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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 3 1239-1244
Copyright © 2000 by The Endocrine Society

Original Studies

Corticotropin-Releasing Hormone Gene Expression in Primary Placental Cells Is Modulated by Cyclic Adenosine 3',5'-Monophosphate1

You-Hong Cheng2, Richard C. Nicholson, Bruce King, Eng-Cheng Chan, John T. Fitter and Roger Smith

Mothers and Babies Research Center, Endocrine Unit, John Hunter Hospital, Newcastle, New South Wales 2310, Australia

Address all correspondence and requests for reprints to: Dr. Roger Smith, Mothers and Babies Research Center, Endocrine Unit, John Hunter Hospital, Newcastle, New South Wales 2310, Australia. E-mail: mdrsm{at}mail.newcastle.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CRH, the principal neuropeptide regulator of pituitary ACTH secretion, is also expressed in placenta. Placental CRH has been linked to the process of human parturition. However, the mechanisms regulating transcription of the CRH gene in placenta remain unclear. cAMP signaling pathways play important roles in regulating the expression of a diverse range of endocrine genes in the placenta. Therefore, we have explored the effect of cAMP on CRH promoter activity in primary cultures of human placental cells. Both forskolin and 8-bromo-cAMP, activators of protein kinase A, can increase CRH promoter activity 5-fold in transiently transfected human primary placental cells, in a manner that parallels the increase in endogenous CRH peptide. Maximal stimulation of CRH promoter activity occurs at 500 µmol/L 8-bromo-cAMP and 10 µmol/L forskolin. Electrophoretic mobility shift assay and mutation analysis combined with transient transfection demonstrate that in placental cells cAMP stimulates CRH gene expression through a cAMP regulatory element in the proximal CRH promoter region and involves a placental nuclear protein interacting specifically with the cAMP regulatory element.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CRH IS A 41-amino acid neuropeptide synthesized in the paraventricular nucleus of the hypothalamus that coordinates many aspects of the response to stress (1, 2). CRH is also expressed in various extrahypothalamic sites of the central nervous system (3, 4, 5) and in peripheral tissues including the placenta (6, 7, 8). In the placenta, CRH biosynthesis in and secretion from trophoblastic cells increase progressively toward the end of human pregnancy (9, 10). Abnormally elevated CRH concentrations in maternal plasma have been associated with preterm delivery (9, 10) and abnormal pregnancy (11). Recent studies suggest that placental CRH is linked to the process of human parturition (9). An understanding of how CRH gene activity is regulated is a prerequisite to gaining an insight into the function of CRH in human physiology. Thus, the mechanisms controlling CRH gene expression in the placenta are of considerable interest.

A variety of endogenous biochemical agents stimulate CRH release from the hypothalamus (12, 13, 14) and placenta (15), including interleukin-1, angiotensin II, oxytocin, arginine vasopressin, norepinephrine, epinephrine, and acetylcholine. These ligands act on various cells through cAMP-dependent protein kinase signal pathways and turn on targeted genes by trans-activation through a consensus DNA sequence, defined as the cAMP regulatory element (CRE), in the promoter region (16). CRE-binding protein (CREB) is a member of the bZIP or leucine zipper family of transcription factors (17, 18, 19), is phosphorylated by several protein kinases, and modulates gene transcription in response to ligand stimulation of the cAMP pathways (20, 21, 22). A CRE has been identified in the human CRH (hCRH) promoter region (23), and regulation of CRH gene expression by cAMP has been demonstrated in several tumor cell lines, including AtT-20 cells (23, 24, 25), PC 12 cells (4), NPLC cells (26, 27), and choriocarcinoma cell lines (28, 29), which were either permanently or transiently transfected with the CRH gene or promoter. Spengler and colleagues (23) reported that cotransfection of CREB-A or -B expression plasmids enhanced forskolin-mediated stimulation of CRH(-666)CAT, whereas cotransfection of the respective antisense constructs significantly inhibited forskolin induction, implying that CREB is directly involved in cAMP stimulation of CRH gene expression in AtT-20 cells. In contrast, very little is known about regulation of the CRH gene in primary placental cells.

The aim of the current study was to investigate the role of cAMP in the regulation of expression of the hCRH gene in primary cultures of human placental cells. Our results indicate that cAMP stimulation of hCRH gene expression is mediated through the CRE, and a nuclear protein in human primary placental cells binds specifically to this DNA sequence.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmid constructs

All constructs used luciferase as the reporter for assaying transcriptional activation of the promoters under study. The orientation and sequence of all constructs were confirmed by DNA sequencing. The human CRH genomic clone, CRH1001+, was a gift from Joseph Majzoub, Harvard University Medical School (Boston, MA) (24). The CRH 5'-flanking DNA was subcloned into the promoterless Photinus (firefly) luciferase reporter vector pGL3-Basic (Promega Corp., Madison, WI). To make pCRH(5500)-GL3, the 5.5-kb upstream region was isolated by XbaI and HaeII double digestion and filled to blunt ends with Klenow enzyme, then ligated into the SmaI site of pGL3-Basic vector with T4 DNA ligase. To create pCRH(4300)-GL3, the 4300-bp fragment was isolated from the pCRH(5500)-GL3 construct with SacI and NheI double digestion, then ligated into the SacI and NheI sites of pGL3-Basic vector in a forward orientation. To construct pCRH(663)-GL3, a 790-bp promoter region (including 663 bp of the hCRH promoter region and 127 bp of the first exon region) was isolated by a PstI digest, then subcloned into pGL3-Basic vector in the same manner.

Additional deletions of the hCRH promoter were made by stepwise removal of 5'-flanking DNA with exonuclease III and S1 nuclease. Briefly, hCRH(663 bp)pGL3-Basic vector was double digested with KpnI and NheI, which generates a 5'-overhang at the NheI site in the vector 5' to the hCRH promoter sequences that is suitable for exonuclease III digestion and a 3'-overhang at the KpnI site, located 15 bp 5' of the NheI site, that is resistant to exonuclease III digestion. After treatment with exonuclease III, S1 nuclease was added to remove the single strand DNA overhangs, and the different length promoter-vector DNAs were recircularized with ligase.

To create a plasmid with the ß-globin promoter driving the luciferase reporter the rabbit ß-globin promoter sequence (-109 to +10 bp) was removed from the GLOB-CAT plasmid (30) by BamHI and BglII double digestion, and ligated into the BglII site of the pGL3 basic vector to make the GLOB-pGL3 vector. To construct the CRE-globin promoter plasmid, the BglII site of a luciferase construct containing the hCRH promoter region from -340 to -215 bp (which had been constructed by linking a hCRH PCR fragment into the BglII and MluI sites of the pGL3-promoter vector) was converted to a XhoI site using a linker oligonucleotide. MluI and XhoI double digestion removed the hCRH promoter sequences from this plasmid, and the fragment was ligated into MluI- and XhoI-digested pGLOB-GL3 to create pCRE-GLOB-GL3.

Mutagenesis

Oligonucleotide-directed mutagenesis of the CRE was carried out in the pCRH(663)-GL3 construct using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Mutated base pairs were confirmed by DNA sequencing. The oligonucleotides used were as follows, with the mutated nucleotides underlined: mtCREf, 5'-ccttccattttagggctcgctgcagtcaccaagaggcg-3'; and mtCREr, 5'-cgcctcttggtgactgcagc gagccctaaaatggaagg-3'.

Placental cell isolation and culture

Human term placentas were obtained from normal pregnant women after spontaneous vaginal delivery or elective cesarean section. Collection of placentas was performed with the approval of the Hunter Area Health Service (Newcastle, Australia) and the University of Newcastle human ethics committees. Cytotrophoblasts were obtained according to Kliman’s method (31). Briefly, chorionic villi tissue obtained from the maternal side of the placenta was dispersed with trypsin and deoxyribonuclease I, a highly purified fraction of cytotrophoblasts was obtained by repeated Percoll gradient centrifugations, and cells were maintained in DMEM (Life Technologies, Inc., Gaithersburg, MD). The purity of the cytotrophoblasts was determined by immunohistochemical staining with markers specific to syncytiotrophoblast (CRH), epithelium (cytokeratin), and endothelium and fibroblasts (vimentin). All experiments were carried out in preparations of cytotrophoblasts with purity above 95%.

Transfection

Standard transfection methods were as follows. Freshly isolated cytotrophoblasts were plated in six-well plates (Falcon, Becton Dickinson, Bedford, MA) at 2.5 x 106 cells/well. Cells were incubated with plasmid-liposome complexes comprised of 20 µg DNA, 0.5 µg control DNA (pRL-TK vector, Promega Corp.), and 20 µg freshly prepared liposomes (1 mg/mL each of dioleyphosphatidylethanolamine and dimethyldioctadecylammonium bromide) (32, 33) in a humidified atmosphere of 5% CO2 at 37 C. Twenty-four hours later, cells were fed with 10% charcoal-stripped FBS with forskolin or 8-bromo-cAMP (in ethanol) or with the same volume of vehicle (ethanol). Luciferase assay was carried out 24 h later with the dual luciferase assay kit (Promega Corp.). Relative luciferase activity is presented as firefly luciferase values normalized to Renilla luciferase activity.

CRH RIA

CRH immunoreactivity in the culture medium was extracted using activated Vycor (Corning, NY) glass. Frozen culture medium samples (1 mL) were thawed at room temperature and adsorbed onto Vycor silica glass powder (200 mg glass powder/1 mL medium sample). The sample was then washed with 3 mL deionized water and 2 mL 1 mol/L HCl (BDH Chemicals, Poole, UK) before the adsorbed material was eluted with 2 mL 60% acetone (BDH Chemicals). The eluate was transferred to polycarbonate tubes, dried, and stored at -20 C for RIA. CRH RIA was performed as previously described (9). Human CRH-(1–41) (Sigma, St. Louis, MO) was used as the standard, and radioligand was prepared with the chloramine-T method and purified by high performance liquid chromatography. The anti-CRH antibody Y2B0 was a gift from Phil Lowry (University of Reading, Reading, UK). The concentration of CRH IR was expressed as picograms per 2.5 x 106 cells/24 h.

Purification of primary human placental cell nuclear extract

Crude primary human placental cell nuclear extracts were prepared following the method of Dignam and colleagues (34). The nuclear extract was aliquoted and stored at -80 C. The protein concentration in nuclear extracts was determined using the Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA).

Electrophoretic mobility shift assay (EMSA)

EMSAs were performed using 32P-labeled, double stranded, oligonucleotide probes generated by annealing two complementary oligonucleotides containing a triple repeat (3 x CRE, 5'-cgcgtgacgtcatgacgtcatgacgtca) of the CRE found in the hCRH promoter region from -228 to -221 bp. The probe (10,000 cpm) was incubated for 30 min at 4 C in 15 µL 10% glycerol-10 mmol/L Tris-HCl (pH 7.8), 100 mmol/L KCl, and 1 mmol/L dithiothreitol containing 10 µg nuclear extract and preincubated for 30 min with 2 µg poly(dI-dC) and 0.5 µg BSA. Competition was performed with 100 pmol unlabeled, double stranded 3 x CRE oligonucleotides, a single CRE (5'-ccttccattttagggctcgttgacgtcaccaagaggcg), or a mutated CRE (5'-ccttccattttagggctcgctgcagtcaccaagaggcg), which were added simultaneously with the labeled probe. The samples were loaded on a 4% polyacrylamide gel in 0.25% Tris-glycine buffer (pH 8.3). The gel was run at 150 V for 3 h at 4 C.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
cAMP stimulates hCRH promoter activity in human placental cell cultures

To investigate whether hCRH promoter activity is modulated by cAMP-dependent signal pathways in the placenta, we transfected plasmids containing 5500 or 663 bp of 5'-flanking DNA sequences of the hCRH gene linked to a luciferase reporter gene into primary cultures of human placental cells in the presence and absence of regulators known to activate the cAMP second messenger system. Exposure of the placental cells to 8-bromo-cAMP or forskolin resulted in an approximately 5-fold increase in hCRH promoter activity (Fig. 1AGo). To assess the endogenous response of the CRH gene in these cells to these stimuli, we measured CRH concentrations in the culture medium of the transfected primary placental cells. Treatment with either 8-bromo-cAMP or forskolin increased the CRH peptide level 5-fold (Fig. 1BGo). Thus, the response of endogenous CRH expression to these agents paralleled the promoter activity increase as measured by luciferase activity in transfected cells.



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  		Figure 1. Effects of forskolin and 8-bromo-cAMP on CRH expression in placental cells. A, Twenty-four hours posttransfection with the indicated hCRH-luciferase plasmids, primary placental cells were treated with forskolin, 8-bromo-cAMP, or ethanol (control) for 24 h, then harvested for luciferase assay. Results represent the mean (±SEM) from four independent experiments. B, The concentration of endogenously synthesized hCRH peptide in the culture medium of cells primary placental cells transfected with pCRH(663)-GL3 was measured by RIA.

 
Dose-response curves indicated that cAMP stimulates hCRH gene expression in a dose-dependent manner. Maximal stimulation of promoter activity (5-fold over basal level) occurred at 500 µmol/L 8-bromo-cAMP or 10 µmol/L forskolin (Fig. 2Go, A and B, respectively).



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  		Figure 2. Dose-response curves for forskolin or 8-bromo-cAMP on CRH promoter activity. Twenty-four hours posttransfection with pCRH(663)-GL3 plasmid DNA, placental cells were exposed to ethanol (control), various forskolin concentrations as indicated (A), or various 8-bromo-cAMP concentrations as indicated (B) for another 24 h before harvesting for luciferase assay. The results represent the mean (±SEM) from four independent experiments.

 
Deletion analysis of the hCRH CRE

To determine whether cAMP stimulation of hCRH promoter activity in placental cells requires a functional CRE, we transfected mutant constructs as well as wild-type constructs into primary human placental cells to measure promoter activities in the presence and absence of cAMP. The study of progressive deletions of 5'-flanking hCRH promoter DNA sequences (see Fig. 3Go) indicates that cAMP responsiveness was lost when the deletion included the region from -342 to -212 bp, thereby removing the CRE. Furthermore, mutation of the CRE not only decreased basal hCRH promoter activity by 30%, but also completely abolished cAMP responsiveness (Fig. 4Go), indicating the requirement for CRE for expression of the CRH gene in placental cells.



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  		Figure 3. Localization of the cAMP regulatory region of the CRH promoter in placental cells. Placental cells were transfected with plasmids containing different lengths of the hCRH gene promoter as shown. Twenty-four hours posttransfection the cells were exposed to 0.5 mmol/L 8-bromo-cAMP or ethanol (control) for 24 h. Results represent the mean (±SEM) from three independent experiments.

 


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  		Figure 4. Effect of mutating the CRE in the CRH promoter. hCRH plasmids containing a wild-type or mutant CRE DNA sequence were transfected into placental cells, and 24 h later the cells were exposed to 0.5 mmol/L 8-bromo-cAMP or ethanol (control) for 24 h before harvesting for luciferase assay. Results represent the mean (±SEM) from three independent experiments.

 
hCRH CRE confers cAMP responsiveness on a heterologous promoter

To test whether hCRH CRE can transfer responsiveness to a heterologous promoter, we constructed plasmids with the hCRH CRE located 5' of the rabbit ß-globin promoter that was linked to the luciferase reporter vector. The effects of cAMP on such chimeric constructs were examined in transiently transfected human primary placental cells (Fig. 5Go). cAMP had no effect on the native rabbit ß-globin promoter driving the luciferase reporter in human primary placental cells, but induced by 6-fold expression of the construct containing a CRE in front of the ß-globin promoter. This supports our observed requirement for CRE in cAMP-mediated gene regulation and shows that CRE alone is sufficient for cAMP-mediated induction of the CRH gene in the placental cell environment.



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  		Figure 5. The hCRH CRE confers cAMP responsiveness to the rabbit ß-globin gene promoter in placental cells. Plasmids containing a luciferase reporter gene under control of a basal rabbit ß-globin promoter with (pCRE-GLOB-GL3) or without (pGLOB-GL3) a linked CRE sequence were transfected into placental cells and then exposed to 0.5 mmol/L 8-bromo-cAMP or ethanol (control). Results represent the mean (±SEM) from three independent experiments.

 
hCRH CRE interacts with a nuclear protein from placental cells

To determine whether nuclear proteins interact with CRE of the hCRH gene, EMSA was performed using crude nuclear extracts of primary human placental cells (Fig. 6Go). A major complex was formed with double stranded 32P-labeled 3 x CRE oligonucleotides as the probe, interacting with placental nuclear protein extracts (Fig. 6Go, lane 2). This DNA-protein complex was prevented from forming by competition with an excess (100-fold) of unlabeled, double stranded 3 x CRE or 1 x CRE oligonucleotides (lanes 3 and 4), but not with the mutated CRE oligonucleotides (lane 5).



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  		Figure 6. EMSA showing placental cell nuclear protein interaction with CRE DNA. A specific complex (arrow) is formed with [32P]CRE probe and nuclear protein (lane 2), but not in the absence of protein (lane 1). This complex is competed away by 3 x CRE (lane 3) or 1 x CRE (lane 4), but not by mutated CRE (lane 5) nonradiolabeled oligonucleotides.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CRH plays a major role in coordinating autonomic, immunological, and behavioral responses of mammalian organisms to stress and is implicated in human reproductive physiology. Our laboratory (9) and others (8) have previously reported that maternal plasma CRH levels rise during pregnancy, in a manner suggesting linkage to the process of human parturition. However, the regulation of hCRH in human placenta and the mechanisms used by hCRH to affect maternal-fetal physiology and parturition remain unclear. The complexity of signaling pathways involved in controlling CRH gene expression make in vivo studies particularly difficult. An alternative approach is to study the mechanisms involved in the regulation of CRH gene expression in cultured cells. Scatena and Adler (28), using as models choriocarcinoma cell lines BeWo and JEG-3 transfected with human and mouse CRH gene constructs, explored the mechanisms controlling species-specific placental CRH gene expression. Their data demonstrated that both the mouse and human CRH promoters are active in human choriocarcinoma cell lines and that their activity is stimulated by cAMP. In contrast, neither promoter is active or cAMP inducible in the rat choriocarcinoma cell line, Rcho-1. They concluded that species-specific trans-acting factors, and not cis-regulatory elements, dictate the species-specific expression of hCRH in the placenta. However, the choriocarcinoma cell lines do not synthesize and secrete CRH. Interpretations of results from transcriptional analyses with cells exhibiting a partially differentiated phenotype to primary placental cells must be made with both caution and an understanding of the cell population used to generate the data. Thus, the mechanisms controlling the endogenous CRH gene expression in primary trophoblasts remain unknown.

The placenta is a unique endocrine organ that elaborates protein, neuropeptides, and steroidal hormones as well as cytokines and growth factors. Placental trophoblasts are the main source of these factors. Cytotrophoblasts in vitro aggregate and fuse to form syncytiotrophoblasts (31), which provide a useful model for examination of the endocrine pathways governing gene expression, processing of ribonucleic acid and translational products, and hormone secretion (8, 35). Primary placental cells may more accurately reflect the behavior of the trophoblast population under investigation, in contrast to choriocarcinoma cell lines, as they synthesize and secrete CRH (8, 9). In the present study, using primary cultures of human placental cells, we have investigated regulation of the CRH gene by the cAMP signaling pathway. Forskolin, which increases intracellular cAMP by up-regulation of adenylate cyclase activity, and the stable analog 8-bromo-cAMP both stimulated hCRH promoter activity in a dose-dependent manner in transfected primary cultures of human placental cells. Incubation for 24 h with 10 µmol/L forskolin or 500 µmol/L 8-bromo-cAMP increased hCRH promoter activity 5-fold. These increases paralleled those in endogenous CRH peptide. The doses of both forskolin and 8-bromo-cAMP at which maximal stimulation of CRH promoter activity occurred in primary placental cells were less than those observed in choriocarcinoma cell lines (28), implying that primary human placental cells are more sensitive to cAMP stimulation. These data confirm the ability of the cAMP pathway to regulate CRH expression in cultured human placental cells.

Furthermore, we report that cAMP stimulation of hCRH gene promoter activity in transfected primary cultures of human placental cells is mediated by a specific nuclear protein interacting with the CRE contained within the hCRH promoter region. cAMP pathways play an important role in the physiology of placental trophoblasts (36). cAMP-regulated pathways have been identified and implicated in control of the expression of a variety of endocrine genes in the placenta (37), including the CRH gene (28). Transcriptional regulation of eukaryotic genes is dictated by the presence and activity of specific nuclear factors that can bind to DNA regulatory sequences and interact with the transcriptional machinery. Some transcription factors are reported to alter their DNA binding and transcriptional activities after phosphorylation by specific protein kinases (17, 18, 21, 22). cAMP, as a second messenger, can activate cAMP-dependent protein kinase A and, in turn, activate nuclear factors that can bind to specific palindromic DNA sequences (consensus TGACGTCA) defined as a CRE (18, 22). CREB binds to the CRE as a homodimer (19), and its activity is modulated by its phosphorylation, not by its intracellular level, because CREB protein appears to be expressed at similar levels in a range of tissues (20). Lee and colleagues (17) have successfully cloned and expressed a CREB cDNA gene from a placental cDNA library, suggesting that CREB is a major transcriptional factor for the cAMP signal pathway in placenta. The hCRH gene has a highly conserved classic CRE within its proximal promoter (23, 38). Here, our EMSAs show that hCRH CRE oligonucleotides can interact with placenta trophoblast nuclear protein and produce a major complex, and formation of this complex can be prevented by competition with excess unlabeled wild-type CRE, but not mutated CRE. These data show that a protein specifically interacts with the conserved CRE centered at -225 bp of the hCRH promoter, indicating that a transcription factor such as CREB is involved in cAMP stimulation of hCRH gene expression in human placental cells.

In summary, we have demonstrated that in primary placental cells cAMP stimulates hCRH promoter activity through the CRE in the hCRH promoter region. This is the first report of CRH reporter gene expression studies in transfected, freshly isolated, human placental cytotrophoblasts.


   Acknowledgments
 
We thank the nursing and medical staff of the delivery suite, John Hunter Hospital, for their cooperation in obtaining placenta, Dr. C. Meldrum (Genetics Laboratory, John Hunter Hospital) for his helpful suggestions and discussions, Prof. J. A. Majzoub (Harvard University Medical School) for the gift of the human genomic CRH clone, and Dr. T. G. Golos (University of Wisconsin Medical School, Madison, WI) for his very helpful suggestions regarding transfection of primary placental cells.


   Footnotes
 
1 This work was supported by the National Health and Medical Research Council of Australia. Back

2 Current address: Division of Endocrinology, Children’s Hospital Research Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039. Back

Received August 11, 1999.

Revised October 18, 1999.

Accepted November 17, 1999.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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