Synergistic Cooperation between the
-Catenin Signaling Pathway and Steroidogenic Factor 1 in the Activation of the Mullerian Inhibiting Substance Type II Receptor*
Anwar Hossain and
Grady F. Saunders
From the
Department of Biochemistry and Molecular Biology, The University of Texas
M. D. Anderson Cancer Center, Houston, Texas 77030
Received for publication, January 24, 2003
, and in revised form, April 25, 2003.
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ABSTRACT
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Mullerian inhibiting substance type II receptor (MISRII) is a member of the
transforming growth factor-
superfamily. Mutations in mullerian
inhibiting substance (MIS) or MISRII cause male sexual
abnormalities, persistent mullerian duct syndrome, and pseudohermaphroditism.
The spatial and temporal regulation of MIS and MISRII is
important for its biological action. Male Wnt7a mutant mice do not undergo
regression of mullerian ducts. Here we showed that the canonical Wnt signaling
pathway regulated MISRII. The promoter MISRII was activated
by
-catenin expression, and this activation was dependent on
TCF4-binding sites. The nuclear receptor superfamily member steroidogenic
factor 1 (SF1) synergistically activated the MISRII promoter with
-catenin. APC, a negative regulator of Wnt signaling, decreased
SF1-mediated activation of the MISRII promoter in the colon carcinoma
cell line SW480. We also showed a direct physical interaction between
-catenin and SF1 by co-immunoprecipitation. Thus, our findings suggest
that MISRII is a developmental target of Wnt7a signaling for
mullerian duct regression during sexual differentiation.
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INTRODUCTION
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One of the essential phases in testis development is differentiation of the
wolffian ducts by androgen and regression of the mullerian ducts, which
normally give rise to oviducts, uterus, and fallopian tubes in females
(1). Sertoli cells secrete a
transforming growth factor-
superfamily member, mullerian inhibiting
substance (MIS),1 also
known as anti-mullerian hormone. Its type II receptor, MIS type II receptor
(MISRII), also known as anti-mullerian hormone receptor, is expressed in the
mesenchymal cells surrounding the mullerian ducts during the regression and in
Sertoli cells in testes and granulosa cells in ovaries
(24).
The MIS type I receptor has not been identified; however, bone morphogenic
protein 1a is a strong candidate to be this receptor
(5). Interaction between MIS
and its receptor initiates a signal that ultimately causes regression of the
mullerian ducts through apoptosis by a paracrine mechanism
(6). Mutations in MIS
or its type II receptor cause the human male sexual abnormalities, persistent
mullerian duct syndrome and pseudohermaphroditism
(7), and deletion of
MIS or the MISRII also results in development of
pseudohermaphroditism in mice
(8,
9). Spatial and temporal
regulation of MIS and its type II receptor is important for its
biological action (10). One
study showed that the MISRII promoter is up-regulated by
steroidogenic factor 1 (SF1), a member of the nuclear hormone receptor family
(11).
The Wnt molecules are a large family of secreted glycoproteins that play an
important role in the developmental program in many organisms
(12). Constitutive activation
of Wnt signaling has been linked to developmental defects and tumorigenesis
(13). Targeted disruption of
Wnt7a in mice revealed that it is required for normal dorsoventral
and anteroposterior polarity in the forming limb
(1416).
Wnt7a-deficient mice are infertile because of retained mullerian ducts and,
ultimately, blocked sperm passages in males and abnormal differentiation of
oviducts and uteri in females
(15). There is no expression
of MISRII in Wnt7a mutant mice. It is not clear, however, that
MISRII is a direct target of the Wnt7a signaling pathway
(15).
The Wnt signaling pathway has been studied extensively, biochemically as
well as genetically. The canonical Wnt/
-catenin or noncanonical
Wnt/Ca2+ pathways transduce extracellular Wnt signals
into the nucleus (17). The
canonical Wnt pathway is mediated by stabilized
-catenin and nuclear
TCF/LEF family members, a high mobility group box containing transcription
factor, distantly related to SOX proteins
(12). In the absence of Wnt
signals, free
-catenin is phosphorylated by GSK3
in the cytosol.
The tumor suppressor APC and axin are part of the large multiprotein complex
that facilitates this phosphorylation process
(18). Phosphorylated
-catenin is ubiquinated and ultimately degraded by the proteosome
(19). In the presence of a Wnt
signal, this phosphorylation of
-catenin is blocked, and the free
cytosolic
-catenin is translocated to the nucleus and heterodimerized
with one of the LEF/TCF family members and can activate Wnt-responsive genes
(12). It is still unclear
whether Wnt7a mediates its signal through the canonical or noncanonical
pathway. In this study, we address the question of whether MISRII is
a direct transcriptional target of the Wnt signaling pathway and, if so,
whether the signal is mediated through the canonical or noncanonical Wnt
signaling pathway.
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EXPERIMENTAL PROCEDURES
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PlasmidsMyc-tagged Xenopus
-catenin in the
vector pCS2 and the reporter constructs TOPflash and FOPflash were provided by
Pierre McCrea (The University of Texas M. D. Anderson Cancer Center). The
expression vectors CMV-APC and CMV-TCF4 were provided by Min-Chie Hung (M. D.
Anderson Cancer Center). The construct pcDNA3SF1 was made by PCR amplification
from human testis RNA. The resulting PCR products were subsequently subcloned
into a modified pcDNA3 vector
(20) harboring the
5'-untranslated region of the herpes simplex virus-thymidine kinase
gene.
The 863-bp MISRII upstream sequence, which drove transcription of
the luciferase gene, was amplified by PCR from human genomic DNA with
MISRII promoter nucleotides 800 to +63 and cloned in front of
the luciferase gene in the vector pGL3basic (Promega). This construct was
designated 800MISRPLuc. Deletion constructs were made by using this
construct as a template. All site-directed mutagenesis of the promoter
constructs was performed with the QuikChange site-directed mutagenesis kit
(Stratagene). All constructs were confirmed by sequencing from both
directions.
Cell Culture and TransfectionHeLa and SW480 cells were
grown at 37 °C in Dulbecco's modified Eagle's/F-12 medium supplemented
with 10% fetal calf serum in an atmosphere containing 5% CO2. The
cells were seeded at a density of 50,00070,000 cells/well in 12-well
plates 1618 h before transfection. The cells were cotransfected with
expression and reporter plasmids as indicated in the figure legends. The
plasmid CMV-
-galactosidase was cotransfected as an internal control to
normalize for differences in transfection efficiency. The transfections were
performed with LipofectAMINE Plus reagent (Invitrogen) according to the
manufacturer's recommendations, and the cells were harvested after 4048
h. Luciferase activity was measured with a luciferase assay kit (Tropix) and a
Lumat LB9507 luminometer (EG&G Berthold).
-Galactosidase was
measured with the Galacto-Light plus kit (Tropix).
Gel Shift AssayGel shift reactions were performed in a
total volume of 20 µl on ice. Radiolabeled probes were prepared by
end-labeling with [
-32P]ATP, and 100 pmol of each labeled
probe and 2.5 µl of in vitro translated protein were used for each
reaction. For competition of wild-type and the WT1-binding site-specific
oligonucleotides, a 100-fold excess of unlabeled oligonucleotides was added to
the reaction mixture before addition of the labeled probe. Thirty minutes
later, the reaction mixture was loaded onto a 5% polyacrylamide gel in Tris
glycine buffer, and electrophoresis was performed at 150 V for 3 h.
Western BlottingWhole cell extracts were prepared with cell
lysis buffer consisting of 50 mM Tris-HCl (pH 8.0), 150
mM NaCl, 1.0% Nonidet P-40, 1 mM dithiothreitol, 1
mM phenylmethylsulfonyl fluoride, and 5 µg/ml each aprotinin,
leupeptin, and benzamidine. Western blots were developed by enhanced
chemiluminescence (Amersham Biosciences). The primary and secondary antibodies
used were rabbit anti-TCF4 at a 1:100 dilution and anti-goat IgG conjugated
with peroxidase (Santa Cruz Biotechnology).
Chromatin Immunoprecipitation AssayFormaldehyde was added
to TCF4-expressing HeLa cells at a final concentration of 1%. Fixation was
allowed to proceed at room temperature for 15 min and was stopped by addition
of glycine to a final concentration of 0.125 M. The cells were then
washed with phosphate-buffered saline and collected by centrifugation. The
cells were incubated with buffer A (10 mM potassium acetate, 15
mM magnesium acetate, and 0.1 mM Tris (pH 7.4) with
Roche protease inhibitor mixture) on ice for 20 min and homogenized with a
Dounce homogenizer. The nuclei were collected by centrifugation, resuspended
in sonication buffer, and incubated on ice for 15 min. The samples were
sonicated on ice with an Ultrasonics sonicator at a setting of 10 for six 20-s
pulses to an average length of
1000 bp (confirmed by electrophoresis) and
microcentrifuged. The chromatin solution was precleared with protein
A-Sepharose (Pierce) for 15 min at 4 °C. Immunoprecipitations (IPs) were
performed overnight at 4 °C with 1 µg of anti-TCF4 antibody. After the
final ethanol precipitation, each IP sample was resuspended in 30 µl of TE.
Total input chromatin samples were resuspended in 30 µl of TE and further
diluted 1:100. Each 50 µl of PCR mixture contained 5 µl of IP sample,
1.5 mM MgCl2, 50 ng of each primer, 300 µM
each dATP, dGTP, dCTP, and dTTP, 1x PCR buffer (PerkinElmer Life
Sciences), and 1.25 unit of TaqDNA polymerase (PerkinElmer Life
Sciences). After 35 cycles of amplification, 5 µl of each of the PCR
products was subjected to electrophoresis on a 1.5% agarose gel, and the DNA
was stained with ethidium bromide and visualized under UV light. The sequences
of primers used for PCR were as follows: MISRP-F1, CAGGCCTCTGCAGTTATG;
MISRP-R1, CATGGTGGTACAGCAAGG; MISRP-F2, CTGGGTTCTCAGCTGGGCCTC; MISRP-R2,
AGCCAGCACAGCTGCCCCTG; MISRII-int10F, TGCCCCATCTGCTCTCCTAATACA; MISRII-int10R,
AGCCTCCCTCCTCTCCCTCTTG.
Co-immunoprecipitation AssayFLAG-tagged SF1 was transfected
in SW480 cells, and whole cell extracts were diluted in modified RIPA buffer
and co-immunoprecipitated with rabbit anti-SF1 (Upstate Biotechnology) or
rabbit control IgG (Santa Cruz Biotechnology) antibody. Immunocomplexes were
collected with Protein A/G-Sepharose (Pierce), separated by 420%
SDS-PAGE, and immunoblotted with rabbit anti-
-catenin antibodies (Santa
Cruz Biotechnology). Western blots were developed using SuperSignal West Femto
Maximum Sensitivity Substrate (Pierce).
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RESULTS
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MISRII Promoter a Direct Target of the Wnt Signaling
PathwayTo determine whether
-catenin and TCF4, the
intracellular mediators of the Wnt signaling pathway, can activate a
luciferase reporter gene driven by the MISRII promoter, we
transfected 800MISRPLuc into HeLa cells. Cotransfection of the
luciferase reporter gene with constitutively active
-catenin resulted in
5-fold greater activation of the reporter gene
(Fig. 1). The constitutively
active
-catenin had mutated phosphorylation sites and therefore escaped
APC-mediated degradation. TCF4 alone cannot activate this promoter, because
-catenin is not constitutively active in HeLa cells. Consistent with
previous work on other Wnt-responsive genes, coexpression of TCF4 with
-catenin increased the reporter gene activity. However, coexpression of
-catenin and TCF4 did not dramatically increase luciferase activity.
This was mostly because of the high level of expression of endogenous TCF4 in
HeLa cells (21). In the
negative control pGL3basic vector,
-catenin and TCF4 did not increase
reporter activity (Fig. 1). This indicates that the MISRII promoter is responsive to the
canonical
-catenin/TCF signaling pathway.
Identification of TCF-binding Sites in the MISRII
PromoterTo identify the binding site in the MISRII
promoter that is responsive to
-catenin, we generated a series of
deletion mutants of the MISRII promoter based on known TCF/LEF-binding sites.
Luciferase activity was up-regulated by severalfold by cotransfection of
-catenin and TCF4 with full-length MISRII promoter containing
luciferase reporter genes (Fig.
2). Progressive deletion of sequences within the promoter
gradually decreased the reporter gene activity. This implies that more than
one TCF4-binding site is present in this promoter. In contrast, that the
construct containing nucleotides 170 to +63 was not responsive to
-catenin and TCF4, which indicates that sequences essential for
activation of the MISRII promoter lie between bases 800 and
170 (Fig. 2).

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FIG. 2. Identification of TCF4-binding sites in the MISRII
promoter. The reporter constructs from the MISRII promoter are
shown on the left. Potential TCF4-binding and SF1-binding sites are
indicated. These promoter deletion constructs were transfected into HeLa cells
as described in the legend to Fig.
1.
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TCF-binding Sites Essential for
-Catenin-mediated
Activation of the MISRII PromoterBetween nucleotides 800
and 170 within the MISRII promoter, there are four potential
TCF-binding sites, designated TCF-RE 14, and one SF1-binding site
(Fig. 3A). These
binding sites are similar to the consensus TCF/Lef-binding sites. To determine
the relative contribution of each of these sites to the
-catenin
responsiveness of the MISRII promoter, we introduced point mutations
in each of these binding sites. We measured luciferase reporter gene activity
driven by each of these mutant-binding sites and by the wild-type promoter in
the presence or absence of TCF4 and
-catenin. Mutation in any one of the
binding sites did not reduce overall responsiveness to
-catenin.
However, mutants TCF-M1 and TCF-M4 reduced MISRII promoter
responsiveness to
-catenin significantly
(Fig. 3B).
Introduction of mutations in both TCF-binding sites 1 and 2 further reduced
the reporter gene activity. However, this reporter construct still showed some
responsiveness to
-catenin. Mutations in binding sites 2 and 3 did not
induce any drastic changes in reporter gene activity, although these two
binding sites did contribute some of the responsiveness to
-catenin.
When mutations were made in all four TCF-binding sites, the reporter gene lost
its responsiveness to
-catenin (Fig.
3B). We concluded from these analyses that the
responsiveness of the MISRII promoter to
-catenin is dependent
on all four TCF-binding sites and that binding sites 1 and 4 are most
critical.

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FIG. 3. Contribution of each TCF4-binding site to activation of the MISRII
promoter. A, potential TCF4- and SF1-binding site sequences in
the MISRII promoter and its mutant derivatives. The numbers indicate
the position of each binding site relative to the transcription start site.
B, the wild-type reporter constructs from the MISRII
promoter and its mutant derivatives are shown in on the left.
Mutant-binding sites are represented by cross-marks. These constructs
were transfected into HeLa cells as described in the legend to
Fig. 1.
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TCF4 Binding in Vitro to the TCF-binding Sites of the MISRII
PromoterTCF-binding sites 1 and 4 were further characterized by
gel shift assays. The gel shift assay in
Fig. 4 shows that TCF4
synthesized in vitro bound to a 32P-labeled
oligonucleotide probe for TCF-binding sites 1 and 4 (TCF-RE 1 and 4). Binding
was competed by unlabeled wild-type probe but not by a WT1-binding site probe
(Fig. 4). These data suggest
that TCF-binding sites 1 and 4 in the MISRII promoter are essential
for the binding of TCF4 to the promoter as well as for
-catenin-mediated
transactivation of the promoter.

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FIG. 4. TCF4 binding to TCF-binding sites 1 and 4 in the MISRII promoter.
Gel mobility shift assays were performed with radiolabeled TCF-binding sites 1
and 4 (TCF-BS 1 and 4) containing oligonucleotides and
incubated with 5 µl of in vitro translated TCF4. DNA-protein
complexes were separated by electrophoresis. Unlabeled oligonucleotides
(TCF-RE 1 and 4) (100x competitor) or the WT1-binding
site oligonucleotides (100x nonspecific competitor) were used as
competitors in some reactions.
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To fully address the possibility that regulation of MISRII gene
expression is mediated by
-catenin, we used the chromatin
immunoprecipitation assay to determine whether TCF4 can bind the endogenous
MISRII promoter. PCR analysis of formaldehyde-cross-linked chromatin
from HeLa cells immunoprecipitated with antibodies specific to TCF4 revealed
that TCF4 can indeed bind to the MISRII promoter sequences, whereas
rabbit IgG antibody controls did not (Fig.
5). Primers for MISRII intron 10 were used as a negative
control to show the specificity of the chromatin immunoprecipitation assay.
The expected 436-bp bands from MISRII intron 10 were not present in the
immunoprecipitated DNA by anti-TCF4 and control antibody, which implies that
TCF4 antibodies specifically immunoprecipitated only the MISRII
promoter-bound TCF4-DNA complex, not a big fragment of genomic DNA
(Fig. 5).

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FIG. 5. TCF4 binding to the MISRII promoter in vivo in HeLa cells.
Cross-linked chromatin from HeLa cells was incubated with antibodies to the
N-terminal region of TCF4. The immunoprecipitated DNA was analyzed by PCR with
primers spanning TCF-binding sites 1 and 4 ( TCF4). PCR was
performed with DNA obtained from IP with rabbit IgG as control (PIS).
As a negative control, PCR was performed with primers for MISRII
intron 10 and the same DNA obtained from IP with TCF4-specific antibody and
with rabbit IgG (PIS) (fifth and sixth lanes). As a
positive control input DNA was amplified by PCR with primers for
MISRII intron 10 along with primers spanning TCF-binding sites 1 and
4 (middle panel). The first lane contains a 100-bp DNA
ladder.
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SF1 and
-catenin-TCF4 Synergistically Activated the
MISRII PromoterThere is an SF1-binding site in the MISRII
promoter. Addition of exogenous SF1 increases luciferase activity driven by
the MISRII promoter in the teratocarcinoma cell line NT2D1
(11). However, SF1 does not
activate luciferase in R2C2, a rat Leydig cell line, although endogenous SF1
protein occupies the SF1-binding sites in these cells
(22). This suggests that SF1
alone cannot activate this promoter in the R2C2 cells and that a necessary
cofactor is missing. To address this possibility, we transfected SF1 with
various combinations of reporter and effector plasmids into HeLa cells. In the
absence of
-catenin expression, SF1 had no effect on the MISRII
promoter, even in the presence of TCF4
(Fig. 6). In the presence of
-catenin, however, SF1 synergistically activated this promoter.
Furthermore, coexpression of
-catenin, TCF4, and SF1 resulted in
superinduction of this promoter (Fig.
6A). However, mutation in the SF1-binding site abrogated
this synergistic activation (Fig.
6B). This result suggests that
-catenin is the
necessary factor for activation of the MISRII promoter.
We also examined the effects of SF1 on TOPflash, a Wnt-responsive reporter
that contains three multimerized TCF-binding sites. In contrast to its
synergistic activation of the MISRII promoter, SF1 had no effect on
-catenin/TCF-mediated induction of luciferase activity from the TOPflash
reporter (Fig. 7). Thus, SF1 is
not a general cofactor for
-catenin/TCF, and SF1 only increases
-catenin signaling from the MISRII promoter.

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FIG. 7. SF1 had no effect on TOPflash or FOPflash reporter gene activation in
HeLa cells. HeLa cells were transfected with 0.5 µg of multimerized
TCF-binding sites containing the luciferase reporter construct (TOPFlash) or
its mutant derivative (FOPFlash). Transfections were performed essentially as
described in the legend to Fig.
6.
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Dominant Negative Action of
N TCF4 To
further characterize the
-catenin- and SF1-mediated transactivation of
the MISRII promoter, we used a dominant negative form of TCF4,
N TCF4, which lacks the N-terminal
-catenin-interacting domain
but has the intact DNA-binding domain. Activation of the MISRII
promoter was abolished by the dominant negative TCF4 construct
(Fig. 8).
-Catenin- and
SF1-mediated synergistic activation of the MISRII promoter was lost
by addition of dominant negative TCF4 (Fig.
8). These observations further show that TCF4-binding sites are
essential for
-catenin- as well as SF1-mediated activation of the
MISRII promoter.
APC Down-regulation of MISRII Promoter Activation in a Colon Cancer
Cell LineIn the colon cancer cell line SW480, the nuclear level of
-catenin is increased because of an inactivating mutation in the tumor
suppressor gene APC, which leads to constitutive activation of the
Wnt-responsive genes by endogenous
-catenin
(23). We transfected the
MISRII promoter-driven luciferase reporter gene with or without
APC and SF1 into SW480 cells. Although SF1 could not
activate this promoter without coexpression of
-catenin in HeLa cells
(Fig. 6A), SF1 was
sufficient to activate this promoter in SW480 cells
(Fig. 9), probably because of
the presence of endogenous
-catenin. This activation was reduced by
transient expression (by cotransfection) of the wild-type APC.
Because of the nature of transient transfection, adding APC did not
dramatically decrease reporter gene activity.

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FIG. 9. Repression of MISRII promoter activation by APC. SW480 cells were
transfected with 0.5 µg of reporter gene and different combinations of
SF1 and APC expression vectors or with 0.5 µg of pcDNA3
as a control. CMV- -galactosidase (50 ng) was cotransfected to control
for differences in the transfection efficiencies.
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Interaction between
-Catenin and SF1We used
in vivo co-immunoprecipitation experiments to determine whether
synergistic activation of the MISRII promoter by
-catenin and
SF1 involves a direct physical interaction between them. We transfected
FLAG-tagged SF1 into SW480 cells, in which a significant amount of free
-catenin is present both in the cytoplasm and in the nucleus
(21). IP of SF1 using anti-SF1
antibody followed by immunoblotting with anti-
-catenin antibody revealed
a
100-kDa band corresponding to
-catenin
(Fig. 10). We found no
evidence for a direct interaction between SF1 and TCF4 by cotransfection or by
mixing in vitro translated proteins followed by IP and immunoblotting
(data not shown).
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DISCUSSION
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Wnt genes play an important role in organogenesis, and aberrant Wnt
signaling is believed to be involved in many types of cancer in humans
(13). Although there are many
Wnt genes in mammals, only a few physiological target genes are known to date
(12). In this study, we have
shown that MISRII, an important component in male sexual differentiation, is a
direct target of the canonical Wnt signaling pathway. Overexpression of mutant
-catenin that cannot be phosphorylated or degraded activated the
MISRII promoter in HeLa cells.
-Catenin itself is not a
DNA-binding factor, but it binds to the TCF/LEF family of DNA-binding factors
and acts as a co-activator
(12). In the absence of
nuclear
-catenin, TCF keeps the target genes in a repressed condition by
virtue of its association with groucho and related co-repressors
(24). Although TCF4 is
abundantly expressed in HeLa cells
(21), expression of TCF4 alone
was not sufficient to activate the MISRII promoter, the addition of
-catenin was necessary to activate the MISRII promoter in these
cells. However, addition of TCF4 with
-catenin did not robustly activate
the MISRII promoter because the levels of endogenous TCF4 in this
cell line were already saturating. TCF family members bind to SOX-related
binding sites, progressive deletions in the MISRII promoter revealed
four TCF-binding sites, two of which were essential for TCF binding as well as
-catenin-mediated activation of this promoter.
Barbara et al.
(11) reported that SF1 can
activate the MISRII promoter in the teratocarcinoma cell line NT2D1.
SF1 can also activate the MISRII promoter in some other cell lines.
In our study, however, SF1 alone did not activate the MISRII promoter
in HeLa cells. This finding indicated that some factor is missing in HeLa
cells. Our results indicate that
-catenin and TCF4 are the missing
factors. It is not surprising that
-catenin and SF1 synergistically
activate the MISRII promoter.
-Catenin interacts with a number
of factors to exert its biological effects, the nuclear hormone receptor RXR
and androgen receptors among them
(25,
26).
MISRII fulfills the requirements of a direct physiological target
gene of Wnt signaling. In Wnt7a mutant mice, which do not express
MISRII, the mullerian ducts do not regress
(15). The MISRII
promoter is regulated in a canonical
-catenin/TCF4-dependent manner.
Moreover,
-catenin is in the nucleus with the lymphoid enhancer factor
during the critical period of mullerian duct regression, this nuclear
accumulation of
-catenin is independent of Wnt7a action
(27). To our knowledge,
however, there are no other signaling pathways that can activate
-catenin/TCF signaling.
Transforming growth factor signaling has been shown to cooperate with Wnt
signaling in Xenopus and Drosophila systems. Because MIS is
a member of the transforming growth factor superfamily, the signal MIS may
cooperate with Wnt in the activation of the MIS receptor. Transforming growth
factor transduces its signal through SMAD-related transcription factors.
However, we did not find SMAD-binding sites within the 800 nucleotides of the
MISRII promoter. On the other hand, we did find that SF1 could
cooperate with
-catenin to activate this promoter. SF1 is an orphan
nuclear receptor for which there is no known ligand. It is known that
phosphorylation modulates the function of SF1 and is mediated by the
mitogen-activated protein kinase signaling pathway
(28). It is likely that
MIS-mediated signal may involve interaction with mitogen-activated protein
kinase signaling (29). This
postulated MIS-mediated mitogen-activated protein kinase activation may
modulate the function of SF1.
Wnt molecules play an important role in sexual determination and
differentiation in species ranging from Drosophila to mice
(30,
31). Our results also showed
that Wnt signaling is important for proper sexual differentiation in human
males. Regression of the mullerian ducts is tightly regulated by the temporal
and spatial expression of genes involved in this process during embryogenesis.
Although both male and female embryos of Wnt7a mutant mice have defects in
proper sex organ development, mullerian duct regression is important only for
the male embryo. Wnt7a is expressed in both male and female gonads, but SF1
expression is sexually dimorphic. Initially, SF1 is expressed in both male and
female embryonic gonads; later, expression of SF1 is up-regulated in male
gonads and down-regulated in female gonads in mice. This higher level of
expression of SF1 cooperates with the intracellular Wnt mediators
-catenin and TCF and up-regulates expression of MISRII during
the critical period of male sexual differentiation.
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FOOTNOTES
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* This work was supported by National Institutes of Health Grants CA 34936
and CA 16672. The costs of publication of this article were defrayed in part
by the payment of page charges. This 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: Dept. of Biochemistry and
Molecular Biology, Box 117, The University of Texas M. D. Anderson Cancer
Center, 1515 Holcombe Blvd., Houston, TX 77030. Tel.: 713-792-2690; Fax:
713-791-9478; E-mail:
gsaunders{at}odin.mdacc.tmc.edu.
1 The abbreviations used are: MIS, mullerian inhibiting substance; IP,
immunoprecipitation; MISRII, MIS type II receptor; SF1, steroidogenic factor
1; CMV, cytomegalovirus. 
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ACKNOWLEDGMENTS
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We gratefully acknowledge Dr. Pierre McCrea and his lab staff for providing
valuable materials and Dr. M. Hung for the APC and TCF4 expression
vectors.
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