trafficking in both fibroblasts and myeloma
cells, using pulse labeling of the plasma membrane with [3H]cholesterol followed by a 1-h chase. In
MDR1-transfected fibroblasts, cholesterol esterification was
not affected by PSC 833 when the drug was added only during the chase.
Because prolonged incubation in PSC 833 has been reported to alter
transmembrane distribution of lipids (10), cells were incubated with
drug for up to 72 h before the assay. Prolonged pretreatment with
PSC 833 also did not affect cholesterol esterification in
MDR1-transfected fibroblasts (Fig.
6A). Conversely, including PSC
833 in the chase buffer inhibited esterification in both myeloma cell
lines, reducing the cholesteryl oleate to 51 and 28% of control in
8226 and Dox 6 cells, respectively (Fig. 6B). The effect of
PSC 833 was immediate in these cells, reducing the percent cholesteryl
oleate to approximately equivalent levels with minimal further
reductions from culturing cells in PSC 833 prior to the experiment.
Inhibition of cholesterol esterification in Pgp-negative cells by PSC
833 may be the result of effects on another unidentified transporter.
For example, PSC 833 has been reported to reduce biliary excretion of
digoxin, another substrate for Pgp, in mice lacking both
mdr1a and mdr1b (42).

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Fig. 6.
Effects of PSC 833 on plasma membrane
cholesterol esterification and Tc-Sestamibi accumulation. NIH 3T3
MDR1 (panel A) or 8226 ( ) and Dox 6 ( )
(panel B) cells were pretreated with vehicle
(veh) or 2 µM PSC 833 (PSC) in
growth medium for 24, 48, or 72 h before labeling plasma membranes
with [3H]cholesterol as described in Fig. 2. After
labeling, cells were incubated for 1 h in MEBSS with or without 2 µM PSC 833 as described in Fig. 5. One set of cells
received vehicle alone during both the pretreatment and chase periods,
and another group was incubated with PSC 833 only during the chase. All
cells that were pretreated with PSC 833 also were incubated with the
modulator during the 1-h chase. The percent
[3H]cholesterol and [3H]cholesteryl oleate
under each condition was determined as described under "Experimental
Procedures." Data are expressed as the mean ± S.E. for three
determinations and are representative of two independent assays. The
inset in panels A and B shows the
steady-state content of Tc-Sestamibi in each cell line after a 30-min
incubation in MEBSS-serum containing vehicle or PSC 833. Data for
Tc-Sestamibi accumulation are the mean ± S.E. for four
determinations under each condition.
|
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In MDR1-transfected fibroblasts, LY335979 decreased
esterification to 60% of control when added only during the chase
period (Fig. 7A). LY335979 had
a greater effect when MDR1-transfected fibroblasts were
cultured with modulator for up to 72 h before the assay. Under
these conditions, esterification was inhibited progressively,
decreasing to only 28% of control after 72 h of pretreatment with
this modulator. In the myeloma cells, LY335979 had a similar effect on
cholesterol trafficking. Esterification decreased to 86% of control
values in Dox 6 cells when the drug was included only in the chase
medium (Fig. 7B). However, inhibition was progressively
greater when this modulator was included in the culture medium of Dox 6 cells for 48-72 h prior to the experiment, with cholesteryl oleate
reduced to approximately 72% of control. LY335979 did not inhibit
esterification in 8226 cells under any of the conditions tested.

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Fig. 7.
Effects of LY335979 on plasma membrane
cholesterol esterification and Tc-Sestamibi accumulation. NIH 3T3
MDR1 (panel A) or 8226 ( ) and Dox 6 ( )
(panel B) cells were pretreated with vehicle or 1 µM LY335979 in growth medium for 24, 48, or 72 h
before labeling plasma membranes with [3H]cholesterol as
described in Fig. 2. After labeling, cells were incubated for 1 h
in MEBSS with or without 1 µM LY335979 as described in
Fig. 6. The percent [3H]cholesterol and
[3H]cholesteryl oleate under each condition was
determined as described under "Experimental Procedures." Data are
expressed as the mean ± S.E. for three determinations and are
representative of three independent assays. The inset in
panels A and B shows the steady-state content of
Tc-Sestamibi in each cell line after a 30-min incubation in MEBSS-serum
containing vehicle or LY335979. Data for Tc-Sestamibi accumulation are
the mean ± S.E. for four determinations under each
condition.
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Unlike the other two modulators, GF120918 had minimal effects on the
esterification of plasma membrane cholesterol, regardless of expression
of Pgp (Fig. 8). In
MDR1-transfected fibroblasts, GF120918 had no effect when
added during the 1-h chase, and incubation with the drug for 72 h
only reduced esterification to 85% of control (Fig. 8A).
The percent cholesteryl oleate was unaffected in 8226 and Dox 6 cells,
even after prolonged incubations with the modulator (Fig.
8B). The same discordant effects of LY335979 and GF120918 were also seen when esterification of plasma membrane cholesterol was
stimulated by sphingomyelinase in the MDR1-transfected
fibroblasts (data not shown).

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Fig. 8.
Effects of GF120918 on plasma membrane
cholesterol esterification and Tc-Sestamibi accumulation. NIH 3T3
MDR1 (panel A), 8226 ( ), and Dox 6 ( )
(panel B) cells were pretreated with vehicle
(veh) or 300 nM GF120918 (GF) in
growth medium for 24, 48, or 72 h before labeling plasma membranes
with [3H]cholesterol as described in Fig. 2. After
labeling, cells were incubated for 1 h in MEBSS with or without
300 nM GF120918 as described in Fig. 6. The percent
[3H]cholesterol and [3H]cholesteryl oleate
under each condition was determined as described under "Experimental
Procedures." Data are expressed as the mean ± S.E. for three
determinations and are representative of four independent assays. The
inset in panels A and B shows the
steady-state content of Tc-Sestamibi in each cell line after a 30-min
incubation in MEBSS-serum containing vehicle or GF120918. Data for
Tc-Sestamibi accumulation are the mean ± S.E. for four
determinations under each condition.
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To determine if the effects of these modulators on esterification were
the result of direct inhibition of ACAT, we measured the activity of
this enzyme in homogenates of fibroblasts. Homogenates were incubated
with vehicle or appropriate drug and an excess of cholesterol before
adding 25 µM [1-14C]oleoyl coenzyme A to
begin esterification. Synthesis of cholesteryl oleate was reduced by
only 18% after treatment of homogenized cells with 100 µM verapamil and was not affected significantly by any of
the specific modulators of Pgp (Table I).
By comparison, cholesterol esterification in the presence of 20 µM progesterone was only 28% of control, which is
approximately equal to inhibition of esterification in whole
fibroblasts and exceeds the effect of this steroid in intact myeloma
cells. Thus, unlike the results with all other modulators tested,
decreases in cholesterol esterification mediated by progesterone may be
caused by inhibition of ACAT rather than cholesterol transport from the
plasma membrane to the ER.
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Table I
ACAT activity in cell homogenates
Monolayers of NIH 3T3 cells were homogenized as described under
"Experimental Procedures." Cell homogenate (200 µg) was
preincubated for 30 min at 37 °C with 10 µg of cholesterol
solubilized in Triton WR-1339 and the indicated concentration of
modulator or vehicle. [1-14C]Oleoyl coenzyme A was added at a
final concentration of 25 µM, and samples were incubated
for 10 min at 37 °C. Cholesteryl oleate was separated by TLC and
quantified by liquid scintillation counting. Data are expressed as
percent of vehicle control ± S.E. from three to six
determinations. Data for enantiomers of DHEA are from two independent
experiments.
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Effects of Enantiomers of DHEA on Esterification--
The
hydrophobicity of steroid hormones has been correlated with inhibition
of both the function of Pgp and esterification of cholesterol derived
from the plasma membrane (16). DHEA has been reported previously to
modulate reduced drug accumulation mediated by Pgp, and it is one of
the most effective steroids at blocking esterification of plasma
membrane cholesterol stimulated by sphingomyelinase (16, 43). To test
directly the effects of hydrophobicity on the accumulation of
Tc-Sestamibi and cholesterol esterification, we synthesized the
(
)-enantiomer of DHEA for comparison with the naturally occurring
(+)-enantiomer. Because these compounds have identical hydrophobicity
(as well as other physical properties), differences in the effects on
accumulation of Tc-Sestamibi or esterification of cholesterol reflect
enantio-specific interactions. MDR1-transfected fibroblasts
and both myeloma cell lines were incubated with Tc-Sestamibi and either
vehicle alone or an enantiomer of this steroid. Low intracellular
accumulation of Tc-Sestamibi (1.1 ± 0.1 and 5.7 ± 0.3 fmol
of Tc-Sestamibi (mg of protein)
1
(nMO)
1 in
MDR1-transfected fibroblasts and Dox 6 cells, respectively) was not increased significantly by either enantiomer of DHEA at concentrations up to 40 µM. DHEA also did not increase
the net cell content of Tc-Sestamibi (141.2 ± 3.6 fmol of
Tc-Sestamibi (mg of protein)
1
(nMO)
1) in Pgp-negative 8226 cells. These data indicate that DHEA is not a modulator of Pgp
transport function in MDR1-transfected fibroblasts or Dox 6 myeloma cells as probed by Tc-Sestamibi.
To determine the effects of DHEA on cholesterol trafficking, the plasma
membranes of MDR1-transfected NIH 3T3 cells were pulse labeled with [3H]cholesterol and then incubated with
sphingomyelinase in the absence or presence of differing concentrations
of each enantiomer of DHEA. Cholesterol esterification was inhibited in
a dose-dependent manner by each steroid (Fig.
9A). At concentrations between 1 and 20 µM, the (
)-enantiomer of DHEA inhibited esterification in the MDR1-transfected fibroblasts less than the
(+)-enantiomer. These effects on esterification were not the result of
direct inhibition of ACAT because the activity of the enzyme in cell homogenates was not affected by a 20 µM concentration of
either enantiomer (Table I). Thus, these data show evidence of
enantio-specific inhibition of cholesterol trafficking in fibroblasts
treated with (+)- or (
)-DHEA.

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Fig. 9.
Effects of enantiomers of DHEA on plasma
membrane cholesterol esterification in response to
sphingomyelinase. NIH 3T3 MDR1 (panel A), 8226 (panel
B), and Dox 6 (panel C) cells were incubated with
[3H]cholesterol to label plasma membranes as described in
Fig. 2. Cells were then incubated for 1 h in buffer containing 50 milliunits/ml sphingomyelinase and vehicle or differing concentrations
of (+)-DHEA ( ) or ( )-DHEA ( ). Cells were harvested and
extracted for quantification of percent [3H]cholesterol
and [3H]cholesteryl oleate as described under
"Experimental Procedures." Each data point is the mean of three
determinations; bars represent ± S.E. when larger than
the symbol. Data for each cell line are representative of
three independent experiments.
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To evaluate further the effects of DHEA on esterification of plasma
membrane cholesterol, we tested the enantiomers of this steroid in both
myeloma cell lines (Fig. 9, B and C). In these cells, cholesterol esterification was inhibited by both enantiomers with no consistent stereo-specific differences over the range of
concentrations tested. The effects of DHEA also were independent of the
expression of Pgp. At 40 µM (+)-DHEA, esterification was reduced to 43 and 36% of control in 8226 and Dox 6 cells,
respectively. Decreased esterification of plasma membrane cholesterol
was not simply the result of ACAT inhibition because esterification in cell homogenates treated with 40 µM steroid was reduced
only to 72-80% of control and did not show enantio-specific effects
(Table I). The mechanisms underlying the differing results seen with enantiomers of DHEA between fibroblasts and myeloma cells are unknown.
Nevertheless, enantio-specific inhibition of esterification in
MDR1-transfected fibroblasts demonstrates that
hydrophobicity alone is not the only determinant of the effects of DHEA
on intracellular cholesterol transport in all cell types.
Sensitivity to Cholesterol Oxidase--
A potential explanation
for the effect of Pgp on cholesterol esterification is changes in
either the overall cholesterol content in the plasma membrane or the
sterol distribution in the membrane domains. To investigate total
cholesterol in the plasma membrane, we probed cells with cholesterol
oxidase. After labeling for 72 h with
[3H]cholesterol, cells were then treated with cholesterol
oxidase according to the method of Porn and Slotte (23). Only
cholesterol in the plasma membranes is oxidized using this method (44). Approximately 80% of total cell cholesterol was oxidized to
cholestenone in both the fibroblast and myeloma cell lines (Table
II). The base-line percent of oxidizable
cholesterol did not differ among the cell lines and was not related to
the levels of Pgp.
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Table II
Susceptibility of plasma membrane cholesterol to cholesterol oxidase in
fibroblast and myeloma cell lines
NIH (3T3 and 3T3 MDR1) cell lines were seeded at 3-4 × 105 cells/well in six-well plates, and myeloma cells (8226 and Dox 6) were incubated at an initial density of 1 × 106 cells/10 ml. Cells were cultured with
[3H]cholesterol in growth medium for 72 h and then
treated with cholesterol oxidase according to either the method of Porn
and Slotte or Smart et al., as described under
"Experimental Procedures." Radiolabeled sterols were extracted and
separated by TLC and quantified by liquid scintillation. Data are the
mean ± S.E. of six samples from two independent experiments for
all conditions except 8226 and Dox 6 cells using the method of Smart
et al., for which n = 3 from one
experiment.
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The cholesterol oxidase technique described by Porn and Slotte uses
fixation with glutaraldehyde prior to treatment with cholesterol oxidase. Because glutaraldehyde has been reported to mask the existence
of different pools of cholesterol in some cell types (45), we also
determined the oxidizable pool of plasma membrane cholesterol in the
absence of fixation, using the protocol of Smart et al.
(24). By this method, the cholesterol sensitive to oxidation localizes
to caveolae, which are membrane specializations enriched in cholesterol
and sphingomyelin (46). Caveolae are reported to mediate cholesterol
efflux from cells (47) and are also the sites at which newly
synthesized cholesterol enters the plasma membrane (48). Although
neither 8226 nor Dox 6 cells are expected to express caveolin,
cholesterol that is oxidized potentially localizes to
detergent-resistant membrane fractions enriched in cholesterol and
glycosphingolipids that can exist without caveolin (49-51). In all
cell lines, approximately 3-5% of cholesterol was oxidized to
cholestenone in both the parental and Pgp-expressing cells (Table II).
These data are comparable to the 5-7% of oxidizable cholesterol
reported previously for human fibroblasts and Chinese hamster ovary
cells (20, 24). Overall, the data show that expression of Pgp does not
confer any change in cholesterol content of the plasma membranes as
examined with cholesterol oxidase.
 |
DISCUSSION |
Cholesterol is distributed asymmetrically among cell membranes,
with up to 90% of free cholesterol located in the plasma membrane (31). However, the enzymes and transcription factors that control cholesterol metabolism are located primarily in the ER, an organelle that is relatively poor in cholesterol (32, 33, 52). Thus, for the bulk
of free cholesterol in cells to communicate with the regulatory pool of
cholesterol in the ER, cholesterol from the plasma membrane and other
intracellular organelles must move to the ER. However, the mechanisms
and regulation of intracellular cholesterol trafficking remain poorly characterized.
In this report, we show that class I Pgp functions to increase
esterification of cholesterol derived from the plasma membrane under
basal conditions and in response to sphingomyelinase, demonstrating that the protein affects a step that is common to both pathways. Because regulation of ACAT by sterols in cultured mammalian cells is
dependent on the supply of cholesterol and not the amount of enzyme or
rate of catalysis (53), the data indicate that Pgp increases
esterification by facilitating cholesterol movement from the plasma
membrane to the ER. The effects of Pgp on transport were not simply the
result of the general expression of an ABC transporter because
esterification was not increased in fibroblasts transfected with
MRP. Furthermore, these effects on cholesterol trafficking
occurred without differences in plasma membrane cholesterol as measured
using cholesterol oxidase with or without glutaraldehyde fixation.
As seen with both steroidal and non-steroidal antagonists, inhibition
of cholesterol trafficking was not always related to reversal of
Pgp-mediated effects on drug accumulation. For example, verapamil
increased the net content of Tc-Sestamibi in Dox 6 cells without
affecting the Pgp-negative 8226 cell line, whereas this drug inhibited
cholesterol esterification in both cell lines, albeit with a greater
effect on Dox 6 cells. Conversely, progesterone and DHEA, which have
been reported to reverse the Pgp-mediated effects on the intracellular
content of some substrates (43, 54), were not effective antagonists of
the reduced accumulation of Tc-Sestamibi mediated by Pgp in these
fibroblast and myeloma cells at the concentrations tested. The reasons
for a lack of effect in the current study are unknown but may relate to
the use of differing cell lines and drug transport substrates for Pgp.
Cell type-specific differences in interactions of Pgp with both
modulators and substrates have been reported previously (for review,
see Ref. 55). Although these steroids decreased cholesterol esterification, the effect of progesterone is likely the result of
direct inhibition of ACAT, whereas the data indicate that DHEA affected
cholesterol trafficking from the plasma membrane to the ER in the
presence or absence of Pgp. Steroid hydrophobicity has been reported to
correlate with the inhibition of cholesterol esterification (16). At
least for DHEA, hydrophobicity alone cannot account for the effects on
cholesterol trafficking in all cell types, as evidenced by the greater
effectiveness of the naturally occurring (+)-enantiomer than the
(
)-enantiomer in fibroblasts. The lack of an enantio-specific effect
of DHEA in myeloma cells likely reflects differences between these
cells and fibroblasts in the mechanisms regulating cholesterol transport.
Specific modulators of Pgp all immediately increased the content of
Tc-Sestamibi in cells expressing class I Pgp and did not significantly
affect accumulation of the radiotracer in Pgp-negative cells. However,
these drugs differed markedly in their effects on esterification of
plasma membrane cholesterol. GF120918 had minimal effects on
esterification in the fibroblast and myeloma cell lines, independent of
Pgp. PSC 833 did not inhibit esterification in
MDR1-transfected fibroblasts, whereas synthesis of
cholesteryl oleate in the presence of this drug was reduced in both
myeloma cell lines. Essentially all of the effect of PSC 833 on
cholesterol trafficking in myeloma cells was observed when the drug was
added during the chase period; prolonged exposure of cells to this
antagonist before the assay did not result in further inhibition of
esterification. Interestingly, LY335979 inhibited esterification
selectively in cells with functional class I Pgp, showing an even
greater effect when used to pretreat cells for a prolonged period prior
to the esterification assay. We cannot exclude that the effects of
LY335979 on cholesterol trafficking may be mediated by a metabolite of this antagonist because the drug is metabolized rapidly when incubated with liver microsomes (56). Nevertheless, these data with LY335979 suggest that part of the effect of class I Pgp on cholesterol trafficking depends on changes in cells which are not immediately reversed. Potentially, the differing effects of the more potent and
selective MDR modulators may be used to probe mechanistic differences
between cholesterol trafficking and drug transport functions mediated
by Pgp. A corollary of this observation is that although all of these
drugs are well characterized as potent reversal agents of MDR, some
possess cross-reactive molecular targets.
The mechanism through which Pgp affects cholesterol trafficking from
the plasma membrane to the ER remains unknown. One possibility is that
Pgp removes an endogenous inhibitor of cholesterol transport. Among the
substrates for Pgp are a wide variety of hydrophobic peptides (57).
Cholesterol esterification in response to lipoproteins or exogenous
cholesterol was significantly decreased in macrophages and Chinese
hamster ovary cells treated with an inhibitor of cysteine proteases,
suggesting that cholesterol trafficking is regulated by an
intracellular peptide (58). Pgp may decrease the intracellular concentration of such a peptide, thus accounting for increased cholesterol esterification.
Another possibility is that Pgp may increase cholesterol esterification
through direct effects on vesicular trafficking. Pgp has been shown to
undergo constitutive recycling through an endosomal compartment
associated with Rab 5 (59), and changes in this endocytic pathway alter
the function of Pgp in MDR. By analogy, the fusion of endosomes
in vivo is stimulated by cystic fibrosis transmembrane
conductance regulator, another ABC transporter related to Pgp (60, 61).
Thus, Pgp may promote cholesterol transport from the plasma membrane to
the ER by increasing endocytic trafficking.
A third possible mechanism is that Pgp alters the organization of the
plasma membrane, perhaps by functioning as a flippase for lipids.
Sphingomyelin, a lipid that interacts strongly with cholesterol (62,
63), is among the lipids that may be translocated by Pgp. PSC 833 inhibited translocation of short chain analogs of sphingomyelin from
the inner to the outer leaflet of the plasma membrane during a 3-h
assay in cells transfected with class I Pgp (8). The distribution of
native sphingomyelin in cells expressing endogenous Pgp also is
reported to be altered by PSC 833, although 72 h of treatment with
antagonist was required to decrease the pool of native sphingomyelin
located in the outer leaflet of the plasma membrane (10). In addition,
PSC 833 is reported to inhibit translocation of both short chain
analogs and full-length phosphatidylcholine (8, 9). By altering the
transbilayer distribution of sphingomyelin or other phospholipids, Pgp
could affect the size or composition of lipid domains within the plasma
membrane. Such domains could promote cholesterol trafficking from the
plasma membrane to intracellular organelles such as the ER without
changing the total amount of cholesterol in the cell membrane.
In conclusion, we have shown that increased expression and function of
class I Pgp in a given cell type correlate with increased esterification of plasma membrane cholesterol, both under basal conditions and in response to sphingomyelinase. However, although it is
a facilitator, functional class I Pgp is not essential for cholesterol
trafficking from the plasma membrane to the ER in all cell lines. The
effect of Pgp on cholesterol transport is not through a mechanism
identical to its function in MDR, as evidenced by discordant inhibition
of cholesterol esterification and modulation of drug transport by many
of the MDR antagonists that were tested. We also demonstrated cell
type-specific differences of inhibitors on cholesterol esterification,
suggesting that mechanisms of sterol trafficking differ between
fibroblast and myeloma cell lines. These data expand on previous
investigations and document a physiologic function for Pgp in
cholesterol trafficking. Thus, although an excretory or protective
function of Pgp in tissues such as liver, kidney, intestine, and brain
capillaries has been documented, our data and the patterns of
expression of Pgp in non-excretory tissues such as adrenal, choroid
plexus, and placenta further point to a role in metabolism of sterols,
perhaps relevant to all organs expressing Pgp. Additional studies are
necessary to define further the mechanism through which class I Pgp
functions in cholesterol homeostasis.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Jay Heinecke for helpful
discussions and Julie Dahlheimer for excellent technical assistance.
 |
FOOTNOTES |
*
This work was supported by United States Department of
Energy Grant D-FG02-94ER61885 (to D. P.-W.), National Institutes of Health Grant GM47696 (to D. F. C.), and Mentored Clinical Scientist Career Development Award K08 HL03683 (to G. D. L.).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: Mallinckrodt
Institute of Radiology, 510 S. Kingshighway Blvd., St. Louis, MO 63110. Tel.: 314-362-9356; Fax: 314-362-0152; E-mail: piwnica-worms{at}mirlink.wustl.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
Pgp, P-glycoprotein(s);
MDR, multidrug resistance;
ACAT, acyl-CoA:cholesterol acyltransferase;
ER, endoplasmic reticulum;
ABC, ATP-binding cassette transporter(s);
DHEA, dehydroepiandrosterone;
MRP, multidrug resistance-associated protein;
MEBSS, modified Earle's
balanced salt solution;
BSA, bovine serum albumin;
PBS, phosphate-buffered saline.
 |
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