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Biology of Reproduction 65, 197-203 (2001)
© 2001 Society for the Study of Reproduction, Inc.

Regular Article

Epidermal Growth Factor Regulation of Equine Glycoprotein Hormone {alpha} Subunit Expression in Trophoblast Cells1

Theingi M. Thwaya, and Michael W. Wolfe2,a

a Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160-7401

ABSTRACT

Primates and equids are the only species known to express the placental glycoprotein hormone, chorionic gonadotropin (CG), a heterodimeric glycoprotein composed of an {alpha} subunit linked to a hormone-specific ß subunit. The regulatory mechanisms involved in the induction of equine glycoprotein {alpha} subunit gene expression have not been identified. Epidermal growth factor (EGF) receptor is known to transduce signals that alter a number of different cellular functions (cell proliferation, differentiation, hormone secretion, and gene regulation). In the present study, we investigated the regulation of the equine {alpha} subunit gene by EGF in trophoblasts. We found that 2800 base pairs of 5' flanking sequence from the equine {alpha} subunit promoter is sufficient for basal expression in human choriocarcinoma cells. Epidermal growth factor and phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), increased transcriptional activity of the equine {alpha} subunit promoter (-2800/+21). These responses were blocked by pretreatment with bisindolylmaleimide-I, an inhibitor of PKC, suggesting an involvement of this pathway downstream of EGF. In addition, PD98059, an inhibitor of the extracellular signal-regulated kinase (ERK) pathway, completely blocked activation of the equine {alpha} promoter by PMA, suggesting that mitogen-activated protein kinase (MAPK) cascade was involved downstream of the PKC pathway. In conclusion, the EGF/PKC/MAPK pathway regulates equine glycoprotein {alpha} subunit gene expression through a distinct regulatory region (-2300 to -1900) in trophoblasts, while essential elements for basal expression appear to exist within the -2800 to -1900 region of the promoter.

gene regulation, placenta, signal transduction, trophoblast

INTRODUCTION

Chorionic gonadotropin (CG) is a member of the heterodimeric glycoprotein family that also includes LH, FSH, and thyroid-stimulating hormone. Chorionic gonadotropin is expressed by the placental trophoblast cells of primates and equids during gestation, and in primates it is necessary to maintain progesterone production by the ovary. Primate CG is synthesized by syncytiotrophoblast cells as early as 6–7 days of gestation and remains in serum at low levels until parturition. Equine CG is secreted by specialized trophoblast cells, chorionic girdle cells, as early as 32 days of gestation and later from endometrial cup cells. Circulating levels of eCG parallel endometrial cup development with eCG first being detected when the girdle cells invade the uterine wall. In horses, levels of eCG gradually decline between 130 and 200 days of gestation as the result of endometrial cup regression [1, 2].

Secretion of hCG is regulated by epidermal growth factor (EGF) [3–6], activin [7], GnRH [8–10], norepinephrine [11], retinoic acid [12, 13], and members of the trophoblast-derived cytokine family [14–16]. However, signaling factors and pathways that regulate secretion and subunit synthesis of eCG in the placenta are largely unknown.

One common factor that stimulates hCG secretion and potentially regulates eCG production is EGF. The EGF family includes structurally related polypeptides that regulate cell proliferation, migration, and differentiation via tyrosine kinase receptors on target cells. In equids, expression of EGF has been shown to increase at 35 days of gestation (total gestation: 320–340 days) [17]. High levels of EGF expression have been localized to the glandular epithelium of the endometrium [17] and are maintained until at least Day 250 of gestation [18]. Specific receptors for EGF were detected in tissue homogenates from the placenta of mares, and increased binding affinities were seen in fetal membranes (allantochorion, Days 30–34) and in the fully developed placenta [18]. This suggests a physiological role for EGF in vivo. However, the expression pattern of the EGF receptors and binding affinity in either the chorionic girdle cells or endometrial cups remains to be elucidated.

The proximal 5'-flanking region of the equine {alpha} subunit (e{alpha}) promoter, like that of the human, contains a putative placenta-specific enhancer (PSE) that harbors two distinct types of cis-acting elements: an upstream-regulatory element (URE) and a cAMP-like response element (CRE) [19]. The URE region can be further subdivided into a trophoblast-specific element (TSE) and an {alpha}-activating element ({alpha}-ACT). The nucleotide sequence within this proximal 5'-flanking region of the {alpha} 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 (TSEB) [20]. In addition, the equine {alpha} subunit promoter contains a single copy of a variant form of the CRE [19, 21] that has significantly reduced affinity for the CRE-binding protein (CREB) [22]. Therefore, the equine {alpha} (e{alpha}) subunit gene may serve as a useful model for delineating the mechanisms regulated by CREB-independent signal transduction pathways in trophoblasts.

In this study, we show that both EGF and phorbol 12-myristate 13-acetate (PMA), a classical protein kinase C (PKC) activator, are able to stimulate an e{alpha} (-2800/+21) promoter/reporter construct three- to fourfold. The responsiveness of the e{alpha} (-2800/+21) promoter to EGF and PMA was completely blocked in the presence of a PKC inhibitor (bisindolylmaleimide-I; Bis-I). In addition, stimulation of the equine {alpha} promoter by EGF/PMA was blocked by the mitogen-activated protein kinase kinase (MAPK) inhibitor, PD98059, and mediated through distally located elements. These findings indicate that the regulation of the equine {alpha} subunit gene by the EGF/PKC/MAPK pathway differs from that of the human {alpha} subunit in that it appears to be a MAPK-dependent (presumably the extracellular signal-regulated kinase, ERK, pathway) and CRE-independent event.

MATERIALS AND METHODS

Epidermal growth factor and PMA were purchased from Sigma Chemical Co. (St. Louis, MO). PD98059 (PD), Deep Vent DNA polymerase, and T4 polynucleotide kinase were obtained from New England Biolabs Inc. (Beverly, MA), and Bis-I was purchased from Calbiochem (La Jolla, CA). All oligodeoxynucleotides were purchased from Gemini Biotech (Alachua, FL). The pGL3 basic vector, restriction enzymes and all other enzymes were purchased from the Promega Corp. (Madison, WI), and Life Technologies (Gaithersberg, MD). All radionuclides were purchased from New England Nuclear Life Science Products Inc. (Boston, MA). All other reagents were obtained from Sigma, Fisher Scientific (Pittsburgh, PA), and Life Technologies Inc.

Cell Culture, Transient Transfections, and Reporter Assay

The JEG-3 choriocarcinoma cell line was cultured in Dulbecco minimal essential medium with 10% FBS and antibiotics at 37°C and 5% CO2. 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 Rous sarcoma virus 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 day the transfection media was replaced with normal growth media. Treatments were administered on the day following transfection if required. In experiments where inhibitors are included, cells were pretreated with media containing particular inhibitors for 1 h prior to stimulation and replaced with media containing both stimulator and inhibitors for the reminder of the experiment. Cells were harvested 2 days after initiation of transfection and assayed for luciferase activity. At the time of harvest, cells were washed briefly with ice-cold PBS and lysed with 150 µl of 1x reporter lysis buffer (Promega Corp.). Relative luciferase activity was measured (20 µl of lysate) using a Microtiter Plate Luminometer (Dynex Technologies Inc., Chantilly, VA) for 10 sec following the addition of 50 µl of luciferase reagent (Promega Corp.). The Galacto-Light ß-galactosidase reporter assay (Tropix, Bedford, MA) was used to measure ß-galactosidase activity to adjust for variation in transfection efficiency and cell number. Briefly, the reaction buffer (50 µl) was added to 20 µl lysate and incubated for 1 h at room temperature. Light emission was measured for 5 sec after addition of 75 µl the light emission accelerator using the Plate Luminometer mentioned above.

Plasmid Constructs

The pGL3 basic luciferase reporter plasmid was used as a vector to make all of the promoter constructs. A previously cloned fragment of the e{alpha} (-2800/+21) was subcloned into the luciferase vector [19]. For the -1100/+21, and -546/+21 constructs, the e{alpha} -2800/+21 fragment was digested with NspV (-1100) or SspI (-546), blunted, and subcloned back into the pGL3 basic vector. For the e{alpha} -261Luc, e{alpha} -546 to +21 was digested with BstEII, blunted, and subcloned into pGL3 basic. The e{alpha} -200,-167, and -146, constructs were generated by PCR. To create e{alpha} (-2300/+21)Luc, e{alpha} (-2800/+21) plasmid was partially digested with VspI, blunted, and subcloned into pGL3 basic. For the e{alpha} (-1900/+21)Luc, e{alpha} (-2800/+21) plasmid was completely digested with VspI, blunted, and subcloned into pGL3 basic. The e{alpha} -2800/-1100 prolactin construct was generated as follows. The e{alpha} -2800/+21 was digested with NspV, blunted, and HindIII linkers were added. The product was digested with XhoI/HindIII and cloned into the XhoI/HindIII sites of a pGL2 basic vector containing a minimal prolactin promoter (a gift from Dr. C. Clay, Colorado State University, Fort Collins, CO). For the e{alpha} -2800/{delta}400/+21 construct, the e{alpha} -2800/+21 fragment was partially digested with Vsp1 to remove the sequence between -2300 and -1900 and was subsequently religated. All constructs generated by polymerase chain reaction (PCR) were sequenced to ensure that random point mutations had not been created.

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 posthoc comparisons of means using Duncan multiple range test.

RESULTS

2800-Base Pair Equine {alpha} Subunit Promoter Is Basally Active in Trophoblast Cells

Farmerie and coworkers previously reported that 2800 base pairs (bp) of e{alpha} 5' flanking sequence was insufficient for placental expression and not responsive to activators of the cAMP-dependent protein kinase (PKA) pathway [19]. In contrast, Steger and coworkers [21] have shown that the e{alpha} (-297/+63) promoter is basally inactive, but responsive to PKA stimulation. The studies by Farmerie and coworkers were conducted in the BeWo choriocarcinoma cell line using chloramphenicol acetyl transferase as the reporter [19], whereas those by Steger and coworkers were conducted in JEG-3 cells [21]. We reexamined the activity of the e{alpha} promoter using a luciferase reporter gene in JEG-3 cells and observed that this promoter construct e{alpha} (-2800/+21)Luc was active (250-fold above that of the promoterless, P < 0.01; Fig. 1). We have also evaluated activity of this construct in the BeWo and JAr human choriocarcinoma cells. In contrast to the studies of Farmerie and coworkers, the e{alpha} (-2800/+21)Luc construct was basally active in both cell lines (data not shown) but less active as compared to JEG-3 cells.



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  		FIG. 1. Basal promoter activity of 5' deletion constructs in JEG-3 cells. Cells were transfected according to the procedures outlined in Materials and Methods. Luciferase activity was adjusted to that of the ß-galactosidase activity. Activity of the e{alpha} (-2800/+21) promoter was significantly higher (200- to 250-fold) than that of the promoterless control vector, while other promoter constructs were only 6- to 10-fold above the promoterless vector

The activities of 5' deletion mutants were also analyzed and compared to that of the promoterless control. Truncation of the promoter from -2800 to -1100 significantly diminished promoter activity. Furthermore, promoters that were 1100 bp in length or shorter had activities that were only five- to sixfold above the promoterless vector (Fig. 1). These data suggest that the region between -2800 and -1100 contains cis-acting elements that are necessary for basal trophoblast expression.

Epidermal Growth Factor and PMA Regulate Equine {alpha} Promoter Activity in Trophoblast Cells

Epidermal growth factor increases the secretion of hCG [3–6] and recently has been shown to induce human {alpha} promoter activity [23, 24]. We investigated the role of EGF in the regulation of equine {alpha} subunit expression. Cells were transfected with the e{alpha} (-2800/+21) construct, serum-starved for 12–16 h following transfection, and then treated with 100 ng/ml recombinant human EGF. A promoter containing seven copies of a consensus AP-1 sequence (TGACTCA, 7X-AP1) was included as a positive control (Fig. 2). Epidermal growth factor induced activity of both the 7X-AP1 construct (6-fold; P < 0.01) and the e{alpha} (-2800/+21) promoter (2.5-fold; P < 0.01; Fig. 2). We next determined whether the PKC pathway was activated downstream of EGF by using a pharmacological inhibitor, Bis-I, that selectively inhibits the PKC pathway by competing at the ATP-binding site. Administration of Bis-I (1 µM) completely inhibited EGF activation of the e{alpha} promoter (Fig. 2), suggesting that EGF activated the equine promoter through the PKC pathway. A much higher concentration of Bis-I (10 µM) was required to completely inhibit EGF activation of the 7X-AP1 construct (Fig. 2). In order to define the role of the PKC pathway in regulation of the e{alpha} promoter, we examined the responsiveness of e{alpha} (-2800/+21) to PMA. A dose (Fig. 3A)- and time (Fig. 3B)-dependent increase in e{alpha} (-2800/+21luc) promoter activity was observed after treatment with PMA. In fact, maximal responsiveness to PMA was observed with 10 nM PMA (P < 0.05; Fig. 3A) and following an overnight treatment (P < 0.01; Fig. 3B). In addition, induction of the e{alpha} (-2800/ +21) promoter by PMA in serum-containing media was inhibited in a dose-dependent manner by treatment with Bis-I (P < 0.01; Fig. 4A). Interestingly, treatment with Bis-I also decreased the basal expression level of the equine promoter activity. However, we did not observe inhibition of basal activity of the e{alpha} promoter by Bis-I when cells were serum starved (Fig. 2). This suggests that growth factors present in the media from the fetal bovine serum may regulate the basal expression of the promoter through a PKC pathway.



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  		FIG. 2. Regulation of e{alpha} promoter activity by EGF in JEG-3 cells. One day after transfection, cells were serum starved for 12–16 h and treated with recombinant human EGF (100 ng/ml) in serum-free media for 8 h. Epidermal growth factor stimulated the equine promoter and 7X-AP1 above basal levels. Cells were pretreated with Bis-I for 1 h prior to stimulation with EGF. Bis-I at two different concentrations completely inhibited EGF activation of both the equine promoter and 7X-AP1 construct



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  		FIG. 3. Dose- and time-dependent activation of e{alpha} (-2800/+21) promoter by PMA. A) Cells were transfected with e{alpha} (-2800/+21) and treated with different concentrations (0, 0.01, 0.1, 1, 10, and 100 nM) of PMA overnight. A maximal response was observed with 10 nM PMA. B) Cells were treated with 10 nM PMA for 2, 4, 8 h, or overnight (16–18 h). PMA stimulation of the e{alpha} (-2800/+21) promoter was maximal following an overnight treatment



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  		FIG. 4. The PKC pathway is involved in regulation of the e{alpha} promoter. A) Cells were transfected with the e{alpha} (-2800/+21) luciferase promoter and subsequently pretreated with the PKC inhibitor Bis-I (1, 5, or 10 µM) for 1 h. Cultures were subsequently stimulated with PMA (10 nM) or DMSO overnight. Bis-I inhibited both basal and PMA-stimulated promoter activity. B) The -2800 to -1100 region of the e{alpha} promoter was placed 5' of a minimal prolactin (Prl) promoter and evaluated for induction by 10 nM PMA and inhibition by the PKC inhibitor Bis-I. The -2800/-1100 fragment of the e{alpha} promoter elevated basal activity and rendered the minimal Prl promoter responsive to PMA. Induction by PMA was blocked by pretreatment with Bis-I

Identification of the EGF/PMA Responsive Region

The previously described (Fig. 1) 5' deletion mutants were used to begin to define the region of the promoter responsible for eliciting EGF/PMA responsiveness. As was the case for basal activity, truncation of the promoter from -2800 to -1100 completely abolished PMA responsiveness (data not shown). This suggested that the PMA responsive region of the e{alpha} promoter might be located within this 1700-bp segment of the promoter. This region of the e{alpha} promoter (-2800 to -1100) was subcloned upstream of a minimal prolactin promoter and reevaluated for PMA responsiveness. The -2800/-1100 fragment of the e{alpha} promoter increased basal expression of the minimal prolactin promoter by 10-fold (Fig. 4B). These data confirmed the fact that high basal expression of the -2800/+21 construct (Fig. 1) was due to this region. In addition, PMA further stimulated this heterologous promoter fragment by fourfold (P < 0.05; Fig. 4B) but had no effect on the minimal prolactin promoter. In order to determine whether induction of promoter activity observed after PMA treatment was specifically due to stimulation of the PKC pathway, cells were pretreated with Bis-I at various concentrations (0, 1, 5, and 10 µM). Bis-I at 5 and 10 µM concentrations completely blocked PMA induction of the heterologous promoter construct (Fig. 4B) as it did with the full-length e{alpha} promoter (Fig. 4A). These data suggest that activation of e{alpha} promoter by the PKC pathway occurs through elements located between -2800 and -1100.

Two additional series of 5' deletion mutants (-2300 and -1900) were generated to delineate further the PKC responsive region within the e{alpha} promoter. Truncation of the promoter to -2300 resulted in a 50–75% loss in basal promoter activity, suggesting that the region located between -2800 and -2300 is required for basal expression. In addition, the e{alpha} (-2300/+21) promoter responded to PMA stimulation (P < 0.01), while the -1900 construct was unresponsive (Fig. 5A). Thus, the region between -2800 and -1900 is required for basal activity while the region from -2300 to -1900 harbors PMA responsive elements.



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  		FIG. 5. Regulation of the e{alpha} promoter by PKC and ERK. Cells were transfected with a -2800,-2300 or -1900 e{alpha} promoter construct (or the promoterless control, pGL3) and evaluated for responsiveness to PMA (10 nM, overnight). A) The -2800 and -2300 e{alpha} promoters retained PMA responsiveness while the -1900 construct was unresponsive. B) Treatment of the e{alpha} promoters with the MEK1 inhibitor PD (50 or 100 µM) prior to PMA stimulation inhibited PMA induction of the -2800 and -2300 constructs, suggesting a PKC/ERK responsive pathway. C) An internal deletion mutant (e{alpha} -2800/{delta}400/+21) that removed the sequence between -2300 and -1900 was constructed and evaluated for PMA responsiveness. Deletion of the bases between -2300 and -1900 eliminated PMA induction of the e{alpha} promoter. while the positive control (7X-AP1) responded to PMA stimulation

MAPK Pathway Is Involved in EGF/PMA Induction of the Equine {alpha} Promoter

The PKC pathway has been demonstrated to stimulate multiple MAPK signal transduction cascades. Therefore, we investigated the involvement of ERK, using the inhibitor PD to specifically inhibit activation of ERK 1 and 2. PMA induction of both the -2800 and -2300 constructs was abolished when cells were pretreated with PD (50 and 100 µM), indicating that the ERK pathway is downstream of PKC and plays a role in PMA induction of the promoter (Fig. 5B). Furthermore, the PKC/ERK responsive element(s) resides within the e{alpha} (-2300/+21) promoter (Fig. 5B). We have also examined the involvement of the p38/MAPK pathway using the inhibitor SB 203580 (SB). Administration of SB did not block PMA induction of the e{alpha} promoter (data not shown). In order to localize further the region responsible for PMA induction, we generated a promoter construct whose internal sequence from -2300 to -1900 bp was deleted (e{alpha} -2800/{delta}400/+21). This construct failed to respond to PMA stimulation, while the positive control vector (7X-AP1; P < 0.01) did respond (Fig. 5C). Together these data suggest that the response element(s) critical for PMA induction of e{alpha} is located between -2300 and -1900 in the promoter. In addition, our data indicate that the region responsible for basal expression is located between -2800 and -2300, while the region between -2300 to -1900 may also contribute to some extent.

Raf Transactivates the Equine {alpha} Promoter in Trophoblast Cells

To this point we have shown pharmacologically that the ERK pathway (but not p38) is critical for EGF/PMA regulation of the e{alpha} promoter. In order to validate further the role of the ERK pathway and evaluate the involvement of the JNK pathway, a different approach was used. Constitutively active kinases known to activate the ERK, JNK, or p38 pathways were cotransfected into cells along with the e{alpha} promoter to determine their ability to regulate promoter activity. First, two different constitutively active forms of Raf kinase were used ({delta}Raf and RafBXB). Raf kinases (c-Raf-1, A Raf, and B-raf) are upstream activators of the MAPK pathway that specifically activate ERK1 and 2 [25, 26]. Both Raf constructs significantly increased activity of e{alpha} -2800 and 7X-AP1 in a concentration-dependent manner (Fig. 6). These Raf kinase vectors had no effect on the promoterless pGL3 basic control vector (data not shown). The {delta}Raf kinase (0.5, 0.75, and 1.0 µg, respectively) induced the -2800 equine promoter (2.4-, 3.8-, and 10-fold; P < 0.01 for 1.0 µg) and 7X-AP1 construct (4-, 6.8-, and 12.5-fold; P < 0.05 for all concentrations). Similarly, RafBXB (0.5, 0.75, and 1.0 µg, respectively) activated the -2800 equine promoter (11-, 18-, and 28-fold; P < 0.05 for 0.75 and 1.0 µg) and 7X-AP1 (37-, 33-, and 51-fold; P < 0.01 for all concentrations). Involvement of the JNK and p38 pathways was investigated by overexpressing JNK and p38 constructs along with a constitutively active upstream activator MKK6. Overexpression of either JNK or p38 (in conjunction with MKK6) had no significant effect on promoter activity, while they activated the 7X-AP1 control vector (data not shown). These data suggest that activation of the ERK pathway by Raf kinase alone is sufficient to stimulate the e{alpha} promoter and that the Raf/ERK pathway functions through the -2300 to -1900 region of the e{alpha} promoter.



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  		FIG. 6. Further delineation of the signal transduction pathways involved in regulation of the e{alpha} subunit. JEG-3 cells were transfected with increasing concentrations of two different constitutively active Raf kinases (0, 0.5, 0.75, and 1 µg) along with 1 µg of either e{alpha} (-2800/+21) or 7X-AP1 reporter constructs. Both Raf constructs transactivated the equine promoter as well as 7X-AP1

DISCUSSION

Chorionic gonadotropin is a unique hormone in that it is only present in primates and equids, secretion occurs transiently during pregnancy, and its role in primates is to maintain the function of the corpus luteum. During the past two decades, much has been learned about the regulation of human CG secretion. The regulation of basal expression of the {alpha} and ß subunits of hCG has been extensively studied. However, our knowledge of the underlying mechanisms involved in regulation of each subunit gene by various signal transduction pathways is limited, especially for equine. Among the aforementioned ligand/receptor systems, several intracellular pathways including PKA [27], PKC [10, 28], Ca2+/calmodulin-dependent kinase [29] and tyrosine kinases [15, 16, 30] have been shown to be involved in the regulation of hCG secretion in the placenta. Limited studies have been done on transcriptional regulation of CG genes by these signal transduction pathways.

Different regulatory elements have been shown to direct expression of the human {alpha} subunit gene in pituitary and placenta [31]. In contrast to equine, the first 180 bp of the human {alpha} subunit promoter are sufficient for placental expression in vitro [32–34], and elements like the TSE, {alpha}-ACT, and CRE have been shown to play a role in basal expression as well as responsiveness to PKA and EGF signal transduction pathways [23, 24, 35, 36]. In addition, more than 500 bp of the {alpha} subunit promoter are necessary to direct its expression to the pituitary [37]. A pituitary glycoprotein hormone basal element, GnRH response element, the {alpha} basal element, and glycoprotein-specific element, all located 5' to PSE, as well as the CRE are required for basal expression in pituitary gonadotropes [31, 37, 38]. Recently, Duan and coworkers [39] have shown that GnRH regulation of {alpha} subunit expression in pituitary occurs through the CRE. In short, the CRE appears to play a critical role in both basal expression and induction via specific signal transduction pathways in placenta and pituitary. Our findings suggest that the EGF stimulus is a common factor regulating the {alpha} subunit promoter of both species in placenta and, perhaps, the existence of a conserved system for stimulating CG genes across species. However, the response elements utilized by EGF to regulate the human and equine promoters in placenta cells appear to differ.

The present study has shown that the e{alpha} -2800/+21 construct contains a region critical for basal expression and appeared sensitive to inhibition of the PKC pathway (Fig. 7). It is likely that factors present within the fetal bovine serum in the media are responsible for stimulating basal expression. Nonetheless, the major DNA regulatory element/region responsible for basal expression in JEG-3 cells resides between -2800 and -2300, while the region from -2300 to -1900 contains additional elements that assist in basal expression (Fig. 5A). Together these data indicate that the region required for basal expression of the e{alpha} promoter is strikingly different from that of the human promoter. It has been shown that a -485-bp human {alpha} promoter is basally active in BeWo cells and that the PSE in the proximal flanking region of the promoter is critical for basal promoter activity [31]. Recently, Knofler and coworkers [40] have reported that basal promoter activity of hCG {alpha} subunit is highly dependent upon the CRE and TSE elements during trophoblast differentation. In short, data presented herein suggest that the regulatory region for basal expression of the e{alpha} promoter is located much further upstream of the transcriptional start site.



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  		FIG. 7. Schematic representation of the regions within the e{alpha} promoter responsible for basal activity and EGF/PMA/ERK induction. PSE, lacenta-specific enhancer; URE, upstream-regulatory element; CRE, a cAMP-like response element; TSE, trophoblast-specific element; and {alpha}-ACT, an {alpha}-activating element

Unlike the EGF/PMA responsive region in the human {alpha} subunit promoter, regulation of the e{alpha} promoter by EGF is mediated through a PKC pathway and a distal region of the promoter that are distinct from the CRE. Our data also indicate that the MAPK signal transduction pathway is activated downstream of PKC. More specifically, the ERK pathway is the essential cascade regulating the e{alpha} promoter in trophoblasts by EGF. These data were further supported by the use of constitutively active Raf constructs. Raf kinases have been shown to activate ERK but not JNK or p38. The equine promoter was stimulated by Raf kinases, unlike the human promoter where overexpression of Raf failed to induce promoter activity [24]. In contrast to the equine promoter, EGF regulation of the human {alpha} promoter also required activation of the JNK pathway [24]. Although we have shown that Raf kinase can activate the equine {alpha} subunit promoter, data presented herein do not specifically implicate the involvement of Ras/Raf in mediating EGF/PMA activation of the equine promoter. Protein kinase C has been shown to activate the MEK-ERK pathway in a Ras-independent and Raf-dependent manner [41]. In addition, PKC activation of Raf-1 has also been shown to be Ras dependent [42]. Therefore, it is possible that Ras may be downstream of EGF and PKC in the regulation of e{alpha} promoter activity.

Recently, two studies have shown that EGF regulates the human {alpha} subunit promoter through two CREs located in the proximal 5' flanking region of the promoter [23, 24]. The CRE in the human {alpha} promoter preferentially binds CREB in trophoblast cells [22]. Matsumoto and coworkers [23] reported that regulation of the human {alpha} subunit promoter (-1500 bp) by EGF occurred through the CRE via CREB phosphorylation in the Rcho-1, rat placental cell line. In contrast, Roberson and coworkers [24] have shown that EGF alone has only a minimal effect on stimulation of human {alpha} subunit promoter (-200 bp) but can synergize with forskolin to recruite AP-1 proteins to the CRE. Unlike the human promoter, the e{alpha} promoter harbors a variant form of the CRE (TGATGTA) with significantly reduced affinity for CREB [22]. We have found that activation of the EGF/PKC pathway in equine chorionic girdle cells increases levels of the {alpha} subunit mRNA, suggesting an involvement of this pathway in vivo [43]. Unfortunately, equine girdle cells are refractory to any conventional transfection method, thus hindering promoter studies in these cells. Similar to Roberson et al. [24], we have observed that the human promoter (-1500 bp) has a minimal response to EGF/PMA, similar to the -1900 or -1100 e{alpha} promoters (unpublished data). Nonetheless, PKC can induce the human {alpha} subunit mRNA two- to threefold [44]. Although it has been reported that different genetic mechanisms underlie basal placental expression of the {alpha} subunit gene in primates and horses [45], limited studies have been done comparing the signal transduction mechanisms that regulate this gene in trophoblasts. Our study presents the possibility that elements/regions similar to those identified in the equine promoter for EGF responsiveness may exist further upstream of -1500 in the human {alpha} subunit promoter.

In conclusion, regulation of e{alpha} subunit expression by the EGF/PKC/ERK signal transduction pathway differs from that of human and is mediated through a distally located element(s). A potential TSE-like element is located within the distal region of the promoter (5' of the proximal TSE) and may be required for basal expression; however, functional significance of this element needs further investigation. Four putative AP-1 like elements have been identified within the -2800 to -1900 bp region of the e{alpha} subunit promoter. The sequences of these sites differ from that of a consensus AP-1 element. Nevertheless, these elements or others may be responsible for EGF/PMA regulation of the e{alpha} subunit promoter in trophoblasts. It will be important to determine whether similar response elements exist upstream in the human {alpha} subunit promoter and that may render the promoter responsive to the EGF/PKC pathway in trophoblasts.

ACKNOWLEDGMENTS

We thank Drs. Leslie Heckert and Michael Soares, University of Kansas Medical Center, for generous supply of JEG-3 cells and various reagents. We also thank Dr. Mark Roberson of Cornell University, Dr. Ulf Rapp of Universitat Wurzburg, Germany, Dr. Dennis Templeton of Case Western Reserve University, Dr. Roger Davis of University of Massachusetts, and Dr. John Nilson of Case Western Reserve University for DNA constructs. Appreciation is extended to Grace Guo of University of Kansas Medical Center for her assistance in the statistical analysis of the data.

FOOTNOTES

First decision: 1 February 2001.

1 This research was supported in part by a grant, R29 DK50668, from the National Institutes of Health (NIH) to M.W.W. and was facilitated by the Cell Culture and Imaging Cores of the NIH-supported Center of Reproductive Sciences (HD 33994) at the University of Kansas Medical Center. T.M.T. is supported in part by the W.S. Sutton Award and Biomedical Research Program fellowship at University of Kansas Medical Center. Back

2 Correspondence: Michael W. Wolfe, Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160-7401. FAX: 913 588 7430;mwolfe2{at}kumc.edu Back

Accepted: February 27, 2001.

Received: December 19, 2000.

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