Molecular Human Reproduction, Vol. 8, No. 3, 262-270,
March 2002
© 2002 European Society of Human Reproduction and Embryology
Implantation and pregnancy |
Control of the human inhibin 
chain promoter in cytotrophoblast cells differentiating into syncytium
Department of Obstetrics and Gynaecological Endocrinology, Université Catholique de Louvain, Brussels, Belgium
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Abstract |
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Inhibins are dimeric proteins consisting of a common
subunit linked to one of the ß subunits, ßA or ßB. During pregnancy, the placenta is the main source of inhibin A production and the in-vitro transformation of cytotrophoblast cells into syncytium is associated with an inhibin subunit mRNA up-regulation. In this study, the 5' region of the human inhibin gene was isolated and sequenced. Three transcription initiation sites were identified. When transiently transfected in trophoblast cells with a luciferase reporter vector, the sequence displayed promoter activity. DNase I footprinting and electrophoretic mobility shift assay (EMSA) analysis showed a specific DNA–protein interaction in the promoter when using cytotrophoblast nuclear proteins. This interaction was weaker with syncytiotrophoblast nuclear proteins. Moreover, the deletion of this DNA–protein interaction region suppressed the promoter activity. In an attempt to identify this factor, the potential binding of known factors 
EF1, AP1 and NFE2 were excluded by competition EMSA experiments. We suggest that it may correspond to an undescribed protein interaction. The identification of the human inhibin promoter could help in understanding the mechanisms modulating inhibin gene transcription. Moreover, the identification of a factor, whose presence is related to the trophoblast cell differentiation state, could help in understanding the transformation of cytotrophoblast cells into syncytium. cytotrophoblast cells/human/inhibin/placenta/promoter
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Introduction |
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Inhibin is a heterodimeric glycoprotein of the transforming growth factor-ß superfamily, whose members regulate cell growth and differentiation (Massague, 1990
; Roberts et al., 1990). Two subtypes of inhibin have been described. They consist of a common subunit disulphide linked to either a ßA subunit (inhibin A) or a ßB subunit (inhibin B), each encoded by separate genes (Vale, 1988). Initially, inhibins were isolated from gonadal tissue and described as inhibitors of FSH production by the anterior pituitary (Ying et al., 1988). Later, investigation of extragonadal sources showed the presence of and ß chain mRNAs in various tissues including placenta, spinal cord, brain and adrenals (Petraglia et al., 1997). During pregnancy, the placenta is the main source of inhibin A (Keelan et al., 1999; Riley et al., 2000). Its concentration in maternal serum increases rapidly during the third trimester, reaching a maximal value at week 36 (Fowler et al., 1998). Moreover, abnormally high concentrations of maternal serum inhibin A are associated with Down's syndrome (Aitken et al., 1996; Spencer et al., 1996) and pregnancies complicated by pre-eclampsia (Muttukrishna et al., 1997; D'Antona et al., 2000).
During placental development cytotrophoblast cells, which are considered to be the undifferentiated stem cells of the placenta, invade maternal tissue and differentiate into hormone-secreting syncytiotrophoblast cells (Fisher and Damsky, 1993; Genbacev and Miller, 2000). The availability of various culture systems (Bloxam et al., 1997) has provided tools to study cytotrophoblast differentiation and function. Indeed, cell culture experiments have demonstrated that placental inhibin decreases the release of GnRH, HCG and progesterone by trophoblast cells (Petraglia et al., 1989). Also, when isolated cytotrophoblast cells differentiate in vitro into a hormone-secreting syncytiotrophoblast, subunit mRNA expression, which correlates with inhibin A synthesis, is stimulated during the fusion of cells into a syncytium (Debiève et al., 2000).
The human inhibin gene is composed of two exons separated by a 1.7 kb intron (Stewart et al., 1986) and its chromosomal location is 2q33–q36 (Barton et al., 1989). The gene codes for preproinhibin , which is subsequently cleaved to provide a mature chain of 20 kDa (Stewart et al., 1986). The availability of the human inhibin promoter would enable investigation of the factors controlling gene transcription and thereby lead to a better understanding of the mechanisms that control placental gene expression during differentiation of the syncytiotrophoblast cells. Previous studies have characterized the rat (Albiston et al., 1990) and mouse (Su and Hsueh, 1992) inhibin promoters. The aims of the present study are (i) to identify the human inhibin gene promoter and (ii) to characterize transcription factors that control inhibin expression during the differentiation of cytotrophoblast cells into syncytium.
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Materials and methods |
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Cytotrophoblast cell isolation and purification
Cytotrophoblast cell isolations were performed as previously described (Debiève et al., 2000
). Briefly, term placentae were collected from uncomplicated pregnancies immediately after elective Caesarean section. All tissues were collected with the approval of the ethical committee at Université Catholique de Louvain. About 60 g of trophoblast tissue was digested in 250 ml of 0.25% dispase II (Boerhinger, Mannheim, Germany) solution in Ca2+-, Mg2+-free Hank's balanced salt solution (GibcoBRL Life Technologies, Paisley, UK) for 45 min at 37°C. A total of 10 mg of deoxyribonuclease I (Boerhinger) was then added and the digestion step was allowed to proceed for another 15 min. The supernatant was decanted off and successively filtered through 100 µm and 40 µm nylon filters. The isolated cells were then subjected to further purification by density centrifugation through a discontinuous gradient of 5–70% Percoll (Amersham Pharmacia Biotech, Buckinghamshire, UK). The purified cytotrophoblasts were collected from the fraction corresponding to a density of 1.048–1.062 g/l, washed and resuspended in 10 ml of 37°C 0.05% trypsin solution (Sigma, St Louis, MO, USA) for 1 min. The cells were washed and filtered through a 40 µm nylon filter. Finally, the cells were resuspended in Iscove's modified Dulbecco medium (IMDM; Gibco) containing 2 mmol/l L-glutamine, 25 mmol/l HEPES (pH 7.4), 50 IU/ml penicillin, 50 µg/ml streptomycin, and 15% fetal calf serum (FCS; Gibco). Cell viability was assessed by Trypan Blue exclusion.
Cloning of the human inhibin promoter
Isolation of the 5' region of the inhibin chain gene was performed using a Genome Walker kit (Clontech, CA, USA). Five human genomic libraries, consisting of genomic DNA fragments produced by five different restriction enzymes and ligated with an adapter to both ends, were provided. This adapter-ligated genomic DNA was used as a template in a PCR amplification using an antisense primer (5'-TTACGTGTGGCTGGGAAAAGGATG-3') located in exon 1 (accession number X04445) and the adapter primer 1 (5'-TAATACGACTCACTATAGGGC-3'). The PCR product was diluted 50-fold and then used as a template for the second nested PCR using an upstream antisense primer (5'-GGGTCAGCAGCAAGAACAGCAGTAGG-3') and the adapter primer 2 (5'-ACTATAGGGCACGCGTGGT-3'). The obtained single band PCR product was then purified using Microcon centrifugal filter device (YM-100; Millipore, Bedford, MA, USA) and sequenced (GenBank accession number AF272341).
To subclone the inhibin promoter region, an antisense primer located upstream of the ATG start codon (5'-AGCTCACCTGGCCCTGCTAGTG-3') and three sense primers (SP1 5'-TGATGACACAGCTGGAGGACAAG-3', SP2 5'-GGGAGAAGGTGTTGTATGTTTGC-3' and SP3 5'-TTCCCAGCCCCTCCCCCACATC-3') were used in PCR amplification with human genomic DNA (a gift from J.-L.Vaerman, Haematological Molecular Biology Laboratory) as a template. Three fragments of the inhibin promoter, INH1, INH2 and INH3, were obtained and subcloned in pGlow-Topo Cloning vector (Invitrogen, Groningen, The Netherlands) and in pGL3 enhancer vector (Promega, Leiden, The Netherlands) upstream of the firefly luciferase reporter gene. Plasmids were propagated in Top10 cells (Invitrogen) and purified with the Endo-free Plasmid Purification Kit (Qiagen Westburg, Leusden, The Netherlands).
Analysis of transcription initiation sites
Total RNA was extracted from term placental tissue obtained immediately after an elective Caesarean section from an uncomplicated pregnancy, using the method of Chomczynski (Chomczynski and Sacchi, 1987). Total RNA was also extracted from freshly isolated cytotrophoblast cells, and from cytotrophoblast cells grown for 3 days in 15% FCS-supplemented IMDM after plating on Primaria Petri dishes (Becton Dickinson, Bedford, MA, USA) at a density of 0.4x106 viable cells/cm2.
The 5' end of the inhibin chain mRNA was determined by rapid amplification of cDNA ends (5'-RACE; Gibco). First strand cDNA synthesis was primed using a specific antisense primer (accession number NM 002191; 5'-CTCCGGAGGCCTCTGCAGCAGGCGCAG-3'). This first strand product was then purified with Microcon centrifugal filter device (YM-100; Millipore) and a C homopolymeric tail was added to the 3' end using terminal deoxynucleotidyl transferase (TdT). Tailed cDNA was then PCR amplified using as primers an upstream antisense primer (5'-TTACGTGTGGCTGGGAAAAGGATG-3') and a complementary homopolymer-containing anchor primer (5'-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3'). To increase the specificity of the amplification, the PCR product was diluted 50-fold and then used as the template for a second nested PCR using a nested antisense primer (5'-GGGTCAGCAGCAAGAACAGCAGTAGG-3') and an abridge universal amplification primer (5'-GGCCACGCGTCGACTAGTAC-3'). The PCR products were then analysed on a 4% agarose ethidium bromide-stained gel, cloned into pGlow-Topo cloning vector (Invitrogen) and sequenced.
Transfections
Isolated cytotrophoblast cells were cultured on 6-well plates at a density of 2x106 viable cells per well. The cells were allowed to attach for 2 h before transfection. Transfection was performed by lipofection as described (Jacquemin et al., 1993). A total of 15 µl of lipofectin (Gibco) diluted to 100 µl with opti-MEM (Gibco) was kept for 45 min at room temperature and mixed with purified plasmid DNA (6 µg of inhibin promoter constructs in pGL3 enhancer vector or pGL3 enhancer vector alone, diluted to 100 µl with opti-MEM). After 10 min incubation at room temperature, 1.3 ml of IMDM supplemented with 10% FCS was added. Attached cells were rinsed twice with phosphate-buffered saline (PBS) pH 7.4 (Gibco) and 750 µl of the transfection mixture was applied in each well. After incubation for 18 h at 37°C, the mixture was removed and replaced with 1.5 ml of 10% FCS IMDM with daily changes.
At 24, 36, 48 and 72 h after the onset of the transfection procedures, cells were rinsed twice with PBS pH 7.4 and lysed with luciferase cell culture lysis reagent (Promega). Protein concentrations were determined (DC Protein assay; BioRad, Hercules, CA, USA) with bovine serum albumin (BSA) as standard. Luciferase activity was measured using luciferase assay reagent (Promega) in a TD 20/20 luminometer. Luciferase activity for each plasmid was related to the protein content of the sample.
Nuclear protein extracts
The purified cytotrophoblast cells were plated on 60 mm Primaria Petri dishes (Becton Dickinson) at a density of 0.4x106 viable cells/cm2 in 4 ml of IMDM supplemented with 15% FCS and cultured at 37°C in a humid atmosphere of 5% CO2. The medium was changed daily and the cells were cultured for 3 days after plating. Cells were rinsed twice with PBS pH 7.4 and harvested by scraping. Nuclear proteins were extracted from freshly isolated cytotrophoblast cells and from cultured cells using NE-PER nuclear and cytoplasmic extraction reagent (Pierce, Rockford, IL, USA) according to the manufacturer's protocol.
Nuclear protein extracts were dialysed on a 7 kDa mini-dialysis unit (Pierce) in a solution containing 20 mmol/l HEPES pH 7.6, 17% glycerol (v/v), 100 mmol/l KCl, 0.1 mmol/l EDTA and 1 mmol/l dithiothreitol (DTT). Protein concentration was determined by absorbance measurement, and a protease inhibitor cocktail for mammalian cells (Sigma) was added. Aliquots were stored at –80°C.
DNase I footprinting
The Spe I–Bgl II human inhibin promoter fragment (–242 to +85) was used as a probe and was purified from the pGlow-Topo vector (Invitrogen). Labelling of the sense strand with 32P dCTP (3000 Ci/mmol; Amersham) was performed at the Spe I site by fill-in reaction with klenow enzyme (MBI Fermentas, Vilnius, Lithuania). The nuclear extract (30 µg of protein) was incubated for 15 min at 4°C in a final volume of 50 µl containing 10 mmol/l HEPES pH 7.6, 50 mmol/l KCl, 0.05 mmol/l EDTA, 8.5% glycerol (v/v), 0.5 mmol/l DTT, 1 µg of poly (dI-dC), 2% polyvinylalcohol and 40 000 cpm of radioactive probe. After 2 min of incubation at 20°C, 50 µl of a 5 mmol/l CaCl2, 10 mmol/l MgCl2 solution was added together with 20 mIU of DNase I (Boerhinger) in 1 µl of water. After 1 min of digestion, the reaction was stopped with 100 µl of 200 mmol/l NaCl, 20 mmol/l EDTA and 1% sodium dodecyl sulphate (SDS). Nucleic acids were extracted with a 1:1 mixture of phenol and chloroform, ethanol precipitated, and analysed on a 7% denaturing polyacrylamide gel. Plasmid pBR322, digested with Hpa II (Sigma) and labelled with 32P dCTP (3000 Ci/mmol; Amersham) using klenow fill-in reaction, was used as a molecular weight marker.
Electrophoretic mobility shift assay (EMSA)
The double stranded oligonucleotide (5'-CATGTGTGAGTCAGGTCGCTTGAGGCGAAATCCTT-3'), corresponding to nucleotides –219 to –185 of the human inhibin promoter, was radiolabelled with T4 polynucleotide kinase (MBI Fermentas) and 
32P ATP (3000 Ci/mmol; ICN Biomedicals, CA, USA) and purified with two successive Sephadex G50 columns (Amersham).
Binding reactions were incubated on ice for 20 min and were performed in 20 µl containing 50 mmol/l NaCl, 10 mmol/l Tris-HCl pH 7.5, 1 mmol/l MgCl2, 0.5 mmol/l EDTA, 0.5 mmol/l DTT, 9% (v/v) glycerol, 1 µg poly (dI-dC), 6 µg nuclear protein extract and 10 000 cpm labelled probe. Competition EMSA was performed in the presence of a 100x molar excess of the specific non-labelled, double-stranded oligonucleotide. The resulting complexes were run at 4°C and 200 V on a 6% native polyacrylamide gel (acryl/bisacrylamide ratio of 29/1) containing 2.5% glycerol, 50 mmol/l Tris-HCl pH 8.5, 0.4 mol/l glycine and 2.7 mmol/l EDTA.
Statistical analysis
Results are presented as mean ± SEM. Comparisons between groups were made using one-way analysis of variance (ANOVA; Statview, Abacus Concepts, Berkeley, CA, USA). In case of heterogeneous distributions (P < 0.05, ANOVA), post-hoc tests (Scheffe) were used to compare subgroups.
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Results |
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To clone the promoter of the human inhibin
chain, we first isolated a genomic region located upstream of the ATG initiator codon. We resorted to PCR amplification, as described in Materials and methods, and obtained a 692 bp fragment whose sequence is shown in Figure 1.
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To determine if this region contains a transcription initiation site, we analysed total RNA by RACE–PCR. Using placental RNA, three fragments of 94, 125 and 143 bp were obtained. Analysis of their sequence was in accordance with the genomic sequence obtained previously. We determined that the three fragments corresponded to the transcription start sites +1, +19 and +50, as indicated in Figure 1
. Interestingly, using the same approach, the same transcription initiation sites were detected in cytotrophoblast cell RNA, but only the site located at +1 was detected in syncytiotrophoblast cell RNA, i.e. in cells grown for 3 days in culture.
Comparison of the human sequence with the published rat and mouse inhibin promoters showed a 67% homology (Figure 1). The mouse and rat share only the first start site. The second one is located in a non-conserved region in these species. Analysis of the sequences upstream of the initiation sites failed to reveal the presence of TATA or CAAT boxes, or of GC-rich sequences. However, the sequences are GA-rich at –43 to –26. Other putative cis-acting elements were found (Table I). In addition, the human sequence contains an alternating purine–pyrimidine (TG) sequence repeat from nucleotide –343 to –242, which is more extended than in the rat or mouse sequences.
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To determine whether the cloned human inhibin
chain gene DNA bears promoter activity, two luciferase reporter constructs were made and tested in transfection assays. The first construct (pINH1-luc) contained a –610 to +82 fragment of the inhibin chain gene upstream of the luciferase gene, and the second construct (pINH2-luc) contained the –242 to +82 region (Figure 2A). pINH1-luc, pINH2-luc or the reporter vector devoid of inhibin fragment (pGL3 enhancer) were transfected in cytotrophoblast cells 2 h after the cells had been plated. Luciferase activities were measured 48 h after the onset of the transfection procedure. Figure 2B shows that the luciferase activities of pINH1-luc and pINH2-luc were significantly higher than that of the empty reporter vector and that pINH2-luc was more active than pINH1-luc (ANOVA, P < 0.01 by Scheffe). When the pINH2-luc luciferase activity was followed over time after transfection (Figure 2C), a significant increase over time was observed until 48 h after transfection, with a subsequent small decrease 72 h after the onset of the transfection procedure (ANOVA, P < 0.01 by Scheffe). Moreover, when comparing the luciferase activities at 24 and 48 h after transfection, pINH2-luc showed a significantly stronger increase (3.2-fold) than the empty reporter vector (2.8-fold) (ANOVA, P < 0.01 by Scheffe). We conclude that pINH2-luc bears promoter activity and that this activity is specifically increased over time.
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To identify the cis-acting elements that control promoter activity, DNase I footprinting experiments were performed with a probe spanning nucleotides –242 to +82 and with nuclear extracts from isolated cytotrophoblast cells and from 72 h-cultured cells transformed into syncytium (Figure 3
). A protected region corresponding to nucleotides –221 to –185 of the human inhibin promoter was observed with nuclear extracts from isolated cytotrophoblast cells (Figure 3,J0). A similar but possibly weaker protection was also seen with nuclear proteins from syncytial cells (Figure 3,J3).
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To identify the factors that are involved in the footprint, a double-strand oligonucleotide corresponding to this protected region was tested in EMSA with the same nuclear extracts previously used in the DNase I footprint experiments. Figure 4A
shows that a specific protein–DNA complex was obtained with extracts from isolated cytotrophoblast cells. This complex was barely detectable with extracts from cells transformed into syncytium.
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The sequence of the binding site matches the consensus of the transcription factors AP1,
EF1 and NFE2. To determine if these factors are involved in the protein–DNA complex, a 100x molar excess of non-labelled oligonucleotide known to bind these factors was used in a competition binding reaction. The sequence of the oligonucleotides used in gel shift experiments are listed in Table II (Angel et al., 1987; Murre et al., 1991; Remacle et al., 1999). No inhibition of complex formation was seen with oligonucleotides that bind AP1 and NFE2 (Figure 4B). EF1 is a zinc finger protein known to bind the ACCT sequence. To investigate if EF1 binds to the ACCT sequence in the inhibin promoter probe, a competing oligonucleotide derived from the E-cadherin promoter and known to bind EF1 was tested (Figure 4C). This competitor inhibited complex formation. However, no inhibition of complex formation was seen in the presence of another EF1 binding competitor, which was derived from the 4-integrin promoter. To test the involvement of the ACCT binding site in the competitor derived from the E-cadherin promoter, three mutated oligonucleotides affecting the binding capacity of EF1-related factors were used. The ability of complex formation inhibition of these mutated oligonucleotides was not altered, excluding the involvement of the ACCT binding region. The zinc chelator agent 1,10-o-phenanthroline was also used to test the involvement of EF1 in the complex formation. This agent can remove zinc ions from zinc finger proteins, abolishing their binding ability. Nevertheless, incubation of nuclear protein extracts with increasing concentrations of this agent does not impair the formation of a protein–DNA complex. We conclude that the AP1, EF1 and NFE2 transcription factors are not involved in the protein–DNA complex observed with the inhibin promoter.
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To determine whether the DNA-binding region identified by DNase I footprint and EMSA has a functional importance in the promoter activity, a luciferase reporter construct without this DNA-binding region was made and tested in transfection assays. This construct (pINH3-luc) contains a –186 to +82 fragment of the inhibin
chain gene and is identical to pINH2-luc except that it lacks 56 bp encompassing the footprinted region. Figure 2B shows that the luciferase activity of pINH3-luc was significantly lower than that of pINH2 luc (ANOVA, P < 0.01 by Scheffe) 48 h after the onset of the transfection procedure. When the pINH3-luc luciferase activity was followed over time after transfection (Figure 2C), a small increase over time until 48 h with a subsequent decrease 72 h after the onset of the transfection procedure was still observed (ANOVA, P < 0.01 by Scheffe). Nevertheless, this increase over time is not different from the increase observed for the empty reporter vector. We conclude that this 56 bp deleted region of the inhibin promoter is essential for its activity. |
Discussion |
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In this paper, we describe the cloning and functional characterization of the sequence upstream from the human inhibin
chain gene. This sequence displays promoter activity in trophoblast cells. The method used to isolate and purify cytotrophoblast cells from term placenta derives from the well-established method of Kliman (Kliman et al., 1986). As previously demonstrated, the isolated cytotrophoblast cells include only ~5% of cells from mesenchymal origin (Debiève et al., 2000). The in-vitro transformation of isolated cytotrophoblast cells into an endocrine active syncytiotrophoblast in 48–72 h has been previously demonstrated by ourselves (Debiève et al., 2000) and by others (Jacquemin et al., 1996; Frendo et al., 2000). During this transformation, we have previously shown that inhibin A synthesis is dependent on subunit mRNA expression, which may be an indicator of cell fusion (Debiève et al., 2000). Indeed, cytotrophoblast cells do not secrete dimeric inhibin A and have a low inhibin mRNA expression level, whereas inhibin mRNA expression level and inhibin A secretion increase significantly as cells transform into syncytium (Keelan et al., 1994; Debiève et al., 2000). In this study, there appears to be a time course increase in inhibin promoter activation during the cytotrophoblast/syncytiotrophoblast differentiation, consistent with an mRNA production increase. This differentiation-related promoter activation is suggested by comparing the time-dependent increase in luciferase activity of the promoter construct with the increase in luciferase activity of the empty reporter vector. However, this relatively small but significant and reproducible luciferase activity increase could also be related to the expectation that luciferase gene expression would increase more over time with a stronger promoter activity, regardless of the differentiation status of the cells.
As in the rat (Albiston et al., 1990) and mouse (Su and Hsueh, 1992) promoters, no TATA box was found in the core promoter. However, a GA-rich sequence, postulated to play a role in defining the start of transcription, is present at the usual position of a TATA element, like in the genes for human Müllerian inhibiting substance (Morikawa et al., 2000) and T3 (Tunnacliffe et al., 1986). The human inhibin gene showed multiple initiation sites, as seen in most genes that lack a TATA box (de Pagter-Holthuizen et al., 1987; Ishii et al., 1987). The main transcription initiation site, which is shared with rat and mouse, is predominantly observed in syncytiotrophoblast differentiated cells. Two other start sites are also present in cytotrophoblast stem cells, which are not able to secrete dimeric inhibin A.
A striking feature of this promoter sequence is the presence of a repeat TG element. They are found in all eukaryotic genomes and occur about every 30000bp in mammals, in 5' regulatory or intronic regions (Hamada and Kakunaga, 1982). They are involved in the DNA transformation from B form (right-handed helical) to non-B form, possibly accounting for altered DNA replication and transcriptional activities (Lancillotti et al., 1987). The physiological intranuclear calcium level could modulate this transformation (Dobi and Agostan, 1998). As intracellular calcium has been shown to positively regulate inhibin production in trophoblast cells (Keelan et al., 1994), this TG repeat could be involved.
The identified promoter region contains several potential binding sites for cis-acting elements. Cyclic AMP analogues have a dose-dependent effect on inhibin gene expression (Li et al., 1994). Moreover, this second messenger plays a significant role in cellular proliferation and differentiation of placental cells (Strauss et al., 1996; Keryer et al., 1998). Two potential cAMP responsive sites were identified: one CRE and one AP-2 element. The presence of an AP1 site suggests an effect of protein kinase C stimulation on inhibin mRNA expression, as shown with GnRH on placental cells (Li et al., 1994; Naor et al., 1995). The presence of AP1, AP2 and CRE sites could possibly account for the increase in the production of mRNA for the inhibin subunit through FSH stimulation (Davis et al., 1988). In addition to that in trophoblast cells, inhibin is also present in many other cell types (Petraglia, 1997). The Wilm's tumour protein (WT1) is described as a repressor of the rat inhibin promoter (Hsu et al., 1995), thereby playing a role in arresting the differentiation of immature follicles. Moreover, WT1 mRNA expression in trophoblast cells declines when cytotrophoblast are transforming into syncytium (Feingold et al., 1998). The delta crystalline enhancer binding protein (EF1) is also a repressor of gene activation (Sekido et al., 1994; Remacle et al., 1999), and could potentially bind to the human inhibin promoter (sites located at nucleotides –546 and –366).
Both DNase I footprinting and gel shift experiments demonstrated the presence of a DNA–protein interaction in a specific region of the identified inhibin promoter. Moreover, this protein–DNA interaction is stronger with cytotrophoblast cell nuclear extracts than with syncytiotrophoblast cell nuclear extracts. Consequently, this protein or protein complex appears to be related to the differentiation of the trophoblast cells from cytotrophoblast to syncytiotrophoblast. The deletion of this DNA binding region leads to the disappearance of promoter activity, indicating that this DNA region is essential for inhibin expression in trophoblast cells. Moreover, this deletion decreases the inhibin promoter activation related to the differentiation of the trophoblast cells from cytotrophoblast to syncytiotrophoblast. Thus, a nuclear factor binding this DNA region should be a potential activator, but its binding decreases with syncytium formation when there is up-regulation of inhibin RNA (Debieve et al., 2000). This discrepancy suggests that another factor could be involved in the activation of the inhibin promoter in syncytial cells. Nevertheless, the factor identified in this study is essential for the activation of the inhibin promoter in both cytotrophoblast and syncytiotrophoblast cells.
Several potential cis-acting elements could bind to the identified sequence: AP1, NFE2 and EF1. Nevertheless, they were all excluded. Moreover, the persistence of a binding in the presence of the zinc chelator o-phenanthroline excludes the possibility of a zinc finger protein. Surprisingly, a sequence of the E-cadherin promoter had the ability to displace the binding of this unknown protein. E-cadherin is also a protein linked to trophoblast cell differentiation, since it disappears with cytotrophoblast cell fusion (Coutifaris et al., 1991). Therefore, it is not surprising that this nuclear factor, which decreases with syncytium formation, could also be involved in the regulation of the E-cadherin promoter. Inhibin and E-cadherin proteins are both related to the differentiation process of cytotrophoblast into syncytium. Further studies will be needed to purify and identify this unknown protein binding to the inhibin promoter and linked to trophoblast cell differentiation.
The identification of the human inhibin promoter could help in understanding the mechanisms modulating inhibin gene transcription and thereby the role of inhibin in endocrine and non-endocrine tissues. Moreover, the identification of a factor whose presence is related to the trophoblast cell differentiation state could help in understanding the physiology of the transformation of cytotrophoblast cells into syncytium and the pathological states implicating placental development.
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Acknowledgements |
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Frédéric Debiève is Research Assistant from the `Fonds National de la Recherche Scientifique' (FNRS, Belgium) and is supported by grant 3.4501.97 from the Fonds de la Recherche Scientifique Médicale (FRSM, Belgium). The authors thank Profs P.Bernard and C.Hubinont (Obstetrical Department) for providing placental tissue, M.Heusterpreut (Haematological Molecular Biology Laboratory) for help with DNA sequencing, and K.Verschueren (University of Leuven) for providing E-cadherin and
4-integrin oligonucleotides. DNA–protein interaction experiments were performed with the kind help of Prof. F.Lemaigre (hormone and metabolism unit). The authors are also grateful to C.Brulet for her excellent technical assistance. |
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1 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecological Endocrinology, Faculty of Medicine, OBST 5330, Avenue E.Mounier 53, B-1200 Brussels, Belgium. E-mail: debieve{at}obst.ucl.ac.be
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Submitted on May 8, 2001; resubmitted on August 7, 2001; accepted on November 27, 2001.
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