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Departments of Molecular and Cellular Pathology (E.L.S., R.H., M.W.H.C.) and Obstetrics and Gynaecology (E.L.S., R.H.), University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland, United Kingdom; and Department of Internal Medicine (T.J.V.), Erasmus University Medical School, 3015 GD Rotterdam, The Netherlands
Address all correspondence and requests for reprints to: Dr. Michael Coughtrie, Department of Molecular and Cellular Pathology, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland, United Kingdom. E-mail: m.w.h.coughtrie{at}dundee.ac.uk
Abstract
Thyroid hormone is essential for normal human development, and disruption of thyroid hormone homeostasis at critical developmental stages can result in severe and often long-term effects on crucial organs such as the brain and lungs. Numerous factors control the bioavailability of receptor active thyroid hormone T3. Sulfation, catalyzed by sulfotransferase enzymes (SULTs), is an important pathway of thyroid hormone metabolism by which T4 is irreversibly converted to inactive reverse T3 rather than active T3. The human fetus and neonate have high levels of circulating sulfated iodothyronines, although the source of these is not clear. The placenta forms the link between the fetus and its mother and is involved in transfer of thyroid hormone early in pregnancy, although its capacity for sulfation is unknown. We therefore examined expression of the SULTs involved in iodothyronine metabolism during human placental development. SULT activity was measured in human placental cotyledon and membranes (amnion, chorion, and decidua basalis) from 13–42 wk of gestation, and Western blot analysis was employed to verify enzyme activity data. Phenol and catecholamine sulfotransferases were expressed at the highest levels and were generally higher in the villous than membranous tissues. SULT1A1 activity showed significant correlation with sulfation of 3,3'-T2, suggesting that this enzyme is primarily responsible for placental T2 sulfation. Estrogen sulfotransferase was present at extremely low levels during early pregnancy, although in mid- and late gestation increased expression in the (predominantly maternal-derived) decidual component of the placenta was observed. Hydroxysteroid sulfotransferase, T3, reverse T3, and T4 SULT activities were also low in all tissues examined, and expression of SULTs 1B1 and 1C2 were essentially undetectable by Western blot analysis. The results highlight a tissue-specific regulation of SULT expression during placental development, demonstrate very low sulfation of iodothyronines suggesting that the placenta is not a major source of circulating sulfated iodothyronines in the fetus.
METABOLISM PLAYS A key role in regulating the biological activity and homeostasis of many hormones. In the case of thyroid hormone, a number of important metabolic pathways interact to regulate synthesis, bioactivation, and deactivation, and the balance among these various pathways influences the response of individual tissues and cells to this hormone (1). This is particularly evident in development, during which thyroid hormone is essential for normal development of the human brain. Numerous factors control levels of receptor-active thyroid hormone (T3), and disruption of any of these at critical phases of human development can lead to severe and persistent cognitive and motor deficits. Transient thyroid dysfunction in preterm infants is common, characterized by low levels of plasma T4 (transient hypothyroxinemia) and T3 but normal levels of TSH. This condition has been recognized as an expression of temporary immaturity of the hypothalamic-pituitary- thyroid axis (2), and recent studies have linked low plasma T4 in preterm infants with later neurodevelopmental deficits in motor and cognitive function (3, 4, 5) and low plasma T3 with reductions in IQ at 8 yr of age (6, 7). Reduced thyroid hormone secretion by the thyroid gland because of immaturity of the hypothalamic-pituitary-thyroid axis or iodine deficiency in the premature neonate may contribute to these low plasma T4 and T3 levels (8). However, supplementation of infants <30 wk of gestation with T4 for up to 6 wk, although increasing total T4 and free T4, does not increase plasma levels of the active hormone T3 (9), nor does this improve long-term neurodevelopmental outcome (10). These clinical observations indicate that disorders within the complex metabolic pathways regulating iodothyronine levels in the developing fetus may play a critical role in transient hypothyroxinemia associated with prematurity.
Sulfation of iodothyronines has major effects on their interconversion mediated by iodothyronine deiodinases and also on their homeostasis (1, 11). Sulfation of T4 completely blocks the metabolic conversion (and bioactivation) of T4 to receptor-active T3 by outer ring deiodination while enhancing the rate of T4 conversion to receptor-inactive reverse T3 (rT3) (11). Sulfation also dramatically accelerates the inner ring deiodination of T3 and the principal product of iodothyronine metabolism 3,3'-diiodothyronine (3,3'-T2) is also extensively sulfated and subsequently deiodinated (11). Sulfation is therefore likely to contribute to the regulation of receptor-active thyroid hormone in target cells. There is a large family of sulfotransferase (SULT) enzymes in humans, comprising at least 11 members (12). It is clear from work that we and others (13, 14, 15, 16) have performed that the sulfation of iodothyronines is carried out by more than one SULT isoform (at least in vitro), including SULTs 1A1, 1A3, 1B1, 1C2, and 1E1. Iodothyronine sulfates are present at very high levels in fetal (cord) blood and persist well into postnatal life, supporting the importance of sulfation as a means of protecting the fetus from excessive stimulation by thyroid hormone (17, 18 ; our unpublished observations). It is not clear whether the placenta plays a role in sulfation of iodothyronines during pregnancy. We have therefore studied the expression of various SULT isoforms proposed to be involved in iodothyronine metabolism in human placenta to determine whether this organ may act as a source of the substantial amounts of sulfated iodothyronines present in the human fetal circulation.
Materials and Methods
Materials
[35S]3'-Phosphoadenosine
5'-phosphosulfate (PAP35S) (1.5–2.54 Ci/mmol),
[3H]17
-ethynylestradiol (41.2 Ci/mmol and
[3H]dehydroepiandrosterone (92.0 Ci/mmol) were
purchased from Du Pont/NEN Life Science Products
(Stevenage, UK) and unlabeled PAPS was purchased from H.R.
Glatt, German Institute of Human Nutrition, Potsdam, Germany. Dopamine,
17-ethynylestradiol, dehydroepiandrosterone (DHEA),
4-nitrophenol, T3, barium acetate, barium
hydroxide, zinc sulfate, rabbit (antigoat IgG) alkaline phosphatase
conjugate (adsorbed with human serum proteins), nitroblue tetrazolium,
and 5-bromo-4-chloro-3-indolyl phosphate (p-toluidine salt)
were purchased from Sigma-Aldrich Corp. (Poole, UK).
Scintillation fluid (Emulsifier Safe) was obtained from Canberra Packard (Pangbourne, UK). (3,
5'-125I)T4,
(3'-125I)T3, were from
Amersham Pharmacia Biotech (Little Chalfont, UK);
T4, rT3 and
3,3'-T2 were obtained from Henning Berlin GmbH & Co. (Berlin, Germany). 3,
(3'-125I)T2 and
(3'5'-125I)rT3 were
prepared and purified as previously described (19). All
other reagents were of analytical grade and purchased from local
suppliers.
Subjects and sample preparation
The study was approved by the Tayside Committee on Medical Research Ethics, and permission was obtained by informed consent. Seventy-eight placentas, distributed over 13–42 wk of gestation, were collected at delivery following either termination induced with Gemeprost vaginal pessaries or caesarean section. Villous and membranous regions were separated, frozen immediately in liquid nitrogen, and stored at -80 C until required. For the purpose of this study, placentas were subdivided into the following compartments: cotyledons (mainly fetal origin, predominantly syncytiotrophoblastic cells); amnion (predominantly fetal origin); chorion/decidua (mixed maternal and fetal origin); and decidua (predominantly maternal origin) (20). For preparation of the cytosolic fraction, tissues (approximately 4 g) were thawed and homogenized in ice-cold buffer (20% [wt/vol] 250 mM sucrose/10 mM HEPES/3 mM 2-mercaptoethanol, pH 7.4). Homogenates were subjected to differential centrifugation as previously described (21), and the resulting 100,000 g supernatants (cytosolic fractions) were stored in 1-ml aliquots at -80 C until use (within 3 months). SULT activity toward a range of substrates, including 4-nitrophenol and dopamine, has been shown to be stable for up to 9 months when stored frozen at -70 C (22). Fetal liver (14 wk gestational age) was used as a positive control for SULT immunohistochemistry (23).
Estimation of protein content
Protein concentration was determined using the method originally described by Lowry et al. (24), using bovine serum albumen as standard.
Assay for SULT enzyme activity
SULT enzyme activity was measured with either PAP35S (25) or radioactively labeled substrates (steroids, iodothyronines) as previously described (19, 26). All assays were optimized with respect to substrate, PAPS and cytosolic protein concentration, buffer composition, and incubation time.
PAP35S was used to assess SULT activity toward dopamine (10 µM) and 4-nitrophenol (5 µM). The incubation mixture (150 µl) comprised: 200 µg placental cytosolic protein, PAPS (20 µM for dopamine, 30 µM for 4-nitrophenol), PAP35S (0.3 µCi), and buffer (10 mM KPO4, pH 7.4). Cytosol samples were assayed in duplicate with a control incubation containing substrate only. The reaction mixture was incubated at 37 C for 20 min and terminated by the addition of 200 µl 100 mM barium acetate. Unreacted PAPS was removed by precipitation with 200 µl 100 mM barium hydroxide and 200 µl 100 mM zinc sulfate followed by centrifugation at 11,000 g for 5 min. Then 500 µl supernatant were removed and mixed with 4 ml scintillation fluid and radioactivity quantified by liquid scintillation spectrometry.
3H-labeled substrates were used to assay SULT
activity toward 17-ethynylestradiol (10 µM) and
DHEA (10 µM). The incubation mixture (250
µl) comprised: placental cytosolic protein (200 µg for
17-ethynylestradiol and 10 µg for DHEA), 0.1 µCi
3H-labeled substrate, unlabeled substrate, PAPS
(10 µM for 17-ethynylestradiol assay; 50
µM for DHEA), and buffer (60 mM
Tris/HCl, 0.7 mM MgCl2, pH 6.0 for
17-ethynylestradiol and 50 mM Tris/HCl, 10
mM MgCl2, pH 7.4 for
DHEA). Assays were performed in duplicate and included a
control incubation containing no PAPS. Following 60-min incubation at
37 C, the reaction was terminated by the addition of 3 ml chloroform
followed by the addition of 300 µl ice-cold dH2O.
The tubes were shaken vigorously and then centrifuged at 3000
g for 3 min. Then 200 µl supernatant were mixed with 4 ml
scintillation fluid and the radioactivity quantified by liquid
scintillation spectrometry.
125I-labeled substrates were used to assess placental SULT activity toward T4, T3, rT3, and 3,3'-T2. Placental cytosol (25 µg) was incubated with 0.1 µM T4, T3, rT3, and 3,3'-T2 and 0.05 µC of the 125I-labeled compound for 60 min (with the exception of the 3,3'-T2 assay with placental cotyledon cytosol, which was incubated for 30 min) at 37 C. Reactions were carried out in the presence or absence (blank) of 50 µM PAPS in 0.2 ml 0.1 M phosphate buffer, containing 2 mM EDTA (pH 7.2). The reaction was terminated by the addition of 0.8 ml 0.1 M HCl and analyzed for SULT formation as previously described (19).
Antibody production
Antibodies against purified, recombinant human SULT1A3 (23, 27), SULT 1E1 (28), SULT 1B1 (14), and SULT 1C2 (to be described elsewhere) were raised in sheep by three separate immunizations (4 wk apart) using 150 µg protein (Diagnostics Scotland, Carluke, UK). Anti-SULT1A3 antiserum was shown to react with human SULTs 1A1, 1A2, and 1A3 by ELISA and Western immunoblot analysis. Cell-free extracts containing expressed SULT enzymes as previously described (27, 29) were used to optimize Western blot analysis with respect to placental cytosol and primary and secondary antibody concentrations to minimize the possibility of cross-reactivity toward other SULTs. Immunoreactivity was quantified using QuantiScan software (Biosoft, Cambridge, UK).
Immunoblot analysis
Placental cytosolic proteins were separated on polyacrylamide gels (11% acrylamide monomer) containing SDS as originally described by Laemmli (30), followed by electrophoretic transfer to nitrocellulose membranes essentially by the method of Towbin et al. (31). Immunoreactive polypeptides were visualized using the alkaline phosphatase system. Primary antibodies used were crude IgG fractions (prepared by ammonium sulfate precipitation) of antisera produced in sheep against SULTs 1A3, 1E1, 1B1, and 1C2, and the secondary antibody was an alkaline phosphatase conjugated antigoat IgG adsorbed against human serum proteins (Sigma, Poole, UK).
Fluorescence immunohistochemistry
Fresh 1-cm3 blocks of fetal liver tissue (positive control (23)) and placental tissue (cotyledon and subdivided placental membranes) were mounted onto circular pieces of cork using OCT embedding medium (Tissue Tek; Sakura, Torrance, CA), sprayed with cryo-jet Lamb’s freezing aerosol (Merck, Poole, UK) and cut into 7-µm sections using an HR cryostat (Slee, London, UK). The slides were left to air dry for 1 h, then wrapped in paper, sealed in Parafilm, and stored at -80 C until use.
Seven-micrometer cryosections (fetal liver, placental cotyledon, and subdivided placental membranes) were removed from the -80 C freezer and allowed to adjust to room temperature for 10 min. The sections were rinsed for 3 x 5 min in PBS, and the tissue was encircled using a PAP pen. Tissues were incubated in PBS containing 1% (wt/vol) BSA for 20 min. The primary antibody was diluted appropriately using antibody diluent (DAKO Corp., Ely, UK), and 200 µl were applied to each section. Slides were placed in a moist chamber at room temperature for 2 h and, following exposure to primary antibody, were washed for 3 x 5 min in PBS. Excess PBS was removed from each section and 200 µl fluorescein isothiocyanate (FITC)-conjugated secondary antibody (diluted 1:200 with antibody diluent) were applied in the dark and the slides returned to the moist chamber for 1 h. The slides were then rinsed for 3 x 5 min in PBS and coverslipped using Fluorosave reagent (Merck). Control experiments with no primary antibody (DAKO Corp. serum free protein block) were performed in parallel. The slides were analyzed using a fluorescence microscope (Carl Zeiss, Jena, Germany) and digitally imaged using a digital camera (Hamamatsu, Welwyn Garden City, UK) and Openlab software (Improvision, Coventry, UK).
Results
SULT enzyme activity during human placental development
To determine the ontogeny and tissue distribution of
sulfotransferase expression in human placenta, we first assayed
cytosolic fractions prepared from various components of placentas
obtained at gestational ages between 13 and 42 wk (Figs. 1
-4). SULT
activity was determined toward substrates
used at concentrations widely accepted to
be selective for the major human SULT
isoforms 1A1 (4-nitrophenol, 5 µM), 1A3 (dopamine, 10
µM), 1E1 (17-ethynylestradiol, 10 µM),
and 2A1 (DHEA, 10 µM). At the time of
writing, substrates selective for SULTs 1B1 and 1C2 have not been
identified. In addition, sulfation of iodothyronines
T2, T3,
rT3, and T4 was quantified
in representative placental samples from both villous and membranous
regions throughout gestation using 125I-labeled
substrates.
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). In
cotyledon, amnion, and decidua, these activities were high at the
earliest time point measured (13–14 wk of gestation), and did not show
any statistically significant trend toward increased or decreased
activity throughout gestation. In chorion/decidua, SULT1A1 and SULT1A3
activities appeared to be lower earlier in gestation, with an increase
toward normal term, although the trend was not statistically
significant. As has been observed for other human tissues, substantial
interindividual variation in enzyme activity was found with substrates
for both SULT1A1 (e.g., approximate 10-fold variation in
cotyledon) and SULT1A3 (e.g., approximate 5-fold variation
in cotyledon). The greater degree of variability in SULT1A1 activity
toward 4-nitrophenol is consistent with the known functional
polymorphism(s) associated with this isoform (32, 33).
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-ethynylestradiol, was also found
in placental cytosols; however, these activities were considerably
lower than for SULT1A1 and SULT1A3 (
Figs. 1–4). Activities in
cotyledon and amnion were substantially lower than in chorion/decidua
and decidua, with activity in chorion/decidua and decidua being
expressed predominantly in third-trimester tissue; activity was
extremely low or absent before 30 wk of gestation. DHEA
sulfotransferase activity, indicative of SULT2A1 expression, was also
present in the placenta, with activity highest in the cotyledon.
To determine whether the placenta has the capacity to sulfate
iodothyronines, we also measured SULT activity toward four
iodothyronines in cytosols isolated from different placental
subfractions, using 125I-labeled substrates
(
Figs. 1–4). Activity toward all iodothyronines was considerably lower
than for dopamine and 4-nitrophenol. 3,3'-T2 was
the best substrate for sulfation by placental cytosols, demonstrating a
similar expression pattern to SULT1A1 in particular. To try and
identify the SULT isoform responsible for the sulfation of
3,3'-T2 in placenta, we performed nonparametric
correlation analysis (Spearman correlation, Prism software,
GraphPad Software, Inc., San Diego, CA) with all the SULT
enzyme activity values determined in the 148 placental cytosol samples
from cotyledon, amnion, chorion/decidua, and decidua. The
3,3'-T2 SULT activity showed a highly significant
correlation (rs = 0.933, 99% confidence limits
0.898–0.956, P < 0.0001) with 4-nitrophenol SULT
activity, strongly suggesting that SULT1A1 is the enzyme primarily
responsible for the sulfation of 3,3'-T2 in human
placenta (Fig. 5). Sulfation of
T3, rT3, and T4
was extremely low in all placental tissues throughout pregnancy, and
there were no highly significant correlation between these
iodothyronine SULT activities and any of the other SULT activities
measured. These results suggest that in placenta
T3, rT3, and
T4 are substrates for multiple SULTs or that the
sulfation of these compounds is carried out by sulfotransferase(s)
other than the 1A1, 1A3, 1E1, and 2A1 isoforms studied here.
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To determine whether SULT enzyme protein expression reflects the enzyme activities measured in placental cytosols and to assess the expression of SULTs 1B1 and 1C2 (for which specific substrates are not available), we carried out semiquantitative immunoblot analysis of placental cytosols, using a panel of antihuman SULT antibodies raised in sheep against purified recombinant enzymes.
Antibodies raised against purified human SULT1A3 have been described
previously (23). This anti-SULT1A3 preparation
cross-reacts with SULTs 1A1 and 1A2 on immunoblot and ELISA analysis
(these three isoforms share >93% amino acid sequence identity); thus,
it was possible to use this antibody preparation to assess the
expression of these three enzymes. Immunoblot analysis with this
antibody preparation confirmed that SULTs 1A1 and 1A3 were expressed in
all placental tissues and that the cotyledon generally exhibited the
highest level of expression, consistent with the enzyme assay data
(Fig. 6). Recombinant SULT1A2 migrates between SULTs 1A1 and 1A3
on SDS-PAGE, and we could detect no expression of this enzyme in any
placental sample studied. The considerable interindividual variability
in SULT1A1 expression was also evident from this immunochemical
analysis. To determine whether SULT enzyme activity was directly
related to enzyme protein expression, we quantified the expression
level of SULT1A1 and SULT1A3 proteins using densitometric analysis and
compared these values with the enzyme activity data determined for 43
placental cytosol samples. There was a strong correlation
(rs = 0.871, 99% confidence limits 0.725–0.942,
P < 0.0001) between SULT1A1 enzyme protein expression
and enzyme activity, with 4-nitrophenol as substrate (Fig. 7). As we
have observed previously for platelets, there was no significant
correlation between SULT1A3 enzyme protein expression and
dopamine sulfotransferase enzyme activity (not shown).
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Localization of placental SULT expression by immunohistochemistry
The placenta is a complex tissue, and we were interested to
localize the site(s) of expression of sulfotransferase. We therefore
performed immunohistochemical analysis using the anti-SULT1A3 antibody
preparation. We first attempted to use tissue sections cut from
formalin-fixed, paraffin- embedded material with the peroxidase
detection system. This gave good localization with our positive control
human fetal liver samples; however, the method did not give
satisfactory immunostaining of placental samples. We were, however,
able to detect expression of SULT1A family members in placenta using
immunofluorescence with frozen sections (Figs. 8 and 9). The use of frozen sections
generally does not provide as high
resolution immunohistochemical staining as formalin-fixed,
paraffin-embedded material; however, we were able to clearly
demonstrate the localized expression of SULT1A protein.
Immunofluorescence was conducted on 7-µm cryosections of fetal liver
(positive control), placental cotyledon, and membrane sections, and the
tissue morphology was slightly disrupted as a result of cryofixation.
The fetal liver positive control revealed immunopositive hepatocytes
and positive hematopoietic cells (Fig. 8A), consistent with previous
immunohistochemical analysis using formalin-fixed, paraffin-embedded
tissue and peroxidase detection (23). Placental cotyledon
showed strong immunopositivity in syncytiotrophoblastic cells and in
the endothelial cells of fetal blood vessels with lesser
immunopositivity in interstitial areas (Fig. 8, C, E, and F). Placental
amnion showed strong positivity in the outer cellular layer (Fig. 9, A
and C), whereas the chorion and decidual layers showed low
immunopositivity (Fig. 9, E and G). Negative controls, in which
preimmune serum was used in lieu of primary antibody, showed no
background staining (Fig. 8, B and D, and Fig. 9, B, D, F, and H).
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The placenta forms a major interface between the developing fetus and its mother and performs many important functions to maintain and terminate pregnancy and to ensure normal growth and development. The human thyroid gland begins to develop at about 10 wk of gestation but may not function normally until at least 20 wk; until this point the fetus is dependent on maternal thyroid hormone (34). The placenta transports T4 to the fetus, where it is converted to T3 by deiodination. Later in pregnancy, the placenta expresses mainly type III deiodinase, which metabolizes T4 to inactive rT3, thus limiting fetal exposure to maternal-derived thyroid hormone. The fetus also has a different pattern of circulating iodothyronines, compared with the adult, with low levels of T3 and high levels of rT3, T4 sulfate, and T3 sulfate (18). Although there is considerable literature on deiodinases in the placenta, there is a paucity of information on sulfation and sulfotransferases in this tissue. To determine whether the placenta is likely to be a significant source of circulating iodothyronine sulfates in the fetus, we have characterized the major SULT isoforms involved in the sulfation of iodothyronines.
Of the SULT1A family, both SULT1A1 and SULT1A3 were expressed in all
placental compartments from the earliest gestational age measured (13
wk), but no evidence for expression of SULT1A2 was found using Western
blot analysis. This is consistent with previous studies that question
the functional relevance of SULT1A2 and that indicate that SULT1A2 RNA
is incorrectly spliced (35). SULT1A1 activity measured
with 4-nitrophenol as substrate correlated strongly with SULT1A1
protein levels determined by quantitative immunoblot analysis,
indicating that the substantial interindividual variation in enzyme
activity observed results primarily from different levels of the
protein. A common functional polymorphism in SULT1A1
(Arg213
His) (32, 33) causes
substantially reduced SULT1A1 enzyme activity and protein stability in
platelets from individuals homozygous for the variant allele
(SULT1A1*2). This is a likely explanation for a major
component of the interindividual variation we observe in SULT1A1
activity, although it is not possible to rule out the influence of
environmental factors that might modulate SULT expression, as
demonstrated for placental cytochrome P450 CYP1A1, which is highly
induced in cigarette smokers (36).
The sulfotransferase enzyme(s) primarily responsible for iodothyronine sulfation in humans in vivo remain to be conclusively determined. SULT1A1 is probably the major enzyme involved in sulfation of 3,3'-T2 in developing human liver (23), and SULT1A1 has the highest specificity constant toward 3,3'-T2 of the major SULT isoforms (15). With placental samples we again found strong correlation between SULT1A1 measured with 4-nitrophenol as substrate and sulfation of 3,3'-T2, reinforcing our view that this enzyme probably provides the majority of the sulfation capacity for this iodothyronine. The identity of the SULT enzymes involved in the sulfation of other iodothyronines is not so clear. All the major SULTs metabolize most iodothyronines in vitro to varying degrees (15). We have demonstrated SULT1E1 to be a major enzyme in vitro for sulfation of rT3, T3, and to a lesser extent T4 (14), although others have claimed SULT1B1 (13) and SULT1C2 (16) to be involved. Our results show that human placenta has a very low capacity for sulfation of T3, T4, and rT3. The absence of significant correlation between SUTL1A1 and 1A3 activities measured with 4-nitrophenol and dopamine, respectively, and the sulfation of rT3, T4, and T3 probably rule out these enzymes in the placenta as having a major role in the appearance of iodothyronine sulfates in the fetal circulation. Similarly, SULT1E1 was expressed at extremely low levels, with the exception of the predominantly maternal decidua toward the end of gestation, and it is again unlikely that this enzyme plays a major role in sulfation of iodothyronines in the fetus. It appeared that neither SULT1B1 nor SULT1C2 was expressed to any significant extent in the placental tissues examined, although we can detect significant expression of both enzymes in fetal tissues, including liver (1B1, 1C2), kidney (1C2), lung (1C2), and small intestine (1B1, 1C2) (37). For SULT1C2, this is in agreement with expression predominantly in the fetus determined by RNA dot blot analysis (38). If the SULT1C2 enzyme does indeed have significant capacity for sulfation of iodothyronines (16), then the expression of this enzyme in the fetus (and neonate) may contribute to the circulating levels of iodothyronine sulfates.
SULT1A1 and SULT1A3 were consistently higher in cotyledon and decidua than in the amnion and chorion/ decidua. These enzymes may provide a metabolic barrier to the transfer of potentially toxic chemicals (either xenobiotics or of endogenous origin) from the maternal to fetal circulation. It is interesting that the SULT1A family appears well developed in the human placenta, as it is in the fetus (23, 39, 40). This is in contrast to many other "drug-metabolizing" enzymes, which are rather poorly represented in fetal and placental tissues. For example, there is little evidence for substantial expression of UDP-glucuronosyltransferase (quantitatively the major conjugating enzyme system involved in xenobiotic detoxication) in human placenta, with the exception of some members of the UGT2B family involved in steroid hormone metabolism (41, 42). Early workers (reviewed in 43, 44) failed to find significant glucuronidation of xenobiotics in human placenta, and this is also the case in the fetus (45). A number of cytochromes P450 (CYPs) are expressed in placenta, particularly the aromatase (CYP19) and cholesterol side-chain cleavage enzyme (CYP11) (36, 46). However, it is generally accepted that the level of CYP-mediated metabolism of xenobiotics by human placenta is low, with the possible exception of substrates for the CYP1A1 enzyme, which is highly induced in placentas from tobacco smokers (36). Thus, sulfation by SULT1A enzymes appears to be a major detoxication pathway in the human fetoplacental unit.
Estrogen sulfotransferase (SULT1E1) was present at very low levels during early pregnancy, although there was significant expression in the predominantly maternal decidua during the third trimester. This observation raises the possibility of hormonal regulation by sulfation within the uterine cavity as parturition approaches because estrogen sulfates are inactive at the estrogen receptors. Human pregnancy is characterized by substantially increased levels of estrogens (particularly estriol, derived from fetal adrenal DHEAS) in the final trimester; however, unlike most other animal species, there is no evidence for a fall in maternal or placental progesterone levels (47). Progesterone is proposed to play an important role in human parturition because the progesterone antagonist RU486 increases uterine activity and induces labor, and many studies (reviewed in 47) suggest that progesterone has direct effects on the expression of genes in the myometrium involved in relaxation of the uterus and the descent of the fetus. This is consistent with well-documented observations that SULT1E1 expression in human endometrium is tightly regulated in relation to the menstrual cycle (28) and that progesterone is able to stimulate SULT1E1 expression in culture models of human endometrium (48, 49, 50, 51). Progesterone-mediated stimulation of estrogen sulfotransferase activity in the decidua may provide a means of modulating local estrogenic stimulation during the crucial last trimester of pregnancy and may contribute to the intricate mechanisms controlling the onset of parturition.
Sulfation of DHEA was also detectable, principally in cotyledons with low expression in other placental tissues. Three SULT isoforms have been proposed as "hydroxysteroid" sulfotransferases, with the ability to sulfate DHEA and other steroids. The most widely studied form, SULT2A1, is expressed at very high levels in fetal adrenal glands. The adrenal enzyme produces the majority of DHEAS, the precursor substrate for estrogen biosynthesis by the placenta (39, 52). The recently discovered SULT2B1 gene on chromosome 19 encodes two sulfotransferase isoforms, SULT2B1a and SULT2B1b (53). Initial experiments indicated that these enzymes could also sulfate DHEA, and that they were expressed in placenta (53). However, recent work indicates that their substrate preference is for cholesterol and other endogenous oxysterols (54), and that their principal site of expression and action is in the skin (35). This suggests that the majority of DHEA sulfotransferase activity measured in human placenta is the result of the action of SULT2A1.
In summary, we have performed the first detailed characterization of sulfotransferase expression in the developing human placenta and have demonstrated that the placenta has significant sulfation capacity, particularly because of the SULT1A family. We failed to find substantial sulfation of the major iodothyronines T3, T4, and rT3, although 3,3'-T2 was extensively metabolized by SULT1A1, which suggests that the placenta is not a major source of very high levels of circulating iodothyronine sulfates in the human fetus. The apparent upregulation of SULT1E1 in the decidua during the third trimester was an interesting observation, suggesting a role for sulfation in modulating estrogenic stimulation of the fetoplacental unit during the important latter stages of pregnancy. We hope these studies will form the basis for greater understanding of the role of sulfation in human development.
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We are grateful to Nicola Rose for assistance with purification of recombinant SULTs 1B1 and 1C2.
Footnotes
This work was supported by grants from Tenovus Scotland and the Scottish Executive Chief Scientist’s Office (to M.W.H.C. and R.H.) and from the Commission of the European Communities (QLG3-CT-2000-00930 to R.H., M.W.H.C., and T.J.V.).
SULT nomenclature used in this paper follows a system devised recently (R. B. Raftogianis et al., submitted for publication). In particular, the human SULT, which is named 1C2 in this paper, corresponds to the human isoform originally named either human SULT1C sulfotransferase 1 (55 ) or human SULT1C1 (38 ).
Abbreviations: CYP, Cytochrome P450; DHEA, dehydroepiandrosterone; FITC, fluorescein isothiocyanate; PAPS, ; PAP35S, [35S]3'-phosphoadenosine 5'-phosphosulfate; rT3, reverse T3; SULT, sulfotransferase enzyme; 3,3'-T2, 3,3'-diiodothyronine.
Received May 31, 2001.
Accepted September 3, 2001.
References
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