(Received for publication, November 12, 1996, and in revised form, March 19, 1997)
From the Division of Endocrinology, Metabolism, and
Molecular Medicine, Northwestern University Medical School, Chicago,
Illinois 60611, the ¶ Department of Endocrinology, Children's
Hospital Medical Center, University of Cincinnati College of Medicine,
Cincinnati, Ohio 45229, and the
Department of Biology, Yale
University, New Haven, Connecticut 06520
Production of the placental
hormone, chorionic gonadotropin (CG), increases dramatically as
cytotrophoblasts fuse to form syncytiotrophoblasts. The CG - and
-promoters are both responsive to cAMP, although the kinetics of
cAMP stimulation are different. In an effort to understand the
mechanisms of coordinate induction of these genes, AP-2 binding sites
were identified in the promoter regions of the
and CG
genes.
AP-2 bound to the upstream regulatory element (
186 to
156 base
pairs (bp)) in the
-promoter and to several different regions of the
CG
promoter, including footprints 2 and 4B (FP2,
311 to
279 bp;
FP4B, 221 to
200 bp). AP-2 antibodies induced supershifts of these
complexes, confirming the identity of the protein-DNA complex. In JEG-3
cells, which contain abundant AP-2, mutations in these CG
AP-2 sites
reduced basal activity and decreased cAMP stimulation. In
AP-2-deficient Hep-G2 cells, co-transfection of AP-2 stimulated
expression of the CG
promoter 10-20-fold, and the
-promoter was
induced by 3-6-fold. Mutations that eliminate AP-2 binding to CG
FP4B reduced AP-2 stimulation by more than 80%, whereas mutations in
FP2 reduced AP-2 stimulation by less than 50%. Analyses of AP-2
mutants revealed a requirement for the DNA binding/dimerization domain
and the amino-terminal proline-rich and acid-rich transactivation
domains for stimulation of the CG
promoter. Primary cultures of
placental cytotrophoblasts were differentiated into
syncytiotrophoblasts in vitro to examine AP-2 expression by
reverse transcriptase-polymerase chain reaction. AP-2 mRNA levels
increased by day 2 and continued to rise in parallel with a marked
increase in
and CG
gene expression. We conclude that both the
and CG
promoters contain binding sites for AP-2 and suggest that
this transcription factor provides a mechanism for coordinating the
induction of these genes during placental cell differentiation.
Human chorionic gonadotropin (hCG)1 is
a heterodimeric placental hormone encoded by separate - and
CG
-subunit genes (1-3). It is a member of a family of hormones that
are expressed in the pituitary (luteinizing hormone (LH),
follicle-stimulating hormone, and thyroid-stimulating hormone) and the
placenta (CG). The
-subunit is common to these hormones, and it is
expressed in both pituitary and placenta. The CG
gene is expressed
almost exclusively in the placenta (3). The function of CG is to
stimulate the corpus luteum in the ovary to produce progesterone during
the early stages of pregnancy.
The dramatic exponential increase in CG expression in early pregnancy correlates with the formation of differentiated placental cells (4, 5). Trophoblast progenitor cells convert to proliferative cytotrophoblasts that invade the endometrium of the uterus. The cytotrophoblasts fuse to form nonmitotic syncytiotrophoblast cells. The production of CG is greatly enhanced upon the formation of syncytiotrophoblasts, which also produce a variety of other hormones including placental lactogen (5).
The cellular pathways that lead to activation of the and CG
genes have not been clearly established, although cAMP is able to
induce expression of these genes in both placental cells (6) and
choriocarcinoma cell lines (7, 8). Cyclic AMP-responsive DNA sequences
have been characterized in the promoters of both genes (for review, see
Ref. 9). In the
-gene, two identical repeats of a consensus cAMP
response element (CRE) are located between
146 and
111 bp of the
promoter (10-12). These CREs bind cAMP response element-binding
protein (12-14) along with other members of the B-Zip family of
transcription factors (15, 16). An adjacent element, termed the
upstream response element (URE,
180 to
151), also contributes to
basal expression and appears to contribute to placenta-specific
expression of the
-promoter (12, 13, 17-20). The URE contains three
overlapping protein binding sites referred to as the
trophoblast-specific element (TSE, or URE2 (
187 to
159)) (12, 13,
18, 21), downstream domain (
172 to
151) (13, 19), and GATA
(
-ACT, URE1) (
165 to
140) (22). Protein binding to the TSE
and downstream domain are mutually exclusive (13, 19).
The cAMP-responsive region in the CG promoter encompasses several
protein binding domains between
311 and
200 bp (23-25). Maximal
expression in placental cell lines and stimulation by cAMP requires
this entire region, suggesting that it functions as a composite
regulatory element (23). Nuclear extracts footprint two major regions
(
311 to
274;
250 to
200) within this domain (23, 24). Neither
region binds transcription factor cAMP response element-binding protein
(23), nor are they competed by the CRE derived from the
-promoter
(23, 24), suggesting distinct pathways for cAMP control of the two
genes. Another B-Zip protein, c-Jun, negatively regulates both
promoters, and it binds to the
-CRE and to the CG
gene promoter
between
245 and
220 bp (16). However, based upon antibody-mediated
supershift studies, c-Jun does not appear to represent the major
protein that binds to this region of the CG
gene (16). Distinct
sequences within the cAMP-responsive region of the CG
promoter may
share common binding proteins, since they cross-compete for protein
interactions (24). In addition, the TSE region from the
-promoter
also competes for these proteins, suggesting that the same
transcription factors might be involved in the coordinate regulation of
the two promoters (24).
Transcription factor AP-2 has been shown to mediate cAMP responses in
other genes including metallothionein IIA (26), acetyl-CoA carboxylase
(27), insulin-like growth factor-binding protein-5 (28), and RII
protein kinase (29) among others (16, 30). AP-2 has also been
implicated in developmentally regulated gene expression in a variety of
cell types including NT-2 and P19 teratocarcinoma cells (31, 32),
primary neural cells (33), keratinocytes (34-36), and adipocytes (27).
AP-2 is a 52-kDa protein that binds to DNA as a homodimer (37) and can
associate with c-Myc, inhibiting transactivation by c-Myc
(38). Several AP-2 variants have been identified (37, 39), some of
which act as inhibitors of AP-2 gene activation through an undetermined
mechanism (37). In this report, we examine a potential role of AP-2 in
the regulation of the
and CG
genes. We find that AP-2 binds to
regulatory elements in both promoters and regulates the expression of
these genes. AP-2 is also induced during placental cell
differentiation.
Nuclear extracts were prepared by the Shapiro method (40) modified by the addition of protease inhibitors (1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 1 µg/ml p-aminobenzamide (Sigma) to the final dialysis buffer. Nuclear extracts (5-10 µg) were added to a 20-µl reaction containing: 2 µl of 10 × buffer (200 mM HEPES, pH 7.9, 400 mM KCl, 10 mM MgCl2, and 1% Nonidet P-40), 500 ng of dI-dC, and 1 µg of AP-2 or Sp-1 antibodies as indicated. Reactions were preincubated on ice for 30 min before the addition of 30 fmol of radiolabeled probe with or without unlabeled competitor DNA. Reactions were incubated at room temperature for 30 min before electrophoresis (180 V, 3 h) through nondenaturing 5% polyacrylamide gels in 0.5 × TBE (45 mM Tris borate, 1 mM EDTA).
Oligonucleotides for electrophoretic mobility shift assays are listed
in Table I. Hybridized oligonucleotides were labeled by Klenow end
filling. Klenow reactions included 5 pmol of annealed oligonucleotides,
1 µl of 10 × Klenow buffer (Promega, Madison, WI), 4 µl of 25 mM deoxynucleoside triphosphates (without dATP), 3 µl of
[-32P]dATP (3000 µCi/ml, DuPont NEN), and 1 µl of
Klenow fragment (Promega). Reactions were incubated at 37 °C for 30 min before the addition of 5 µl of 25 mM dNTPs and
incubation for an additional 10 min. After stopping the reactions, the
solution was passed through Centricep columns (Adelphia, NJ) to remove
unincorporated nucleotides.
|
The and CG
luciferase constructs in the pA3LUC plasmid have been
described previously (23, 25). Site-directed mutagenesis was performed
using sequences that correspond to the mutant oligonucleotides used in
gel mobility shift assays (Table I). Polymerase chain reactions were
used to incorporate the mutations within the
and CG
promoters
(25). All site-directed mutations were sequenced to verify the mutation
as well as the correct native promoter sequence. Expression vectors
containing wild type AP-2 and AP-2 mutants were driven by the Rous
sarcoma virus promoter (41).
Transient transfections were
performed using either the CaPO4 (42) or lipid-mediated
(43) methods. CaPO4 reactions consisted of 4.5 µg of
reporter plasmid, 250 µl of HEPES-buffered saline (137 mM
NaCl, 5 mM KCl, 0.7 mM
Na2PO4, 6 mM dextrose, 21 mM HEPES, pH 7.05) and 10 µl of 2 M
CaCl2. Expression vectors (300-600 ng) were used as
indicated, and equal amounts of empty vector were added to keep the
total amount of expression vector constant in different reactions.
Lipid transfections were performed using L--phosphatidylethanolamine, dioleoyl (Sigma) and
dimethyldioctadecyl-ammonium bromide (Sigma) lipids prepared by the
ethanol injection method (44). Cells were harvested for luciferase
assays 18-24 h after transfection (45).
Nuclear extracts are described above (40). Whole cell placental extracts were kindly provided by Dr. T. Woodruff (Northwestern University). Primary antibodies included 1:1000 AP-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and 1:1000 Sp-1 (Santa Cruz Biotechnology) and were added to nitrocellulose membranes for 60 min in solution containing albumin (5 mg/ml). After washing three times in 0.1% Tween phosphate-buffered saline, membranes were incubated with 1:10,000 secondary anti-rabbit antibody (Santa Cruz Biotechnology) in 3% milk phosphate-buffered saline. After washing, membranes were subjected to enzyme-linked chemiluminescence as described by the manufacturer (Amersham Corp.).
RT-PCR Assays for mRNA Expression in Placental CellsCytotrophoblast cells were isolated from human term
placenta and cultured under conditions that allow fusion into
syncytiotrophoblast cells (46). RNA was isolated from placental cells
every 2 days over a 12-day period of culture (46). Total RNA (1 µg)
was reverse transcribed (37 °C, 2 h) by the addition of 15 units of reverse transcriptase (Promega) in the presence of 10 pmol of
random hexamer primers, 25 mM deoxynucleoside triphosphates
(dNTPs) in 1 × MI buffer (67 mM Tris, pH 8.8, 6.7 mM MgCl2, 16 mM
(NH4)2SO4, 10 mM
-mercaptoethanol) in a total volume of 20 µl. PCR reactions included specific primers for the
and CG
genes, AP-2, and
internal controls, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and ribosomal protein 19 (RPL19). The final PCR reaction (100 µl) included a 1-µl aliquot of the RT reaction product, 50 pmol of sense
and antisense primers for AP-2,
, and CG
, along with one of the
controls, GAPDH or hRPL19, in 1 × MI buffer (25 µM
dNTPs, 10 µl of Me2SO, 0.5 µl of Taq DNA
polymerase (Promega), and 0.1 µl of [32P]dATP (DuPont
NEN). Cycle conditions were 96 °C for 30 s, 94 °C for 1 min,
58 °C for 1 min, and 72 °C for 1 min. An aliquot (20 µl) of
each reaction was subjected to polyacrylamide (6%) gel electrophoresis, and the specific products were quantitated using a
Fujix 2000 phosphoimager (Fuji Medical Systems, Stamford, CT). Data
were analyzed by one-way analysis of variance using Dunnett's test.
Primers (Life Technologies, Inc.) were designed to span exon-intron
boundaries to avoid amplification of genomic DNA. The primers include
the following: sense, 5
-CCAGAATGCACGCTACAG-3
,
antisense,
5
CGCCGTGTGGTTCTCCAG3
, product 222 bp; hCG
sense, 5
-GTGGAGAAGGAGGGCTGC-3
, hCG
antisense, 5
-GGCGGCAGAGTGCACATT-3
, product 232 bp; AP-2 sense, 5
-CTGCCAACGTTACCCTGC-3
, AP-2 antisense 5
-TAGTTCTGCAGGGCCGTG-3
, product 339 bp; GAPDH sense,
5
-GAGCCACATCGCTCAGAC-3
, GAPDH antisense, 5
-CTTCTCATGGTTCACACCC-3
,
product 430 bp.
The and CG
regulatory elements that have been
shown to bind proteins in JEG-3 nuclear extracts are depicted in Fig.
1A (12, 13, 23, 24). Previous studies have
shown cross-competition by a subset of these elements, including the
URE/TSE and several of the CG
footprinted elements, suggesting
that these sequences may share common transcription factors (24, 47).
The size of the URE/TSE binding protein (24) and the GC-rich nature of the DNA sequences shared by these elements raised the possibility that
AP-2 might interact at these sites.
Electrophoretic mobility shift assays were performed using JEG-3
nuclear extracts to assess whether a consensus AP-2 sequence competed
for binding to the and CG
elements (Fig. 1B). Excess (100-fold) unlabeled AP-2 oligonucleotide inhibited binding to the
URE and CG
FP4B. The lower part of the complex that binds to
CG
FP2 was also reduced by the AP-2 competitor. As a positive control, the AP-2 competitor was shown to compete well for protein binding to the homologous AP-2 sequence derived from the human metallothionein IIA (hMTIIA) promoter (
185 to
167) (26, 48). However, excess unlabeled AP-2 oligonucleotide did not compete with
complexes that bind to the Sp-1 binding site or to CG
FP4A, indicating that the competition is specific. These results suggest that
AP-2 binds to sequences within both the
and CG
promoters.
AP-2 antibody was used in supershift assays to confirm whether AP-2 was
present in the protein complexes that bind to the and the CG
promoter elements (Fig. 2A). The AP-2
antibody supershifted the major complex binding to the
URE and to
CG
FP4B as well as proteins that bind to the control AP-2 element,
hMTIIA. In contrast, the AP-2 antibody had no effect on protein binding
to an Sp-1 element. The Sp-1 antibody had no effect on protein binding to the putative AP-2 sites, whereas it caused a supershift of the Sp-1
complex.
Protein interactions with different domains within CG FP2 are
illustrated in Fig. 2B. When the full-length FP2 fragment
was used, multiple protein complexes were observed. The addition of either AP-2 or Sp-1 antibody, but not preimmune AP-2 sera, appeared to
shift part of the major protein complex, suggesting that both proteins
might bind to FP2. The FP2 fragment was divided into overlapping
segments FP2A, FP2B, and FP2C. AP-2 competitor DNA inhibited protein
binding to the FP2A and FP2C fragments, and these complexes were also
supershifted by AP-2 antibody. On the other hand, Sp-1 competitor
inhibited protein binding to FP2B, and Sp-1 antibody supershifted this
complex. Additional experiments confirmed that these interactions were
specific, since the Sp-1 antibody did not alter binding to FP2A and
FP2C and the AP-2 antibody had no effect on the FP2B complex (data not
shown). These experiments indicate that AP-2 binds to the 5
- and
3
-ends of the FP2, whereas Sp-1 binds to the central region (
306 to
285 bp).
The interaction of AP-2 with the FP2 region was resolved further by performing combinations of oligonucleotide competitions and antibody supershift experiments (Fig. 2C). In the presence of Sp-1 competitor oligonucleotide, several FP2 protein complexes were diminished, and the amount of the putative AP-2 complex was increased. The addition of AP-2 antibody in the presence of Sp-1 competitor supershifted the residual protein complex. These findings support the idea that both Sp-1 and AP-2 bind to FP2 and raise the possibility that these binding sites overlap partially.
AP-2 Is Present in JEG-3 Nuclear Extracts and in PlacentaWestern blots were performed to determine whether AP-2
is present in cells that express the and CG
genes (Fig.
3). AP-2 antibody detected a 52-kDa protein in JEG-3
cells and extracts from whole placenta and trophoblasts. As controls,
the same band was seen in HeLa cells but not in Hep-G2 cells, which
have been shown previously to be deficient in AP-2 (41). The same blot was reprobed with an Sp-1 antibody and verified that the nuclear extracts from each of the cell lines contained similar levels of Sp-1
protein (Fig. 3B). However, little Sp-1 was detected in the
whole cell extracts from placenta or trophoblast cells (Sp-1 was seen
in these extracts with longer exposure, data not shown). These results
indicate that AP-2 is abundant in the placenta.
Role of the AP-2 Binding Sites in the Function of the
Transient expression assays were performed in Hep-G2
cells to examine the effects of co-transfected AP-2 on the and
CG
promoters (Fig. 4) (41). AP-2 stimulated
3700
CG
promoter activity 22-fold (Fig. 4A). Deletions to
1700 or
345 bp decreased stimulation to 10-fold, and subsequent
deletions to
248 bp essentially eliminated AP-2 stimulation. The
deletion between
345 and
248 bp includes FP2 but not FP4. An
internal deletion of FP4 (345 d-
FP4) also eliminated AP-2
stimulation.
AP-2 increased 846
-promoter activity by 6.8-fold. This effect was
reduced to 3-4-fold by deletion to
290 or
180 bp. Deletion to
156 bp eliminates the
URE and the AP-2 binding site. Deletion to
132 bp eliminates one of the two CREs. These deletions both reduced
basal expression and also reduced AP-2 stimulation to 1.3- and
2.5-fold, respectively. A single point mutation (172M
), shown
previously to disrupt binding to the URE (19) (see below), decreased
AP-2 stimulation further (0.9-fold).
Because deletion of CG FP2 has been shown previously to eliminate
cooperative interactions with more proximal sequences (23, 25), the
roles of CG
FP2 and FP4B were examined further by creating point
mutations within the individual domains. Electrophoretic mobility shift
assays were used to determine the effects of the mutations on protein
binding (Fig. 5A). Relative to the wild-type FP4B sequence, each of the CG
FP4B mutants (Table I)
competed poorly for AP-2 binding. FP4B-m1 and FP4B-m3 showed little or no competition, whereas FP4B-m2 competed partially for AP-2 binding. The effects of mutations in CG
FP2 or the
URE are shown in Fig. 5B. Consistent with the AP-2 supershift studies in Fig.
2B, fragments FP2A and FP2C, but not FP2B competed for AP-2
binding. The FP2-m1 mutation, which alters the binding site in fragment
FP2A did not compete for AP-2 binding. The 172 M in the
URE eliminated AP-2 binding to this fragment of the
-promoter
(Fig. 5B).
The effects of the CG FP4 and FP2 mutations were examined in the
context of the
345 bp construct (Fig. 6A).
In Hep-G2 cells, each of the point mutations in FP4 reduced AP-2
induction by 80% or more, confirming that AP-2 exerts functional
effects through the FP4 element (Fig. 6B). In contrast to
the FP4B mutations, the FP2 mutations reduced basal activity, but AP-2
induction was reduced by only 30%.
JEG-3 cells have been used extensively for studies of and CG
gene expression (9). Because JEG-3 cells express abundant amounts of
AP-2 (Fig. 3), they were used to assess the effects of the AP-2
mutations in the presence of the endogenous protein (Fig.
7). Each of the FP4 mutations greatly reduced basal
activity, consistent with a role for endogenous AP-2 in the regulation
of this site (Fig. 7A). The FP2 mutations caused an even
greater decrease in basal activity, suggesting that the AP-2 and/or the Sp-1 sites in this region are also involved in basal expression.
AP-2 has been implicated in cAMP regulation of gene expression
(26-28), and cAMP is known to induce the CG gene. The effect of
mutations of the AP-2 binding site on cAMP stimulation of CG
promoter activity were assayed after 18 h of treatment (Fig.
7B). The wild-type
345 CG
promoter was induced 42-fold
by cAMP (Fig. 7B). The AP-2 mutations in FP4 reduced cAMP
stimulation to a variable extent (50-75% decrease). The FP2-m1
mutation, which eliminates AP-2 binding to the FP2A sequence, also
decreased cAMP stimulation. In contrast, the FP2-m2 and FP2-m3
mutations, which disrupt Sp-1 binding, reduced basal activity but had
little effect on cAMP stimulation. The AP-2 mutant in the
-promoter
did not affect cAMP stimulation (data not shown), consistent with the
presence of other consensus CREs in this promoter (9).
Several functional domains have been delineated in AP-2,
including a DNA binding domain, a dimerization domain, and
transactivation domains (41, 49). Transient co-transfection studies
were conducted in Hep-G2 cells comparing wild type and mutant AP-2
expression vectors using the 345 hCG
promoter as the reporter gene
(Fig. 8). Deletion of the amino-terminal 50 amino acids
of AP-2 (
N51) did not affect transactivation, but further deletion
of the amino-terminal 165 amino acids (
N165) decreased CG
promoter activation by 70%. The region of difference between the two
deleted stretches includes the proline-rich and acidic activation
domains. A further deletion that also removes the DNA binding domain
(
N278) was inactive. Carboxyl-terminal deletion from 437 to 413 (
C413) had no effect, whereas deletion into the dimerization domain
(
C390) completely eliminated AP-2 induction of the CG
promoter.
These results suggest that AP-2 induction of the CG
promoter
requires dimerization and DNA binding together with the transactivation
domains, similar to studies performed with the hMTIIA AP-2 site
(41).
AP-2 Gene Expression Increases during in Vitro Differentiation of Trophoblast Cells
Cytotrophoblast cells were isolated from human
placenta and induced to undergo differentiation into
syncytiotrophoblasts (46). RNA was extracted over the course of 12 days
of differentiation, and the levels of AP-2 and of and CG
mRNA were analyzed by RT-PCR (Fig. 9). AP-2 mRNA
levels increased 3-fold after 2 days of differentiation and continued
to increase gradually during the 12-day period (8-fold increase). The
and CG
mRNA levels also increased during the first 2 days
and showed more marked stimulation between days 2 and 4 before reaching
a plateau (maximal -fold increase for
was 16-fold and for CG
was
45-fold). GAPDH and hRPL19 were used to normalize expression of the
other mRNAs, and they did not change substantially during the
differentiation process (data not shown). The finding that AP-2
mRNA levels increase in conjunction with the stimulation of the
and CG
genes is consistent with a role for AP-2 in the regulation of
these genes in the placenta.
Chorionic gonadotropin gene expression in the placenta is a
relatively recent evolutionary event, since its expression occurs almost exclusively in higher primates (9). In the case of the -gene,
modifications in the CRE sequence and in adjacent upstream regulatory
elements appear to account for the ability of the
-gene to be
expressed in the placenta as well as in the pituitary gland (17, 18).
The CG
genes appear to have duplicated and diverged from an
ancestral LH
gene (2). Although LH
expression is restricted to
the pituitary gland, the CG
genes are expressed preferentially in
the placenta, presumably reflecting the acquisition of new regulatory
DNA sequences that direct placenta-specific expression (50).
The DNA regulatory elements that control CG gene expression have
been challenging to define. Mutational studies have suggested that
several distinct elements may interact in an interdependent manner, a
phenomenon that has made it difficult to clearly delineate discrete
functional domains (23). Protein binding studies have therefore proven
quite helpful for defining potential regulatory elements. DNase I
footprinting analyses delineated several discrete binding sites,
particularly between
311 and
200 bp (23, 24). More recently, it was
found that several of these sites competed with one another, raising
the possibility that a common protein was binding to multiple sites
(24). Moreover, evidence that a key regulatory element (URE/TSE) for
placental expression of the
-promoter also competed for binding to
the CG
elements suggested that this protein might be involved in the
coordinate expression of the two genes (24). These findings have
underlined the importance of identifying the factors that bind to the
CG
regulatory elements.
In this report, we provide several lines of evidence that a
transcription factor that is immunoreactive with AP-2 antibodies binds
to the CG regulatory elements. Consensus AP-2 elements compete for
binding to FP4B and for two of the complexes that bind to FP2. The
identity of this protein as AP-2 is strengthened by the fact that an
AP-2 antibody supershifts the complexes that bind to these sites.
Similar data were found for the
URE site that competes for protein
interactions with the CG
elements, supporting the notion that this
protein is shared in common by these sequences. The molecular mass of
AP-2 is similar (52 kDa) to the size of the protein previously purified
by affinity chromatography using the
URE sequence (24). Last, in
AP-2-deficient Hep-G2 cells, co-expression of AP-2 stimulated
expression of the
-promoter and, to a greater degree, the CG
promoter. Taken together, these data suggest AP-2 may be a regulator of
and CG
gene expression in the placenta.
AP-2 appears to interact with several CG elements, including FP2 and
FP4B. Additional AP-2 sites were also identified several kilobase pairs
upstream in the CG
promoter.2 The
proximal part of the CG
promoter is very G-C-rich and has yet to be
tested for AP-2 binding. FP2 is also bound by Sp-1, and it appears that
AP-2 and Sp-1 may bind to partially overlapping elements, since
competition for Sp-1 facilitated the binding of AP-2 (Fig.
2C). Previous studies revealed that c-Jun binds close to
FP4A, which is adjacent to one of the AP-2 sites (FP4B) (25). Thus,
Jun, AP-2, and Sp-1 have now been shown to interact with the CG
promoter. Given evidence for combinatorial interactions among these
sequences, an important question for future studies is to understand
the mechanisms by which these regulatory elements interact. One
possibility is that they may share transcriptional co-activators.
AP-2 has been suggested to mediate cAMP responsiveness in a variety of
promoters (26-30). cAMP regulation of hCG was partially reduced by
AP-2 mutants in FP4B (50-75% decrease). In FP2, the mutation that
eliminates AP-2 binding (FP2-m1) decreased cAMP stimulation (65%
decrease), whereas the mutations within the Sp-1 binding site (FP2-m2,
FP2-m3) had less effect on cAMP stimulation. These findings support the
idea that AP-2 plays a role in cAMP stimulation of the CG
promoter.
However, the fact that cAMP responsiveness is not eliminated by these
mutations indicates that other sequences are probably involved in cAMP
stimulation of the CG
promoter (50).
AP-2 has been implicated in developmental regulation in several cell
types, and it is intriguing to consider the possibility that AP-2 may
participate in a developmental cascade during trophoblast differentiation. For example, the expression of keratin genes in the
developing epidermis correlates with the presence of cells that express
high levels of AP-2 (35). In NT-2 teratocarcinoma cells, retinoic acid
induces AP-2 expression as these cells undergo differentiation (31). We
found that AP-2 mRNA levels increased early in the process of
trophoblast differentiation in vitro, and AP-2 protein
levels are high in normal placenta. Because the CG genes are expressed
very early during embryogenesis and implantation, it is of interest to
determine whether AP-2 is already expressed at this time of
development. Recently, the AP-2 gene was disrupted by targeted
mutagenesis in mice, revealing that it is necessary for neural tube
closure and craniofacial development (51, 52). Potential effects on
placental development were not evaluated, but one can speculate that
there were no severe abnormalities as pregnancies proceeded to
completion. It should be noted, however, that two additional
AP-2-related genes (AP-2, AP-2
) have recently been identified
(37, 39), and it is possible that these genes may exert redundant
functions in the placenta and other tissues. In addition, because the
CG
genes are not expressed in mice, it is not possible to evaluate
potential effects of the null mutants on CG
gene expression in the
murine model.
In conclusion, we have shown that AP-2 expression increases as
trophoblasts differentiate in an in vitro model. AP-2
appears to be a major regulator of the CG gene, and to a lesser
degree, the
-gene. As such, it may represent one of several factors
that coordinate the expression of these genes. Future studies will help
to define other factors that function in conjunction with AP-2 to
regulate the combinatorial elements in these genes.