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Physiol. Genomics 18: 99-107, 2004. First published March 23, 2004; doi:10.1152/physiolgenomics.00181.2003
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Received 21 October 2003; accepted in final form 4 March 2004.
Physiological Genomics 18:99-107 (2004)
1094-8341/04 $5.00 © 2004 American Physiological Society

Critical role for transcription factor AP-2{alpha} in human trophoblast differentiation

You-Hong Cheng , Bruce J. Aronow , Shaikh Hossain , Bruce Trapnell , Sue Kong and Stuart Handwerger

Departments of Endocrinology and Molecular and Developmental Biology, Children’s Hospital Research Foundation and Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To examine whether AP-2{alpha} is a critical component of the genetic program that directs human trophoblast differentiation, we used DNA microarray analyses to characterize the effects of a dominant-negative form of the AP-2 protein upon in vitro differentiating cytotrophoblast cells. Human cytotrophoblast cells (>95% pure) were cultured for 3 days in the presence of control medium or medium containing an adenovirus that expresses a dominant-negative mutant of AP-2 (Ad2.AP-2D/N) or an adenovirus lacking the AP-2 mutant gene (Ad.WT). DNA microarray analyses using Affymetrix human U95Av2 GeneChips were performed on RNA extracted from the three groups of cells immediately prior to and after 3 days of cell culture. Cells infected with Ad2.AP-2D/N or Ad2.WT underwent morphological differentiation similar to that of uninfected cells, with greater than 90% of the cells in each group fusing to form multinucleated syncytiotrophoblast cells. However, Ad2.AP-2D/N markedly inhibited the induction or repression of many genes that were regulated in the noninfected and Ad2.WT-infected cells during differentiation. Eighteen of the 25 most induced genes and 17 of the 20 most repressed genes during differentiation were AP-2 dependent, with the majority of these related to extracellular organization, cellular communication, and signal transduction. Taken together, these findings strongly suggest that AP-2 plays a critical role for both the induction and repression of genes that comprise postsyncytialization gene expression programs of trophoblast differentiation and maturation. AP-2, however, is not required for the fusion of cytotrophoblast cells to form a syncytium or the expression of syncytin.

placenta; gene expression; DNA microarray


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE TROPHOBLAST LAYER of the human placenta villus is composed of multinucleated syncytiotrophoblast cells and mononuclear cytotrophoblast cells that are separated from the interior of the villus by a basement membrane (3). The syncytiotrophoblast cells are involved in most of the differentiated functions of the placenta, including hormone production and gas and substrate exchange between the maternal and fetal circulations. The cytotrophoblast cells serve as the precursor cells for the syncytiotrophoblast cells (3). During the initial phase of human trophoblast differentiation, the cytotrophoblast cells proliferate and fuse to form a multinucleated syncytium. As the cells begin to fuse, the cells undergo biochemical differentiation and express many genes that are not expressed in the undifferentiated state, including placental lactogen (hPL) and chorionic gonadotropin (hCG{alpha} and hCGß).

Many aspects of the dynamic processes occurring during trophoblast differentiation have been studied using trophoblast cell cultures as a model system. Numerous studies have shown that mononucleated cytotrophoblast cells isolated by enzymatic dispersion of placental tissue and cultured in medium containing fetal calf serum or bovine serum will aggregate and fuse to form a multinucleated syncytiotrophoblast that synthesizes and secretes hCG, progesterone, and many other proteins and steroids that are secreted by syncytiotrophoblast cells in vivo (13, 26). To date, this in vitro model has provided considerable information about the expression of cell adhesion molecules and other factors during the early differentiation process and has demonstrated that the inductions of hPL and hCG gene expression are linked to the process of trophoblast differentiation (5–7, 17, 39). Studies using the in vitro model of trophoblast differentiation have identified many factors that induce the differentiation of human cytotrophoblast cells, including IGF-I (4), leptin (28), and cAMP (25, 42). Similar in vitro studies in the mouse have identified several transcription factors that are implicated in the regulation of trophoblast growth and differentiation, including HOXB6, HOXC5, HOXC6, HOX3E, HB24, GAX, MSX2, DLX4, Pit-1, MASH2, Hand1, TF-1, TEF5, and c-Ets1 (11, 12, 22, 32, 37). ID-2, a member of a family of inhibitors of basic helix-loop-helix (bHLH) binding, acts in human trophoblast cells as a dominant-negative bHLH transcription factor (21). Constitutive overexpression of ID-2 prevents differentiation of the cells. However, little is known about the transcription factors involved in human trophoblast differentiation.

Several lines of evidence strongly suggest that AP-2 family members are involved in the regulation of human villous cytotrophoblast differentiation. Two of the isoforms, AP-2{alpha} and AP-2{gamma}, are expressed in the human placenta. In earlier experiments, we observed that AP-2{alpha} mRNA levels increase significantly during trophoblast differentiation and that AP-2{alpha} induces the expression of the syncytiotrophoblast-specific proteins hPL (36), hCG{alpha} (23), hCGß (23), and human corticotropin-releasing hormone (hCRH) (9). Moreover, AP-2{alpha} also stimulates expression of the genes for aromatase cytochrome P-450 (CYP11A1)(45), germ cell alkaline phosphatase (40) 17ß-hydroxysteroid dehydrogenase type 1 (34), and leucine aminopeptidase/oxytocinase (20, 24) in trophoblast cell lines; and AP-2{gamma} transactivates the adenosine deaminase (ADA) gene in murine placenta (38). In addition, AP-2 binding sites are present on the promoters of other genes in the placenta that affect placental function, such as TGFß, vascular endothelial growth factor, matrix metalloproteinases, tissue inhibitor of metalloproteinases, and the estrogen receptor. Numerous investigations have also shown that AP-2{alpha} is important in differentiation of many cell types, including the differentiation of keratinocytes and neural crest cells, as well as the differentiation of 3T3-LI fibroblasts to adipocytes (46).

AP-2 family members bind to the same consensus DNA sequence to activate transcription (29). However, their effects on development are different. Disruption of the genes encoding AP-2{alpha} and AP-2ß results in perinatal mortality without affecting murine placental development (2). AP-2{alpha} disruption affects multiple developmental programs, including formation of the limbs, eye, cranium, body wall, skeleton, and cardiovascular system, whereas AP-2ß has a primary role in kidney development. AP-2{gamma} has no autonomous role in development of the embryo proper, but the presence of AP-2{gamma} in the extra-embryonic membranes is required for normal development of this compartment and survival of the mouse embryo. Null knockout mice lacking the AP-2{gamma} gene have a paucity of giant cells. However, the mutant embryos express placental lactogen I, a hormone that is normally secreted by giant cells, and ADA, which is activated by an AP-2 binding site in its promoter. The reason why these genes are expressed in the absence of AP-2{gamma} is unknown, but Auman and coworkers (2) postulate that other AP-2 family members may compensate for the loss of AP-2{gamma}.

Using cDNA microarray analyses, we recently identified groups of genes that are regulated during in vitro differentiation of human trophoblast cells, with 141 of 6,918 genes induced and 256 repressed by twofold or more (1). The genes were classified according to their biological functions, with different kinetic patterns of expression for the regulated genes identified using a K-means algorithm. Three distinct induction and repression patterns were identified that varied in the onset of change from predifferentiation levels. Within each functional group, some genes underwent marked repression while others with a similar molecular function underwent marked induction, a process referred to as "categorical reprogramming."

The occurrence of distinct temporal behaviors for gene activation and repression within different functional categories correlated with the progressive morphological changes that underlie trophoblast differentiation. During early stages of differentiation, when cells are aggregating and fusing to form a syncytium, many specialized adhesion genes were induced. Following initial syncytial formation, there was marked induction of genes involved in intercellular communication, including many protein hormones and growth factors. From these findings, we hypothesized that there are two gene regulatory programs responsible for villous cytotrophoblast differentiation. The first is characterized by early induction of genes involved in cell adhesion and cell fusion and the formation of multinucleated cells. The second is characterized by the induction of genes involved in hormone production, transport, metabolism, and other functions of fully differentiated syncytiotrophoblast cells.

Analysis of the DNA sequences of the promoters of many of the genes regulated during trophoblast differentiation indicates that many of these promoters contain consensus binding sites for members of the AP-2 transcription factor family (1). Since this finding suggests that AP-2 may important in the regulation of trophoblast differentiation, we have examined in this study whether blocking AP-2 action in human cytotrophoblast cells during differentiation inhibits the differentiation process. Cultured cytotrophoblast cells undergoing spontaneous differentiation were infected with an adenovirus that expresses a dominant-negative mutant of AP-2, and DNA microarray analyses were performed to ascertain which genes regulated during trophoblast differentiation are AP-2 dependent. We observed that AP-2 is not required for cytotrophoblast cells to form a syncytium but is critical for the induction and repression of many genes that are regulated in trophoblast cells later in differentiation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparation of adenoviruses.
Plasmids containing the human AP-2{alpha} gene and a dominant-negative mutant of the human AP-2{alpha} gene were kindly provided by Dr. Trevor Williams, University of Colorado Health Sciences Center, Denver, CO. After demonstrating that the expression plasmid inhibited the induction of hPL and hCG{alpha} expression in trophoblast cells exposed to AP-2{alpha} (data not shown), the AP-2{alpha} D/N gene was excised from the plasmid by double digestion and ligated into the EcoRV site of the shuttle plasmid pAd2. The shuttle plasmid was then used to construct replication defective, recombinant adenoviral vectors, Ad2.AP-2D/N and Ad2.WT, as previously described by Cheng and Handwerger (10). An adenovirus of identical structure except expressing a mammalianized green fluorescent protein (GFP) was used in preliminary studies to determine transduction efficiency (47).

Cell culture.
Third trimester placentas were obtained from women with normal pregnancies and deliveries, and cytotrophoblast cells were isolated by enzymatic disaggregation and cultured as described previously (10). The cytotrophoblast cells were purified to >98% homogeneity by negative CD-9 selection. The protocol for obtaining placentas was approved by the Human Investigation Committees of the University of Cincinnati and the Children’s Hospital Medical Center. The cytotrophoblast cells were plated in six-well culture plates at 2.5 x 106 cell/well in DMEM medium containing 10% fetal bovine serum (FBS). Four hours later, the cells were infected with 500 pfu per cell of either Ad2.AP-2D/N or Ad2.WT in DMEM with 2% FBS and 2 mM glutamine. Two hours after the cells were infected, the medium was changed to DMEM containing 10% FBS (time = 0 day). The cultures were terminated 72 h later (time = 3 days) by withdrawing the medium, and RNA was extracted from the cells with TRIzol reagent (Invitrogen, New York, NY). A transduction efficiency of ≥98% was noted in preliminary studies in which cytotrophoblast cells were infected under identical experimental conditions with the adenovirus expressing GFP (data not shown).

Microarray analysis.
Total RNA was isolated from the cells of three culture plates for each time and transfection condition. Two independent starting condition ("time 0") RNA preparations were used from two different human placenta. Ten micrograms RNA from each sample was subjected to reverse transcription using random hexamers. Samples were then biotinylated and hybridized to the Affymetrix U95Av2 GeneChips using the Affymetrix-recommended protocol. Affymetrix Microarray Suite version 5.0 was used to scan and quantitate GeneChips using default scan settings to produce "*.cel" files that were then subjected to the "robust multiple array" (RMA) analysis algorithm as developed and described by Irizarry et al. (19). Relative gene expression was determined from the Affymetrix *.cel files by using the RMA analysis protocol. RMA signal strength was then transformed from a log base 2 to linear values. Relative gene expression changes across the sample series were then obtained by dividing each measurement for each gene in each specific sample by the mean of that gene’s measurements in the two day 0 control samples. Normalized log signal strength data were collected from each array, and the results were further analyzed using GeneSpring 6.0 (Silicon Genetics, Redwood City, CA). Genes expressed similarly in the two day 0 samples were examined for induction or repression at day 3 using a Welch t-test ANOVA with P < 0.05. Multiple testing correction was not applied based on too few replicates, but the genes passing the ANOVA were further filtered for those that differed from the day 0 reference by more than a factor of 2. These genes were subjected to cluster analysis using Pearson correlation. Genes were categorized using the Simplified Gene Ontology tool in GeneSpring 6.0 (Silicon Genetics) using functional and biochemical associations obtained from Medline, GenBank, Locus Link, OMIM, Swiss-Prot, and Gene Ontology Consortium (http://geneontology.org) resources. In selected instances, the relative amounts of mRNAs for hPL (1), hCG{alpha} (1), hCGß (1), and syncytin (see below) were determined by semiquantitative RT-PCR using GAPDH mRNA as an internal standard. All raw and normalized experimental data as well as the gene lists and hierarchical gene trees are viewable and downloadable from the GeNet web site (http://genet.cchmc.org). Login as "guest" and choose the Affymetrix Human U95Av2 genome ("AffyHumanU95a" platform) and navigate to the HandwergerAP2DN subdirectories within the directories labeled "Experiments," "Gene Lists," and "Gene Trees."

Syncytin mRNA.
Total RNA was isolated from normal trophoblast cells. First-strand cDNA synthesis was performed using SuperScript II reverse transcriptase (Invitrogen) and oligo dT according to the manufacturer’s protocol. Primer sequences used for detection of syncytin transcripts were 5'-TCT ATG GAG AAT GCA GCG TCC CG-3' and 5'-TAG GCT TAC CAG GCG AGT ATG GG-3', which resulted in a predicted product of 391 bp. Primers for human GAPDH were 5'-CCA TGG AGA AGG CTG GGG-3' and 5'-CAA AGT TGT CAT GGA TGA CC-3', which resulted in a predicted PCR product of 192 bp. The PCR reactions were spiked with 0.1 µl of [{alpha}-32P]dCTP (3,000 Ci/mM). Optimal PCR cycles required for linear amplification for each set of primers were determined. Total amplification in each reaction primer set (syncytin plus GAPDH) was kept below saturation levels to permit the two products to remain within the exponential range. GAPDH required 18 cycles while syncytin required 20 cycles. The radiolabeled PCR products were separated by a 6% polyacrylamide gel electrophoresis at 225 V for 3 h. The gel was then transferred to 3M paper, dried, and quantified using a PhosphorImager and ImageQuant 1.2 software (Molecular Dynamics, Sunnyvale, CA).

Immunocytochemistry.
Cultured trophoblast cells were washed three times (5 min each wash) with PBS and then fixed by incubation with 95% ethanol for 10 min. The cells were blocked for 30 min in 5% FBS which was dissolved in PBS. Biotinylated monoclonal antibody to desmosomal protein (Sigma-Aldrich, St. Louis, MO) was diluted 1:20 in FBS-PBS and incubated for 1.5 h at 37°C. The cells were washed three times (5 min each wash) with PBS, incubated for another 1 h with secondary antibody (ExtrAvidin-peroxidase, Sigma-Aldrich) that was diluted 1:20 in FBS-PBS. Following a 10-min incubation with 0.3% hydrogen peroxide, the cells were washed three times with PBS and incubated for 5–15 min with 3-amino-9-ethylcarbazole and 0.2% H2O2 dissolved in 0.05 M acetate buffer, pH 5.0. The cells were washed with distilled water and counterstained with hematoxylin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of Ad2.AP-2D/N on hPL and hCG mRNA levels.
To determine whether Ad2.AP-2D/N inhibits the induction of AP-2{alpha}-dependent genes during trophoblast differentiation, we initially examined the effects of Ad2.AP-2D/N on hPL, hCG{alpha}, and hCGß mRNA levels, all three of which are known to be AP-2 dependent (23, 36) (Fig. 1). As anticipated, hPL, hCG{alpha}, and hCGß mRNA levels increased markedly in the noninfected and Ad2.WT-infected cells during 3 days of culture. The relative amounts of the three mRNAs in the Ad2.WT-exposed cells were nearly identical to that of uninfected cells. In contrast, hPL, hCG{alpha}, and hCGß mRNA levels in the cells infected with Ad2.AP-2D/N showed little or no induction during the 3 days of culture. Nearly identical results were observed in two additional experiments using cells from different placentas. In addition, Ad2.AP-2D/N markedly inhibited hPL and hCGß promoter activity in transduced trophoblast cells (data not shown). Taken together, these experiments demonstrate that Ad2.AP-2D/N markedly inhibits hPL and hCG gene expression.



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  		Fig. 1. Effect of Ad2.AP-2D/N on placental lactogen (hPL) and chorionic gonadotropin (hCG{alpha} and hCGß) mRNA levels during trophoblast differentiation. RNA was isolated from the three groups of cells. HPL, hCG{alpha}, and hCGß mRNA levels were then determined by RT-PCR. Note that hPL, hCG{alpha}, hCGß, and GAPDH mRNA levels increased markedly in the control and Ad2.WT-infected cells. Ad2.AP-2D/N cells, however, had little or no increase in these mRNAs.

 
DNA microarray analysis of trophoblast cells infected with Ad2.AP-2D/N.
DNA microarray analyses significantly extended the RT-PCR findings. Of the 12,651 gene elements analyzed on the Affymetrix U95Av2 GeneChip, we identified 1,145 genes in the noninfected cells that exhibited varied expressions during the 3 days of in vitro differentiation (P < 0.05). Of these genes, 205 were induced by twofold or more during differentiation; 229 genes were repressed by twofold or more (Fig. 2).



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  		Fig. 2. Graphic depiction of the changes in AP-2-dependent genes during trophoblast differentiation. To provide a measure of relative gene expression, the expression value of each gene is shown in relation to the mean of each gene’s expression at day 0 with and without Ad2.WT infection. Samples for GeneChip analysis were obtained from two different sets of cells from different placentas, after 3 days without treatment, after 3 days following infection with Ad2.WT, and after 3 days following infection with Ad2.AP-2D/N. A: a gene cluster that is induced during 3 days of differentiation and inhibited by Ad2.AP-2D/N. B: genes repressed during differentiation that were stimulated by Ad2.AP-2D/N. C: repressed genes that were further inhibited by Ad2.AP-2D/N. D: induced genes that were further stimulated by Ad2.AP-2D/N. E: the large number of genes unaffected by differentiation and Ad2.AP-2D/N.

 
The gene expression profile of placental trophoblast cells infected with unmodified Ad2.WT virus was nearly identical to that of the noninfected cells. Only 13 genes in the Ad2.WT-infected cells were expressed at levels that were twofold greater (2.1- to 3.2-fold) than that of uninfected cells, and only 19 genes were expressed at levels that were twofold less (2.3- to 2.8-fold) than those observed in uninfected cells.

The expression profile of cells infected during differentiation with Ad2.AP-2D/N was markedly different than that of the noninfected and Ad2.WT-infected cells. After 3 days of differentiation, Ad2.AP-2D/N blocked induction or repression of 125 genes that were significantly regulated by differentiation (Fig. 2). Ninety-one of these genes were from groups of genes that were induced by twofold or more during differentiation. The expression levels of 84 of these induced genes were less in the Ad2.AP-2D/N-infected cells than in uninfected or wild-type virus-infected cells, strongly suggesting that AP-2 normally plays a strong role in the induction of their expression. The other seven induced genes were expressed at greater levels in the Ad2.AP-2D/N-infected cells than in the other two groups, suggesting that AP-2 exerts a repressive effect on their expression. The expression of 34 genes normally repressed by twofold or more during differentiation was also affected by infection with Ad2.AP-2D/N. For 29 of these, Ad2.AP-2D/N-infection activated expression, suggesting that during differentiation, AP-2 plays a role in their repression. The other five repressed genes were expressed at even lower levels following Ad2.AP-2D/N infection, suggesting that AP-2 can also attenuate repression during trophoblast differentiation.

The impact of Ad2.AP-2D/N on genes regulated during trophoblast differentiation was much greater than those that are not regulated by differentiation. The expression patterns of 91 of the 205 genes (44.4%) induced by twofold or greater during differentiation and 34 of the 229 genes (14.9%) repressed by twofold or greater during differentiation were AP-2 dependent. In contrast, only 260 of the genes that were unaffected by differentiation were AP-2 dependent.

The most induced and repressed genes during differentiation exhibited the most AP-2 dependency. Thus 18 of the 25 most induced genes (72%) and 17 of the 20 most repressed genes (85%) during trophoblast differentiation were AP-2 dependent. Table 1 shows a list of the 50 most induced AP-2-dependent genes, and Table 2 shows a list of the 25 most repressed AP-2-dependent genes. Most of the regulated AP-2-dependent genes are involved in signal transduction, cell communication, cell growth and maintenance, and hormone synthesis and release (Table 3).


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  		Table 1. The 50 most induced AP-2-dependent genes during trophoblast differentiation

 

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  		Table 2. The 25 most repressed AP-2-dependent genes during trophoblast differentiation

 

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  		Table 3. Functional category distribution of AP-2-dependent induced and repressed genes

 
The reliability of the microarray quantitative data was independently corroborated by the use of RT-PCR analysis of selected mRNAs used in the microarray experiments, as well as in replicate analyses using two additional cell and mRNA preparations from independent placentas. In all three experiments, the profiles of pregnancy-specific ß1-glycoprotein 1, CRH, retinoblastoma-like protein, hPL, laminin, and GAPDH mRNA levels during differentiation determined by semiquantitative RT-PCR analyses were similar to those determined by microarray analyses (data not shown).

Effect of Ad2.AP-2D/N on syncytium formation and syncytin mRNA levels.
To determine whether AP-2 is critical for the fusion of cytotrophoblast cells to form a syncytium, we examined whether Ad2.AP-2D/N prevents and Ad2.AP-2{alpha} enhances syncytialization. Trophoblast cells that had been infected with Ad2.AP-2D/N, Ad2.AP-2{alpha}, or Ad2.WT, as well as uninfected control cells, were cultured for 3 days. The cells were then incubated with an antiserum to desmosomal protein to visualize cell membranes and counterstained with hematoxylin to visualize nuclear membranes. As shown in Fig. 3, cells infected with Ad2.AP-2D/N and Ad2.AP-2{alpha}, as well as the AD.WT-infected and control cells, showed morphologic changes characteristic of cytotrophoblast cells undergoing differentiation. All three groups of trophoblast cells showed similar kinetics for the formation of a syncytium in vivo. Twelve hours following plating, greater than 90% of the cells in each group were attached to the culture plates (time 0). By day 1, the cytotrophoblast cells in each group had formed large aggregates, and ~20% of the cells had fused to form multinucleated cells that contained two or three nuclei. By day 2, ~50% of the cells were multinucleated containing five or more nuclei. By day 3, greater than 90% of the cells were multinucleated, and large syncytiotrophoblast cells appeared to form a network with numerous nuclei arranged in linear arrays (Fig. 3). Some clusters consisted of cells with only a few nuclei; most were in larger clusters of 10 or more nuclei. These findings strongly suggest that inhibition of AP-2 function does not interfere with the fusion of cytotrophoblast cells to form a multinucleated syncytium.



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  		Fig. 3. Effect of on cytotrophoblast fusion and syncytialization. Control (noninfected) cytotrophoblast cells and cytotrophoblast cells infected with Ad2.AP-2D/N or Ad2.WT were cultured for 3 days. The cells were incubated with a monoclonal antibody to desmosomal protein to visualize the cell membranes and counterstained with hematoxylin to visualize cell nuclei. Note that Ad2.AP-2D/N did not decrease syncytium formation.

 
Earlier investigations demonstrated that syncytin, the envelope protein of a retrovirus (HERV-W) incorporated into the human genome, is critical for the fusion of cytotrophoblast cells to form a syncytium (16, 30). Syncytin mRNA levels increase during in vitro differentiation of human trophoblast cells, and treatment of cytotrophoblast cells with antisense oligonucleotides to syncytin blocks syncytialization (16). To determine whether infection of cytotrophoblast cells with Ad2.AP-2D/N inhibits syncytin mRNA levels, syncytin mRNA levels were determined in noninfected trophoblast cells after 1 and 3 days of culture and in trophoblast cells that had been infected for 3 days with Ad2.AP-2D/N or Ad2.WT. As shown in Fig. 4, syncytin mRNA levels increased in the uninfected cells during the 3 days of culture, and Ad2.AP-2D/N and Ad2.WT had no effect. In a separate experiment, Ad2.AP-2{alpha} also had no effect on syncytin mRNA levels (data not shown). These findings therefore support the hypothesis that AP-2 is not a master regulator but is responsible for a subcomponent of the differentiation response.



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  		Fig. 4. Effect of on syncytin mRNA levels during trophoblast differentiation. Control (noninfected) cytotrophoblast cells and cytotrophoblast cells infected with Ad2.AP-2D/N or Ad2.WT were cultured for 3 days. RNA was then isolated from the 3 groups of cells, and syncytin mRNA levels were then determined by RT-PCR.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results of this study indicate that a large number of genes that are regulated during trophoblast differentiation are AP-2 dependent, including many of the most induced and repressed genes. Using Affymetrix Human U95 microarrays that recognize 12,651 gene elements, we observed that 205 genes are induced and 229 genes are repressed by twofold or more in trophoblast cells undergoing spontaneous differentiation over a 3-day period. Overexpression of a dominant-negative AP-2 protein in trophoblast cells during differentiation significantly inhibited the induction of 91 of the 205 induced genes (44.4%) and blocked the repression of 34 of the 229 downregulated genes (14.9%). In fact, 18 of the 25 most induced genes and 17 of the 20 most repressed genes were AP-2 dependent. The expression profiles of the genes affected by Ad2.AP-2D/N infection were not affected by Ad2.WT infection, indicating the specificity of AP-2 dominant-negative action. Overexpression of the dominant-negative AP-2 had no effect on the ability of cytotrophoblast cells to fuse to form a syncytium, indicating that AP-2 has little or no effect on trophoblast cells during the early stage of differentiation. After 3 days in culture, the morphological appearance and syncytin mRNA levels of cells infected with Ad2.AP-2D/N were identical to those of untreated cells and cells infected with Ad2.WT. Instead, the effects of AP-2 were on the later stages of differentiation following syncytialization, particularly on genes involved in differentiated functions such as cell growth and maintenance, hormone synthesis, and substrate transport.

In our earlier studies of gene expression during trophoblast differentiation using Incyte microarrays that recognized 6,918 genes, we noted similar proportions, with 141 genes induced and 256 genes repressed over a 6-day culture period, with nearly 88% of the regulated genes showing significant regulation by 3 days (1). There were 3,245 genes on the Affymetrix arrays that were also present on the Incyte arrays. Of the 85 genes, 57 that were induced on day 3 using the Incyte arrays were also present on the Affymetrix arrays. Of these 57 genes, 52 were also measured as induced using the Affymetrix arrays. Of the 147 genes, 105 that were repressed on day 3 using the Incyte arrays were also measured as repressed on Affymetrix arrays. These findings indicate strong concordance in gene expression among different preparations of trophoblast cells undergoing differentiation and relatively good consistency among different microarray platforms. However, to establish a significance measure associated with the platform and sample variables, a considerably larger study design will be needed.

Although AP-2 was critical for the induction of genes involved in the differentiated functions of syncytiotrophoblast cells, AP-2 does not appear to be important for the induction of syncytium formation earlier in the differentiation process. Immunohistochemical studies indicated that infection of cytotrophoblast cells with Ad2.AP-2D/N has no effect on spontaneous syncytium formation. In addition, Ad2.AP-2D/N did not inhibit and Ad2.AP-2{alpha} did not stimulate the induction of syncytin mRNA levels. Since the transduction efficiency of the adenovirus is ≥98%, it is unlikely that syncytium formation is rescued by AP-2 expressed by the untransduced cells. Furthermore, since all AP-2 isoforms bind to the same consensus DNA binding site, it is unlikely that different AP-2 isoforms are resistant to antagonism by Ad2.AP-2D/N. Earlier studies by Mi and coworkers (30) demonstrated that syncytin, the envelope gene of a recently identified human endogenous defective retrovirus, HERV-W, is markedly induced in BeWo choriocarcinoma cells undergoing syncytialization in response to cAMP and that the fusion can be inhibited by an anti-syncytin antiserum. Furthermore, overexpression of recombinant syncytin in a wide variety of cell types induced the formation of giant syncytia.

Most studies suggest that AP-2{alpha} and AP-{gamma}, the two AP-2 isoforms present in the human placenta, bind to the same DNA element (GCCNNNGGC) (43). Since Ad2.AP-2D/N blocks AP-2 action by competing with AP-2 for binding to DNA, the dominant-negative mutant inhibits the action of both AP-2 isoforms, and it is not possible from studies with Ad2.AP-2D/N to distinguish between the actions of AP-2{alpha} and AP-{gamma}. Although the two isoforms bind to the same DNA elements, there appear to be significant differences in the biological actions mediated by the two isoforms. For example, AP-2{alpha} knockout mice die at birth, whereas AP-2{gamma} knockout mice die at about 7.5 days of gestation before complete formation of the placenta (2). This finding suggests that the two isoforms are activating different targets, perhaps due to the binding of the transcription factors of different partners, coactivators, and/or corepressors (18).

At present, the factors that regulate AP-2 gene expression in the placenta are poorly understood. AP-2{alpha} gene expression in some, but not all cell types, is induced by cAMP, prostaglandins, retinoic acid, thyroid hormone, inflammatory cytokines, and estrogen (8, 15, 31, 33, 41). In addition, the transcription factor Sp1, the retinoblastoma (Rb) protein, and the glucocorticoid receptor (GR) form complexes with AP-2 in some cells to synergize or repress the activity of AP-2{alpha} (14, 44). However, the effects of these factors on the activity of AP-2{gamma} in the placenta and other cell types are unknown. The recent observation that forskolin induces AP-2{alpha} but not AP-2{gamma} expression in JEG-3 choriocarcinoma cells suggests that the two AP-2 family members are differentially regulated in normal trophoblast cells (27). Our studies of the ontogeny of the AP-2 isoforms in human cytotrophoblast cells during differentiation reveal markedly different patterns for AP-2{alpha} and AP-2{gamma}. AP-2{alpha} mRNA levels significantly increase during cytotrophoblast differentiation, with the increase paralleling the increases in hPL, hCG{alpha}, and hCGß mRNA levels (35). AP-2{gamma} mRNA levels, on the other hand, markedly decrease during differentiation, suggesting that the AP-2{gamma} is the predominant AP-2 isoform in cytotrophoblast cells and AP-2{alpha} is the predominant isoform during cytotrophoblast differentiation and in fully differentiated syncytiotrophoblast cells.

Since AP-2{alpha} induces many syncytiotrophoblast marker genes and other genes that are induced during cytotrophoblast differentiation, we hypothesize that AP-2{alpha} is a critical component of the cascade of transcription factors and signaling molecules that induce villous cytotrophoblast differentiation. The cascade includes other transcription factors, coactivators, and corepressors required to induce downstream trophoblast genes. Since inhibition of AP-2 action in differentiating cytotrophoblast cells inhibits the expression of hPL, hCG, and other syncytiotrophoblast-specific genes but not the earlier events of cell fusion and syncytium formation strongly suggests that morphological differentiation alone is insufficient to confer biochemical and endocrine maturation.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grant HD-07447.


   ACKNOWLEDGMENTS
 
We thank Sarah Williams and Kristen Stanley in the Children’s Hospital Medical Center Bioinformatics Core and Sara Rankin in the Affymetrix GeneChip Core for assistance. We also thank Dr. Trevor Williams, University of Colorado Health Sciences Center, Denver, CO, for providing the plasmid that expresses the dominant-negative AP-2 protein.

A. S. Greene served as the review editor for this manuscript submitted by Editor B. J. Aronow.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: Y.-H. Cheng, Division of Endocrinology, Children’s Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: cheny0{at}cchmc.org).


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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