The Apolipoprotein E-dependent Low Density Lipoprotein Cholesteryl Ester Selective Uptake Pathway in Murine Adrenocortical Cells Involves Chondroitin Sulfate Proteoglycans and an alpha 2-Macroglobulin Receptor*

Snehasikta SwarnakarDagger , Jeanette Beers§, Dudley K. Strickland§, Salman Azhar, and David L. WilliamsDagger ||

From the Dagger  Department of Pharmacological Sciences, University Medical Center, State University of New York, Stony Brook, New York 11794, § American Red Cross, Holland Laboratory, Rockville, Maryland 20855, and  Geriatric Research, Education, and Clinical Center, Veterans Affairs, Palo Alto Health Care System, Palo Alto, California 94304

Received for publication, February 23, 2001, and in revised form, March 22, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells acquire lipoprotein cholesterol by receptor-mediated endocytosis and selective uptake pathways. In the latter case, lipoprotein cholesteryl ester (CE) is transferred to the plasma membrane without endocytosis and degradation of the lipoprotein particle. Previous studies with Y1/E/tet/2/3 murine adrenocortical cells that were engineered to express apolipoprotein (apo) E demonstrated that apoE expression enhances low density lipoprotein (LDL) CE uptake by both selective and endocytic pathways. The present experiments test the hypothesis that apoE-dependent LDL CE selective uptake is mediated by scavenger receptor, class B, type I (SR-BI). Surprisingly, SR-BI expression was not detected in the Y1/E/tet/2/3 clone of Y1 adrenocortical cells, indicating the presence of a distinct apoE-dependent pathway for LDL CE selective uptake. ApoE-dependent LDL CE selective uptake in Y1/E/tet/2/3 cells was inhibited by receptor-associated protein and by activated alpha 2-macroglobulin (alpha 2M), suggesting the participation of the LDL receptor-related protein/alpha 2M receptor. Reagents that inhibited proteoglycan synthesis or removed cell surface chondroitin sulfate proteoglycan completely blocked apoE-dependent LDL CE selective uptake. None of these reagents inhibited SR-BI-mediated LDL CE selective uptake in the Y1-BS1 clone of Y1 cells in which LDL CE selective uptake is mediated by SR-BI. We conclude that LDL CE selective uptake in adrenocortical cells occurs via SR-BI-independent and SR-BI-dependent pathways. The SR-BI-independent pathway is an apoE-dependent process that involves both chondroitin sulfate proteoglycans and an alpha 2M receptor.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

LDL1 is a major carrier of cholesterol in the plasma, and the concentration of plasma LDL cholesterol is regulated mainly by the LDL receptor pathway in the liver (1, 2). On the other hand, LDL receptor-independent pathways mediate about one-third of LDL removal in normal individuals and are responsible for most of the LDL removal in individuals homozygous for familial hypercholesterolemia who lack functional LDL receptors (3). The LDL receptor processes LDL via receptor-mediated endocytosis in which the LDL particle is delivered to lysosomes where the CE is hydrolyzed to free cholesterol for use by the cell. In addition to receptor-mediated endocytosis, LDL CE also can be taken up by cells via a selective uptake pathway in which lipoprotein CE is transferred into the cell membrane without the uptake and degradation of the LDL particle. Although the selective uptake pathway has been studied predominantly with HDL, numerous studies have shown the selective uptake of LDL CE in steroidogenic cells of rodents (4-9). In cultured human ovarian granulosa cells, ~33% of total LDL CE uptake occurs via the selective uptake pathway (10). Furthermore, LDL CE selective uptake occurs in human fibroblasts (11), rat and human liver cells (6, 11-13), and a murine macrophage cell (11). Thus, the selective uptake of LDL CE is a widespread process that occurs in a variety of species and cell types.

Little is known about the mechanism of LDL CE selective uptake or the cell surface receptors responsible for this process. In the Y1-BS1 clone of murine adrenocortical cells, SR-BI mediates a major fraction of LDL CE selective uptake as judged by inhibition with antibodies directed to the extracellular domain of SR-BI (14). Expression of SR-BI in transfected COS-7 cells (14) or Chinese hamster ovary cells (15) showed directly that SR-BI mediates LDL CE selective uptake but with a much reduced efficiency compared with HDL CE selective uptake (14). Thus, SR-BI may contribute to LDL CE selective uptake in some cell types.

In a previous study, we found that expression of apoE in murine Y1 adrenocortical cells enhanced the selective uptake of LDL CE by 2-2.5-fold (16). The clone of Y1 cells used in that work, Y1/E/tet/2/3, was engineered to express human apoE4 under control of a tetracycline-regulated promoter, permitting apoE expression to be induced some 20-fold by removal of tet from the culture medium. Experiments in the present study were designed to explore the mechanism by which apoE alters LDL CE selective uptake and, in particular, to test the hypothesis that the enhancement by apoE occurred via SR-BI-mediated LDL CE selective uptake. Surprisingly, we found that the Y1/E/tet/2/3 adrenocortical cell line does not express detectable levels of SR-BI. Furthermore, characterization of the LDL CE selective uptake pathway in Y1/E/tet/2/3 cells showed its properties to be distinct from that mediated by SR-BI and to involve cell surface chondroitin sulfate proteoglycan, apoE, and a member of the LDL receptor family. Thus, these findings identify a new pathway for the selective uptake of LDL CE.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Y1-BS1 cells were maintained in a 37 °C humidified 95% air, 5% CO2 incubator in Ham's F-10 medium supplemented with 12.5% heat-inactivated horse serum, 2.5% calf serum, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin as described (17). Y1/E/tet/2/3 cells were maintained in the same fashion except that the medium also contained 100 µg/ml G418 (Geneticin, Life Technologies, Inc), 200 µg/ml hygromycin (Calbiochem), and 2 µg/ml tetracycline (16). MEF cells were maintained in a 37 °C humidified 95% air, 5% CO2 incubator in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin (18).

Western Blotting-- One day before assay, medium was changed to serum-free medium plus or minus tet containing 2 mM Bt2cAMP where indicated. Postnuclear supernatants were isolated as previously described (19). Proteins (10-20 µg) were resolved by SDS, 10% polyacrylamide gel electrophoresis (PAGE) or SDS, 4-15% PAGE and transferred to nitrocellulose membranes. The membranes were blocked for 1 h at room temperature in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.02% Tween 20 (TBST) containing 7% nonfat milk. The blocked membranes were then incubated with one of the following primary antibodies in TBS-Tween containing 1% nonfat milk: anti-SR-BI C-terminal tail (1:5000) (20), anti-SR-BII C-terminal tail (1:5000) (21), anti-LRP (2 µg/ml, R777) (22), anti-VLDL receptor (4 µg/ml, R2623) (23), anti-gp330 (4 µg/ml, R784) (24), anti-LDL receptor (1:500, R4526) (a generous gift from J. Herz), and anti-LR8B/apoER2(1:1000, alpha -20) (a generous gift of J. Nimpf). The membrane was washed three times with TBS containing 0.05% Tween 20 and incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature in TBS containing 1% nonfat milk and 0.05% Tween. Bands were visualized by enhanced chemiluminescence (Pierce).

Preparation of 125I-Dilactitol Tyramine-[3H]cholesteryl Oleolyl Ether Human HDL3 (Human 125I-Labeled [3H]HDL3) and 125I-Dilactitol Tyramine-[3H]cholesteryl Oleolyl Ether Human LDL (Human 125I-Labeled [3H]LDL)-- Human HDL3 (1.125 g/ml < rho  <1.210 g/ml) and human LDL (1.019 g/ml < rho  <1.063 g/ml) were double-labeled with 125I-dilactitol tyramine and [3H]cholesteryl oleolyl ether as described (5). The specific activity of the human 125I-labeled [3H]HDL ranged from 46 to 70 dpm/ng of protein for 125I and from 6 to 28 dpm/ng of protein for 3H. The specific activity of the human 125I-labeled [3H]LDL ranged from 25 to 75 dpm/ng of protein for 125I and from 3-30 dpm/ng of protein for 3H.

Determination of Selective Uptake of LDL CE and HDL-CE-- Y1/E/tet/2/3cells were cultured in 6-well plates in complete medium plus or minus tet for 4 days with changes of medium on days1 and 3. On day 4, the medium was changed to serum-free medium plus or minus tet containing 2 mM Bt2cAMP. After 24 h, 125I-labeled [3H]LDL at 50 µg/ml protein or 125I-labeled [3H]HDL at 50 µg/ml protein (except where indicated) was added, and the incubation was continued for 4 h at 37 °C. For Y1-BS1 cells, 48 h after plating, medium was changed to serum-free medium supplemented with 100 nM Cortrosyn (Organon), a synthetic (1-24) adrenocorticotropic hormone analogue. After 24 h, medium was changed to serum-free medium lacking Cortrosyn, doubly labeled lipoprotein particles were added, and the incubation was continued for 4 h at 37 °C. Cells were placed on ice, washed three times with cold phosphate-buffered saline containing 1% bovine serum albumin and once with phosphate-buffered saline, and lysed with 1.5 ml of 0.1 N NaOH. The lysate was processed to determine trichloroacetic acid-soluble and -insoluble 125I radioactivity and organic solvent-extractable 3H radioactivity (5) and cell protein (25). The trichloroacetic acid-soluble 125I radioactivity represents endocytosed and degraded lipoprotein. Trichloroacetic acid-insoluble 125I radioactivity represents cell-associated undegraded apolipoproteins. The sum of trichloroacetic acid-soluble and -insoluble 125I radioactivity expressed as CE equivalents was subtracted from the CE measured as extractable 3H radioactivity to determine the selective uptake of CE from HDL or LDL (16).

Degradation of 125I-alpha 2M*-- Y1/E/tet/2/3 cells were cultured in complete medium in the absence of tet for 4 days. At day 4, the medium was changed to serum-free medium containing 2 mM Bt2cAMP. After 24 h, cells were washed twice with assay medium (Dulbecco's modified Eagle's medium containing Neutridoma serum supplement, 20 mM HEPES, and 1.5% bovine serum albumin) and incubated in assay medium containing 1 nM 125I-alpha 2M* plus or minus RAP or alpha 2M* for different time periods. MEF cells were plated at a density of 2 × 10 5 cells/well in 12-well plates and grown overnight in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37 °C until 80% confluent. Cells were washed twice with assay medium and incubated in assay medium containing 1 nM 125I-alpha 2M* plus or minus RAP or alpha 2M*. After incubation, the medium was removed, precipitated with trichloroacetic acid, and centrifuged, and the supernatant was counted for the measurement of 125I-alpha 2M* degradation (18). Degradation was defined as the amount of radioactivity in the medium that was soluble in 15% trichloroacetic acid. Non-cell-mediated degradation was measured by incubating 125I-alpha 2M* in the absence of cells. Cellular degradation of 125I-alpha 2M* was determined as fmol of 125I-alpha 2M*/105 cells. Inhibition of degradation was performed by incubating Y1/E/tet/2/3 cells with different concentrations of RAP or alpha 2M* for 1 h before the addition of 125I-alpha 2M*.

Inhibition of Proteoglycan Synthesis and Sulfation-- To inhibit proteoglycan synthesis (26), Y1/E/tet/2/3 and Y1-BS1 cells were pre-incubated with 1 mM or 2 mM p-nitrophenyl-beta -D-xylopyranoside (beta -xyloside) for 20 h at 37 °C in serum-free medium before lipoprotein uptake assays. To inhibit sulfation (27) of glycosaminoglycans, cells were preincubated for 20 h at 37 °C in serum-free medium containing 30 mM NaClO3 before the addition of lipoprotein particles. For selective removal of cell surface heparan sulfate proteoglycans, cells were treated for 3 h at 37 °C with 3 unit/ml heparinase I (EC 4.2.2.7) or heparinase I and III together each at 3 units/ml before the addition of the double-labeled lipoprotein particles. For selective removal of chondroitin sulfate and dermatan sulfate proteoglycans, cells were treated with chondroitinase ABC (EC 4.2.2.4) for 3 h at 37 °C with 1 unit/ml before the addition of the double-labeled particles. Each enzyme (Sigma) was dissolved in F-10 medium and used immediately.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Detection of SR-BI Expression in Y1/E/tet/2/3 and Y1-BS1 Cells-- Y1/E/tet/2/3 cells were prepared from the Y1 parent cell line by two rounds of cloning to place apoE expression under control of the tet-off system described by Gossen and Bujard (28). Y1/E/tet/2/3 cells express apoE at 2-2.5 µg/ml in the medium in the absence of tet and suppress apoE expression to 0.1 µg/ml in the presence of tet (16). To examine the effect of Bt2cAMP on SR-BI expression in Y1/E/tet/2/3 cells and to compare with the level of SR-BI expression in Y1-BS1 cells, postnuclear supernatant was used for Western blot analysis using antibody raised against the C-terminal tail of murine SR-BI (20). Fig. 1 shows that Y1/E/tet/2/3 cells plus or minus tet in the presence or absence of Bt2cAMP did not show detectable SR-BI expression. As expected, SR-BI expression was readily detected in Y1-BS1 cells and showed the expected increase in the presence of Bt2cAMP (19). These results indicate that Y1/E/tet/2/3 cells do not make detectable SR-BI in the presence or absence of apoE expression or upon activation of the protein kinase A pathway by Bt2cAMP. A Western blot using an antibody to the C-terminal tail of SR-BII also failed to detect this splice variant of the class B scavenger receptors in the presence or absence of apoE expression (Fig. 2). Note that SR-BI expression was detected in the parent Y1 cell line, from which the Y1/E/tet/2/3 clone was generated, at ~14% of the expression level in the Y1-BS1 cell line as determined by quantitative Western blotting (14). The basis for the loss of SR-BI expression in the Y1/E/tet/2/3 cell line is unknown, but a wide range of SR-BI expression levels has been observed in unselected subclones of the parent Y1 cell line isolated by limited dilution cloning (14).


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Fig. 1.   Immunoblotting of SR-BI in Y1/E/tet/2/3 and Y1-BS1 cells Ten µg of post-nuclear supernatant of each cell extract was electrophoresed on a 10% SDS-polyacrylamide gel under reducing conditions and electroblotted onto nitrocellulose. The blot was probed with antibody raised against the C-terminal tail of murine SR-BI. The mobilities of molecular weight markers are shown on the right side of the gel.


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Fig. 2.   Immunoblotting of SR-BII and LDL receptor family members Post-nuclear supernatant (20 µg of protein) of 2 mM Bt2cAMP-treated Y1/E/tet/2/3 cells grown in the presence or absence of tet were analyzed by SDS, 4-15% PAGE under reducing conditions and electroblotted to nitrocellulose membranes. Membrane strips were probed with the indicated antibody for 3 h at room temperature followed by incubation with secondary antibody and development via chemiluminescence. Positive controls (cont) were provided by alpha -SR-BII (mouse testis extract), alpha -LR8B (extract of COS-7 cells transfected with expression vector for LR8B), alpha -LRP (extract of mouse embryo fibroblast cells), alpha -VLDLR (extract of Y1-BS1 cells).

Effect of RAP on LDL CE Uptake-- Since neither SR-BI nor SR-BII seemed likely to be the basis for the apoE-mediated enhancement of LDL CE selective uptake in Y1/E/tet/2/3 cells, we surveyed the cells for other apoE receptors that are members of the LDL receptor superfamily. As shown in Fig. 2, the LRP/alpha 2M receptor and the VLDL receptor were both detected in Y1/E/tet/2/3 cells with no reproducible differences in expression in the presence or absence of apoE expression. As expected, the LDL receptor was also detected in other Western blots (data not shown). The presence of each of these receptors in normal adrenal cells was confirmed with Western blots of extracts from Y1-BS1 cells and from mouse adrenal glands (data not shown). Western blots with antibody against the LR8B/apoER2 receptor failed to detect this receptor in Y1/E/tet/2/3 cells (Fig. 2), Y1-BS1 cells, or mouse adrenal glands, although this antibody against chicken LR8B/apoER2 readily detected this receptor in extracts of mouse Neuro2A cells (29). Antibody against gp330 detected a faint band in extracts of Y1/E/tet/2/3 cells, but this was not seen with Y1-BS1 cells or mouse adrenal glands (data not shown). Thus, the LDL receptor, the LRP/alpha 2M receptor, and the VLDL receptor were the most prominent members of the LDL receptor family detected in adrenal cells.

To determine whether these members of the LDL receptor family might play a role in apoE-dependent LDL CE selective uptake, Y1/E/tet/2/3 cells were incubated with RAP, which inhibits the binding of apoE as well as other ligands to many members of this receptor family (30, 31). As shown in Fig. 3A, increasing concentrations of RAP blocked the apoE-dependent component of LDL CE selective uptake in Y1/E/tet/2/3 cells but had no effect on LDL CE selective uptake in the absence of apoE expression. In a separate experiment with Y1/E/tet/2/3 cells induced to express apoE, RAP inhibited LDL CE selective uptake but had no effect on HDL CE selective uptake (Fig. 3B). Fifty percent of the apoE-dependent LDL CE selective uptake component was inhibited at a RAP concentration of ~50 nM.


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Fig. 3.   Effect of RAP on LDL CE and HDL CE selective uptake in Y1/E/tet/2/3 cells. Panel A, Y1/E/tet/2/3 cells were cultured in complete medium in the presence or absence of tet for 4 days. At day 4, medium was changed to serum-free medium containing 2 mM Bt2cAMP plus or minus tet. After 24 h RAP was added to the indicated concentrations and incubated for 3 h at 37 °C, after which 50 µg/ml 125I-labeled [3H]LDL was added for another 4 h. Cells were processed to determine LDL CE selective uptake as described under "Experimental Procedure." Panel B, in a separate experiment after the addition of RAP for 3 h, cells were incubated with 50 µg/ml 125I-labeled [3H]LDL or 125I-labeled [3H]HDL for another 4 h. Cells were processed to determine CE selective uptake. Data are shown as the percent of CE selective uptake in the absence of RAP. Values represent mean ± S.E. (n = 3).

The effect of RAP on both LDL CE selective uptake and HDL CE selective uptake was also tested in Y1-BS1 adrenocortical cells in which the majority of LDL CE and HDL CE selective uptake is due to SR-BI (14, 32). At neither of two lipoprotein concentrations (10 µg/ml or 100 µg/ml) did RAP at 1 µM have an appreciable effect (<10% inhibition) on LDL CE selective uptake or HDL CE selective uptake in Y1-BS1 cells (data not shown). Thus, SR-BI-mediated LDL CE and HDL CE selective uptake is not sensitive to RAP inhibition in Y1-BS1 cells, whereas apoE-dependent LDL CE selective uptake in Y1/E/tet/2/3 cells that do not express SR-BI is completely sensitive to RAP.

Role of LRP/a2M Receptor in LDL CE Uptake-- Based on the RAP inhibition data, we asked whether the LRP/alpha 2M receptor might be responsible for apoE-dependent LDL CE selective uptake. Functional activity of the LRP/alpha 2M receptor in Y1/E/tet/2/3 cells in the presence of apoE was first tested by monitoring the degradation of 125I-alpha 2M*. As shown in Fig. 4A, Y1/E/tet/2/3 cells degraded 125I-alpha 2M* at a rate similar to that of mouse embryo fibroblast cells, which are known to express the LRP/alpha 2M receptor. Both RAP and alpha 2M* blocked the degradation of 125I-alpha 2M* in Y1/E/tet/2/3 cells. In addition, RAP inhibited 50% of alpha 2-M* degradation by Y1/E/tet/2/3 cells at a concentration of 50-100 nM (Fig. 4B), which is similar to the concentration dependence for inhibition of apoE-dependent LDL CE selective uptake (Fig. 3B).


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Fig. 4.   Effect of RAP and alpha 2M* on degradation of 125I-alpha 2M* in Y1/E/tet/2/3cells and MEF cells. Panel A, Y1/E/tet/2/3 cells were cultured in complete medium in the absence of tet for 4 days. At day 4, medium was changed to serum-free medium containing 2 mM Bt2cAMP. After 24 h, cells were washed twice with assay medium and incubated with 1 nM 125I-alpha 2M* plus or minus RAP or alpha 2M*. After incubation, medium was removed for the measurement of 125I-alpha 2M* degradation. MEF cells were plated at a density of 2 × 10 5 cells/well in a 12-well plate and grown overnight in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37 °C. Cells were then washed twice with assay medium and used for the degradation assay as described above. Panel B, inhibition of 125I-alpha 2M* degradation was performed by incubating Y1/E/tet/2/3 cells with different concentrations of RAP for 1 h before the addition of 125I-alpha 2M*. Cells were incubated for 12 h at 37 °C and processed to determine 125I-alpha 2M* degradation. Samples containing no RAP were taken as 100% of control. Error bars represent the range of triplicate determinations.

Inhibition by RAP is consistent with the participation of the LRP/alpha 2M receptor but is not diagnostic for this receptor alone, since RAP interacts with other members of the LDL receptor family (30, 31). In contrast, alpha 2M* is specific for the LRP/alpha 2M receptor. The experiment in Fig. 5A shows that alpha 2M* inhibited ~30% of the total LDL CE selective uptake activity in apoE-expressing Y1/E/tet/2/3 cells. The enhancement of LDL CE selective uptake by apoE expression in this experiment was ~2-fold (Fig. 5B); hence, the 30% inhibition of total LDL CE selective uptake by alpha 2M* reflects a 60% inhibition of apoE-dependent LDL CE selective uptake. In three such experiments, alpha 2M* inhibited an average of 68% of apoE-dependent LDL CE selective uptake but inhibited less than 10% of LDL CE selective uptake in Y1/E/tet/2/3 cells not induced to express apoE or in Y1-BS1 cells that do not express apoE (data not shown). These data suggest that the LRP/alpha 2M receptor is responsible for a substantial fraction of the apoE-dependent LDL CE selective uptake in Y1/E/tet/2/3 cells. We attempted to confirm the role of the LRP/alpha 2M receptor by blocking the activity with antibody against human LRP/alpha 2M receptor. Under conditions where the antibody effectively blocked 125I-alpha 2M* uptake, we obtained inconsistent inhibition of apoE-dependent LDL CE selective uptake. In two experiments, 50-60% of the apoE-dependent component was blocked by anti-LRP/alpha 2M receptor, but in two other experiments, no inhibition was seen.


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Fig. 5.   Inhibition of LDL CE selective uptake by alpha 2M*. Panel A, Y1/E/tet/2/3 cells were cultured in complete medium in the absence of tet for 4 days. At day 4, medium was changed to serum-free medium containing 2 mM Bt2cAMP minus tet. After 24 h, RAP or alpha 2M* was added to the indicated concentration and incubated for 3 h at 37 °C. Fifty µg/ml 125I-labeled [3H]LDL was then added and incubated for another 4 h. Cells were processed to determine selective CE uptake. Panel B, in the same experiment, one set of Y1/E/tet/2/3 cells was cultured in complete medium in the presence of tet for 4 days. At day 4, medium was changed to serum-free medium containing 2 mM Bt2cAMP plus tet. After 24 h, 50 µg/ml 125I-labeled [3H]LDL was added for 4 h. Cells were processed to determine selective CE uptake. Values represent mean ± S.E. (n = 3).

Role of Cell Surface Proteoglycan in ApoE-dependent LDL CE Selective Uptake-- Cell surface proteoglycans are known to be important for the endocytic uptake of apoE-containing beta -VLDL particles by the LRP/alpha 2M receptor (33, 34). In addition, direct internalization of lipoproteins bound to the cell surface via heparan sulfate proteoglycans, particularly syndecan and perlecan, may occur without the participation of an LDL receptor family member (35, 36). To test the role of proteoglycan in apoE-dependent LDL CE selective uptake in Y1/E/tet/2/3 cells, we used two approaches to inhibit proteoglycan formation. In the first, the sulfation of glycosaminoglycan side chains was inhibited by treatment of cells with 30 mM sodium chlorate for 20 h before the addition of 125I-labeled [3H]LDL. In three experiments, treatment of Y1/E/tet/2/3 cells with sodium chlorate caused about 75% inhibition of the apoE-dependent LDL CE selective uptake (Fig. 6A) but had no effect on SR-BI-mediated LDL CE selective uptake in Y1-BS1 cells that do not make apoE (Fig. 6B). In the second approach, cells were treated with 1 or 2 mM p-nitrophenyl-beta -D- xylopyranoside (beta -xyloside) to inhibit the incorporation of glycosaminoglycan side chains into cell surface proteoglycans before incubation with 50 µg/ml 125I-labeled [3H]LDL. The results in Fig. 7A show that beta -xyloside completely blocked apoE-dependent LDL CE selective uptake in Y1/E/tet/2/3 cells but had no effect on LDL CE selective uptake in Y1/E/tet/2/3 cells when apoE expression was suppressed. Similarly, this inhibitor had no effect on SR-BI-mediated LDL CE selective uptake in Y1-BS1 cells that do not make apoE (Fig. 7B). These results indicate that cell surface proteoglycan is essential for apoE-dependent LDL CE selective uptake in mouse adrenocortical cells.


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Fig. 6.   Effect of sodium chlorate treatment on LDL CE selective uptake in Y1/E/tet/2/3 cells and Y1-BS1 cells. Panel A, Y1/E/tet/2/3 cells were maintained in complete F-10 medium minus tet for 4 days. At day 4, medium was changed to serum-free medium containing 2 mM Bt2cAMP plus 30 mM sodium chlorate and incubated for 20 h at 37 °C. The double-labeled LDL (100 µg/ml) was then added and incubated for 4 h. Cells were processed to determine LDL CE selective uptake. The percent inhibition by chlorate of the apoE-dependent component of LDL CE selective uptake was determined by subtracting the -apoE value from the +apoE value in both treatment groups and then expressing the difference between the resultant values as a percent of the apoE-dependent value for the control group. Panel B, Y1-BS1 cells were cultured in serum-free medium plus 30 mM sodium chlorate for 20 h at 37 °C. Double-labeled LDL (50 µg/ml) was then added and incubated for 4 h. Cells were processed to determine LDL CE selective uptake. Values represent the mean ± S.E. (n = 3).


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Fig. 7.   Effect of p-nitrophenyl-beta -D-xyloside on selective uptake of LDL CE by Y1/E/tet/2/3 cells and Y1-BS1 cells. Panel A, Y1/E/tet/2/3 cells were maintained in complete F-10 medium minus tet for 4 days. At day 4, medium was changed to serum-free medium containing 2 mM Bt2cAMP plus the indicated concentration of beta -D-xyloside and incubated for 20 h at 37 °C. Double-labeled LDL (50 µg/ml) was then added for 4 h. Cells were processed to determine LDL CE selective uptake. Panel B, Y1-BS1 cells in serum-free medium were incubated with the indicated concentration of beta -D-xyloside for 20 h at 37 °C. Double-labeled LDL (50 µg/ml) was then added for 4 h. Cells were processed to determine LDL CE selective uptake. Values represent the mean ± S.E. (n = 3).

Effect of Heparinase and Chondroitinase on ApoE-dependent LDL CE Uptake-- To test the specific effect of heparan sulfate proteoglycan and chondroitin sulfate proteoglycan in apoE-dependent LDL CE selective uptake, Y1/E/tet/2/3 cells were subjected to enzymatic digestion of glycosaminoglycan side chains by proteoglycan lyases. When cells were treated with 3 units/ml heparinase I or heparinase I and III together for 3 h at 37 °C there was at most a 20% reduction of apoE-dependent LDL CE selective uptake (Fig. 8). In addition, both heparinase II and III had an inhibitory effect of less than 20% on apoE-dependent LDL CE selective uptake (data not shown). However, upon treatment of Y1/E/tet/2/3 cells with 1 unit/ml chondroitinase ABC at 37 °C for 3 h, apoE-dependent LDL CE selective uptake was inhibited by greater than 90%, suggesting the participation of chondroitin sulfate and/or dermatan sulfate proteoglycans. Neither chondroitinase ABC nor the heparinases had any effect on the apoE-independent component of LDL CE selective uptake in Y1/E/tet/2/3 cells (Fig. 8). Note in the above studies with proteoglycan synthesis inhibitors and proteoglycan lyases the reductions in apoE-dependent LDL CE selective uptake were paralleled by similar reductions in the cell association of LDL particles (data not shown).


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Fig. 8.   Effect of heparinase and chondroitinase treatment on LDL CE selective uptake in Y1/E/tet/2/3 cells. Y1/E/tet/2/3 cells were cultured in complete medium in the absence or presence of tet for 4 days. At day 4, medium was changed to serum-free medium containing 2 mM Bt2cAMP plus or minus tet. After 24 h heparinase I (3 unit/ml) or heparinase I and III (3 unit/ml each) or chondroitinase ABC (1 unit/ml) was added for 3 h at 37 °C. Double-labeled LDL (50 µg/ml) was then added for another 4 h, and cells were processed to determine LDL CE selective uptake. Each data point represents the mean ± S.E. (n = 3).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The major finding in this study is the identification of an LDL CE selective uptake pathway in murine adrenocortical cells that requires apoE but is independent of SR-BI. The apoE-dependent LDL CE selective uptake pathway involves both an alpha 2M receptor and cell surface proteoglycans. As judged by quantitative comparisons of apoE-dependent LDL CE selective uptake in Y1/E/tet/2/3 cells with SR-BI-dependent LDL CE selective uptake in Y1-BS1 cells (Figs. 6 and 7, for example), both pathways have the capacity to take up similar amounts of LDL CE (3-6 µg of CE/4 h/mg of cell protein at 50 µg/ml LDL). Interestingly, with both the apoE-dependent pathway (16) and the SR-BI-dependent pathway (14) the majority of LDL CE is taken up by cultured adrenal cells by the selective pathway as opposed to endocytic processing of LDL particles. LDL CE selective uptake has been observed in steroidogenic cells and the liver in vivo (4, 6) as well as in a variety of cultured cells such as human fibroblasts (11), rat and human liver cells (6, 11-13), and a murine macrophage cell (11). The contributions of the SR-BI-dependent versus apoE-dependent LDL CE selective uptake pathways in each of these cell types in culture and in vivo is currently unknown. However, many of these cell types including macrophage, hepatocytes, and steroidogenic cells synthesize substantial quantities of apoE (37-48), raising the possibility that apoE-dependent LDL CE selective uptake is a widespread process in LDL metabolism.

The finding that RAP inhibited the apoE-dependent component of LDL CE selective uptake suggested that a member of the LDL receptor family might be involved. Western blots showed that Y1/E/tet/2/3 cells expressed three members of the LDL receptor family, two of which (LRP/alpha 2M receptor and VLDL receptor) are inhibited by RAP concentrations similar to those that inhibit alpha 2M* degradation. Furthermore, approximately two-thirds of the apoE-dependent LDL CE selective uptake was inhibited by alpha 2M*. Since the LRP/alpha 2M receptor is the only known receptor for alpha 2M*(30, 31), these data provide strong evidence that the LRP/alpha 2M receptor participates in the apoE-dependent selective uptake of LDL CE. These results, however, do not eliminate the potential participation of other LDL receptor family members, some of which have only recently been identified (49).

The mechanism through which the LRP/alpha 2M receptor participates in LDL CE selective uptake is unclear. Results of studies with inhibitors of proteoglycan biosynthesis clearly show that proteoglycans are necessary for apoE-dependent LDL CE selective uptake. This result is further supported by the finding that treatment of cells with chondroitinase ABC completely abolished apoE-dependent LDL CE selective uptake. Thus, the participation of the LRP/alpha 2M receptor in the LDL CE selective uptake process requires cell surface proteoglycans. In addition it appears likely that a portion of the apoE-dependent LDL CE selective uptake occurs as a result of proteoglycan interactions independently of the LRP/alpha 2M receptor since alpha 2M* inhibited only 68% of the apoE-dependent LDL CE uptake, whereas RAP and inhibitors of proteoglycan synthesis completely blocked apoE-dependent CE uptake. RAP binds with high affinity to most LDL receptor family members but also binds with lower affinity to cell surface proteoglycan with a KD of greater than 200 nM (50, 51). The more gradual inhibition of LDL CE selective uptake at higher RAP concentrations (100-2000 nM, Fig. 3) may reflect the inhibition of LDL CE selective uptake due to direct interaction of apoE-enriched LDL with proteoglycan.

Previous studies showed that an LDL particle acquires approximately one molecule of apoE upon incubation with Y1/E/tet/2/3 cells (16). In addition, the enhancement of LDL CE selective uptake by apoE also requires apoE expression by Y1/E/tet/2/3 cells (16), suggesting that both LDL-bound apoE and cell surface-bound apoE are necessary. A likely scenario is that apoE serves a bridging function to localize LDL particles to the cell surface via interactions with proteoglycans via the LRP/alpha 2M receptor or to a ternary complex of apoE-enriched LDL, the LRP/alpha 2M receptor, and proteoglycans. The observation that inhibition of proteoglycan synthesis or treatment of cells with chondroitinase ABC blocks apoE-dependent LDL cell association as well as LDL CE selective uptake suggests that the primary role of apoE is at the step of LDL binding to the cell surface. The findings reported here on apoE-dependent LDL CE selective uptake have striking parallels to previous studies on the apoE-mediated enhancement of chylomicron remnant uptake into liver cells in response to apoE enrichment of beta -VLDL particles (33, 34, 51-54). Very similar proposals have been made to explain the roles of the LRP/alpha 2M receptor and proteoglycans in chylomicron remnant uptake by hepatocytes. In that case, however, the uptake of beta -VLDL CE is believed to occur primarily via endocytic uptake of intact beta -VLDL particles (33).

Irrespective of the details of how the apoE-enriched LDL particle is bound to the cell surface, it seems likely that the apoE-dependent selective uptake of LDL CE differs in important ways from the process mediated by SR-BI. For example, SR-BI is believed to be localized to cholesterol/sphingolipid-enriched membrane microdomains or caveolae that appear to be the site of CE uptake from HDL particles (9, 55, 56). In contrast, the LRP/alpha 2M receptor and other members of the LDL receptor family are targeted for endocytosis via coated pits (57). As recently discussed (33), cell surface binding of remnant-like particles in the livers of LDL receptor-deficient mice is very rapid, but endocytosis via an apoE-dependent pathway (presumably the LRP/alpha 2M receptor) is very slow compared with that mediated by the LDL receptor (58). This suggests that the LRP/alpha 2M receptor is relatively inefficient at endocytosis and that the residence time on the cell surface for an apoE-enriched LDL bound to the LRP/alpha 2M receptor may be very long. It is also of note from other studies that LDL bound to cell surface proteoglycan occurs in two kinetic pools, one of which appears to be a sequestered pool that is internalized slowly, if at all (59). Although speculative, we suggest that a prolonged residence time of apoE-enriched LDL on the cell surface, whether bound to the LRP/alpha 2M receptor or proteoglycan, will lead to selective CE transfer to the cell membrane. Whether this occurs because some of the bound LDL is directed to caveolae or this process is independent of these membrane domains is unknown. Interestingly, the recently reported enhancement of LDL CE selective uptake by lipoprotein lipase (60) also requires cell surface proteoglycan as well as interaction of lipoprotein lipase with both LDL and the proteoglycan. These data as well as previous results (16) and the results of the present study suggest the hypothesis that bridging proteins such as apoE or lipoprotein lipase or hepatic lipase facilitate the selective uptake of lipoprotein CE by holding the lipoprotein particle on the cell surface for prolonged periods of time.

The present results on apoE-dependent LDL CE selective uptake show an interesting difference in comparison to the secretion/capture hypothesis for apoE-mediated chylomicron remnant uptake into hepatocytes due to the LRP/alpha 2M receptor and/or proteoglycans (33). In the case of beta -VLDL uptake into hepatocytes, heparan sulfate proteoglycans appear to be of major importance for the enhancement by apoE (34). In contrast, in the Y1/E/tet/2/3 adrenal cells, lyases specific for heparan sulfate proteoglycans had only modest effects, whereas chondroitinase completely abolished apoE-dependent LDL CE selective uptake. This result suggests that apoE-enriched LDL interacts with chondroitin sulfate or dermatan sulfate proteoglycan on adrenocortical cells. This difference with the results of beta -VLDL uptake in hepatoma cells (33) could reflect cell-type differences in proteoglycan composition or differences in how apoE-enriched LDL versus apoE-enriched beta -VLDL interact with different proteoglycans. Interestingly, Burgess et al. (61) noted that a major pool of HepG2 cell surface apoE is associated with chondroitin sulfate proteoglycans. Similarly, there is ample documentation of binding of chondroitin/dermatan sulfate proteoglycan to LDL both in vivo and in vitro (62-64). In this regard, it is of interest that a basal level of LDL CE selective uptake occurs in the Y1/E/tet/2/3 cells in the absence of apoE expression (Figs. 3A, 5B, 6A, 7A, and 8). Although the nature of this pathway is not understood, we have observed that heparin blocks this apoE-independent process (data not shown). This result is consistent with the possibility that the apoE-independent LDL CE selective uptake in the Y1/E/tet/2/3 cells is due to an apoB100/proteoglycan interaction that serves to localize LDL particles to the cell surface.

In summary, we report here that delivery of LDL CE by the selective uptake process occurs via multiple pathways in mouse adrenocortical cells. One pathway is apoE-dependent and involves the LRP/alpha 2M receptor and proteoglycans. A second pathway is SR-BI-dependent, occurs in the absence of apoE, and does not require cell surface proteoglycan. Additional studies will be required to assess the contributions of these pathways to LDL CE selective uptake in various cell types both in culture and in vivo.

    ACKNOWLEDGEMENTS

We thank J. Herz and J. Nimpf for providing antibodies and Margery Connelly for purification of cholesteryl ester transfer protein.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL 58012, HL 32868, HL 50784, and HL 54710 and by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Pharmacological Sciences, University Medical Center, State University of New York, Stony Brook, NY 11794. Tel.: 516-444-3083; Fax: 516-444-3218; E-mail: dave@pharm.som.sunysb.edu.

Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M101691200

    ABBREVIATIONS

The abbreviations used are: LDL, low density lipoprotein; VLDL, very LDL; HDL, high density lipoprotein; apo, apolipoprotein; SR-BI, scavenger receptor class B type I; CE, cholesteryl ester; alpha 2M, alpha 2-macroglobulin; alpha 2M*, methylamine-activated alpha 2M; LRP/alpha 2M receptor, LDL receptor-related protein/alpha 2M receptor; tet, tetracycline; RAP, receptor-associated protein; Bt2cAMP, dibutyryl cyclic AMP; TBS, Tris-buffered saline; human 125I-labeled [3H]HDL3, 125I-dilactitol tyramine-[3H]cholesteryl oleolyl ether human HDL3; human 125I-labeled [3H]LDL, 125I-dilactitol tyramine-[3H]cholesteryl oleolyl ether human LDL.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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