(Received for publication, August 23, 1995; and in revised form, October 23, 1995)
From the
Cells acquire cholesterol through endogenous synthesis and through receptor-mediated uptake of cholesterol-rich low density lipoprotein (LDL). Esterification of LDL-derived cholesterol is catalyzed by acyl-CoA:cholesterol acyltransferase (ACAT) in the endoplasmic reticulum (ER). Progesterone inhibits esterification, and, although the mechanism of inhibition is not completely understood, this inhibition results from progesterone's ability to inhibit the activity of multiple drug resistance (MDR) P-glycoproteins (P. DeBry and J. E. Metherall, submitted for publication). In the current manuscript, we demonstrate that progesterone inhibits cholesterol biosynthesis resulting in the accumulation of a number of sterol precursors. In Chinese hamster ovary (CHO) cells, high concentrations (100 µM) of progesterone completely blocked cholesterol production, resulting in the accumulation of lanosterol and a lanosterol precursor. Lower concentrations (40 µM) of progesterone cause plasma membrane accumulation of several sterol products. The majority of these sterols are precursors of cholesterol since they were efficiently converted to cholesterol upon removal of progesterone from the culture medium. Although very high concentrations (>200 µM) of progesterone killed CHO cells, their growth was restored by the addition of cholesterol to the growth medium, indicating that progesterone toxicity resulted from cholesterol auxotrophy. The effect of progesterone was not unique to CHO cells; progesterone also inhibited cholesterol biosynthesis in all human cell lines tested. These observations suggest that a common progesterone-sensitive pathway is involved in both cholesterol biosynthesis and the processing of LDL-derived cholesterol.
Active mechanisms of cholesterol transport and sorting are
required for maintaining proper distributions and levels of cholesterol
within the cell. Approximately 90% of cellular cholesterol is found in
the plasma membrane(1, 2) , and most of the remainder
is found in endocytic vesicles derived from the plasma
membrane(3) . The endoplasmic reticulum (ER) ()and
other organelle membranes are relatively devoid of cholesterol. Despite
this distribution, a number of important enzymatic reactions involving
sterols occur at the ER. Sterols must be transported from the plasma
membrane to the ER to function as substrates for these reactions. The
mechanisms of this transport are poorly understood.
When excess
cholesterol accumulates in the plasma membrane, cholesterol is
transported to the ER where it is esterified by acyl-CoA:cholesterol
acyltransferase (ACAT). ACAT catalyzes the transfer long-chain fatty
acyl residues from acyl-CoA to the -hydroxyl group of cholesterol.
This esterification reaction contributes to cholesterol homeostasis;
while free cholesterol is toxic, cholesteryl esters can accumulate to
relatively high levels as cytosolic lipid droplets. Metabolic labeling
studies demonstrate that the cholesterol substrate pool for ACAT
derives from the plasma membrane (4) and that, under most
conditions, ACAT is not saturated with cholesterol
substrate(5, 6) . These findings indicate that
regulation of cholesterol esterification occurs at the level of
cholesterol substrate availability and suggest that cholesterol
transport to the ER may be a regulated process.
Progesterone has
long been known to inhibit esterification of low density lipoprotein
(LDL)-derived cholesterol(7) . This inhibition does not involve
direct inhibition of ACAT activity since it has no effect on the
esterification of cholesterol that has been solubilized in
detergent(8) . Rather, progesterone appears to inhibit delivery
of LDL-derived cholesterol to ACAT. Progesterone inhibits the movement
of LDL-derived cholesterol from the lysosome to the plasma membrane (9) and the movement of cholesterol from the plasma membrane to
the ER(8) . Progesterone's effects on esterification
appear to be mediated through its ability to inhibit the activity of
one or more of the multidrug resistance (MDR) families of
P-glycoproteins. ()
The movement of sterols from the plasma membrane to the ER is also required for cholesterol biosynthesis. Enzymes involved in the late stages of cholesterol biosynthesis reside in the ER. However, newly synthesized substrates for these enzymes (lanosterol and zymosterol) are found in the plasma membrane(11, 12) . These substrates are rapidly and efficiently transported to the ER for conversion to cholesterol (13, 14) .
In the current report, we demonstrate that progesterone inhibits cholesterol biosynthesis, resulting in the accumulation of a number of cholesterol precursors. We suggest that this inhibition results from progesterone's ability to prevent plasma membrane-derived sterol precursors from reaching ER-resident enzymes. These findings suggest that cholesterol precursors utilize a common transport system to reach the ER as does cholesterol in the process of cholesterol esterification. In the accompanying paper(15) , we provide evidence that the effect of progesterone on cholesterol biosynthesis requires the multidrug resistance family of P-glycoproteins.
Figure 1:
Effect of progesterone
on the incorporation of [C]acetate into cellular
sterols in CHO cells. CHO cells were plated at 5
10
cells/well in a 24-well Linbro plate in medium A containing 5%
(v/v) newborn calf lipoprotein-deficient serum. On day 3, cells were
refed 0.5 ml of medium A containing 2 mg/ml bovine serum albumin (BSA)
and the indicated additions of progesterone. After incubation at 37
°C for 2 h, [
C]acetate (9.98 dpm/pmol) was
added to a final concentration of 0.5 mM and cells were
incubated at 37 °C for the indicated time. Additions were made in a
staggered fashion so that cells were harvested simultaneously for
measurement of [
C]sterol synthesis. Radiolabeled
sterols were extracted and resolved by thin layer chromatography as
described under ``Experimental Procedures.'' TLC plates were
exposed to Amersham Hyperfilm for 4 days at -70 °C, and the
bands corresponding to lanosterol and cholesterol were identified by
comparing the iodine-stained TLC sheet with the
autoradiogram.
To test if the lack
of cellular cholesterol resulted from increased release of sterols into
the medium, we monitored the appearance of
[C]sterols in the medium for extended periods of
time following the addition of progesterone (Fig. 2A).
In untreated cells, virtually all of the sterols produced were found
intracellularly (upper left); very few sterols were found in
the culture medium (upper right). Progesterone treatment
resulted in a very slight increase in the production of sterols into
the medium. This minor increase in sterols released into the media does
not explain the drastic decrease in cholesterol produced within cells.
At 40 µM progesterone, approximately 4% of the sterols
were found in the medium as compared to 1% in untreated cells. Two of
the products, B and D, were preferentially released into the medium (Fig. 2B), suggesting that this release was not due to
general cell death or lysis. As a control, most fatty acids produced
from [
C]acetate remained within the cell (lower left) while very few reached the medium (lower
right).
Figure 2:
Incorporation of
[C]acetate into cellular and media lipids in
progesterone-treated cells. Cells were plated and grown through day 2
as described in the legend to Fig. 1. On day 3, duplicate dishes
were refed 0.5 ml of medium A containing 2 mg/ml BSA and the indicated
additions of progesterone. After incubation at 37 °C for 2 h,
[
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 the indicated time. Additions were made in a staggered
fashion so that cultures were harvested simultaneously. In A,
cultures were assayed for [
C]sterol (top) and
C-fatty acid (bottom) content
in both cells (left) and culture medium (right).
Sterols and fatty acids were extracted and resolved by TLC as described
under ``Experimental Procedures.'' Bands corresponding to
lanosterol, cholesterol, and products A-F (see text) were pooled and
counted in a liquid scintillation counter for determination of total
[
C]sterol content. In B, individual
sterols produced in the absence or presence of 40 µM progesterone are shown in an autoradiograph of a TLC sheet, as
described in the legend to Fig. 1.
Figure 3:
Effect of cholesterol synthesis inhibitors
on incorporation of [C]acetate into cellular
sterols in progesterone-treated cells. 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 2 mg/ml BSA and the
indicated additions of 30 nM SKF 104976, 1 µM AY-9944, and 40 µM progesterone. After incubation at
37 °C for 2 h, [
C]acetate (95.4 dpm/pmol)
was added to a final concentration of 0.5 mM and cells were
incubated at 37 °C for an additional 2 h. Cells were harvested, and
sterols were extracted and resolved by TLC as described under
``Experimental Procedures.'' TLC plates were exposed to Kodak
XAR film for 4 days at -70 °C. The indicated locations of
recovery-derived lanosterol and cholesterol were determined by staining
with iodine vapor.
The conversion of lanosterol to cholesterol is a complicated process requiring at least five enzymatic modifications; some of the key intermediates in this process are shown in Fig. 3B. The order of these modifications is not well established, and, in fact, the order may not be strictly processive. Saturation at the C-24(25) position distinguishes the intermediates shown on the left from those on the right. Many, if not all, of the enzymes involved in modifications to the steroid nucleus recognize both C-24(25) saturated and unsaturated substrates, resulting in two parallel pathways for conversion; one pathway processing C-24(25) saturated intermediates (left) and the other processing C-24(25) unsaturated intermediates (right).
The first step in modification of the steroid nucleus involves three demethylations; one at position 14 and two at position 4. The removal of these methyl groups is catalyzed by the enzyme lanosterol demethylase. Reduction at the C-24(25) position most likely occurs following the removal of a single methyl group at position 14. SKF 104976 is a specific inhibitor of lanosterol demethylase and thereby prevents demethylation of lanosterol and dihydrolanosterol. Treatment of CHO cells with SKF 104976 completely blocked the conversion of lanosterol to cholesterol and resulted in the specific accumulation of lanosterol (lane 3). Treatment of CHO cells with SKF 104976 in the presence of progesterone resulted in the production of only lanosterol and the product designated D (lane 6). These findings suggest that product D is a precursor of lanosterol, and that products C, E, and F are products of lanosterol.
The final step in the modification of
the steroid nucleus involves reduction of the C-7(8) double bond. This
step is catalyzed by 7-dehydrocholesterol (7-DHC) reductase and results
in the conversion of cholest-5,7,24-trien-3-ol to desmosterol and
7-DHC to cholesterol. AY-9944 is an inhibitor of 7-DHC reductase.
Treatment of CHO cells with AY-9944 resulted mainly in the accumulation
of products B and F (lane 2). Our observation that product F
comigrates with authentic 7-DHC (data not shown), has resulted in our
tentative assignment of product F as 7-DHC and product B as
cholest-5,7,24-trien-3
-ol. AY-9944 had no effect on the spectrum
of labeled intermediates produced in the presence of progesterone
(compare lanes 4 and 5). These findings demonstrate
that all of the products generated in progesterone-treated cells are
derived from sterol intermediates upstream of 7-DHC reductase.
Interestingly, while one of these products appears to be 7-DHC itself,
there is little or no cholest-5,7,24-trien-3
-ol produced,
suggesting that reduction of the C-24(25) position must occur
efficiently in the presence of progesterone.
The metabolic
inhibition experiments described above demonstrate that the abnormal
products observed in the presence of progesterone must be direct
precursors of cholesterol or products derived from these precursors. To
determine if these metabolites were in fact precursors of cholesterol,
we monitored their conversion to cholesterol following removal of
progesterone from the medium. CHO cells were labeled for 1 h with
[C]acetate in either the absence (Fig. 4, lane 1) or presence (lanes 2-7) of 40
µM progesterone. The label was removed and the cells were
chased with cold acetate for different lengths of time. As previously
shown, progesterone treatment nearly completely inhibited cholesterol
production (lane 2). These products diminished rapidly; within
a few hours of chase, after removal of progesterone (lanes
3-7). This disappearance correlated with the appearance of
two new bands that corresponded to cholesterol and a novel product,
designated G. In addition, some lanosterol remained resistant to
metabolic conversion. These results demonstrate that the block in
cholesterol synthesis caused by progesterone is reversible and that
many of the sterols produced are precursors of cholesterol.
Figure 4:
Pulse-chase analysis of incorporation of
[C]acetate into cellular sterols in the absence
or presence of progesterone. Cells were plated and grown through day 2
as described in the legend to Fig. 1. On day 3, duplicate dishes
were refed 0.5 ml of medium A containing 2 mg/ml BSA in either the
absence (-) or presence (+) of 40 µM progesterone. After incubation at 37 °C for 2 h,
[
C]acetate (9.9 dpm/pmol) was added to a final
concentration of 0.5 mM, and the cells were incubated at 37
°C for an additional 1 h. Cells were then washed 3 times with
phosphate-buffered saline and refed 2 ml of medium A containing 5% LPPS
and 2 mM sodium acetate. Cells were further incubated at 37
°C for the indicated amount of time. Additions were made in a
staggered fashion so that cells were harvested simultaneously for
measurement of [
C]sterol content as described in
the legend to Fig. 1.
Figure 5:
Subcellular location of sterols. Cells
were plated, grown, and labeled on day 3 with
[C]acetate for 2 h at 37 °C as described in
the legend to Fig. 1. Cells were harvested, homogenized and
fractionated into P10, P100, and S100 fractions as described under
``Experimental Procedures.'' Radiolabeled sterols were
extracted from each fraction and resolved by TLC as described in the
legend to Fig. 1. TLC plates were exposed to Amersham Hyperfilm
for 7 days at -70 °C. The bands corresponding to lanosterol
and cholesterol were identified by comparing the iodine-stained TLC
sheet with the autoradiogram.
As a control for the mixing of sterols
between various fractions during homogenization, we simultaneously and
routinely monitored the distribution of
[H]cholesterol in cells labeled with
[
H]cholesterol at 4 °C. Under these
conditions, labeled cholesterol is only present in the plasma membrane
and fails to reach intracellular membranes(26) . Cells labeled
at 4 °C with [
H]cholesterol were mixed with
cells labeled at 37 °C with [
C]acetate,
prior to homogenization. The [
H]cholesterol
distribution was very similar to that observed for the
[
C]acetate-labeled sterols, with 87-88% of
the [
H]cholesterol found in the P10, 9% in the
P100, and 4% in the S100. As further controls, we also monitored the
distribution of enzyme markers known to reside predominantly within
specific subcellular compartments. Nearly 70% of the alkaline
phosphodiesterase activity, an enzyme known to reside in the plasma
membrane, was found in the P10. Approximately 55% of the hexosaminidase
activity, an enzyme known to reside in the lysosome, similarly
fractionated with the P10, indicating that this fraction was also rich
in lysosomes. A significant amount of hexosaminidase activity (36%) was
found in the S100, most likely due to breakage of the lysosome during
homogenization; significantly, little or no cholesterol was found in
this fraction. Lactate dehydrogenase, a cytosolic enzyme marker, was
found predominantly in the S100 fraction. These findings demonstrate
that the endogenously produced sterols that accumulate in the presence
of progesterone cofractionate with sterols known to reside in the
plasma membrane and that the distribution of these sterols is
distinctly different from that of lysosomal enzyme markers.
Figure 6:
Growth of CHO cells in progesterone. On
day 0, CHO cells were plated at 3 10
cells/60-mm
dish in medium A. On day 1, cells were refed medium A containing 5%
LPPS and the indicated additions of progesterone and cholesterol. Cells
were refed every 2-3 days with medium of identical composition.
On day 12, cells were washed, fixed, and stained with crystal
violet.
Figure 7:
Effect of progesterone in human cell
lines. Cells were plated on day 0 in their respective growth media. On
day 1, all cells were refed medium A containing 5% LPPS. On day 3,
duplicate dishes were refed 0.5 ml of medium A containing 2 mg/ml
bovine serum albumin in either the absence(-) or presence
(+) of 100 µM progesterone. After incubation at 37
°C for 2 h, [C]acetate (95.4 dpm/pmol) was
added to a final concentration of 0.5 mM, and cells were
incubated at 37 °C for an additional 2 h. Cells were harvested, and
sterols were extracted and resolved by TLC as described under
``Experimental Procedures.'' TLC plates were exposed to Kodak
XAR film for 4 days at -70 °C. The indicated locations of
recovery-derived lanosterol and cholesterol were determined by staining
with iodine vapor.
In the current report, we demonstrate that progesterone inhibits cholesterol biosynthesis by preventing conversion of sterol intermediates to cholesterol. We demonstrate that the ability of progesterone to inhibit cholesterol synthesis is a general phenomenon, seen not only in CHO cells but in many different human cell lines, as well as in human primary tissues. Therefore, it is likely that this progesterone-sensitive process is a general aspect of normal cell function and not a unique characteristic of differentiated cells.
Since progesterone is known to block cellular cholesterol transport, we postulate that progesterone blocks cholesterol biosynthesis by inhibiting intracellular transport of cholesterol precursors. Enzymes involved in the conversion of lanosterol to cholesterol reside in the ER(13, 14) . Progesterone may prevent sterol precursors from reaching these ER-resident enzymes, thereby preventing their conversion to cholesterol. Consistent with this hypothesis is our observation that the sterols that accumulate in progesterone-treated cells accumulate in cell fractions containing the plasma membrane.
The concentration of progesterone required to inhibit cholesterol
synthesis is relatively high and is similar to the concentration
previously shown to be required to inhibit cholesterol
esterification. Such high concentrations prevent growth of
CHO cells in prolonged culture. Since cell growth can be rescued by
addition of cholesterol to the medium, it appears that progesterone
toxicity results from inhibition of cholesterol biosynthesis;
progesterone induces cholesterol auxotrophy. The increase in sterol
production observed in the presence of high concentrations of
progesterone (see Fig. 1and Fig. 2A) provides
further evidence that progesterone induces cholesterol auxotrophy.
Normal mechanisms of feedback suppression allow cells to up-regulate
cholesterol production as cellular cholesterol levels
fall(28) . We find that in cells treated with 100 µM progesterone, total sterol production increases, resulting in an
increased accumulation of lanosterol and products C and D. These
findings suggest that the cells have up-regulated the enzymes
controlling the early steps in the biosynthetic pathway and demonstrate
that the cells recognize the decreased cholesterol production caused by
progesterone. Furthermore, these findings indicate that regulatory
metabolites involved in feedback suppression must be downstream of the
metabolites that accumulate in progesterone-treated cells. Further
studies will be required to determine the exact mechanism of this
derepression, since the regulation of the pathway involves many
distinct but coordinated mechanisms(28) .
Most of the sterols that accumulate in the presence of progesterone have the capacity to continue through the pathway to cholesterol, once progesterone is removed from the media. These studies provide evidence that many of the sterols produced in progesterone-treated cells are cholesterol precursors. There are two notable exceptions to this relationship. First, a novel minor product (product G) is produced upon removal of progesterone, suggesting that some metabolites irreversibly leave the cholesterol biosynthetic pathway to form a novel product. Although the exact nature of this product is not known, it may be one of the 4-carboxysterols recently shown to accumulate in a mutant CHO cholesterol auxotroph(10) . Second, a portion of lanosterol that is produced in progesterone-treated cells is refractory to conversion. Presumably, this lanosterol has entered a metabolic pool no longer capable of being converted to cholesterol. The nature of this pool is not known; however, it does not appear to result from fatty acid esterification of lanosterol (data not shown).
Mammalian cells utilize two distinct sources of cholesterol: LDL-derived cholesterol and endogenously synthesized cholesterol. Cells maintain exquisite control over the level of free cholesterol by coordinately regulating these two pathways through transcriptional regulation of genes required in both processes(28) . The current observations suggest that a single, progesterone-sensitive process is required for cells both to synthesize cholesterol and to esterify LDL-derived cholesterol. Regulation of this process could provide an additional mechanism for coordinately regulating these pathways. In the process of cholesterol esterification, delivery of cholesterol substrate to the ER is rate-limiting(6) . Since the same progesterone-sensitive process seems to be involved in cholesterol synthesis, it seems possible that, under some circumstances, delivery of sterol precursors to the ER may be limiting to cholesterol biosynthesis.
Progesterone
is unlikely to be a physiologic regulator of sterol synthesis since the
concentration of progesterone required to inhibit cholesterol synthesis
exceeds normal physiologic levels. Other steroids might be more potent
regulators of the process and may represent physiologic regulators of
this process. In the accompanying manuscript (15) , we
investigate the effects of additional steroid hormones on cholesterol
biosynthesis and demonstrate that, like cholesterol
esterification, multidrug resistance P-glycoprotein
activity is required for cholesterol biosynthesis.