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The Expression of 11ß-Hydroxysteroid Dehydrogenase Type 2 Is Induced during Trophoblast Differentiation: Effects of Hypoxia

Canadian Institutes of Health Research Group in Fetal and Neonatal Health and Development, Child Health Research Institute, and Lawson Health Research Institute, St. Joseph’s Health Care London, Departments of Obstetrics and Gynecology and Physiology, University of Western Ontario, London, Ontario, Canada N6A 4V2

Address all correspondence and requests for reprints to: Dr. K. Yang, Lawson Health Research Institute, Grosvenor Campus, 268 Grosvenor Street, London, Ontario, Canada N6A 4V2. E-mail: . kyang{at}uwo.ca

Abstract

The intracellular enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) catalyzes the unidirectional conversion of bioactive glucocorticoids to their inert metabolites. In the human placenta, 11ß-HSD2 is highly expressed in syncytiotrophoblasts, although cytotrophoblasts also express this enzyme at lower levels. Given that cytotrophoblasts will differentiate into syncytiotrophoblasts in vivo and in vitro, the present study was designed to examine the hypothesis that the expression of 11ß-HSD2 is induced during in vitro trophoblast differentiation. When Percoll-purified human cytotrophoblast cells were cultured under standard (20% oxygen) conditions, they aggregated and fused to form syncytiotrophoblasts. Within the first 24 h during differentiation, levels of 11ß-HSD2 protein and activity were increased by 2- to 3-fold, but they did not increase further thereafter. However, when the cells were exposed to hypoxic (1% oxygen) conditions, both the induction of 11ß-HSD2 and trophoblast differentiation were prevented. Taken together, these results demonstrate for the first time that the expression of 11ß-HSD2 is induced early during trophoblast differentiation, and hypoxia prevents this induction, indicating that placental 11ß-HSD2 expression is subjected to regulation by the local oxygen environment. If placental villi respond to hypoxia in a similar fashion in vivo, the present findings would suggest that hypoxia might be a factor contributing to the previously reported decreases in placental 11ß-HSD2 in pregnancies complicated by intrauterine growth restriction and preeclampsia.

WITHIN THE HUMAN placenta, trophoblasts play a critical role in controlling metabolic and endocrine functions throughout pregnancy. The outer syncytiotrophoblastic layer, covers the chorionic villi and is bathed in maternal blood; it is positioned to regulate nutrient and gas exchange between the mother and the fetus (1). In addition, the syncytiotrophoblastic layer is considered the primary endocrine unit of the placenta, producing a wide variety of peptide and steroid hormones (2, 3). In vivo, these multinucleated syncytiotrophoblasts are formed by the maturation and fusion of the underlying mononucleated cytotrophoblasts (1). As pregnancy progresses, cytotrophoblasts continually differentiate, and by late gestation the placenta becomes largely dominated by syncytiotrophoblasts. In vitro trophoblast cell cultures have been used to examine the dynamic events that accompany trophoblast differentiation (4, 5, 6, 7, 8, 9). Under various culture conditions, isolated cytotrophoblasts will aggregate, fuse, and undergo morphological and biochemical changes that are consistent with syncytium formation (4). Furthermore, several in vitro studies have demonstrated that the differentiation of cytotrophoblasts to syncytiotrophoblasts is accompanied by the generation of a cascade of regulatory signals that result in the expression of genes encoding a variety of peptide hormones and steroid-metabolizing enzymes (4, 5, 6, 7, 8, 9). However, the molecular events that initiate and sustain syncytiotrophoblast differentiation and lead to expression of various genes are poorly understood.

Previous in vitro studies have provided evidence that hypoxia limits trophoblast differentiation, which is characterized by defects in cell fusion and diminished hormone production [e.g. human chorionic gonadotropin (hCG) and progesterone; Refs. 10, 11 ]. A number of clinical conditions, most notably preeclampsia and intrauterine growth restriction (IUGR), expose placental villi to hypoxia (12, 13). Histologically, placentas from these pathological pregnancies display cytotrophoblast prominence and abnormalities in syncytiotrophoblast formation (12, 13). Furthermore, preeclampsia and IUGR are also associated with a decreased expression of the placental enzyme, 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2; Refs. 14, 15, 16, 17, 18), which is responsible for the unidirectional conversion of bioactive glucocorticoids (cortisol and corticosterone) to their inert metabolites (cortisone and 11-dehydrocorticosterone; Ref. 19). Given that it is predominantly expressed in the syncytiotrophoblast layer (20, 21, 22), the site of fetal-maternal exchange, placental 11ß-HSD2 serves as a functional barrier to control fetal exposure to maternal glucocorticoids (23, 24). The physiological significance of this functional barrier is evident from both human and animal studies in which excessive exposure to glucocorticoids during fetal life was shown to be associated with reduced birth weight (25, 26, 27). Therefore, the maintenance of this placental glucocorticoid barrier by 11ß-HSD2 appears to be crucial for normal fetal development.

Despite the critical role of placental 11ß-HSD2 in pregnancy/fetal development, the molecular events that culminate in the high expression of 11ß-HSD2 in syncytiotrophoblasts remain unexplored. Given that cytotrophoblasts (expressing lower levels of 11ß-HSD2) differentiate into syncytiotrophoblasts (expressing higher levels of 11ß-HSD2) both in vivo and in vitro, we hypothesized that the expression of 11ß-HSD2 would be induced during trophoblast differentiation. Furthermore, because hypoxia has been shown to impair trophoblast differentiation, we further hypothesized that hypoxia would block the induction of 11ß-HSD2 during trophoblast differentiation. Therefore, the present study was designed to test these two hypotheses using the well established human trophoblast cell culture model.

Materials and Methods

Reagents and supplies

[1,2,6,7-³H(N)]-Cortisol (80 Ci/mmol) was purchased from DuPont Canada Inc. (Markham, Ontario). Nonradioactive steroids were obtained from Steraloids Inc. (Wilton, NH). Polyester-backed thin-layer chromatography plates were obtained from Fisher Scientific Ltd. (Nepean, Ontario). All solvents used were OmniSolve grade from VWR Canlab (Mississauga, Ontario). Cell culture supplies were obtained from Canada Life Technologies, Inc. (Burlington, Ontario) or Fisher Scientific.

Placental trophoblast cell cultures

Placental trophoblast cells were prepared using a modification of the method of Kliman (4), as described (28). Briefly, human placentas were obtained from uncomplicated pregnancies at term after elective cesarean section. Villous tissues were dissected free from fetal membranes and blood vessels, rinsed in 0.9% NaCl₂, and digested with 0.125% trypsin (Sigma-Aldrich Canada Ltd., Oakville, Ontario) and 0.02% deoxyribonuclease-I (Sigma) in DMEM containing 0.05% streptomycin and gentamicin (Canada Life Technologies, Inc.) three times for 30 min each. The placental cells were loaded onto a 5–70% Percoll gradient at step increments of 5% Percoll, and centrifuged at 2500 g for 20 min to separate different cell types. Cytotrophoblasts between the density markers of 1.049 and 1.062 g/ml were collected and plated in 35-mm dishes at a density between 2.5 x 10⁵ and 5.0 x 10⁵ cells/cm² in DMEM containing 10% fetal calf serum (Canada Life Technologies, Inc.). The cells were maintained at 37 C in humidified 5% CO₂-95% air (20% O₂) for 72 h. For hypoxic experiments, cells were exposed immediately after plating to 1% O₂, 5% CO₂, and balanced N₂ in humidified sealed chambers (Billups-Rothenburg, Del Mar, CA) at 37 C for the same time period. In some experiments, cells were cultured under normoxic conditions (20% O₂) for 6 h or for 48 h after plating, before being cultured under hypoxic conditions (1% O₂).

Assay of 11ß-HSD2 activity–radiometric conversion assay

The level of 11ß-HSD2 activity in intact cells was determined at various time points by measuring the rate of cortisol to cortisone conversion, as described previously (29). Briefly, the cells were incubated for 1 h at 37 C in serum-free medium containing approximately 50,000 cpm [³H]-cortisol and 100 nM unlabelled cortisol. At the end of incubation, the medium was collected, and steroids extracted. The extracts were dried, and the residues were resuspended. A fraction of the resuspension was spotted on a thin-layer chromatography plate that was developed in chloroform/methanol (9:1, vol/vol). The bands containing the labeled cortisol and cortisone were identified by UV light of the cold carriers, cut out into scintillation vials, and counted in Scintisafe Econo 1 (Fisher Scientific). The rate of cortisol to cortisone conversion was calculated, and the blank values (defined as the amount of conversion in the absence of cells) were subtracted and expressed as picomoles of cortisone formed per 1.0 x 10⁵ cells per hour. Results are shown as mean + SEM.

Determination of hCG

hCG concentration in cell culture media was determined by microparticle enzyme immunoassay (MEIA) using a specific monoclonal antibody directed against the ß-subunit of hCG (Abbott Laboratories, Abbott Park, IL). The values are shown as mean ± SEM.

Protein extraction and Western blot analysis

Cells were lysed with cold lysis buffer (100 mM NaCl, 50 mM NaF, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.1 mM phenylmethylsulfonylfluoride, 1 mM orthovanadate, and 50 mM Tris HCl, pH 7.5) for 5 min at room temperature. The total cell lysates were collected with a cell scraper, vortexed vigorously, and centrifuged at 10,000 g for 20 min at 4 C. The supernatant was collected, and the protein content determined by the Bradford method using a protein assay kit (Bio-Rad Laboratories, Inc., Missasauga, Ontario) with BSA as a standard.

Western blot analysis was conducted as described previously (30). Briefly, 20 µg of the protein extracts were subjected to a standard 12% SDS-PAGE. After electrophoresis, proteins were transferred to nitrocellulose using a Bio-Rad Mini Transfer Apparatus. The 11ß-HSD2 protein was detected on the nitrocellulose filter using an enhanced chemiluminescence Western Blotting Analysis System (Amersham Pharmacia Biotech, Inc.; Baie D’Urte, Quebec) following the manufacturer’s instructions. Briefly, the nitrocellulose filter was blocked overnight at 4 C with 10% Blotto in TTBS (0.1% Tween-20 in TBS) and incubated with primary antibody (HUH23; 0.25 µg/ml in TTBS) for 1 h at room temperature. The primary antibody was a polyclonal rabbit antihuman 11ß-HSD2 antibody (a generous gift from Dr. Z. Krosowski, Baker Medical Research Institute, Melbourne, Australia). After three 5-min washes with TTBS, the filter was incubated with horseradish peroxidase-labeled second antibody and developed in enhanced chemiluminescence detection reagents. The filter was then exposed to x-ray film (Eastman Kodak Co., Rochester, NY) for 1–5 min.

Results

Induction of 11ß-HSD2 during trophoblast differentiation

When cultured under standard conditions, the isolated human placental cytotrophoblasts differentiated, over the course of 72 h, into syncytiotrophoblasts. This was reflected by a progressive increase in the release of hCG, a biochemical marker of differentiation (Ref. 3 ; Fig. 1). Appreciable levels of 11ß-HSD2 activity were detected in isolated cytotrophoblasts at 6 h in culture, then increased 2- to 3-fold by 24 h, but did not increase further thereafter (Fig. 2). To determine whether increases in enzyme activity are due to changes in 11ß-HSD2 expression, the level of 11ß-HSD2 protein was assessed by Western blot analysis. There was a corresponding increase in 11ß-HSD2 protein levels during trophoblast differentiation (Fig. 3).

View larger version (13K): [in this window] [in a new window]   Figure 1. Time course of hCG secretion during trophoblast differentiation. The isolated trophoblast cells were cultured, immediately after plating, in either normoxic (20% oxygen) or hypoxic (1% oxygen) conditions for a total of 72 h. The culture medium was renewed at each time point as indicated, and the level of hCG in the medium was determined by MEIA, as described in Materials and Methods. Values represent the mean ± SEM from four independent experiments, each conducted in duplicate.

View larger version (15K): [in this window] [in a new window]   Figure 2. Induction of 11ß-HSD2 activity during trophoblast differentiation. The isolated trophoblast cells were cultured, immediately after plating, in either normoxic (20% oxygen) or hypoxic (1% oxygen) conditions for a total of 72 h. The culture medium was renewed at each time point as indicated, and the level of 11ß-HSD2 activity in intact cells was determined by a standard radiometric conversion assay, as described in Materials and Methods. Values represent the mean ± SEM from six independent experiments, each conducted in duplicate.

View larger version (39K): [in this window] [in a new window]   Figure 3. Induction of 11ß-HSD2 protein during trophoblast differentiation. The isolated trophoblast cells were cultured, immediately after plating, in either normoxic (20% oxygen) or hypoxic (1% oxygen) conditions for a total of 72 h. The cell lysate was collected at each time point as indicated, and the level of 11ß-HSD2 protein was assessed by Western blot analysis, as described in Materials and Methods. This experiment was repeated three times with similar results. Shown is an autoradiograph of a representative Western blot.

In contrast, when they were exposed immediately after plating to hypoxic conditions, the isolated cytotrophoblasts exhibited a markedly diminished hCG release over the course of 72 h, indicative of impaired trophoblast differentiation (Fig. 1). Furthermore, the 2- to 3-fold increase in 11ß-HSD2 activity was blocked (Fig. 2). Similar to the effect on 11ß-HSD2 activity, hypoxia also prevented increases in 11ß-HSD2 protein (Fig. 3). It is possible that the hypoxia-induced blockade of 11ß-HSD2 induction may be attributed to a decrease in cell viability. We reasoned that if this were the case, trophoblast cells that had been exposed to hypoxic conditions would not be able to recover and exhibit their normal endocrine functions, such as the release of hCG, once they have been switched from hypoxic to normoxic conditions. However, there was a progressive increase in hCG in trophoblasts that were switched after 3 d of hypoxia to normoxic conditions (Fig. 4), thus arguing against such a possibility.

View larger version (13K): [in this window] [in a new window]   Figure 4. Time course of hCG secretion after 3 d of hypoxia. The isolated trophoblast cells were cultured, immediately after plating, in hypoxic (1% oxygen) conditions for 3 d, and were then either maintained under hypoxic or switched to normoxic (20% oxygen) conditions for an additional 3 d. The culture medium was renewed daily, and the level of hCG in the medium after the initial 3 d of hypoxia was determined by MEIA, as described in Materials and Methods. Values are mean of one experiment, which was repeated with similar results.

As a first step in exploring the role of 11ß-HSD2 induction and hypoxia in trophoblast differentiation, the effect of a delay in the onset of hypoxia on 11ß-HSD2 and trophoblast differentiation was examined. When cytotrophoblast cells were initially exposed to normoxic conditions for 6 h, and then switched to hypoxic conditions for the remainder time in culture, trophoblast differentiation was impaired, although the induction of 11ß-HSD2 activity was maintained (Fig. 5).

View larger version (13K): [in this window] [in a new window]   Figure 5. Effects of delayed hypoxia on hCG secretion and 11ß-HSD2 activity. The isolated trophoblast cells were cultured immediately after plating in normoxic (20% oxygen) conditions for 6 h and were then either maintained under normoxic or switched to hypoxic (1% oxygen) conditions for up to 72 h. The culture medium was renewed at each time point as indicated, and the level of hCG in the medium (A) and the level of 11ß-HSD2 activity in intact cells (B) were determined by MEIA and a standard radiometric conversion assay, respectively, as described in Materials and Methods. Values are mean of one experiment, which was repeated with similar results.

To study the effects of hypoxia on 11ß-HSD2 in trophoblast cells after 48 h of differentiation, cytotrophoblast cells were exposed to normoxic conditions for 48 h, and then transferred to hypoxic conditions for 24 h. Although it dramatically attenuated progression of hCG release from 647 to 130 mIU/ml, hypoxia had no effect on 11ß-HSD2 activity or 11ß-HSD2 protein (Fig. 6).

View larger version (19K): [in this window] [in a new window]   Figure 6. Effects of hypoxia on hCG secretion and 11ß-HSD2 in trophoblast cells after 48 h of differentiation. The isolated trophoblast cells were cultured in normoxic (20% oxygen) conditions for 48 h and were then switched to hypoxic (1% oxygen) conditions for 24 h. The culture medium was renewed daily, and the level of hCG in the medium (A), the level of 11ß-HSD2 activity (B), and the level of 11ß-HSD2 protein (C) in intact cells were determined by MEIA, standard radiometric conversion assays, and Western blot analysis, respectively, as described in Materials and Methods. Values are mean of one experiment, which was repeated with similar results.

In the present study, we used a well established primary cell culture model of trophoblasts to examine the sequence of events leading to the high expression of 11ß-HSD2 in syncytiotrophoblasts. We demonstrated for the first time that freshly isolated cytotrophoblasts express appreciable levels of 11ß-HSD2 protein and activity. Furthermore, within the first 24 h during in vitro trophoblast differentiation, there is a 2- to 3-fold induction in 11ß-HSD2 expression, and hypoxia prevents this induction. These novel observations, when confirmed by 11ß-HSD2 promoter studies, may provide a molecular basis for the previously reported higher 11ß-HSD2 expression in syncytiotrophoblasts (31). Given that hypoxia is often associated with complicated pregnancies such as IUGR and preeclampsia (12, 13), the present findings also suggest that hypoxia may contribute to the previously reported decreases in placental 11ß-HSD2 expression and activity in these two clinical conditions (14, 15, 16, 17, 18).

Because a number of syncytiotrophoblast-specific genes are induced during trophoblast differentiation (4, 5, 6, 7, 8, 9), it is conceivable that the higher level of 11ß-HSD2 expression occurs in association with the spontaneous differentiation of cytotrophoblasts to syncytiotrophoblasts. We demonstrated that the induction of 11ß-HSD2 occurred early during trophoblast differentiation, because its maximal expression occurred in the first 24 h when the cells were predominantly mononucleated. This induction preceded biochemical evidence of syncytial formation, leading us to postulate that the induction of 11ß-HSD2 may play a crucial role in trophoblast differentiation. Obviously, further studies will be required to elucidate the mechanism of 11ß-HSD2 induction and to determine whether this induction is linked to, or necessary for, trophoblastic differentiation.

Although the physiological significance of the induction of 11ß-HSD2 during trophoblast differentiation remains to be defined, it is possible that this induction may have a profound impact on local glucocorticoid actions within trophoblast cells. Given that syncytiotrophoblasts produce a wide range of enzymes, growth factors, peptide hormones, and steroids (2, 3), the induction in placental 11ß-HSD2 during trophoblast differentiation will likely play an important role in glucocorticoid-mediated endocrine functions in differentiated syncytiotrophoblasts.

In a variety of complicated pregnancies such as IUGR and preeclampsia, the placental villi and the fetus are exposed to hypoxia (12, 13). Histologically, placentas from pregnancies complicated by IUGR and preeclampsia also display cytotrophoblast prominence and syncytiotrophoblast damage, indicating impaired trophoblast differentiation (12, 13). Hypoxia is also known to limit trophoblast differentiation in vitro (10, 11). Furthermore, in IUGR and preeclampsia, placental 11ß-HSD2 expression and activity are markedly reduced (14, 15, 16, 17, 18). Taken together, these findings suggest that hypoxia may regulate the expression of 11ß-HSD2 during trophoblast differentiation. In the present study, we examined this hypothesis using a well-established in vitro model of trophoblast differentiation, and 1% oxygen to mimic hypoxic conditions in vivo, because it is widely accepted that a low oxygen (1–2%) environment mimics the placental defects associated with preeclampsia and IUGR (32). When the isolated trophoblast cells were cultured in 1% oxygen, both the induction of 11ß-HSD2 and trophoblast differentiation were prevented, demonstrating for the first time that placental 11ß-HSD2 expression is subjected to regulation by the local oxygen environment. Furthermore, these findings may provide a potential explanation for the previous findings that the attenuated placental 11ß-HSD2 activity and expression in IUGR is not attributed to decreases in total placental mass, imprinting, or mutations in the 11ß-HSD2 gene (15).

The demonstration of the hypoxia-induced blockade in the induction of placental 11ß-HSD2 expression during trophoblast differentiation will likely have far-reaching implications for our understanding of pathological pregnancies. To gain insight into the role of 11ß-HSD2 induction and hypoxia in trophoblast differentiation, we examined the effect of a delayed onset of hypoxia on 11ß-HSD2 expression during trophoblast differentiation. When it was delayed by 6 h (i.e. trophoblast cells were cultured in 1% oxygen 6 h postplating), hypoxia was ineffective in blocking the induction of 11ß-HSD2 although it was able to limit trophoblast differentiation, as reflected by the lack of a progressive increase in hCG. Furthermore, when cytotrophoblast cells were cultured for 48 h under normoxic conditions to induce cellular differentiation and 11ß-HSD2 expression, and then transferred to hypoxic conditions, hypoxia did not alter levels of 11ß-HSD2 activity or 11ß-HSD2 protein. However, hypoxia greatly attenuated the progression of hCG release, indicative of an effect on trophoblast differentiation. Taken together, these observations indicated that the molecular events leading to the induction of 11ß-HSD2 during trophoblast differentiation have been initiated within the first 6 h in culture, and once initiated, these events cannot be reversed or altered by hypoxia. Moreover, these results also suggested that the induction of 11ß-HSD2 is unlikely to play a fundamental role in trophoblast differentiation, because when the onset of hypoxia was delayed by 6 h, the induction of 11ß-HSD2 was maintained, even though trophoblast differentiation was prevented. Obviously, further studies will be required to define the role, if any, of 11ß-HSD2 induction in trophoblast differentiation.

In summary, we have demonstrated that 11ß-HSD2 expression is induced during in vitro trophoblast differentiation and hypoxia prevents this induction. Given that in a number of clinical conditions, both the placenta and the fetus are exposed to hypoxia, the present findings will have far-reaching implications for our understanding of the intricate relationship between hypoxia and compromised placental function in these clinical conditions.

Acknowledgments

We are indebted to Jonathan van Beek for his critical review of this manuscript.

Footnotes

This work was supported by the Canadian Institutes of Health Research (CIHR). D.B.H. is a recipient of the Doctoral Research Award from CIHR, and K.Y. is an Ontario Ministry of Health Career Scientist.

Abbreviations: hCG, Human chorionic gonadotropin; 11ß-HSD2, 11ß-hydroxysteroid dehydrogenase type 2; IUGR, intrauterine growth restriction; MEIA, microparticle enzyme immunoassay; TTBS, Tween-20 in TBS.

Received December 20, 2001.

Accepted April 22, 2002.

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