(Received for publication, October 13, 1994; and in revised form, January 5, 1995)
From the
Mechanisms and pathways of excess cholesterol removal from
intracellular sites of accumulation to extracellular cholesterol
acceptors remain poorly defined. To gain further insights, compounds
known to affect cellular protein transport pathways were tested for
their effects on high density lipoprotein (HDL)-mediated cholesterol
efflux from cultured cells enriched with cholesterol. Monensin,
nigericin, and brefeldin A inhibited the ability of HDL to decrease
cellular cholesterol esterification, stimulate sterol biosynthesis, and
promote the efflux of labeled cholesterol and cholesterol mass from
fibroblasts and smooth muscle cells. HDL-mediated decrease in cell
cholesterol esterification was inhibited up to 80% by these compounds
compared with control incubations over an HDL concentration of
5-100 µg/ml and up to 18 h of incubation. Up-regulation of
sterol biosynthesis after depletion of cell cholesterol by HDL
increased over 10-fold; however, inclusion of monensin or brefeldin A
during the incubation completely prevented the increase of sterol
biosynthesis by HDL. Efflux of [H]cholesterol to
HDL from prelabeled cells was inhibited up to 40% by these compounds,
and this effect persisted when cholesterol esterification was blocked.
Similarly, monensin and brefeldin A inhibited up to 50% of HDL-mediated
cholesterol mass efflux relative to controls. Treatment of cells with
cholesterol oxidase demonstrated an increase of intracellular
cholesterol after exposure to monensin or nigericin and to a lesser
extent with brefeldin A. These data show that monensin, nigericin, and
brefeldin A sequester cholesterol from sites normally available for
efflux by HDL. Since these compounds act by disruption of Golgi complex
structure and function, a role for this intracellular organelle in
transport of cholesterol between intracellular sites and the plasma
membrane for eventual removal by extracellular acceptors such as HDL is
suggested.
Efflux of cholesterol by high density lipoproteins (HDL) ()from extrahepatic tissue is thought to account for the
protective effect of elevated HDL levels against
atherosclerosis(1) . However, mechanisms by which HDL remove
excess cholesterol from cells remain poorly understood, especially
transport pathways involving removal of intracellular cholesterol.
Removal of unesterified cholesterol from the plasma membrane has been
adequately explained by the aqueous diffusion model. By this mechanism,
cholesterol present in the plasma membrane desorbs from the cell
surface to the surrounding aqueous space, diffusion occurs against a
concentration gradient and the sterol molecule is absorbed by an
appropriate extracellular acceptor, such as
HDL(2, 3, 4) . The rate of desorption from
cell membranes appears to be an intrinsic property of the membrane but
also may be influenced by the cholesterol
acceptor(3, 5, 6) . Kinetic analysis of
cholesterol efflux data demonstrated that membrane cholesterol
available for efflux exists in up to three kinetic pools with fast,
intermediate, and slow desorption rates, depending on the cell type
studied (reviewed in (7) ). These pools were proposed to arise
from cholesterol distribution within different microdomains of the
plasma membrane. Efflux of cholesterol from these pools appears to be a
passive process although rates of cholesterol desorption from cell
membranes could be modulated by apolipoproteins, primarily
A-I(5, 8) , by altering distribution of
cholesterol between the postulated domains. However, similar pools were
shown to exist when phospholipid vesicles were used as the acceptors,
suggesting that desorption of cholesterol from these domains is
primarily a function of the membrane(7) . Such models for
cholesterol efflux do not account for removal of cholesterol from other
cellular compartments such as the pool(s) of sterol involved in the
cholesteryl ester cycle or in the regulation of
3-hydroxy-3-methylglutaryl coenzyme A reductase and low density
lipoprotein (LDL)-receptor activity(9) . Indeed, the cellular
identities of such pools are poorly defined. Intracellular pools of
cholesterol involved in the regulation of cholesterol homeostasis and
the relationship between these pools and the various membrane pools
have not been established.
Recent studies have examined the identity of the acyl coenzyme A:cholesterol acyl transferase (ACAT) substrate pool in cultured cells and have suggested that sterol available for esterification is derived from, or is in equilibrium with, plasma membrane cholesterol(10, 11) . These data imply transport of sterol from plasma membrane to sites of ACAT-mediated esterification in the endoplasmic reticulum must occur. Such results are consistent with data of Johnson et al.(12) , demonstrating that the majority of LDL-derived cholesterol is not directly esterified but instead rapidly transported to the plasma membrane from lysosomes, and little of the lysosomal cholesterol passes through the endoplasmic reticulum during transport to the cell membrane. Certain intracellular pools of cholesterol (i.e. not plasma membrane associated) can be depleted by HDL through a process that requires intact HDL apolipoproteins but are not affected by apolipoprotein-depleted HDL or artificial (non-apolipoprotein containing) acceptors(13, 14, 15, 16) . Additionally, several studies have shown that transport and efflux of cholesterol from intracellular sites can be activated by various signaling pathways, including activation of protein kinase C (17, 18) or protein kinase A (19, 20) under conditions that do not influence plasma membrane cholesterol desorption rates. These data suggest a dissociation between efflux of cholesterol present in the plasma membrane and intracellular pools based on sensitivity to different acceptor types or changes in cell metabolism.
These results suggest a transport pathway exists to deliver cholesterol from the plasma membrane to intracellular sites of cholesterol esterification and accumulation. Conversely, a pathway should also exist for the transport of free cholesterol (derived from cholesteryl ester hydrolysis and other sites of overaccumulation) within intracellular pools to the plasma membrane. Such a pathway has been shown to operate for HDL-mediated efflux of newly synthesized cholesterol in cholesterol overloaded cells(13, 18) . Also in support of this concept, a vesicular transport pathway has been demonstrated to exist for newly synthesized sterol molecules from the endoplasmic reticulum to the plasma membrane in growing cells(21) . Whether a similar mechanism(s) transports newly synthesized and excess cholesterol stored intracellularly to the plasma membrane for eventual efflux is unknown.
To better understand mechanisms of cholesterol transport between cellular compartments, the effects of various agents, known to affect cell protein transport pathways, were examined for their influence on cell cholesterol homeostasis and HDL-mediated cholesterol efflux. Results showed that monensin and brefeldin A caused a redistribution of cell cholesterol from the plasma membrane to intracellular compartments, and inhibited efflux of cholesterol available for removal by HDL. These compounds are known to alter the structure and function of the Golgi apparatus, and thus may implicate this intracellular organelle in cholesterol transport pathways influenced by HDL.
To label cell cholesterol pools to
constant specific activity, subconfluent cultures were maintained in
DMEM containing 10% FBS and 0.2 µCi/ml
[H]cholesterol until confluent (usually 3 days).
Labeled cells were subsequently loaded with non-lipoprotein cholesterol
by incubation with DMEM containing 2 mg/ml fatty acid free bovine serum
albumin (BSA) and 30 µg/ml cholesterol (added from an ethanol stock
solution) for 24 h. Cultures were incubated for an additional 48 h in
DMEM containing 1 mg/ml BSA, after which free and esterified pools of
cholesterol attained constant specific activity (data not shown). Cells
were also enriched with cholesterol by incubation with LDL.
Subconfluent cultures were incubated with DMEM containing 2%
lipoprotein-deficient FBS (LPDS, prepared by ultracentrifugation of FBS
at density 1.25 g/ml and adjusted to equal protein content as FBS)
containing 100 µg/ml LDL protein. After 48 h, by which time cells
reached confluence, cells were rinsed with phosphate buffered saline
(PBS) and incubated for an additional 24 h in DMEM containing 1 mg/ml
BSA to allow equilibration of cholesterol pools. During incubation of
cells with test compounds, vehicle alone (ethanol) was added to control
dishes at equal concentrations (never exceeding 0.25%).
The dose response of
monensin and brefeldin A on cholesterol esterification was examined (Fig. 1). Increasing concentrations of monensin or brefeldin A
had no effect on the basal rate of cholesterol esterification compared
with controls during the 6-h incubation. In contrast, when HDL was
present, monensin and brefeldin A inhibited the ability of HDL to
decrease cellular cholesterol esterification. The effects of monensin
were apparent at the lowest concentration tested and increased with
dose, completely blocking the decrease in cholesterol esterification by
HDL at 50 µM. Brefeldin A also prevented the decrease of
cholesterol esterification by HDL at the lowest dose examined (0.2
µM) without any further effect up to 36 µM,
inhibiting up to 70% of the decrease in cholesterol esterification by
HDL obtained in the absence of brefeldin A. The dose response of HDL on
cell cholesterol esterification in the presence or absence of these
compounds was examined (Fig. 2). Under control conditions, HDL
decreased ACAT activity in a concentration dependent manner. When
monensin or brefeldin A was present, the decrease in cholesterol
esterification by HDL was prevented at nearly all concentrations.
Cholesterol esterification was inhibited an average of 56 ± 13%
and 63 ± 9% for monensin and brefeldin A, respectively, over the
tested concentration range. Removal of these compounds by washing the
cells restored the ability of HDL to deplete ACAT substrate to control
levels, demonstrating the effects of these compounds were reversible
(data not shown). In these and other experiments, neither monensin or
brefeldin A showed any cytotoxic effects compared with control cells
based on recovery of cell protein, lactate dehydrogenase release to the
medium, trypan blue dye exclusion, or incorporation of
[C]oleate into phospholipids (not shown).
Monensin, nigericin, and brefeldin A had similar effects on
HDL-mediated ACAT inhibition when tested in porcine smooth muscle cells
(data not shown), with the notable exception that monensin and
nigericin decreased basal esterification rates by about 25% compared
with controls.
Figure 1:
Dose
response of monensin and brefeldin A on cholesterol esterification in
cholesterol-loaded fibroblasts in the presence or absence of HDL. Human
skin fibroblasts were grown to confluence then loaded with
non-lipoprotein cholesterol as described under ``Methods.''
Cultures were then incubated in serum-free DMEM containing 1 mg/ml BSA
(, SFM) alone or with 50 µg/ml HDL protein (
, +HDL) and the indicated concentrations of monensin or
brefeldin A for 6 h at 37 °C. Cells were rinsed with PBS and
incubated in DMEM containing 9 µM [
C]oleate and 3 µM BSA for 1 h
to measure cholesterol esterification as described under
``Methods.'' Results are expressed as picomoles of
[
C]oleate incorporated into
[
C]cholesterol esters/mg of cell protein. In A results are the mean ± S.D. of three dishes, and in B results are the mean of duplicate dishes, and the
coefficient of variation was 7.8%.
Figure 2:
Effect of monensin and brefeldin A on the
decrease in cholesterol esterification by HDL in cholesterol-loaded
fibroblasts. Human skin fibroblasts were grown and enriched with
cholesterol as described in the legend to Fig. 1. Cultures were
then incubated in serum-free DMEM containing 1 mg/ml BSA alone, 25
µM monensin, or 3.6 µM brefeldin A and the
indicated concentrations of HDL for 16 h at 37 °C. Cholesterol
esterification was subsequently measured as described in the legend to Fig. 1and expressed as picomoles of
[C]oleate incorporated into
[
C]cholesterol esters/mg of cell protein. A:
, control;
, 25 µM monensin.
Results are the mean of duplicate dishes, and the coefficient of
variation was 5.6%. B:
, control;
, 3.6 µM brefeldin A. Results are the mean ± S.D. of three dishes,
and missing error bars are contained within the
symbols.
The time that monensin addition would inhibit the HDL-mediated decrease in ACAT activity was studied (Fig. 3). Addition of monensin at the start of the incubation or after 1 h maximally inhibited the decrease in cholesterol esterification due to HDL (70% of control values). Addition of monensin after 2 or 3 h showed partial inhibition (46 and 28%, respectively), and if monensin was added during the last hour of incubation with HDL, no effects were observed. Monensin had no statistically significant effect on the basal rate of cholesterol esterification in cultures incubated without HDL. These results show that monensin can effectively block HDL-mediated ACAT inhibition even after stimulation of this process had already begun.
Figure 3:
Effect of addition time on the ability of
monensin to prevent HDL-mediated decrease in cholesterol esterification
in cholesterol-loaded human skin fibroblasts. Fibroblast cultures were
grown and enriched with cholesterol as described in the legend to Fig. 1. Cultures were then incubated in serum-free DMEM
containing 1 mg/ml BSA (, SFM) alone or containing 50
µg/ml HDL protein (
, HDL) for a total of 6 h at 37
°C. Monensin was added to a final concentration of 25 µM at the indicated times. After incubation cells were incubated in
DMEM containing [
C]oleate as described in the
legend to Fig. 1. Results are expressed as picomoles of
[
C]oleate incorporated into
[
C]cholesterol esters/mg of cell protein and are
the mean ± S.D. of three dishes, missing error bars are
contained within the symbols. The asterisk indicates p < 0.05 compared with control incubations containing
HDL.
Cholesterol esterification in cells depleted of cholesterol and the ability of LDL to increase cholesterol esterification rates were examined after incubating cells with monensin, nigericin, or brefeldin A (Table 2). In cholesterol-depleted cells, HDL had a limited capacity to decrease cholesterol esterification (<10% compared with control medium) and was not examined. When ACAT substrate was limited, monensin and nigericin decreased cholesterol esterification by about 50%, suggesting that treatment of cells with these drugs diverts cholesterol out of the pool available for esterification. Brefeldin A increased esterification by 1.4-fold, possibly by increasing the availability of cholesterol to ACAT. Inclusion of LDL during the incubation caused a 6-fold increase in cholesterol esterification. As expected, monensin and nigericin blocked the uptake of LDL derived cholesterol(31) . Brefeldin A-treated cells processed LDL-derived cholesterol to a similar extent as control cells, suggesting that brefeldin A did not alter the uptake and lysosomal processing of LDL-cholesterol to sites of ACAT-mediated esterification under these conditions.
Figure 4:
Effects
of monensin and brefeldin A on stimulation of sterol synthesis by HDL
in cholesterol-loaded human skin fibroblasts. Preconfluent fibroblast
cultures were incubated for 48 h with DMEM containing 2% LPDS and 100
µg/ml LDL protein to enrich cholesterol pools followed by
incubation for 24 h in DMEM containing 1 mg/ml BSA to allow
equilibration of cholesterol pools. Cultures were incubated with DMEM
containing 1 mg/ml BSA and the indicated concentrations of HDL protein
alone (, Control), with 4 µM brefeldin A
(
) or 25 µM monensin (
) for 24 h at 37 °C.
Cells were then rinsed with PBS and incubated with DMEM containing 2
µCi/ml [
C]acetate for 2 h at 37 °C. Cell
lipids were extracted and saponified as described under
``Methods,'' and incorporation of radioactivity into sterols
was measured after TLC separation of the non-saponified lipid fraction.
Results are expressed as picomoles of [
C]acetate
incorporated into [
C]sterols/mg of cell protein
and are the mean ± S.D. of three dishes. Missing error bars are contained within the symbols.
Figure 5:
Effects of monensin and brefeldin A on
HDL-mediated cholesterol efflux and cell cholesterol distribution
examined with cholesterol oxidase in cholesterol-loaded human skin
fibroblasts. Fibroblast cultures were labeled with
[H]cholesterol then loaded with non-lipoprotein
cholesterol as described under ``Methods.'' Cells were then
incubated with DMEM containing 1 mg/ml BSA and the indicated
concentrations of HDL protein alone (
, Control), with 4
µM brefeldin A (
, BFA) or 25 µM monensin (
, Mon) for 24 h at 37 °C. After
incubation, efflux medium was collected, and cells were treated with
cholesterol oxidase as described under ``Methods.'' Cell
lipids were extracted and separated by TLC to isolate cholesterol,
cholestenone (the cholesterol oxidase product), and cholesterol esters.
Results were calculated as the percent of
H in each
fraction relative total
H. A, efflux. B,
oxidase-resistant cholesterol. C, oxidase-accessible
cholesterol. D, cholesterol ester. Results are the means of
duplicate incubations, representative of at least three experiments for
each compound tested. Total
H recovered was (mean ±
S.D., n = 21) 55,137 ± 2384 cpm/dish, and there
were no differences between groups.
Since these agents both affected cell
[H]cholesterol esters, although in opposite
directions, we examined whether the observed effects on efflux and cell
distribution were due to these changes. A similar experiment as above
was conducted in the presence of an ACAT inhibitor (Table 3).
Monensin and brefeldin A induced qualitatively similar changes in cell
distribution and HDL-mediated efflux of cholesterol under these
conditions. Inhibition of ACAT increased cholesterol oxidase-accessible
[
H]cholesterol, with no appreciable change in
oxidase-resistant cholesterol in control cells. Addition of monensin
increased oxidase-resistant cholesterol similar to results without ACAT
inhibition, whereas a small increase (from 10 to 14%) occurred in
brefeldin A-treated cells that was not apparent without ACAT
inhibition. These data suggest that inhibition of cholesterol efflux to
HDL by these agents was independent of changes in the cholesterol ester
content of cells.
Effects of monensin, nigericin, and brefeldin A on
cholesterol efflux to HDL and cholesterol oxidase sensitivity were
tested in porcine smooth muscle cells. Interestingly, and different
from what was observed in fibroblasts, monensin and nigericin
significantly increased efflux from cells to HDL-free medium (from 2.1%
for controls to 9.4 and 10.5% for monensin and nigericin), although the
reason for this has not been explored further. Efflux of cell
[H]cholesterol to HDL was significantly inhibited
(by approximately 40%) by all three compounds, and changes in cell
cholesterol distribution were similar to those observed in fibroblasts
for monensin and nigericin (data not shown). Brefeldin A increased
oxidase-resistant and esterified cholesterol with a concomitant
decrease in oxidase-accessible cholesterol in smooth muscle cells (data
not shown), although the increase in oxidase-resistant sterol was less
than observed for monensin and nigericin. Thus, the effects of these
drugs on HDL-mediated cholesterol efflux and cell distribution were not
identical in smooth muscle cells and fibroblasts and several
similarities were noted, suggesting that these drugs exert their
effects through common mechanisms in both experimental models.
The
time course of cell [H]cholesterol efflux and
cholesterol oxidase sensitivity of in the presence of absence of
monensin and brefeldin A were examined ( Fig. 6and Fig. 7). In both studies, control dishes incubated without HDL
showed no appreciable efflux of [
H]cholesterol to
medium (less than 2% of total cell radioactivity) and relatively no
change among the various pools of cholesterol over the times examined,
suggesting that these pools were in isotopic equilibrium. Efflux of
cholesterol to medium containing HDL occurred in a time-dependent
manner ( Fig. 6and Fig. 7). The majority of
[
H]cholesterol efflux was accounted for by
depletion of cholesterol oxidase-accessible
[
H]cholesterol, especially at earlier times, and
to depletion of cell [
H]cholesterol esters, most
obvious after 16 h. In these studies, similar to data in Fig. 5,
HDL did not influence the levels of oxidase-resistant
[
H]cholesterol in control incubations. When
monensin was present, [
H]cholesterol efflux to
HDL was similar to controls after 2 h, but decreased at all other times (Fig. 6). Inhibition of HDL-mediated efflux was maximal by 6 h
(39% inhibition compared with controls) and similarly inhibited after 8
and 16 h (38 and 39%, respectively). Monensin decreased cholesterol
oxidase-accessible sterol at all times, attaining a new basal level
after 6 h (coincident with the maximal decrease in HDL-mediated efflux
in monensin-treated cells) and paralleled by an increase in oxidase
resistant [
H]cholesterol.
[
H]Cholesterol esters decreased over time,
relative to controls, also contributing to the increase in
oxidase-resistant [
H]cholesterol. Similar changes
were observed when HDL and monensin were present together, except for a
greater decrease in the cholesterol oxidase-accessible pool accounted
for by [
H]cholesterol appearing in medium. HDL
was unable to decrease [
H]cholesterol esters in
monensin-treated cells. As observed previously, efflux of cell
cholesterol to HDL in the presence of monensin was limited to removal
of plasma membrane (i.e. cholesterol oxidase-accessible)
cholesterol.
Figure 6:
Effect of monensin on the time course of
HDL-mediated efflux and cell distribution of
[H]cholesterol in cholesterol-loaded human skin
fibroblasts. Fibroblast cultures were labeled and enriched with
cholesterol as described in the legend to Fig. 5. Cultures were
incubated with DMEM containing 1 mg/ml BSA alone (
), 50 µg/ml
HDL (
), 25 µM monensin (
), or 25 µM monensin and 50 µg/ml HDL (
) for the indicated times.
After incubation, medium was collected and cells were treated with
cholesterol oxidase and results calculated as described in the legend
to Fig. 5. A, efflux. B, oxidase-resistant
cholesterol. C, oxidase-accessible cholesterol. D,
cholesterol esters. Results are the means of duplicate incubations,
representative of at least three experiments for each compound tested.
Total
H recovered was (mean ± S.D., n = 42) 31,485 ± 1,401 cpm/dish, and there were no
differences between groups.
Figure 7:
Effect of brefeldin A on the time course
of HDL-mediated efflux and cell distribution of
[H]cholesterol in cholesterol-loaded human skin
fibroblasts. Fibroblast cultures were labeled and enriched with
cholesterol as described in the legend to Fig. 5. Cultures were
incubated with DMEM containing 1 mg/ml BSA alone (
), 50 µg/ml
HDL (
), 4 µM brefeldin A (
), or 4 µM brefeldin A and 50 µg/ml HDL (
) for the indicated
times. After incubation, medium was collected, and cells were treated
with cholesterol oxidase and results calculated as described in the
legend to Fig. 6. A, efflux. B,
oxidase-resistant cholesterol. C, oxidase-accessible
cholesterol. D, cholesterol esters. Results are the means of
duplicate incubations, representative of at least three experiments for
each compound tested. Total
H recovered was (mean ±
S.D., n = 42) 56,453 ± 2,847 cpm/dish, and there
were no differences between groups.
When brefeldin A was included during incubation with
cells (Fig. 7), [H]cholesterol efflux to
medium containing HDL was not appreciably different from controls until
6 h of incubation (12% inhibition); approximately 30% inhibition was
seen at 6 and 8 h, and maximum inhibition of HDL-mediated efflux
occurred at 16 h (46% decrease compared with control). The effects of
brefeldin A on cholesterol oxidase sensitivity of cell
[
H]cholesterol was again different from changes
observed with monensin. Brefeldin A caused a transient decrease in
cholesterol oxidase accessible sterol, paralleled by a rise in the
oxidase-resistant pool, which returned to near control levels by 8 h.
This trend was not apparent in the presence of brefeldin A and HDL
together, which did not demonstrate any measurable differences relative
to control incubations containing HDL in either the cholesterol
oxidase-accessible or -resistant pools. These data also demonstrate
that brefeldin A did not alter efflux of plasma membrane
(oxidase-accessible) cholesterol to HDL. In brefeldin A-treated cells
[
H]cholesterol esters were slightly increased
over time relative to controls, apparent after 6 h and continuing to
increase during the course of the experiment. When HDL was also
present, this increase was attenuated, but when compared with control
incubations with HDL, brefeldin A blocked HDL-mediated depletion of
[
H]cholesterol esters.
Monensin, nigericin, and brefeldin A blocked HDL-mediated efflux of intracellular cholesterol based on the following observations. First, these compounds prevented HDL from depleting the substrate pool of cholesterol available for esterification by ACAT. Since the enzyme resides within the endoplasmic reticulum(33) , the cholesterol substrate must be available to the same intracellular site. Second, monensin and brefeldin A prevented HDL to increase sterol biosynthesis compared with control cells. Third, treatment with monensin, nigericin, and brefeldin A inhibited HDL-mediated efflux of radiolabeled cholesterol and cholesterol mass from cholesterol-enriched cells.
We propose the following model for cholesterol flux through the cell (Fig. 8), making the following assumptions. First, cholesterol esters and oxidase-resistant cholesterol reside within intracellular compartments. Second, oxidase-sensitive cholesterol resides in the plasma membrane. Third, cholesterol esters participate in continuous hydrolysis and re-esterification (i.e. the cholesterol ester cycle). Fourth, cholesterol removed from cells is unesterified and passes through the plasma membrane before removal by an extracellular acceptor. In control cells overloaded with cholesterol, and incubated without an exogenous cholesterol or acceptors, a steady state is attained in which cholesterol oxidase-sensitive, -resistant and -esterified cholesterol remain at constant levels. These pools may be in equilibrium, and exchange among these pools may occur (indicated by reversible arrows, Fig. 8A). Results in cells incubated with an ACAT inhibitor indicate that hydrolyzed cholesterol esters transport to the plasma membrane without accumulating in the oxidase-resistant pool. When HDL is present, efflux is due to removal of plasma membrane (pathway a) and esterified cholesterol pools (pathway b). Decreases in cell cholesterol esters occur after hydrolysis, followed by transport and then uptake of free cholesterol from the plasma membrane. The oxidase-resistant cholesterol pool remains unchanged after incubation with HDL, suggesting that this pool of cholesterol is not available for efflux to HDL or, if depleted, is rapidly replenished by other cholesterol pools. Cholesterol ester hydrolysis does not cause the accumulation of free cholesterol in the oxidase-resistant or -sensitive pools, suggesting that transport of hydrolyzed cholesterol, once stimulated by appropriate extracellular acceptors, is not rate-limiting and rapidly removed from cells.
Figure 8: Pathways of cellular cholesterol transport and efflux. Potential pathways involved in cellular cholesterol transport and efflux to HDL in cholesterol-loaded cells and effects of monensin and brefeldin A. A, control conditions. B, monensin-treated cells. C, brefeldin A-treated cells. C, unesterified cholesterol; CE, cholesterol esters.
Monensin treatment of cells caused a redistribution of cell cholesterol (Fig. 8B). Oxidase-sensitive and -esterified cholesterol pools decrease, resulting in an increase in the cholesterol oxidase-resistant pool, and all pools attain an apparent new steady state over time. Re-distribution of cell cholesterol by monensin had little or no effect on the extent of plasma membrane cholesterol depletion by HDL (pathway a), in spite of a decrease in the size of this pool. In contrast, monensin blocks the ability of HDL to deplete intracellular cholesterol ester pools (pathway b), and similar to control conditions, the oxidase-resistant cholesterol pool is not available for efflux to HDL. Thus, monensin causes the redistribution of cholesterol into intercellular compartments and blocks transport to sites available for efflux to HDL.
Brefeldin A treatment of cells had different effects
on cell cholesterol distribution than monensin. This compound caused a
slight increase in the cell cholesterol ester pool due to a decrease in
the oxidase-sensitive cholesterol pool (most notable in
cholesterol-depleted cells). Efflux of plasma membrane cholesterol to
HDL (pathway a) was comparable with control conditions.
However, HDL could not deplete cholesterol ester pools (pathway
b) in brefeldin A-treated cells, suggesting that transport of
hydrolyzed cholesterol is blocked, and this pool of cholesterol was
then efficiently re-esterified. Cholesterol ester hydrolysis rates were
the same in brefeldin A-treated and controls cells, ()implying that changes in cholesterol ester hydrolysis did
not cause the observed effects.
Based on this model, we suggest that HDL promotes cholesterol efflux from cells by two distinct pathways. Efflux of cholesterol from the plasma membrane (oxidase-sensitive) pool probably occurs by desorption and diffusion of cholesterol already present in this compartment, and some of the efflux from this pool may represent exchange between the cell and lipoprotein(2, 7) . Cholesterol efflux from cells by this mechanism does not depend on a functional and intact Golgi apparatus. A second pathway must also exist for the transport of intracellular cholesterol, derived from the hydrolysis of cholesterol esters, by a pathway that requires an intact and functional Golgi apparatus, revealed by sensitivity to monensin and brefeldin A. This pathway promotes cholesterol efflux in addition to efflux from the plasma membrane, and transport from intracellular sites to extracellular acceptors is rapid without causing the accumulation of cholesterol within any cellular pools.
Monensin affects the trans-cisternae of the Golgi apparatus in those regions primarily associated with the final stages of secretory vesicle maturation and in post-Golgi structures associated with endocytosis and membrane/product sorting (29) and preventing secretory vesicle production(33) . These effects of monensin have been used as one criterion for verifying passage of molecules through the Golgi apparatus ( (29) and references therein). These processes may account for the observed effects of monensin on cell cholesterol transport. Thus, monensin treatment of cells may prevent antegrade transport of cholesterol through the Golgi apparatus for delivery to the plasma membrane. Additionally, monensin may prevent cholesterol transport from the trans-Golgi region into the endoplasmic reticulum, effectively blocking cholesterol entry into the cholesteryl ester cycle. Retrograde transport of plasma membrane cholesterol to other intracellular sites may continue, but might accumulate at those sites if antegrade transport back to the plasma membrane depends on a functional trans-Golgi network.
Brefeldin A causes the disassembly of the Golgi apparatus, primarily the cis- and medial Golgi cisternae(30) , in contrast to monensin that primarily affects the trans-Golgi cisternae. The cis- and medial Golgi membranes redistribute with the endoplasmic reticulum, whereas components of the trans-Golgi network do not(30) . Retrograde transport of cis- and medial Golgi membrane back to the endoplasmic reticulum by the action of brefeldin A would maintain or increase substrate pools of cholesterol available for esterification. Disassembly of the Golgi apparatus would effectively block antegrade transport of cholesterol derived from hydrolysis of esters, resulting in the inability of cells to become depleted of intracellular cholesterol pools by efflux to an acceptor particle. However, cholesterol transport into cells and back to the plasma membrane at sites distal to the cis- and medial Golgi apparatus may not be affected by brefeldin A.
Previous studies have examined the effects of brefeldin A on cholesterol homeostasis in cultured cells. Stein et al.(34) showed that brefeldin A increased cholesterol esterification rates in cultured cells, attributed to increased substrate availability to sites of esterification due to the collapse of the Golgi apparatus into the endoplasmic reticulum without a direct effect on ACAT activity. Results from Hasumi et al.(35) using cultured macrophage cell lines showed that brefeldin A increased cell cholesteryl esters, at the expense of free cholesterol, but without affecting sterol biosynthetic rates. These authors reported that brefeldin A did not alter cholesterol oxidase sensitivity of cell cholesterol, however, without showing data. Neither of these studies examined the effects of brefeldin A on cholesterol transport to extracellular acceptors. More recently, Azhar et al.(37) demonstrated that okadaic acid prevented steroid hormone production from cholesterol in cultured cells and suggested this could result from the effect of this compound on disruption of the Golgi complex structure, implicating this organelle in providing cholesterol substrate to the mitochondria for steroid hormone production. Simoni and colleagues examined the effects of monensin (37) and brefeldin A (21) on the transport of newly synthesized cholesterol from intracellular sites of biosynthesis to the plasma membrane in Chinese hamster ovary cells. These studies did not demonstrate any effect on cholesterol transport by these compounds. Although these results were different to those of the present study, this may be accounted for by different pathways involved in transport of newly synthesized cholesterol in growing, non-cholesterol-loaded cells compared with transport of excess cholesterol in quiescent, cholesterol-loaded cells. These authors also showed that newly synthesized cholesterol was transported by a unique class of low density vesicles, apparently a post-endoplasmic reticulum intermediate (21, 37) , consistent with the idea that newly synthesized sterols are transported by novel vesicular transport pathways that bypass the Golgi apparatus. An additional caveat to those studies is that most of the newly synthesized sterol labeled during short pulse incubations are not cholesterol, but more polar sterol precursors(36) . Whether such molecules are transported in an identical manner as authentic cholesterol has not yet been addressed in that experimental system.
Cholesterol has been shown to accumulate
in membranes enriched with sphingomyelin(39, 40) . As
suggested by Shiao and Vance(41) , one possibility is that
these lipids travel together in specialized vesicles to the plasma
membrane. Newly synthesized sphingomyelin transport to the plasma
membrane of hepatocytes was not affected by brefeldin A or
monensin(41) , similar to effects of these compounds on
transport of newly synthesized sterols(21, 37) .
Brefeldin A also failed to prevent sphingomyelin transport in
CaCO cells, although a redistribution of sphingomyelin
between the apical and basolateral membranes did occur(42) .
Based on those results and the present data, one would conclude that
sphingomyelin and newly synthesized cholesterol are transported by
Golgi-independent pathways (21, 37, 41, 42) distinct from
transport of excess intracellular cholesterol that appears to be
dependent on a functional Golgi complex.
In contrast, other studies have shown that brefeldin A and monensin block cellular transport of newly synthesized sphingomyelin(43, 44, 45) . Brefeldin A had no effects on the steady state levels of sphingomyelin mass, but reduced the proportion of plasma membrane sphingomyelin by 25% in baby hamster kidney cells(43) . In the same cell type, monensin stimulated degradation of plasma membrane sphingolipid and inhibited transport of newly synthesized sphingomyelin to the plasma membrane (44) . Monensin also inhibited transport of a fluorescent sphingolipid analog to the plasma membrane of Chinese hamster ovary cells(45) . Thus, depletion of plasma membrane sphingomyelin by monensin or brefeldin A may stimulate the internalization of membrane cholesterol. This concept is supported by studies of Slotte et al. (46, 47) demonstrating that degradation of plasma membrane sphingomyelin with exogenous sphingomyelinase promoted transport of cholesterol to intracellular compartments. These results suggest that plasma membrane sphingomyelin content directly influences cholesterol distribution. Internalization or degradation of plasma membrane sphingomyelin and intracellular accumulation, induced by brefeldin A or monensin, may result in the sequestration of cholesterol within those sphingomyelin-enriched membranes, and such effects may occur even if transport of these lipids occurs by different pathways. The relationship between sphingomyelin and cholesterol transport, especially under conditions of excess cholesterol accumulation and removal, deserve further investigation.
Pathways of intracellular cholesterol transport and their role in removal of excess cholesterol to extracellular acceptors remain incompletely understood. We have shown that agents that disrupt Golgi apparatus structure and function prevent efficient cholesterol removal by HDL and alter cellular cholesterol distribution. Such evidence strongly suggests a role for the Golgi apparatus in maintaining cell cholesterol distribution and in transport from intracellular sites to the plasma membrane for eventual removal. These studies did not directly address the mechanisms by which HDL promotes cholesterol efflux, such as by desorption and diffusion events (2, 3) or interactions with cell surface binding sites(13, 17, 18) , instead were designed to explore cellular pathways involved in cholesterol transport during efflux to an appropriate cholesterol acceptor, in this case HDL. The Golgi apparatus is composed of multiple subcompartments with continuous membrane exchange occurring between them(48) . The Golgi apparatus contains substantial amounts of cholesterol, and there is evidence for a cholesterol gradient in the cis to trans direction(49) . One may speculate that cholesterol is transported from the cis- to trans-Golgi (or antegrade transport) and possibly accumulates within trans-Golgi vesicles until the plasma membrane can accommodate more cholesterol, such as after depletion of plasma membrane cholesterol by appropriate acceptors or stimulation by signaling molecules(17, 18, 19, 20) . Alternatively, there may be continuous recycling of cholesterol between the plasma membrane and the Golgi complex, such that a ``transport equilibrium'' exists between these two membrane systems and possibly between the Golgi apparatus and other intracellular compartments such as the endoplasmic reticulum or mitochondria. Removal of cholesterol from the plasma membrane by acceptors would shift the equilibrium to deliver more cholesterol from Golgi membranes to the plasma membrane. Influx of cholesterol, which initially accumulates in the plasma membrane(10, 11, 12) , would shift the equilibrium to deliver more cholesterol to the Golgi apparatus and eventually transport cholesterol to the endoplasmic reticulum for esterification and storage. In either scheme the Golgi apparatus would be involved in various aspects of cellular cholesterol transport and targeting, not only to the plasma membrane but also to other sites of cholesterol utilization or storage. Further studies are needed to examine the possible steps in Golgi-mediated intracellular cholesterol transport, and elucidation of these pathways will increase our understanding of the mechanisms involved in excess cholesterol accumulation and removal from cells.