Trophoblast-like human choriocarcinoma cells serve as a
suitable
in vitro
model for selective cholesteryl ester uptake
from high density lipoproteins
Christian Wadsack
1
, Andelko Hrzenjak
1
, Astrid Hammer
2
, Birgit Hirschmugl
1
, Sanja Levak-Frank
1
,
Gernot Desoye
3
, Wolfgang Sattler
1
and Ernst Malle
1
1
Institute of Medical Biochemistry and Molecular Biology,
2
Institute of Histology and Embryology,
3
Clinic of Obstetrics
and Gynecology, Karl-Franzens University Graz, Austria
As human choriocarcinoma cells display many of the
biochemical and morphological characteristics reported for

H]CE-uptake in excess of HDL
3
–holoparticle
association, and cAMP–mediated enhanced cell associ-
ation of [
3
H]CE-HDL
3
in JAr and Jeg3 suggested the
scavenger receptor class B, type I (SR-BI) to be respon-
sible 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 immunocyto-
chemical technique. Adenovirus-mediated overexpression
of SR-BI in all three choriocarcinoma cell lines resulted in
an enhanced capacity for cell association of [
3
H]CE-HDL
3
(20-fold in BeWo; fivefold in JAr and Jeg3). The fact that
exogenous HDL
3
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.
Keywords: BeWo; JAr; Jeg3; placenta; SR-BI.

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:[
3
H]CE, [1,2,6,7–
3
H]-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; HDL
3
,highdensity
lipoprotein subclass 3; LDL, low density lipoprotein; LPDS, lipo-
protein-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)
Eur. J. Biochem. 270, 451–462 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03394.x
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 biosyn-

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 syncytiotrophoblast-
like 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 choriocar-
cinoma 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 resul-
ted 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 ¼ 1.035–1.065 gÆmL
)1
) and HDL subclass 3
(HDL
3
, lacking apolipoprotein E, a ligand for the LDL-
receptor, d ¼ 1.125–1.21 gÆmL
)1

preparations were monitored by SDS/PAGE to ensure that
the preparations were free of radiolytic or oxidative damage.
(b) HDL
3
was labeled with [cholesteryl-1,2,6,7–
3
H]-palmi-
tate ([
3
H]CE, NEN) by cholesteryl ester transfer, protein-
catalyzed transfer from donor liposomes exactly as described
[34]. Subsequently, the labeled HDL
3
-fractions were reiso-
lated in a TLX120 bench-top ultracentrifuge in a TLA100.4
rotor (Beckman, Austria). The HDL
3
-band was aspirated
and dialyzed against 10 m
M
NaCl/P
i
,pH7.4.
Double-labeling. Labeling of HDL
3
with [cholesteryl-
1,2,6,7–
3
H]-oleate (NEN) was performed by incubation of
the lipoprotein with palmityl oleyl phosphatidylcholine-

proliferation, the cells were seeded into 6-well plates at a
starting density of 2.5 · 10
4
cells in 2 mL 10% lipoprotein-
deficient serum (LPDS) medium for BeWo, 5 · 10
4
cells for
JAr and 1 · 10
5
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 (CASY
Ò
1, Scha
¨
rfe,
Reutlingen, Germany). Fresh medium containing lipopro-
tein-deficient serum or lipoprotein-deficient serum plus
100 lgÆmL
)1
HDL
3
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
452 C. Wadsack et al. (Eur. J. Biochem. 270) Ó FEBS 2003

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
125
I-labeled HDL
3
-protein,
the cells were incubated at 37 °C for 4 h as described above
with the same amounts of labeled and unlabeled HDL
3
.
Subsequently, the medium was aspirated and the cells were
rinsed as described above [34]. Degradation of
125
I-labeled
HDL
3
by choriocarcinoma cells was estimated by measur-
ing the nontrichloroacetic acid-precipitable radioactivity in
the medium after precipitation of free iodine with AgNO
3
exactly as described [38]. Briefly, 0.5 mL of medium was

[
3
H]CE-HDL
3
by choriocarcinoma cells was measured by
increasing concentrations of unlabeled competitors (HDL
3
and ox-LDL) as well as in the absence or presence of 300 l
M
8-(4-Chlorophenylthio)adenosine 3¢,5¢-cylic monophosphate
(8-CPTcAMP, Sigma, Vienna).
Selective CE-uptake
To calculate the selective CE-uptake from HDL
3
during the
same experiment, choriocarcinoma cells were incubated
with
125
I-labeled and [
3
H]CE-HDL
3
. In case of
125
I-labeled
HDL
3
, the cell-associated and nontrichloroacetic acid-
precipitable radioactivity in the medium was counted. The
sum of cell-associated and degraded

H-tracers on the same basis.
In a parallel set of experiments, the selective CE-uptake
from HDL
3
was calculated from double-labeled HDL
3
as
the difference between [
3
H]cholesteryl oleate- and
125
I-
labeled lipoprotein [35]. The sum of specific degradation
(measured as described above) and specific cell association of
125
I-labeled HDL
3
reflects HDL
3
–holoparticle association.
SDS/PAGE and Western blotting
Detergent extracts of solubilized membrane protein frac-
tions [33] or total cell proteins of choriocarcinoma and CHO
cells were separated on 8% SDS/PAGE. Protein transfer to
nitrocellulose membranes was performed electrophoreti-
cally 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,

Ó FEBS 2003 Human choriocarcinoma cells and SR-BI (Eur. J. Biochem. 270) 453
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, Zu
¨
rich,
Switzerland) which was originally inserted into pcEXV-3
vector was partially restricted with EcoRIandthe2.5kb
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, pro-
pagated, 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

) in 1 mL of infection media
(DMEM medium containing 2% FBS, 50 UÆmL
)1
penicil-
lin, and 50 lgÆmL
)1
streptomycin) for 2 h (37 °C, 5% CO
2
,
humidified atmosphere). After removing infection-media,
the cells were supplied with 2 mL of S12K medium
(containing 5% FBS, 2 m
M
glutamine, 50 UÆmL
)1
penicil-
lin and 50 lgÆmL
)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.
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 m
M

Pictures were taken with an Axiophot microscope (Zeiss,
Oberkochen, Germany).
Results
Binding of HDL to choriocarcinoma cells
Specific binding of HDL
3
to all three choriocarcinoma cell
lines at 4 °C was saturable at the highest lipoprotein
concentrations (100 lgproteinÆmL
)1
) used. Calculation of
binding parameters yielded similar K
d
and b
max
values for all
three cell lines (Table 1). Also the specific cell association of
125
I-labeled HDL
3
(the sum of binding and internalization)
measured at 37 °C was comparable for all three cell lines
(Fig. 1A–C). The specific degradation of
125
I-labeled HDL
3
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

-proteinÆmL
)1
)
b
max
(ng HDL
3
-proteinÆmg
)1
cell protein)
BeWo 41.9 ± 10.1 161.3 ± 17.3
JAr 34.1 ± 3.9 145.9 ± 6.9
Jeg3 29.7 ± 7.4 186.9 ± 21.2
454 C. Wadsack et al. (Eur. J. Biochem. 270) Ó FEBS 2003
HDL
3
). However, calculating the selective CE-uptake from
HDL
3
as the difference between both tracers revealed that
this pathway is not a preferential routing to supply BeWo
with CEs from HDL
3
. To confirm the low capacity of
BeWo cells for selective CE-uptake, the HDL
3
-particle was
double-labeled and values obtained for specific cell associ-
ation of [
3

sixfold (1537 vs. 267 ng lipoproteinÆmg
)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 HDL
3
-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

bound and internalized radioactivity). To determine specific degrada-
tion (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
describedinMaterialsandmethods(ÔdegradedÕ). Results are given as
means ± SD of three independent experiments.
Ó FEBS 2003 Human choriocarcinoma cells and SR-BI (Eur. J. Biochem. 270) 455
immunocytochemistry (Fig. 4A). To further verify specifi-
city 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 HDL
3
and
ox-LDL (a high affinity ligand for SR-BI [10]) were equally
effective to compete for cell association of [
3
H]CE-HDL
3
in
BeWo cells, HDL
3
lowered cell association of [

3
H]CE-HDL
3
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 [
3
H]CE-HDL
3
. 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 [
3
H]CE–HDL
3
and cell
association of [
3
H]CE-HDL
3
was measured. While mock-

H]CE-HDL
3
at 37 °C. To determine spe-
cific cell association of [
3
H]CE-HDL
3
(closed circles) the cells were
incubated in the absence (total association) or presence of a 20-fold
excess (nonspecific association) of unlabeled HDL
3
. 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
[
3
H]CE-HDL (closed circles) and
125
I–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.
456 C. Wadsack et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Effects of exogenous HDL on growth rates
To investigate whether (a) the pronounced cell association
of [
3

low rate of selective CE-uptake from HDL
3
in these cell
lines (Fig. 2) demonstrating that exogenous HDL
3
may not
significantly alter CE synthesis in these cell lines [45].
Discussion
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 choriocarcino-
mas 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 tropho-
blast behaviour in vitro.
Exogenous sources for cholesterol supply to fetal tissues
involve receptor-mediated uptake of maternal LDL [2,17].

3
to tropho-
blast 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 HDL
3
in excess of holoparticle association in
JAr and Jeg3 (but not in BeWo) cells was indicative for
SR-BI-mediated selective CE-uptake from HDL
3
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 expres-
sion 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 HDL
3
does act as cholesterol
delivery vehicle to these cells via the SR-BI-mediated
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

humans, lacking functional LDL-receptor, fetal develop-
ment 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 CE-
uptake 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 pro-
tein was found to be associated with syncytiotrophoblasts
(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]).
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
chamberslidesasdescribedinMaterialsand
methods. Labeling was peformed with poly-
clonal rabbit anti-(SR-BI) IgG (dilution of
1 : 1000) followed by goat anti-rabbit cyanine-
3 secondary antibody (dilution 1 : 500).
Table 2. Effect of HDL and ox–LDL on cell association of [
3
H]CE-
HDL at 37 °C. Choriocarcinoma cells were incubated for 4 h at 37 °C
with 10 lgÆmL
)1
of [
3

carcinoma cells were incubated for 4 h with 10 lgÆmL
)1
of [
3
H]CE-
labeled HDL
3
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 m
M
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.
458 C. Wadsack et al. (Eur. J. Biochem. 270) Ó FEBS 2003
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 consid-
ered as an essential agent in embryonic development.

37 °C as described in Fig. 5. Thereafter, the incubation medium was
removed, the cells were washed and analyzed for cell-associated
radioactivity of [
3
H]CE-HDL
3
. 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.
Fig. 7. Effect of exogenous HDL on cellular growth rates and choles-
terol content. Cells were seeded in 6-well trays and incubated in F12K
(BeWo) and DMEM (JAr and Jeg3) containing 10% lipoprotein-
deficient serum (open symbols) or in medium containing lipoprotein-
deficient serum and 100 lgÆmL
)1
HDL
3
(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.
Ó FEBS 2003 Human choriocarcinoma cells and SR-BI (Eur. J. Biochem. 270) 459
trophoblast–lipoprotein interactions in vitro. In BeWo the
LDL-receptor seems to be the predominant pathway
supplying these cells with cholesterol/CE via holoparticle-
uptake [10]. The fact that BeWo, a cell line with many

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.).
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