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(Received for publication, August 23, 1995; and in revised form, October 23, 1995) From the
Multidrug resistance (MDR) P-glycoproteins were first recognized
for their ability to catalyze ATP-dependent efflux of cytotoxic agents
from tumor cells when overexpressed. Despite extensive study, little is
known about the normal substrate(s) and normal cellular function of
these proteins. In the accompanying manuscript (Metherall, J. E.,
Waugh, K., and Li, H. (1996) J. Biol. Chem. 271,
2627-2633), we demonstrate that progesterone inhibits cholesterol
biosynthesis, causing the accumulation of a number of cholesterol
precursors. In the current manuscript, we use several criteria to show
that the progesterone receptor is not involved in this inhibition.
Rather, we demonstrate that progesterone inhibits cholesterol
biosynthesis by interfering with MDR activity. We show that a steroid
hormone's ability to inhibit cholesterol biosynthesis is
correlated with: 1) its general hydrophobicity and 2) its ability to
inhibit MDR activity. The only exception to this finding is
Mammalian cells grown in the presence of a single cytotoxic
drug, such as vincristine or dactinomycin, can result in the selection
cells that are resistant to that drug as well as a broad spectrum of
structurally and functionally distinct compounds(1) . This
phenomenon, termed multidrug resistance (MDR), ( P-glycoproteins are encoded by a small gene family, which consists
of three members in rodents and two members in
humans(2, 3) . The MDR proteins are highly homologous,
containing 12 predicted membrane-spanning domains and two ATP-binding
cassettes. The three rodent genes appear to have arisen from a common
ancestor through two successive gene duplication events, the most
recent event producing mdr1 and mdr3. The human MDR1 gene is the counterpart of rodent mdr1 and mdr3(4) , while human MDR2 is the counterpart
of rodent mdr2(5) . Human MDR1 and MDR2 are encoded by linked genes on chromosome 7q that are often
co-amplified in drug-resistant cell lines. The human MDR1 gene
and the rodent mdr1 and mdr3 genes are primarily
responsible for the drug efflux, while human MDR2 and rodent mdr2 lack drug efflux activity(6) . The MDR gene
family is part of a larger superfamily of ATP-binding cassette membrane
transporters. This superfamily includes: the STE6 protein from Saccharomyces cerevisiae(7) , the cystic fibrosis
transmembrane conductance regulator chloride channel(8) , and
the TAP1 and TAP2 proteins involved in peptide translocation and
antigen presentation in T lymphocytes(9) . The ability of
P-glycoproteins to catalyze the efflux of unnatural drugs has led to
the proposal that MDR normally functions in detoxification. Consistent
with this proposal is the recent finding that mice homozygous for
disruption of the mdr3 gene, which is primarily expressed in
brain capillaries that define the blood-brain barrier, are
phenotypically normal unless challenged with drugs; they demonstrate
increased sensitivity to drugs, especially in the brain (10) .
However, these mice also demonstrated increased expression of the mdr1 mRNA. The normal physiologic role of MDR might be not be
evident in these studies if mdr1 activity substitutes for mdr3 activity in these tissues. Other recent studies have
suggested that the normal physiologic role of MDR proteins is to
catalyze the transport of normal cellular lipids across membranes. mdr2 is expressed predominantly in bile
cannicula(11) , and mice with a homozygous disruption of mdr2 develop severe liver disease due to a lack of
phosphatidylcholine and cholesterol production into bile(12) .
More recently, the mdr2 gene has been shown to catalyze the
ATP-dependent translocation of phosphatidylcholine across secretory
vesicles when expressed in yeast(13) . Targeted disruption of
the mdr1 (mdr1b) gene in a mouse adrenal cell line
decreased the stimulated secretion of steroid hormones from the
cell(14) . These experiments strongly suggest the MDR proteins
normally function to facilitate the transport of normal cellular lipids
across membranes. We have recently demonstrated that MDR activity is
required for the esterification of LDL-derived cholesterol. ( In the accompanying manuscript(18) , we demonstrate
that progesterone also inhibits late steps in cholesterol biosynthesis,
steps that also occur in the ER(19) . In the current manuscript
we demonstrate that MDR activity is required for cholesterol
biosynthesis and suggest a model in which MDR functions in the movement
of cholesterol and cholesterol precursors from the plasma membrane to
the ER.
Figure 1:
Effect of progesterone and RU 486 on
cholesterol biosynthesis in CHO cells. CHO cells were plated at 5
We next tested for the characteristic
lag time associated with progesterone receptor-mediated events. CHO
cells were pretreated with 40 µM progesterone for various
amounts of time prior to labeling with
[
Figure 2:
Effect of time of preincubation in
progesterone on cholesterol biosynthesis. Cells were plated and grown
through day 2 as described in the legend to Fig. 1. On day 3,
cells were preincubated for the indicated periods of time in 40
µM progesterone (lanes 3-10). Additions
were made in a staggered fashion so that cultures could be labeled
simultaneously with 0.5 mM [
Figure 3:
Effect of other steroids on cholesterol
biosynthesis. Cells were plated and grown through day 2 as described in
the legend to Fig. 1. On day 3, cells were preincubated at 37
°C for 1 h in medium containing the indicated additions of either
40 or 100 µM steroid hormone. Cells were then labeled with
0.5 mM [
Figure 4:
Correlations between inhibition of
cholesterol biosynthesis and steroid hydrophobicity. The incorporation
of [
A
strong correlation was observed for 11 of the 12 steroid hormones
tested (open circles); trend lines and correlation
coefficients for this relationship are indicated. One steroid (closed circle) appeared to be a more potent inhibitor than
would be expected based solely on hydrophobicity. This steroid was
Figure 5:
Correlations between
[
To determine whether
the effect of the
Figure 6:
Effect of progesterone,
To test definitively the role of the
estrogen receptor in these events, we assayed the effect of tamoxifen
on cholesterol biosynthesis. Tamoxifen is a known antagonist of the
estrogen receptor and an inhibitor of MDR(29) . If the effects
of
Figure 7:
Effect of nonsteroidal MDR inhibitors on
cholesterol biosynthesis. Cells were plated and grown through day 2 as
described in the legend to Fig. 1. On day 3, cells were
preincubated for the indicated periods of time in 40 µM progesterone (lanes 3-10). Additions were made in a
staggered fashion so that cultures could be labeled simultaneously with
0.5 mM [
Figure 8:
Effect of lanosterol demethylase and ACAT
inhibitors on cholesterol biosynthesis. Cells were plated and grown
through day 2 as described in the legend to Fig. 1. On day 3,
duplicate dishes were refed medium A containing the indicated additions
of 30 nM SKF 104976 and 30 µM progesterone. After
incubation at 37 °C for 2 h, [
Volume 271,
Number 5,
Issue of February 2, 1996 pp. 2634-2640
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
-estradiol, which is a more potent inhibitor of cholesterol
biosynthesis than expected based solely on hydrophobicity and MDR
inhibition. We further demonstrate that nonsteroidal inhibitors of MDR
also inhibit cholesterol biosynthesis. Since MDR activity is required
for esterification of LDL-derived cholesterol (P. DeBry and J. E.
Metherall, submitted for publication), we investigated the relationship
between these phenomena and show that inhibition of cholesterol
esterification does not cause inhibition of cholesterol biosynthesis
and that inhibition of cholesterol biosynthesis does not cause
inhibition of cholesterol esterification. We propose a model in which
MDR is required for transport of sterols from the plasma membrane to
the endoplasmic reticulum (ER). Inhibiting this transport prevents
cholesterol esterification and cholesterol biosynthesis by preventing
sterol substrates from reaching ER-resident enzymes.
)often
results from amplification and overexpression of genes encoding
integral plasma membrane proteins known as MDR P-glycoproteins. These
proteins function as ATP-dependent efflux pumps that reduce drug
cytotoxicity by decreasing their intracellular accumulation. 
)Cholesterol esterification, catalyzed by
acyl-CoA:cholesterol O-acyltransferase (ACAT), is an important
cellular process involved in maintaining cholesterol homeostasis. When
plasma membrane cholesterol levels rise, cholesterol is transported
from the plasma membrane to the endoplasmic reticulum (ER) where it is
esterified by ACAT. While excess cholesterol is toxic to cells,
cholesteryl esters can accumulate to relatively high levels as
cytosolic lipid droplets. Progesterone inhibits cholesterol
esterification by causing the accumulation of cholesterol in the
lysosome (16) and by preventing the transport of cholesterol
from the plasma membrane to the ER(17) . The ability of
progesterone to inhibit this process results from its ability to
inhibit the activity of MDR P-glycoproteins, suggesting
that MDR activity is required for proper cholesterol trafficking within
cells.
Materials
CHO cells (CHO-7) are a previously
described subline (20) of CHO-K1 cells. Newborn calf
lipoprotein-deficient serum (d > 1.215 g/ml; cholesterol
content of 33-61 µg/ml), human LDL (d 1.019-1.063 g/ml), and rabbit -very low density
lipoproteins were prepared by ultracentrifugation as described
previously(21) . RU 486 was the kind gift of Raymond Daynes
(University of Utah). Lovastatin was provided by Alfred Alberts (Merck
Sharp & Dohme, Rahway, NJ), CL 283,796 was provided by Elwood
Largis (American Cyanamid, Pearl River, NY), and SKF 104976 was
provided by Julia Christie (SmithKline Beecham, King of Prussia, PA).
Cholesterol was obtained from Alltech Chemicals. Progesterone and
methyl--cyclodextrin were obtained from Sigma. Other steroid
hormones were purchased from Steraloids Inc. DL-Mevalonic acid
lactone was purchased from Fluka Chemical Co. and was converted to the
sodium salt as described(22) .
[2-
C]Acetic acid (52-55 mCi/mmol) was
purchased from Amersham. Other materials were obtained from previously
reported sources(18, 20, 23, 24) .MDR Nomenclature
MDR1 refers to the human
gene whose product catalyzes drug efflux, while MDR2, also
known as MDR3(25) , refers to the human gene whose
product does not catalyze drug efflux(2) . mdr1 and mdr3 refer to the rodent homologues of the human MDR1 gene. In mice, the mdr1 gene is also called mdr1b, and the mdr3 gene is called mdr1a(10) . In hamsters, the mdr1 gene is
also called pgp3, the mdr3 gene is called pgp1, and the mdr2 gene is called pgp2(26) .Cell Growth
Cells were grown in monolayer at 37
°C in an atmosphere of 5% CO. CHO-7 cells were grown in
medium A (a 1:1 mixture of Ham's F12 medium and Dulbecco's
modified Eagle's minimum essential medium containing 100 units/ml
penicillin, 100 µg/ml streptomycin, and 5% (v/v) newborn calf
lipoprotein-deficient serum). Sterols and progesterone were added to
the culture medium in ethanol; the final ethanol concentration did not
exceed 0.5% (v/v).Assays
Steroid hydrophobicity was estimated by
measuring retention fraction (R
) on Silica Gel G
(Analtech) thin layer chromatography sheets developed in
chloroform:methanol (12:1). For cellular assays, cells were plated and
grown as described in the individual figure legends. The incorporation
of [C]acetate into cellular lipids by cell
monolayers was measured using modifications (18) of the
procedure of Brown et al.(22) . Vinblastine
accumulation was measured as described previously. The
amount of [
H]vinblastine accumulated is reported
as pmol per mg of total cellular protein.Cell Fractionation
Cell fractionation was
performed as described previously by Lange and Muraski (27) with minor modifications. Briefly, cell monolayers were
harvested into ice-cold phosphate-buffered saline. Cell pellets were
then washed in cold phosphate-buffered saline and resuspended in 5
mM sodium phosphate, pH 7.5, containing 0.25 M sucrose. After centrifugation at 1,700 
g, the
recovered pellet was resuspended in 0.5 mM sodium phosphate,
pH 7.5, containing 0.25 M sucrose. A second pellet, obtained
following centrifugation at 3,000 g, was resuspended
in 5 volumes of 0.5 mM sodium phosphate, pH 7.5, containing
0.25 M sucrose. Following a 10-min incubation on ice, sodium
phosphate was added to a final concentration of 5 mM, and
MgCl was added to a final concentration of 0.3 mM.
Protease inhibitors were added to final concentrations of 0.2 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, and 1
µg/ml aprotinin. The suspension was then homogenized by 25 strokes
with a Dounce homogenizer using a tight-fitting pestle. The suspension
was cleared of unlysed cells and nuclei by two sequential spins at
1,700 g. High density pellet (P10) and supernatant
(S10) fractions were generated after centrifugation at 10,000 g. Microsomal pellet (P100) and cytosolic (S100) fractions
were generated after centrifugation of the S10 fraction at 100,000
g. The P10 and P100 pellets were resuspended in buffer
containing 100 mM Tris-Cl, pH 7.4, 0.25 M sucrose, 1
mM EDTA, 0.2 M NaCl, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 µg/ml pepstatin, 0.5
µg/ml aprotinin, and 0.5 µg/ml leupeptin.
The Progesterone Receptor Is Not Involved in
Cholesterol Biosynthesis
Progesterone is known to interact
directly with two proteins within the cell: the progesterone receptor
and MDR P-glycoproteins. The progesterone receptor is a member of the
steroid hormone receptor family of transcription factors that upon
activation enters the nucleus to promote transcription of a class of
progesterone-sensitive genes. Progesterone receptor-mediated events
typically: 1) occur at nanomolar progesterone concentrations, 2)
require a lag time after receptor activation due to the time required
for transcriptional changes induced by the receptor to manifest as
phenotypic changes, and 3) are prevented by a specific antagonist of
the receptor, such as RU 486(28) . To test if
progesterone's effects on cholesterol biosynthesis were mediated
through the progesterone receptor, we first determined the
concentration of progesterone required to inhibit synthesis. CHO cells
were preincubated with various concentrations of progesterone for 1 h
prior to the addition of [C]acetate. Following a
2-h labeling, radiolabeled sterols were harvested, resolved using thin
layer chromatography (TLC), and visualized by autoradiography (Fig. 1). Untreated CHO cells produce mainly cholesterol and
only small quantities of lanosterol as well as two other sterols,
designated A and B (lane 1). Increasing concentrations of
progesterone inhibited the formation of cholesterol and resulted in the
accumulation of a number of unusual sterol precursors (lanes
2-6). Cholesterol synthesis was inhibited 78% with 5
µM progesterone (lane 2); greater than 95%
inhibition was observed only with progesterone concentrations of 10
µM and above (lanes 3-6). This
concentration is much higher than that which is typically required for
progesterone receptor-mediated events. We next tested the ability of a
progesterone receptor antagonist to prevent this inhibition. CHO cells
were pretreated for 1 h with RU 486 and increasing concentrations of
progesterone prior to a 2-h [C]acetate labeling.
RU 486, a specific antagonist of the progesterone receptor, had no
detectable effect on cholesterol biosynthesis alone (lane 7)
and failed to prevent the inhibition observed with progesterone (lanes 8-12).
10
cells/well in a 24-well Linbro plate in medium
A. On day 3, duplicate dishes of cells were refed 0.5 ml of the same
medium containing the indicated additions of progesterone and 2
µM RU 486. After incubation at 37 °C for 1 h,
[C]acetate (99.8 dpm/pmol) was added to a final
concentration of 0.5 mM, and the cells were incubated at 37
°C for 2 h. Radiolabeled sterols were extracted and resolved by
thin layer chromatography (TLC) as described under ``Experimental
Procedures.'' TLC plates were exposed to Amersham Hyperfilm for 3
days at -70 °C. One set of the duplicate incubations is
shown; the other set demonstrated identical results. The positions of
recovery-derived lanosterol and cholesterol were determined by
comparing the autoradiogram with the TLC sheet that had been stained
with iodine vapor.
C]acetate. An autoradiogram of one such
experiment is shown in Fig. 2. Again, untreated cells produced
mainly cholesterol, some lanosterol, and small amounts of products A
and B (lane 1). Cells treated with progesterone produced some
lanosterol and products C-F. In this experiment, product E appears to
comigrate with cholesterol; however, we have been able to distinguish
product E from cholesterol using TLC sheets containing silver nitrate.
The same products were formed whether the cells were pretreated with
progesterone (lanes 5-10) or progesterone was added
simultaneously with the addition of the
[C]acetate (lanes 3 and 4).
These findings demonstrate that very little, if any, time is required
for progesterone to inhibit cholesterol biosynthesis. Taken together,
these findings demonstrate that progesterone has a direct effect on
cholesterol biosynthesis and that the progesterone receptor is not
involved.
C]acetate
(95.4 dpm/pmol). Cells were then incubated at 37 °C for an
additional 2 h. Radiolabeled sterols were then isolated and analyzed by
TLC as described in the legend to Fig. 1. TLC plates were
exposed to Amersham Hyperfilm for 4 days at -70 °C. Both sets
of the duplicate incubations are shown.
Effect of Other Steroid Hormones on Cholesterol
Biosynthesis
We next tested the ability of other steroid
hormones to inhibit cholesterol biosynthesis. Cells were pretreated
with various steroid hormones at either 40 or 100 µM for 1
h prior to the addition of [C]acetate. An
autoradiogram of the sterols formed in a 2-h labeling is shown in Fig. 3. Most of the steroids tested inhibited cholesterol
biosynthesis to some degree. Dehydroepiandrosterone (DHEA; lanes 6 and 7), -estradiol (lanes 12 and 13), pregnenolone (lanes 16 and 17),
progesterone (lanes 22 and 23), and
deoxycorticosterone (lanes 24 and 25) were potent
inhibitors, while androstenediol (lanes 2 and 3),
DHEA sulfate (lanes 8 and 9), and cortisol (lanes
14 and 15) had very little effect. High concentrations of
the more potent inhibitors resulted in the accumulation of product D,
while lower concentrations resulted in the production of product B. We
have previously provided evidence that product D is a precursor of
lanosterol and product B is 7-dehydrocholesterol(18) , the
immediate precursor of cholesterol. Precise identification of these
compounds requires further study.
C]acetate (95.4 dpm/pmol) for 2
h at 37 °C. Radiolabeled sterols were isolated and analyzed by TLC
as described in the legend to Fig. 1. TLC plates were exposed to
Amersham Hyperfilm for 2 days at -70
°C.
Role of Steroid Hydrophobicity
We have found
previously that the general hydrophobicity of steroids correlates with
their ability to: 1) inhibit MDR activity and 2) inhibit the
esterification of LDL-derived cholesterol. We, therefore,
tested whether steroid hydrophobicity is a predictor of
steroid-mediated inhibition of cholesterol biosynthesis. We evaluated
the production of cholesterol, lanosterol, and product D in cells
treated with various steroids at either 40 or 100 µM (Fig. 4). The rate of sterol production was plotted against
the hydrophobicity of the steroid, as measured by its migration (R) in TLC. A correlation was observed between the
ability of a steroid to inhibit cholesterol synthesis and its
hydrophobicity at both 40 and 100 µM. Steroids with high R values were more effective at both decreasing
cholesterol production (panels A and B) and
increasing lanosterol production (panels C and D).
Only steroids with very high R values resulted in
the accumulation of product D (panels E and F).
C]acetate into cholesterol (A and B), lanosterol (C and D) and band D (E and F) are reported as the amount of label incorporated
into the individual sterol as a percentage of total labeled sterols.
Digital reproductions of autoradiograms including and similar to that
shown in Fig. 3were generated on a Howtek Scanmaster 3+
using transmitted light. Densiometric analyses of the digital images
were performed using the Millipore Bio Image software package. Steroid
hydrophobicity was estimated by measuring retention fraction (R) on Silica Gel G (Analtech) thin layer
chromatography sheets developed in chloroform:methanol (12:1). Each
value represents the average of duplicate
incubations.
-estradiol, the active metabolite of estrogen.Effect of Estrogen on Cholesterol
Biosynthesis
Since -estradiol was a more potent inhibitor
than expected based on hydrophobicity, we first considered the
possibility that cells metabolically convert -estradiol to a more
hydrophobic compound and, therefore, a more potent MDR inhibitor. To
test this hypothesis, we directly measured MDR activity in parallel
cultures using a [H]vinblastine accumulation
assay (Fig. 5). Vinblastine is an MDR substrate that passively
enters cells and is pumped out by MDR. Steady-state intracellular
levels of [H]vinblastine are dictated by MDR
activity; inhibition of MDR increases intracellular accumulation. Cells
treated with either 40 µM (panel A) or 100
µM steroid (panel B) were assayed for the
accumulation of cell-associated vinblastine which was plotted against
the general hydrophobicity of the steroid (R). A
strong correlation was observed between vinblastine accumulation and
hydrophobicity at both steroid concentrations. This correlation
included -estradiol (closed circles); -estradiol
inhibited vinblastine accumulation as would be expected by its relative
hydrophobicity. These results suggest that the cells do not
metabolically convert -estradiol to a more hydrophobic compound
and consequently a more potent MDR inhibitor.
H]vinblastine accumulation and steroid
hydrophobicity. Cells were plated and grown through day 2 as described
in the legend to Fig. 1. On day 3, the medium was replaced with
medium A containing 20 mM HEPES (pH 7.2). One h later, cells
were refed 0.5 ml of identical medium containing 50 nM vinblastine sulfate (0.1 µCi/ml) and the indicated
concentrations of steroid hormones. The steroids used in this analysis
were identical with those described in Fig. 3. Following a 1-h
incubation, cell monolayers were washed rapidly 3 times with an
ice-cold 1:1 mixture of Ham's F12 medium and Dulbecco's
modified Eagle's minimum essential medium containing 100 units/ml
penicillin, 100 µg/ml streptomycin, and 20 mM HEPES (pH
7.2). The monolayers were then solubilized in 0.1 N NaOH and
analyzed for protein and [H]vinblastine content. R values used are those reported in Fig. 4. Each value represents the average of duplicate
incubations.
-estradiol on cholesterol biosynthesis was due
to estrogen receptor-mediated events, we first measured the
concentration of -estradiol required to inhibit cholesterol
biosynthesis (Fig. 6A). The concentration of
-estradiol required to completely inhibit cholesterol biosynthesis
was greater than 10 µM (lane 10). This
concentration was similar to the concentration of progesterone required
to inhibit cholesterol biosynthesis (lane 5) and was higher
than the concentrations typically required for estrogen
receptor-mediated events. At 100 µM, -estradiol
completely blocked cholesterol production. However, unlike progesterone (lane 6), -estradiol resulted only in lanosterol
accumulation (lane 11); band D was not apparent. In addition,
the effect of -estradiol was immediate (data not shown), not
requiring any lag time for the transcriptional changes mediated by the
receptor to be expressed. These findings suggest that the estrogen
receptor is not involved in the ability of -estradiol to inhibit
cholesterol biosynthesis.
-estradiol,
tamoxifen, and mixtures of -estradiol and tamoxifen on cholesterol
biosynthesis. Cells were plated and grown through day 2 as described in
the legend to Fig. 1. On day 3, cells were preincubated at 37
°C for 2 h in medium containing the indicated additions. In A, the additions of progesterone, -estradiol, and
tamoxifen were made to medium A. In B, the indicated additions
of -estradiol were made to medium A containing 1 µM tamoxifen. Cells were then labeled with 0.5 mM [C]acetate (95.4 dpm/pmol) for 2 h at 37
°C. Radiolabeled sterols were isolated and analyzed by TLC as
described in the legend to Fig. 1. TLC plates were exposed to
Amersham Hyperfilm for 5 days at -70
°C.
-estradiol are mediated through the estrogen receptor, then
tamoxifen should prevent inhibition. If the effects of -estradiol
are mediated through MDR, tamoxifen itself should inhibit cholesterol
biosynthesis, even in the absence of -estradiol. Concentrations of
tamoxifen (1 µM), known to antagonize the estrogen
receptor, had no effect on the ability of -estradiol to inhibit
cholesterol biosynthesis (Fig. 6B). In addition,
tamoxifen was capable of inhibiting cholesterol biosynthesis at
concentrations similar to those required for -estradiol and
progesterone (panel A, lanes 12-16). These results
suggest that the ability of -estradiol to inhibit cholesterol
biosynthesis is not mediated through the estrogen receptor.Nonsteroidal MDR Inhibitors Also Inhibit Cholesterol
Biosynthesis
With the exception of -estradiol, the observed
correlations between cholesterol biosynthesis, vinblastine
accumulation, and steroid hydrophobicity raised the possibility that
MDR activity is required for cholesterol biosynthesis. To test this
more directly, we measured the ability of nonsteroidal MDR inhibitors
to prevent cholesterol biosynthesis (Fig. 7). Verapamil, a
calcium channel blocker and MDR inhibitor, inhibited cholesterol
production at similar concentrations (lanes 1-4) and
resulted in the production of lanosterol and product B
(7-dehydrocholesterol). Low concentrations of Triton X-100, an MDR
inhibitor, also inhibited cholesterol production and increased the
production of lanosterol and product B (lanes 9-12).
These findings demonstrate that nonsteroidal inhibitors of MDR also
inhibit cholesterol biosynthesis.
C]acetate (95.4 dpm/pmol).
Cells were then incubated at 37 °C for an additional 2 h.
Radiolabeled sterols were then isolated and analyzed by TLC as
described in the legend to Fig. 1. TLC plates were exposed to
Amersham Hyperfilm for 4 days at -70
°C.
Inhibition of Cholesterol Esterification Does Not Inhibit
Cholesterol Biosynthesis
The finding that MDR activity is
required for cholesterol biosynthesis and our previous findings that
MDR activity is required for cholesterol esterification, raise the possibility that the effects of MDR inhibitors on
cholesterol biosynthesis result from decreased cholesterol
esterification. To test whether an inability to synthesize cholesterol
esters interferes with cholesterol biosynthesis, we monitored the
incorporation of acetate into sterols in cells treated with CL 283,796,
a specific inhibitor of acyl-CoA:cholesterol O-acyltransferase
(ACAT), the enzyme involved in cholesterol esterification. Untreated
CHO cells produced mainly cholesterol with small amounts of lanosterol
and products A and B (Fig. 8, lane 1). As a control, 40
µM progesterone inhibited cholesterol synthesis and
resulted in the accumulation of lanosterol and products C-F (lane
2). As a second control, treatment with SKF 104976, a specific
inhibitor of lanosterol demethylase, resulted in the accumulation of
only lanosterol (lane 3). Treatment with CL 283,796, which
inhibited cholesterol ester formation by >10-fold (data not shown),
resulted in only minor changes in cholesterol biosynthesis. There was
an accumulation of lanosterol, products A and B, and a major product
with mobility only slightly altered from that of cholesterol (lane
4).
C]acetate
(95.4 dpm/pmol) was added to a final concentration of 0.5 mM and the cells were incubated at 37 °C for an additional 2 h.
Cells were harvested, and sterols were extracted and resolved by thin
layer chromatography (TLC) as described in Fig. 1. TLC plates
were exposed to Kodak XAR film for 2 days at -70
°C.
Inhibition of Cholesterol Biosynthesis Does Not Inhibit
Cholesterol Esterification
To test the reciprocal hypothesis,
that preventing normal lanosterol metabolism inhibits cholesterol
esterification, we monitored cholesterol esterification in cells grown
in the presence of the lanosterol demethylase inhibitor SKF 104976. In
the presence of LDL, progesterone inhibited cholesterol oleate
formation by 6.5-fold (Table 1). In contrast, SKF 104976 had
essentially no effect on cholesterol ester formation, even though it
completely blocked the conversion of lanosterol to cholesterol (Fig. 8, lane 3). In addition, progesterone inhibited
esterification in the presence of SKF 104976 (Table 1). As a
control, cholesteryl ester formation was 5.5-fold lower in the absence
of LDL. These results demonstrate that neither the inhibition of
cholesterol synthesis nor the accumulation of lanosterol is responsible
for the decreased cholesterol ester production observed in
progesterone-treated cells.
The Progesterone Receptor Is Not Involved in
Progesterone's Inhibition of Cholesterol Biosynthesis
We
present three lines of evidence that the progesterone receptor is not
involved in progesterone's ability to inhibit cholesterol
biosynthesis. First, the concentration of progesterone required for
inhibition is much higher than typically required for progesterone
receptor-mediated events. While progesterone receptor-mediated events
are typically elicited by nanomolar progesterone concentrations,
inhibition of cholesterol biosynthesis occurs at micromolar
concentrations. Since CHO cells are derived from steroidogenic tissue,
these cells may require unusually high concentrations of progesterone
to elicit progesterone receptor-mediated events due to increased
catabolism, decreased uptake or increased efflux of progesterone as
compared to non-steroidogenic cells. However, we have found that
similar concentrations of progesterone inhibit cholesterol biosynthesis
in a large number of cell types not derived from steroidogenic
tissue(18) . The second line of evidence arguing against a role
for the progesterone receptor is that RU 486, a specific antagonist of
the progesterone receptor, fails to prevent progesterone-mediated
inhibition of cholesterol biosynthesis. Although RU 486 typically
functions as an antagonist of the progesterone receptor, RU 486 can
also act as a progesterone agonist in the presence of
cAMP(30, 31) . Our experiments, however, were
performed under conditions where cAMP levels are low(32) . The
third observation arguing against a role for the progesterone receptor
is that the effect of progesterone is immediate. The only known
function of the progesterone receptor is to activate transcription
following hormone binding. Secondary events resulting from progesterone
receptor activation demonstrate a characteristic lag due to the time
required for transcriptional changes to present as phenotypic changes.
Since no lag time is observed in progesterone's ability to
inhibit cholesterol biosynthesis, progesterone must have a direct role
in mediating these events and that the progesterone receptor is not
involved.Role of MDR in Cholesterol Biosynthesis
In
addition to the progesterone receptor, progesterone physically
interacts with one other class of cellular protein: members of the MDR
family of P-glycoproteins. Progesterone has a photoactivatable bond
that allows its direct cross-linking to proteins with which it
interacts. Studies in multidrug resistance human leukemic lymphoblasts
have demonstrated a direct physical interaction between progesterone
and MDR(33) . Our current studies demonstrate a strong
correlation between MDR activity and cholesterol biosynthesis. This
correlation is observed with both steroidal and nonsteroidal MDR
inhibitors. Since a number of these inhibitors are known to interact
with MDR directly, the most likely explanation for our observations is
that changes in cholesterol metabolism are a consequence of the effect
of these inhibitors on MDR activity and not that cholesterol
biosynthesis is required for MDR activity.
We demonstrate a
strong correlation between a steroid's general hydrophobicity and
its ability to inhibit cholesterol biosynthesis. The only exception to
this correlation is -Estradiol Is an Exception-estradiol, which is a more potent inhibitor
than expected based solely on hydrophobicity. We have excluded the
possibility that cells metabolically convert -estradiol to a more
potent MDR inhibitor by demonstrating a direct correlation between MDR
activity and steroid hydrophobicity that includes -estradiol.
Estrogen has been shown to stimulate cholesterol biosynthesis in
estrogen-sensitive cells (34) by activating
3-hydroxy--methylglutaryl-CoA reductase activity(35) .
This stimulation appears to involve activation of the estrogen receptor
since stimulation: 1) takes many hours, 2) occurs at nanomolar estrogen
concentrations, and 3) occurs only in estrogen-sensitive cells. This
increased cholesterol biosynthesis induced by -estradiol is
associated with increased cell proliferation and may be secondary to
the increased cholesterol requirements of proliferating cells. Our
studies did not reveal estrogen receptor-mediated increases in
cholesterol biosynthesis, since our studies were performed following
relatively short treatments with -estradiol. Tamoxifen is both an
antagonist of the estrogen receptor and an inhibitor of MDR. Our
findings that tamoxifen alone inhibits cholesterol biosynthesis, and
that tamoxifen fails to prevent the inhibition of cholesterol synthesis
induced by -estradiol demonstrates that estrogen receptor-mediated
events are not involved in the processes described in the current
report.Relationship between Cholesterol Biosynthesis and
Esterification
Since MDR activity is required for both
cholesterol biosynthesis and cholesterol esterification, we
investigated whether defects in one of these processes causes defects
in the other. Inhibiting cholesterol esterification with ACAT
inhibitors had no effect on the late steps of cholesterol biosynthesis.
Similarly, inhibiting a late step in cholesterol biosynthesis had no
effect on cholesterol esterification. These findings suggest that MDR
independently affects each of these processes, and that MDR activity
may be required for a common process involved in both cholesterol
esterification and cholesterol biosynthesis. Since both cholesterol
esterification and the late steps in cholesterol biosynthesis occur in
the ER and the precursors to these reactions reside in the plasma
membrane, we propose that MDR activity is required for the movement of
sterols from the plasma membrane to the ER. In fact, progesterone, one
of the more potent inhibitors used on our studies, blocks the movement
of cholesterol from the plasma membrane to the ER(17) . The
nature of this transport process is very poorly understood.MDR Isoforms
Although the current studies
demonstrate that MDR activity is required for cholesterol biosynthesis,
they do not define which MDR isoform is required. Since cholesterol
biosynthesis occurs in virtually all cells of the body, the MDR isoform
involved should be expressed widely. The direct correlation between
cholesterol biosynthesis and vinblastine accumulation suggests that an
isoform capable of catalyzing drug efflux is involved. In humans,
isoform-specific mRNA probes have demonstrated that MDR1 is
expressed in all of the tissues tested, while MDR2 expression
is limited to the liver, kidney, and spleen(36) , suggesting
that MDR1 may play a role in the general process of
cholesterol biosynthesis. We cannot exclude a possible role of mdr2 in cholesterol metabolism since a biologic assay for mdr2 activity has been devised only recently(13) , and further
studies will be required to determine if the inhibitors used in our
studies also affect mdr2 activity. Definitive identification
of the isoform involved will come from studies of cholesterol
biosynthesis in cells transfected with individual MDR genes.Mechanism of MDR Action in Cholesterol
Metabolism
How MDR activity influences cholesterol metabolism is
also not clear. Disruption of one of the mouse MDR genes (mdr2) results in an inability to secrete
phospholipids(12) , and the mdr2 gene demonstrates the
ability to translocate phospholipids across the lipid bilayer when
expressed in yeast secretory vesicles(13) . One possibility is
that MDR-mediated phospholipid translocation is required for movement
of cholesterol from the plasma membrane to the ER. If vesicular
transport is involved(37) , then phosphatidylcholine
translocation across the bilayer may be required for generation of the
vesicles. Another possibility is that one of the MDR genes may directly
catalyze the translocation of cholesterol across cellular membranes.
Although cholesterol has many of the structural characteristics of MDR
substrates, catalyzed translocation seems unnecessary since cholesterol
moves relatively freely across bilaminar membranes(38) . A
third possibility is that MDR catalyzes the efflux of a metabolite that
normally regulates intracellular cholesterol trafficking; inhibiting
efflux of this metabolite would cause its intracellular accumulation
and consequent alterations in cholesterol metabolism. The MDR gene
family belongs to a superfamily of ATP-binding cassette membrane
transporters that includes the yeast STE-6 gene. The STE-6 gene
catalyzes the secretion of the prenylated 
-factor peptide
pheromone from yeast cells(7) . Mammalian prenylated proteins
have been proposed to regulate cholesterol metabolism(39) ,
and, recently, prenylcysteine methyl esters, the degradative breakdown
products of these prenylated proteins, have been shown to be substrates
of MDR1(40) . These observations raise the possibility that MDR
indirectly participates in cholesterol transport by extruding a
regulatory prenylated protein or amino acid from the cell.Implications to Coronary Heart Disease
A number of
the more potent MDR inhibitors used in our studies previously have been
shown to be clinically beneficial in the treatment of coronary heart
disease. Premenopausal women have reduced risk for coronary heart
disease as compared to men and postmenopausal women. Estrogen and
progesterone replacement therapy can prevent the increased risk
associated with menopause(41) , suggesting that female-specific
steroid hormones function to protect women from heart disease. In
addition, post-menopausal breast cancer patients treated with tamoxifen
demonstrate a reduced risk of heart disease(42) . Serum levels
of DHEA and its sulfated derivative (DHEA-S) are inversely correlated
with cardiovascular death in
men(43, 44, 45) . DHEA was one of the most
potent inhibitors of cholesterol biosynthesis in our studies. Studies
in hypercholesterolemic rabbits demonstrate that administration of DHEA
decreases the incidence of atherosclerosis (46) and the
deposition of cholesterol in the arteries(15) . These findings
suggest that elevated serum DHEA levels may protect against heart
disease by preventing cholesterol deposition, a process that requires
cholesterol esterification. Our findings that these agents affect
cellular cholesterol metabolism through their ability to inhibit MDR
provide a possible mechanistic basis for these observations and raise
the possibility that inherited abnormalities at the MDR locus may
contribute to coronary heart disease.
), retention fraction;
DHEA, dehydroepiandrosterone.
)
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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