© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Apolipoprotein AII Enrichment of HDL Enhances Their Affinity for Class B Type I Scavenger Receptor but Inhibits Specific Cholesteryl Ester Uptake
From INSERM U325, Institut Pasteur de Lille et Université Lille 2, Lille, France.
Correspondence to V. Clavey, INSERM U325, Institut Pasteur de Lille, 1 rue du Professeur Calmette, BP 245, F-59019 Lille Cédex, France. E-mail Veronique.Clavey{at}pasteur-lille.fr
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Abstract |
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Abstract—Apolipoproteins of high density lipoprotein (HDL) and especially apolipoprotein (apo)AI and apoAII have been demonstrated as binding directly to the class B type I scavenger receptor (SR-BI), the HDL receptor that mediates selective cholesteryl ester uptake. However, the functional relevance of the binding capacity of each apolipoprotein is still unknown. The human adrenal cell line, NCI-H295R, spontaneously expresses a high level of SR-BI, the major apoAI binding protein in these cells. As previously described for murine SR-BI, free apoAI, palmitoyl-oleoyl-phosphatidylcholine (POPC)-AI, and HDL are good ligands for human SR-BI. In vitro displacement of apoAI by apoAII in HDLs or in Lp AI purified from HDL by immunoaffinity enhances their ability to compete with POPC-AI to bind to SR-BI and also enhances their direct binding capacity. The next step was to determine whether the higher affinity of apoAII for SR-BI correlated with the specific uptake of cholesteryl esters from these HDLs. Free apoAII and, to a lesser extent, free apoAI that were added to the cell medium during uptake experiments inhibited the specific uptake of [3H]cholesteryl esters from HDL, indicating that binding sites on cells were the same as cholesteryl ester uptake sites. In direct experiments, the uptake of [3H]cholesteryl esters from apoAII-enriched HDL was highly reduced compared with the uptake from native HDL. These results demonstrate that in the human adrenal cell line expressing SR-BI as the major HDL binding protein, efficient apoAII binding has an inhibitory effect on the delivery of cholesteryl esters to cells.
Key Words: adrenal cells • scavenger receptor, class B type I • cholesteryl esters • apolipoprotein AI • apolipoprotein AII
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Introduction |
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Liver and adrenal selective uptake of HDL cholesteryl esters (CEs) appears to be a receptor-mediated process. The class B type I scavenger receptor (SR-BI), a molecularly well-defined cell surface HDL receptor for selective cholesterol uptake, has recently been identified.1 First described in rodents, its human analogue, initially called CLA-1, has a protein sequence identity close to that of the rodent receptor.2 3 Selective uptake of CE does not involve endocytic uptake and lysosomal degradation of lipoprotein particles. The first step in the cellular mechanism of this pathway involves HDL binding and then the incorporation of HDL-derived CE into the plasma membrane.4 After uptake, CEs are directed intracellularly to a nonlysosomal destination for degradation.5
In mice, SR-BI is largely expressed in liver, and overexpression of the SR-BI level in vivo on hepatocytes induces the disappearance of plasmatic HDL and the doubling of biliary cholesterol6 ; inversely targeted disruption of the SR-BI gene induces a 2.2-fold increase in plasma cholesterol concentration.7 All these results strongly suggest that SR-BI plays a key role in hepatic HDL metabolism in rodents. Also in rodents, SR-BI seems to play an important role in the maternal-fetal lipoprotein transport system during embryogenesis.8 Azhar et al9 have shown that the induction of the SR-BI receptor and the HDL-selective cholesterol uptake pathway in rat granulosa cells appear to be linked morphologically, biochemically, and functionally. Last, Temel et al10 provided the first evidence that SR-BI was involved directly in mediating selective CE uptake in cultured mouse adrenocortical cells.
In humans, SR-BI is expressed in the liver and mostly in steroidogenic tissues: adrenal gland, testis,11 ovary,3 adrenal tumors, and cultured adrenal cells.12 These data support the hypothesis that SR-BI is involved in selective CE uptake in human adrenocortical cells even if the respective relative importance of the LDL and HDL pathway is not demonstrated.
The detailed mechanism of HDL–SR-BI interaction in CE uptake is not yet well defined. Although previous studies have shown that murine SR-BI can bind anionic phospholipid liposomes, LDL, HDL, and modified lipoproteins with high affinity and have hypothesized that lipids on the surface of lipoproteins are the principal determinants of binding to SR-BI,13 the protein-protein interactions seem to play an important role in the first step of the pathway. It has been demonstrated that HDL and LDL do not compete to bind to SR-BI14 and that individual apolipoproteins from HDL, eg, AI, AII, and CIII, can all mediate binding to SR-BI, either free or incorporated in lipid complexes.15 Human SR-BI seems to have the same broad multiligand binding capacity.11 16
Although several distinct apolipoproteins are associated with HDL, apoAI and apoAII together represent >85% of total HDL protein. In plasma, different populations of HDLs have been identified, and HDLs containing apoAI without apoAII (LpAI) promote cholesterol efflux from peripheral cells, whereas HDLs simultaneously containing apoAI and apoAII (LpAI-AII) have an antagonist effect on this process.17 Recent results from Rinninger et al4 have demonstrated that in human fibroblasts and hepatic cells, CEs are selectively taken up to a higher extent from LpAI than from LpAI-AII.
We previously demonstrated that the NCI-H295R human adrenal cell line, which synthesizes steroid hormones, also strongly expresses SR-BI in an cAMP-dependent way.18
In the present study, we have established that in these cells SR-BI is the major apoAI binding protein and that these cells can be used to study the different ligands of this receptor. We have focused on the respective role of the 2 major HDL proteins, apoAI and apoAII, regarding binding to SR-BI and the consequences for CE uptake. We have provided the evidence that apoAII is a better ligand than is apoAI but that it has an antagonist effect on CE uptake.
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Methods |
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Materials
Antibodies against apoAI were an equimolar mixture of mouse monoclonal antibodies against apoAI (A03, A51, A17, and A05).19 Antibodies against SR-BI were rabbit polyclonal antibodies against the 470 to 509 COOH terminal peptide corresponding to the cytoplasmic tail of the human CLA-1/SR-BI protein.2 Antibodies were purified by affinity chromatography on a Sepharose-peptide column. Peroxidase-labeled goat anti-mouse antibodies and goat anti-rabbit antibodies came from Sanofi Diagnostics Pasteur, SDS-PAGE (4% to 12%) and native PAGE (4% to 20%) precast gradient NuPage gels were obtained from Novex, and molecular mass standards were from Bio-Rad.
Palmitoyl-oleyl-phosphatidylcholine (POPC) and dimyristoyl-phosphatidylcholine (DMPC) were from Sigma Chemical Co.
Lipoprotein and Apolipoprotein Isolation and Labeling,
Reconstituted and Modified HDL Preparation
LDLs and apoE free HDL fraction 3
(HDL3) were prepared from human plasma by
sequential ultracentrifugation at a density (d) of
1.030 g/mL<d<1.053 g/mL and 1.12 g/mL<d<1.21 g/mL,
respectively.20 Lack of apoE in HDL3
was tested by SDS-PAGE electrophoresis. HDL3
molecular weight determined by native 4/20% PAGE was 
180 kDa.
Lipids from HDL were measured by enzymatic methods. ApoAI and apoAII were prepared from HDL3 as previously described.21
Reconstituted HDL, DMPC-AI, POPC-AI, and POPC AII were prepared by the
cholate dialysis method22 at a POP/apoAI or POP/apoAII
ratio of 100/1 and a DMP/apoAI ratio of 150/1 (mole/mole). The majority
of the particles (>80%) exhibited a homogeneous
electrophoretic mobility in native 4/20% PAGE, and molecular mass,
which was determined by comparison with a calibration curve constructed
with albumin (7.1 nm), lactate dehydrogenase (8.16 nm),
ferritin (12.2 nm), and thyroglobulin (17.0 nm), was 220 kDa for
DMPC-AI (10.1 nm), 201 kDa for POPC-AI (9.55 nm), and 188 kDa for
POPC-AII (9.33 nm). Because the POPC/protein ratio was 100/1 and
because the molecular weight was 188 kDa for POPC-AII and 201 kDa for
POPC-AI, we presumed that these complexes contained 2 apolipoproteins
per particle and were expected to be discoidal.23
LpAI was prepared from HDL3 by immunoaffinity. The unretained fraction on anti-apoAII immunosorber was purified on anti-apoAI immunosorber, and the retained fraction was eluted by 3 mol/L NaSCN.4
Apolipoproteins in lipoproteins (native or reconstituted HDL) were radiolabeled with [125I]iodine.24 The final specific activities varied between 400 and 1500 disintegrations per minute (dpm) per nanogram of protein and were homogeneous in a same experiment. HDL3 was also labeled with [3H]CE by incubation overnight at 37°C with a plasma fraction enriched with cholesteryl ester transfer protein (d>1.21 g/mL).25 The final specific activities varied between 45 and 400 dpm/ng of cholesterol and were homogeneous in the same experiment. Concentrations of labeled HDL were expressed in protein concentration or in total cholesterol concentration.
Displacement of apoAI by apoAII in lipoproteins was obtained by incubating HDL3 (native or labeled) or LpAI fractions for 2 hours at 37°C with different concentrations of apoAII by use of the procedure of Lagrost et al,26 modified as indicated. ApoAII was added to lipoproteins in AII/HDL protein mass ratios from 0.5 to 6. After incubation, lipoproteins were centrifuged at d=1.21 g/mL and then dialyzed in a Centricon 100 (Amicon) to remove free apolipoproteins (absence of free apolipoprotein was checked by nondenaturing PAGE). As previously described,26 these enriched HDLs had a slight increase in the mean apparent diameter (from 8.8 to 9.3 nm). Apolipoprotein composition of modified lipoproteins was checked by SDS-PAGE.
SDS-PAGE
Apolipoproteins of HDL3, native or
modified by incubation with apoAII, were analyzed by
electrophoresis in nonreducing conditions on 4/12% precast
polyacrylamide gels. Proteins were stained with Coomassie
brilliant blue.
Cell Culture and Membrane Preparation
Adrenocortical carcinoma NCI-H295R adherent cells were obtained
from the American Type Culture Collection (ATCC CRL-2128). Cells were
maintained in DMEM/F12 (1/1) medium containing 2% FCS as previously
described.27 Cell membranes were prepared according to
Basu et al.28 Briefly, cells were scraped and pelleted by
centrifugation, homogenized with a Bioblock
375W ultrasonic homogenizer, and centrifuged
for 10 minutes at 10 000g. The supernatant was
centrifuged at 100 000g for 1 hour at 4°C, and
the pellet was resuspended in Laemmli buffer29 and
stored frozen before analysis.
Cross-Linking of ApoAI and SR-BI in NCI-H295R Cells and
Immunoblot Assay
Cells were cultured in 2% Ultroser SF medium (free of
lipoproteins) for 24 hours to dissociate or internalize lipoproteins
bound to the plasma membrane. Then cells were incubated for 1 hour at
37°C without (control cells) or with 10 µg/mL POPC-AI. After
washing, cells were incubated for 1 hour at 4°C in HEPES buffer
(10 mmol/L HEPES and 150 mmol/L NaCl, pH 8.0) containing
0.5 mmol/L dithio-bis(succinimidyl propionate)
(DSP).30 Cell membranes were prepared as described earlier
and analyzed by SDS-PAGE and immunoblotting.
After transfer onto nitrocellulose sheets, the SR-BI/apoAI complexes
were revealed either with the monoclonal antibodies against apoAI, or
with the polyclonal antibodies against SR-BI. After incubation with the
primary antibodies, the sheets were incubated with peroxidase-labeled
goat anti-mouse or goat-anti rabbit antibodies and revealed by enhanced
chemiluminescence (Amersham).
Immunoprecipitation experiments with 10 µg anti–SR-BI antibodies were performed on 150-µg membranes from cells incubated with POPC-AI and then DSP. Immuno-complexes were then precipitated with protein A–Sepharose beads. After SDS-PAGE and transfer, the sheets were revealed with antibodies against apoAI or with peroxidase-labeled anti–SR-BI antibodies.
Binding of 125I-Labeled Lipoproteins and Competition
With Unlabeled Apolipoproteins and Lipoproteins
Cells cultured in 12-well plates were washed and preincubated
for 1 hour at 37°C in serum-free medium. For direct binding, cells
were incubated for 2 hours at 4°C or 1 hour at 37°C with labeled
lipoproteins without (total binding) or with a 40-fold excess of
unlabeled HDL3 (nonspecific binding). Specific
binding was calculated as the difference between total and nonspecific
binding. Cells were washed and dissolved with 1 mol/L NaOH, and
cell-associated radioactivity was counted. An aliquot was used to
quantify cell proteins.31 Results were expressed as
nanograms of bound or degraded proteins per milligram of cellular
protein. Kd and Bmax
values were calculated from Scatchard plots, and curves were linearized
by regression analysis on a 1-site model basis. Apolipoprotein
degradation was measured in cell medium after incubation at 37°C and
corresponds to the non–trichloroacetic acid–precipitable
material.32 Competition studies were performed with
[125I]POPC-AI at 2 µg/mL and increasing
concentrations of the different unlabeled apolipoproteins or
lipoproteins for 1 hour at 37°C. Cells were preincubated, washed, and
then counted as in direct binding studies. Results were expressed as a
percentage of the binding measured without competitor.
Uptake of [3H]CE-Labeled Lipoproteins
Cells cultured in 12-well plates were washed and preincubated
for 1 hour at 37°C in DMEM/F12 serum-free medium. They were then
washed and incubated for the indicated times with
[3H]CE-labeled lipoproteins without (total
uptake) or with a 50-fold excess of unlabeled
HDL3 (nonspecific uptake).33 Cells
were then washed and dissolved with 1 mol/L NaOH, and cell-associated
radioactivity was counted. An aliquot was used to quantify cell
proteins.31 It was verified by isopropanol extraction that
all the cell-associated radioactivity was lipid-associated. Results are
expressed as nanogram or microgram cholesterol incorporated
per milligram cellular protein.
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Results |
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POPC-AI Binding to NCI-H295R Cells
Membrane preparations of NCI-H295R cells expressed an 80-kDa protein that was revealed with anti–SR-BI antibodies (Figure 1A
, lane 1). When cells were incubated
with DSP, a bifunctional cross-linking reagent, complexes were formed
that reacted with anti–SR-BI (Figure 1A, lane 2, upper band).
These complexes could represent SR-BI association with any
unknown cellular protein. Incubation with POPC-AI in the presence of
DSP prevented SR-BI complex formation with cellular proteins and made
way for the formation of high molecular weight complexes containing
SR-BI and apoAI, which reacted with anti–SR-BI (Figure 1A, lane
3) and with anti-apoAI antibodies (Figure 1A, lane 4). The 3
bands of higher molecular weight containing apoAI also contained SR-BI.
Others complexes of 100 and 120 kDa were present but were
perhaps apoAI aggregates, although weak SR-BI–containing bands with
the same molecular weight can be observed. All these complexes did not
appear in the absence of apoAI. To confirm these results,
immunoprecipitation of SR-BI complexes with anti–SR-BI antibodies was
performed on membranes from cells incubated with POPC-AI and DSP. After
SDS-PAGE and immunotransfer onto nitrocellulose, the immunoprecipitate
was revealed with anti-apoAI antibodies (Figure 1B, lane 2).
Numerous complexes with high molecular weights were revealed,
indicating that apoAI was covalently linked to SR-BI and coprecipitated
with SR-BI during specific immunoprecipitation. No band was revealed in
control immunoprecipitation with preimmune immunoglobulins (not shown).
All complexes revealed with anti-apoAI without immunoprecipitation
(Figure 1B, lane 1) were present after immunoprecipitation
(Figure 1B, lane 2). Cross-linking experiments are not
quantitative, and it cannot be excluded that a protein that interacts
with POPC-AI was not cross-linked with apoAI in our experimental
conditions; however, SR-BI was probably the major apoAI binding protein
in these physiological relevant cells. Thus, a
study of ligand specificity could be performed on this model.
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Binding of Lipoproteins Containing ApoAI and/or ApoAII to
NCI-H295R Cells
Direct binding of native and reconstituted
125I-HDL to NCI-H295R cells at 4°C is shown in
Figure 2. Reconstituted HDL (POPC-AI or
DMPC-AI), generated with phospholipids and apoAI, the major protein of
HDL, bound to cells with better affinity than did native HDL. Affinity
depends on the nature of the phospholipids of reconstituted HDL, with
POPC-AI being more efficient than DMPC-AI. Nonspecific binding obtained
in the presence of a 40-fold excess of unlabeled HDL is 25% of
total binding for 125I-HDL and always lower than
9% for 125I-reconstituted HDL (not shown).
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),
or DMPC-AI (
) without (total) or with a 40-fold excess of unlabeled
HDL (nonspecific). Curves are the specific binding obtained by the
difference between total and nonspecific binding. Results shown are
mean±SD of triplicate assays. Data shown are
representative of 2 (HDL and DMPC-AI) or 4 (POPC-AI)
independent experiments. Kd and
Bmax calculated from these curves were, respectively, 34
µg/mL and 138 ng/mg cellular protein for HDL, 13 µg/mL and 154
ng/mg cellular protein for DMPC-AI (mean of 2 independent experiments),
and 1.87 µg/mL and 150 ng/mg cellular protein for POPC-AI (mean of 4
independent experiments).
At 37°C, the apparent Bmax of POPC-AI is higher
than that at 4°C, as shown in Figure 3
(432 versus 150 ng/mg cellular protein). To evaluate the degradation of
apoAI, we measured it in the culture medium after the binding
experiment at 37°C. The degradation was very low and <3% of the
specific binding for the lower ligand values; it increased to 20%
at the higher ligand concentrations (Figure 3). In consequence,
the degradation cannot account for the higher binding of POPC-AI at
37°C, and higher Bmax values at 37°C were
probably due to the higher mobilization of SR-BI up to the cell
surface.
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) without (total) or with a
50-fold excess of unlabeled HDL (nonspecific). Curves are the specific
binding obtained by the difference between total and nonspecific
binding (solid lines) or degradation (dotted lines) measured in the
incubation medium, as indicated in Methods. Results shown are mean±SD
of triplicate assays and are representative of 2
independent experiments. Kd and
Bmax calculated from the binding curves were, respectively,
1.42 µg/mL (25 nmol/L) and 432 ng/mg cellular protein for POPC-AI and
1.6 µg/mL (45 nmol/L) and 465 ng/mg cellular protein for POPC-AII
(means of 2 independent experiments).
To test the binding of apoAII, the second major protein of HDL, we used
125I-labeled POPC-AII. Compared with POPC-AI,
direct binding to cells at 37°C (Figure 3
) was nearly the
same, also with very low degradation. However, when expressed on a
molar basis, Kd for POPC-AII was higher
than for POPC-AI (45 versus 25 nmol/L).
To compare the different SR-BI ligands in competition experiments, we
used 125I-labeled POPC-AI at 2 µg/mL and
increasing concentrations of competitors. This choice was justified
because POPC-AI is a good SR-BI ligand and is more
homogeneous than HDL. As previously demonstrated by
different authors studying murine SR-BI,13 15 cationic
phospholipids such as POPC did not compete with POPC-AI for binding to
SR-BI, and LDL poorly inhibited POPC-AI binding (Figure 4A). Native HDL containing apoAI
and apoAII did not compete with POPC-AI to the same extent as POPC-AI.
When associated with POPC, apoAI and apoAII were not highly different
competitors (Figure 4A), but free apoAII was a better competitor
than free apoAI (Figure 4B). These differences remained when
apoAI and apoAII concentrations were expressed as molar concentrations
instead of mass protein concentrations (Figure 4B, insert).
ApoAII was also a better competitor than apoAI when
125I-labeled HDL was used as an SR-BI ligand (not
shown). These first results indicated that apoAI and apoAII had
different affinities for SR-BI, depending also on the lipid
environment.
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), HDL (•), POPC-AI (
) and apoAII (
) in panel B.
After the cells were washed, cell-associated radioactivity was
measured. In these conditions, POPC-AI degradation was 
3% of total
binding. Competitor concentrations are expressed in micrograms per
milliliter or in nanomoles per liter (B, insert). Results are
expressed as a percentage of binding in absence of competitor and are
mean±SD of duplicate assays (100% was 151±7 and 169±8 ng POPC-AI
bound per milligram cellular protein for panels A and B, respectively).
Data shown are representative of 2 independent
experiments.
Effect of ApoAII Enrichment of Native Lipoproteins on Binding on
NCI-H295R Cells
To study the specific role of these 2 apolipoproteins in an
HDL3 environment, we prepared
HDL3 for which enrichment in apoAII was obtained
by apoAI displacement.26 ApoAII was also used to displace
apoAI in LpAI lipoproteins purified from HDL3 by
immunoaffinity. Free apolipoproteins were eliminated by
ultracentrifugation (d=1.21 g/mL) and then
ultrafiltration on membranes, with a cutoff of 100 kDa; absence of free
apolipoprotein was checked by nondenaturing PAGE (Figure 5A). As previously
described,26 the mean apparent molecular mass of these
enriched HDLs or LpAI was increased. After incubation of
HDL3 or LpAI with free apoAII, the lipoproteins
became enriched in apoAII, and for the higher apoAII concentration,
only traces of apoAI could be observed in the resulting HDL (Figure 5B).
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In Figure 6, we have compared the
capacity of these modified HDLs to displace
[125I]POPC-AI bound to NCI-H295R cells.
Increasing the content of apoAII in HDLs increased the competition with
labeled POPC-AI (Figure 6A). Direct binding of apoAII-enriched
125I-HDL also demonstrated the better affinity of
these modified HDLs compared with native HDLs (Figure 6B). At
37°C, Kd values for HDL of 32 µg/mL
were decreased to 22 µg/mL after apoAII enrichment, and this decrease
was found in 4 independent experiments. Lipoproteins isolated by
immunoaffinity (LpAI) were a slightly better competitors than were HDLs
in spite of the absence of apoAII; however, displacement of apoAI by
apoAII in these lipoproteins also increased their affinity for SR-BI in
NCI-H195R cells (Figure 6C).
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All these results suggested that displacement of apoAI by apoAII in lipoproteins increased their interaction with SR-BI in NCI-H295R cells, but it was important to test the correlation between this higher interaction and CE-selective uptake by cells.
CE Uptake by NCI-H295R Cells From Lipoproteins Containing ApoAI
and/or ApoAII
First, we tested in competition studies the inhibitory
effect of free apoAI and apoAII on [3H]CE
uptake from HDLs. Figure 7 shows that
apoAI and apoAII inhibit CE uptake from HDLs, confirming that binding
sites for free apolipoproteins were the same as binding sites for
[3H]CE uptake from HDLs. Free apoAII was a more
potent inhibitor of CE uptake compared with free apoAI,
either results were expressed in mass or in molar concentrations. In
the conditions used for uptake inhibition, 26% of apoAI in the HDLs
was displaced by the higher concentration of free apoAII added as a
competitor. So the inhibitory effect of free apoAII
addition on CE uptake can be due to a combined process: (1) a
competition effect due to the high affinity of apoAII for SR-BI and (2)
a direct uptake inhibition due to apoAI replacement by apoAII in HDLs
during the experiment.
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Then we labeled HDLs with [3H]CE and displaced
apoAI by apoAII in these lipoproteins before testing CE uptake by cells
(Figure 8). The
[3H]CE uptake by NCI-H295R cells was
concentration dependent and was 50-fold higher than the calculated
internalization of CE deduced from protein degradation measured after
125I labeling of proteins, shown in Figure 3 (it was assumed that when 1 milligram of HDL protein was
internalized and degraded, 0.25 mg CE was taken into the cell in the
same time); thus, it can be concluded that CE uptake is independent of
protein degradation. Figure 8A shows that the lower
concentrations of apoAII (0.5/1 apoAII to HDL proteins) added to
displace apoAI had no effect on [3H]CE uptake,
but higher ratios (1/1 and 2/1) strongly inhibited CE uptake by cells
(up to 65%).
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Kinetic experiments were then performed with
[3H]CE-labeled HDLs modified with large apoAII
excess (1/1 and 4/1 apoAII to HDL proteins, Figure 8B). With the
larger excess of apoAII, HDLs failed to transfer CE to cells, with a
residual uptake from 20% to 30% compared with native HDLs. Inhibition
did not vary over 1 to 5 hours. The low uptake is inversely correlated
with high binding capacity of these modified HDLs. Control HDLs
obtained after incubation of apoAI with HDL in a large excess (4/1 mass
ratio) and elimination of free apoAI had the same CE transfer capacity
as native HDL (not shown).
These experiments demonstrate that the human adrenal cell line NCI-H295R takes up CE from HDL3 and that uptake is higher from native HDL than from HDLs enriched in apoAII.
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Discussion |
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When Acton et al1 identified SR-BI as an HDL receptor, they underlined its high expression in the adrenal gland. Temel and al10 then demonstrated that SR-BI was the major route for the delivery of HDL CEs to the steroidogenic pathway in cultured mouse adrenocortical Y1-BS1 cells. In humans, the SR-BI/CLA-1 gene is also highly expressed in adult and fetal adrenal3 and in human adrenal tumors12 suggesting that SR-BI is involved in the selective uptake of CE from HDL in human adrenocortical cells. In this perspective, to study the role of different SR-BI ligands on uptake efficiency, we used an NCI-H295R human adrenocortical cell line, a good physiological model of human steroidogenesis.27 In a recent study, we demonstrated upregulation of SR-BI expression and CE uptake by cAMP in these cells.18 In the present study, we show in Figure 1
that reconstituted HDLs containing apoAI as the sole
apolipoprotein bind to NCI-H295R cells. In cross-linking experiments,
we also demonstrate that SR-BI is probably the major binding site for
apoAI in these cells. After immunoprecipitation of cross-linked
complexes by anti–SR-BI antibodies, the same bands were revealed by
anti-apoAI antibodies as before immunoprecipitation. In addition,
incubation of POPC-AI with cells prevents complex formation between
SR-BI and other membrane proteins. Moreover, in these cells, free apoAI
and apoAII, which are ligands for SR-BI,15 compete with
HDLs to bind to cells and also to take up CE. This suggests that in
NCI-H295R cells, the same SR-BI binding site is responsible for apoAI
binding and is involved in apoAI-dependent CE-selective uptake.
Ligand specificity of SR-BI has been studied on rodent and human SR-BI
in transfected cultured cells.15 Adrenal cell lines highly
expressed SR-BI but also the LDL receptor, and they do not allow for
the study of common ligands except in competition study. In the
present study, we did not investigate apoE- and apoB-containing
lipoproteins and only checked the absence of apoE in all our HDL
preparations. We verified, as previously demonstrated by Rigotti et
al14 on rodent SR-BI and Calvo et al16 on
human SR-BI, that LDL was a poor competitor for HDL binding (Figure 4A). Affinity of HDLs for human SR-BI
(Kd 34 µg/mL) was of the same order of
magnitude as that previously published for murine
SR-BI.15 Competition by free apoAI and apoAII, also
demonstrated by Xu et al15 in rodents, was confirmed
(Figure 4B), indicating an analogy in ligand specificity between
rodent and human SR-BI and consistence with the high sequence homology
between the 2 receptors.
In the present study, our intention was to analyze the
respective roles of apoAI and apoAII in binding to SR-BI and as
mediators of CE-selective uptake by SR-BI. In competition experiments,
apoAII was a better ligand than was apoAI. When incorporated in
reconstituted HDLs, POPC-AI, and POPC-AII, these 2 apolipoproteins are
good ligands, but no difference in affinity could be displayed either
in competition or in direct bindings when expressed in mass
concentration (Figure 4), but when affinity is expressed on a
molar basis, POPC-AI seems to have more affinity than does POPC-AII.
This suggests the importance of the lipid environment in
SR-BI–lipoprotein interaction. Reconstituted HDLs generated with
phospholipids and apoAI or apoAII bound to cells with better affinity
than did native HDLs; this was also shown by Xu et al15 in
phosphatidyl choline/cholesterol/apoAI or apoAII reconstituted
HDL with murine SR-BI. Lipoprotein prepared by immunoaffinity (LpAI)
was a better ligands than was native HDL3, from
which it was prepared. However, displacement of apoAI by apoAII, either
in native HDL or in LpAI, induced higher affinity for the SR-BI of the
apoAII-enriched lipoproteins, with that affinity being evaluated either
by competition or by direct binding (Figure 6). These results
indicate that if apoAII has better affinity for SR-BI, it depends not
only on the amino acid sequence of the ligand but also on the lipid and
protein environment in lipoproteins.
A surprising result was that in spite of that higher affinity, apoAII-enriched HDLs displayed a lower capacity to deliver CE to NCI-H295R cells. This CE uptake inhibition by apoAII enrichment of HDLs is dose dependent and did not depend on the incubation time of up to 5 hours.
These opposite effects of apoAII, an agonist for binding and an antagonist for CE uptake, may imply a "2-scale" mechanism for CE delivery to cells. High affinity for a cell surface receptor is not sufficient to ensure efficient cellular CE uptake from HDLs. ApoAII could promote the first binding step and inhibit the second step, corresponding to CE internalization. A recent study of Gu and al34 came to the same conclusion when they compared the abilities of murine SR-BI and human CD36, a scavenger receptor with large sequence analogy with SR-BI, to bind HDLs and mediate the cellular uptake of lipids. They concluded that CD36 can bind HDLs but cannot mediate efficient lipid uptake and that the distinctive ability is primarily a consequence of the extracellular loop of SR-BI.
Previous experiments by Lagrost et al26 demonstrated that it was possible to replace apoAI with apoAII in HDL3 without modifying the structure and lipid composition of HDL particles but with a slight increase in molecular weight. By use of an analogous protocol of apoAI displacement by apoAII in HDLs, antagonist effects of apoAII have been demonstrated in vitro in different functions in which apoAI plays a major activating role: cholesterol efflux from cells,17 lecithin-cholesterol acyltransferase activation,35 CE transfer protein activity,26 and specific CE uptake by hepatic cells.4 In vivo studies in apoAI-, apoAII-, or apoE-deficient mice also suggest that the lack of apoAI has a major impact on adrenal gland physiology and that apoAI is essential for the selective uptake of HDL-CE.36
Our results demonstrate that apoAII impairs efficient CE uptake in
adrenal cells, although it promotes a high level of HDL binding. In
this antagonist effect of apoAII, changes in HDL size
(Figure 5A), even if low, can play an important structural role.
The effect of apoAII is probably due to a distinct specific requirement
for binding and lipid uptake via the scavenger receptor SR-BI. This
could aid in the understanding of the mechanism of the differential
antiatherogenic effects of human apoAI and apoAII in transgenic mice.
Overexpression of human apoAI protects mice with diet-induced
atherosclerosis.37 This antiatherogenic
effect is attenuated by overexpression of apoAII.38 There
is controversy over the atherogenicity or the protective effect of
apoAII in mice overexpressing only apoAII.39 In these
mice, high apoAII overexpression could inhibit the SR-BI pathway and
yet maintain a high level of HDL-CE. It would be interesting to test
the SR-BI pathway in these mice and to correlate it with apoAII
expression.
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Acknowledgments |
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Antibodies against SR-BI were produced by J. Najib-Fruchart. B. Delfly is thanked for excellent technical assistance.
Received February 8, 1999; accepted November 29, 1999.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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