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INTRODUCTION |
Although the
HDL-mediated1 steps of
cholesterol transfer from the cell surface membrane and subsequent fate
of cholesterol in the vasculature have been extensively studied, much
less is known about intracellular components of cholesterol efflux
(reviewed in Refs. 1-5). Plasma membrane cholesterol is distributed
into multiple pools or domains (reviewed in Refs. 6 and 7). It is now
recognized that there may be a connection between such domains and HDL
receptor-mediated reverse cholesterol transport (reviewed in Refs.
7-9). The transbilayer distribution of cholesterol in plasma membranes
is asymmetric, with the cholesterol enriched 400% in the cytofacial
leaflet versus exofacial leaflet (reviewed in Refs. 10-14).
Transbilayer movement of cholesterol across the plasma membrane appears
fast (t1/2 = 1-6 min; reviewed in Ref. 10). Plasma
membrane cholesterol is also distributed into lateral cholesterol-rich
and -poor membrane domains (reviewed in Refs. 6 and 10). Most of the
cholesterol in the plasma membrane is localized in lateral domains that
are, for the most part, relatively inert in terms of transfer kinetics (i.e. t1/2 = hours to days), and movement
between such domains is also slow. However, a small pool of plasma
membrane cholesterol appears highly dynamic (reviewed in Ref. 6) and is
associated with cholesterol-rich, HDL receptor containing microdomains
called caveolae (reviewed in Refs. 8, 9, 15, and 16). Molecular details
of cholesterol entry/exit, cholesterol organization, and mechanism(s)
of cholesterol transbilayer movement in caveolae remain to be
determined. Likewise, the relationships between caveolae, "rafts,"
and other cholesterol-rich plasma membrane microdomains is not yet
clear (reviewed in Ref. 9).
The intracellular steps preceding cellular cholesterol efflux include
transfer of cholesterol from the Golgi, endoplasmic reticulum, and
lipid droplets to the plasma membrane (reviewed in Refs. 1, 9, and 17).
The time frame of bidirectional vesicular transfer of cholesterol
between plasma membranes and Golgi has a t1/2 of
10-20 min (reviewed in Refs. 8, 18, and 19). Alternately, molecular
cholesterol transfer, mediated by cholesterol-binding proteins in the
cytoplasm, occurs much faster (t1/2 near 1-2 min)
from the lysosome (exogenous cholesterol) to the plasma membrane (20,
21) and from the endoplasmic reticulum (newly synthesized cholesterol)
to the plasma membrane (reviewed in Refs. 7, 8, and 22). Which one (or
both) of the processes participate in cholesterol trafficking from
lipid droplets, a primary site of cholesterol storage, is presently unknown.
The purpose of this investigation was to visualize three aspects of
cholesterol efflux in intact cells. (i) To determine the role of SCP-2
in HDL-mediated cholesterol efflux. Previously it was shown that SCP-2
stimulates biliary cholesterol secretion (23-25) but inhibits
lipoprotein secretion (22). (ii) To address the lack of information
regarding the lipid droplet as a source for cholesterol efflux via the
HDL pathway. Lipid droplets contain a surface coat of cholesterol and
phospholipid surrounding a cholesterol ester/triglyceride core (26,
27). Further, the lipid droplet surface monolayer contains unique
proteins including perilipin and adipose differentiation-related
protein (ADRP), neither of whose function is well understood (28).
Because ADRP binds cholesterol, this suggests a potential role of this
protein in cholesterol trafficking from the lipid droplet (29). (iii)
To examine the effect of SCP-2 expression on HDL-mediated cholesterol
efflux from the lipid droplet. This possibility was suggested by the fact that SCP-2 stimulates cholesterol transfer from lipid droplets to
mitochondria in adrenal cells (reviewed in Ref. 27). The results
presented herein provide fresh insights into the efflux process and for
the first time establish new information demonstrating that SCP-2
expression: (i) inhibits HDL-mediated cholesterol efflux and (ii)
inhibits cholesterol efflux from a little understood, subcellular
compartment, i.e. lipid droplets.
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EXPERIMENTAL PROCEDURES |
Materials--
[3H]Cholesterol was from
PerkinElmer Life Sciences. Bovine serum albumin (essentially fatty acid
free) and fetal bovine serum (FBS) were from Sigma. HDL was from
Calbiochem (San Diego, CA). Lab-Tek Coverglass slides were from Fisher.
NBD-cholesterol and Nile red were from Molecular Probes (Eugene, OR).
Sandoz 58-035 was a gift from T. Y. Chang (Dartmouth Medical
School, Hanover, NH). Rabbit polyclonal antisera to ADRP were prepared
(30) using ADRP generously provided by Dr. G. Serrero (University of
Maryland, Baltimore, MD). Monoclonal anti-caveolin-1 was from
Transduction Laboratories (Lexington, KY). Rabbit polyclonal
anti-scavenger receptor B1 antisera were from Novus Biologicals
(Littleton, CO). All reagents and solvents used were of the highest
grade available and were cell culture tested.
L-cell Culture--
Murine L-cells (L
arpt
tk) were grown to confluency in Higuchi
medium (31) supplemented with 10% fetal bovine serum (Hyclone, Logan,
UT) as described (30). Development of mock-transfected cells
(designated as control) and cells transfected with cDNA corresponding to the 15-kDa pro-SCP-2 protein was as described earlier
(30, 32). In high expression cells, SCP-2 comprised 0.036 + 0.002% of
the total cytosolic proteins, whereas levels in the control and mock
transfected cells were below the level of detectability. Cellular
cholesterol and cholesterol ester mass were increased 20 and 100%,
respectively, in the SCP-2 expression clones. For radiolabeled
experiments, control and SCP-2 expressors were plated to 24-well plates
at approximately 5 × 105 cells/well. For fluorescence
imaging experiments, cells were seeded at a density of 50,000 cells/chamber onto Lab-Tek Chamber Coverglass slides (Nunc, Naperville,
IL). To ensure a monolayer, samples analyzed by imaging were examined
within 20 h of seeding. Cells for radiolabeled experiments were
treated as described below.
Measurement of Cellular [3H]Cholesterol
Efflux--
Cholesterol efflux was done as described earlier (33).
Briefly, mock-transfected and SCP-2 expressors were plated in medium containing 1 µCi/ml of [3H]cholesterol and 2.5% fetal
bovine serum (1 ml/well) overnight. The medium was then replaced with
medium containing 0.3% bovine serum albumin to allow equilibration
between different cholesterol pools. After 24 h, cells were washed
twice with phosphate-buffered saline. HDL at indicated concentrations
was then added, and radiolabeled aliquots (50 µl) were removed at
indicated time points and counted. At the end of the time course, the
monolayers were washed three times with phosphate-buffered saline,
cells were dissolved in 0.1 N NaOH (500 µl) overnight,
and protein was determined (34). Unless otherwise stated, Sandoz 58-035 was included during labeling and equilibration to eliminate competition
by intracellular [3H]cholesterol esterification. Extra
wells without acceptor were harvested at the beginning of the time
course to determine the initial [3H]cholesterol for each
cell line.
Efflux was determined from the fraction of
[3H]cholesterol remaining in the cell at different time
points. Data points were fitted to the single exponential decay curve
y = Aekt + C, where
y is the fraction of initial cellular
[3H]cholesterol remaining in the cells at time
t; A is the cholesterol pool size available for
efflux; and k is the apparent rate constant. Half-times
(t1/2) were determined from the equation
t1/2 = ln2/k. Half-time and k values were
apparent values because of dependence on HDL acceptor concentrations.
Laser Scanning Confocal Microscopy--
Confocal fluorescence
imaging was performed on a Bio-Rad MRC-1024 Laser Scanning Confocal
Imaging System. Confocal and co-localized images were acquired using
multiple photomultiplier tubes under the control of LaserSharp v.3.2
software (Bio-Rad). For probe excitation, the MRC-1024 system utilized
a 15 mW krypton-argon laser (American Laser Corp., Salt Lake City, UT)
with a 5 mW output. Cells cultured on coverslips were placed on the
stage of a Zeiss Axiovert 135 inverted epifluorescence microscope
(Zeiss, Thornwood, NY) and examined with a 63× oil immersion, infinity
objective (numerical aperture 1.4). Fluorescence emission was detected
using 488 nm excitation and a 540/32 band path emission filter for
NBD-cholesterol (green channel) and 568 nm excitation with a HQ598/40
band path emission filter for Nile red (red channel). Samples were
exposed to the light source for minimal time periods to minimize
photobleaching effects. Image files were analyzed using either
Metamorph software (West Chester, PA) or NIH Image, a program written
by W. Rasband and available by anonymous FTP.
NBD-cholesterol Efflux Measurement by Laser Scanning Confocal
Microscopy--
To perform the efflux experiments, cells were first
loaded with NBD-cholesterol (0.35 µM) in medium
containing 2.5% FBS for 1 h in a 37 °C CO2
incubator. Because only 7.8% of NBD-cholesterol is esterified at
24 h (29), the acyl-CoA:cholesterol O-acyltransferase inhibitor was not used for the fluorescence experiments for the comparatively short (40 min) incubation time used herein. After loading, the cells were washed twice with Puck's buffer (1 mM Na2HPO4, 0.9 mM
H2PO4, 5.0 mM KCl, 1.8 mM CaCl2, 0.6 mM MgSO4, 6 mM glucose, 138 mM NaCl, and 10 mM HEPES) and placed in serum-free medium. A medial section
passing through 5-10 cells was selected, and the section was scanned
for initial fluorescence intensity of NBD-cholesterol. HDL was added to
start the efflux experiment. The fluorescence in the total area of each
cell was measured over time and was used to gauge the extent of
NBD-cholesterol efflux from the cell.
NBD-cholesterol Co-localization with Lipid Droplets--
Three
types of co-localization studies were performed to determine the
identity of high intensity regions of NBD-cholesterol visible within
the cell. First, NBD-cholesterol was co-localized with Nile red, a
lipid stain. Second, Nile red was co-localized with ADRP, a protein
closely associated with lipid droplets. Third, NBD-cholesterol was
co-localized with ADRP. Cells overexpressing SCP-2 were incubated with
both NBD-cholesterol (0.35 µM) and Nile red (0.4 µM) for 30 min in a 37 °C CO2 incubator.
Although Nile red is known to stain other nonlipidic structures, care
was taken to titer both Nile red and NBD-cholesterol to low levels to
give a low cytoplasmic background with emphasis on lipid droplet
staining in the co-localization experiments. However, it should be
noted that Nile red and NBD-cholesterol are also located in sites
outside lipid droplets where they also co-localize to a large degree. After incubation, cells were washed with Puck's buffer and then simultaneously imaged for NBD-cholesterol (excitation, 488 nm; emission, 540/30; green channel) and Nile red (excitation, 568 nm;
emission, HQ598/40; red channel). The confocal images from the green
and red channels were merged and appeared yellow where superimposition
occurred (red and green are additive and yield yellow to orange in RGB
color space). Pixel fluorograms were constructed, and correlation
coefficients generated from the fluorograms were derived from the
following equations as described earlier (35).
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(Eq. 1)
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(Eq. 2)
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where Ri, coloc is the sum of
intensities of all red pixels which also have a green component;
Ri is the sum of intensities of all red pixels in
the image; Gi, coloc is the sum of
intensities of all green pixels, which also have a red component; and
Gi is sum of intensities of all the green pixels in
the image.
To co-localize ADRP with Nile red or with NBD-cholesterol, SCP-2
expressors were plated on 8-well Lab-Tek chamber slides (Nunc, Naperville, IL) at subconfluency. The cells were washed with
serum-free medium and incubated with 0.005% of either Nile red or
NBD-cholesterol in serum-free medium for 30 min, at 37 °C. Cells
were washed with Hanks' solution, fixed with 70:30 (v/v)
acetone:ethanol for 10 min at 4 °C, permeabilized with 0.05%
saponin in Hanks' for 5 min at room temperature, and then blocked with
10% FBS in Hanks' for 1 h at room temperature. Anti-ADRP (rabbit
polyclonal anti-mouse ADRP, affinity purified, concentrated to 0.5 mg/ml; dilution, 1:20) was incubated in 5% FBS in Hanks' for 1 h
at room temperature, followed by extensive washing with 2% FBS in
Hanks' buffer. Next, the cells were incubated with either goat
anti-rabbit IgG conjugated to fluorescein (for the ADRP/Nile red
co-localization) or goat anti-rabbit IgG conjugated to Texas red (for
the ADRP/NBD-cholesterol co-localization) for 1 h at room
temperature. After a final wash with Hanks', cells were mounted in
SlowFade (Molecular Probes) according to the manufacturer's procedure
and were visualized with the MRC1024 laser scanning system as described above.
NBD-cholesterol Efflux from Subcellular Compartments: Cytoplasm
and Lipid Droplets--
NBD-cholesterol efflux from the whole cell was
compared with that arising from two compartments designated as lipid
droplets (based on the above co-localization data) or cytoplasmic
(defined as the nonlipid droplet, lower intensity, diffuse pattern of
the remaining NBD-cholesterol fluorescence in the cell). Cells were imaged and analyzed by Metamorph software to graphically determine the
separate contributions of the efflux process. Data points were fitted
to either a biexponential (y = Aebt + Cedt) or multiparameter
(y = (AebT0 + CedT0)(eh(tT0))
exponential decay equation where y was the fraction of
initial cellular NBD-cholesterol remaining in the cells at time
t; A and C were the size of the
cholesterol pools available for efflux; b, d, and
h were the apparent rate constants; and
T0 was the time at which depletion of the pools
increased to completion. The multiparameter equation was employed to
adjust for an inflection point observed in the time course data
obtained from both control and SCP-2 overexpressors. It appeared that
at some time T0, the rate of efflux was altered, which effected a decrease in the half-life. To compare the data, a
model was developed that allowed for the initial decay curve (Aebt + Cedt) to be modified at some
T0 to become
(AebT0 + CedT0)
eh(tT0). Using a
nonlinear least squares routine in Sigma Plot 4.0, all parameters were
obtained simultaneously from the fitted data. Unique fittings were
obtained individually for each time course involving individual cells.
This produced excellent results for the cytoplasmic compartment
contribution from the cell. Statistical methods involving the paired
t test were used to compare the control and SCP-2
expressors. However, the lipid droplet contribution was quite
"noisy," and an inflection point could not be determined for these
data, which were subsequently fit to the equation
Aebt + Cedt for
all times. This also contributed some complication in determining the
inflection point of the whole cell because it is the combination of
lipid droplet plus cytoplasm. To overcome this difficulty, the time
courses of n individual cells for the specified
concentration and cell type were averaged to produce a single time
course, which was then fitted to the multiparameter equation.
Isolation of Mouse Lipid Droplets from L-cell
Fibroblasts--
Lipid droplets from control cells and SCP-2
expressors were isolated as described by Chanderbhan et al.
(36). Briefly, cells scraped from 10 confluent trays (245 × 245 × 25 mm each; Nunc, Naperville, IL) were homogenized in 50 mM NaH2P04 buffer, pH. 7.4, containing 154 mM NaCl and 5 mM
MgCl2 and centrifuged at 800 × g for 10 min. The supernatant was then centrifuged at 5,000 × g
for 20 min to sediment mitochondria. This was followed by centrifuging
at 35,000 rpm in a SW 40.1 rotor for 2 h at 4 °C. The lipid
droplet fraction, forming a distinct, white band on the surface of the
preparation, was removed, and protein was quantified (34).
Quantitative Western Blotting for ADRP, Caveolin, and
SRB1--
Lipid droplets and cell homogenates were analyzed by Western
blot analysis to determine ADRP content in control and SCP-2 expressors. Cell homogenates were analyzed for presence of caveolin and
SRB1. For all quantitative Western blots, protein samples (17-20 µg)
were run on Tricine gels (12%) and transferred to nitrocellulose membranes. The blots were blocked in 3% gelatin in 10 mM
Tris-HCl, pH 8, 100 mM NaCl, 0.055 Tween-20 before
incubation with the polyclonal rabbit antibodies against either ADRP,
caveolin, or SRB1. Alkaline-phosphatase conjugates of goat anti-rabbit
IgG and Sigma Fast 5-bromo-4chloro-3-indolyl phosphate/nitro blue
tetrazolim tablets (Sigma) were used to visualize and quantitate bands
of interest (37).
Statistics--
All values were expressed as the means ± S.E. with n and p indicated under "Results."
Statistical analyses were performed using Student's t test
(GraphPad Prism, San Diego, CA). Values with p < 0.05 were considered statistically significant.
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RESULTS |
Dependence of the Extent of HDL-mediated Efflux of
[3H]Cholesterol on HDL Concentration of HDL and on SCP-2
Expression--
When L-cell clones were prelabeled with
[3H]cholesterol followed by incubation in medium without
HDL, very little [3H]cholesterol effluxed from either
mock transfected control cells (Fig. 1,
filled circles) or SCP-2-expressing cells (not shown). Although HDL (98 µg HDL protein/ml) dramatically stimulated efflux of
[3H]cholesterol from both mock transfected control (Fig.
1, filled triangles) and SCP-2-overexpressing cells (Fig. 1,
open triangles), SCP-2 expression decreased the extent of
HDL-mediated [3H]cholesterol efflux by 61%
(p < 0.015, n = 4). However, this effect was highly dependent on the HDL concentration. In mock transfected control cells, the extent of HDL-mediated
[3H]cholesterol efflux was increased proportional to
medium HDL concentration up to a maximum at 98 µg HDL protein/ml
(Fig. 2A, open
bars). Further increasing the medium HDL concentration to 175 µg
HDL protein/ml did not additionally increase
[3H]cholesterol efflux, whereas higher HDL concentrations
actually inhibited rather than increased [3H]cholesterol
efflux. In contrast, in SCP-2-expressing cells the extent of
HDL-mediated [3H]cholesterol efflux was increased in
proportion to medium HDL concentration up to a maximum at 175 µg HDL
protein/ml (Fig. 2A, solid bars) and then
decreased at 250 µg HDL protein/ml. Thus, the HDL concentration
required to achieve maximal HDL-mediated [3H]cholesterol
efflux was increased 78% in SCP-2-expressing cells. In summary, SCP-2
expression decreased the extent of HDL-mediated [3H]cholesterol efflux and shifted the concentration of
HDL required to achieve maximal efflux of [3H]cholesterol
without further stimulating maximal HDL-mediated [3H]cholesterol efflux above the level observed in mock
transfected controls.
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Fig. 1.
Efflux of [3H]cholesterol from
L-cell fibroblasts expressing SCP-2. Mock transfected control and
transfected L-cells expressing SCP-2 were labeled with
[3H]cholesterol before incubation with HDL (98 µg
protein/ml) to start the efflux process. Aliquots were removed at timed
intervals and counted to determine the fraction of
[3H]cholesterol remaining in the cells. Values represent
the means ± S.E. from 3-5 cell wells. If not clearly seen,
error bars are obscured by the markers. Filled
circles, medium without HDL; filled triangles, mock
transfected cells + HDL; open triangles, SCP-2-expressing
cells + HDL.
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Fig. 2.
[3H]Cholesterol efflux
dependence on HDL concentrations. Mock transfected control cells
and SCP-2 expressors were labeled with [3H]cholesterol to
show the extent of efflux (A) and initial rates
(B) after 4 h of incubation at various concentrations
of HDL (54-250 µg protein/ml). Values are the means ± S.E.
from 3-5 samples and represent the percentage of
[3H]cholesterol released to the medium. *, indicates
significance, p < 0.047. **, indicates significance,
p < 0.017, as compared with the control.
Open and filled bars refer to mock transfected
control and SCP-2-expressing cells, respectively.
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Effect of SCP-2 Expression on Kinetics of HDL-mediated Efflux of
[3H]Cholesterol--
In the absence of HDL, the initial
rate of [3H]cholesterol efflux from both mock transfected
cells (Fig. 1, filled circles) and SCP-2-expressing cells
(not shown) was essentially near zero. Although addition of HDL (98 µg protein/ml) increased the initial rate of
[3H]cholesterol efflux dramatically from both mock
transfected and SCP-2 overexpressing cells (Fig. 1), analysis of
multiple experiments revealed that SCP-2 expression decreased the
initial rate of HDL-mediated [3H]cholesterol efflux by
20% (p < 0.05, n = 4) as compared
with mock transfected control cells. In mock transfected control cells, the initial rate of HDL-mediated [3H]cholesterol efflux
increased in near linear (slope 0.65) proportion with increasing medium
HDL concentration up to 250 µg HDL protein/ml (Fig. 2B,
open bars). In SCP-2-expressing cells, the initial rate of
HDL-mediated [3H]cholesterol efflux similarly increased
with medium HDL, except that the slope of the increase was 40% higher
(Fig. 2B, filled bars). Thus, SCP-2 expression
inhibited the initial rate of HDL-mediated [3H]cholesterol efflux at low, but not high, HDL
concentration. However, even at 250 µg HDL/ml the initial rate of
HDL-mediated [3H]cholesterol efflux in SCP-2-expressing
cells was not increased over that in mock transfected controls.
Further analysis of the HDL-mediated [3H]cholesterol
efflux curves in Fig. 1 revealed that the efflux curve fit single
exponential kinetics that allowed resolution of two parameters: the
time at which half the cholesterol pool was depleted
(t1/2) and the pool size of HDL-mediated
[3H]cholesterol (A). SCP-2 expression
increased the t1/2 of HDL-mediated
[3H]cholesterol efflux by 61% (p < 0.019, n = 4). The longer half-time was consistent with
the inhibitory effect of SCP-2 on the initial rate of cholesterol
efflux (Figs. 1 and 2). Finally, SCP-2 expression significantly reduced
the pool size of HDL-mediated [3H]cholesterol efflux by
23% (p < 0.03, n = 3) as compared
with that in mock transfected control cells.
Direct Visualization of HDL-mediated Cholesterol Efflux from Living
Cells by Laser Scanning Confocal Microscopy: Effect of SCP-2 Expression
on Extent of HDL-mediated NBD-cholesterol Efflux--
Although the
above studies with [3H]cholesterol indicated that
HDL-mediated [3H]cholesterol efflux was inhibited by
SCP-2 expression, radiolabeled studies did not provide direct
visualization of this process. Therefore, cells were preloaded with
NBD-cholesterol and washed, and NBD-cholesterol was imaged by laser
scanning confocal microscopy as described under "Experimental
Procedures." The total fluorescence intensity/cell was then
determined individually for a large number of cells. The
time-dependent disappearance of NBD-cholesterol fluorescence of each cell was then determined in the presence and
absence of HDL (see "Experimental Procedures"). As with
[3H]cholesterol efflux, NBD-cholesterol fluorescence
intensity/cell (corrected for a small amount of photobleaching over the
time period studied) was essentially zero in the absence of HDL for both mock transfected control and SCP-2-expressing cells (not shown).
The addition of HDL dramatically stimulated NBD-cholesterol efflux from
both the SCP-2 expressor and mock transfected control cells (Fig.
3). However, the extent of this effect
was highly dependent on the expression of SCP-2 and the HDL
concentration in the medium. HDL-mediated NBD-cholesterol efflux from
mock transfected control cells was greater than 90% by 40 min (Fig. 3,
filled triangles). In contrast, SCP-2 expression decreased
the extent of HDL-mediated NBD-cholesterol efflux over the same time
period studied (Fig. 3, open triangles). Examination of a
large number of cells showed that the extent of HDL-mediated
NBD-cholesterol efflux was 40% less than that from mock transfected
control cells (p < 0.006, n = 11-14).
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Fig. 3.
Efflux of NBD-cholesterol from L-cell
fibroblasts expressing SCP-2. Single cell efflux of
NBD-cholesterol was examined from mock transfected control
(filled triangles) and SCP-2-expressing cells (open
triangles). Efflux was initiated by addition of HDL (10 µg
protein/ml) to cells prelabeled with NBD-cholesterol. Values from cells
in medium only were subtracted to correct for minimal losses because of
photobleaching effects. Values represent the means ± S.E. from
11-14 cells.
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The extent of NBD-cholesterol efflux was dependent on the HDL
concentration. The extent of efflux increased proportional to medium
HDL concentration in near linear fashion for both mock transfected
control (slope, 0.74; Fig. 4A,
open bars) and for SCP-2-expressing cells (slope, 1.04; Fig.
4A, solid bars). SCP-2 expression inhibited
HDL-dependent NBD-cholesterol 157% (p < 0.006, n = 6-9) at low HDL concentration (Fig.
4A) but not at high HDL concentration. However, even when
the SCP-2-mediated inhibition was abolished at high HDL concentration,
the extent HDL-mediated NBD-cholesterol efflux in SCP-2-expressing
cells was not increased above that exhibited by mock transfected
control cells (Fig. 4A).
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Fig. 4.
NBD-cholesterol efflux dependence on HDL
concentrations. The extent of efflux (A) and initial
rates (B) are shown after 10 min of incubation at several
concentrations of HDL (5-30 µg protein/ml) in control (open
bars) and SCP-2-expressing cells (filled bars)
prelabeled with NBD-cholesterol. Values are the means ± S.E. of
cells from several dishes and represent the percentage of
NBD-cholesterol released to the medium. * indicates significance,
p < 0.006, as compared with the control.
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Comparison of the Effect of SCP-2 Expression on Extent of
HDL-mediated NBD-cholesterol and [3H]Cholesterol
Efflux--
To compare the results from efflux experiments using
either [3H]cholesterol or NBD-cholesterol, the amount of
HDL added to initiate efflux was adjusted with regard to the number of
cells present during the experiment. The number of cells present in the
[3H]cholesterol experiments was approximately 10 times
greater than with the NBD-cholesterol probe. However, the amount of HDL
used to initiate efflux was also 10 times greater than with the
[3H]cholesterol experiments so that the ratio of HDL to
cell number remained constant regardless of which probe was used. The
results from Fig. 5 show that
HDL-mediated efflux of [3H]cholesterol and
NBD-cholesterol were inhibited to the similar degree at the same ratios
of HDL/number of cells. Thus, even though HDL was more effective in
mediating efflux of NBD-cholesterol (Fig. 4A) than
[3H]cholesterol (Fig. 2A), SCP-2 expression
inhibited HDL-mediated sterol efflux at low HDL levels to the same
degree (65-75%), and this inhibition was abolished at similar high
ratios of HDL/number of cells.
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Fig. 5.
Inhibition of efflux from SCP-2
overexpressing cells loaded with [3H]cholesterol and
NBD-cholesterol. The extent of inhibition with cells loaded with
[3H]cholesterol (open bars) and
NBD-cholesterol (filled bars) was compared. The inhibition
was comparable at similar ratios of HDL/cell number over the range of
HDL concentrations examined (1.0-2.0 × 104 HDL
µg protein/number of cells).
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Effect of SCP-2 Expression on Kinetics of HDL-mediated
NBD-cholesterol Efflux in Living Cells--
SCP-2 expression decreased
the initial rate of HDL-mediated NBD-cholesterol efflux as much as
220% at low HDL concentration in the medium (Fig. 4B).
Increasing HDL concentration abolished the inhibitory effect of SCP-2
expression on HDL-mediated NBD-cholesterol efflux. Visual examination
of the curves in Fig. 3 indicated that the kinetics of HDL-mediated
NBD-cholesterol efflux were not simple exponentials. Attempts at
fitting these curves to simple exponential kinetics for either mock
transfected controls or SCP-2-expressing cells were unsuccessful (see
"Experimental Procedures"). In contrast, the efflux curves in Fig.
3 fit very well (R2 = 0.999) to a
multiparameter exponential decay equation, y = (AebT0 + CedT0)
(eh(tT0)) (see
"Experimental Procedures"). This equation describes two pools of
cholesterol (a large, slow pool and a smaller, faster pool) showing
increased efflux after some time point T0. In
mock transfected control cells, the respective half-times of these cholesterol pools (tb1/2 and
td1/2) were 1.2 and 13.1 min, respectively (Table I). The longer half-time was
increased 100% (p < 0.0025) in SCP-2-expressing
cells. In mock transfected control cells, the pool sizes (A
and C) corresponding to these half-times were 0.23 and 0.77, respectively (Table I). Although the slowly effluxing NBD-cholesterol
pool was 230% larger than the rapidly effluxing pool, SCP-2 expression
did not affect the respective pool sizes. Overexpression of SCP-2
resulted in a 59% decrease in the half-life associated with
h. T0 was not affected by SCP-2
expression. In summary, these data suggested that SCP-2 expression
inhibited NBD-cholesterol efflux from the cells primarily by inhibiting the half-time rather than altering the pool size of
NBD-cholesterol.
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Table I
Kinetic analysis of HDL-mediated NBD-cholesterol efflux from intact
cells
Parameters were derived from the following multi-parameter exponential
decay equation. When t < T0,
y = (Aebt + Ce dt); when t  T0, y = (AebT0 + CedT0) × e h(tT0) where
A and C represent the fraction of available
NBD-cholesterol in each rate differentiated pool; b,
d, and h are the apparent rate constants;
t is time in minutes; and T0 (derived
from the fitted curve) is the time at which depletion of the pools
increased to completion (18.2 ± 0.6 and 16.8 ± 0.9 min for
the control and SCP-2 expressor, respectively).
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Intracellular Localization of the NBD-cholesterol: Confocal Laser
Scanning Microscopy of L-cells Expressing SCP-2--
Although the
NBD-cholesterol fluorescence in cells preloaded with NBD-cholesterol
appeared localized throughout the cell, distinct highly fluorescent
regions resembling lipid droplets in size and shape were also prominent
(Fig. 6). To establish whether the
intensely fluorescent regions were indeed lipid droplets, three
co-localization experiments were performed.
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Fig. 6.
NBD-cholesterol efflux by HDL in SCP-2
overexpressing cells. Transfected L-cells expressing the SCP-2
protein were labeled with NBD-cholesterol (0.35 µM) and
incubated with HDL (10 µg protein/ml). Cells at 2 min (A),
28 min (B), and 58 min (C) during the efflux
process were examined with the Bio-Rad MRC-1024 confocal system as
described under "Experimental Procedures." Arrows
indicate high retention lipid droplets during the time course.
Objective, 63×.
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First, cells preloaded with NBD-cholesterol were stained with Nile red,
a selective lipid droplet stain, and emission of the two fluorophores
was simultaneously determined through separate photomultipliers:
NBD-cholesterol (green) and Nile red (red). Upon superposition of such
images (Fig. 7A),
co-localization appeared as yellow/orange areas (red plus green = yellow/orange), whereas lack of co-localization appeared as separate
green and red areas. A graphical representation of the superposition is
shown in the form of a pixel fluorogram (Fig. 7B). High
co-localization in pixel fluorograms is indicated both by the
localization of many pixels along the diagonal, the yellow/orange
color, and the green and red correlation coefficients approaching
values near 1.0. The green correlation coefficient (0.81 in Fig.
7B) is the ratio of all the green (NBD-cholesterol)
intensities that showed a red (Nile red) component divided by the sum
of all the green (NBD-cholesterol) intensities. The red correlation
coefficient (0.92 in Fig. 7B) is ratio of all the red (Nile
red) intensities showing a green (NBD-cholesterol) component divided by
the sum of all the red intensities. It should be noted, however, that
although a significant population of data points fell along the
diagonal line, NBD-cholesterol also appeared throughout the cell (green
in the superimposed image in Fig. 7A). Morphometric analysis
on intensity measurements using Metamorph software (see "Experimental
Procedures") revealed the percentage of NBD-cholesterol outside the
lipid droplets (i.e. designated as the cytoplasmic
compartment of the cell) ranged from 80 to 96% for both control and
SCP-2 expressors. Although the correlation coefficients given in Fig.
7B determined the degree of co-localization of Nile red in
NBD-cholesterol (and vice versa), they did not measure the
amount of NBD-cholesterol in the lipid droplets versus that
in the rest of the cell.
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Fig. 7.
Intracellular distribution of NBD-cholesterol
and Nile red in transfected L-cells. Co-localization patterns of
NBD-cholesterol and Nile red were shown using pseudo-coloring derived
from confocal image acquisition from red- and green-specific PMT
channels. A 24-bit RGB image was created from the red plus green plus
blue (null) channels. Red and green are additive in RGB color space
yielding yellow-orange. L-cells expressing the SCP-2 protein stained
with NBD-cholesterol and Nile red were combined to yield yellow-orange
(A) where co-localization occurred. Superimposition of the
probes was graphically demonstrated in a pixel fluorogram
(B) where co-localization of NBD-cholesterol (green) and
Nile red (red) resulted in yellow to orange points falling along the
diagonal line in the fluorogram. The correlation coefficients
corresponding to red and green were proportional to the degree of
fluorescence probe co-localizing in each component of the image
relative to the total fluorescence and indicated the extent of overlap
between the probes. The cells were examined using the Bio-Rad MRC-1024
confocal system. Objective, 63×.
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Second, the lipid droplet specific stain, Nile red, was co-localized
with ADRP, a protein closely associated with lipid droplets. Cells were
simultaneously labeled with Nile red (red) and anti-ADRP antibodies
(green). Confocal fluorescence images for ADRP (Fig. 8B) highly resembled those
obtained for Nile red (Fig. 8A), the lipid droplet specific
stain. Magnification of a representative lipid droplet showed uniform
staining with Nile red throughout (Fig. 8A,
inset). In contrast, magnification of the same lipid droplet
stained with anti-ADRP showed much more intense staining at the lipid
droplet surface (Fig. 8B, inset). Superposition
of the two images (Fig. 8, A and B) yielded a
yellow-orange color where co-localization occurred. Although both
stains co-localized to lipid droplets, the magnified image of the lipid
droplet revealed that ADRP (green) was present on the surface of the
lipid droplet, whereas the Nile red stain was seen both at the surface
(orange/yellow) and throughout (red) the lipid droplet (Fig.
8C).
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Fig. 8.
Double-label immunofluorescence with ADRP and
Nile red or NBD-cholesterol in transfected L-cells expressing
SCP-2. Cells were simultaneously labeled for Nile red
(A) and ADRP with fluorescein isothiocyanate-conjugated goat
anti-rabbit IgG (B) or ADRP with Texas Red-conjugated goat
anti-rabbit IgG (D) and NBD-cholesterol (E).
Co-localization (C and F) were shown using
pseudo-coloring resulting in yellow to orange where superimposition
occurred. Insets were lipid droplets located within the
larger frame.
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Third, NBD-cholesterol was co-localized with ADRP by simultaneous
labeling with anti-ADRP antibodies (red) and NBD-cholesterol (green).
Simultaneous acquisition of confocal images for ADRP (Fig.
8D) and NBD-cholesterol (Fig. 8E) showed that
ADRP and NBD-cholesterol were both present in intense staining areas,
i.e. lipid droplets shown in superposed images (Fig.
8F). Magnified images of representative lipid droplets (Fig.
8F, inset) showed that ADRP again appeared on the
lipid droplet surface (distinct red region on left side of lipid
droplet), whereas NBD-cholesterol was present at the surface
co-localized with a portion of ADRP (yellow) as well as throughout the
interior of the lipid droplet (green).
Effect of SCP-2 Expression on ADRP Levels in Transfected L-cell
Fibroblasts--
Because ADRP binds cholesterol and NBD-cholesterol
(29) and is significantly co-localized with ADRP, the inhibition of
HDL-mediated cholesterol efflux by SCP-2 may occur in part by altered
level of ADRP. Because the mock-transfected and untransfected cell
lines had similar levels of ADRP (not shown), Western blots of cell homogenates and lipid droplets, isolated as described under
"Experimental Procedures," were run for control and
SCP-2-expressing cells samples (Fig. 9).
Although SCP-2 was not detected in lipid droplets, ADRP was enriched
140% in lipid droplets as compared with cell homogenates for both
control (Fig. 9, lane 2 versus lane 1) and SCP-2-expressing cells (Fig. 9, lane 4 versus lane 3). SCP-2 expression
reduced the level of ADRP by 60% (±0.36, n = 11) in
the cell homogenate and by 70% (±0.08, n = 11) in the
isolated lipid droplets. These data suggest an important role for ADRP
as well as SCP-2 in regulating cellular cholesterol levels.
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Fig. 9.
Western blot analysis of SCP-2 expression
clones showing expression levels of ADRP, caveolin, and SRB1.
Samples from control and SCP-2-expressing cells were run on 12%
Tricine gels. A, lane 1, untransfected L-cell
homogenate; lane 2, lipid droplet fraction isolated from
L-cells; lane 3, SCP-2 cell homogenate; lane 4,
lipid droplet fraction isolated from SCP-2-expressing cells.
B, lane 1, untransfected L-cell homogenate;
lane 2, SCP-2 cell homogenate. C, lane
1, untransfected L-cell homogenate; lane 2, SCP-2 cell
homogenate. The blots were probed with polyclonal rabbit antibodies
against ADRP (A), caveolin (B), or SRB1
(C) as described under "Experimental Procedures."
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Effect of SCP-2 Expression on Scavenger Receptor B1 and Caveolin
Levels in Transfected L-cells--
It is possible that SCP-2
inhibition of HDL-mediated cholesterol efflux was due to alterations in
plasma membrane components of the cholesterol efflux pathway, caveolin
and the HDL receptor (SRB1). However, Western blotting of cell
homogenates; Fig. 9, B and C) from control and
SCP-2 expression clones showed no significant change in the levels of
caveolin (panel B) or SRB1 (panel C).
Relative Contributions of the Intracellular Compartments to
HDL-mediated Cholesterol Efflux Kinetics--
The relative
contributions of the low intensity, diffuse pattern typical of
distribution throughout the cytoplasm and the high intensity pattern
localized to lipid droplets to HDL-mediated cholesterol efflux kinetics
were examined over time: 2 min (Fig. 6A), 28 min (Fig.
6B), and 58 min (Fig. 6C). After 58 min, the lipid droplet NBD-cholesterol intensity (indicated by
arrows) was still visible, whereas that in cytoplasmic areas
of the cell was almost depleted. Cholesterol was retained in the lipid
droplets; this suggested either that the rate-limiting step of
NBD-cholesterol efflux from the cell was efflux from lipid droplets or
that the concentration of NBD-cholesterol in the lipid droplets was
higher than in the cytoplasm. To resolve these possibilities, cells
were imaged and graphically partitioned (see "Experimental
Procedures") to separate the lipid droplet contribution from the
cytoplasmic component of the cell. Surprisingly, NBD-cholesterol efflux
from lipid droplets (Fig.
10B, filled
triangles) was initially faster than from cytoplasm (Fig.
10A) but quickly reached an equilibrium level.
Multicomponent kinetic analysis of NBD-cholesterol fluorescence decrease confirmed this observation. The data points derived from the
lipid droplet compartment (Fig. 10B, filled
triangles) best fit (R2 = 0.99) to a
biexponential decay equation, whereas the cytoplasmic com- partment
(Fig. 10A, filled triangles) best fit to a
multi-parameter decay equation (R2 = 0.99),
suggesting that multiple pools of NBD-cholesterol were available for
efflux from each compartment. In control cells, the half-times of
NBD-cholesterol efflux from the lipid droplets were
tb1/2 = 0.66 and
td1/2 = 8.4 min, significantly smaller (p < 0.005) than the respective half-time from the
cytoplasm tb1/2 = 1.9 and
td1/2 = 14.7 min (Table
II). Because Pool A, the rapid efflux
component, was nearly 200% larger than that of cytoplasm (0.56 versus 0.18), the larger pool size further contributed to
the more rapid efflux of NBD-cholesterol from the lipid droplet than
from the cytoplasm. Thus, the efflux of NBD-cholesterol from the lipid
droplet was not the rate-limiting step in HDL-mediated cholesterol
efflux from the cell, and efflux kinetics from the two components in
the whole cells (Table I) basically reflected those from the two
cytoplasmic components rather than the lipid droplets (Table II). Lower
apparent loss of NBD-cholesterol from the lipid droplets
versus cytoplasm was apparently due to a higher concentration of NBD-cholesterol in the lipid droplets than in the
cytoplasm rather than a slower rate of NBD-cholesterol efflux from the
lipid droplet.
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Fig. 10.
NBD-cholesterol efflux from the cytoplasmic
and lipid droplet compartments. Single cell efflux of
NBD-cholesterol from the lipid droplet and the cytoplasmic compartments
of the cell was shown plotted against time. Cells were loaded with 0.35 µM NBD-cholesterol and incubated with 10 µg protein/ml
of medium to start the efflux process. Cells were imaged using the
Bio-Rad MRC-1024 confocal system and analyzed by Metamorph software as
described under "Experimental Procedures." Symbols refer to mock
transfected control (filled triangles) and SCP-2-expressing
L-cells (open triangles).
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Table II
Effect of SCP-2 expression on HDL-mediated NBD-cholesterol efflux
kinetics from cytoplasm and lipid droplets
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Effect of SCP-2 Expression on HDL-mediated Cholesterol Efflux
Kinetics from Cytoplasm and Lipid Droplets--
In SCP-2-expressing
cells, efflux of NBD-cholesterol from lipid droplets (Fig.
10B, open triangles) was initially faster than from cytoplasm but did not reach an equilibrium level in either cytoplasm or lipid droplets as quickly as observed with control cells.
Multicomponent kinetic analysis of NBD-cholesterol fluorescence decrease showed that efflux from the lipid droplet compartment of
SCP-2-expressing cells (Fig. 10B, open triangles)
again best fit (R2 = 0.99) to a biexponential
decay equation, whereas from the cytoplasmic compartment (Fig.
10A, open triangles) best fit to a
multi-parameter decay equation (R2 = 0.99).
Examination of the relative half-times and pool sizes of
NBD-cholesterol efflux from cytoplasm and lipid droplets of SCP-2
expression showed: (i) The half-times of NBD-cholesterol efflux from
the lipid droplets of SCP-2-expressing cells were shorter (faster) than
those from cytoplasm (Table II), consistent with the NBD-cholesterol
efflux from the lipid droplet not being the rate-limiting step in
HDL-mediated NBD-cholesterol efflux from SCP-2-expressing cells. (ii)
In SCP-2-expressing cells the half-times and pool sizes of
NBD-cholesterol efflux from the whole cells (Table I) reflected those
of the cytoplasmic compartment (Table II) rather than those observed
for the lipid droplets (Table II).
SCP-2 expression differentially affected the two half-times of
NBD-cholesterol efflux from the cytoplasm as compared with those in the
mock transfected control cells. The half-time
tb1/2 for rapid NBD-cholesterol efflux from
the cytoplasmic compartment was significantly shorter (faster) in
SCP-2-expressing cells than that observed for control cells, 1.4 ± 0.1 versus 1.9 ± 0.1 min, p < 0.005 (Table II). In contrast, the slower half-time
td1/2 was 113% longer in SCP-2-expressing cells as compared with control cells (31.4 ± 2.0 versus 14.7 ± 0.8 min, p < 0.005)
(Table II). Although SCP-2 expression altered the half-times of
NBD-cholesterol efflux from the cytoplasmic compartment, there was
little effect of SCP-2 expression on the respective pool sizes. The
pool size C associated with the slower half-time
td1/2 was 460% greater than that of pool
size A associated with the rapid half-time
tb1/2 (Table II). However, neither pool size
differed significantly from those in mock transfected control cells. In
summary, SCP-2 expression enhanced efflux from the rapidly transferring
NBD-cholesterol pool in the cytoplasm. Concomitantly, SCP-2 expression
inhibited the efflux from the slowly transferring, but much larger,
NBD-cholesterol pool in the cytoplasm.
SCP-2 expression also altered NBD-cholesterol efflux from lipid
droplets. SCP-2 expression did not alter the rapid half-time of
NBD-cholesterol efflux from the lipid droplets,
tb1/2 = 0.56 but decreased (slowed) the
half-time for the slower efflux pool, td1/2 = 18.1 min, by 220% (Table II). SCP-2 expression increased the pool
size C (0.65 versus 0.44, p < 0.005) of the
slower effluxing component while at the same time decreasing pool size
A (0.34 versus 0.56, p < 0.005) of the more
rapidly effluxing component.
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DISCUSSION |
Because cholesterol accumulation inside mammalian cells is
detrimental to cell survival and contributes significantly to
cholesterol deposition in atherosclerosis, cellular cholesterol
homeostasis is tightly regulated by balancing of the influx and efflux
of cholesterol (38). Influx of exogenous unesterified and esterified cholesterol occurs by both the classic LDL receptor pathway (39) as
well as by the more recently demonstrated HDL receptor pathway (reviewed in Refs. 1, 29, 38, and 40). In contrast, efflux of
cholesterol occurs by the HDL receptor pathway but not LDL receptor
pathway. HDL-mediated cholesterol efflux utilizes unesterified cholesterol, but not cholesterol ester, transferred from intracellular sites to the plasma membrane by both vesicular and cytosolic pathways (reviewed in Refs. 1, 8, and 38). Despite the presence of the
HDL-mediated cholesterol efflux pathway, some tissues retain cholesterol for steroidogenesis (adrenal, testis, and ovary), for
secretion as very low density lipoprotein or Chylomicrons (liver and
intestine), or for secretion as biliary cholesterol (liver). Because of
the paucity of knowledge regarding the sources of intracellular
cholesterol and the pathways for transfer of intracellular cholesterol
to the cell surface, it is not clear whether cholesterol retention is
due to intracellular mechanisms opposing HDL-mediated cholesterol. The
data herein show fundamental new observations regarding the role of
SCP-2 in cholesterol efflux.
First, overexpression of SCP-2 in transfected L-cell fibroblasts
inhibited the extent and rate of HDL-mediated
[3H]cholesterol and NBD-cholesterol efflux. SCP-2
expression inhibited the maximal extent of HDL-mediated
[3H]cholesterol and NBD-cholesterol efflux to the same
degree at equivalent, nonsaturating ratios of HDL/cell in the culture
medium. SCP-2 expression also increased half-times (longer) and
decreased cholesterol pool size available for HDL-cholesterol efflux.
Second, SCP-2 expression within the cell opposed the effects of
extracellular HDL on cholesterol efflux. The major effect of SCP-2 was
to retain cholesterol in the cell, whereas HDL, an excellent
cholesterol acceptor, promoted cholesterol efflux. At nonsaturating
conditions of HDL, the predominant effect was that of inhibition by
SCP-2 expression, whereas at higher HDL level maximal efflux (but not
above controls) occurred and overcame inhibition by SCP-2 expression.
Substantial evidence in vivo and in vitro is
consistent with the interpretation that SCP-2 promotes retention of
cholesterol in cells (7, 22, 30, 41-50) for utilization in
steroidogenesis (27, 51), secretion in bile (reviewed in Refs. 23-25,
52, and 53), or secretion as lipoproteins (22).
Third, confocal imaging of living cells allowed graphic delineation of
HDL-mediated cholesterol efflux into at least two components: lipid
droplets and outside lipid droplets. Because of its diffuse distribution, the latter was defined as cytoplasmic. Efflux curves of
NBD-cholesterol were best fit to bi-exponential and multi-parameter exponential decay equations, indicating the presence of a small, rapid
pool with half-time near 1 min and a larger, slow effluxing pool with
half-time near 13 min. These data were consistent with data reported
elsewhere wherein two pools of intracellular cholesterol transfer with
similar half-times were observed and attributed to protein-mediated and
vesicular cholesterol transfer, respectively (18, 20, 21, 50, 50, 53).
Furthermore, the efflux kinetics from the cytoplasmic compartment
largely reflected that of the whole cell. This suggested that transfer
of cholesterol through the cytoplasm, rather than desorption from lipid
droplets, was the rate-limiting step in HDL-mediated cholesterol efflux.
Fourth, SCP-2 expression differentially affected cholesterol efflux
from the cytoplasmic compartment: (i) SCP-2 stimulated the smaller,
fast pool of protein-mediated cholesterol through the plasma membrane
to the HDL acceptor. In the absence of an extracellular cholesterol
acceptor, SCP-2 expression in transfected L-cells was shown to
stimulate the rapid transfer of plasma membrane derived cholesterol to
the endoplasmic reticulum for esterification by acyl-CoA:cholesterol
O-acyltransferase (49). This was consistent with data from
transfected hepatoma cells overexpressing SCP-2 wherein rapid
cholesterol cycling of plasma membrane and intracellular cholesterol
was observed (22). Also pertinent were observations that the fast pool
of protein-mediated cholesterol transfer was abolished by treatment of
human fibroblasts with antisense DNA to SCP-2 (50). We speculate that
this fast pool represents, at least in part, SCP-2-bound
NBD-cholesterol because SCP-2 has been shown to bind NBD-cholesterol
with high affinity (7, 54, 55). Parallel studies on the intracellular
diffusion of NBD-stearic acid, a ligand that SCP-2 also binds with high
affinity, also showed that SCP-2 expression increased the rapid
diffusion coefficient of this ligand in the cytoplasm (30, 56, 57).
Lipid-binding proteins enhance the intracellular diffusion of poorly
water soluble molecules (i.e. lipids) by increasing the
desorption membranes and increasing their aqueous solubility (reviewed
in Refs. 58 and 59). (ii) SCP-2 expression increased the half-time of
the slow vesicular pool of cholesterol transfer. In contrast, treatment of human fibroblasts with antisense DNA to SCP-2 stimulated the slower,
vesicular component of cholesterol transfer through the cytoplasm (50).
Likewise, HDL cholesterol secretion (mediated by vesicular transport)
was decreased in transfected rat hepatoma cells overexpressing SCP-2
(22).
Fifth, cholesterol efflux from the lipid droplet was characterized by
both fast and slow components that were distinct from those observed in
the cytoplasm. The data presented herein and earlier (29) demonstrated
that lipid droplet cholesterol is present in at least three forms:
esterified and localized primarily in the core of the lipid droplet;
unesterified and localized in the surface lipid of the lipid droplet;
and unesterified and bound to ADRP, a cholesterol-binding protein
present in the surface of the lipid droplet. The effect of SCP-2
expression on lipid droplet cholesterol pool size, taken together with
the observation that ADRP binds cholesterol, suggests that the slowly
effluxing component was the unesterified lipid. Pool sizes in lipid
droplets were dramatically affected by SCP-2 expression where the
larger cholesterol pool (C) was increased, whereas that of
the smaller rapidly transferable cholesterol pool (A) was
decreased. The 60% decrease in the rapidly effluxing pool size in the
lipid droplet of SCP-2-expressing cells correlated with a 70% decrease
in ADRP level in lipid droplets from SCP-2-expressing cells as compared with the control cells. It should be noted that the two pools were not
due to ADRP-bound versus unbound NBD-cholesterol that was
not esterified. Although NBD-cholesterol is esterified at least as well
as cholesterol in L-cells, CaCo2 cells, and hamster intestinal
enterocytes (29, 40), after 24 h less than 8% of the
NBD-cholesterol was observed to be esterified in L-cells (29). Because
the time frame of the efflux experiments using NBD-cholesterol was less
than an hour, little to no esterification of the probe was expected.
Thus, the fact that ADRP binds cholesterol with nM
Kd (29) taken together with data showing
NBD-cholesterol co-localizes with ADRP suggests a role for ADRP in
determining the lipid droplet cholesterol pool size available for
efflux. Because this observation might be correlative, work is in
progress to further delineate the role of ADRP in efflux and other
intracellular processes in cells overexpressing ADRP.
In summary, NBD-cholesterol proved a useful probe for monitoring the
relative effects of SCP-2 expression on HDL-mediated cholesterol
efflux. However, this does not imply that the behavior of
NBD-cholesterol is identical to that of cholesterol. One important distinction is that NBD-cholesterol efflux achieved equilibrium in
20-40 min, significantly faster than [3H]cholesterol
(20-24 h). Conversely, HDL-mediated uptake of NBD-cholesterol is also
faster than that of [3H]cholesterol in L-cells, hamster
intestinal enterocytes, and CaCo2 cells (29, 60). Although the
mechanism by which HDL facilitates the removal of cholesterol from the
cell is not yet fully resolved, the more rapid dynamics of
NBD-cholesterol efflux/uptake may simply reflect the higher aqueous
solubility of NBD-cholesterol versus cholesterol (55). For
example, in the sterol efflux pathway HDL interacts with the scavenger
receptor (SRB1) at the plasma membrane surface. The existing evidence
favors a mechanism whereby plasma membrane cholesterol then desorbs and
diffuses through the aqueous medium to be taken up by the acceptor HDL
(reviewed in Ref. 61). Thus, the higher aqueous solubility of
NBD-cholesterol favors an increased rate of HDL-mediated efflux or, in
the reverse direction, uptake. Substantial evidence suggests that
NBD-cholesterol undergoes metabolism and utilizes the same
intracellular trafficking pathways as cholesterol. NBD-cholesterol
distributes in L-cells similarly as dehydroergosterol, a naturally
occurring fluorescent sterol (29). NBD-cholesterol is esterified
similarly as cholesterol and dehydroergosterol in L-cells, intestinal
enterocytes, and CaCo2 cells (29, 60). Finally, NBD-cholesterol
trafficks by similar uptake, intracellular, and secretory pathways as
was shown herein and previously by others in studies with L-cell
fibroblasts and CaCo2 cells (29, 60). However, there are limitations of the use of NBD-cholesterol that include the inability to incorporate to
as high a degree as cholesterol in some membranes (such as the plasma
membrane) (62). Notwithstanding, although NBD-cholesterol does not
accurately reflect all aspects of cholesterol, the above studies taken
together with those presented herein indicate that NBD-cholesterol is
an acceptable probe for examining relative effects of factors such as
HDL concentration and SCP-2 expression on cholesterol uptake, efflux,
and metabolism.
The physiological importance of the findings reported herein is that
SCP-2 may participate in maintaining high intracellular pools of
cholesterol. This is especially significant to tissues such as liver,
intestine, and steroidogenic tissues wherein cholesterol utilization is
high and SCP-2 expression greatest (reviewed in Refs. 7 and 27). The
observation that SCP-2 expression stimulated cholesterol uptake in
transfected cells is also consistent with SCP-2 affecting the balance
of influx/efflux in favor of influx/retention of cholesterol (reviewed
in Refs. 7 and 32). The ability of SCP-2 cells to retain cholesterol,
along with longer half-times and changing pool size contributed to the
overall effect of inhibition of HDL-mediated cholesterol efflux.
Interestingly, the data suggest that the SCP-2 protein may have an
opposite effect on cellular cholesterol balance as compared with
caveolin, a cholesterol-binding protein also enriched in tissues where
cholesterol utilization is high, i.e. liver and
steroidogenic tissues (reviewed in Ref. 8). Thus, the opposite effects
of caveolin and SCP-2 on HDL-mediated cholesterol efflux suggest that
these two proteins may participate in fine tuning cellular cholesterol
efflux versus uptake.
,
TOP
ABSTRACT
INTRODUCTION
