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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 7 2503-2508
Copyright © 1998 by The Endocrine Society

Original Studies

Expression of Wilms’ Tumor Suppressor Gene (WT1) in Term Human Trophoblast: Regulation by Cyclic Adenosine 3',5'-Monophosphate1

Michael Feingold2, Moshe Zilberstein2, Rajesh K. Srivastava, Machelle M. Seibel, Shalom Bar-Ami and Edouard Hambartsoumian

Department of Obstetrics and Gynecology, Boston University School of Medicine (M.F., M.Z., E.H.); and the Faulkner Center for Reproductive Medicine (M.Z., R.K.S., M.M.S., S.B.-A., E.H.), Harvard/Deaconess Surgical Service, Harvard Medical School, Boston, Massachusetts 02130

Address all correspondence and requests for reprints to: Michael Feingold, M.D., Boston Medical Center, Division of Obstetrics and Gynecology, Department of Maternal-Fetal Medicine, One Boston Medical Center Place, Harrison Avenue Campus, Dowling 3 South, Boston, Massachusetts 02118.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Wilms’ tumor suppressor gene (WT1), which is deleted in some Wilms’ tumors, encodes a zinc finger transcription factor. We studied WT1 messenger ribonucleic acid (mRNA) in human term placenta and cytotrophoblasts differentiating into syncytiotrophoblasts in vitro by RT-PCR. The results suggest that WT1 mRNA is expressed in the trophoblasts in a cell-specific fashion. WT1 mRNA expression has been observed to decline remarkably in trophoblast cells after 72 h, when these cells are morphologically differentiated into multinucleated syncytiotrophoblasts. As it is well known that cAMP as a second messenger plays a significant role in cellular proliferation and differentiation of placental cells, we examined the effect of 8-bromo-cAMP on WT1 mRNA expression in undifferentiated cytotrophoblasts and differentiated syncytiotrophoblasts. We observed that cAMP enhanced WT1 mRNA expression in cytotrophoblasts, but remained ineffective in altering WT1 mRNA in syncytiotrophoblasts. In summary, the results of this investigation demonstrate that the WT1 gene is developmentally regulated during trophoblast differentiation. An involvement of the cAMP-mediated system in regulating the WT1 gene in the trophoblast is suggested.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MAINSTAY of placental development is a dramatic spatiotemporal cellular and tissue reorganization. Cytotrophoblast cells invade maternal tissue, where they differentiate into hormonally active syncytiotrophoblasts. Recent data suggest that what seemed to be a straightforward process, where an undifferentiated cytotrophoblast matures into a terminally differentiated syncytiotrophoblast, is far more complex. The cytotrophoblasts emerge as the undifferentiated stem cells of the placenta. They possess the potential to transform into nontrophoblast cells depending on their localization, either inside or outside of the villi (1, 2). It has also been suggested that cytotrophoblast differentiation into syncytiotrophoblasts is a continuum, and cells may be extracted from the placenta for culture at different stages of the process depending on the stage of the pregnancy (3, 4). These multifaceted differentiation processes challenge any simple experimental design, and therefore, the factors that promote or limit cytotrophoblast differentiation are poorly understood. Notwithstanding the intricacy of this process, the development of various culture systems, not unlike the one used for this study, allowed invaluable insight, enhancing the understanding of the now classical basic elements of trophoblast differentiation (3, 4).

WT1 is a tumor suppressor gene that encodes a transcription-regulating protein and bears homology to the prototypic transcription factor family of early growth response genes (5, 6, 7, 8, 9). It has been shown that WT1 protein binds to the same sequences as early growth response proteins and represses the promoter activity of insulin-like growth factors (IGFs) and their receptor genes (10, 11). IGFs, in turn, have been implicated as important regulators of placental growth and differentiation (12, 13). Several other transcription factors have been implicated to have a possible role in placental ontogeny and function. Transcription factors, such as members of the GATA family, Rex-1, and basic helix-loop-helix (14, 15, 16), have been shown to be expressed in placenta and have been suggested to regulate trophoblast differentiation and function. Recently, overexpression of p53, a tumor suppressor protein that negatively regulates the cell cycle, has been described in human cytotrophoblast, thus implicating p53 as a repressor of cytotrophoblast proliferation and invasion (17, 18).

WT1 messenger ribonucleic acid (mRNA) expression is specifically observed in the urogenital system, i.e. kidneys, uterus, and gonads (19, 20, 21), and it is suggested that in these tissues WT1 might be functioning as a regulator of proliferation and differentiation. The rat trophoblast is devoid of WT1 mRNA, which is, however, abundant in rat decidua (22). WT1 transcripts have recently been demonstrated in early human placenta, where the gene also appears to undergo imprinting (23).

We as well as others have previously established that cAMP analogs and activators of adenyl cyclase strongly affect the morphology and function of trophoblastic cells (3, 24). Specifically in cell preparations (similar to the one used in this study), mononucleated cells aggregate and fuse to produce syncytial cells. Concomitantly with these morphological changes, intracellular levels of cAMP increase, and the cells express genes that delineate differentiation (3). It has been universally accepted that cAMP has a critical role in heralding the terminal stages of trophoblast cell differentiation. Many of the differentiated functions of the syncytiotrophoblasts can be stimulated by cAMP analogs in vitro even before they mature into syncytium (24, 25, 26, 27). For these reasons this study also investigated the possible effect of cAMP on the production of WT1 mRNA in cytotrophoblasts and syncytiotrophoblasts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Hanks’ Balanced Salt Solution without Ca2+ and Mg2+, 8-bromo-cAMP, deoxyribonuclease (DNase) type IV, ammonium chloride, ethylenediamine tetraacetate, and Percoll were obtained from Sigma Chemical Co. (St. Louis, MO). DMEM with 25 mmol/L HEPES and L-glutamine, trypsin, trypsin-ethylenediamine tetraacetate, antibiotic (gentamicin), FBS, TRIzol reagent, Taq DNA polymerase, a 100-bp DNA ladder, and the Superscript II preamplification system were purchased from Life Technologies (Gaithersburg, MD).

Isolation and culture of trophoblast

The protocol was approved by institutional review board. Placentas were obtained from normal term deliveries after receiving patients’ consents. For demonstrating WT1 mRNA expression in human placenta, tissue from the fetal part of the placenta was used. Cytotrophoblasts were isolated by trypsin-DNase digestion and purified on a discontinuous Percoll gradient with a modification of a previously described procedure (28, 29). Briefly, about 30 g soft villi from the placenta were scrapped off, minced, and subjected to three sequential 30-min digestions by 0.25% trypsin and 500 U DNase I. The resultant cell suspension was filtered through nylon mesh to remove undigested tissue and layered on the discontinuous Percoll gradient (5–70%) and centrifuged. The Percoll-purified cells were washed once in DMEM and resuspended in DMEM containing antibiotic and 10% FBS. Cell viability was more than 90%, as determined by the trypan blue dye exclusion method. Trophoblasts were cultured in 24-well Falcon culture dishes at a concentration of 8 x 105 cells/well at 37 C in an atmosphere of 5% CO2-95% air. Before plating, an aliquot of cells (~5 x 106) was snap-frozen in liquid nitrogen and stored at -80 C until total RNA was isolated. This is referred to as day 0 cytotrophoblasts. Cells were allowed to attach overnight, and medium was replaced with fresh medium. Cell culture was terminated at the designated time. Cells were washed repeatedly by ice-cold phosphate-buffered saline and stored at -80 C until total RNA was prepared.

In another experiment, cytotrophoblasts were treated with different doses of 8-bromo-cAMP (0, 10, 100, and 1000 µmol/L) after overnight attachment of the cells. For determining the effect of cAMP on syncytiotrophoblasts, the cytotrophoblast cells were cultured for 72 h until they were differentiated into syncytiotrophoblasts and then incubated with medium containing different doses of 8-bromo-cAMP.

RNA isolation and RT-PCR

Total RNA was isolated from the cells and placental tissue using TRIzol reagent. RNA was quantified by absorbance at 260 nm and stored at -80 C until being subjected to RT-PCR. Total RNA (1–3 µg) was reverse transcribed at 42 C in the presence of random hexamer primers (100 pmol) and treated with Superscript II reverse transcriptase in a 20-µL reaction. Two microliters of complementary DNA were used for the first PCR amplification using WT1-specific primers (5'-CAT GAC CTG GAA TCA GAT GAA C-3' and 5'-CAG TCC TTG AAG TCA CAC TGG-3'). To enhance the specificity of the PCR amplification step, nested PCR was employed, using primers 5'-GAG GCA TTC AGG ATG TGC-3' and 5'-GTT TCT CAC CAG TGT GCT TC-3' and 2 µL of the first PCR product. A pair of oligonucleotides of human ribosomal protein S16 (5'-CGT TCA CCT TGA TGA GCC CAT T-3') and (5'-TCC AAG GGT CCG CTG CAG TC-3') was also included in each reaction as an internal control (30). A mix containing 10 x PCR buffer, MgCl2, and Taq polymerase was used in a total volume of 50 µL. Amplification was carried out for 25 cycles using a 50 C annealing temperature in a Perkin-Elmer/Cetus thermal cycler (Norwalk, CT). The conditions were such that amplification of the product was in the exponential phase, and the assay was linear with respect to the amount of input RNA. The samples were resolved on a 2% (wt/vol) agarose gel containing ethidium bromide. A 100-bp DNA ladder mol wt marker was run on the same gel to determine product size. The primers used for PCR amplification gave a product size of 170 bp for WT1 and 100 bp for S16. The gel was analyzed with a GS-700 imagining densitometer using Molecular Analyst software (Bio-Rad Laboratories, Hercules, CA). The intensity of the signal was normalized using S16 as an internal standard, and the amount of WT1 was expressed as a ratio of WT1/S16. Two types of negative controls were prepared: 1) RT was performed with total RNA without reverse transcriptase to detect possible contamination in RNA samples by genomic DNA; and 2) total RNA was omitted in the RT. These controls confirmed that no contamination occurred during the course of the RT-PCR procedure.

Statistics

Data were calculated using one-way ANOVA and Student’s paired t test. Differences were considered significant for P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
WT1 mRNA production in human placental tissue was examined and compared to that in purified trophoblasts. Total RNA prepared from placental tissue and freshly isolated trophoblasts were subjected to nested RT-PCR as described in Materials and Methods. As shown in Fig. 1Go, WT1 mRNA was expressed in the placental tissue as well as in the trophoblasts. However, purified trophoblast cells showed higher levels of WT1 mRNA.



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  		Figure 1. Wilms’ tumor (WT1) mRNA expression in placental tissue and freshly isolated cytotrophoblast cells. Total RNA was isolated from whole placental tissue and from freshly isolated cytotrophoblast cells. RNA was subjected to RT-PCR (Nested) as described in Materials and Methods. A, The upper panel shows band intensity in agarose gel on which the PCR products have been separated. A human ribosomal protein S16 was used as an internal standard in each reaction. B, The lower panel represents quantification of the data (mean ± SEM; n = 3).

 
To determine the morphological changes that occur during cytotrophoblast differentiation in vitro, trophoblasts were isolated and purified from the term placental tissue and cultured for 72 h. As is evident in Fig. 2BGo, within 24 h in culture the cytotrophoblasts begun to aggregate and started to fuse. The process of syncytium formation was completed within 72 h when the cells appeared fully differentiated (Fig. 2DGo). Mononuclear cells in culture up to 24 h were considered cytotrophoblasts, whereas the multinucleated cells beyond 72 h in culture were considered syncytiotrophoblasts.



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  		Figure 2. Growth and differentiation of cytotrophoblast into syncytiotrophoblast cells in vitro. The cytotrophoblast cells were cultured for 72 h. At 12 h of culture, cytotrophoblast cells attached to the culture dishes and remained scattered (A; magnification, x20). After 24 h of culture, the mononuclear trophoblasts started to aggregate (B; magnification, x40). At 48 h, multinucleated cells with or without membrane separating nuclei appeared (C; magnification, x40). Cultures at 72 h showed large groups of multinucleated cells forming conspicuous syncytium (D; magnification, x100). Staining was performed using hematoxylin and eosin.

 
Expression of WT1 during trophoblast differentiation was examined. mRNA was isolated from trophoblast cells cultured for 0, 12, 24, 48, and 72 h. The results revealed that WT1 is expressed in cultured cytotrophoblasts at a constant level during the first 48 h (Fig. 3Go, A and B). However, after 72 h, WT1 mRNA levels dropped significantly, at a time when the culture contained predominantly syncytiotrophoblasts.



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  		Figure 3. WT1 mRNA expression in cytotrophoblast cells differentiating into syncytiotrophoblast cells in vitro. Freshly isolated cytotrophoblast cells were cultured as described in Materials and Methods. WT1 mRNA expression was determined in the trophoblast cells cultured for 12, 24, 48, and 72 h. A, The upper panel shows the band intensity of PCR product in agarose gel. B, The lower panel represents the quantification of data of four experiments presented as the mean ± SEM. The asterisk refers to significant differences from 12, 24, and 48 h values (P < 0.05).

 
Figure 4Go demonstrates the effective dose of cAMP on cytotrophoblasts. 8-Bromo-cAMP significantly enhanced WT1 expression in a dose of 1000 µmol/L. However, in some experiments this effect was seen with a dose of 100 µmol/L. The maximum effect of 8-bromo-cAMP was reached within 24 h (time course not shown). However, 8-bromo-cAMP in doses that increased WT1 expression in cytotrophoblasts had no effect on syncytiotrophoblasts (Fig. 5Go).



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  		Figure 4. Effects of various doses of 8-bromo-cAMP on WT1 mRNA expression in cytotrophoblast cells in vitro. Cytotrophoblast cells were cultured for 24 h with different doses of cAMP. A, The upper panel shows the band intensity in agarose gel. B, The lower panel shows the quantification of data from four experiments, presented as the mean ± SEM. The asterisk refers to significant differences from 0 µmol/L cAMP values (P < 0.05).

 


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  		Figure 5. Effect of 8-bromo-cAMP on WT1 mRNA expression in cytotrophoblast cells in vitro. Cytotrophoblast cells were cultured for 72 h until they formed syncytium and then were treated with 1000 µmol/L 8-bromo-cAMP for 24 h. A, The upper panel shows the PCR products on 2% agarose gel. B, The lower panel shows the quantification of data from three experiments, presented as the mean ± SEM. The data did not differ significantly (P > 0.1).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Placental ontogeny entails a well orchestrated process in which cytotrophoblast cells divide and invade the decidua. This incursion into the maternal tissue is circumscribed to mold the typical finger-like villi. Syncytiotrophoblast cells that are terminally differentiated trophoblast cells are embedded in the villi and fulfill structural and endocrinological functions. The gene expression repertoire of villous cytotrophoblasts differs from that of syncytiotrophoblasts, and they are the primary source of putative paracrine agents that regulate the trophoblasts (24, 31). In vivo cytotrophoblast cells are mitotically active; they stop dividing and change their cellular characteristics just before fusion. Furthermore, it has been recently shown that cytotrophoblast cells may encounter a different fate and convert to mature endothelial cells when they reach the maternal vasculature, underscoring the multipotent nature of these cells (2). Therefore, studying the processes that bring about cytotrophoblast differentiation holds the key to understanding proper placental development and function. It is still controversial whether human cytotrophoblasts in culture maintain their ability to replicate. Cells derived from early pregnancy readily divide in defined conditions, whereas little division was observed in term placental cells (4). It is unfortunate that very few studies were performed on cells from early pregnancy. There is some evidence to suggest that early gestation cytotrophoblasts may indeed present different qualitative and quantitative characteristics. Very little is known, for example, about the response of these cells to cAMP. It could be, therefore, that these cells with their mitotic capacity represent a more proximal predifferentiation state. Taken together, the data suggest a stepwise perpetuity of the cytotrophoblasts toward differentiation. Enriched term placental cytotrophoblasts in culture as used in this study probably represent a distal stage just before terminal cell differentiation. Nonetheless, term placentas are readily available, and since it was first developed, this model conferred numerous breakthroughs in the understanding of differentiation processes in the placenta (24).

This report shows for the first time that human term placenta contains WT1 transcripts similar to what has been previously shown in early placenta (23). The expression of WT1 in freshly prepared cytotrophoblast surpassed the levels in crude placental tissue. Hence, the results here also demonstrate for the first time that human term trophoblasts express WT1 mRNA in a cell-specific manner.

Others (28) and ourselves (24) have recently demonstrated that cytotrophoblasts in culture undergo a well described morphological transformation reminiscent of those that occur in vivo. This process coincides with the initiation of copious production of hormones such as estrogen, progesterone, hCG, and human placental lactogen. Using this model we have demonstrated here that the expression of WT1 occurs in both cytotrophoblast and syncytiotrophoblast cells. In fully differentiated syncytiotrophoblasts, WT1 transcripts are maintained at a much lower, albeit detectable, level. These results may indicate a role for WT1 gene product in both cytotrophoblast growth restriction and maintainence of trophoblast differentiation. WT1 product is a prototypic transcription factor that has been shown to determine transcription regulation of pluripotent mesenchymal cells into highly differentiated epithelial cells in the fetal kidney (8, 9). In the ovary, on the other hand, WT1 has been suggested to play an important role in inducing atresia (20). Two alternative splice sites are used to produce four forms of WT1 mRNA. One splice site introduces 17 amino acids into the protein just proximal to the first zinc finger. The second splicing alternative generates a protein with three amino acids (lysine, threonine, and serine, hence KTS) inserted between the third and fourth zinc fingers. The existence of alternative isoforms of WT1 protein with potentially different tissue-specific activity confers a wide repertoire of possible modes of action. Moreover, these protein variants can be variably regulated in different stages of differentiation (27). However, it has already been shown in the ovary that the ratio of the four WT1 splicing products does not vary during development (20).

The well documented target of WT1 gene products in various in vitro models is the IGF system (13, 15, 16). WT1 gene products have been shown to repress the IGF-II gene and its receptor gene, namely the IGF-I receptor. It has been shown that IGF-II, in turn, plays an important role to promote placental growth (17, 18). Thus, WT1 may inhibit the production of both IGF-II and IGF-I receptors to induce cytotrophoblast division arrest and direct them toward differentiation. Interestingly, both IGF-II and WT1 are imprinted genes, and as such are implicated in regulating placental and embryonic growth (32). WT1 also suppresses growth factor-encoding genes such as colony-stimulating factor-1, platelet-derived growth factor A chain, inhibin-{alpha}, and transforming growth factor-ß1 (33, 34, 35, 36). These growth factors are important for placental development (37, 38, 39). In addition to these effects, WT1 protein is a powerful repressor of its own gene (40).

Recently, it has also been shown that administration of equine CG to rats suppresses WT1 mRNA in the ovary (41). It is tempting, therefore, to suggest that hCG, which is produced abundantly by syncytiotrophoblasts, plays a similar role in the suppression of WT1 in the human. However, the copious constitutive production of hCG by the human cells impedes any traditional treatment study that adds hCG to the culture.

It is very well established that cAMP has a profound effect on the DNA synthesis, morphology, and function of many primary and malignant cells. In most of the instances, specifically activation of the cAMP system in trophoblasts, this leads to the induction of a differentiated state. Most of the available data regarding cAMP action on human trophoblast cells stem from cell systems similar to the one employed here. Treatment of term cytotrophoblasts with 8-bromo-cAMP induces immediate changes in their appearance, and they round up. This may be an acute response to changes in the cytoskeleton induced by the analog. Within 24 h, striking intracellular changes occur, with an increase in the endoplasmic reticulum and an enlargement of the Golgi apparatus. The secretory capacity of the cells is enhanced even before fusion into syncytium. Because term placental cytotrophoblasts hardly divide, it is difficult to appreciate effects on their invasiveness. Nonetheless, it has been shown that cAMP thwarts trophoblast penetration into extracellular matrix (3).

The results of this study have also demonstrated that 8-bromo-cAMP treatment of cytotrophoblasts increased the level of WT1 mRNA. Hence, cAMP may be regulating WT1 mRNA production in a manner that is yet unexplained. It could well be that this cell-specific increase in WT1 mRNA is brought about indirectly, through one of the numerous biological actions of cAMP in these cells. It is noteworthy that the timing of action as well as the effective dose of 8-bromo-cAMP in our experiments are similar to what have been reported for the induction of hCG in cytotrophoblast cells (25, 26). It is, therefore, suggested that WT1 is part of the cascade that brings forth the cAMP-induced differentiation and endocrine function. However, once the cells are fully differentiated, they lose their capacity to respond to cAMP with increased WT1 mRNA. Interestingly, activation of protein kinase A by cAMP analog in WT1-transfected cells resulted in WT1 protein translocation to the cytoplasm and the reduction of its transcription activity (27). This observation suggests that cAMP may also regulate the posttranslational function of WT1 when its restrictive activity is less desired. Concomitantly, WT1 product may be capable of autorepression in the syncytium similar to what has been shown in other models. The virtual nature of the apparent constitutive production of WT1 mRNA in the trophoblasts and the suggested regulation by cAMP are far from elucidation, and these deserve further investigation.

In summary, the results of this study demonstrate that WT1 mRNA is expressed in human trophoblast and that this expression is reduced as cytotrophoblasts differentiate into syncytium. It suggests that in human trophoblasts, WT1 may function as a growth and invasion suppressor. The expression of the WT1 gene seems to be mediated through cAMP as a second messenger in cytotrophoblasts, but not in the differentiated syncytiotrophoblasts. Further studies on the role of WT1 gene in human trophoblasts may elucidate the mechanisms of placental physiology and pathology as well as obstetrical disorders.


   Acknowledgments
 
We acknowledge the support for this study given by the Chairman, Dr. Phillip Stubblefield, and the clinical team at the Department of Obstetrics and Gynecology, Boston City Hospital.


   Footnotes
 
1 This work was supported by a grant from the Boston University School of Medicine and the Faulkner Institute for Reproductive Medicine. Back

2 M.F. and M.Z. contributed equally to this study and to the manuscript. Back

Received December 22, 1997.

Revised April 3, 1998.

Accepted April 9, 1998.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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