Tài liệu Báo cáo khoa học: Trophoblast-like human choriocarcinoma cells serve as a suitable in vitro model for selective cholesteryl ester uptake from high density lipoproteins pdf

Eur. J. Biochem. 270, 451–462 (2003) Ó FEBS 2003

doi:10.1046/j.1432-1033.2003.03394.x

Trophoblast-like human choriocarcinoma cells serve as a
suitable in vitro model for selective cholesteryl ester uptake
from high density lipoproteins
Christian Wadsack1, Andelko Hrzenjak1, Astrid Hammer2, Birgit Hirschmugl1, Sanja Levak-Frank1,
Gernot Desoye3, Wolfgang Sattler1 and Ernst Malle1
1

Institute of Medical Biochemistry and Molecular Biology, 2Institute of Histology and Embryology, 3Clinic of Obstetrics
and Gynecology, Karl-Franzens University Graz, Austria

As human choriocarcinoma cells display many of the
biochemical and morphological characteristics reported for
in utero invasive trophoblast cells we have studied cholesterol supply from high density lipoproteins (HDL) to
these cells. Binding properties of 125I-labeled HDL subclass 3 (HDL3) at 4 °C were similar for BeWo, JAr, and
Jeg3 choriocarcinoma cell lines while degradation rates at
37 °C were highest for BeWo. Calculating the selective
cholesteryl ester (CE)-uptake as the difference between
specific cell association of [3H]CE-labeled HDL3 and
holoparticle association of 125I-labeled HDL3 revealed that
in BeWo cells, the selective CE-uptake was slightly lower
than holoparticle association. However, the pronounced
capacity for specific cell association of [3H]CE-HDL3 and
selective [3H]CE-uptake in excess of HDL3–holoparticle
association, and cAMP–mediated enhanced cell association of [3H]CE-HDL3 in JAr and Jeg3 suggested the

scavenger receptor class B, type I (SR-BI) to be responsible for this pathway. Abundant expression of SR-BI (but
not SR-BII, a splice variant of SR-BI) could be observed

in JAr and Jeg3 but not in BeWo cells using RT-PCR,
Northern and Western blot analysis, and immunocytochemical technique. Adenovirus-mediated overexpression
of SR-BI in all three choriocarcinoma cell lines resulted in
an enhanced capacity for cell association of [3H]CE-HDL3
(20-fold in BeWo; fivefold in JAr and Jeg3). The fact that
exogenous HDL3 remarkably increases proliferation in
JAr and Jeg3 supports the notion that selective CE-uptake
and subsequent intracellular generation of cholesterol is
coupled to cellular growth. From our findings we propose
that JAr and Jeg3 cells serve as a suitable in vitro model to
study selective CE-supply to human placental cells.

Mammalian fetal nutrition during development is wholly
dependent on the transport of nutrients by the placenta.
Cholesterol is not only a major component of cell
membranes but is also necessary for the maintenance of
fetal growth and serves as the initial substrate for steroid
hormone synthesis [1]. There are two sources of cholesterol
in the fetus, as in any other tissues. The first source is
endogenous and constitutes cholesterol synthesized within

the fetus. The second source of fetal cholesterol is exogenous
[2,3]. Exogenous cholesterol could be derived from cholesterol synthesized in the yolk sac and placenta or by
transport of lipoprotein-associated cholesterol across the
placenta from the maternal circulation [4–8].
The fact that the placenta binds and internalizes maternal
lipoproteins both in vivo and in vitro [8] suggests the
existence of functionally intact lipoprotein receptors [2–11].
The presence of active lipoprotein receptors in the yolk sac,
the placenta and placenta-derived cells support internalization of cholesterol-rich maternal lipoproteins [8]. In addition

to low-density lipoprotein (LDL) that are taken up by the
classical receptor-mediated endocytosis pathway (the LDLor apolipoprotein B/E-receptor), the high clearance rate of
high-density lipoprotein (HDL) in rodents suggested specific
HDL receptors [8] contributing to sterol metabolism in fetal
tissues. Among these gp280 preferentially mediates HDLholoparticle-uptake [9] while scavenger receptor class, B,
type I (SR-BI), the predominant receptor mediating selective uptake of cholesteryl esters (CEs) without concomitant
holoparticle-uptake [12,13], has been identified in steroidogenic tissue and the liver [12] as well as in human [14] and
rodent placental tissues [15,16]. Immunohistochemistry
revealed strong induction of SR-BI expression in murine
giant trophoblast cells that surrounded the developing
embryo [16]. The fact that murine SR-BI (mSR-BI) is

Correspondence: E. Malle, Karl-Franzens University Graz,
Institute of Medical Biochemistry and Molecular Biology,
Harrachgasse 21, A)8010 Graz, Austria;
Fax: +43 316 380 9615; Tel.: +43 316 380 4208;
E-mail:
Abbreviations: [3H]CE, [1,2,6,7–3H]-cholesteryl palmitate; CE(s),
cholesteryl ester(s); CHO, Chinese hamster ovary; 8-CPTcAMP,
8-(4-chlorophenylthio)adenosine 3¢:5¢-cyclic monophosphate;
DMEM, Dulbecco’s minimum essential medium; FBS, fetal bovine
serum; HBSS, Hank’s balanced salt solution; HDL3, high density
lipoprotein subclass 3; LDL, low density lipoprotein; LPDS, lipoprotein-deficient serum; mSR-BI, murine scavenger receptor class B,
type I; TCA, trichloroacetic acid.
(Received 22 July 2002, revised 10 October 2002,
accepted 26 November 2002)

Keywords: BeWo; JAr; Jeg3; placenta; SR-BI.

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452 C. Wadsack et al. (Eur. J. Biochem. 270)

expressed on the side of the tissue that faces the maternal
blood expression supports the notion that this protein could
act as a candidate receptor for supplying cholesterol to
developing embryonic tissues for placental steroid biosynthesis.
The process of binding and uptake of maternal lipoproteins is carried out by placental trophoblast [17], which lines
the chorionic villi and represents the epithelial layer
separating the maternal and fetal circulation [18]. In the
human placenta chorionic villi, a layer of syncytiotrophoblasts lies over the cytotrophoblasts, and surrounds the
internal mesoderm and fetal capillaries [18]. Trophoblasts
may also give rise to choriocarcinoma which are malignant
tumors of epithelial origin but have been shown to display
characteristics of invasive trophoblasts [19]. Choricarcinoma cells are morphologically similar to their cell of
origin, the trophoblast of the normal first trimester placenta
and may serve as a valid and convenient in vitro model
system for studying the cellular activities and regulation of
transplacental transport and uptake mechanisms. To date,
only limited information on binding and holoparticleuptake of lipoproteins by choriocarcinoma cell lines is
available [20,21]. However, no studies have been performed
to clarify whether choriocarcinoma cell lines may serve a
suitable in vitro model for supplying cholesterol to placental
cells via selective CE-uptake from HDL.
We therefore have studied holoparticle association of and
selective CE-uptake from HDL by three human trophoblast-like choriocarcinoma cell lines, i.e. BeWo, JAr, and
Jeg3. The BeWo cell line which is heterogenous on several
criteria [22], is comprised of cytotrophoblasts with no
differentiation to syncytium [23] under non activated

conditions [24]. This is in contrast to primary cultures of
term cytotrophoblasts which aggregate and form syncytia.
In contrast to BeWo and Jeg3 cell lines, JAr cells share many
of the characteristics of early placental trophoblasts, such as
synthesis of human chorionic gonadotropin and steroids
[25], and the ability to differentiate into syncytiotrophoblastlike cells in vitro [19]. Jeg3, derived originally from the BeWo
[26], express abundant human chorionic gonadotropin and
placental lactogen – hallmarks of trophoblasts [27] – and
form large, multinucleated syncytia in culture [28] which
resemble that of syncytiotrophoblasts in vivo.
Results from the present study demonstrate that the
capacity of selective CE-uptake by all three choriocarcinoma cell lines is closely related to the expression level of
SR-BI. Only JAr and Jeg3 could serve as suitable in vitro
models to study selective CE supply to human placental
cells. High level adenoviral-overexpression of SR-BI resulted in an enhanced and comparable capacity for selective
CE-uptake by all three choriocarcinoma cell lines. The lack
of SR-BII protein suggested that this splice variant of SR-BI
[29] does not contribute to selective CE-uptake by BeWo,
JAr, and Jeg3 cells.

Experimental procedures
Human lipoproteins
LDL (d ẳ 1.0351.065 gặmL)1) and HDL subclass 3
(HDL3, lacking apolipoprotein E, a ligand for the LDLreceptor, d ẳ 1.1251.21 gặmL)1) was prepared by

discontinuous density ultracentrifugation from plasma
obtained from normolipidemic blood donors [30]. Purity
of lipoprotein preparations was checked by SDS/PAGE.
The protein concentration was determined as described [31]
using bovine serum albumin as a standard. Oxidation of

LDL (ox-LDL) was performed with 1.66 lM of Cu2+ in
NaCl/Pi as described [32].
Lipoprotein labeling procedures
Single-labeling. (a) Iodination of HDL3 with 125I-Na
(NEN, Vienna, Austria) was performed using N-Br-succinimide as the coupling agent [33]. This procedure resulted in
specific activities between 300 and 500 d.p.m.Ỉng)1 protein
with less than 3% lipid associated activity. All 125I-HDL3preparations were monitored by SDS/PAGE to ensure that
the preparations were free of radiolytic or oxidative damage.
(b) HDL3 was labeled with [cholesteryl-1,2,6,7–3H]-palmitate ([3H]CE, NEN) by cholesteryl ester transfer, proteincatalyzed transfer from donor liposomes exactly as described
[34]. Subsequently, the labeled HDL3-fractions were reisolated in a TLX120 bench-top ultracentrifuge in a TLA100.4
rotor (Beckman, Austria). The HDL3-band was aspirated
and dialyzed against 10 mM NaCl/Pi, pH 7.4.
Double-labeling. Labeling of HDL3 with [cholesteryl1,2,6,7–3H]-oleate (NEN) was performed by incubation of
the lipoprotein with palmityl oleyl phosphatidylcholine[3H]cholesteryl oleate vesicles exactly as described [35].
Following separation from the vesicles by ultracentrifugation, the labeled lipoproteins were subsequently labeled with
125
I-Na as described above using N-Br-succinimide.
Cell culture studies
Choriocarcinoma cells. The BeWo cell line (ATCC, Manassas, VA, USA) was cultured in F12K (Kaighn’s Modification) Nutrient Mixture (Gibco BRL, Vienna, Austria)
supplemented with 10% (v/v) heat inactivated fetal bovine
serum (FBS, hyClone, Utah, USA) containing 100 mL)1
penicillin/streptomycin and 2 mM L-glutamine at 37 °C
under 5% CO2. The JAr and Jeg3 cell lines (ATCC) were
maintained in Dulbecco’s minimum essential medium
(DMEM, supplemented with 10% [v/v] FBS and
100 mL)1 penicillin/streptomycin) at 37 °C under 5%
CO2. To assess influences of exogenous HDL3 on cell
proliferation, the cells were seeded into 6-well plates at a
starting density of 2.5 · 104 cells in 2 mL 10% lipoproteindeficient serum (LPDS) medium for BeWo, 5 · 104 cells for
JAr and 1 · 105 cells for Jeg3 cells, respectively. After 24 h,

media was removed, the cells were washed with Hank’s
balanced salt solution (HBSS), trypsin-treated and counted
with a cell counter and analyzer system (CASYề1, Scharfe,
ă
Reutlingen, Germany). Fresh medium containing lipoprotein-deficient serum or lipoprotein-deficient serum plus
100 lgỈmL)1 HDL3 was added and the cells were incubated.
At the indicated time points the cells were washed twice with
HBSS, trypsin-treated and counted.
Chinese hamster ovary (CHO) cells. LdlA7 (clone 7, a
LDL receptor-negative CHO cell line) which express
minimal levels of SR-BI were cultured in HAM’s


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Human choriocarcinoma cells and SR-BI (Eur. J. Biochem. 270) 453

F12 medium containing 5% (v/v) FBS, 2 mM glutamine,
50 mL)1 penicillin and 50 lgỈmL)1 streptomycin [36];
ldlA(mSR-BI) (ldlA7 stably transformed with mSR-BI)
were maintained in medium containing 0.5 mgỈmL)1 G-418.
Both cell lines were kindly provided by Dr M. Krieger
(MIT, Cambridge, MA, USA).
Binding studies
Binding studies of human HDL3 to choriocarcinoma cells
were performed at 4 °C in medium (F12K/DMEM without
FBS) with increasing amounts of 125I-labeled HDL3 (1, 5, 10,
25, 50, and 100 lg proteinỈmL)1) in the absence (total
binding) or presence of a 20-fold excess of unlabeled HDL3
(nonspecific binding) [37]. Following this incubation the

medium was aspirated and the cells were washed twice with
HBSS containing 5% (v/w) bovine serum albumin followed
by two washes in HBSS. Cells were lysed with 0.3 M NaOH
(1 mL, 1 h at 4 °C) to determine bound-radioactivity and cell
protein in the lysate. Protein measurement was performed as
described [31]. Specific binding (4 °C) was calculated as the
difference between total and nonspecific binding [37].
To determine cell-associated (the sum of binding and
internalization) and degraded 125I-labeled HDL3-protein,
the cells were incubated at 37 °C for 4 h as described above
with the same amounts of labeled and unlabeled HDL3.
Subsequently, the medium was aspirated and the cells were
rinsed as described above [34]. Degradation of 125I-labeled
HDL3 by choriocarcinoma cells was estimated by measuring the nontrichloroacetic acid-precipitable radioactivity in
the medium after precipitation of free iodine with AgNO3
exactly as described [38]. Briefly, 0.5 mL of medium was
removed, mixed with 100 lL bovine serum albumin
(30 mgỈmL)1) and 1 mL trichloroacetic acid (3 M, 4 °C)
and left at 4 °C for 30 min. Subsequently 250 lL of AgNO3
(0.7 M) was added, mixed, and the samples were centrifugated at 1500 · g at 4 °C for 15 min. One milliliter of the
supernatant was counted on a c-counter.
To determine cell association of tritiated-HDL3, the cells
were incubated with [3H]CE-HDL3 at 37 °C for 4 h as
described above with the same amounts of labeled and
unlabeled HDL3 [34]. After removing the medium and
washing the cells, cell association was estimated by measuring the radioactivity and protein content of the cell lysates,
respectively. Specific cell association was calculated as the
difference between total and nonspecific cell association.
In a parallel set of experiments, the cell association of
[3H]CE-HDL3 by choriocarcinoma cells was measured by

increasing concentrations of unlabeled competitors (HDL3
and ox-LDL) as well as in the absence or presence of 300 lM
8-(4-Chlorophenylthio)adenosine 3¢,5¢-cylic monophosphate
(8-CPTcAMP, Sigma, Vienna).

particles only specific cell-associated radioactivity (the
difference between total and nonspecific cell association of
[3H]CE-HDL3) was counted. To facilitate quantitative
comparison of 125I-labeled and [3H]CE association, results
are expressed in terms of HDL3-protein equivalents [39]
calculated on the basis of the specific activity of the
correspondingly labeled HDL preparation used that would
be necessary to deliver the observed amount of tracer. These
calculations are performed to compare uptake of 125I- and
3
H-tracers on the same basis.
In a parallel set of experiments, the selective CE-uptake
from HDL3 was calculated from double-labeled HDL3 as
the difference between [3H]cholesteryl oleate- and 125Ilabeled lipoprotein [35]. The sum of specific degradation
(measured as described above) and specific cell association of
125
I-labeled HDL3 reflects HDL3–holoparticle association.
SDS/PAGE and Western blotting
Detergent extracts of solubilized membrane protein fractions [33] or total cell proteins of choriocarcinoma and CHO
cells were separated on 8% SDS/PAGE. Protein transfer to
nitrocellulose membranes was performed electrophoretically at 150 mA, 4 °C, 50 min [34]. Immunochemical
detection of SR-BI and SR-BII (a splice variant of SR-BI
lacking the C-terminal portion [29]) was performed with a
sequence-specific rabbit anti-SR-BI-peptide (496–509) and
anti-SR-BII peptide (491–506) IgG (dilutions 1 : 1500,

Abcam, Cambridge, UK). Immunoreactive bands were
visualized with peroxidase-conjugated goat anti-rabbit IgG
and ECL development [30].
Reverse-transcriptase-polymerase chain reaction
Total RNA from choriocarcinoma cell lines was isolated by
using RNeasy kit (Qiagen, Vienna, Austria). Three micrograms of total RNA were treated with RQ1 RNase-free
DNase I (Promega, Mannheim, Germany) for 15 min at
37 °C and subsequently used as a template for first strand
cDNA synthesis in a 30-lL reaction. The PCR conditions applied were described in detail elsewhere [40]. The
following oligonucleotides were used: forward primer
(A) 5¢-TCTACCCACCCAACGAAGGCT-3¢ (nucleotides
1007–1027), SR-BI reverse primer (B) 5¢-CCTGAATGGC
CTCCTTATCCT-3¢ (1514–1534) and SR-BII reverse
primer
C-5¢-AGAAGCGGGGTGTAGGGACTGG-3¢
(1655–1676) (MWG Biotech, Ebersberg, Germany). By
using the primers A and B, a 527-bp fragment was obtained.
By using the primers A and C, two fragments were obtained:
a 669-bp-fragment for SR-BI and a 540-bp-fragment for
SR-BII (129 bp shorter than SR-BI). All fragments were
subcloned in TOPO-TA cloning vector and sequenced.

Selective CE-uptake

Northern blot analysis

To calculate the selective CE-uptake from HDL3 during the
same experiment, choriocarcinoma cells were incubated
with 125I-labeled and [3H]CE-HDL3. In case of 125I-labeled
HDL3, the cell-associated and nontrichloroacetic acidprecipitable radioactivity in the medium was counted. The

sum of cell-associated and degraded 125I-labeled HDL3
reflects holoparticle association. For the [3H]CE-HDL3-

Total RNA was isolated from choriocarcinoma and human
liver tissues (used as a positive control) by the RNA-easy kit
(Qiagen) exactly as described [40]. A 553-bp fragment,
amplified by RT-PCR [forward primer 5¢-TCGCTCAT
CAAGCAGCAGGT-3¢ (nucleotides 169–188), reverse primer 5¢-GCCCAGAGTCGGAGTTGTTG-3¢ (nucleotides
702–721)] was used as a probe [30].


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454 C. Wadsack et al. (Eur. J. Biochem. 270)

Construction of recombinant human SR-BI adenovirus
The adenoviral plasmid shuttle vector (pAvCvSv) and
pJM17 vector were kindly supplied by L. Chan (Baylor
College of Medicine, Houston, Texas). Human SR-BI
cDNA (kindly provided by H. Hauser, ETH, Zurich,
ă
Switzerland) which was originally inserted into pcEXV-3
vector was partially restricted with EcoR I and the 2.5 kb
band was eluted from the gel. This band was subcloned into
pBluescript using the EcoRI site, amplified, restricted with
Kpn-I and this fragment was finally partially restricted with
BamHI. The plasmid shuttle vector was opened using Kpn-I
and BglII, and Kpn-I/BamHI restricted SR-BI cDNA was
inserted. These modifications were necessary to enable
insertion of SR-BI cDNA under the control of the CMV

promoter in the plasmid shuttle vector. Recombinant
vector (pAvCvSv/human SR-BI) was used to transform
E. coli DH5-a competent cells in order to amplify the
recombinant plasmid. Positive clones were confirmed by
restriction analysis and DNA-sequencing. The resulting
recombinant shuttle plasmid (5 lg) was cotransfected with
5 lg supercoiled pJM17 into 293 cells by calcium-phosphate
coprecipitation. Two weeks after transfection, infectious
recombinant adenoviral vector plaques were picked, propagated, and screened for SR-BI sequences by PCR.
Adenoviral vectors containing SR-BI were further amplified
in 293 cells and the expression was determined by Western
blotting analysis. Large-scale production of high-titer
recombinant adenovirus was performed as described [41].
The virus was purified twice by caesium chloride density
gradient centrifugation and dialyzed for 14 h at 4 °C against
a buffer containing 10 mM Tris/HCl, pH 7.5, 1 mM MgCl2,
10% glycerol and stored at )70 °C. Virus-titer was determined by plaque-assay on 293 cells and was found to be
2 · 1010 pfmL)1. Control b-galactosidase (b-gal) and
viruses were amplified and purified as described above.
Adenovirus infection of choriocarcinoma cells
Choriocarcinoma cells were cultivated in 12-well culture
dishes. At a density of 5 · 104 cells per cm2 they were rinsed
once with NaCl/Pi and infected with recombinant adenoviruses (MOI ẳ 30 pfuặmL)1) in 1 mL of infection media
(DMEM medium containing 2% FBS, 50 mL)1 penicillin, and 50 lgỈmL)1 streptomycin) for 2 h (37 °C, 5% CO2,
humidified atmosphere). After removing infection-media,
the cells were supplied with 2 mL of S12K medium
(containing 5% FBS, 2 mM glutamine, 50 mL)1 penicillin and 50 lgỈmL)1 streptomycin) and the incubation was
continued for 36 h without rocking. Control cells were
infected with b-gal virus as described for SR-BI infected
cells. The expression rate of SR-BI was determined by

Northern and Western blot techniques as described above.

cultured on Laboratory-Tek chamber slides (Nunc, USA).
After 24 h the cells were washed with HBSS, air dried (2 h
at 22 °C), and acetone-fixed (5 min) [42]. Fixed chamberslides were incubated for 30 min with rabbit anti-SR-BI IgG
(dilutions of 1 : 1000, NB 400–104, Novus Biologicals,
USA) followed by a 30-min incubation with cyanine3-labeled goat anti-rabbit IgG (dilution 1 : 400, Jackson
Dianova). NaCl/Pi was used for washing sections between
different incubation steps. Sections were mounted with
Moviol (Calbiochem-Novabiochem, La Jolla, CA) and
analyzed on a confocal laser scanning microscope (Leica
TCS NT, Heidelberg, Germany) equipped with an argonkrypton laser. Signals were detected with a double dichroitic
beam splitter (488/568 nm), using a 590-nm long pass filter
for cyanine-3 [42].
Control experiments encompassed immunocytochemistry (a) without primary detection antibodies (b) with
polyclonal nonimmune antibodies as primary antibodies
(c) without secondary antibodies, or (d) using ldlA(mSR-BI)
membrane protein fractions as competing antigen; the
competitor (20-fold molar excess) was preincubated with the
primary antibody for 20 min before adding to the section.
Pictures were taken with an Axiophot microscope (Zeiss,
Oberkochen, Germany).

Results
Binding of HDL to choriocarcinoma cells
Specific binding of HDL3 to all three choriocarcinoma cell
lines at 4 °C was saturable at the highest lipoprotein
concentrations (100 lg proteinỈmL)1) used. Calculation of
binding parameters yielded similar Kd and bmax values for all
three cell lines (Table 1). Also the specific cell association of

125
I-labeled HDL3 (the sum of binding and internalization)
measured at 37 °C was comparable for all three cell lines
(Fig. 1A–C). The specific degradation of 125I-labeled HDL3
determined in a parallel set of experiments was highest for
BeWo and decreased in the following order: BeWo >
JAr > Jeg3.
Selective HDL-CE uptake by choriocarcinoma cells
For BeWo cells, the specific cell association of [3H]CEHDL3 increased up to 1200 ng HDL3Ỉmg)1 cell protein
(Fig. 2A) and is approximately 1.8-fold higher than
125
I-labeled HDL3–holoparticle association (the sum of
specific cell association and degradation of 125I-labeled

Table 1. Specific binding of 125I-labeled HDL to choriocarcinoma cell
lines at 4 °C. Binding constants were calculated by non linear regression analysis (GRAPHPAD). Values are given as means ± SD of three
independent experiments.

Immunofluorescence and confocal
laser scanning microscopy
Immunofluorescence was performed on choriocarcinoma
cells (24 h in DMEM containing 10% FBS, 1% glutamine
and 1% penicillin/streptomycin) and CHO cells (HAM’s
F12 medium containing 5% (v/v) FBS, 2 mM glutamine, 50 mL)1 penicillin and 50 lgỈmL)1 streptomycin)

Kd
(lg HDL3-proteinỈmL)1)
BeWo
JAr
Jeg3

bmax
(ng HDL3-proteinỈmg)1
cell protein)

41.9 ± 10.1
34.1 ± 3.9
29.7 ± 7.4

161.3 ± 17.3
145.9 ± 6.9
186.9 ± 21.2


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Human choriocarcinoma cells and SR-BI (Eur. J. Biochem. 270) 455

Fig. 1. Specific cell association and degradation of 125I-labeled HDL by
choriocarcinoma cells. Following preincubation in F12K, [BeWo (A)],
or DMEM [JAr (B) and Jeg3 cells (C) without FBS (12 h)], the cells
were incubated for 4 h (37 °C) in the presence of increasing amounts of
125
I-labeled HDL3. To determine specific cell association of 125I-labeled HDL3 (closed triangles), the cells were incubated in the absence
(total cell association) or presence of a 20-fold excess of unlabeled
HDL3 (non specific cell association). The cells were washed and lysed
with 0.3 M NaOH to determine the cell-associated fraction (the sum of
bound and internalized radioactivity). To determine specific degradation (closed circles – the difference between total and nonspecific
degradation) the cells were incubated under the same conditions as
described above. After 4 h the cellular supernatant was collected to

determine the nontrichloroacetic acid-precipitable radioactivity as
described in Materials and methods (ÔdegradedÕ). Results are given as
means ± SD of three independent experiments.

HDL3). However, calculating the selective CE-uptake from
HDL3 as the difference between both tracers revealed that
this pathway is not a preferential routing to supply BeWo
with CEs from HDL3. To confirm the low capacity of
BeWo cells for selective CE-uptake, the HDL3-particle was
double-labeled and values obtained for specific cell association of [3H]cholesteryl oleate- and 125I-labeled HDL3 were
compared with those obtained by single labeling experiments. Comparable results were obtained with both labeling
techniques (data no shown). In contrast to BeWo, JAr cells
showed a pronounced capacity for specific CE tracer uptake
from HDL3 in excess of holoparticle association, exceeding
holoparticle association by approximately fivefold
(Fig. 2B). In parallel, the selective CE–uptake exceeded
holoparticle association by approx. 4-fold (2703 vs. 652 ng
lipoproteinỈmg)1 cell protein) at the highest HDL3-concentrations used (Fig. 2B). Although specific cell association
of [3H]CE-HDL3 in Jeg3 cells (Fig. 2C) was similar to
BeWo (Fig. 2A), the capacity for selective CE-uptake from
HDL3 exceeded holoparticle association by approximately
sixfold (1537 vs. 267 ng lipoproteinỈmg)1 cell protein) in
these cells.
Identification and characterization of SR-BI
Based on the pronounced capacity of JAr and Jeg3 cells
(Fig. 2B,C) for selective uptake of HDL3-associated CEs it
was reasonable to assume that this pathway is caused by
high expression levels of SR-BI. Using specific primers for
SR-BI the corresponding 527 bp product (primer A and B,
Fig. 3A) and 669 bp product (primer A and C, not shown)

was abundantly present in JAr and Jeg3 cells while only
negligible amounts became apparent in BeWo cells. In
parallel (using primers A and C) a faint 540 bp band
(indicative for SR-BII) was found in all three cells lines (not
shown). Northern blotting experiments confirmed data
obtained by RT-PCR (Fig. 3B). Western blot experiments
finally revealed high expression of SR-BI in JAr and Jeg3
cells (Fig. 3C). In BeWo cells, marginal SR-BI expression
was observed on RNA level but no SR-BI protein was
detectable under the conditions described (Fig. 3). Western
blot experiments of detergent solubilized membrane protein
fractions revealed no expression of SR-BII on all three
choriocarcinoma cell lines (not shown).

Using specific antibodies, pronounced staining for SR-BI
could be observed primarily on the plasma membrane of
JAr and Jeg3 cells and to a much lesser degree in the
cytoplasma of both cell lines (Fig 4B,C). Omission of the
primary antibody (data not shown) or replacement of
primary antibodies with an IgG isotype control (data not
shown) eliminated all staining. Competition experiments of
antiSR-BI IgG preabsorbed with SR-BI-enriched membrane protein fraction from ldlA(mSR-BI) cells at a molar
ratio of 1 : 20 prevented antibody binding, demonstrating
that staining was specific for SR-BI. The lack of SR-BI
expression in BeWo cells (Fig. 3C) could be confirmed by


456 C. Wadsack et al. (Eur. J. Biochem. 270)

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Fig. 2. Specific cell association of [3H]CE-HDL and selective
CE-uptake from HDL by human choriocarcinoma cells. Following
preincubation in F12K [BeWo (A)], or DMEM [JAr (B) and Jeg3
cells (C) without FBS (12 h)], the cells were incubated for 4 h with
increasing amounts of [3H]CE-HDL3 at 37 °C. To determine specific cell association of [3H]CE-HDL3 (closed circles) the cells were
incubated in the absence (total association) or presence of a 20-fold
excess (nonspecific association) of unlabeled HDL3. Subsequently,
the cells were washed and lysed in 0.3 M NaOH to measure
associated radioactivity. The selective CE-uptake (closed triangles)
was calculated as the difference between specific cell association of
[3H]CE-HDL (closed circles) and 125I–labeled HDL holoparticle
association (closed squares, the sum of specific cell association and
degradation, Fig. 2). Results are given as means ± SD of three
independent experiments.

immunocytochemistry (Fig. 4A). To further verify specificity of the primary antibody on choriocarcinoma cells,
CHO cells were used as controls. While faint staining was
observed on ldlA7 cells (expressing minimal levels of
SR-BI, Fig. 4E), bright staining could be observed on
ldlA(mSR-BI) cells (Fig. 4D).
SR-BI expressed on choriocarcionoma cells contributes
to CE-uptake from HDL
To further confirm that SR-BI accounts for the high rates of
selective CE-uptake by JAr and Jeg3 cells, a series of

competition experiments were performed. While HDL3 and
ox-LDL (a high affinity ligand for SR-BI [10]) were equally
effective to compete for cell association of [3H]CE-HDL3 in
BeWo cells, HDL3 lowered cell association of [3H]CEHDL3 by approx 25% in JAr and Jeg3 cells (Table 2).
Further inhibition (up to 60%) was achieved by increasing

the competitor concentration of unlabeled HDL3 to
500 lgỈmL)1. The addition of ox-LDL led to a pronounced
inhibition in JAr and Jeg3 cells, findings in line with
previous results [43] performed on liver cells that express
high levels of SR-BI.
Next, cell association from [3H]CE-HDL3 was studied in
cells preincubated with 8-CPTcAMP, a direct activator of
SR-BI via cAMP-dependent protein kinase pathway
[10,11]. In line with findings shown in Fig. 2, cell association
of [3H]CE-HDL3 is lowest in BeWo compared with JAr and
Jeg3 cells (Fig. 5). Following 8-CPTcAMP treatment, cell
association of [3H]CE-HDL3 was unaltered in BeWo but
increased in JAr and Jeg3 cells. These changes were
paralleled by SR-BI mRNA levels (Fig. 5).
Finally, we tested whether transient overexpression of
SR-BI would restore the ability of BeWo for cell
association of [3H]CE-HDL3. Therefore, choriocarcinoma
cell lines were transiently transfected with the human
SR-BI gene and expression was followed by Northern blot
(data not shown) and Western blot experiments (Fig. 6).
The SR-BI protein expressed was predominantly localized
at the plasma membrane as determined by immunoblot
analysis of plasma membrane preparations. In order to
test the functionality of the adenoviral SR-BI construct,
the cells were incubated with [3H]CE–HDL3 and cell
association of [3H]CE-HDL3 was measured. While mocktransfection of all three choriocarcinoma cells did not
change the capacity for cell association of [3H]CE-HDL3
compared with wild type cells, adenoviral overexpression
increased the capacity for cell association of [3H]CE-HDL3
approximately fivefold (JAr and Jeg3) and 20-fold

(BeWo), respectively. From this set of experiments, we
conclude that transfection of all three choriocarcinoma cell
lines results in functionally active SR-BI protein, and that
transfection in BeWo (a cell line with low SR-BI
expression comparable to ldlA7 cells [44]) results in cell
association of [3H]CE-HDL3 to an extent similar as shown
with JAr and Jeg3 under the same conditions.


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Human choriocarcinoma cells and SR-BI (Eur. J. Biochem. 270) 457

cellular cholesterol content of BeWo cultures was unaffected
by the presence of HDL3; findings which are in line with the
low rate of selective CE-uptake from HDL3 in these cell
lines (Fig. 2) demonstrating that exogenous HDL3 may not
significantly alter CE synthesis in these cell lines [45].

Discussion

Fig. 3. Identification of SR-BI on mRNA and protein level in human
choriocarcinoma cells. (A) RT-PCR analysis. cDNA for SR-BI was
amplified using specific forward and reverse oligonucleotide primers as
described in Materials and methods. The PCR product was separated
on a 1.5% agarose gel: lane 1 (BeWo), lane 2 (JAr), lane 3 (Jeg3), lane 4
(bp standard). (B) Northern blot analysis. Total RNA (15 lg) was
subjected to agarose gel electrophoresis and hybridized using the
553 bp fragment as a probe: lane 1 (BeWo), lane 2 (JAr), lane 3 (Jeg3),
lane 4 (total liver tissue). The blot was then stripped and reprobed by

using a fragment from the human glyceraldehyde 3-phosohate dehydrogenase cDNA (Clontech). Conventional Northern blotting did not
distinguish between SR-BI and SR-BII isoforms [27]. (C) Western blot
analysis. Immunoblotting experiments of detergent solubilized membrane protein fractions (lane 1–3, 50 lg protein; lane 4, 10 lg protein)
were separated on 8% SDS/PAGE. Immunoreactive bands were
visualized with rabbit anti-(SR-BI peptide) IgG (1 : 1500 dilution),
peroxidase-conjugated goat anti-rabbit IgG, and the ECL-detection
system. Arrow indicates the 84-kDa position of SR-BI: lane 1 (BeWo),
lane 2 (JAr), lane 3 (Jeg3), lane 4 [ldlA(mSR-BI)].

Effects of exogenous HDL on growth rates
To investigate whether (a) the pronounced cell association
of [3H]CE-HDL3 and the high capacity for selective CEuptake from HDL3 can be shown during culture conditions
and (b) to assess the role of exogenous cholesterol pool on
cell proliferation of choriocarcinoma cells, the time-dependent effect of exogenous HDL3 on cellular cholesterol levels
was measured (Fig. 7A–C). While HDL3 had no significant
effect on cell proliferation in BeWo cells, cell numbers were
significantly (P > 0.05) increased in JAr and Jeg3 cells in
the presence of HDL3 after 48 h. Analysis of the cellular
cholesterol content revealed that HDL3 led to a remarkable
increase in cellular cholesterol levels in JAr and Jeg3 cells
compared to cells cultured in the absence of HDL3. The

During placental development the trophoblast cells develop
along a cell lineage forming the villous cytotrophoblast with
the overlaying syncytiotrophoblast, both responsible for
hormone production and fetomaternal exchange (reviewed
in ref [18]). In addition, another trophoblast population, the
extravillous trophoblast of cell island and cell columns, is
formed which maintains the ability of proliferation and
invasion. Choriocarcinoma is a malignant neoplasm that

represents the early trophoblast of the attachment phase or
as later invasive stage [46–48]. Thus, in most cases,
choriocarcinoma has the appearance of trophoblast, being
predominantly syncytiotrophoblastic or cytotrophoblastic.
Some cytotrophoblastic choriocarcinomas secrete little
human chorionic gonadotropin and some choriocarcinomas also secrete human placental lactogen [22–27]. A
number of choriocarcinoma cell lines have been established;
these replicating trophoblasts, derived from the malignant
tumor or produced by viral transformation of normal
trophoblasts, are appropriate systems to mimick trophoblast behaviour in vitro.
Exogenous sources for cholesterol supply to fetal tissues
involve receptor-mediated uptake of maternal LDL [2,17].
Both trophoblasts [17,49] and trophoblast-like cells have
been reported to bind LDL [17,20,21]. Simpson et al. [21]
further reported that HDL3 was taken up and degraded
by BeWo cells in a time- and concentration-dependent
fashion, but the rate of degradation was considerably less
than was the rate of degradation of LDL. HDL serves as
a physiological cholesterol/CE-carrier during reverse cholesterol transport from peripheral tissue to the liver and/or
steroidogenic tissues. As HDL may exert biological action
in human trophoblast cells [50] we have investigated
binding, cell association, and holoparticle association of
HDL3 as well as selective CE-uptake from HDL3 in three
different choriocarcinoma cells lines. Binding of 125Ilabeled HDL3 (at 4 °C) to BeWo, JAr, and Jeg3 cells
studied here revealed Kd and bmax values (Table 1)
comparable to those when binding of HDL3 to trophoblast membrane protein fractions [3,51] or to intact first
trimester trophoblasts was investigated [52]. However, the
pronounced capability for lipid tracer uptake from
CE-labeled HDL3 in excess of holoparticle association in
JAr and Jeg3 (but not in BeWo) cells was indicative for

SR-BI-mediated selective CE-uptake from HDL3 in these
cell lines. SR-BI has been identified in human placental
tissues by Northern blot experiments [14]. Initial attempts
to explore a possible role of SR-BI on murine trophoblast
cells have involved immunofluorescence microscopy to
define the temporal and spatial pattern of SR-BI expression during rodent embryogenesis [16]. Here, we provide
evidence that SR-BI is highly expressed on human
trophoblast-like choriocarcinoma cell lines JAr and Jeg3
and we further propose that HDL3 does act as cholesterol
delivery vehicle to these cells via the SR-BI-mediated


Ó FEBS 2003

458 C. Wadsack et al. (Eur. J. Biochem. 270)

Fig. 4. Immunocytochemical evidence for
SR-BI on choriocarcinoma cells. (A) BeWo,
(B) Jar, (C) Jeg3, (D) ldlA(mSR-BI) and (E)
ldlA7 cells were cultured on Laboratory-Tek
chamber slides as described in Materials and
methods. Labeling was peformed with polyclonal rabbit anti-(SR-BI) IgG (dilution of
1 : 1000) followed by goat anti-rabbit cyanine3 secondary antibody (dilution 1 : 500).

pathway. The fact that the C-terminal cytosolic domain
of SR-BI which is a critical domain involved in selective
CE-uptake [29,53] is lacking in SR-BII and the lack of
SR-BII protein in all three choriocarcinoma cell lines
investigated here suggested that this receptor does not play
a specific role for CE-supply during fetal development.

SR-BI may probably exert more profound capabilities
than the common LDL-receptor pathway mediating holoparticle-uptake of apolipoprotein B- or E-containing lipoprotein particles. This must be seen in light of the fact that
expression of LDL-receptor mRNA decreases from first
trimester to term tissues [54] and thus SR-BI could provide
an alternative pathway for cholesterol/CE-supply during
fetal development. As SR-BI is critical for cholesterol
transport its regulation has been addressed in a number of
placental tissues involved in steroidogenesis [55–57]. In
humans, lacking functional LDL-receptor, fetal development and cholesterol supply is normal compared to
controls. Also in mice with a targeted deletion of the
LDL-receptor gene, the up-regulation of selective CEuptake suggests that SR-BI can compensate for the loss of
LDL-receptor function [58]. Recently, the LDL-receptor
related protein has also been addressed to mediate, at least
in part, selective lipid uptake [59]. LDL-receptor related protein was found to be associated with syncytiotrophoblasts

Table 2. Effect of HDL and ox–LDL on cell association of [3H]CEHDL at 37 °C. Choriocarcinoma cells were incubated for 4 h at 37 °C
with 10 lgỈmL)1 of [3H]CE-labeled HDL3 in the absence (control) or
presence of 100 lg of proteinỈmL)1 of unlabeled competitor (HDL or
ox-LDL) in F12K (BeWo) or DMEM (JAr and Jeg3). The 100% value
for cell association of [3H]CE-HDL was 105 ± 8 ng (BeWo),
255 ± 17 (JAr) and 286 ± 21 ng HDL3-protein/mg cell protein
(Jeg3). The cell association is expressed as the percentage of the radioactivity measured in the absence (100%) or presence of competitor.
Results are given as means ± SD of three independent experiments.
[3H]CE-HDL
BeWo
JAr
Jeg3

Control HDL

ox-LDL

100
100
100

43.5 ± 6.6
70.5 ± 9.5
79.4 ± 8.7

37.6 ± 5.7
21.2 ± 3.2
28.4 ± 4.3

Fig. 5. Effect of 8–CPTcAMP on cell association of [3H]CE-HDL and
mRNA SR-BI expression in choriocarcinoma cells. Following preincubation in F12K and DMEM without FBS (12 h) in the absence (open
squares) or presence (full squares) of 0.3 mM 8-CPT-cAMP, choriocarcinoma cells were incubated for 4 h with 10 lgỈmL)1 of [3H]CElabeled HDL3 at 37 °C. The washed cells were lysed with 0.3 M NaOH
to determine the cell-associated fraction. To determine the in vitro
effect of 8-CPT-cAMP on SR-BI mRNA expression, choriocarcinoma
cells were incubated as described above. Total RNA was isolated, and
Northern blot analysis was performed using radiolabeled SR-BI
cDNA probe for each cell line (top panel) in the absence (lane 1, 3, 5)
or presence of 0.3 mM 8-CPT-cAMP (lane 2, 4, 6). Radiolabeled
cDNA for GAPDH was used to monitor RNA loading (bottom
panel). Results are given as means ± SD of three independent
experiments.

(but not cytotrophoblasts) and BeWo cells [10]. However,
cAMP-treatment decreases expression of LDL-receptor
related protein [10] but increases expression of SR-BI in

parallel (Fig. 7 [13]).


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Human choriocarcinoma cells and SR-BI (Eur. J. Biochem. 270) 459

Fig. 6. Transfection of choriocarcinoma cells. Untransfected (1, 4,
and 7), mock-transfected (b-gal-transfected, 2, 5, and 8), and SR-BItransfected (3, 6, and 9) BeWo (1–3), JAr (4–6), and Jeg3 cells (7–9)
were incubated for 4 h with 10 lgỈmL)1 protein of [3H]CE-HDL3 at
37 °C as described in Fig. 5. Thereafter, the incubation medium was
removed, the cells were washed and analyzed for cell-associated
radioactivity of [3H]CE-HDL3. Results are given as means ± SD of
three independent experiments. Immunoblot experiments of detergent
solubilized membrane protein fractions (50 lg protein per lane) of
untransfected (1, 4, and 7) and SR-BI-transfected cells (3, 6, and 9)
were performed as described in Fig. 3C.

Different lines of evidence are of support that SR-BI is a
physiologically relevant HDL receptor, studies which are
primarily addressing its role in cholesterol metabolism
[12,13]. Further studies demonstrated that SR-BI-deficiency
in rodents leads to defective erythroid maturation and
abnormalities in the female reproductive system [13,60].
Evidence is accumulating that cholesterol must be considered as an essential agent in embryonic development.
Histochemical analysis of ovaries from superovulated
females showed reduced oil red O staining of lipids in the
ovarian copora lutea of SR-BI knock out relative to those of
wild-type animals suggesting reduced CE storage as a source
for steroid hormone production [61]. Also plasma progesterone levels between pseudopregnant controls and

knock out females 6 days after mating were slightly
although not significantly impaired. Finally, the majority
of embryos from SR-BI knock out females at harvesting
showed abnormal, nonrefractile morphology of oocytes and
embryos [61] similar to wild-type females that had been
treated with cholesterol-binding drugs that can perturb
membrane structure. Female mice with a targeted null
mutation of the SR-BI gene are infertile [61], a fact
underscoring the importance of the SR-BI pathway during
embryonic development. As SR-BI is expressed on the
maternal–fetal interface [16], this could indicate defective
cholesterol/CE-supply to the growing embryo.
From our studies we propose that some choriocarcinoma
cell lines can be used as a suitable model to mimick

Fig. 7. Effect of exogenous HDL on cellular growth rates and cholesterol content. Cells were seeded in 6-well trays and incubated in F12K
(BeWo) and DMEM (JAr and Jeg3) containing 10% lipoproteindeficient serum (open symbols) or in medium containing lipoproteindeficient serum and 100 lgỈmL)1 HDL3 (closed symbols). At the
indicated time points the cells were washed with HBBS, trypsin-treated
and the cell number was counted. The cellular cholesterol content of
lysed cells was analysed with enzymatic cholesterol reagent. Data
shown represent means ± SD of three independent experiments. (A)
BeWo, (B) JAr, (C) Jeg3.


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460 C. Wadsack et al. (Eur. J. Biochem. 270)

trophoblast–lipoprotein interactions in vitro. In BeWo the
LDL-receptor seems to be the predominant pathway

supplying these cells with cholesterol/CE via holoparticleuptake [10]. The fact that BeWo, a cell line with many
characteristics of cytotrophoblast, are almost lacking SR-BI
and SR-BI-mediated selective CE-uptake from HDL3,
supports the assumption that JAr and Jeg3 resembling
syncytiotrophoblasts-like properties are suitable in vitro
models to study directed cholesterol/CE-transport in polarized cells. Invasiveness of trophoblast cells in vivo and
choriocarcinoma cells in vitro apparently is linked to celldifferentiation and proliferation [47] similar to that reported
for other tumors [62,63].
In line with previous findings using various differentiation-modulating agents [47,48] we also observed pronounced differences between BeWo and JAr/Jeg3 cells
regarding proliferation rates. The fact that exogenous
HDL3 remarkably increases proliferation in JAr and Jeg3
supports a receptor-mediated specific and selective CEuptake and cellular growth in parallel. The results of this
study demonstrate that JAr and Jeg3 cells, replicating
trophoblast cells derived from a malignant tumor are
appropriate cellular systems to mimick cholesterol supply
from maternal lipoproteins to developing embryonic tissues
via SR-BI-mediated CE-uptake from HDL3.

9.

10.

11.

12.

13.

14.

15.

Acknowledgements
The authors thank Dr Vardon (local blood bank) providing human
plasma. This work was supported by the Austrian Science Fund (FWF,
15404 to E. M. and SFB 007–716 to W. S.) and the Austrian National
Bank OENB 8840 (to A. H.), and 8778 and 9962 (to E. M.).

16.

17.

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