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Endocrinology Vol. 141, No. 10 3737-3748
Copyright © 2000 by The Endocrine Society

ARTICLES

Promoter Elements and Transcription Factors Involved in Differentiation-Dependent Human Chorionic Gonadotrophin-{alpha} Messenger Ribonucleic Acid Expression of Term Villous Trophoblasts1

Martin Knöfler, Leila Saleh, Sandra Bauer, Richard Vasicek, Georg Griesinger2, Heinz Strohmer, Hanns Helmer and Peter Husslein

Department of Obstetrics and Gynecology, University of Vienna, Währinger Gürtel 18–20, A-1090 Vienna, Austria

Address all correspondence and requests for reprints to: Martin Knöfler, Department of Obstetrics and Gynecology, University of Vienna, Waehringer Guertel 18–20, A-1090 Vienna. E-mail: martin. knoefler{at}akh-wien.ac.at


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Differentiation of primary villous cytotrophoblasts into syncytia is associated with increasing production of {alpha} and ß human CG subunits, which is predominantly governed at the level of messenger RNA expression. Here, we present a detailed study on the mechanisms involved in the differentiation-dependent regulation of the trophoblast-specific CG{alpha} gene promoter. Site-directed mutations in each of the five DNA-elements of the composite enhancer were performed to investigate the contribution of the individual regulatory sequences to the overall transcriptional activity of the promoter at two different stages of trophoblast in vitro differentiation. We show that deletion of one cyclic AMP response element (CRE) did not affect CG{alpha} promoter activity in cytotrophoblasts; however, it reduced transcription by 33% in differentiating cultures. Removal of both CREs almost abolished transcription at early and later stages of in vitro differentiation. Upon mutation the enhancer elements {alpha}ACT, JRE, and CCAAT significantly decreased luciferase reporter transcription; however their contribution to the total promoter activity did not change during in vitro differentiation. Contrary to that, mutated TSE diminished promoter activity by 19% during 12 and 48 h of cultivation but reduced luciferase expression by 78% between 48 and 84 h of differentiation. In electrophoretic mobility shift assay, the TSE interacted with activating protein (AP)-2{alpha} in both primary trophoblasts and choriocarcinoma cells. While CRE-interacting proteins were detectable 12 h after isolation, the TSE-binding complex did not appear before 36 h of in vitro differentiation. During syncytium formation increasing protein expression of activating transcription factor (ATF)-1, cAMP response element-binding protein (CREB)-1, and AP-2{alpha} was observed on Western blots. Moreover, phosphorylated CREB-1 and ATF-1 accumulated between 24 and 78 h of trophoblast cultivation. By fluorescence immunohistochemistry, we show that CREB-1 was predominantly expressed in syncytiotrophoblasts, whereas ATF-1 and AP-2{alpha} localized to the syncytium and some cytototrophoblasts as well as to stromal and endothelial cells of the placental villus. Phosphorylated CREB-1/ATF-1 and the coactivator protein CBP were primarily detected in syncytial nuclei, suggesting the presence of functional, cAMP-dependent transcriptional complexes in the differentiated tissue. In agreement to the in vivo situation, phosphorylated CREB-1/ATF-1 were observed in nuclei of the differentiated trophoblast cultures. The activity of the CG{alpha} promoter as well as CREB-1/ATF-1 phosphorylation increased upon elevation of cAMP levels and overexpression of the catalytic subunit of protein kinase A. Additionally, we demonstrate that overproduction of the enzyme enhanced protein expression and binding of AP-2{alpha} to the TSE. We conclude that differentiation-dependent transcription of the CG{alpha} gene in villous trophoblasts is mainly governed by increasing expression of AP-2{alpha} and PKA-dependent phosphorylation of CREB-1 and ATF-1.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HUMAN CG, a member of the family of glycoprotein hormones, is composed of a specific ß-subunit and an {alpha}-subunit that are common to FSH, LH, and TSH (1). Its main function is to maintain the corpus luteum during early pregnancy; however, the biological properties of CG signaled through the CG/LH receptor in several tissues suggest that the hormone has a more general role in establishment and maintenance of pregnancy (2, 3, 4, 5). The major source of CG is the villous trophoblast of the human placenta (6). Maternal serum levels of CG peak at the ninth to tenth weeks of gestation and fall to a plateau at low concentrations thereafter (7). Because concentrations of the free {alpha}-subunit increase until parturition, levels of holo-CG are determined by selective expression of ß-protein (8). Clinically, elevated serum levels of CG of the second trimester are known to be associated with chromosomal abnormalities of the fetus such as Down’s syndrome (9, 10); however, the mechanisms controlling gestation-dependent expression of CG in normal and pathological pregnancies remain poorly defined (11).

Because little CG is stored intracellularly, expression/secretion of the hormone is thought to be mainly triggered by de novo synthesis of its subunits (12). Numerous studies have focused on the role of DNA-regulatory elements controlling CG{alpha} and CGß messenger RNA (mRNA) transcription in trophoblastic tumor cells (13). Six genes encoding CGß and a single copy of the LHß gene are arranged in a gene cluster on chromosome 19 (14, 15). The CGß5 gene is most abundantly transcribed in the placenta and choriocarcinoma cells (16, 17) and expression of individual CGß mRNAs varies during the first trimester of gestation (18). An extended promoter region of the CGß gene interacting with broadly expressed proteins is required to direct cAMP-dependent and trophoblast-specific expression (19, 20, 21). CG{alpha} mRNA expression in choriocarcinoma cells is controlled by a composite enhancer harboring five cis-acting elements located between (-180 and -80) of the 5' flanking region of the single-copy CG{alpha} gene, trophoblast-specific element (TSE), {alpha}-activating element ({alpha}ACT), tandem cAMP response element (CRE), junctional regulatory element (JRE), and the CCAAT box (Fig. 1AGo). The {alpha}ACT element is recognized by GATA factors (22) and comprises the placenta-specific enhancer together with TSE and the CREs (23, 24). The CREs provide the strongest contribution to transcription (25, 26) and members of the ATF/CREB (activating transcription factor/cAMP response element-binding protein) family were shown to interact with the palindromic sequences in trophoblastic tumor cells (27). The JRE is located between the proximal CRE and the CCAAT box, binds an unknown 50-kDa polypeptide, and augments cell-specific CG{alpha} expression (28). The CCAAT box is recognized by {alpha}-CCAAT binding factor ({alpha}CBF), a protein that could be involved in tissue-restricted expression of a small number of genes (29, 30). Coordinate expression of CG{alpha} and ß mRNA may involve activating protein-2 (AP-2), which interacts with the TSE of CG{alpha} and various sequences of the CGß promoter (20, 21, 31). However, no trophoblast-specific DNA-binding protein(s) regulating CG{alpha}- and CGß-transcription have been characterized, suggesting that combinatorial mechanisms may determine tissue-restricted expression of both subunit mRNAs.



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  		Figure 1. Transfection of purified villous trophoblasts with CG{alpha} gene promoter constructs. A, Schematic representation of the CG{alpha} wild-type (wt) and mutant promoter regions. Open and filled boxes indicate wt and mutated sequences of the composite enhancer, respectively. Positions relative to the transcriptional start site (+1) are depicted. B, Transcription of wild-type and mutant CG{alpha} promoter during trophoblast differentiation. Cells were cotransfected with luciferase-expressing plasmids and pCMV-ßGal at 12 and 48 h of in vitro differentiation. Protein lysates were prepared after an additional 36 h, respectively. Luciferase activity (normalized to the respective ß-Gal activity of each extract) was determined as described in Materials and Methods. Two- to 3-fold differences in overall transfection rates of primary cells were observed. For comparison of different trophoblast preparations (calculation of mutants), values of CG{alpha} wt promoter were arbitrarily set to 100% in each transfection experiment. Bars represent the mean values of 10 independent transfections of trophoblasts isolated from five different placentae; error bars indicate SD (*): P < 0.05, ns, not significant.

 
CG expression has been widely studied in mononuclear cytotrophoblastic tumor cells; however, the in vivo source of the hormone is the syncytium, a nonproliferating multinuclear epithelial cell layer generated by continuous cellular fusion of underlying cytotrophoblast stem cells (32). CGß is exclusively expressed in the syncytium and can only be detected in cytotrophoblasts before the sixth week of gestation (33). CG{alpha} mRNA is predominantly found in the syncytiotrophoblast but can be detected in a small number of cytotrophoblasts (34). Thus, induction of CG{alpha} and CGß mRNA expression in vivo is coupled to differentiation of cytotrophoblasts to syncytiotrophoblasts. Little information, however, is available on the mechanisms that control the differentiation-dependent synthesis of CG subunit mRNAs. In vitro, CG expression can be studied in mononuclear, villous cytotrophoblasts isolated from term placenta that aggregate and form syncytial-like structures in a time-dependent manner (35, 36, 37). In these cultures, syncytialization is promoted by a variety of growth factors using cAMP as a second messenger (38). Indeed, cAMP analogs induced cell fusion in vitro involving protein kinase A-dependent phosphorylation (39, 40). During differentiation, an increase in CG secretion as a consequence of CG{alpha} and CGß mRNA de novo synthesis was observed reflecting the in vivo situation (41, 42). CG{alpha} rose earlier than CGß during spontaneous differentiation or upon cAMP stimulation, suggesting that production of the latter is more strictly dependent on syncytium formation (43, 44, 45). We have recently demonstrated that the promoter of the CG{alpha} gene is active in isolated cytotrophoblasts and increases during in vitro differentiation (42). Concomitantly, accumulation of CRE-binding activity harboring phosphorylated CREB-1/ATF-1 was observed. Here, we extended our studies on the differentiation-dependent regulation of the CG{alpha} promoter. In transient transfection studies, we investigated the contribution of each DNA-binding site of the CG{alpha} composite enhancer to differentiation-dependent transcription and studied the temporal expression, binding activity and placental localization of transcription factors involved in CG{alpha} gene regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Purification, cultivation, and transfection of villous term trophoblast cells
JEG-3 and JAr choriocarcinoma cell lines were cultivated as described recently (46). Villous trophoblast cells were isolated from selected placental tissue between 38 and 40 weeks of gestation after spontaneous delivery or cesarean section. Placental material was processed using the method of Kliman et al. (35). Trophoblasts were immunopurified and characterized as previously described (42, 47). Pure cells (trophoblast content of = 97%) were seeded on plastics at a density of 4 x 105 cells/cm2 and cultivated in keratinocyte growth medium (KGM+10% FBS) to perform in vitro differentiation (37). For reporter studies, cells were seeded in 24-well plates and transiently transfected after 12 and 48 h of cultivation using ProFection Mammalian Transfection System-Calcium Phosphate (Promega Corp., Madison, WI) as described elsewhere (42). Briefly, trophoblasts were incubated in the presence of 2.5 µg pGL-3 basic plasmid (Promega Corp.) harboring CG{alpha} wild-type (wt) or mutant promoters and 0.5 µg pCMV-ß-Gal (CLONTECH Laboratories, Inc., Palo Alto, CA) as a normalization standard. Two parallel transfections per construct were performed. Precipitates were left on cells for 12 h and medium was changed. After an additional 24 h, supernatants were aspirated and cellular protein lysates were prepared using reporter lysis buffer (Promega Corp.). For analyses of protein kinase A-mediated effects, cells were transfected with 1 µg plasmid pMtC encoding the catalytic subunit of protein kinase A (48), 2.5 µg pGL-3 basic carrying CG{alpha} wt promoter and 0.5 µg pCMV-ß-Gal after 12 h of cultivation.

Reporter assays
Luciferase activity and ß-Gal activity were determined in 200 µl of protein extract which had been stored at -70 C in reporter lysis buffer (Promega Corp.). Luciferase activity was determined on a luminometer (Lumat LB 9507, EG&G Berthold, Bad Wildbad, Germany) by using Luciferase Assay System (Promega Corp.). Activity of ß-Gal was quantitated by photometrically assaying the conversion of the chromogenic substrate chlorophenol red-ß-D-galactopyranoside (CPRG) (Roche Molecular Biochemicals, Mannheim, Germany) at 570 nm as described (49). ß-Gal values were determined in 40 µl of protein lysate incubated 15 min at 37 C. Luciferase- and ß-Gal assays were performed in duplicate and mean values were calculated. Concentration of protein lysates was determined by Bio-Rad Laboratories, Inc. Assay Reagent according to the manufacturer’s instructions (Bio-Rad Laboratories, Inc., Hercules, CA).

Fluorescence immunohistochemistry and immunocytochemistry
For paraffin embedding, cubes of third trimester placental tissue (15 x 15 x 5 mm) were fixed in a neutral, phosphate-buffered 4% formaldehyde solution for a maximum of 24 h at 4 C. The samples were dehydrated in a graded series of ethanol and embedded in paraffin (melting point 52 C, Merck, Darmstedt, Germany). Serial sections (3–4 µm) were cut on a rotation microtome (Microm, Walldorf, Germany) mounted on glass slides and deparaffinized using xylene and a decreasing series of ethanol (5 min each step). The slides were heated in microwave oven (2 x 5‘; 850W) in 10 mM citrate buffer (pH 6) for antigen retrieval pretreatment and the endogenous peroxidase activity was blocked with 3% H2O2 at RT for 15 min. For immunocytochemistry, purified trophoblasts were plated on chamber slides and fixed in methanol (-20 C) after 60 h of cultivation. The staining procedure was performed using Tyramide Signal Amplification Kit (TSA) for Fluorescence Immunohistochemistry according to the manufacturer’s instructions (NEN Life Science Products, Boston, MA). Briefly, slides were incubated 30 min in TNB blocking buffer and exposed to primary antibodies for 1 h at 37 C in a humidity chamber. The following mouse monoclonal antihuman antibodies were used: cytokeratin 7 IgG1 (clone OV-TL 12/30, DAKO Corp., Glostrup, Denmark, 0.3 µg/ml), vimentin IgG2a (clone 3B4, DAKO Corp., 0.5 µg/ml), antidesmosomal protein IgG1 (Sigma, St. Louis, MO, 18 µg/ml), CG{alpha}2 IgG1 detecting CG{alpha} subunit in CG and free {alpha}-subunit (Serotec, Oxford, UK, 5 µg/ml), ATF-1 IgG1 (clone Fl-1, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, 20 µg/ml), CBP IgG1 (clone C-1, Santa Cruz Biotechnology, Inc., 20 µg/ml). For negative control, the primary antibody was replaced by isotype IgGs (IgG1, IgG2a, Serotec, 50 µg/ml). Rabbit antihuman antibodies (final concentrations) used were: CGß (DAKO Corp., A0231, 7.6 µg/ml), AP-2{alpha} (C-18, Santa Cruz Biotechnology, Inc., 10 µg/ml), CREB-1 (240, Santa Cruz Biotechnology, Inc., 10 µg/ml), antiphosphorylated CREB detecting both phospho-CREB-1 and -ATF-1 (Upstate Biotechnology, Inc., Lake Placid, NY, 1:10), p300 (N-15, Santa Cruz Biotechnology, Inc., 20 µg/ml). Subsequently, peroxidase-linked antimouse IgG (NA 931, Amersham Pharmacia Biotech, Buckinghamshire, UK, 1:100) or peroxidase linked antirabbit Ig (NA 934, Amersham Pharmacia Biotech, 1:100), respectively, were added. For costaining of cytokeratin 7 with p300 or antiphosphorylated CREB, sections were simultaneously incubated with primary antibodies and, subsequently, with peroxidase linked antirabbit Ig and antimouse R-phycoerythrin-conjugated goat antibody (DAKO Corp., 10 µg/ml). After 1 h, slides were incubated 8 min. in the presence of fluorophore tyramide and mounted using fluoromount-G (Southern Biotechnology Associates Inc., Birmingham, AL). To reveal in vitro syncytium formation, slides that had been incubated with antidesmosomal AB were counterstained with Hoechst-dye. Finally, all slides were analyzed by fluorescence microscopy (Olympus Corp., Hamburg, Germany, BX50) and photographs were taken on Ektachrome 100HC (Eastman Kodak, Rochester, NY).

Construction of mutant luciferase plasmids
PCR-amplification and subcloning of the CG{alpha} gene promoter (-300 to +63) into pGL-3 basic (Promega Corp.) has been described recently (42). A schematic picture of the 5' flanking region of the gene carrying the placenta-specific composite enhancer is shown (Fig. 1AGo). Deletion of CREs of the CG{alpha} promoter was performed as described (23). Briefly, CG{alpha} wt promoter was digested with AatII, cutting palindromic CREs, and religated. In a second step, constructs harboring a single functional CRE (CRE-mutant) were digested with AatII, blunted with mungbean exonuclease and ligated ({Delta}CRE-mutant). CCAAT-, JRE-, {alpha}ACT- and TSE-pGL-3 constructs were mutated using Transformer Site-directed Mutagenesis Kit as specified by the supplier (CLONTECH Laboratories, Inc.). Point mutations of TSE, {alpha}ACT, which abolish DNA-protein interactions without affecting binding to neighboring sequences, were described (20, 22). Similarly, bases that were shown to be critical for binding to CCAAT and JRE sequences were altered (28, 30). Mutations introduced in JRE did not affect binding to the proximal CRE in electrophoretic mobility shift assay (EMSA) experiments (not shown). The following oligonucleotides were used (mutated bases with respect to the wild-type are depicted in bold letters, recognition sequences are marked by brackets): TSE, 5'GCT CCA AAC [AAA AAT GAT CTG AGA G]TT GAA ACA AGA TAA GAT C3', {alpha}ACT, 5'GGG TTG AAA [CAA GAC ATG ATC A]AA TTG ACG3', JRE, 5'GGT AAA AAT TGA CGT CAT G[AC GGC CAC A]CC AAG TAC CCT TC3', CCAAT, 5'CCA AGT ACC CTT GT[A TCT GGC CAT] GGA ATT TCC TGT TGA TCC3'. All mutants were confirmed by sequencing using the nonradioactive ABI PRISM Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Corp., Foster City, CA). Plasmids were purified using EndoFree Plasmid Maxi Kit (QIAGEN, Hilden, Germany).

EMSA
Protein extraction from villous trophoblasts, annealing/labeling of complementary oligonucleotide sequences, binding reactions, and EMSA were performed as described elsewhere (42). Extracts were prepared by freezing (liquid nitrogen)-thawing in buffer containing 20 mM HEPES (pH 7.9), 0.4 M NaCl, 2.5% glycerol, 1 mM EDTA, 1 mM PMSF, 0.5 mM NaF, 0.02 µg/ml leupeptin, 0.02 µg/ml aprotinin, 0.1 µg/ml trypsin inhibitor, and 0.5 mM DTT. Nuclear extracts from choriocarcinoma cell lines were performed using Dignam’s method (50) as described earlier (30). Twenty micrograms of total protein extract or 5 µg of nuclear extract were used in binding reactions. Electrophoresis of protein-DNA complexes was carried out on 5% polyacrylamide gels in the cold (10 V/cm). Gels were dried and exposed to films (Hyperfilm MP, Amersham Pharmacia Biotech). Oligonucleotides were derived from known DNA-binding sequences of the CG{alpha} promoter (20, 51). Sequences of sense oligonucleotides harboring wild-type (wt) or mutant (mut) DNA-elements were as follows (mutated bases are depicted in bold letters, recognition sequences are marked by brackets): TSE-wt, 5'AC[A AAA ATG ACC TAA GGG] TTG AAA3', TSE-mut, 5'AC[A AAA ATG ATC TGA GAG] TTG AAA3', CRE-wt, 5'GAT CAA AT[T GAC GTC A]TG GTA A3', CCAAT-wt, 5'AAG TAC CCT TCA [ATC ATT GGA T]GG AAT TTC CT3', CCAAT-mut, 5'AAG TAC CCT TGT [ATC TGG CCA T]GG AAT TTC CT3', AP-2-wt (consensus sequence), 5'GAT CGA ACT GAC CGC CCG CGG CCC GT3'. In supershift experiments, antibody was added after 30 min of binding reaction and incubated for an additional 15 min at RT. Antibodies used were: antiphosphorylated CREB (Upstate Biotechnology, Inc., 1:10), AP-2{alpha} (C-18, Santa Cruz Biotechnology, Inc., 100 µg/ml), AP-2ß (C-20, Santa Cruz Biotechnology, Inc., 100 µg/ml), AP-2{gamma} (T-17, Santa Cruz Biotechnology, Inc., 100 µg/ml).

Western blot analysis
Cells were cultured for 24, 48, and 72 h, and proteins were extracted as mentioned above. Equal amounts of protein (200 µg) were separated on 10% SDS/polyacrylamide gels and transferred to nitrocellulose (Protran, Schleicher & Schuell, Inc., Dassel, Germany) by electroblotting (Trans-Blot SD, Bio-Rad Laboratories, Inc.). For detection of CGß protein 12.5% gels were used. Equal loading of protein was monitored by Ponceau S staining of membranes. Subsequently, immunodetection was performed using standard procedures. Final dilutions of primary and secondary antibodies were as follows: antiphosphorylated CREB (Upstate Biotechnology, Inc., 1:1000), 15.2 µg/ml CGß (A0231, DAKO Corp.), 0.1 µg/ml ATF-1/CREB-1 detecting both CREB-1 and ATF-1 (25C10G, Santa Cruz Biotechnology, Inc.), 0.4 µg/ml AP-2{alpha} (C-18, Santa Cruz Biotechnology, Inc.), peroxidase-linked antimouse IgG (NA 931, Amersham Pharmacia Biotech, 1:100 000), antirabbit Ig horseradish peroxidase (NA 934, Amersham Pharmacia Biotech, 1:50,000). Signals were developed by using the Enhanced Chemiluminescence System (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. For reprobing, blots were stripped 10 min in a buffer containing 100 mM 2-mercaptoethanol, 2% SDS and 62.5 mM Tris·Cl, pH 6.7 at 50 C and subsequently incubated with a different primary antibody. Autoradiographs were densitometrically scanned and quantification of signals was done by using Pdi Analysis Software for Biological Data (Pharmacia Biotech, Uppsala, Sweden). Values obtained from 24 h cultures were arbitrarily set at 100%. Mean values (n = 3) at 48 and 72 h were calculated as relative percentage ± SD.

Statistical analysis
Statistical analyses were performed with Sigma Stat Statistical Software (Jandel Corp., Chicago, IL) using Student’s paired t test. A P value <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We recently showed that small amounts of CG{alpha} mRNA were detectable in immuno-purified trophoblasts after 12 h of cultivation while CGß mRNA was absent (42, 47). We concluded that cytotrophoblasts that have recovered from the isolation procedure are present. Both transcripts were abundantly expressed after 60 h of culturing, indicating functional differentiation. Therefore, cells were transfected with CG{alpha} promoter constructs after 12 h and at a later time point (48 h) when numerous syncytia are detectable in culture (47). Site-directed mutations in the five DNA elements of the CG{alpha} composite enhancer were performed to study their contribution to promoter activity (Fig. 1AGo). Compared with the wt promoter, deletion of one CRE did not significantly decrease luciferase expression in early cultures; however, it reduced reporter activity to 67% in 48 h transfected cells (Fig. 1BGo). Mutation of both CREs diminished luciferase activity to 9% (12 h) and 4% (48 h). Inactivation of the {alpha}ACT decreased transcription to 12% and 9%, while mutation of the JRE reduced reporter expression to 64% and 46% after 12 and 48 h of transfection, respectively. Mutation of the CCAAT box decreased promoter activity to 44% in early trophoblast cultures and to 48% in differentiated cells. After transformation with the TSE-mutant 81% and 22% of luciferase activity were detected in extracts prepared after 48 and 84 h of cultivation, respectively.

In EMSA, the CRE interacts with increasing amounts of phosphorylated CREB-1/ATF-1, suggesting a role in differentiation-dependent transcription of the CG{alpha} promoter (42). JRE, {alpha}ACT, and CCAAT contributed significantly to promoter activity in early and late cultures; however, the above data do not strongly support a role in differentiation-dependent regulation of the gene. Transfection of the mutated TSE-reporter at 48 h diminished luciferase expression to a greater extent than at 12 h, suggesting that the TSE-binding activity might be required at later stages of in vitro differentiation. Therefore, we examined DNA-protein interactions of the TSE in more detail (Fig. 2Go). TSE was previously shown to interact with AP-2 from JEG-3 cells (31). To investigate whether similar complexes can be found in primary trophoblasts, EMSA using radiolabeled TSE was performed (Fig. 2AGo). A low-mobility complex of similar size was identified in tumor cells and villous trophoblasts. Binding activities of both cell types were competed by an excess of unlabeled TSE-wt oligonucleotide, but not in the presence of cold TSE-mutant oligonucleotide. Interaction with the TSE was abolished upon addition of an unlabeled AP-2 consensus sequence. The AP-2 complex of JEG-3 cells migrated at the same position as the TSE-complex and could not be competed by an excess of the mutated TSE oligonucleotide, suggesting that the two binding activities may contain similar DNA-binding proteins. Several members of the AP-2 protein family were described, of which AP-2{gamma} was shown to be involved in placenta-specific expression of the murine adenosine deaminase gene (52). To test which AP-2 proteins might be present in the TSE-complex, supershift analysis was performed (Fig. 2BGo). We found that AP-2{alpha} was abundant in the TSE-complexes of JEG-3 and villous trophoblasts, whereas AP-2ß was absent. Weak signals were detected in the presence of AP-2{gamma} antibodies after long exposure of films (not shown). We failed to detect AP-2ß and {gamma} by fluorescence immunohistochemistry, suggesting that these proteins are either absent or expressed at very low levels in the human placenta (not shown). Next, we examined the differentiation-dependent accumulation of the trophoblast-specific element binding protein (TSEB, Fig. 2CGo). CRE-binding activity was readily detectable in 12 h cultures and further increased as previously described (42). The TSE complex, however, was absent at 12 h of culturing and steeply increased between 24 and 36 h, suggesting that appearance of the TSE-binding activity is more closely coupled to the in vitro differentiation process.



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  		Figure 2. Interaction of DNA-binding proteins with the TSE. Representative experiments are shown. Binding reactions containing 32P-labeled TSE/CRE oligonucleotide or radiolabeled AP-2 consensus sequence, supershift experiments and EMSA were performed as described in Materials and Methods. Extracts of villous trophoblasts were isolated after 48 h of cultivation (A, B). Sequences of wild-type and mutant oligonucleotides are mentioned in Materials and Methods. Supershifted complexes are marked by arrows, unspecific binding activities are indicated by open circles. A, EMSA of TSE complexes from JEG-3 and villous trophoblasts. Binding reactions using radiolabeled TSE (villous trophoblasts, JEG-3) or AP-2 (JEG-3) were incubated in the absence (-) or presence of a 100-fold molar excess of unlabelled competitor oligonucleotide. +wt, +mut and +wt AP-2 indicate the nonlabeled competitor oligonucleotides TSE-wt, TSE-mutant and AP-2-consensus, respectively. B, Identification of TSEB as AP-2{alpha}. Extracts of villous trophoblasts and JEG-3 cells were incubated with 32P-labeled TSE and various AP-2 antibodies. Competition using TSE-wt (+wt) or TSE-mutant (+mut) is shown. C, Discordant induction of CRE- and TSE-binding complexes during in vitro differentiation. Protein extracts were isolated from villous cells after 12, 24, and 36 h of cultivation. Equal amounts of proteins (20 µg) of different time points were used in the binding reactions. The same extract was incubated either with labeled CRE or TSE oligonucleotide. As a control, CRE- and TSE-complexes from JAr cells are shown in the absence (-) or presence (+) of an 100-fold molar excess of cold CRE- or TSE-wt oligonucleotides, respectively.

 
In comparison to that, the CCAAT-binding activity was abundant in early trophoblast cultures and varied little during in vitro differentiation (Fig. 3AGo). Complexes of similar sizes were detected in villous trophoblasts, JEG-3 cells, and JAr cells, which disappeared in the presence of an excess of CCAAT-wt but not mutant oligonucleotide (Fig. 3Go, A and B).



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  		Figure 3. CCAAT-binding activity in JEG-3, JAr and villous trophoblasts. Unspecific DNA-binding activities are marked by open circles. A, Differentiation-dependent expression of CCAAT complexes in villous trophoblasts. Extracts used in Fig. 2CGo (12, 24 and 36 h of differentiation) were supplemented with radiolabeled CCAAT-wt oligonucleotide and analyzed by EMSA. Nuclear extracts from JEG-3 or JAr cells were incubated in the absence (-) or presence of a 100-fold molar excess of CCAAT-wt sequence. B, Specificity of CCAAT-binding activity. Incubation of proteins isolated from villous trophoblasts (12 h) or JEG-3 cells was performed in the absence (-) or presence of a 100-fold molar excess of unlabeled CCAAT-wt (+wt) or CCAAT-mutant (+mut) oligonucleotides.

 
Subsequently, we analyzed abundancies of transcription factors that might be involved in differentiation-dependent regulation of the CG{alpha} promoter (Fig. 4AGo). Both ATF-1 (35 kDa) and CREB-1 (43 kDa) were expressed in early villous trophoblasts. In three experiments, CREB-1 protein levels increased to 320% ± 90 and 550% ± 210 at 48 and 72 h, respectively, compared with the amount at 24 h of cultivation (100%). ATF-1 rose to 260% ± 80 (48 h) and 480% ± 180 (72 h). The AP-2{alpha} antibody recognized a single band at 49 kDa in 24 h villous trophoblasts, which increased during the in vitro differentiation process (440% ± 190 at 48 h and 510% ± 130 at 72 h). Phosphorylated CREB-1 and ATF-1 appeared in the presence of an antibody reacting with modified Ser 133 of the kinase inducible domain of CREB-1. The modified proteins were weakly expressed in cytotrophoblasts. Phosphorylated CREB-1 and ATF-1, however, increased to 840% ± 350 and 310% ± 140, respectively, between 48 h (100%) and 72 h of cultivation. Endogenous CGß protein (27 kDa) was absent in 24 h extracts but could be detected after 48 h of cultivation (Fig. 4BGo). Expression of the ß-subunit further increased at 72 h, indicating functional differentiation of trophoblasts.



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  		Figure 4. Differentiation-dependent expression of trophoblast proteins. Immunopurified cells were cultured for 24, 48, and 72 h. Protein extraction and Western blot analyses were performed as described in Materials and Methods. Specific signals were visualized by enhanced chemiluminescence and densitometrically scanned. Molecular weights of the marker (Rainbow, Amersham Pharmacia Biotech) are depicted. Data in (A) are from one of three experiments shown with similar results. A, Differentiation-dependent expression of CG{alpha} promoter-interacting transcription factors. After incubation with the phospho-specific CREB-1 antibody, the blot was stripped and reprobed with the AP-2{alpha} antibody. CREB-1/ATF1 of the same trophoblast preparation were analyzed on a different gel using an antibody directed against a common epitope. B, Production of CGß subunit at different times of trophoblast differentiation.

 
To study the in vivo localization of the transcription factors, fluorescence immunohistochemistry of term placental sections was performed (Fig. 5Go). Both CG{alpha} and CGß were exclusively detected in the trophoblast epithelium (Fig. 5Go, C and D). AP-2{alpha} protein was strongly expressed in nuclei and cytoplasm of the syncytium and to a lesser extent in some of the cytotrophoblasts. Positive signals were also observed in the majority of stromal and endothelial cells of the villous core (Fig. 5EGo). ATF-1 localized to nuclei of syncytia, cytotrophoblasts, and endothelia and was also expressed in some of the mesenchymal cells (Fig. 5FGo). CREB-1 was found in the cytosol and some nuclei of syncytiotrophoblasts, but only in a minority of cytotrophoblasts and stromal cells (Fig. 5GGo). CREB-1 was undetectable in endothelial cells. Phosphorylated CREB-1 and ATF-1 were detected in nuclei of the syncytium, but only in few cytotrophoblasts, underlining the importance of the active transcription factors in the differentiated villous tissue (Fig. 5HGo). Strong expression of the modified proteins was also detectable in nuclei of stromal cells, whereas nuclei of endothelial cells were negative. Additionally, we analyzed the adapter proteins p300 and CBP, which function as coactivators of CREB/ATF-mediated transcription (53, 54). CBP was strongly expressed in nuclei of syncytiotrophoblasts and villous stromal cells and to a lesser extent in nuclei of endothelial cells (Fig. 5IGo). p300 was primarily expressed in cytotrophoblasts but could also be detected in a low number of syncytial nuclei (Fig. 5JGo). The coactivator was not produced in cells of the villous core.



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  		Figure 5. Immunohistochemical detection of transcription factors in term placenta. Placental tissues of week 38 of pregnancy were embedded in paraffin and serial sections were taken. Fluorescence immunohistochemistry was performed as described in Materials and Methods. Photomicrographs (1000-fold magnification) are from one of three experiments shown with similar results. Large arrows mark the syncytium, whereas cytotrophoblasts are depicted by small arrows. Endothelial cells of the villous core are indicated by arrowheads. Expressing as well as nonexpressing cell types are depicted. Please note that photographs in H and J were obtained after costaining of phosphorylated ATF-1/CREB-1 or p300, respectively, with cytokeratin 7 (large arrow) to mark the trophoblast epithelium. A, cytokeratin 7, B, vimentin (reacting with villous stromal and endothelial cells), C, CG{alpha}, D, CGß, E, AP-2{alpha}, F, ATF-1, G, CREB-1, H, phosphorylated ATF-1/CREB-1, I, CBP, J, p300.

 
To compare in vivo localization of the transcription factors to the in vitro expression pattern, CREB-1 and phosphorylated CREB-1/ATF-1 were analyzed by TSA-enhanced immunofluorescence in the syncytializing trophoblast cultures (Fig. 6Go). At 60 h of differentiation, CREB-1 was detected in nuclei and cytoplasm, whereas phosphorylated CREB-1/ATF-1 was preferentially detected in nuclei of cells reflecting the in vivo situation.



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  		Figure 6. Immunocytochemical analysis of transcription factors in differentiated cultures. Immunopurified villous trophoblasts, seeded in chamberslides, were fixed after 60 h of culturing and stained with specific antibodies as described in Materials and Methods. Pictures were taken at a 400-fold magnification under the fluorescence microscope. Sncytialized areas with absent desmoplakin-staining (indicated by arrows), containing numerous nuclei, were detected. A, cytokeratin 7, B, vimentin, C, desmosomal protein, D, visualization of nuclei in (C) using Hoechst-dye, E, CREB-1, F, phosphorylated CREB-1/ATF-1.

 
We recently showed that elevation of cAMP levels increased CG{alpha} promoter activity in early villous trophoblast cultures (42). Enhanced expression of luciferase was abolished by a specific inhibitor of protein kinase A (PKA), suggesting that the activity of the enzyme is required for cAMP-dependent CG{alpha} mRNA synthesis. Here, we investigated whether overexpression of the catalytic subunit of PKA could modulate phosphorylation of CREB-1/ATF-1 and promoter activity of the CG{alpha} gene (Fig. 7Go). Cotransfection of a PKA-expressing plasmid and the CG{alpha} wt promoter reporter increased luciferase expression to 430% compared with transfections with CG{alpha} wt promoter alone (Fig. 7AGo). Concomitantly, we observed an increase in phosphorylated CREB-1 and ATF-1, respectively, on Western blots (Fig. 7BGo). However, inducibility of promoter was abolished upon deletion of the tandem CRE (Fig. 7AGo). Similar results were obtained upon elevation of cAMP levels by forskolin, an activator of the adenylate cyclase (Fig. 7Go, A and B). Interestingly, we also detected an increase in the protein abundance of AP-2{alpha} upon overexpression of PKA (Fig. 8AGo). In agreement, the binding activity of TSE rose upon PKA overexpression (Fig. 8BGo).



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  		Figure 7. The effects of cAMP and protein kinase A-overexpression in villous trophoblasts. A, Transcription of the CG{alpha} promoter in the absence (-) or presence of forskolin or PKA- overproduction. Cells were transfected after 12 h of cultivation using wt (open bars) or mutant luciferase construct lacking functional CREs (filled bars). Additionally, cells were cotransfected with a PKA-expressing plasmid as described in Materials and Methods or supplemented with 10 µM forskolin. Cell lysates were prepared after an additional 36 h and luciferase activity was determined. Bars represent the mean of three different experiments/trophoblast preparations; error bars indicate SD. Values of CG{alpha} wt promoter were arbitrarily set to 100% in each experiment. (*): P < 0.05, ns, not significant. B, Western blot analyses of phosphorylated ATF-1/CREB-1 in the absence (-) or presence of forskolin or PKA-overproduction. Representative experiments are shown. Equal amounts of protein extract (200 µg) were separated on gels, transferred to filters and incubated with the phosphorylated CREB-1/ATF-1 antibody as described in Materials and Methods. Molecular weights of the marker are indicated.

 


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  		Figure 8. PKA-dependent expression and binding activity of AP-2{alpha}. Data in A and B are derived from the same protein extract. Transfection, Western blot analysis and EMSA were performed as described in Materials and Methods. Equal amounts of protein extract were loaded. A, Western blot analysis of AP-2{alpha} in the absence (-) or presence of PKA-overexpression. As a positive control, 40 µg of nuclear extract from JEG-3 cells were used. Marker bands are indicated. B, EMSA of TSE-binding complexes after PKA-transfection. EMSA of protein extract from nontransfected (control) or PKA-transfected cells was performed in the absence (-) or presence (+AB) of AP-2{alpha} antibodies. Arrows mark the supershifts containing AP-2{alpha}. Notice the increase in signal intensity after PKA overexpression. Open circle indicates an unspecific complex.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In vivo expression of CG{alpha} is restricted to a few cell types, gonadotropes, thyrotropes, and trophoblasts, in accordance with its coexpression with the different ß-subunit genes of various glycoprotein hormones (13). Transcription of the CG{alpha} gene is controlled by the DNA-elements of the composite enhancer, which contribute differently to pituitary- and trophoblast-specific expression (27, 51, 55). CRE and {alpha}ACT sequences are required for CG{alpha} production in JEG-3 and BeWo choriocarcinoma cells as well as in nontrophoblastic cell lines, whereas the function of JRE and TSE is restricted to trophoblastic tumor cells (22, 51). Similar to that, the CCAAT element exhibits limited cell specificity because the particular sequence was shown to be dispensable for CG{alpha} transcription in {alpha}T3–1 gonadotrope cells (51). Regarding CG{alpha} mRNA expression in choriocarcinoma cell lines, extensive studies were performed to characterize the role of individual DNA elements of the composite enhancer in basal and cAMP-inducible transcription. Mutations in {alpha}ACT and JRE sequences were shown to decrease transcriptional activity to 6.8% and approximately 42%, respectively, compared with the wt promoter (22, 51). Deletion of a single CRE reduced transcription of a CAT-reporter 6-fold (23), whereas removal of both CREs almost abolished promoter activity (23, 55). Inactivation of the TSE and CCAAT, respectively, decreased basal reporter expression in BeWo cells to approximately 63% and 43% of wt promoter activity (51).

Similar to the data obtained in choriocarcinoma cells, we show that inactivation of {alpha}ACT and JRE reduced reporter expression to 12% and 64% in early primary cultures confirming their role in CG{alpha} expression of cytotrophoblasts. At a later stage, mutations diminished transcription to 9% and 46%, respectively, suggesting that the contribution of the two elements to CG{alpha} promoter activity does not largely vary during trophoblast in vitro syncytialization. Different to choriocarcinoma cells, deletion of one CRE had no impact on basal CG{alpha} transcription in early trophoblasts, but reduced luciferase activity to 67% between 48 and 84 h of cultivation, suggesting that both copies of the CRE are required for optimal expression of the gene in differentiated trophoblasts. Inactivation of the tandem CRE resulted in very low promoter activities (9% and 4%) underscoring the importance of the tandem element in CG{alpha} transcription at different stages of trophoblast differentiation. Similar to tumor cells, mutation of the CCAAT box reduced transcription to 44% in early cultures. In late cultures, a reduction to 48% was observed, suggesting that the element is not involved in differentiation-dependent CG{alpha} gene expression. This would also be in accordance with the data obtained in EMSA because we detected abundant CCAAT-binding in cytotrophoblasts that remained unchanged during in vitro differentiation.

Contrary to that, a role of TSE in differentiation-dependent transcription of the CG{alpha} gene is suggested, because inactivation of the recognition sequence contributed little to reporter expression in early cultures but decreased activity to 22% between 48 and 84 h of cultivation. Concomitantly, an increase in TSE-binding activity was observed, which was only detectable at later stages of differentiation, suggesting that the TSEB fulfills a particular role in cells undergoing syncytialization. Coordinate expression of CG{alpha} and CGß genes may require TSEB because the purified protein was shown to recognize TSE and different footprint regions of the CGß5 promoter with equal affinities (20). It has also been shown that TSE is bound by AP-2, which might be identical to TSEB (20, 31). A role of AP-2 in coordinating CG expression has been suggested because overexpression of its mRNA stimulated CG{alpha} and CGß transcription in AP-2 deficient cells (31). Here, we demonstrate that the TSE predominantly interacted with one particular member of the AP-2 family, AP-2{alpha}, both in primary trophoblast cultures and choriocarcinoma cells suggesting that the factor could play a major role in TSE-mediated transcription. Also, protein expression of AP-2{alpha} increased during differentiation, which would be compatible with its requirement in CG expression of differentiated trophoblast cultures.

CRE-binding activities could be detected in cytotrophoblasts and an increase between 12 and 36 h of cultivation was noticed preceding the induction of TSE-complexes. The protein levels of the cAMP-dependent transcription factors CREB-1 and ATF-1, which we recently detected in CRE-complexes of the primary cultures (42), increased during cultivation supporting their role in differentiation-dependent CG{alpha} transcription. Phosphorylated, active forms of CRE-binding proteins were previously identified in EMSA of early trophoblast extracts using antibodies against phosphorylated Ser 133 of the kinase inducible domain of CREB-1 (42). The importance of Ser 133 of CREB-1 in CG{alpha} transcription has been described in BeWo cells: compared with the wt, a mutated GAL4-CREB-1 fusion protein was shown to reduce the activity of a promoter in which the tandem CRE has been replaced by two GAL4 binding sites (51). Here, we show by Western blot analyses that both CREB-1 and ATF-1 are phosphorylated in early cultures (24 h), suggesting that the active forms of both proteins could be involved in CG{alpha} transcription of cytotrophoblasts. Upon differentiation, the phosphorylated forms of CREB-1 and ATF-1 increased supporting the view that the modified proteins could be necessary for enhanced CG{alpha} expression in syncytializing cells. This would also fit to the observation that the CG{alpha} promoter required both CREs for full activity at later stages while a single CRE was dispensable in early cultures. On tissue sections and in the in vitro cultures phosphorylated CREB-1/ATF-1 accumulated in nuclei of the syncytium suggesting the assembly of active transcriptional complexes. Indeed, the adapter protein CBP was also found in nuclei of syncytiotrophoblasts, whereas the co-activator p300 mainly localized to nuclei of residual cytotrophoblasts. From our results, one may conclude that different cofactors are used by cyto- and syncytiotrophoblasts to mediate CG{alpha} expression and transcription of other genes, which require the intrinsic histone acetyltransferase activity of the proteins (56). The distinct roles of the two coactivators in differentiation were previously noticed in other systems (57, 58).

Signaling through cAMP as a second messenger is central to hormone expression and formation of the syncytium. In primary cultures, CG{alpha} and ß mRNA synthesis and secretion as well as transcription from the CG{alpha} promoter could be stimulated in response to cAMP (42, 43, 45). Addition of exogenous cAMP analogs was also shown to promote in vitro cell fusion of villous trophoblasts and resulted in down-regulation of the regulatory subunit of PKA type I (39, 40). Because endogenous cAMP levels spontaneously rise 6-fold during in vitro syncytium formation (59), increasing amounts of active forms of the enzyme might be generated by dissociation/down-regulation of the inhibitory subunits and translocation of the catalytic protein to the nucleus, the rate limiting step in transactivation of cAMP-dependent genes (60). Our data support the role of cAMP/PKA in CG{alpha} expression of primary cytotrophoblasts because elevation of cAMP levels and overexpression of PKA enhanced transcription of the CG{alpha} wt reporter as well as steady state levels of phosphorylated CREB-1/ATF-1. The steeper increase in CREB-1 phosphorylation compared with ATF-1 (Fig. 7BGo), which has also been noticed in nontransfected, syncytializing cultures (Fig. 4AGo), may suggest that phospho-CREB-1 could be the primary mediator of differentiation-/PKA-dependent CG{alpha} transcription. Phosphorylated ATF-1, however, may augment or attenuate the effects of CREB-1. Indeed, in cAMP-responsive cells ATF-1 can also antagonize CREB-1-dependent transcription by the formation of heterodimers (61).

Recent data supported the role of AP-2 in mRNA expression of the CGß gene (31). Three AP-2 sites of the 5' enhancer region (-318 to -188) are involved in basal transcription since mutation of the elements reduced promoter activity by 87% (21). The authors also showed that cAMP-treatment increased binding to the most proximal AP-2 recognition sequence of the CGß enhancer. Similar to the CGß promoter, we show that PKA-overexpression augmented binding to the TSE of the CG{alpha} composite enhancer and, in correlation to that, protein expression of AP-2{alpha}. We assume that PKA-dependent induction of TSEB-complexes in primary trophoblasts is mainly caused by an increase in AP-2{alpha} transcript levels. Indeed, cAMP was recently shown to stimulate AP-2{alpha} mRNA expression in choriocarcinoma cells (62).

Phosphorylation of AP-2 by cAMP/PKA-signaling can induce promoter activity without affecting protein expression and DNA-binding of the transcriptional activator (63). In tumor cells, however, it was shown that the AP-2 sites of the CGß enhancer contributed little to inducible transcription because mutation of the three recognition sequences only resulted in a 2-fold reduction of cAMP-stimulation (21). Similar to that, the TSE does not seem to play a major role in cAMP/PKA-induction of the CG{alpha} gene in primary cytotrophoblasts. Because we failed to detect inducible promoter activity of the tandem CRE-mutant, it is assumed that the CREs are the primary mediators of cAMP/PKA-responsiveness of early cultures. In agreement to that, we found that the relative increase of luciferase activity was similar after coexpression of PKA with CG{alpha} wt or TSE-mutant, respectively (not shown).

In conclusion, our data suggest that discrete DNA-elements of the composite enhancer may contribute differently to basal CG{alpha} transcription in cyto- and syncytiotrophoblasts. We suspect that in differentiating cultures CG{alpha} promoter activity is mainly governed by elevation of cAMP and functional PKA which increase CREB-1/ATF-1 phosphorylation and AP-2{alpha} expression.


   Acknowledgments
 
The authors wish to thank Dr. S. Goodbourn for providing the pMtC plasmid and G. Puller for help with graphics and statistical analyses.


   Footnotes
 
1 This work was supported by Grant No. 8122 of the "Jubiläumsfond" of the Austrian National Bank. Back

2 Funded by Grant No. 6795 of the "Jubiläumsfond"of the Austrian National Bank. Back

Received March 13, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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