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Regular Article |
Subunit Expression in Trophoblast Cells1
a Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160-7401
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit linked to a hormone-specific ß subunit. Previously, we have reported that epidermal growth factor (EGF) regulates the equine glycoprotein hormone subunit promoter through a protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) signal transduction pathway in trophoblasts. The current study investigates the regulatory element/factors involved in the induction of equine glycoprotein subunit gene expression by EGF. Using 5' deletion mutagenesis, we have delineated the primary EGF/PKC responsive region of the equine subunit gene to be located between -2039 to -2032 base pairs upstream of the transcriptional start site. The sequence within this region contains an activator protein 1 (AP-1)-like response element (TGAATCA) and is similar to a consensus AP-1 (TGAC/GTCA) response element. This element appeared to preferentially interact with a c-fos/JunD heterodimer. Stimulation by EGF induced the binding of c-fos and JunD to this element and subsequently elevated promoter activity. In conclusion, an EGF/PKC/MAPK signal transduction pathway regulates equine glycoprotein subunit gene expression through a distinct regulatory element(s) that lies between -2039 to -2032 of the equine glycoprotein subunit promoter in trophoblasts and involves an AP-1 complex.
gene regulation, placenta, signal transduction, trophoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit noncovalently linked to a specific ß subunit [2]. CG is expressed by the trophoblast cells of primates and equids during gestation and is necessary to maintain progesterone production from the ovary [3]. Human CG (hCG) secretion is regulated by epidermal growth factor (EGF) [4, 5], activin [6], interleukin 6 [7], interleukin 1 [8], gonadotropin-releasing hormone [9, 10], norepinephrine [11], macrophage colony-stimulating factor [12], retinoic acid [13], keratinocyte growth factor [14], and cAMP stimulators [15]. Signaling factors and pathways that regulate secretion and subunit synthesis of equine CG (eCG) in the placenta are largely unknown.
The subunit, encoded by a single copy gene, is expressed in pituitary of all mammals and in placenta of equids and primates [1, 16]. The ß subunit of primate CG is encoded by six genes [16], whereas the equine CG is encoded by a single copy gene [17]. The proximal 5'-flanking region (200 base pairs [bp]) of the equine and primate/human subunit promoter contains a placenta-specific enhancer (PSE) that can be subdivided into two distinct regions: an upstream-regulatory element (URE) and a cAMP responsive region containing two cAMP response elements (CRE) [18–23] (Fig. 1). The URE region can be further subdivided into a trophoblast-specific element (TSE) and an -activating element (-ACT). In humans, the first 180 bp of the subunit promoter is sufficient for trophoblast expression. The nucleotide sequence within the proximal 5'-flanking region of the subunit promoter is highly conserved between humans and equids. However, the equine TSE contains a single base pair change that disrupts binding of the TSE-binding protein [24]. In addition, the equine subunit promoter harbors a single copy of a variant form (TGAtGTCA) of the CRE [21, 25]. The variant CRE has significantly reduced affinity for CRE-binding protein (CREB) and preferentially binds CRE modulator (CREM), activator transcription factor 2 (ATF-2) and c-jun transcription factors [26]. In contrast with humans, we have shown that the major regulatory element responsible for basal expression of the equine promoter resides between -2800 and -2300 [27]. Additional element(s) located between -2300 and -1900 appear to assist in basal expression in the JEG-3 human choriocarcinoma cell line [27].
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Recently, two different laboratories have shown that EGF regulation of the human subunit promoter is mediated through its CRE elements [28, 29]. Matsumoto and coworkers reported that EGF regulation of the human subunit promoter occurred via phosphorylation of CREB and binding to the CRE element in the Rcho-1 rat placental cell line [28]. In contrast, Roberson and coworkers have shown that EGF alone has only a minimal effect on stimulation of human subunit promoter (-845/+49) but can synergize with forskolin. Treatment with EGF plus forskolin resulted in the recruitment of a heterodimer of c-fos and c-jun (members of the activator protein 1 [AP-1] family) to the CRE [29]. Under basal conditions, this element has been shown to interact with CREB, CREM, activator transcription factor (ATF-1 and ATF-2), and c-jun [26]. Furthermore, regulation of human subunit by EGF required activation of both the extracellular-signal regulated kinase (ERK) and Jun N terminal kinase (JNK) mitogen activated protein kinase pathways (MAPK). Although EGF activates the p38 MAPK signal transduction pathway, it was not involved in EGF induction of the human promoter in the JEG-3 cells [29].
It is evident that EGF also plays an important role during equine implantation (Days 30–34). Prior to Day 38 of gestation, low levels of EGF are present in the endometrium [30]. Expression of EGF was markedly upregulated in the epithelium of endometrial glands from Day 38 onward, which coincides with the period of placentation [30]. In addition, EGF binding was observed in equine allantochorion from Day 30 to Day 34 (implantation) as well as in the fully developed placenta at Days 150–250 [30]. Previously, we have shown that both EGF and phorbol 12-myristate 13-acetate (PMA), a classical PKC activator, induce activity of the e(-2800/+21) promoter in JEG-3 cells [27]. Regulation of equine promoter by EGF/PMA was mediated by the ERK signal transduction pathway. The region of the promoter between -2300 and -1900 bp was determined to be responsible for this induction. In addition, we have reported that both activators of cAMP dependent protein kinase A and protein kinase C induce the steady state mRNA level of equine subunit in equine trophoblast cell lines [31]. Unfortunately, we have been unable to use these cell lines to study regulation of the equine subunit promoter due to the fact that these cell lines are refractory to common transfection methods. In the current study, we have used the JEG-3 human choriocarcinoma cell line to identify the regulatory element and factors that mediate EGF induction of the equine subunit promoter.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture, Transient Transfections, and Reporter Assays
Immortalized equine girdle cell line (eCG500.1) was cultured in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 5% horse serum, and antibiotics at 37°C and 5% CO2 in 100-mm tissue-culture plates. The JEG-3 choriocarcinoma cell line was cultured and transfected as previously described [27]. Briefly, cells were plated a day before transfection at a density of 1.1 x 105 cells per well in 6-well plates. The next day, the cells were transfected with 1.0 µg of reporter plasmid, 200 ng of RSV ß-galactosidase (as an internal control for transfection efficiency), and 5 µl of LipofectAmine (Life Technologies Inc.) according to the manufacturer's recommendations. The following morning, the transfection media was replaced with normal growth media (DMEM with 10% FBS and antibiotics). The cells were serum starved the following evening. The EGF (100 ng/ml) or PMA (10 nM) treatment (serum-free media) was administered the next morning and cultured for 8 h or overnight, respectively, in serum-free media. The cells were harvested and assayed as previously described [27]. The luciferase value for each sample was divided by its corresponding ß-galactosidase level to give an adjusted luciferase.
RNA isolation and analysis
ECG 500.1 cells were cultured in 100-mm plates until they reached 80%–85% confluency as stated above and were treated with various concentrations of human recombinant EGF (50, 100, and 200 ng/ml) for 20 h. Total RNA was isolated using Trizol Reagent (Life Technologies). Total RNA (10–15 µg) was separated by electrophoresis through a 1.0% formaldehyde-agarose gel and Northern analysis was performed according to the protocol described previously [31]. An equine cDNA was used to synthesize random primer-generated 32P-labeled cDNA probes. Following autoradiography, the blots were stripped and rehybridized with a radiolabeled cDNA probe for equine ribosomal protein L7 as an internal control. Specific signals were quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA), and these values were adjusted by taking the ratio of specific signals (such as e) to that of the control L7.
Plasmid Constructs
The pGL3 basic reporter plasmid was used as a vector to make all promoter constructs. The promoter constructs for e-2800/+21, -2300/+21, and -1900/+21 were previously reported [27]. The e-2110, -2045, and -2020 constructs were generated by PCR. The following strategy was used to generate the e(-2045/+21) µA construct. A sense oligodeoxynucleotide encompassing bases -2045 to -2015 with a mutation in the A element (-2039/-2032) and an antisense oligodeoxynucleotide encompassing bases +21 to +5 were used for PCR. The upstream oligodeoxynucleotide mutated the A element from TGAATCA to gtcgaCA, generating a SalI site. The PCR product was kinased, digested with NdeI, and subsequently subcloned into a SmaI/NdeI-digested wild type promoter construct. A different strategy was used to generate the e (-2045/+21) µB construct. An antisense oligodeoxynucleotide encompassing bases -2022 to -2053 and an upstream oligodeoxynucleotide (RV3, located upstream of multiple cloning site of the pGL3 vector) were used in a first PCR reaction while a sense oligodeoxynucleotide (-2045 to -2015) and an antisense oligodeoxynucleotide (+21 to +5) were used in a second PCR reaction. The PCR fragments mutated the B element (-2012/-2005) from GCGTCAGT to agcTCgag, generating an XhoI site. These two PCR products were used as template in a third PCR reaction using the RV3 and an antisense oligodeoxynucleotide (+21 to +5) primers to generate the full PCR product. This PCR product was digested with MluI/NdeI and subsequently subcloned into the MluI/NdeI sites of the wild-type promoter. All constructs generated by the PCR method were sequenced to confirm that the appropriate mutation had been made and to ensure that a random point mutation had not been made in the native sequence. The Raf BXB and 
Raf expression constructs were provided by Dr. Ulf Rapp (Universitat Wurzburg, Germany) and Dr. Dennis Templeton (Case Western Reserve University), respectively, and have been reported previously [27]. The 7X-AP1 construct has been described previously [27].
Nuclear Extract Preparation and Electrophoretic Mobility Shift Assays
Cells were cultured until 70–85% confluent in 150-mm plates using conditions described above. Cells were subsequently serum starved for 16–20 h, followed by stimulation with 100 ng/ml EGF (0, 1, 2, and 4 h). Nuclear extracts were prepared according to Daggett and coworkers [32] and stored at -80° C. Oligodeoxynucleotides were end-labeled with 32P-ATP using T4 polynucleotide kinase, purified through a 6% polyacrylamide gel, and used as electrophoretic mobility shift assay (EMSA) probes. The nuclear extracts (10–20 µg) were incubated for 15 min at 4°C in the binding reaction buffer (10 mM HEPES pH 7.9, 20 mM KCl, 5 mM MgCl2, 10 µM ZnCl2, 1 mM EDTA, and 10% glycerol) containing 0.15 µg of poly(dI-dC) and 0.1 µg of salmon sperm DNA in total reaction volume of 20 µl. Competitor and radiolabeled probe (25 fmol) were added and incubated for an additional 15 min at room temperature. The DNA-protein complexes were resolved on a 4% nondenaturing polyacrylamide gel (prerun for 15 min at 240 V) run at 4°C using 0.5x Tris-borate-EDTA buffer. The gel was transferred to blotting paper (Schleicher & Schuell Inc., Keene, NH), dried, and exposed to x-ray film. Experiments utilizing antibodies were conducted as follows: 1–2 µl of antibody was added to the binding reaction and incubated for 30 min at 4°C prior to the addition of the probe. The sequences of probes (e-2045, e-2045µA, and e-2020) used in EMSA are shown in Figure 3A. Consensus AP-1 (cAP-1) oligo was purchased from Promega Corp. The hCRE (palindromic) and TGAT (variant CRE) were described previously [26]. The sequence of the equine CRE used as a competitor was 5'-TCAATTGATGTCATATAATT-3'.
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Western Analysis
JEG-3 cells were cultured until 60–70% confluent in 100-mm plates. Cells were serum starved overnight and subsequently treated with 100 ng/ml human recombinant EGF for 0, 1, 2, and 4 h in serum-free media. Nuclear proteins were isolated according to the method described by Daggett and coworkers [32]. Proteins (100 µg) were resolved on a 10% SDS-PAGE gel and transferred to an Immobilin-P membrane (Millipore, Bedford, MA). Equivalent protein loading was determined by staining the blot with Ponceau-S. Subsequently, the membrane was immunoblotted with anti c-fos (sc-052) and JunD (sc-74) and signals were detected using enhanced chemiluminescence.
Statistical Analysis
All transient transfection experiments were performed in triplicate and repeated a minimum of three times. Statistics were performed where noted, using one-way ANOVA followed by Duncan multiple range test. A P value 
0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previously, we reported that regulation of the equine promoter by EGF is mediated through a PKC pathway and that the critical EGF/PMA responsive region was located between -2300 and -1900 bp of the promoter. We have subsequently examined the role of EGF in regulating the expression of the equine subunit gene in immortalized equine chorionic girdle cells (eCG 500.1). Treatment with EGF (100 ng/ml) for 20 h induced equine -mRNA expression 2.9 ± 0.5-fold (data not shown). This further strengthens the role of EGF in regulation of equine CG genes.
To identify the EGF/PMA responsive element(s), we generated a series of deletion constructs (e-2110/+21, -2045/+21, and -2020/+21) and evaluated them in transient transfection assays (Fig. 2A). The e-2300/+21 and 7X-AP1 luciferase promoter constructs were included as positive controls while the pGL3 basic and e(-1900/+21) vectors were included as negative controls. The transfected cells were serum starved and treated with either EGF (100 ng/ml) for 8 h or 10 nM PMA overnight. Both the e(-2110/+21) and (-2045/+21) promoter constructs responded to EGF stimulation (P < 0.05), while the e(-2020/+21) construct was unresponsive and similar to the e(-1900/+21) promoter construct described previously (Fig. 2A). Similar results were obtained with PMA (data not shown). These data suggest that the PMA/EGF responsive element(s) must reside within a 25-bp region (-2045 to -2020) of the promoter. The DNA sequence in this distal region of the promoter is distinct from the proximal variant CRE element as well as a palindromic CRE element in the human promoter. These data suggest that the regulatory mechanism used by EGF to regulate the equine promoter is quite distinct from that of human promoter.
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Raf Kinases Stimulate the Equine Promoter
We have previously shown that EGF/PMA regulation of the equine promoter was mediated through a Raf/ERK signal transduction pathway in JEG-3 cells [27]. The response to EGF involved the activation of the ERK but not the JNK and p38 MAPK signaling pathways. To determine whether the region between -2045 and -2020 was also regulated by the Raf/ ERK pathway, we cotransfected cells with the e-promoter constructs along with two different constitutively active forms of Raf kinase, Raf and RafBXB. Raf kinases (c-Raf-1, A Raf, and B-raf) are upstream activators of the MAPK pathway that specifically activate ERK 1 and 2 [33, 34]. Both Raf kinases transactivated the longer e constructs but failed to activate the e-2020/+21 and -1900/+21 constructs (Fig. 2B). The Raf and RafBXB kinases induced the e-2300 promoter (6.65 ± 0.95- and 13.4 ± 0.61-fold, respectively; P < 0.005), e-2110 (4.8 ± 0.2- and 12.9 ± 0.7-fold, respectively; P < 0.005), and -2045 (5.2 ± 0.8- and 7.9 ± 0.78-fold, respectively; P < 0.005). The Raf and RafBXB kinases induced the control 7X-AP1 promoter 31 ± 4.0- and 45 ± 2.3-fold, respectively. Together with the deletion mutagenesis studies, these data suggest that EGF/ERK regulation of the equine promoter occurs primarily through this 25-bp region located between -2045 and -2020 bp (Fig. 3A). It is interesting to note that the e-2020/+21 retained two AP-1-like elements (B and C in Fig. 3A) and yet it was unresponsive to EGF/PMA stimulation or Raf overexpression.
A Heterodimeric Protein Complex Is Involved in Regulation of the Equine Promoter
To analyze and identify the transcription factor(s) activated by EGF and PMA, we used the 25-bp region spanning -2045 to -2020 of equine promoter as a probe in an EMSA. This 25-bp region harbors an AP-1-like element (Fig. 3A). Time-dependent induction of protein binding to this DNA sequence was observed following stimulation by EGF (Fig. 3B). Maximum protein binding to the e-2045/-2020 probe was evident using extracts from cells treated with EGF for 4 h (Fig. 3B). Interestingly, significant protein binding to the cAP-1 probe was observed at 1 h (Fig. 3B). In order to determine the binding specificity of this protein complex, we performed a series of competition assays. Protein binding to the e-2045/-2020 was entirely competed off by a 50-fold molar excess of unlabeled wild-type oligodeoxynucleotide, indicating specific binding of this complex to the radiolabeled oligodeoxynucleotide (Fig. 3C). We also competed with an oligodeoxynucleotide probe in which site A (Fig. 3A) was mutated. This mutant oligodeoxynucleotide failed to compete for protein binding, suggesting that this protein complex interacts specifically with the A site within the -2045/-2020 sequence. This element had homology to a consensus AP-1 (cAP-1) site, with only a single base pair change (Fig. 3A). Addition of a cAP-1 oligodeoxynucletide effectively competed for protein binding (Fig. 3C), suggesting that the complex may consist of AP-1-like proteins.
To further delineate the protein(s) binding to this site and DNA sequence preferences, we performed competition assays using various response elements from the subunit gene that have been shown to interact with AP-1 proteins (Fig. 4). In terms of the AP-1 family of proteins, the hCRE interacts with c-jun and c-fos, while hTGAT and the eCRE preferentially bind to c-jun [26, 29]. We also included the cAP-1 oligo, the e 2045 wild-type probe, and a downstream segment of the e promoter (e2020) that contained two AP-1-like elements (B and C in Fig. 3A). A 25- and 250-fold molar excess of competitor were used to compete the complex. The unlabeled wild-type oligodeoxynucleotide (e2045) completely competed at 25-fold molar excess. It is interesting to note that cAP-1, hCRE, and e2020 competed moderately well while hTGAT and eCRE competed very weakly. The relative binding affinity of this complex to these sequences appears as e2045 > cAP-1, hCRE and e2020 > hTGAT and eCRE. This suggests that this complex binds more efficiently to the A site of e 2045 sequence than it does to a cAP-1, CRE site or the B and C sites downstream of site A.
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The nature of the protein complex was investigated by supershift EMSAs using a series of antibodies that bind AP-1 proteins (Fig. 5A). Other antibodies (USF-1, USF-2, SP-1, and Ets-1) were included as negative controls. Among these antibodies, c-fos effectively shifted the complex. Approximately 60% of complex was shifted with the c-fos antibody (Fig. 5A), while c-jun and JunB failed to supershift the complex. In addition, the Jun* antibody, which recognizes all members of the Jun family of transcription factors (cjun, JunB, and JunD, recognizes DNA binding domain of these transcriptions) disrupted protein binding as effectively as did the c-fos antibody. Because this antibody inhibits the binding of the jun proteins to DNA and the c-jun and JunB antibodies had no effect, it suggested that JunD was in the complex. Additional EMSAs were performed using antibodies to c-fos and the Jun family members, including JunD (Fig. 5B). C-fos alone supershifted, while Jun* and JunD depleted the complex, suggesting that this complex is predominantly composed of a c-fos/JunD heterodimer that interacts with element A.
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EGF Regulation of c-fos and JunD
We have shown that a heterodimer of c-fos and JunD preferentially interacts with element A in the equine promoter following stimulation with EGF. Previously, it had been shown that c-fos and c-jun are induced shortly after treatment with EGF in JEG-3 cells [29]. We examined the expression of c-fos and JunD in response to EGF stimulation in JEG-3 cells using Western blot analysis. Consistent with previous reports, c-fos was induced following a 1-h exposure to EGF and was maintained until 4 h (Fig. 6). In contrast, JunD was induced at 4 h (Fig. 6) and was maintained until 8 h (data not shown). These data suggest that EGF differentially induces c-fos and JunD in JEG-3 cells.
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Functional Analysis of AP-1 Elements in the Promoter
To determine the functional significance of element A (referred to as eAP-1 element) in EGF/PMA/ERK stimulation of the equine promoter, we generated a promoter construct that contained a mutation in this element (same mutation as that used in the EMSA studies). Because the B/C elements had some affinity for the c-fos/JunD complex (Fig. 4), we reasoned that the presence of these sites may play a role in EGF/PMA/ERK stimulation of the promoter in conjunction with the eAP-1 element. Therefore, we created a mutation in the B/C elements in the context of the -2045 to +21 promoter. These constructs were evaluated for responsiveness to EGF (Fig. 7A) and Raf kinase (Fig. 7B). EGF induced activity of the e-2300 (2.1 ± 0.2-fold), e-2045 (2.1 ± 0.26-fold), and e-2045µB/C (2.2 ± 0.14-fold) promoters (P < 0.01), while the e-2020 and e-1900 constructs were unresponsive to EGF. Mutation of the eAP-1 site (e-2045µA) blocked EGF stimulation of the promoter, suggesting that the eAP-1 element has functional significance. The Raf and RafBXB kinases transactivated the e-2045 (4.5 ± 0.9- and 5.6 ± 1.0-fold, respectively; P < 0.001) and e-2045µB/C promoters (3.2 ± 0.2- and 4.4 ± 0.7-fold, respectively; P < 0.005). Mutation of the eAP-1 site severely attenuated Raf activation of the e promoter (2.1 ± 0.3- and 2.6 ± 0.6-fold, respectively), confirming the functional significance of this element (Fig. 7B). This also supports the loss of responsiveness of the e-2020/+21 promoter to EGF/PKC/ERK stimulation. Mutation of the DNA sequence representing the B/C sites slightly reduced the ability of Raf kinase to activate the promoter. This is not entirely surprising because B/C elements have some affinity for the c-fos/JunD heterodimer (Fig. 7B). However, this reduction was not evident following stimulation with EGF (Fig. 7A).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Many studies have shown the importance of EGF signaling during implantation and placentation in rodent models. Synchronized expression of the EGF receptors is necessary for successful implantation of embryos in mice [40]. Targeted disruption of the EGF-R gene in mice resulted in trophoblast defects and death at midgestation [41]. In addition, EGF has been shown to play an important role in trophoblast proliferation, invasion, and differentiation [4, 42–44]. Furthermore, EGF also promotes amino acid transport in the rat placenta, influencing fetal growth [45], and regulates the transplacental supply of glucose [46].
In humans, EGF has been shown to block the cytokine-induced apoptosis of human cytotrophoblasts and syncytiotrophoblasts from normal term placentas in vitro [47, 48]. EGF also lowered the apoptotic effect induced by hypoxia in cultured trophoblasts [49]. Furthermore, EGF increases secretion of human CG in differentiating cytotrophoblasts [4, 5]. Cao and coworkers have shown that EGF increased the stability of the hCG and ß mRNAs in JEG-3 cells [50].
The mechanisms involved in regulation of the CG subunit genes by various signal transduction pathways is not clear, especially for equids. The EGF stimulus is a common factor in regulating the subunit gene of both humans and horses, and perhaps represents the existence of a conserved signaling system for stimulating the CG genes. Our studies have shown that the regulation of the equine subunit by EGF is mediated through a PKC/Raf/ERK signal transduction cascade in trophoblasts [27]. Activation of this signal transduction cascade induced the binding of a heterodimeric AP-1 complex to a cis-acting eAP-1 element located in an upstream region of the equine promoter (-2039 to -2032). This increased binding activity correlates with an increase in promoter activity. Furthermore, Raf kinase transactivates the equine subunit promoter through this response element. One member of this heterodimeric complex is c-fos, and it appears to heterodimerize with Jun-D. Heterodimerized cfos/JunD preferentially interacted with this eAP-1 element as compared with a cAP-1 element. It is interesting to note that the hCRE has a similar binding affinity to this complex, as does the cAP-1 response element. Interestingly, the B/C element could compete for protein binding to the eAP-1 site, and its relative affinity was similar to that of cAP-1 and hCRE. However, in the absence of the -2045 to -2020 segment of the promoter, the B/C elements failed to respond to stimulation by EGF, PKC, or Raf overexpression. Although sequences of the eAP-1 and B/C are similar, the B/C elements do not appear to have a central role in the context of the -2045 promoter.
Our data support previous findings where the human subunit promoter (1500 bp) failed to respond to EGF alone in JEG-3 cells [29]. Truncation of the equine subunit promoter (either 2020 or 1900) resulted in a failure to respond to EGF/ERK activation. The current data suggest that EGF regulation of the equine subunit is distinct from that of the human subunit gene and opens the possibility that similar elements to those identified in the equine promoter may exist upstream of -1500 in the human subunit promoter. Nonetheless, it is evident that members of the AP-1 family of transcription factors play important roles in EGF regulation of the glycoprotein subunit gene of both equids and humans.
It is very interesting to note that some AP-1 transcription factors such as Fra-1 and JunB have been shown to be essential for placental development in mice. Fetal mice lacking Fra-1 were severely growth retarded and died between E10.0 and E10.5 as the result of a reduced labyrinth layer [51]. Similarly, mice lacking JunB die between E8.5 and E10.0 due to lack of a vascularized labyrinth layer [52]. Although other AP-1 factors such as c-fos have been disrupted, such mice do not show severe placental phenotypes as compared with Fra-1 and JunB. Interestingly, it has been shown that Fra-1 can maintain the functional equivalence of c-fos during vertebrate evolution [53]. However, the Fra-1 knockout data suggest that c-fos cannot functionally replace Fra-1 during placental vascularization. Unlike the aforementioned AP-1 proteins, much less is known about JunD. The JunD gene has been disrupted and is reported to lead to a defect in spermatogenesis, but these mice were viable [54]. Transcriptional activation by JunD has been shown to be blocked by the tumor suppressor protein menin [55–57]. Its role in placenta is unknown. Furthermore, heterodimerized JunD/Fra2 has been shown to bind AP-1 and CRE elements to trigger gene expression in PC12 cells [58], and induction of JunD/Fra2 was observed in differentiating granulosa cells [57]. The current study is the first to document a role for JunD in trophoblast function.
The expression profile for some of these immediate early genes during the process of human trophoblast differentiation has been reported. Dakour and coworkers [59] have shown that expression of c-fos and JunB increases in response to EGF stimulation in spontaneously differentiating cytotrophoblast cells. In addition, levels of CREB and ATF-1 have been shown to increase during syncytium formation [60]. However, the role of JunD in the process of trophoblast differentiation remains to be investigated. Nonetheless, numerous AP-1 transcription factors play very important roles during placental development. Their role in equine placental development and function require further investigation.
In summary, the regulation of equine subunit expression by the EGF/PKC/ERK signal transduction pathway is mediated by a heterodimeric protein complex, which was identified to contain c-fos and JunD. In contrast to studies of the human subunit promoter and its proximal CREs, this complex interacts with a distally located element in the equine promoter. This element mediates EGF regulation of the promoter. Furthermore, it appears that similar response elements exist within the human subunit promoter and the possibility exists that these elements may mediate EGF and PKC regulation in human trophoblasts.
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ACKNOWLEDGMENTS |
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Raf construct, and Dr. John Nilson (Case Western Reserve University) for the e-2800/+21 CAT construct. |
FOOTNOTES |
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1 This research was supported in part by grant R29 DK50668 from the National Institutes of Health to M.W.W. and was supported by NICHD/NIH through cooperative agreement U54 HD 33994 as part of the Specialized Cooperative Center Program in Reproductive Research. T.M.T. is supported in part by a fellowship from Biomedical Research Program at University of Kansas Medical Center. 
2 Correspondence: Michael Wolfe, Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7401. FAX: 913 588 7430; mwolfe2{at}kumc.edu
Accepted: April 4, 2002.
Received: November 2, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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