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doi:10.1016/j.placenta.2004.10.005
Liver X Receptors Mediate Inhibition of hCG Secretion
in a Human Placental Trophoblast Cell Line
M. S. Weedon-Fekjaer, A. K. Duttaroy* and H. I. Nebb
Department of Nutrition, University of Oslo, POB 1046 Blindern, N-0316 Oslo, Norway
Paper accepted 6 October 2004
Liver X receptors (LXR) a and b are important regulators of lipid homeostasis in liver, adipose and other tissues. However, no
such information is available for the human placenta. We determined expression of both LXR a and b in placental trophoblast cell
lines, BeWo and JAR. Exposure of BeWo cells to a synthetic LXR agonist, T0901317, resulted in an increase in the amount of
mRNA of LXR target genes, sterol regulatory element-binding protein-1 and fatty acid synthase. T0901317 also increased the
synthesis of lipids. Moreover, T0901317 resulted in a reduced secretion of hCG during differentiation of these cells. Our data for
the first time demonstrate a new role for LXRs in the human placenta.
Placenta (2004), jj, jjjejjj
Ó 2004 Elsevier Ltd. All rights reserved.
Keywords: LXR; Placenta; Lipid synthesis; hCG; SREBP-1; BeWo cells
INTRODUCTION
Placental synthesis of hormones and transport of nutrients are
critical for fetal growth and development [1]. Despite its
importance in growth and development of the feto-placental
unit, very little is known regarding regulation of fatty acid
synthesis, transport and metabolism in the placenta [2]. Fatty
acids are used by both the fetus and placenta to form
triglyceride for energy stores, for membrane structure, to
synthesize complex lipids, cell signaling, and gene expression
[1,2]. The placenta is composed of villi with highly specialized
trophoblast cells, the villous trophoblasts, that form a barrier
between the maternal uterus and fetus [3]. These cells are
involved in the nutrient transport and metabolism in the
human placenta [2]. The villous trophoblasts consist of
cytotrophoblasts and syncytiotrophoblasts. The villous cytotrophoblasts proliferate and differentiate by fusion to form
syncytiotrophoblasts that covers the entire surface of the villi
[4].
A sign of cytotrophoblast fusion in vitro is the increased
production of the placental hormone hCG as syncytiotrophoblasts are the primary cells involved in placental hormone
production. Placental synthesis of hCG is necessary for the
maintenance of pregnancy in humans during the first trimester
and it is directly involved in the stimulation of cytotrophoblast
differentiation into the syncytiotrophoblast, in an autocrine
* Corresponding author. Tel.: C47 22 85 15 47; fax: C47 22 85 13
41.
E-mail address: a.k.duttaroy@medisin.uio.no (A.K. Duttaroy).
0143e4004/$esee front matter
process [5,6]. Recently, peroxisome proliferator-activated
receptor gamma (PPARg) and retinoid X receptors (RXR)
have been shown to be involved in the regulation of hCG [7,8].
hCG is a heterodimer comprising an alpha subunit (hCGa),
common to all glycoprotein hormones, and a distinct beta
subunit (hCGb) responsible for biological specificity of the
hormone. PPARg/RXRa heterodimers directly increase the
secretion and transcript levels of hCGb. Preliminary data
indicate that elements located in the regulatory region of the
hCGb gene bind RXRa/PPARg heterodimer suggesting that
the heterodimer directly regulates human hCGb transcription
[7,8]. In human placenta, PPARg/RXRa heterodimers are
functional units during cytotrophoblast differentiation into the
syncytiotrophoblast in vitro. PPARg is also involved in cell
differentiation of different cell types [8,9]. Barak et al. [10]
demonstrated that PPARg null mice die during embryogenesis
because of interference of terminal differentiation of the
trophoblast and placental vascularization. Unlike the PPARa
knockout mice, PPARg knockout mice did not survive
gestation and they had an embryonic lethal phenotype at day
10 of gestation [10]. Schaiff et al. [11] also reported, in
trophoblasts cells, that two types of PPARg agonists have
opposing effects, one promoting differentiation and the other
hindering differentiation and promoting apoptosis. All these
observations indicate that PPARg is involved in trophoblast
differentiation.
Several transcription factors are known to regulate lipid
flux, storage and utilization, of which some of the most
important are SREBP, PPAR and LXR. The LXRs belong to
a subclass of nuclear hormone receptors that form obligate
Ó 2004 Elsevier Ltd. All rights reserved.
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heterodimers RXR and are bound and activated by oxidized
derivatives of cholesterol [12,13]. There are two known LXR
isoforms; LXRa (NR1H3) and LXRb (NR1H2). LXRa is
known to have a relative restricted expression pattern (liver,
kidney, intestine, adipose tissue, adrenals and macrophages),
whereas LXRb is ubiquitously expressed. RXR/LXR heterodimers activate their target genes by binding to specific
responsive elements termed LXREs, consisting of a hexanucleotide direct repeat separated by four nucleotides (DR4)
[14].
Studies of LXR target genes in LXR deficient mice indicate
a pivotal role for the LXRs in the regulation of hepatic bile
acid synthesis and intestinal cholesterol absorption [15,16].
LXRs have also been shown to be involved in cholesterol efflux
and reverse cholesterol transport in macrophages and other
peripheral tissues [17,18]. In liver and adipose tissue, LXRs
are involved in lipogenesis by direct regulation of key target
genes, including SREBP-1c [19], the master transcriptional
regulator of fatty acid synthesis, and fatty acid synthase (FAS),
a key enzyme in the de novo biosynthesis of fatty acids [20].
LXRaÿ/ÿ knock out mice showed impaired expression of
several hepatic genes while LXRbÿ/ÿ knock out mice failed to
the phenotype as observed in LXRaÿ/ÿ knock out mice [21].
These studies indicated a more prominent role of LXRa than
LXRb as a regulator of LXR target genes in hepatocytes.
Since the LXRs are key regulators of lipid metabolic
signaling pathways in several tissues, these may also play an
important role in lipid metabolism in placenta and thus may
regulate lipid metabolism in pregnancy. Normal human
pregnancies result in a pronounced physiologic hyperlipidemia
involving a gestational rise in plasma lipids [22]. Hyperlipidemia is the most common complication of pregnancy, remains
a source of maternalechild morbidity and mortality, however,
the etiology of this disorder is still not understood. Maternal
hyperlipidemia is a characteristic feature during pregnancy and
corresponds to an accumulation of triglycerides not only in
very low-density lipoprotein but also in low- and high-density
lipoprotein. In preeclampsia, there is a further increase in
plasma lipids compared to normal pregnancy [22,23]. Maternal
liver may play major roles in lipid homeostasis, however,
placenta’s role in the development of hyperlipidemia has been
increasingly evident [24,25]. As triglycerides do not cross the
placental barrier, the presence of lipoprotein receptors in the
placenta, together with lipoprotein lipase, phospholipase A2,
and intracellular lipase activities, allows the release to the fetus
of polyunsaturated fatty acids transported as triglycerides in
maternal plasma lipoproteins. The expression of both VLDL
and LDL receptor mRNAs was reduced in the placentas of
women whose pregnancies were complicated by preeclampsia
[26]. The decrease in these placental lipoprotein receptors may
result in reduced receptor-mediated uptake of plasma lipoproteins by the placenta and hence a reduced clearance of
these lipoproteins from the maternal plasma.
Placental regulatory proteins and their integration for
maintenance of cholesterol and lipid homeostasis need to be
maintained optimally for both the mother and the developing
fetus [24,25]. The presence of PPARs and RXRs in human
trophoblasts and their importance in feto-placental development is reported [8e10]. However, no such information is
available on LXRs and their roles in lipid metabolism in the
placenta. In order to understand their role in placenta, we
investigated LXRs in human placental trophoblast cells. In
this study we have used BeWo as a model for lipid metabolism.
BeWo cells are a good experimental model for placental
trophoblasts to study lipid metabolism, as these cells express
several lipid metabolic genes [27e31].
We demonstrate, for the first time, that both LXRa and
LXRb are expressed in BeWo and JAR cells, and that they
regulate expression of LXR target genes (FAS and SREBP)
involved in lipogenesis. Moreover, we also report that the
LXRs mediate inhibition of hCG secretion during trophoblast
differentiation. Together these data indicate a potential role for
the LXRs in the regulation of lipid metabolism and a possible
involvement in differentiation of trophoblast cells.
MATERIALS AND METHODS
Materials
Dual luciferase assay and pTK renilla luciferase were obtained
from Promega Corp. (Madison, USA). The LXR ligand
T0901317 was purchased from Alexis Biochemicals (Carlsbad,
USA). Hybond-N nylon membrane and ImageQuantÔ,
software for quantifying the Northern blots by phosphorimaging, were obtained from Amersham Biosciences (Buckinghamshire, UK) and [a-32P]dCTP from Perkin Elmer (Boston,
USA). Scintillation solution was obtained from Packard
BioSciences B.V. (Groningen, The Netherlands), [2-14C]acetic
acid sodium salt (55 mCi/mmol) from ARC (St Louis, MO,
USA), TLC plates from Merck (Darmstadt, Germany) and
lipid standards from Larodan Fine Chemicals (Malmö,
Sweden). Reagent for measuring protein was from Uptima
Inerchim (Montlucon, France). Nutrient Mixture F12 HAM
and RPMI-1640 medium, penicillin, trypsin, streptomycin,
fetal bovine serum, L-glutamine, 22(R) HC and other chemicals
used were obtained from Sigma (St Louis, MO, USA).
Cell culture
The BeWo (p195) and JAR (p175) cell lines, human
choriocarcinoma cell lines, were obtained from American
Type Culture Collection (Manassas, USA). BeWo and JAR
cells were maintained in HAM’s F12 and RPMI-1640
medium, respectively. Both media were supplemented with
2 mM L-glutamine, penicillin (50 units/ml), streptomycin
(50 mg/ml) and 10% heat-inactivated fetal bovine serum,
and trypsinated approximately every 4th day. Cell cultures
were maintained at 37 (C in a 5% CO2 atmosphere.
Differentiation of BeWo cells was performed by incubating
the cells for 48 h with 100 mM forskolin. Differentiation
efficiency was verified by measuring hCG secretion with
ELISA (DRG Instruments, Germany). Cell viability was
determined by measuring LDH (Roch, Mannheim, Germany).
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Ligand treatment
Wherever necessary, BeWo cells were treated with different
ligands (T0901317 or 22(R) hydroxycholesterol) dissolved in
DMSO or vehicle (DMSO) as a control. These agents were
added at the same time and subsequently incubated under
identical experimental conditions.
RNA extraction and Northern blot analysis
Total RNA from BeWo or JAR cells was extracted with
TRIzolÒ Reagent (Invitrogen, Paisley, UK) as recommended
by the manufacturer. Total RNA (20 mg) was separated on
a 1.5% agarose formaldehyde gel and blotted and hybridized as
described previously [32]. Blots were hybridized with cDNA
probes from human (h) and rat (r), rLXRa, hLXRb, hCG,
rSREBP-1 and rFAS. Hybridization with the human ribosomal protein L27 (Manassas, USA) was used as a control for
equal RNA loading. The membranes were probed with
[a-32P]dCTP radiolabeled cDNA, prepared using Megaprime
DNA labeling system (Amersham Biosciences, Inc., Buckinghamshire, UK). Specific activities from 2 to 6 ! 108 cpm/
mg DNA were used. The sizes of the mRNA transcripts were
calculated on the basis of the migration of the 18 and 28 S
rRNA, which were visualized by ethidium bromide. Northern
blots were analyzed by phosphorimaging.
Transient transfection and luciferase assay
BeWo cells (20,000 cells/cm2) were seeded in six-well plates.
The cells were transfected using 4 ml LipofectamineÔ Reagent
(Invitrogen, Carlsbad, USA) and 1.5 mg of 2 ! LXRE plasmid
fused to a luciferase reporter gene (a gift from J.A. Gustafsson,
Karolinska Institute, Sweden) and 100 ng pTK renilla
luciferase (as a control of transfection efficiency). The transient
transfection was carried out as recommended by the
manufacturer. Three hours after transfection, cells were
cultured in medium containing serum and incubated for 24 h
in the same medium containing appropriate agents, as
indicated. Cells were harvested in 250 ml reporter lyses buffer,
and the luciferase activities were measured using the DualLuciferaseÒ Reporter Assay System (Promega, Madison,
USA) as recommended by the manufacturer.
Thin-layer chromatography (TLC)
BeWo cells (15,000 cells/cm2) were seeded in six-well plates.
After 24 h, these cells were stimulated with 1 mM T0901317 or
with DMSO for 48 h. Medium was then replaced with serum
free medium and incubated for 12 h prior (in order to reduce
the amount of cellular lipids) to the addition of 100 mM Na
acetic acid containing 1.5 mCi/ml [2-14C]acetic acid, and
subsequently incubated for 8 h in serum free medium. Lipids
were extracted from the medium using chloroform:methanol
as described previously [33]. Total radioactivity was counted
by liquid scintillation to determine the incorporation of
radiolabeled acetic acid into lipids. In addition, incorporation
of radioactivity into different lipid fractions (fatty acids, FA,
3
monoacylglycerol, MAG, diacylglycerol, DAG, triacylglycerol,
TAG, and phospholipids, PL) was determined by TLC
analysis using hexane/diethyl ether/acetic acid (65:35:1, vol/
vol/vol) as solvent [33]. The various lipids were visualized by
iodine, the TLC plates scraped and the samples were counted
by liquid scintillation. Appropriate lipid standards were used
for identification of lipids. Cells from each well were harvested
in 400 ml distillated water and protein measured as an internal
control.
ELISA of hCG
BeWo (9000 cells/cm2) cells were seeded in six-well plates.
After 72 h, the cells were then incubated with vehicle (DMSO)
or increasing concentrations of T0901317, together with
100 mM of forskolin as indicated elsewhere. Medium was
collected after incubation for 48 h. Free bhCG was then
measured in the medium without concentration by ELISA, as
recommended by the manufacturer (DRG Instruments
GmbH, Germany).
Statistical analysis
Differences in 2 ! LXRE reporter gene activation were tested
with independent samples T-test, and related confident
intervals were calculated using Student T-distribution. The
effect of different levels of T0901317 was studied by either
linear regression on log scale (hCG) or linear regression on
square root (SREBP-1 and FAS). Regarding the TLC lipid
data, a mixed effect model [34] was used to account for the two
independent experiments with unequal number of measurements. All calculations were done using either the R or SPSS
statistical packages.
RESULTS
Expression of LXRs in trophoblast cell lines
Expression of LXR a and b genes was studied in undifferentiated and differentiated BeWo using Northern blot
analyses (Figure 1A). Expression of LXRa and LXRb was also
determined after differentiation of these cells with 100 mM
forskolin for 48 h to form a syncytium. Syncytium formation
was monitored by measuring hCG secretion (data not shown).
A higher LXRb mRNA expression was observed in the
differentiated BeWo cells compared to undifferentiated cells,
whereas the reverse expression pattern was observed in the
case of LXRa. JAR cells (undifferentiated) also showed the
presence of LXR isomers.
BeWo cells were then transfected with a luciferase reporter
construct containing 2 ! LXRE and incubated with 5 mM of
an LXR oxysterol agonist 22(R) hydroxycholesterol [22(R)
HC] and a synthetic selective LXR agonist T0901317 (1 mM)
[35]. Consistent with the LXRa and LXRb mRNA data, there
was an approximately 10-fold increase with T0901317 and a 6fold increase with the 22(R) HC in reporter gene activity. This
indicates that the BeWo cells contain endogenous LXR and
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A
Udiff.
BeWo
Diff.
BeWo
JAR
LXRα
LXRβ
L27
B
statistically significant increase in FA, TAG and DAG
(p ! 0.001) and PL/MAG (p ! 0.05) compared to untreated
cells (Table 1).
In order to explore whether LXR lipogenic target genes
(SREBP-1 and FAS) were involved in increased synthesis of
lipids, we examined the mRNA expression of SREBP-1 and
FAS genes in BeWo cells under these conditions. A dose
dependent increase in SREBP-1 and FAS expression was
observed with increasing concentrations of T0901317. The
highest expression of both SREBP-1 (7-fold) and FAS (5-fold,
p-values for linear trend !0.00001) was observed at 0.1 mM of
the agonist in undifferentiated cells (Figure 2A). Similarly, the
highest expression of both SREBP-1 (2- and 9-fold) and FAS
(2- and 6-fold, p-values for linear trend !0.001) was observed
at 0.1 mM of the agonist in differentiated cells (Figure 2B).
Use of T0901317 at concentrations O0.1 mM did not produce
further expression of these genes (data not shown). The
SREBP-1 cDNA probe used in these Northern blot analyses
does not distinguish between SREBP-1 and SREBP-1a.
However, only SREBP-1c is shown to be induced by LXR
agonists [19], and is therefore likely to be the transcript
observed in this study.
Effect on hCG production during
cell differentiation
Since the level of hCG increases during differentiation of
cytotrophoblasts to syncytiotrophoblasts, we wanted to
examine if activated LXRs could affect hCG mRNA and
protein synthesis during differentiation. Simultaneous incubation with T0901317 and forskolin throughout cell
differentiation produced a dose dependent reduction of hCG
mRNA transcript (p-value for trend !0.0001) (Figure 3A)
and hCG secretion (p-value for trend !0.001) (Figure 3B).
Figure 1. Expression and activation of LXRa and LXRb in placental cells.
(A) Total RNA from undifferentiated and differentiated BeWo cells and JAR
cells (undifferentiated) were harvested, and the LXRa, LXRb and L27 mRNA
levels were measured by Northern blot analysis. (B) Undifferentiated BeWo
cells were transiently transfected with a 2 ! LXRE luciferase reporter plasmid
and a control reporter plasmid (renilla luciferase) as described in Materials and
methods. Cells were incubated for 24 h with vehicle (DMSO), 5 mM 22(R)
HC or 1 mM T0901317. The results are expressed as relative luciferase
activity. Values are given as mean with 95% confidence interval of three
independent experiments. A star (*) indicates significant difference (p ! 0.01)
from the control.
RXR proteins that are able to transactivate LXR target genes
(Figure 1B).
LXR mediated lipid synthesis and target genes
To investigate whether the LXRs are involved in lipid
synthesis, BeWo cells were incubated with 14C-acetate after
stimulation with T0901317 for different time periods. An
incubation for 8e10 h produced greatest increase in lipid
synthesis (2.6-fold) when compared with those of untreated
cells (data not shown). At 8 h there was a 2.5- to 4.5-fold
DISCUSSION
We demonstrated that both LXRa and LXb genes are
expressed in human placental trophoblast cell lines, BeWo
and JAR, and these can be regulated by LXR agonist
treatment. We further demonstrated that regulation of fatty
acid synthesis is possibly mediated via LXR target genes in
BeWo cells.
Placenta is metabolically active in lipid metabolism, and our
data for the first time support such a regulatory role for LXR
in placental trophoblasts. LXR has similar lipid metabolic
effect in placenta as observed in liver and adipose tissues
[19,35e37].
The gene expression patterns of LXRa and LXRb mRNA
in differentiated BeWo cells are not similar. LXRa was
expressed in greater amounts in undifferentiated than in
differentiated cells, while this was opposite for LXRb. In
addition, LXRa but not LXRb mRNA expression was
increased by T0901317 treatment in BeWo cells (data not
shown). This indicates possible different metabolic roles yet
unknown for the two isoforms of LXR in the cytotrophoblasts
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Table 1. LXR induced synthesis of lipids in BeWo cells expressed as (pmol ( C) acetic acid incorporated/mg protein)
FFA
PL/MG
DG
TG
Control
1 mM T0901317
54.8
209.5
103.8
83.4
177.4
554.6
251.9
363.4
Difference w/95% confidence interval
122.7
345.0
148.1
280.0
p-Value
(61.9, 183.5)
(75.0, 615.1)
(96.2, 199.9)
(139.7, 420.4)
0.00063
0.01572
0.00002
0.00069
BeWo cells were incubated with vehicle (DMSO) or T0901317 for 48 h, followed by 8 h with 14C acetate. Medium and cells were collected and lipids
extracted and separated. Values were normalized to protein and given as mixed effect model estimates built on two independent experiments with 3 and 6
parallels.
and syncytiotrophoblasts. However, this observation is not
without precedent as differential expression of LXRa and
LXRb genes is also observed in adipocytes and myoblasts. In
contrast to BeWo cells, expression of LXRa in adipocytes and
myoblasts was much more pronounced in differentiated cells
compared with undifferentiated cells, whereas no such
difference of gene expression of LXRb was observed [20,37].
In LXRb deficient mice there was an increase in LXRa
expression, while no such increase was seen in LXRb mRNA
A
B
T0901317 (µM)
Control
0.0001
0.001
levels in LXRa deficient mice (unpublished data). This
indicates that LXRb has the potential to regulate basal LXRa
expression, and giving an explanation for a reduced expression
of LXRa where LXRb is dominant as in BeWo cells. The
physiological implications of these differential roles of LXR in
placental biology are yet to be explored. Most recent data
suggest that LXRb is involved in inhibitory effects of oxLDL
on trophoblast invasion in vitro, however, mechanisms are not
known yet [38]. LXR thus may play a role in preeclampsia,
0.01
T0901317 (µM)
Control
0.1
SREBP-1
SREBP-1
FAS
FAS
L27
L27
0.0001
0.001
0.01
0.1
Figure 2. SREBP-1 and FAS are target genes for LXR in BeWo cells. BeWo cells were incubated with vehicle (DMSO) or increasing concentrations of
T0901317. SREBP-1 (drawn line and squares) and FAS (dotted line and triangles) mRNA levels were measured by Northern blot analysis and quantitated by
phosphorimager analysis, normalized to L27. The values are related to control (set to Z 1). Statistical significance was tested by linear regression. (A)
Undifferentiated BeWo cells incubated for 24 h with T0901317. p-Values !0.00001. (B) BeWo cells incubated for 48 h with T0901317 and 100 mM forskolin.
p-Values !0.001.
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A
T 0901317 (µM)
Control
0.0001
0.001
0.01
0.1
hCG
L27
B
Figure 3. The expression of hCG mRNA and protein is reduced by
T0901317 during BeWo cell differentiation. BeWo cells were incubated with
vehicle (DMSO) or increasing concentrations of T0901317, together with
100 mM of forskolin. Statistical significance was tested by linear regression. (A)
Cells were harvested after 48 h. hCG mRNA levels were measured by
Northern blot analysis and quantitated by phosphorimager analysis, normalized to L27. The values are related to control (set to Z 1). (B) Medium and
cells were harvested after 48 h and free hCGb levels measured in media by
ELISA. Values were normalized to protein levels in each well and related to
control (set to Z 1). Values were given as mean of triplicates (p Z 0.00017),
and the data is a representative of three independent experiments.
a condition in which lipid peroxidation is increased and
invasion trophoblast cells are reduced.
Compared with the liver, placenta secretes 50e200 times
more FA [39], indicating the importance of FA as a mode of
delivery of lipids to the fetus [2]. The increased FA secretion
by BeWo cells mediated by LXRs indicates its importance in
placental lipid transport. This important process is probably
mediated at least in part by the LXR target genes SREBP-1
and FAS, as these genes were up-regulated by LXR agonist
treatment in BeWo cells. The presence of SREBP-1 and FAS
in human trophoblasts as well as in human fetal tissues was
reported [40]. High levels of expression of these genes in
human fetal tissues indicate their roles in proliferation in fetal
tissues. It is therefore interesting to speculate that SREBP-1
and FAS have an important function in rapidly developing
tissues like placenta where increased supply of lipids is
required in order to support optimum fetal growth. LXR may
play an important regulatory role in this regard.
It is interesting to note that a pathological increase in TAG
and FA during pregnancy is an independent risk factor for the
development of preeclampsia [41]. Preeclampsia is also
associated with elevated plasma concentrations of oxidized
low density lipoproteins (oxLDL) [42]. The receptors for
oxLDL in trophoblast cells [18,43] may supply oxysterols
through uptake of lipoproteins containing oxLDL for
activation of LXR [12,18] and subsequent increased fatty acid
synthesis. Fatty acid synthesized in the trophoblasts may opt
for fetal or maternal circulation, or both. Our experimental
conditions were designed to determine lipid synthesis by LXR
agonist in these cells, not the lipid secretion [39]. Therefore at
the present moment we cannot speculate whether increased
synthesis of fatty acids contribute to the elevated maternal or
fetal lipids. Further work is required using a transwell cell
culture model to determine the contribution of placental lipids
to maternal or fetal circulation.
LXRs significantly reduced hCG production in BeWo cells
in contrast to PPARg which increased hCG secretion in
a ligand specific way [8]. Complicating the picture, another
study showed a different effect of PPARg on hCG production
depending on which PPARg agonist was used [10,11]. This is
despite the fact that PPARg is important in the development of
placenta [10]. Interestingly, there was an induction of LXRa
transcript when BeWo cells were incubated with 1 mM of the
synthetic PPARg ligand darglitazone (data not shown),
indicating that LXRa is a PPARg target gene in these cells.
LXRa is also observed to be a PPARg target gene in adipocytes
[37] and in macrophages [44]. Although the mechanisms of
LXR mediated inhibition of hCG secretion in BeWo cells is not
known at present, we can speculate that LXRa may have a new
regulatory role in trophoblasts, countervailing the effect of
PPARg on hCG production and thereby contributing to
balance the production of hCG during pregnancy. Recent
observations indicate that apart from the activators of several
lipogenic genes, LXRs are involved in down regulation of genes
associated with carbohydrate metabolism and other functions
[45e47]. Further work needs to be done to understand the
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mechanisms involved in inhibition of hCG secretion mediated
by the LXRs in BeWo cells.
In conclusion, for the first time, we demonstrate that both
LXR a and b are expressed in human trophoblast cell lines,
7
and that these are involved in lipogenesis as well as
inhibition of hCG production in BeWo cells. LXR may
therefore play an important role, hitherto unknown, in fetoplacental biology.
ACKNOWLEDGMENT
We thank Harald Weedon-Fekjaer for help with statistics and figures. We also thank the Norwegian Sudden Infant Death for supporting this work.
REFERENCES
[1] Coleman RA. The role of the placenta in lipid metabolism and transport.
Semin Perinatol 1989;13:180e91.
[2] Duttaroy AK. Transport mechanisms for long-chain polyunsaturated
fatty acids in the human placenta. Am J Clin Nutr 2000;71:315Se22S.
[3] Knipp GT, Audus KL, Soares MJ. Nutrient transport across the
placenta. Adv Drug Deliv Rev 1999;38:41e58.
[4] Evain-Brion D, Malassine A. Human placenta as an endocrine organ.
Growth Horm IGF Res 2003;13(Suppl A):S34e7.
[5] Shi QJ, Lei ZM, Rao CV, Lin J. Novel role of human chorionic
gonadotropin in differentiation of human cytotrophoblasts. Endocrinology 1993;132:1387e95.
[6] Cronier L, Bastide B, Herve JC, Deleze J, Malassine A. Gap junctional
communication during human trophoblast differentiation: influence of
human chorionic gonadotropin. Endocrinology 1994;135:402e8.
[7] Guibourdenche J, Roulier S, Rochette-Egly C, Evain-Brion D. High
retinoid X receptor expression in JEG-3 choriocarcinoma cells: involvement in cell function modulation by retinoids. J Cell Physiol 1998;
176:595e601.
[8] Tarrade A, Schoonjans K, Guibourdenche J, Bidart JM, Vidaud M,
Auwerx J, et al. PPAR gamma/RXR alpha heterodimers are involved in
human CG beta synthesis and human trophoblast differentiation.
Endocrinology 2001;142:4504e14.
[9] Rosen ED, Spiegelman BM. PPAR{gamma}: a nuclear regulator of
metabolism, differentiation, and cell growth. J Biol Chem 2001.
[10] Barak Y, Nelson MC, Ong ES, Jones YZ, Ruiz-Lozano P, Chien KR,
et al. PPAR gamma is required for placental, cardiac, and adipose tissue
development. Mol Cell 1999;4:585e95.
[11] Schaiff WT, Carlson MG, Smith SD, Levy R, Nelson DM, Sadovsky Y.
Peroxisome proliferator-activated receptor-gamma modulates differentiation of human trophoblast in a ligand-specific manner. J Clin Endocrinol
Metab 2000;85:3874e81.
[12] Janowski BA, Willy PJ, Devi TR, Falck JR, Mangelsdorf DJ. An
oxysterol signalling pathway mediated by the nuclear receptor LXR
alpha. Nature 1996;383:728e31.
[13] Lehmann JM, Kliewer SA, Moore LB, Smith-Oliver TA, Oliver BB,
Su JL, et al. Activation of the nuclear receptor LXR by oxysterols defines
a new hormone response pathway. J Biol Chem 1997;272:3137e40.
[14] Willy PJ, Umesono K, Ong ES, Evans RM, Heyman RA,
Mangelsdorf DJ. LXR, a nuclear receptor that defines a distinct retinoid
response pathway. Genes Dev 1995;9:1033e45.
[15] Peet DJ, Turley SD, Ma W, Janowski BA, Lobaccaro JM, Hammer RE,
et al. Cholesterol and bile acid metabolism are impaired in mice lacking
the nuclear oxysterol receptor LXR alpha. Cell 1998;93:693e704.
[16] Repa JJ, Turley SD, Lobaccaro JA, Medina J, Li L, Lustig K, et al.
Regulation of absorption and ABC1-mediated efflux of cholesterol by
RXR heterodimers. Science 2000;289:1524e9.
[17] Repa JJ, Berge KE, Pomajzl C, Richardson JA, Hobbs H,
Mangelsdorf DJ. Regulation of ATP-binding cassette sterol transporters
ABCG5 and ABCG8 by the liver X receptors alpha and beta. J Biol Chem
2002;277:18793e800.
[18] Venkateswaran A, Laffitte BA, Joseph SB, Mak PA, Wilpitz DC,
Edwards PA, et al. Control of cellular cholesterol efflux by the nuclear
oxysterol receptor LXR alpha. Proc Natl Acad Sci U S A 2000;97:
12097e102.
[19] Repa JJ, Liang G, Ou J, Bashmakov Y, Lobaccaro JM, Shimomura I,
et al. Regulation of mouse sterol regulatory element-binding protein-1c
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
gene (SREBP-1c) by oxysterol receptors, LXRalpha and LXRbeta. Genes
Dev 2000;14:2819e30.
Muscat GE, Wagner BL, Hou J, Tangirala RK, Bischoff ED, Rohde P,
et al. Regulation of cholesterol homeostasis and lipid metabolism in
skeletal muscle by liver X receptors. J Biol Chem 2002;277:40722e8.
Steffensen KR, Gustafsson JA. Putative metabolic effects of the liver X
receptor (LXR). Diabetes 2004;53(Suppl 1):S36e42.
Hubel CA, McLaughlin MK, Evans RW, Hauth BA, Sims CJ,
Roberts JM. Fasting serum triglycerides, free fatty acids, and
malondialdehyde are increased in preeclampsia, are positively correlated,
and decrease within 48 hours post partum. Am J Obstet Gynecol 1996;
174:975e82.
Potter JM, Nestel PJ. The hyperlipidemia of pregnancy in normal and
complicated pregnancies. Am J Obstet Gynecol 1979;133:165e70.
Connor WE, Lin DS. Placental transfer of cholesterol-4-14C into rabbit
and guinea pig fetus. J Lipid Res 1967;8:558e64.
Lin DS, Pitkin RM, Connor WE. Placental transfer of cholesterol into
the human fetus. Am J Obstet Gynecol 1977;128:735e9.
Murata M, Kodama H, Goto K, Hirano H, Tanaka T. Decreased verylow-density lipoprotein and low-density lipoprotein receptor messenger
ribonucleic acid expression in placentas from preeclamptic pregnancies.
Am J Obstet Gynecol 1996;175:1551e6.
Campbell FM, Bush PG, Veerkamp JH, Duttaroy AK. Detection and
cellular localization of plasma membrane-associated and cytoplasmic fatty
acid-binding proteins in human placenta. Placenta 1998;19:409e15.
Duttaroy AK, Taylor J, Gordon MJ, Hoggard N, Campbell FM.
Arachidonic acid stimulates internalisation of leptin by human placental
choriocarcinoma (BeWo) cells. Biochem Biophys Res Commun 2002;299:
432e7.
Duttaroy AK, Crozet D, Taylor J, Gordon MJ. Acyl-CoA thioesterase
activity in human placental choriocarcinoma (BeWo), cells: effects of fatty
acids. Prostaglandins Leukot Essent Fatty Acids 2003;68:43e8.
Schmid KE, Davidson WS, Myatt L, Woollett LA. Transport of
cholesterol across a BeWo cell monolayer: implications for net transport
of sterol from maternal to fetal circulation. J Lipid Res 2003;44:1909e
1918.
Wadsack C, Hrzenjak A, Hammer A, Hirschmugl B, Levak-Frank S,
Desoye G, et al. Trophoblast-like human choriocarcinoma cells serve as
a suitable in vitro model for selective cholesteryl ester uptake from high
density lipoproteins. Eur J Biochem 2003;270:451e62.
Dalen KT, Ulven SM, Bamberg K, Gustafsson JA, Nebb HI. Expression
of the insulin-responsive glucose transporter GLUT4 in adipocytes is
dependent on liver X receptor alpha. J Biol Chem 2003;278:48283e91.
Rustan AC, Halvorsen B, Huggett AC, Ranheim T, Drevon CA. Effect
of coffee lipids (cafestol and kahweol) on regulation of cholesterol
metabolism in HepG2 cells. Arterioscler Thromb Vasc Biol 1997;17:
2140e9.
Chaan A, Laird NM, Slasor P. Tutorial in biostatistics; using the general
linear mixed model to analyse unbalanced repeated measures and
longitudinal data. Stat Med 1997;16:2349e80.
Schultz JR, Tu H, Luk A, Repa JJ, Medina JC, Li L, et al. Role of LXRs
in control of lipogenesis. Genes Dev 2000;14:2831e8.
Tobin KA, Ulven SM, Schuster GU, Steineger HH, Andresen SM,
Gustafsson JA, et al. LXRs as insulin mediating factors in fatty acid and
cholesterol biosynthesis. J Biol Chem 2002.
Juvet LK, Andresen SM, Schuster GU, Dalen KT, Tobin KA,
Hollung K, et al. On the role of liver x receptors in lipid accumulation
in adipocytes. Mol Endocrinol 2003;17:172e82.
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Placenta (2004), Vol. jj
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[38] Pavan L, Hermouet A, Tsatsaris V, Therond P, Sawamura T, EvainBrion D, et al. Lipids from oxidized-LDL modulate human trophoblast
invasion: involvement of LXR nuclear receptors. Endocrinology 2004.
[39] Coleman RA, Haynes EB. Synthesis and release of fatty-acids by human
trophoblast cells in culture. J Lipid Res 1987;28:1335e41.
[40] Wilentz RE, Witters LA, Pizer ES. Lipogenic enzymes fatty acid
synthase and acetyl-coenzyme A carboxylase are coexpressed with sterol
regulatory element binding protein and Ki-67 in fetal tissues. Pediatr Dev
Pathol 2000;3:525e31.
[41] Clausen T, Djurovic S, Henriksen T. Dyslipidemia in early second
trimester is mainly a feature of women with early onset pre-eclampsia.
BJOG 2001;108:1081e7.
[42] Gratacos E. Lipid-mediated endothelial dysfunction: a common factor to
preeclampsia and chronic vascular disease. Eur J Obstet Gynecol Reprod
Biol 2000;92:63e6.
[43] Wadsack C, Hammer A, Levak-Frank S, Desoye G, Kozarsky KF,
Hirschmugl B, et al. Selective cholesteryl ester uptake from high density
[44]
[45]
[46]
[47]
lipoprotein by human first trimester and term villous trophoblast cells.
Placenta 2003;24:131e43.
Chawla A, Boisvert WA, Lee C, Laffitte BA, Barak Y, Joseph SB, et al. A
PPARgamma-LXR-ABCA1 pathway in macrophages is involved in
cholesterol efflux and atherogenesis. Mol Cell 2001;7:161e71.
Cao G, Liang Y, Broderick CL, Oldham BA, Beyer TP, Schmidt RJ,
et al. Antidiabetic action of a liver x receptor agonist mediated by
inhibition of hepatic gluconeogenesis. J Biol Chem 2003;278:1131e
1136.
Stulnig TM, Oppermann U, Steffensen KR, Schuster GU,
Gustafsson JA. Liver X receptors downregulate 11beta-hydroxysteroid
dehydrogenase type 1 expression and activity. Diabetes 2002;51:2426e
2433.
Stulnig TM, Steffensen KR, Gao H, Reimers M, Dahlman-Wright K,
Schuster GU, et al. Novel roles of liver X receptors exposed by gene
expression profiling in liver and adipose tissue. Mol Pharmacol 2002;62:
1299e305.