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INTRODUCTION |
The glycoprotein hormones are a family of heterodimeric peptides
that consist of an
subunit noncovalently linked to a
hormone-specific
subunit (1). Within the glycoprotein hormone
family, the
subunit is a common component of all members, and it is
the
subunit of these hormones that confers biological specificity to the heterodimer. Glycoprotein hormones derived from the anterior pituitary gland include luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone, produced by cells of the
gonadotrope (luteinizing hormone and follicle-stimulating hormone) and
thyrotrope lineages (thyroid-stimulating hormone). These peptide
hormones play an integral role in the regulation of reproduction and
metabolic homeostasis in mammals. In humans and nonhuman primates, an
additional member of the glycoprotein hormone family is synthesized and
secreted from the placenta. Human
CG1 is produced and secreted
by the cells of the trophoblast lineage during the first trimester of
pregnancy and is necessary for the maintenance of progesterone
secretion from the ovarian corpus luteum to ensure the establishment of
an appropriate uterine environment and pregnancy (2-5). Furthermore,
CG may play a paracrine role in the uterine endometrium to facilitate
uterine receptivity to implantation in primates (6).
The expression of the
subunit gene is restricted to cells of
pituitary and placental origin. The regulatory region of the
subunit gene is unique in that it confers cell-specific expression via
a complex array of cis-acting elements and their cognate binding factors (7, 8). In the gonadotrope of the anterior pituitary gland,
expression of the human
subunit gene requires dual CREs that bind
CREB family members, an element that binds a LIM-homeobox factor, and an SF-1 binding site, all of which contribute to
cell-specific expression. Two additional elements, referred to as
basal elements 1 and 2, also contribute to pituitary-specific
subunit expression; however, the factors that bind these sites have not
yet been identified (9, 10). An additional element within the murine
subunit promoter has also been identified as an Ets factor-binding
site that contributes to inducible expression of the
subunit by
phorbol esters and gonadotropin-releasing hormone (11, 12). Together, these elements and the transcription factors that bind them represent a
combinatorial code required for expression of the
subunit in the
anterior pituitary gland. An overlapping but separate combinatorial code exists for expression of the
subunit in cells of placental trophoblast origin. The dual CREs, in conjunction with an upstream regulatory element, a junctional regulatory element, and a unique CCAAT
box provide for trophoblast-specific expression (13-19). Portions
of the URE and JRE putatively bind cell-specific factors; however, the
identity of these factors is not currently known (19). We have recently
demonstrated that the dual CREs are a focal point for regulation via
multiple signal transduction cascades including the protein kinase A
and multiple mitogen-activated protein kinase pathways in
choriocarcinoma cells (20). Thus, with the exception of the dual CREs,
we know relatively little about the cell-specific factors that bind to
and transactivate the
subunit gene in trophoblast cells. The aim of
our studies was to determine the identity and role of the
transcriptional regulator that binds to the JRE.
Regulation of morphogenic changes and differentiation of cell lineages
within many endocrine organs occurs in association with complex
expression patterns of cell-specific transcriptional regulators
including homeodomain-containing transcription factors. Two notable
examples are the organogenesis of the anterior pituitary gland and the
pancreas. Of the many transcription factors described in the ontogeny
of pituitary gland development (21), the LIM homeobox factor Lhx3 (20,
22) and the POU-homeobox factor Pit 1 (23) play key roles in early
determination of pituitary cell lineages that give rise to multiple
endocrine cell types. Furthermore, the homeobox factor Pdx 1 is
necessary for differentiation of multiple cell lineages within the
pancreas (24, 25). Targeted gene disruption of Lhx3 and Pdx 1 lead to
developmental failure of the anterior pituitary gland and pancreas,
respectively, underscoring the importance of these factors to
organogenesis. Naturally occurring mutations in Pit 1 have generated
important mouse models for pituitary dwarfism (26-28). Interestingly,
these homeobox factors also contribute to cell-specific regulation of
gene products that define the differentiated character of cell lineages
within the developing endocrine organ. For example, Lhx3 and Lhx2 have
been shown to regulate the
subunit gene in the anterior pituitary
(29, 30). Pit 1 is a key regulator for the growth hormone, prolactin,
and thyroid-stimulating hormone
genes (23, 31-33). Pdx 1 functions
in the transactivation of the insulin gene (34). The studies presented
in this investigation of trophoblast-specific gene regulation are
completely consistent with this model. It has been previously reported
that the Distal-less class homeobox factor, Dlx 3, is necessary for
normal placental development in a mouse knockout model (35). Mouse
knockout models are not sufficient to determine a role for Dlx factors
in the cell-specific regulation of CG subunit genes since the mouse
does not express a CG. Our studies demonstrate that Dlx 3 is localized to the trophoblast layer of human chorionic villus, and Dlx 3 binds to
and transactivates the human
subunit gene via interaction at the
JRE. JRE/Dlx 3 interactions contribute functionally to the
combinatorial code necessary for placental-specific regulation of the
subunit gene.
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MATERIALS AND METHODS |
Plasmids--
All plasmids were prepared by two cycles through
cesium chloride using standard methodologies. The glycoprotein hormone
subunit reporter has been described previously (20). Mutagenesis of
this reporter was carried out using oligonucleotide-directed mutagenesis as described (20). The JRE mutation consisted of a
four-nucleotide substitution (i.e. wild type 5'-TAATTACA-3'; the JRE mutation 5'-TGGCCACA-3'). This mutation was
confirmed by nucleotide sequence analysis. The Dlx 3 coding sequence
was amplified using High Fidelity polymerase chain reaction (Roche Molecular Biochemicals). The polymerase chain reaction product was
subcloned into the TA cloning vector (Invitrogen, Carlsbad, CA), and
orientation and fidelity were corroborated by DNA sequencing. A FLAG
epitope (DYKDDDDK) was inserted at the 3' end of the coding sequence
immediately upstream of the TGA stop codon using the site direct
mutagenesis kit (Stratagene, La Jolla, CA). The Dlx 3 sequence was then
excised by specific restriction endonuclease digestion
(EcoRI-NotI) and cloned into the pBK-CMV vector
(Stratagene, La Jolla, CA) to create the pCMV/Dlx 3 FLAG construct. DNA
sequence analysis was performed to confirm the Dlx 3 sequence.
Cell Culture and Transfection Studies--
The human
choriocarcinoma cell line, JEG3, was cultured in monolayers using
Dulbecco's modified Eagle's medium supplemented with fetal bovine
serum (10%). All culture media were purchased from Sigma. Fetal bovine
serum was purchased from Life Technologies, Inc. Before all studies,
cell cultures were split to fresh medium, and sub-confluent cultures
were used. All transient transfection studies were conducted using
electroporation as described previously (20). In transfection studies
requiring agonist administration, cells were treated with forskolin (1 µM) and/or EGF (50 ng/ml) for 6 h before collection
(18 h after electroporation). Forskolin was purchased from Sigma and
resuspended at a stock concentration of 1 mM in dimethyl
sulfoxide. Epidermal growth factor was purchased from Life
Technologies, Inc. and resuspended at a stock concentration of 1 mg/ml
in Dulbecco's phosphate-buffered saline. After cell collection,
lysates were prepared by three freeze-thaw cycles and clarified by
centrifugation, and luciferase activity was determined as described
(11, 20, 36). Luciferin was purchased from Promega (Madison, WI).
Luciferase activity was standardized by total protein amount, and all
transfection studies were conducted in triplicate on at least three
separate occasions with similar results. Data shown are reported as a
mean (n = 3) ± S.E of the mean.
Preparation of Recombinant Lhx2 Homeodomain, Dlx 3, and JEG3 Cell
Nuclear Extracts--
The homeodomain for Lhx2 (coding sequence from
lysine 147 through the carboxyl termini of the protein) was prepared as
a polyhistidine fusion protein in bacteria as described (29). Lhx2
homeodomain was partially purified from bacterial lysates using a
nickel chelate-agarose. Full-length Dlx 3 was prepared using a rabbit
reticulocyte lysate system. A transcription and translation
reticulocyte lysate kit was purchased from Promega and was used as per instructions.
Subconfluent JEG3 cells were used for the preparation of nuclear
extracts. Plates were placed on an ice bed, and cells were washed with
ice-cold 10 mM Hepes (pH 7.4) and 150 mM NaCl
(Hepes-buffered saline). Cells were collected by scraping in ice-cold
Hepes-buffered saline and pelleted by centrifugation (2000 rpm for 15 min). Cells were resuspended in a hypotonic buffer and lysed by
douncing, and nuclei were isolated using the sucrose cushion method as
described previously (20). Nuclei were resuspended in a buffer
containing 10 mM Tris (pH 7.5), 50 mM NaCl, 5%
glycerol, 1 mM EDTA, 1 mM dithiothreitol, 5 mM benzamadine, and 0.2 mM phenylmethylsulfonyl fluoride. This buffer was referred to as binding buffer. Nuclear proteins were extracted by adding additional NaCl (in binding buffer)
to a final concentration of 450 mM and incubating the extract for 30 min at 4 °C with constant rocking. Nuclear debris was
removed by centrifugation, and protein concentration was determined by
Bradford assay. Nuclear extracts were separated into aliquots and
stored at
80 °C until later use.
Electrophoretic Mobility Shift Assay--
Electrophoretic
mobility shift assays were conducted essentially as described (11, 29).
Briefly, recombinant Lhx2 homeodomain, recombinant Dlx 3, or binding
activity from nuclear extracts was mixed with binding buffer, 1 µg of
poly(dI-dC), and in some reactions, antiserum or competition
oligonucleotides in a total reaction volume of 25 µl. Reactions were
maintained at room temperature for 20 to 30 min. JRE oligonucleotides
were radiolabeled using polynucleotide kinase and
[
-32P]ATP. Labeled JRE probe (~10,000-20,000 cpm)
was then added, and the incubation was continued for 20-30 min at room
temperature. The binding reactions were resolved on native
polyacrylamide gels. The gels were dried, and DNA-protein complexes
were visualized by autoradiography. All DNA binding studies were
conducted at least twice with similar results.
Western Blot Analysis--
JEG3 or
T3-1 cell nuclear
extracts were suspended in an equal volume of 2× SDS loading buffer
(100 mM Tris (pH 6.8), 4% sodium dodecyl sulfate, 20%
glycerol, and 200 mM dithiothreitol). Protein samples were
boiled for 5 min and chilled briefly on ice before loading on gels.
Proteins were resolved by SDS-polyacrylamide gel electrophoresis and
transferred to polyvinylidine difluoride membranes by electroblotting.
Membranes were blocked with nonfat dried milk (5%) in Tris-buffered
saline (10 mM Tris (pH 7.5), 150 mM sodium
chloride) containing 0.1% Tween 20 (TBST). Lhx2 antiserum was obtained
from rabbits immunized with a purified glutathione
S-transferase fusion of the homeodomain region of Lhx2 as
previously reported (37). The Lhx2 antibody was used at 1:15,000 in
TBST, 5% nonfat dried milk. Rabbit anti-Dlx3 polyclonal antiserum was
generated by Berkeley Antibody Company (Berkley, CA), against a
16-mer synthetic peptide containing amino acids 242 through 256 of the
murine Dlx3 protein. Sequence comparison with other Dlx family members
of several species showed homology in this area only between Dlx 3 orthologs. There is a very high amino acid conservation between the
mouse Dlx 3 and human Dlx 3. There are only eight amino acid
substitutions throughout the entire protein when comparing murine and
human Dlx 3. IgG anti-Dlx3 antibodies were obtained by running the
polyclonal antiserum through a protein A column, washing, and eluting
with a change in pH and salt concentration as directed by Pierce
(ImmunoPure IgG Purification Kit). The Dlx 3 antibody was used at 1:500
in TBST, 5% nonfat dried milk. Proteins were visualized by
chemiluminescence using reagents purchased from PerkinElmer Life Sciences.
Immunocytochemical Analysis--
JEG3 cells were plated on
poly-L-lysine-coated glass slides and cultured overnight.
The cells were fixed in ice-cold methanol and allowed to dry for ~20
min. Human chorionic villi from placenta obtained at 8 weeks of
gestation were fixed in 3% paraformaldehyde, infiltrated with 5-15%
sucrose, placed in embedding medium, and frozen in liquid
nitrogen. Sections (5-7 µm) were cut using a cryostat and
transferred to charged glass slides as described (38). The slides were
stored at
80 °C until use. For immunocytochemistry, slides and
sections were allowed to dry for ~20 min, then dehydrated using 70%
ethanol for 10 min at room temperature. Slides and sections were
blocked for endogenous peroxidase in a solution of 1.5% hydrogen peroxide in absolute methanol for 10 min at room temperature. Antigen
retrieval was not necessary for slides of fixed JEG3 cells. Antigen
retrieval for chorionic villus sections included boiling the sections
in 0.01 M sodium citrate (pH 6.0) for ~5 min. The sections were cooled for 20 min and then washed 3 times (5 min each)
with deionized water. The sections were then treated with a trypsin
solution (0.1% trypsin, 0.1% CaCl2, 20 mM
Tris (pH 7.8)) for 5 min at room temperature followed by washing in
deionized water as described above. The sections were equilibrated in
0.01 M PBS (pH 7.5) for 5 min.
The slides and sections were then incubated with serum-blocking
solution (Histostain SP Kit, Zymed Laboratories Inc.,
South San Francisco, CA) for 10 min at room temperature to eliminate nonspecific background staining. Rabbit-derived anti-Dlx3 in a dilution
of 1:50 in PBS was used as a primary antibody. In chorionic villus
sections, murine-derived pan-cytokeratin antibody (Zymed Laboratories Inc.) was used as a marker for cells of the
trophoblast lineage. Normal rabbit serum (preimmune) was used as
negative control. Incubation with primary antisera was carried out for 4 h at room temperature followed by washing with 0.01 M PBS (pH 7.5) three times (2 min each). The samples were
next incubated with biotinylated secondary antibodies for 20 min at
room temperature and then washed with PBS as described above. After
exposure to secondary antibody, the samples were incubated with a
strepavidin peroxidase conjugate for 10 min at room temperature and
washed with PBS as described above. A chromogen solution was applied to
the samples. The reaction was observed under a microscope and blocked
with deionized water after 8 min.
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RESULTS |
Mutations within the Junctional Regulatory Element Reduce Basal
Expression of an
Subunit Reporter Gene--
The regulatory regions
of the human
subunit gene promoter necessary for expression in
trophoblast cell models such as choriocarcinoma cells are depicted in
Fig. 1A. The dual CREs play a
central role in basal and cAMP-induced regulation of the
subunit
promoter. However, the contribution of the dual CREs to basal
expression of the
subunit also depends upon interaction among
several other cis regulatory elements including the URE and the JRE
(10, 19). Portions of the URE and the JRE putatively bind
tissue-specific factors thought to be necessary for placental-specific
gene activation (10, 19, 39). A four-nucleotide mutation was placed
within the JRE and used in the context of an
subunit reporter in
transient transfection studies in JEG3 choriocarcinoma cells. These
studies revealed that the JRE was required for full basal expression of the
subunit reporter gene despite the presence of wild type dual
CREs (Fig. 1B).

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Fig. 1.
A mutation in the junctional regulatory
element reduces basal expression of the subunit promoter in choriocarcinoma cells. Panel
A depicts the 5'-flanking region of the human subunit gene.
These sequences reflect the subunit reporter gene used. Key
features of the promoter include the URE, dual CRE, JRE, and the CAAT
box. The TATAA box is underlined. In panel B,
JEG3 cells were transiently transfected with either wild type or a
mutant form (JRE Mut) of the human subunit reporter gene
(500 ng). Approximately 24 h later, luciferase activity was
determined. The representative data shown are from one of three
experiments conducted in triplicate at different times. Data are
reported as means ± S.E.
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The Junctional Regulatory Element Can Serve as a Homeobox
Factor-binding Site--
Examination of the nucleotide sequence of the
JRE revealed marked similarity to the downstream half-site of the
pituitary glycoprotein hormone basal element (Fig.
2A). The PGBE is an imperfect palindromic sequence required for basal and GnRH-induced activation of
the human and murine
subunit genes in the anterior pituitary gland
(10, 12, 29). The PGBE is a binding site for a LIM class of homeobox
factor, where Lhx3 and Lhx2 have been shown to bind and induce
transcriptional activation (29, 30). Based upon this similarity, we
speculated that the JRE could serve as a homeobox-binding site. To test
this hypothesis, EMSA was performed using a radiolabeled JRE
oligonucleotide and the Lhx2 homeodomain (recombinant DNA binding
domain) prepared and partially purified from a bacterial expression
system (Fig. 2B; Ref. 29). Nonradioactive competitor
oligonucleotides were added to some reactions at a 200-fold molar
excess. Competitors included wild-type JRE, a JRE probe containing a
four-nucleotide substitution mutation that disrupts basal expression of
the
subunit reporter (Fig. 1B) or the PGBE. Competition
studies revealed that Lhx2 homeodomain binding to the JRE was specific
since the wild type JRE and PGBE oligonucleotides successfully competed
for binding, whereas the mutant JRE was ineffective. The JRE-Lhx2
homeodomain complex was confirmed using an Lhx2-specific antibody (37)
that resulted in the formation of a "supershift" (Fig.
2B). These studies provide direct evidence that the JRE can
serve as a homeobox-binding site in vitro.

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Fig. 2.
The JRE can serve as a homeobox-binding site
in vitro. Panel A depicts the
similarity between the nucleotide sequences of the JRE
(JREwt), the PGBE, and a mutant JRE containing a
four-nucleotide substitution mutation (JREmut). EMSA
was used to determine whether the JRE could bind a recombinant
homeodomain from Lhx2 (Lhx2homeobox; panel
B). Lhx2homeobox was prepared in bacteria and
partially purified. Shown are binding reactions containing radiolabeled
JRE (JRE Probe) and Lhx2homeobox in the
absence or presence of a 200-fold molar excess of unlabeled
oligonucleotides for wild type JRE (JREWT), the
PGBE, or the JRE mutant (JREmut). In some binding
reactions, antiserum directed against Lhx2 (Lhx2 ab) was
used to determine the specificity of JRE binding compared with NRS.
Lhx2 antibody induced a supershifted complex. Endogenous JEG3 cell
nuclear extracts were used with the labeled JRE to determine binding
activity present in choriocarcinoma cells (panel C). Similar
competition studies were conducted using the unlabeled oligonucleotides
described above. The JRE binding reactions resulted in the formation of
two discrete complexes, complex 1 and 2, identified by the
arrows.
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We characterized the JRE binding activity in JEG3 cells by EMSA.
Nuclear extracts were prepared from JEG3 cells and used in DNA binding
reactions containing the radiolabeled JRE binding site. Again,
competition studies were used to determine the specificity of binding
as described above. EMSA revealed that JRE binding with JEG3 cell
nuclear extracts resulted in the formation of two distinct DNA-protein
complexes (designated complex 1 and 2). Competition with wild type JRE
or the PGBE oligonucleotides abolished formation of both complexes. In
contrast, the mutant JRE failed to disrupt JRE binding to JEG3 cell
nuclear extracts, suggesting that the complexes formed were specific to
the central core of the JRE binding site (Fig. 2C).
Dlx3 Is Present in Human Choriocarcinoma Cells--
A number of
homeobox transcription factors have been localized to the developing
murine and human placenta and are thought to be important in placental
cell differentiation and function (40, 41). Recently, targeted deletion
of a member of the Distal-less class of homeobox factors, Dlx 3, resulted in embryonic lethality characterized by a failure in the
development of the murine placenta (35). The reported consensus-binding
site for Dlx 3 is identical to the JRE (42). Fig.
3A depicts the known
structural domains of Dlx 3 including the homeodomain that likely
serves as the DNA binding domain and two putative transcriptional
activation domains (42). Dlx 3 is expressed in JEG3 cells as measured
by Western blot analysis of nuclear extracts but not in nuclear
extracts from
T3-1 cells, a clonal gonadotrope cell line that
expresses the
subunit gene (Fig. 3B). As a positive
control for
T3-1 nuclear extracts, Lhx2 immunoreactivity was
determined and was readily apparent in Western blots. Dlx 3 has an
apparent molecular mass of 38-39 kDa, which is slightly larger than
predicted by the amino acid sequence. Additional immunocytochemical
studies revealed that Dlx 3 expression in JEG3 cells was
compartmentalized to the nucleus of this trophoblast cell model (Fig.
3C).

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Fig. 3.
Dlx 3 is present in choriocarcinoma
cells. Dlx 3 is a homeodomain-containing transcription factor
characterized by the presence of the consensus WFXNXR motif
in the third helix of the homeodomain (HD; panel
A). Two putative activation domains (AD) have been
identified (AD-1 and AD-2; Ref. 42). Western blot
analysis was conducted using JEG3 and T3-1 cell nuclear extracts to
determine the expression of Dlx 3 and Lhx2 (identified by the
arrows; panel B). Molecular size standards are
denoted on the left side of the blots. Cultured JEG3 cells were fixed
and examined for expression of Dlx 3 using immunocytochemistry
(panel C). The JEG3 cell morphology was visualized by
hematoxylin staining (left panel). Dlx 3 primarily stained
the nuclear compartment of JEG3 cells (center panel;
identified by the arrow). Preimmune normal rabbit serum
(right panel; preimmune serum) was used as a control.
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Dlx 3 Expression Is Restricted to the Nuclear Compartment of
Trophoblast in 8-Week Human Chorionic Villus--
We sought to
localize expression of Dlx 3 in human chorionic villus obtained during
the first trimester of gestation (Fig. 4), a time when
subunit expression
and CG secretion are relatively high. Eight-week human chorionic villus
samples were obtained, fixed, and sectioned. The chorionic villus was
visualized by hematoxylin staining, demonstrating a
cytokeratin-positive trophoblast layer surrounding the central villus
core. Dlx 3 expression was restricted primarily to the trophoblast of
the villus and was clearly compartmentalized to the nucleus (Fig. 4).
Villi stained with preimmune normal rabbit serum were essentially
devoid of signal. Consistent with the murine model (35), these
immunocytochemical studies provide evidence that Dlx 3 is present in
human trophoblasts at a time during early gestation when CG is
synthesized and secreted.

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Fig. 4.
Dlx 3 is compartmentalized to the nucleus in
trophoblast cells in first-trimester human chorionic villus.
Immunocytochemistry was used to determine the subcellular
compartmentalization of Dlx 3 in first trimester chorionic villus
samples. Sections from human chorionic villus samples obtained at 8 weeks of gestation were assessed using hematoxylin stain (upper
left panel), antibodies against cytokeratin (a marker for the
trophoblast lineage; upper right panel), or Dlx3
(lower left panel). Control sections (lower right
panel) were incubated with normal rabbit serum. Arrows
identify staining in the cytokeratin-positive trophoblast cell
layer of the villus and the Dlx 3-positive nuclei within the
trophoblast.
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Dlx 3 Binds to the Junctional Regulatory Element of the
Subunit
Promoter--
Recombinant full-length Dlx 3 was synthesized in
reticulocyte lysates and subjected to EMSA to determine whether Dlx 3 could bind to the JRE of the
subunit promoter. Dlx 3 formed a
single DNA-protein complex over a range of doses of recombinant Dlx 3 (Fig. 5A). Additionally, EMSA
performed with the JRE probe and control reticulocyte lysates
expressing luciferase did not produce any DNA-protein complexes (data
not shown), suggesting that JRE binding is specific to recombinant Dlx
3 expression in this system. Competition studies in EMSA using
recombinant Dlx 3 (Fig. 5B) revealed a similar pattern of
DNA-protein interactions compared with EMSA conducted with JEG3 cell
nuclear extracts (Fig. 2). Unlabeled, wild type JRE and PGBE
effectively competed for Dlx 3 binding to the radiolabeled JRE. The
mutant JRE oligonucleotide did not compete for JRE binding. These
studies suggested that Dlx 3 binding to the JRE was specific. Direct
comparison of JRE binding to recombinant Dlx 3 and nuclear extracts
from JEG3 cells revealed that the recombinant Dlx 3-JRE complex
migrated with the same relative electrophoretic mobility as complex 1 from JRE binding to JEG3 cell nuclear extracts (Fig.
5C).

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Fig. 5.
Recombinant Dlx 3 specifically binds to
the JRE. Recombinant Dlx 3 was synthesized in rabbit reticulocyte
lysates and subjected to EMSA. Panel A depicts the result of
addition of increasing doses of recombinant Dlx 3 into the binding
reaction. Competition studies were used to determine the specificity of
JRE binding by recombinant Dlx 3 (panel B). Binding
reactions containing radiolabeled JRE and recombinant Dlx 3 received no
additional oligonucleotide or a 200-fold molar excess of unlabeled wild
type JRE (JREWT), mutant JRE
(JREmut), or PGBE. The experiment depicted in
panel C compared JRE binding to recombinant Dlx 3 and JRE
binding activity in JEG3 cell nuclear extracts. Recombinant Dlx 3 was
added at a single dose, whereas two doses of JEG nuclear extracts were
used. JRE complexes using nuclear extracts are denoted as Complex
1 and 2 and are indicated by arrows. The
lanes marked No Protein represent binding
reactions in the absence of recombinant Dlx 3 or JEG3 cell nuclear
extracts, and unbound JRE probe is denoted as JRE probe in
each panel.
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The Dlx 3 Antibody Recognizes an Epitope within the Endogenous JRE
Binding Complex--
The appropriateness of the Dlx 3 antibody for use
in EMSA was initially investigated using recombinant Dlx 3 and the JRE. Binding reactions included normal rabbit serum (NRS), the Dlx 3 antibody, or an antibody directed against Lhx2 (37). Consistent with
previous control studies, interactions between the JRE, recombinant Dlx
3, and NRS resulted in the formation of a single complex (Fig. 6A). Replacement of NRS with
the Dlx 3 antibody generated a complex with slower electrophoretic
mobility, characterized as a marked supershift in the JRE-Dlx 3 complex. The addition of the Lhx2 antibody gave results similar to the
NRS control. These studies provide evidence for the specificity of the
Dlx 3 antibody in EMSA. We then examined whether the Dlx 3 antibody
would alter JRE-nuclear protein interactions using extracts from JEG3
cells. Again, JRE-nuclear protein interactions resulted in the
formation of two complexes (Fig. 6B). The addition of the
Dlx 3 antibody specifically disrupted the formation of complex 1. The
addition of either Lhx2 antibody or NRS did not reduce complex 1 and,
in the case of Lhx2 antibody, slightly enhanced binding. It is unclear from these studies whether a supershift formed in the presence of the
Dlx 3 antibody since the supershifted complex that formed with
recombinant Dlx 3 had a similar electrophoretic mobility as complex 2 from JEG3 nuclear extracts. There did appear to be a slight increase in
activity present in complex 2 in the presence of the Dlx 3 antibody,
suggesting that this may have been due to a supershift of complex 1. These experiments provide compelling evidence that Dlx 3 or a highly
related epitope is present in the JRE binding complex associated with
complex 1.

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Fig. 6.
Dlx 3 is present in the JRE binding complex
in JEG3 cell nuclear extracts. The Dlx 3 antibody was used in
control experiments to determine the efficacy of the antibody in EMSA.
Recombinant Dlx 3 was used in binding reactions with radiolabeled JRE
in the presence of NRS, Dlx 3 antibody (Dlx 3 ab), or Lhx2
antibody (Lhx2 ab) (panel A). The Dlx 3 antibody
induced a supershift as denoted. In a separate experiment (panel
B), JEG3 cell nuclear extracts (JEG NE) were used in
EMSA in the absence or presence of Dlx 3 antibody, Lhx2 antibody, or
NRS. Two JRE protein complexes are denoted as Complex 1 and
2. The lanes marked No Protein
represent binding reactions in the absence of recombinant Dlx 3 or JEG3
cell nuclear extracts, and unbound JRE probe is denoted as JRE
probe in each panel.
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Overexpression of Dlx3 Is Sufficient to Activate the
Subunit
Promoter in JEG3 Cells--
To determine whether Dlx 3 could function
as a transcriptional regulator in JEG3 cells, overexpression studies
were conducted using an expression vector for full-length murine Dlx 3. Overexpression of Dlx 3 has been reported to activate transcription of
a multimer of a consensus Dlx 3 binding site cloned upstream of a
minimal promoter in a heterologous system (42). JEG3 cells were
transiently cotransfected with a wild-type
subunit reporter (wild
type) or an
subunit reporter containing a mutation within the JRE (Fig. 7A, JRE Mut) and control plasmid or
increasing doses of the Dlx 3 expression vector. The following day,
luciferase activity was determined (Fig.
7A). Transcription of the
wild-type
subunit reporter increased with increasing doses of the
Dlx 3 expression vector. Similar increases were not evident using the
subunit reporter containing the mutation within the JRE. Thus,
these studies provide evidence of the potential for Dlx 3 to regulate
transcription of the
subunit promoter via the JRE. These studies
certainly do not discount the possibility that in addition to
endogenous Dlx 3, other uncharacterized factors (contributing to
complex 2) may also be involved in the regulation of the
subunit
promoter in JEG3 cells.

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Fig. 7.
Overexpression of Dlx 3 induces activation of
the subunit reporter. Transient
cotransfection studies in JEG3 cells were used to determine the effects
of Dlx 3 overexpression on subunit reporter gene activity
(panel A). JEG3 cells were cotransfected by electroporation
with either wild type or a mutant form (JRE Mut) of the
human subunit reporter gene (500 ng). Some transfections included
control vector or increasing doses (2.5 or 5.0 µg) of the Dlx 3 expression vector. All transfections contained similar amounts of total
DNA (5 µg) standardized by adding control vector to the
electroporation cuvette. Approximately 24 h later, luciferase
activity was determined. Data are reported as fold induction ± S.E. Panel B depicts similar transfection studies, conducted
using the human subunit reporter in the absence (control) or
presence of 5 µg of Dlx 3 expression vector. Approximately 18 h
later, transfected cells received control solution, EGF (50 ng/ml),
forskolin (1 µM), or the combination of EGF and forskolin
as denoted by the legend. Luciferase activity was determined after
6 h of agonist treatment. Transient transfection in JEG3 cells was
used to determine the effects of a mutation in the JRE on activation of
the subunit reporter gene by EGF and forskolin (panel
C). JEG3 cells were transfected by electroporation with either
wild type or a mutant form (JRE Mut) of the human subunit reporter gene (500 ng). Cells were treated as described in
panel B. The representative data shown are from one of three
experiments conducted in triplicate at different times. Data are
reported as means ± S.E. Fold induction relative to control
values for this experiment is listed on the horizontal
axis.
|
|
We have recently demonstrated that expression of the
subunit
promoter is potentiated by activation of multiple signal transduction pathways induced by EGF and forskolin (20). The dual CREs of the
subunit promoter are located immediately upstream of the JRE (separated
by two nucleotides; see Fig. 1A) and are required for
activation of the
subunit by EGF and forskolin (20). Our aim was to
determine whether overexpression of Dlx 3 would interfere with
transcriptional activation of the
subunit reporter via inducible
factors that bind to the immediately adjacent CREs. Overexpression of
Dlx 3 increased basal expression of the
subunit reporter but did
not markedly alter response to EGF, forskolin, or the combination of
these two agonists (Fig. 7B). These studies support the
conclusion that Dlx 3 could function as a transcriptional regulator in
this system concurrent with inducible transcriptional activation of the
subunit reporter via the immediately adjacent CREs. Thus, the JRE
and dual CREs can be functionally occupied at the same time.
A Mutation within the JRE Alters EGF- and Forskolin-stimulated
Subunit Gene Regulation--
The previous overexpression studies
provide evidence that Dlx 3 has the potential to serve as a
transcriptional regulator in transfected JEG3 cells. We sought to
determine whether the JRE is required for transcriptional activation of
the
subunit by EGF and forskolin. JEG3 cells were transiently
transfected with a wild type
subunit reporter or a reporter
containing the mutation within the JRE. Transfected cells then received
control solution, EGF, forskolin, or the combination of EGF and
forskolin (Fig. 7C). With the wild-type
subunit
reporter, EGF administration induced a 90% increase in
subunit
promoter activity. Forskolin administration resulted in a 3.7-fold
induction of the
subunit reporter. The combined actions of EGF and
forskolin on the
subunit reporter resulted in a 7.2-fold
activation. A mutation within the JRE of the
subunit promoter
reduced basal activity as observed previously. Interestingly, the JRE
mutation resulted in a reduction in activation by the combined effects
of EGF and forskolin. Thus, JRE interactions with Dlx 3 and other
potential factors (JRE-binding proteins in complex 2) appear to
contribute to activation of the
subunit gene by EGF and forskolin.
The effects of Dlx 3 are likely permissive since overexpression of Dlx
3 did not enhance the effects of EGF and forskolin on the
subunit reporter.
 |
DISCUSSION |
Expression of Dlx 3 is required for development of a normal murine
placenta (35). Our studies provide novel evidence to support the
conclusion that Dlx 3 is likely involved in placental-specific activation of the gene encoding the human
subunit of CG, a hormone critical for maintenance of early pregnancy. Establishment of pregnancy
in mammals requires appropriately timed endocrine communication between
the mother and conceptus. In human and nonhuman primates, secretion of
CG in early pregnancy is critical as a luteotropic signal to maintain
progesterone secretion from the ovarian corpus luteum. Mistimed or
reduced rate of CG synthesis and secretion have been associated with
increased potential for early pregnancy failure and repeated
miscarriage (3, 5, 43-46). The promoter of the
subunit gene
contains a complex array of cis-acting elements that define a
transcription factor "code" required for cell-specific and
hormone-inducible gene regulation. Among the elements required for
expression of the
subunit in cells of the trophoblast lineage are
the URE, dual tandem CREs, and the JRE (10, 19). The present studies
extend our understanding of the transcription factor code by providing
direct evidence that the JRE supports basal expression of the
subunit gene. Furthermore, Dlx 3 is an excellent candidate as a
homeobox factor that binds to and contributes to the transactivation of
the
subunit promoter by direct interaction with the JRE.
The Distal-less family of homeobox factors currently has six members,
Dlx 1-6 (for a comprehensive review, see Ref. 47). A unique quality of
this family of homeobox factors is that they are linked as contiguous
pairs within the genome. For example, Dlx 3 and Dlx 4 colocalize on
human chromosome 17q21, a region closely linked with the HOXB cluster
(48, 49). In mammals, expression of the Dlx 3 gene has been shown to be
restricted to the branchial arches, dental tissues, epithelial
derivatives, and the placenta (47). The Dlx 3 expression pattern
partially overlaps with Dlx 4, which is also found in the placenta (40, 50). Dlx 3 promoter activity was induced by calcium coincident with
keratinocyte differentiation (51). In addition, overexpression of Dlx 3 to the epidermis induced cessation of proliferation and premature or
accelerated differentiation, supporting a role for Dlx 3 during
epidermal differentiation (52). Recently, a naturally occurring
mutation in Dlx 3 was identified and correlated with a condition known
as tricho-dento-osseus (TDO) syndrome (53). This frameshift mutation
resulted in premature termination of Dlx 3, leading to compromised function.
Within the developing mouse placenta, expression of Dlx 3 is restricted
to the ectoplacental cone cells, the chorionic plate and the
labyrinthine trophoblast layer. Targeted deletion of Dlx 3 resulted in
embryonic death between embryonic day 9.5 and 10, due to failure in the
appropriate morphogenesis of the placenta (35). Interestingly, Dlx 3
/
mice also have reduced expression of a paired class homeobox
factor, Esx 1 (35). In normal mice, Esx 1 has been shown to be
expressed in the labyrinthine trophoblasts in a pattern consistent with
Dlx 3 expression, suggesting that Dlx 3 expression may be a
prerequisite for up-regulation of Esx 1 (41). Our immunocytochemical
studies document expression of Dlx 3 compartmentalized to the nucleus
in human choriocarcinoma cells and human trophoblasts during the first
trimester of pregnancy, providing evidence that in vivo Dlx
3 is expressed at the appropriate time and location to contribute to
subunit gene regulation via the JRE. It is tempting to speculate a
developmental role for Dlx 3 in the differentiation of the early human placenta.
A hierachary of regulatory "importance" exists among the array of
cis elements required for
subunit gene transcription in placental
cells (10, 19). The dual CREs are the principal regulatory elements
that confer basal and cAMP inducibility to the
subunit gene in
placental cells (12, 14, 18, 54, 55). Mutations within the CREs reduce
basal expression quite dramatically. The JRE was first described nearly
a decade ago (14). At that time, the JRE binding activity was believed
to be required for placental regulation of the
subunit gene;
however, the binding activity was not believed to be specifically
expressed in the trophoblast cell lineage. Subsequent Southwestern blot analysis revealed that the JRE binding activity was characterized as a
39-40-kDa protein and was specific to choriocarcinoma cells and not
cells of pituitary lineage that also express the
subunit (19). Our
identification of Dlx 3 as a JRE binding factor is completely
consistent with this estimate of molecular size and cell-specific
expression. The Dlx 3 antibody predominantly recognizes a 38-39-kDa
protein in JEG3 cells. Furthermore, expression of Dlx 3 appears to be
restricted to cells of the trophoblast lineage but not
T3-1 cells
that are derived from the gonadotrope lineage of the pituitary gland.
Mutations within the JRE reduced basal
subunit expression despite
the presence of intact dual CREs (19). Thus, despite a principal role
for the dual CREs, basal expression via the CREs requires potential
contributions from the JRE. Overexpression studies revealed that
JRE/Dlx 3 interactions increased basal expression but did not interfere
with agonist-induced
subunit activation via CREB family member
dimers present on the dual CREs immediately upstream of the JRE. These
studies suggest that the JRE and CREs can be functionally occupied at
the same time. Interestingly, mutations within the JRE interfered with
induction of
subunit transcription by the combined actions of EGF
and forskolin. After activation by EGF and forskolin, the CRE binding
complex consists of multiple transcriptional regulators including CREB
and AP-1, which are required for activation of the
subunit by this
combination of agonists (20). In contrast, treatment with
forskolin alone results in recruitment of CREB alone to the CRE
binding complex. It is reasonable to speculate that the formation of
the CRE binding complex associated with the combined actions of EGF and
forskolin on
subunit gene expression may require recruitment of
additional factors such as coactivators, whose presence may depend at
least in part on JRE interactions with factors such as Dlx 3. This
study underscores the notion that the JRE binding complex may influence transcriptional mechanisms mediated at adjacent cis elements
(i.e. the dual CREs). A similar mechanism has been described
in the regulation of the murine
subunit promoter by GnRH in cells
of pituitary lineage (11, 12). Basal expression of the mouse
subunit gene in the pituitary depends on a Lhx homeobox factor binding
to the PGBE. Regulation of mouse
subunit expression by
gonadotropin-releasing hormone requires the PGBE and a more distal
site, the GnRH-responsive element. The GnRH-responsive element is an
Ets factor binding site capable of binding a putatively ubiquitous Ets
factor(s) that is inducible via signaling cascades activated by GnRH.
Thus, a composite transcriptional unit exists where the PGBE binds to a
cell-specific factor (Lhx homeobox factor), and the GnRH-responsive
element binds a non-cell-specific factor, whose activity depends on
activation via signaling molecules induced by GnRH. In the present
studies, the JRE serves as the cell-specific homeobox binding site,
whereas the dual CREs bind more general factors (like CREB and AP-1)
that are regulated by signaling cascades induced by EGF and forskolin.
This conserved strategy for gene regulation may reflect an important
mechanism by which homeobox proteins contribute to inducible,
cell-specific gene regulation in differentiated cells.
Our studies have only accounted for a single factor, Dlx 3, within the
JRE binding complex. However, EMSA reveals that two complexes form at
this site, suggesting that proteins in addition to Dlx 3 likely
contribute to
subunit gene regulation via the JRE. Thus, we cannot
yet rule out the possibility of additional factors involved in the JRE
binding complex that may have critical importance to the regulation of
the
subunit gene. Several possibilities may account for two
complexes. A key observation that led to our studies of Dlx 3 was that
the consensus binding site for Dlx 3 ((A/C/G)TAATT(A/G)(C/G))
was identical to the JRE (bold; Ref. 42). The central core of the Dlx 3 binding site is essentially the preferred recognition sequence for
another family of homeobox factors, the Msx proteins (for review, see Ref. 47). Msx 1, Msx 2, and Dlx 3 are putatively capable of binding
identical nucleotide sequence motifs. Msx 2 is expressed in regions of
the placenta consistent with Dlx 3 during mouse development (40). Most
importantly, Msx proteins have been shown to be transcriptional
repressors that functionally antagonize transcriptional activation via
Dlx proteins (56). Based upon this, the possibility exists for
potential antagonistic interactions between Msx factors and Dlx 3 in
placental cells. In our studies, overexpression of Dlx 3 may have
simply altered this functional antagonism to favor transcriptional
activation. Alternatively, the possibility exists that additional
factors are capable of binding the JRE directly or indirectly by
physical interaction with Dlx 3, independent of DNA binding. Our
studies provide clear evidence that Dlx 3 or a highly related epitope
represents the binding activity in complex 1. JRE binding complex 2 may
reflect binding of a second protein(s) directly with the JRE or
facilitated by protein-protein interactions with Dlx 3. Since the Dlx3
antibody was developed against a peptide, it is possible that Dlx3 may be present in complex 2 but with the epitope functionally blocked due
to protein-protein interactions. Additional studies are necessary to
resolve these possibilities.
The present studies support the conclusion that Dlx 3 or a highly
related epitope binds to and contributes to trophoblast-specific regulation of the
subunit gene. Consistent with findings for the
murine placenta, Dlx 3 is expressed in the cells of the trophoblast lineage within the human placenta during the first third of gestation, a time when expression of CG subunit genes and CG secretion is high.
Our studies provide novel evidence that overexpression of Dlx 3 can
serve to increase basal transcription of the
subunit gene in JEG3
cells. Furthermore, JRE interactions with factors such as Dlx 3 contribute to
subunit gene regulation after administration of EGF
and forskolin to induce potentiated activation. Consistent with
developmental determination of the anterior pituitary gland and the
pancreas, key homeobox proteins (such as Dlx 3) that direct early
developmental decisions in the placenta are also important to
differentiated endocrine cell function by contributing to expression of
cell-specific target genes (such as the
subunit) that define the
differentiated character of that cell type.