From the Department of Obstetrics and Gynecology, Osaka University Medical School, Suita, Osaka 565, Japan
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ABSTRACT |
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The purpose of this study was to analyze the
mechanism of transcriptional activation of human chorionic
gonadotropin- (hCG
) gene by epidermal growth factor (EGF) in
trophoblast cells. We stably transfected hCG
promoter-chloramphenicol acetyltransferase constructs into Rcho-1
trophoblast cells and monitored the promoter activities.
290-base
pair hCG
promoter containing a tandem repeat of cAMP response
element (CRE) was activated by EGF in a dose- and
time-dependent manner. Deletion analysis of hCG
promoter suggested an involvement of CRE in EGF-induced hCG
transcriptional activation. Moreover, the hCG
promoter, of which both CREs were mutated, did not respond to EGF. These results indicate that EGF activates the hCG
gene transcription through CRE. Although EGF did
not alter the amount of CRE-binding protein (CREB), EGF induced CREB
phosphorylation. We next examined the mechanism of CREB phosphorylation by EGF. Protein kinase C inhibitors (H7, staurosporin, and
chelerythrine) inhibited EGF-induced CREB phosphorylation, whereas
either mitogen-activated protein kinase kinase-1 inhibitor (PD98059) or
protein kinase A inhibitor (H8) showed no effect. Furthermore, H7 and
staurosporin but not H8 inhibited hCG
promoter activation by EGF. In
conclusion, EGF promotes hCG
gene transcription via the CRE region
probably by phosphorylating CREB mainly through the protein kinase C
pathway in trophoblast cells.
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INTRODUCTION |
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Epidermal growth factor (EGF),1 which consists of 53 amino acids, stimulates proliferation and differentiation in many kinds of cells and tissues (1). An important role of EGF has been suggested in mouse (2-5) and human (6-9) pregnancies. Human placentas are extremely rich in EGF receptors (6-8), suggesting that EGF is important in placental functions (9). EGF has been shown to promote functional differentiation of human trophoblast cells (10); EGF treatment of choriocarcinoma cells (11-13) and normal human trophoblasts (9, 14, 15) results in an increase in human chorionic gonadotropin (hCG) secretion. Human chorionic gonadotropin plays a critical role at the early stage of pregnancy and is progressively produced as human trophoblasts differentiate into syncytiotrophoblasts (10).
EGF has been shown to increase hCG and hCG
mRNA level and
their stability (16); however, it remains unclear whether or not EGF
increases the transcriptional activity of hCG genes. hCG consists of
hCG
and hCG
subunits (17). hCG
gene is present as a single
copy gene on chromosome 6q21.1-23 (18), whereas hCG
consists of six
closely spaced genes (19). The structure of hCG
promoter is simpler
and is well studied (19). Therefore, in this study we analyzed the
effect of EGF on the hCG
gene.
In this study, we used Rcho-1 cells to examine the molecular mechanism
of EGF effect on the hCG gene transcription. The Rcho-1 cell line
was established from a transplantable rat choriocarcinoma (20) and can
be manipulated to proliferate or differentiate along the trophoblast
giant cell pathway (21). Several genes have been shown to be
transcriptionally activated during Rcho-1 cell differentiation (22,
23). Using Rcho-1 cells stably transfected with hCG
promoter-CAT
construct, we studied the EGF effect on the transcriptional activity of
hCG
gene and analyzed the mechanism of EGF action.
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EXPERIMENTAL PROCEDURES |
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Cell Culture-- The Rcho-1 cell line was routinely maintained in subconfluent conditions with NCTC-135 medium (Sigma) supplemented with 20% fetal bovine serum (20% FBS/NCTC) as reported previously (21). Differentiation was induced by growing to confluence in FBS-supplemented culture medium and then replacing the serum supplementation with 1% horse serum (1% HS/NCTC).
Plasmid Construction--
290h
CAT was kindly provided by
Dr. John Nilson (24). Deletion mutants of
290h
CAT were generated
by the polymerase chain reaction. Polymerase chain reaction products
were inserted into pSV0CAT (Promega). CRE-tk-CAT and CRE-CRE-tk-CAT
were generated by inserting one or two copies of CRE in the native
orientation directly up-stream of the tk-CAT.
290h
(mCRE)CAT,
both CREs of which were mutated, was generated using a polymerase chain reaction-based site-directed mutagenesis kit (Stratagene). All constructs were sequenced using T7 DNA polymerase sequencing kits (Amersham).
Stable Transfections and CAT Assays-- Rcho-1 cells were transfected using a liposome-mediated delivery system (Life Technologies, Inc.) as described previously (23). Stable transfectants were established by co-transfecting 9 µg of promoter-reporter construct plasmid DNA with 1 µg of pSV2Neo DNA. Selection of stable transfectants was performed by growth in the presence of G418 (250 µg/ml).
Stably transfected Rcho-1 cells were incubated in 20% FBS/NCTC medium for 2 days, and then culture was shifted to 1% HS/NCTC. EGF from mouse submaxillary glands (Toyobo) was used for the EGF stimulation experiments. EGF treatment was started when the medium was replaced with 1% HS/NCTC. Protein concentrations of the whole cell extracts were determined by the Bio-Rad protein assay system. CAT reactions were carried out with 50 µg of protein for 3 h at 37 °C. The acetylated and nonacetylated forms of [14C]chloramphenicol were separated by a thin layer chromatography, autoradiographed, and quantitated by an image analyzer system (BAS2000, FUJIX). All experiments were repeated at least three times with consistent results.Oligonucleotides-- Synthesized oligonucleotides were obtained from Vector Research (Osaka, Japan). The following oligonucleotides were used in this study. CRE upper strand, 5'-AAATTGACGTCATGGTAA-3'; CRE lower strand, 5'-TTACCA TGACGTCAATTT-3'; mCRE upper strand, 5'-AAATTGATCTCA TGGTAA-3'; and mCRE lower strand, 5'-TTACCATGAGATCAATTT-3'. The complementary oligonucleotides were annealed to form double-stranded DNA, which contained 5'-AGCT or -TCGA overhangs to facilitate labeling.
Electrophoretic Mobility Shift Assays-- Nuclear extracts were prepared from Rcho-1 cells according to a previously described procedure (25). The extraction buffer contained an additional phosphatase inhibitor (NaF) at 10 mM. Protein concentrations of extracts were determined using the Bio-Rad protein assay system. Nuclear extracts (5 µg/lane) were incubated for 10 min at room temperature with 2 µg poly(dI-dC)-poly(dI-dC) in a reaction mixture containing 10 mM HEPES, pH 7.8, 50 mM KCl, 1 mM EDTA, 5 mM MgCl2, 1 mM dithiothreitol, and 10% glycerol. A 32P-labeled oligonucleotide probe (1 × 104 cpm) was added, and the reaction mixture was incubated for 30 min at room temperature. To demonstrate binding specificity, unlabeled CRE or mCRE was used. DNA-protein complexes were resolved on 5% polyacrylamide gels in 0.5× TBE and visualized by autoradiography.
Western Blot Analysis-- Nuclear extracts (150 µg) were resolved by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes. After a blocking reaction (5% nonfat dry milk in Tris-buffered saline, pH 7.4, 0.05% Tween-20) for 1 h, membranes were incubated in a blocking buffer with antisera against rat CREB (1:3,000 dilution) or against rat phosphorylated CREB (1:2, 500 dilution) for overnight at 4 °C. After incubation with horseradish peroxidase-linked rabbit IgG (Life Technologies, Inc., 1:3,000), the membranes were developed by using Enhanced Chemiluminescence System (Amersham) according to the manufacturer's instructions. For reprobing, the membranes were submerged in a stripping buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) and incubated at 50 °C for 30 min with occasional agitation. After washing twice in Tris-buffered saline for 10 min at room temperature, the membranes were blocked for 1 h and incubated with a rat CREB antiserum (1:3,000). CREB and phosphorylated CREB antisera were raised in rabbits against a synthetic peptide (amino acids 1-205) and a synthetic phosphopeptide (amino acids 123-136), respectively (Update Biotechnology, Inc.).
Southwestern Blot Analysis-- Nuclear extracts were resolved and transferred in the same way as above. Membranes were initially incubated in TNE-50 (10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol) containing 5% nonfat dry milk for 2 h at room temperature, washed briefly in TNE-50 without milk, and then incubated in TNE-50 containing a 1 × 106 cpm/ml CRE as a probe and 10 µg/ml poly(dI-dC)-poly(dI-dC). After the incubation, the blots were washed three times for 5 min each with TNE-50, air dried, and then exposed to a Kodak XAR film.
Statistics-- Statistical analysis was performed by unpaired t test. All experiments were performed in triplicate or quadruplicate and repeated at least three times with similar results.
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RESULTS |
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EGF Promotes Differentiation-dependent Increase in
hCG-CAT Activity in Rcho-1 Cells--
Rcho-1 cells morphologically
and functionally differentiate in the differentiation medium (1% HS)
throughout the culture period as reported previously (21).
290h
CAT
activity showed a differentiation-dependent increase in
stably transfected Rcho-1 cells (Fig.
1B, Control). To
study the effect of EGF on the differentiation-dependent
increase in hCG
promoter activity, Rcho-1 cells stably transfected
with
290h
CAT were treated with various concentrations of EGF
(0-10 nM) for days 2-8 of culture. Cells were harvested
on day 8, and CAT activities were determined. EGF enhanced the hCG
promoter activity in a dose-dependent manner with a maximal
effect at 10 nM (2.7-fold enhancement) (Fig.
1A). EGF at 30 nM or more did not show a further
promotion (data not shown).
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CRE on the hCG Gene Promoter Is the Region Responsible for EGF
Effect--
The
290-base pair region of the hCG
promoter
contains several consensus sequences: trophoblast-specific element,
GATA element, and CRE (16). To determine the region responsible for EGF
effect, various deletion mutants were generated and stably transfected into Rcho-1 cells. Fig. 2
(left) shows a diagram of deletion mutants used in the
study. Stable transfectants were cultured with or without EGF (10 nM) on days 2-8 of culture. CAT activities in EGF-treated
and untreated cells were compared (Fig. 2, right). Although
transcriptional activities in deletion mutants up to
142 base pairs
were promoted by EGF to an extent (2.6-2.8-fold) similar to that in
290h
CAT,
128h
CAT, which contains only one CRE, showed a
substantially decreased response to EGF.
110h
CAT, which does not
contain CRE, did not respond to EGF.
290h
CAT, and its deletion
mutants up to
142 base pairs also responded to forskolin.
128h
CAT showed an impaired response to forskolin, and
110h
CAT
did not respond to forskolin (data not shown), suggesting that CRE was
functional in Rcho-1 cells. These results imply that CRE may be a
response element for EGF.
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EGF Does Not Alter the CRE-binding Nuclear Proteins-- To analyze the possible change by EGF in the transcriptional factors associating with CRE, we performed electrophoretic mobility shift assays using nuclear proteins from EGF-treated and untreated Rcho-1 cells (Fig. 4A). CRE formed several major DNA-protein complexes in both untreated (lane 2) and 10 nM EGF-treated (lane 5) Rcho-1 cells. These binding complexes seemed specific because these complexes were competed by excess amount of cold CRE (lanes 3 and 6) but not by excess amount of cold mutated CRE (lanes 4 and 7). No obvious differences were found in CRE-binding nuclear proteins between EGF-treated and untreated cells. These results suggest that EGF might not alter CRE-binding proteins.
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EGF Phosphorylates CREB--
It is known that CREB is mainly
phosphorylated at Ser133 and that the phosphorylation is
essential for gene activation by CREB (27). To elucidate the mechanism
of hCG promoter activation by EGF through CRE, we determined whether
or not EGF phosphorylates CREB protein. Nuclear extracts were obtained
from EGF-treated (10 nM for 5 or 30 min) and untreated
cells, and Western blot analysis was performed using
anti-phosphorylated CREB antibody (Fig.
5A). Although phosphorylated
CREB was not observed in nuclear proteins from untreated cells, it was
present in cells treated for 5 min with EGF, and the amount of
phosphorylated CREB decreased by 30 min of EGF treatment (Fig.
5A, upper panel). Anti-CREB antibody detected a
similar amount of CREB in nuclear protein samples from untreated and
EGF-treated (5 and 30 min) cells (Fig. 5A, lower panel), showing that the changes in CREB phosphorylation by EGF were specific. We examined the time dependence of EGF effect on the
CREB phosphorylation throughout 3 h (data not shown) and observed that the EGF effect was the most at 5 min. The results suggest that EGF
promotes hCG
gene transcriptional activity through CRE, at least
partly, by phosphorylating CREB.
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Induction of CREB Phosphorylation Depends on PKC-- To further analyze the mechanism of CREB phosphorylation by EGF, we tested whether protein kinase A (PKA) and/or PKC pathways are involved. Rcho-1 cells were left untreated or pretreated for 30 min with PKC inhibitors (H7 (10 µM), staurosporin (50 nM), chelerythrine (5 µM)), or a PKA inhibitor (H8 (10 µM)) and then stimulated with EGF (10 nM, 5 min) or with forskolin (10 µM, 1 h). Nuclear extracts were prepared, and Western blot analysis was performed using anti-phosphorylated CREB antibody. Pretreatment with the PKC inhibitors inhibited EGF-induced phosphorylation of CREB (Fig. 6, upper panel). Pretreatment with a PKA inhibitor H8 did not inhibit EGF-induced phosphorylation of CREB. H8 inhibited forskolin-induced phosphorylation of CREB, showing the efficiency of the PKA inhibitor used in the study. Either PKC or PKA inhibitors did not affect CREB expression (Fig. 6, lower panel). These results suggest that EGF phosphorylates CREB mainly through the PKC-dependent pathway in trophoblast cells.
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PKC Inhibitors Decrease EGF-promoted hCG Transcriptional
Activity--
To study the effects of PKC and PKA inhibitors on the
EGF-enhanced hCG
promoter activity, Rcho-1 cells stably transfected with
290h
CAT were treated with EGF (10 nM) in the
absence or the presence of PKC inhibitors (H7 (10 µM),
staurosporin (50 nM)), or a PKA inhibitor (H8 (10 µM)) for days 2-8 of culture. Although H8 did not reduce
the EGF-promoted hCG
promoter activity (2.6-fold), PKC inhibitors
(H7 and staurosporin) significantly reduced the enhancement by EGF
(Fig. 7). These PKC inhibitors alone did
not reduce hCG
promoter activity (data not shown), indicating that they may specifically inhibit the EGF effect. In addition, H7 (10 µM) did not affect P-450 side chain cleavage (P-450scc)
gene transcription but rather promoted progesterone secretion by Rcho-1 cells (31). Therefore, the effect of the PKC inhibitors may not be due
to cell toxicity. All these results suggest that phosphorylation of
CREB through the PKC pathway may be involved in the hCG
promoter activation by EGF.
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DISCUSSION |
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Many studies have shown the importance of EGF in the maintenance
of pregnancy and in fetal development. EGF deficiency during pregnancy
causes abortion in mice (4). Fetal mice lacking the EGF receptors are
retarded in growth and die at midgestation in a 129/Sv genetic
background (5). In humans, the amounts of placental EGF receptors are
decreased in intrauterine growth-retarded pregnancy (32). The promotion
of hCG gene expression by EGF might be a part of the
pregnancy-supporting system. It has been known that EGF stimulates hCG
secretion by trophoblast cells (33). Cao et al. showed that
EGF increased hCG and hCG
mRNA level by stabilizing them in
JEG-3 cells (16); however, it has not been known whether or not EGF
promotes hCG gene transcription. In this report, we first showed that
EGF increased the transcriptional activity of hCG
gene using Rcho-1
cells. Rcho-1 cells are derived from a rat choriocarcinoma. Although
rodent placentas do not express chorionic gonadotropin, the hCG
gene
promoter when expressed as part of a transgene is active in placentas
and pituitary of transgenic mice (24). Also we observed
differentiation-dependent transcriptional activation of
hCG
gene (Fig. 1B). Therefore, the Rcho-1 cell line seems
to possess the activation system of hCG
gene as well as human
trophoblast cells. The EGF effect to promote the hCG
transcriptional
activity was dose- and time-dependent; however, EGF was
less effective on day 10 of culture than on day 8, suggesting that
terminally differentiated cells have a reduced response to EGF.
EGF increases the transcriptional activities in various genes; EGF
stimulation of gastrin transcription is mediated through a GC-rich
gastrin EGF response element (34). Expression of the ovine P-450 side
chain cleavage enzyme gene (CYP11A1) is stimulated by EGF through
AP-1-like site (35). An AP-1-binding site in the c-fos gene
can mediate the induction by EGF in HeLa cells (36). We showed that CRE
was an EGF response element in the hCG promoter in trophoblast
cells. It has been known that CRE is essential for basal promoter
activity and cAMP responsiveness of hCG
gene (37-40). Some cAMP
responsive genes also respond to EGF (35, 41-43). Although both cAMP
and EGF activate the ovine CYP11A1 promoter in JEG-3 cells through
distinguishable regions (35), both factors activated the hCG
gene
via the same region (CRE) in this study. The mechanism of
transcriptional activation by EGF is complicated and might be
gene-specific.
Several proteins binding to CRE have been identified, which include CREB, cAMP response modulator, activating transcription factor-1, and cAMP response element-binding protein-1 (identical to activating transcription factor-2). These proteins are known as members of bZIP proteins (44). It has been known that these proteins form homodimers or heterodimers to bind to the CRE (45, 46). We focused on CREB in this study because in Southwestern blot analysis we detected a 43-kDa CRE-binding protein, the migration of which in SDS-PAGE was the same as that of a 43-kDa immunoreactive CREB. However, we also observed other associating proteins (Fig. 4B). The possible involvement of other CRE-binding proteins remains to be investigated.
The activity of many transcription factors is regulated by
posttranslational modification. Such modifications include
phosphorylation and dephosphorylation of serine and threonine residues
and oxidation and reduction of cysteines (47). We showed that CREB was
phosphorylated by EGF. A most probable candidate for the
phosphorylation site is Ser133 (27). The importance of the
phosphorylation of CREB in generating its transcriptional functions has
been shown in transgenic mice expressing a CREB with a serine to
alanine substitution mutation at Ser133 (48).
Phosphorylation can alter protein function by introducing an allosteric
conformational change in the protein or by allowing (or blocking)
specific electrostatic interactions with other molecules. These changes
are thought to be involved in the regulation of transcription. The
structural properties of CREB and phosphorylated CREB were analyzed by
the method of CD, and it was shown that the phosphorylation at
Ser133 did not alter the secondary structure of CREB and
the DNA binding affinity of CREB to CRE sequences (49). From these
results the phosphorylation of CREB might induce the production of the
specific interactions with proteins such as CREB-binding protein (CBP) rather than the conformational change or increased DNA binding affinity. Furthermore other studies suggest that although
phosphorylation of CREB is required to form the CREB-CBP complexes,
other events are also involved for activation of CREB-mediated
transcription (50). Biophysical evaluation of phosphorylated CREB-CBP
complexes will help to further understand the hCG transcriptional
activation.
Whereas much research on the regulation of CREB transactivation has
been directed toward the mechanisms of phosphorylation, relatively
little is known about the phosphatase-mediated inactivation of CREB.
Hagiwara et al. provided evidence that protein phosphatase-1 selectively dephosphorylates Ser133 in CREB and
correspondingly attenuates the transcriptional activity of CREB (51).
There is another study focused on the intracellular processes that
regulate the phosphorylation state of CREB in the hippocampal neurons
(52). In the study it is shown that synaptic activity simultaneously
influences both phosphorylation and dephosphorylation of CREB. During a
brief stimulus, simultaneous activation of both kinase and phosphatase
ensures that pCREB elevation will be large but brief. Longer stimuli
cause prolongation of nuclear pCREB, possibly by hampering the
phosphatase pathway. A possible involvement of the CREB
dephosphorylation mechanism in the EGF effect on the hCG gene
remains to be studied.
The phosphorylation of CREB at Ser133 is mediated by PKA in
pheochromocytoma cells (53) and by PKC in B lymphocytes (54). In this
study, we showed that EGF-induced phosphorylation of CREB may be mainly
mediated by PKC. In Rcho-1 cells forskolin also phosphorylated CREB
(Fig. 6, upper panel), suggesting that there may be several
pathways (at least two pathways, PKC and PKA) to mediate CREB
phosphorylation. EGF has been shown to activate Ras (55, 56) and to
induce members of the MAP kinase family including the extracellular
signal-regulated kinases and the stress-activated protein kinases, also
referred to as c-Jun N-terminal kinases (57-59). EGF and c-Jun act via
a common DNA regulatory element to stimulate transcription of the ovine
CYP11A1 (35), and induction of the CYP11A1 promoter by EGF involves a
ras/MEK1/AP-1-dependent pathway (60). Several
reports have shown that PKC activates Raf, suggesting cross-talk
between MAP kinase and PKC pathways (61-63). We showed that MAP kinase
pathway might not be essential for EGF-induced phosphorylation of CREB
in trophoblast cells. Although our data do not exclude possible
cross-talk between MAP kinase and PKC pathways, all these results
suggests that EGF induces CREB phosphorylation mainly through the PKC
pathway in trophoblast cells, resulting in transcriptional activation
of the hCG gene.
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ACKNOWLEDGEMENTS |
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We thank Dr. John Nilson for a 290h
CAT
promoter-reporter construct and Dr. Michael Soares for Rcho-1 cells and
critical review. We also thank Drs. Masahide Ohmichi and Kanji Masuhara for assisting MAP kinase assay.
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FOOTNOTES |
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* This work was supported in part by a grant-aid for scientific research from the Ministry of Education, Science, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 81-6-879-3351;
Fax: 81-6-879-3359; E-mail: yamamoto{at}gyne.med.osaka-u.ac.jp.
1 The abbreviation used are: EGF, epidermal growth factor; hCG, human chorionic gonadotropin; FBS, fetal bovine serum; HS, horse serum; CAT, chloramphenicol acetyltransferase; PAGE, polyacrylamide gel electrophoresis; CRE, cAMP-response element; CREB, CRE-binding protein; mCRE, mutant CRE; MAP, mitogen-activated protein; MEK, MAP kinase kinase; PKC, protein kinase C; PKA, protein kinase A; CBP, CREB-binding protein.
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REFERENCES |
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