Characterization and identification of steroid sulfate
transporters of human placenta
Bernhard
Ugele1,
Marie
V.
St-Pierre2,
Monika
Pihusch3,
Andrew
Bahn4, and
Peer
Hantschmann1
1 I. Frauenklinik Innenstadt and
3 Medizinische Klinik II Großhadern, Klinikum der
Universität München, D-80337 Munich; and
4 Zentrum für Physiologie und
Pathophysiologie, D-37073 Gottingen, Germany; and
2 Division of Clinical Pharmacology, Department of
Internal Medicine, University Hospital, CH-8091 Zürich,
Switzerland
 |
ABSTRACT |
Human trophoblasts depend on the supply of
external precursors, such as dehydroepiandrosterone-3-sulfate (DHEA-S)
and 16
-OH-DHEA-S, for synthesis of estrogens. The aim of the present
study was to characterize the uptake of DHEA-S by isolated
mononucleated trophoblasts (MT) and to identify the involved
transporter polypeptides. The kinetic analysis of DHEA-35S
uptake by MT revealed a saturable uptake mechanism
(Km = 26 µM,
Vmax = 428 pmol · mg
protein
1 · min
1),
which was superimposed by a nonsaturable uptake mechanism (diffusion constant = 1.2 µl · mg
protein
1 · min
1).
Uptake of [3H]DHEA-S by MT was Na+
dependent and inhibited by sulfobromophthalein (BSP), steroid sulfates,
and probenecid, but not by steroid glucuronides, unconjugated steroids,
conjugated bile acids, ouabain, p-aminohippurate (PAH), and
bumetanide. MT took up [35S]BSP,
[3H]estrone-sulfate, but not 3H-labeled
ouabain, estradiol-17
-glucuronide, taurocholate, and PAH. RT-PCR
revealed that the organic anion-transporting polypeptides OATP-B, -D,
-E, and the organic anion transporter OAT-4 are highly expressed, and
that OATP-A, -C, -8, OAT-3, and Na+-taurocholate
cotransporting polypeptide (NTCP) are not or are only lowly expressed
in term placental tissue and freshly isolated and cultured
trophoblasts. Immunohistochemistry of first- and third-trimester
placenta detected OAT-4 on cytotrophoblast membranes and at the basal
surface of the syncytiotrophoblast. Our results indicate that uptake of
steroid sulfates by isolated MT is mediated by OATP-B and OAT-4 and
suggest a physiological role of both carrier proteins in placental
uptake of fetal-derived steroid sulfates.
estrogen synthesis; isolated trophoblasts; organic anion
transporter; dehydroepiandrosterone sulfate
 |
INTRODUCTION |
IN PRIMATES, PLASMA
CONCENTRATIONS of unconjugated estrone, estradiol, and estriol
increase linearly with advancing gestation. After approximately
week 9 of human pregnancy, the placenta becomes the main
source of maternal estrogens. However, because the placenta lacks the
enzyme system 17
-hydroxylase/17-20-lyase, or CYP17, and thus is
unable to convert cholesterol into estrogen, its estrogen synthesis is
highly dependent on the supply of C-19 steroids for their conversion
into estrogens. It was demonstrated that sulfated C-19 steroids of
fetal and maternal origin serve as precursors for the placental
estrogen biosynthesis and that the resulting estrogens are mainly
released into the maternal blood (for review see Refs. 2,
20, and 32).
Dehydroepiandrosterone-3-sulfate (DHEA-S) of maternal and fetal origin
contributes about equally to the placental formation of estrone and
estradiol, whereas 16
-hydroxydehydroepiandrosterone-sulfate (16
-OH-DHEA-S) supplied by the fetus contributes to >90% of
placental estriol synthesis. Therefore, the concept of a functional
fetal placental unit for estrogen synthesis was established, providing the basis for maternal estriol measurement to assess fetal well-being, placental function, and/or uteroplacental blood flow (for review see
Ref. 20).
The conversion of sulfated C-19 steroid precursors to estrogens
involves the action of four enzymes located predominantly intracellularly in the syncytiotrophoblast (7), which is
formed from mononucleated cytotrophoblasts by cell fusion and
differentiation. Therefore, the substrates must enter the cells. The
mechanism of their uptake by trophoblast cells is unknown. Recently, we have shown that uptake of DHEA-S by isolated mononucleated trophoblasts (MT) is very likely mediated by one or more carriers that are not
functional in choriocarcinoma cell lines (38). In the
present study, we examined the kinetic characteristics of this
transport activity. Because the results obtained for MT share
properties with those identified in hepatocytes, kidney, and other
tissues (16, 17), we investigated the expression of these
organic anion carriers in human placental tissue and freshly isolated MT by RT-PCR. Furthermore, we studied the expression of these carriers
during cultivation of the MT, which have been shown to fuse within 4 days to syncytia (13, 35, 37).
 |
MATERIALS AND METHODS |
Materials.
The sodium salts of estradiol-17
-sulfate and estradiol-3,
17
-disulfate were obtained from Leo (Helsingborg, Sweden).
Indocyanine green was purchased from ICN Biomedicals (Eschwege,
Germany) and ouabain (G-strophanthin) from Boehringer (Mannheim,
Germany). All other substrates and inhibitors were purchased from Sigma (Deisenhofen, Germany). Materials used for isolation and cultivation of
trophoblasts and uptake studies have been described previously (35, 36, 38).
Radiochemicals.
[7-3H(N)]dehydroepiandrosterone sulfate sodium salt,
[3H(G)]taurocholic acid, [6,7-3H(N)]estrone
sulfate ammonium salt,
[6,7-3H(N)]estradiol-17
-D-glucuronide,
[3H(G)]ouabain, and
p-[glycyl-2-3H]aminohippuric acid were
purchased from NEN DuPont (Bad Homburg, Germany).
Dehydroepiandrosterone [35S]sulfate and
[35S]sulfobromophthalein were synthesized as described
earlier (8, 19).
Isolation of MTs.
Human term placentas from uncomplicated pregnancies were obtained after
spontaneous vaginal delivery and elective caesarean sections and
immediately processed. Human MTs were isolated from placental villous
tissue, purified, and cultured as described earlier (13, 35,
36).
Transport studies.
Cell suspensions, obtained as described above, were washed one time in
transport buffer containing (in mM) 142.9 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 1.8 CaCl2, and 20 HEPES, pH 7.4, and were suspended to 1.5 × 106 cells/ml in cold transport buffer and kept on ice.
For Na+-free transport buffer, NaCl was replaced by an
equimolar concentration of choline chloride; for
SO
-free transport buffer, MgSO4 was
omitted. The uptake of the 3H- and/or
35S-labeled substrates by isolated trophoblasts in
suspension was studied by use of the silicone oil centrifugation
technique, as previously described in detail (38).
RT-PCR.
Total RNA was extracted from tissues of three different human term
placentas and from isolated and cultivated trophoblasts of three
different cell preparations by use of the TRIzol reagent from
Invitrogen (Karlsruhe, Germany). One microgram of each RNA sample
was reverse transcribed using 0.5 µl of an oligo(dT) primer and 8 U/µl of SuperScript II RT (Invitrogen).
cDNA equivalents to 200 ng (transporter proteins) and 80 ng (
-actin)
RNA were subjected to PCR amplification with specific primers (300 nmol/l) and 0.05 U/µl AmpliTac Gold DNA polymerase (Applied
Biosystems, Weiterstadt, Germany). For amplification of the organic
anion-transporting polypeptides (OATP)-A, -C, and -8, the organic anion
transporter (OAT)-3, and Na+-taurocholate cotransporting
polypeptide (NTCP) transcripts, a nested PCR technique was used with 22 cycles in the first PCR reaction and 22 (OATP-A), 25 (OATP-8), 24 (NTCP), and 30 (OATP-C, OAT-3) cycles, respectively, in the second PCR
reaction. OATP-B, -D, and -E, OAT-4, and
-actin were amplified using
one single PCR reaction with 28, 32, 32, 28, and 18 cycles,
respectively. PCR products were separated on an agarose gel and
visualized under ultraviolet light by ethidium bromide staining.
Primers used in the present study were as follows.
NTCP: (SLC10A1, GenBank accession no. L21893)
OATP-A: (SLC21A3, GenBank accession no. NM_005075)
OATP-B: (SLC21A9, GenBank accession no. NM_007256, AB020687)
OATP-C: (OATP-2, LST-1, SLC21A6, GenBank accession no.
NM_006446)
OATP-D: (SLC21A11, GenBank accession no. AB031050)
OATP-E: (SLC21A12, GenBank accession no. AB031051)
OATP-8: (SLC21A8, GenBank accession no. NM_019844)
OAT-3: (SLC22A6, GenBank accession no. AB042505)
OAT-4: (SLC22A7, GenBank accession no. NM_018484)
-Actin: (ACTB, GenBank accession no. X00351.1)
The primer sequences were obtained from references as indicated
or designed by us, partly by use of the computer program OMIGA (Oxford Molecular).
Immunohistochemistry.
Paraffin-embedded sections from three first- and third-trimester
placentas were processed for immunohistochemistry. Sections were
deparaffinized in xylol, rehydrated through a graded series of ethanol,
and pretreated in 100 mM sodium citrate buffer, pH 6.0, for 5 min in a
pressure cooker. The affinity-purified polyclonal rabbit anti-human
OAT-4 antiserum raised against an 18-amino acid residue peptide near
the cytoplasmic COOH terminus of OAT-4 (Alpha Diagnostic, San Antonio,
TX) was diluted 1:50, and the tissue sections were incubated 45 min at
room temperature. For visualization, the avidin-biotin peroxidase
detection method (Vectastain ABC kit, Vector Laboratories) and
3-amino-9-ethylcarbazole (Sigma, Munich, Germany) as chromogenic
substrate were used. Controls were performed by preadsorption of the
antiserum with 1 µM antigenic peptide (Alpha Diagnostic, San Antonio, TX).
Protein determinations.
Cellular protein was determined using a modification of the method
described by Lowry et al. (22).
Calculation of kinetic parameters and statistics.
Uptake of all substrates was linear up to >60 s, and the initial
uptake velocity (v) was estimated by linear regression
analysis of substrate uptake after 0.25, 0.5, 0.75, and 1.0 min. To
take care of different uptake mechanisms and carriers, the data were fitted to different equations, ranging from simple saturable
Michaelis-Menten (2 parameters, Eq. 1), Michaelis-Menten in
the presence of a nonsaturable system, denoted by
Pdiff (3 parameters, Eq. 2), and two
Michaelis-Menten components (4 parameters, Eq. 3). A fitting
to equations with five or more parameters (e.g., 2 Michaelis-Menten
components in the presence of a nonsaturable system) did not converge.
Fitting was completed with a weighted nonlinear least square fit
computer program (SigmaPlot; SPSS, Chicago, IL), on the basis of the
Marquart-Levenberg algorithm. The weighting factor was
w = 1/variance.
|
(1)
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|
(2)
|
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(3)
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Statistics.
An F-test (23, 29) and Akaike's information
criterion (12) were used to select the appropriate model
equation(s). The quality of the fitting was scrutinized by analyzing
the residuals with the RUNS test (23, 31). To test the
significance of differences between data sets, double-sided, paired
t-tests were performed.
 |
RESULTS |
Uptake of DHEA-S by isolated trophoblasts.
Kinetic analysis of both the initial uptake rates of
DHEA-35S (see Fig. 1) and
[3H]DHEA-S (data not shown) revealed a saturable uptake
mechanism that was superimposed by a nonsaturable component, presumably simple diffusion, or a second saturable uptake mechanism with very low
affinity. Fitting the model with four parameters (Eq. 3) to
the data led to extremely large or small absolute values of both
Km and Vmax for the
second class of Michaelis-Menten components, which are biologically not
meaningful or did not converge. On the other hand, the model with three
parameters (Eq. 2) was statistically more appropriate than
the simpler model (Eq. 1). For DHEA-35S, the
mean parameter values of five cell preparations were, for the apparent
Km, 26 ± 7.6 µM; for
Vmax, 428 ± 98 pmol · mg
protein
1 · min
1; and
for Pdiff, 1.2 ± 0.5 l · mg
protein
1 · min
1.

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Fig. 1.
Kinetics of dehydropiandrosterone [35S]sulfate
(DHEA-35S) uptake by isolated mononucleated trophoblasts.
Suspended cells (1.5 × 106 cells/ml) were incubated
in the presence of increasing substrate concentrations. After 15, 30, 45, and 60 s, aliquots were taken, and the cells were centrifuged
through silicone oil. The radioactivity of the pellet was measured, and
the initial slope of the specific amount of DHEA-S, which was
sedimented with the cells, was calculated by linear regression
analysis. Each point represents the arithmetic mean of the values of 5 cell preparations. The curve through the data points (dashed line)
represents the nonlinear regression analysis, as described in
MATERIALS AND METHODS.
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Uptake of [3H]DHEA-S by MT was significantly
(P < 0.01) decreased in Na+-free transport
buffer (12.5 ± 21.1% of control, n = 5) but was not changed in SO
-free transport buffer (92.5 ± 31.6% of control, n = 3). Uptake of
[3H]DHEA-S by MT was strongly inhibited by BSP, steroid
sulfates (DHEA-S, estrone sulfate, estradiol-17
-sulfate,
estradiol-3,17
-disulfate, vitamin D3-sulfate), and
probenecid (see Figs. 2 and
3). Uptake of [3H]DHEA-S by
MT was not inhibited by indocyanine green, p-aminohippurate (PAH), bumetanide, steroid glucuronides (estradiol-17
-glucuronide, estrone-glucuronide), unconjugated steroids (DHEA, 16
-OH-DHEA, dexamethasone), conjugated bile acids (taurocholate,
tauroursodeoxycholate), and ouabain (see Figs. 2 and 3).

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Fig. 2.
Uptake of [3H]DHEA-S by isolated
mononucleated trophoblasts: inhibition by different organic anions.
Suspended cells (1.5 × 106 cells/ml) were incubated
with 5 µM [3H]DHEA-S and different inhibitors. PAH,
p-aminohippurate. After 15, 30, 45, and 60 s, aliquots
were taken, and the cells were centrifuged through silicone oil. The
radioactivity of the pellet was measured, and the initial slope of the
specific amount of DHEA-S, which was sedimented with the cells, was
calculated by linear regression analysis. Each bar represents the
arithmetic mean (±SD) of the values of 4-6 cell preparations.
*P < 0.05; **P < 0.01.
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Fig. 3.
Uptake of [3H]DHEA-S by isolated mononucleated
trophoblasts: inhibition by different steroids. Suspended cells
(1.5 × 106 cells/ml) were incubated with 5 µM
[3H]DHEA-S and different inhibitors. After 15, 30, 45, and 60 s, aliquots were taken, and the cells were centrifuged
through silicone oil. The radioactivity of the pellet was measured, and
the initial slope of the specific amount of DHEA-S, which was
sedimented with the cells, was calculated by linear regression
analysis. Each bar represents the arithmetic mean (±SD) of the values
of 4-6 cell preparations. *P < 0.05;
**P < 0.01.
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Uptake of different organic anions and ouabain by isolated
trophoblasts.
The initial uptake rate of [35S]BSP was about three to
five times higher than the uptake rate of [3H]DHEA-S and
estrone sulfate (E1S). Initial uptake rate of BSP was
decreased by 100 µM rifamycin and low temperature (data not shown).
In contrast, there was no significant initial uptake velocity detectable for 3H-labeled estradiol-17
-glucuronide
(E217
G), ouabain, taurocholate, and PAH (see Table
1).
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Table 1.
Slope of initial uptake of different radiolabeled organic anions and
ouabain by isolated mononucleated trophoblasts
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Detection of steroid sulfate transporter mRNAs by use of RT-PCR.
OATP-B was highly expressed in term placental tissue and freshly
isolated MT. During cultivation, expression levels decreased after 1 day and increased significantly after 4 days, in parallel with syncytia
formation. In contrast, the expression level of the "housekeeping
gene"
-actin did not change during cultivation (Fig.
4). OATP-D, OATP-E, and OAT-4 were highly
and equally expressed in term placental tissue and in freshly isolated
and cultured trophoblasts (Fig. 4). OATP-A and NTCP were lowly
expressed in term placental tissue and freshly isolated MT and could be
detected only by nested RT-PCR. During cultivation, expression levels
of OATP-A and NTCP did not change significantly. OATP-C (LST) was lowly
expressed in term placental tissue but was not detectable in isolated
and cultured trophoblasts (Fig. 4). OATP-8 was intermediately expressed
in placental tissue and could be detected with normal PCR after 40 cycles (data not shown). When the nested PCR technique was used, OATP-8
could be detected in two of three cell preparations in cultured
trophoblasts after 1 day in culture (see Fig. 4) and in one preparation
only in freshly isolated MT.

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Fig. 4.
RT-PCR analysis of organic anion-transporting peptide (OATP)-A, -B,
-C, -D, -E, -8, Na+-taurocholate cotransporting polypeptide
(NTCP), organic anion transporter (OAT)-3, -4, and -actin in human
placental tissue and isolated and cultured trophoblasts. RT-PCR with
total RNA isolated from placental tissue (lane 1), isolated
mononucleated trophoblasts (lane 2), cultivated trophoblasts
after 1 day (lane 3), and cultivated trophoblasts after 4 days (lane 4) was performed as described in MATERIALS
AND METHODS. PCR products were separated by agarose gel
electrophoresis and visualized by ethidium bromide staining.
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Immunohistochemistry.
At term, OAT-4 was abundantly expressed at the basal surface of the
syncytiotrophoblast in terminal and intermediate villi (Fig. 5,
A and C). The
specificity of the antiserum was verified by repeating the staining
after preadsorption of the antiserum with 1 µM antigenic peptide
(Fig. 5B). In the first trimester, OAT-4 was also abundantly
expressed at the basal surface of the syncytiotrophoblasts, and,
additionally, strong staining of the cytoplasma membrane and
perinuclear region of cytotrophoblasts was detectable (Fig.
5D).

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Fig. 5.
Immunohistochemical detection of OAT-4 in human first-
and third-trimester placentas. A-C,
third-trimester placentas; D, first-trimester placenta.
Staining for OAT-4 was strong at the basal surface of the
syncytiotrophoblast (arrow in C). Staining decreased to
background levels after adsorption of the antibody with the antigenic
peptide (B). Cytotrophoblasts of first-trimester placenta,
which have not fused with the syncytiotrophoblasts, reveal distinct
staining of the cytoplasma membrane (D, arrowhead) and of
the perinuclear region. Bars, 25 µm.
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 |
DISCUSSION |
Recently, different carrier proteins for steroid sulfates have
been cloned mainly from liver and kidney. In Table
2, the substrate specificities of these
carrier polypeptides are summarized and compared with the substrate
specificity of isolated MT of the present study. Table 2 demonstrates
that only the substrate specificities of OATP-B and OAT-4 are in
accordance with the substrate specificity of isolated MT. In contrast
to isolated MT, all other carrier polypeptides transport taurocholate
and/or ouabain, and/or E217
G, and/or PAH. However,
concerning OATP-D and -E, only limited data are available.
Furthermore, the RT-PCR analysis (see Fig. 4) has shown that OATP-B,
OATP-D, OATP-E, and OAT-4 are highly expressed and that all other
carrier polypeptides are not or are only very lowly expressed in
placental tissue and isolated MT. This observation is in accordance
with reports of other groups who have detected expression of OATP-B and
OAT-4 in placental tissue with Northern blot technique (4,
18), and of OATP-D and OATP-E with the RT-PCR technique
(10, 33). Our results indicate that uptake of steroid
sulfates by isolated trophoblast cells is predominantly mediated by
OATP-B and OAT-4. However, the uptake of steroid sulfates by OATP-D,
OATP-E, and other so-far-unknown transporting polypeptides with a
similar substrate specificity cannot be excluded. The function of the
very low expression of other transporting polypeptides in isolated
trophoblast cells is unclear and may be due to a meaningless "illegitimate" physiological transcription of the respective genes (5). Independently, the fact that OATP-C was lowly
expressed in placenta tissue but not detectable in isolated
trophoblasts may indicate that OATP-C is only expressed in
nontrophoblast cells, e.g., endothelial cells, fibroblasts, and the like.
With use of the Xenopus oocyte expression system, the
Km value of OAT-4 was calculated to be 0.63 µM
for DHEA-S and 1.26 µM for E1S (4) and that
of OATP-B to be 9.0 or 6.3 µM for E1S (18,
34). Thus it seems that, compared with OATP-B, the affinity of
OAT-4 to steroid sulfates is ~5-10 times higher. However, with use of isolated MT, a kinetic discrimination of these two transporting polypeptides was not possible. The calculated apparent
Km value of DHEA-S uptake by isolated MT was
higher (26 µM) than that of the transporting polypeptides expressed
in Xenopus oocytes but similar to that of E1S
uptake by basal membrane vesicles from the placental
syncytiotrophoblast (30).
Uptake of DHEA-S by MT was strongly inhibited only by compounds with
one or more sulfate residues (see Figs. 2 and 3). Thus the sulfate
residue independent of the position in the molecule, e.g., C-3 or C-17
of the steroid ring, seems to be essential for inhibition and may also
be for transport. On the other hand, uptake of DHEA-S by MT was not
influenced by 1.2 mM SO
of the transport buffer,
indicating that SO
is not an inhibitor/substrate of
the steroid sulfate transporters and that the carbon backbone of the
steroid sulfates is a prerequisite for inhibition/transport.
Uptake of E1S (data not shown) and DHEA-S by isolated MT
was partly dependent on extracellular Na+ ions. In
contrast, uptake of E1S by OAT-4 expressed in
Xenopus oocytes was sodium independent (4).
Kullak-Ublick et al. (17) showed that uptake of DHEA-S by
human OATP-A was also partly sodium dependent, whereas, to our
knowledge, sodium dependency of steroid sulfate uptake by OATP-B
remains to be tested. Thus the interpretation that the
Na+-dependent part of DHEA-S uptake by MT is mediated by
OATP-B is highly speculative.
In cultured trophoblasts, the expression of OATP-B decreased after 1 day and then increased after 4 days compared with freshly isolated
cells. These changes occur in parallel with syncytia formation. The
former observation may be explained by an adaptation of the cells to
the culture condition; the latter observation may indicate an increase
during differentiation of MT to the syncytiotrophoblast. Expression of
OAT-4 and the other more highly expressed transporting polypeptides did
not change significantly during syncytium formation in vitro. Thus our
results demonstrate that OATP-B and OAT-4 are highly expressed not only
in MT, i.e., cytotrophoblast, but also in the multinucleated
syncytiotrophoblast formed in vitro and may also contribute to the
uptake of steroid sulfates of the syncytiotrophoblast in vivo. The
difference in expression of OATP-B and OAT-4 during cultivation may
indicate a different regulation of transcription of the respective genes.
Very recently, we have shown that immunohistochemical staining
for OATP-B is abundant in the placenta throughout gestation, with
strong reactivity in the cytotrophoblast membranes and at the
basolateral surface of the syncytiotrophoblast (30). A
very similar staining pattern was observed for OAT-4 in the present study (see Fig. 5). These results demonstrate a physiological role of
OAT-4 and OATP-B in the placental uptake of fetus-derived steroid
sulfates only. In contrast, the identity and characteristics of
carriers for uptake of maternal steroid sulfates at the microvillous membrane of the syncytiotrophoblast (see introduction) are still unknown. Our results probably indicate either that these microvillous carriers show a substrate specificity similar to OATP-B and OAT-4, or
that these transporter polypeptides are not expressed in isolated MT,
or that they have been destroyed during the enzymatic isolation procedure. The binding of the OAT-4 antibodies to the perinuclear region in cytotrophoblasts may reflect a strong synthesis (translation) of the OAT-4 polypeptide in the rough endoplasmatic reticulum of
cytotrophoblasts but not in syncytiotrophoblasts. Intracellular transport to the plasma membrane of the cytotrophoblast and fusing of
these cells with the preexisting syncytiotrophoblast add the OAT-4
polypeptide to the basal membrane of the syncytiotrophoblast. However,
to test this hypothesis, further experiments are necessary.
The preponderance of 16
-hydroxyestrogens during pregnancy is
greater than might be expected from the ratio of 16
-OH- to 16-deoxy-C-19 steroid sulfates in fetal plasma (for review see Ref.
26). Thus, presumably, precursor abundance is not the sole regulator of the relative amounts of the 16
-OH- and
16-deoxyestrogene formed in the placenta. An explanation of the
discrepancy between the ratio of 16
-OH- and 16-deoxy-C-19 substrates
in fetal plasma and that of the products in the urine is that the
16
-hydroxy-C-19 sulfate precursors might be converted more
efficiently to estrogens than the corresponding 16-deoxy precursors. In
the placenta, both sulfated precursors follow the same pathway, because
the tissue lacks steroid 16
-hydroxylase. This pathway involves
sequential desulfation at C-3
, oxidation and isomerization to the
corresponding 4-en-3-oxosteroids, and aromatization. Reduction at C-17
catalyzed by 17
-hydroxysteroid dehydrogenase yields a mixture of
estrone and estradiol-17
from DHEA-S and 16
-hydroxyestrone and
estriol from 16
-OH-DHEA-S. The utilization of these precursors has
been investigated using purified enzymes (9, 27),
microsomal preparation, and tissue slices (26). However,
the results of these studies have demonstrated that formation by the
intracellular enzymatic pathway of 16-deoxyestrogen from the sulfate
precursors is more efficient than that of 16
-hydroxyestrogens. In
contrast, nothing is known about the efficiency of the transport
systems responsible for the uptake of the 16
-hydroxy-C-19 and
16-deoxy-C-19 sulfate precursors. Now, one may speculate whether the
discrepancy of the placental synthesis of 16-hydroxyestrogens vs.
16-deoxyestrogens mentioned above is due to different efficient uptake
of the respective sulfated precursor from fetal and maternal blood. It
will take further studies to characterize the uptake of these
substances by isolated and cultured trophoblasts and cells expressing
cloned placental carrier proteins, which have been identified to be
involved in the uptake steroid sulfates in the present study.
In conclusion, we have shown that uptake of steroid sulfates by
isolated mononucleated trophoblasts is mediated by OATP-B and OAT-4.
Our results suggest a physiological role of both carrier polypeptides
in placental uptake of fetus-derived steroid sulfates.
 |
ACKNOWLEDGEMENTS |
We acknowledge the significant scientific contributions to this
work of Professor Dr. Erich Kuss.
 |
FOOTNOTES |
We also thank Karin Regemann and Angelika Bolle for excellent technical
assistance and Jasmin Winiker for help in preparing the manuscript.
This work was supported in part by Friedrich-Baur-Stiftung der
Medizinischen Fakultät, Munich, Germany, Grant no. 26/95 (to B. Ugele) and the National Science Foundation Switzerland, Grant no. 31-056020.98 (to M. V. St-Pierre).
Address for reprint requests and other correspondence: B. Ugele, Klinikum der Ludwig-Maximilians-Universität
München, I. Frauenklinik Innenstadt, D-80337 München,
Germany (E-mail:
ugele{at}helios.med.uni-muenchen.de).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published October 29, 2002;10.1152/ajpendo.00257.2002
Received 11 June 2002; accepted in final form 16 October 2002.
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