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Molecular Endocrinology, doi:10.1210/me.2004-0259
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Molecular Endocrinology 18 (12): 3035-3049
Copyright © 2004 by The Endocrine Society

Canonical Wnt Signaling Is Critical to Estrogen-Mediated Uterine Growth

Xiaonan Hou, Yi Tan, Meiling Li, Sudhansu K. Dey and Sanjoy K. Das

Departments of Pediatrics (X.H., Y.T., M.L., S.K.De., S.K.Da.), Cancer Biology (X.H., Y.T., M.L., S.K.Da.), Cell & Developmental Biology (S.K.De.) and Pharmacology (S.K.De.), Vanderbilt University Medical Center, Nashville, Tennessee 37232; and Laboratory Animal Center (Y.T.), Chongqing University of Medical Sciences, Chongqing 400016, People’s Republic of China

Address all correspondence and requests for reprints to: Dr. Sanjoy Das, Division of Reproductive and Developmental Biology, Department of Pediatrics, D-4105 Medical Center North, Vanderbilt University Medical Center, 1161 21st Avenue South, Nashville, Tennessee 37232-2678. E-mail: sanjoy.das{at}vanderbilt.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Major biological effects of estrogen in the uterus are thought to be primarily mediated by nuclear estrogen receptors, ER{alpha} and ERß. We show here that estrogen in an ER-independent manner rapidly up-regulates the expression of Wnt4 and Wnt5a of the Wnt family and frizzled-2 of the Wnt receptor family in the mouse uterus. One of the mechanisms by which Wnts mediate canonical signaling involves stabilization of intracellular ß-catenin. We observed that estrogen treatment prompts nuclear localization of active ß-catenin in the uterine epithelium. We also found that adenovirus mediated in vivo delivery of SFRP-2, a Wnt antagonist, down-regulates estrogen-dependent ß-catenin activity without affecting some of the early effects (water imbibition and angiogenic markers) and inhibits uterine epithelial cell growth, suggesting that canonical Wnt signaling is critical to estrogen-induced uterine growth. Our present results provide evidence for a novel role of estrogen that targets early Wnt/ß-catenin signaling in an ER-independent manner to regulate the late uterine growth response that is ER dependent.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DIVERSE BIOLOGICAL EFFECTS of estrogen are primarily mediated via activation of its nuclear estrogen receptors ER{alpha} and ERß, which are ligand-inducible transcription factors (1, 2). However, increased uterine expression of specific genes after estrogen exposure in mice missing the ER{alpha} gene or in which both ER{alpha} and ERß functions have been silenced by an ER antagonist ICI 182,780 (ICI) suggested an alternative pathway for specific estrogen functions. For example, we have previously shown that that uterine expression of secreted frizzled-related protein-2 (SFRP-2), a negative regulator of Wnt signaling, is rapidly down-regulated by estrogen in wild-type or ER{alpha}(–/–) mice in the presence of ICI or a protein synthesis inhibitor cycloheximide (3). The result suggested that a rapid onset of Wnt signaling plays a role in mediating estrogen actions in the mouse uterus.

The Wnt family of genes encodes a large group of highly conserved secreted glycoproteins. They play crucial role in embryonic developmental processes (4, 5, 6) and are also involved in tumorigenesis (6, 7, 8). To be effective in autocrine or paracrine signaling, Wnt proteins must associate with their extracellular surface receptors frizzled (Fz) to mediate intracellular signal transduction pathways (9). Fzs constitute a large family of seven transmembrane G protein-coupled receptors and possess an extracellular cysteine-rich domain (CRD) for Wnt binding (10, 11). Another family of proteins, termed secreted Fz-related proteins (SFRPs) produced by different genes, is structurally similar to Fzs with respect to CRD, but lacks the seven transmembrane and the intracellular signaling domains (12, 13). SFRPs primarily exert inhibitory effects on Wnt signaling by competing with Wnt ligands for the Fz receptor and forming a nonfunctional complex with Fzs in a dominant-negative manner (14, 15, 16).

Wnt functions are executed at least via three intracellular signaling pathways in the context of cell types (17, 18, 19). Most widely studied, the canonical Wnt signaling pathway involves regulation of ß-catenin. The activation of Wnt signaling stabilizes intracellular ß-catenin by inhibiting serine-threonine kinase activity of GSK3ß (glycogen synthase kinase 3ß). In the absence of Wnt signaling, GSK3ß binds to axin (a bridging molecule), adenomatous polyposis coli and ß-catenin complex, leading to ß-catenin phosphorylation and its degradation by ubiquitination. The active intracellular ß-catenin is considered to be the stabilized dephosphorylated from (20, 21, 22), which translocates to the nucleus and forms complex with lymphoid enhancer factor (Lef)/T-cell factor (Tcf) family of transcription factors to stimulate transcription of Wnt target genes (23, 24). Two other noncanonical Wnt signaling pathways include Wnt/Ca2+ and Wnt/ras homolog gene family, member A/c-Jun NH2-terminal kinase pathways that primarily affect actin cytoskeleton and planar polarity of cells (17, 19, 25). Activation of each signaling pathway depends on the type of ligands and receptors involved because Wnt proteins possess preferential binding to specific receptors (26). Moreover, distinct Fzs are also known to exhibit differential activation of these various signaling pathways (27, 28, 29), although the pathway specificity appears to be developmentally regulated.

It is well known that Wnt signaling plays roles in epithelial-mesenchymal interactions and cellular organization during embryonic and postembryonic development that involve cell proliferation and differentiation, cell fate specification and cell-to-cell communication (4, 5, 6). Wnt signaling is also considered important for female reproductive functions (30, 31) and has been described as a target for endocrine disruptors (32). Our present study shows for the first time that several genes of the Wnt signaling pathway are regulated by estrogen in the mouse uterus in an early responsive manner without involving classical ERs. Furthermore, we show here that ß-catenin-mediated rapid canonical Wnt signaling is activated in uterine epithelial cells under the direction of estrogen in both wild-type and ER{alpha}(–/–) mice. Most importantly, in vivo delivery of adenovirally expressed SFRP-2 (a Wnt antagonist) in ovariectomized wild-type mice markedly inhibits estrogen-dependent uterine growth and ß-catenin activity. Collectively, these studies demonstrate that estrogen-dependent control of Wnt/ß-catenin signaling that is mediated in a nonclassical manner regulates late uterine growth response that is ER dependent.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen Regulates Early Wnt Signaling in the Mouse Uterus in the Absence of ER Function
Our previous studies demonstrated that estrogen regulates the expression of several early responsive genes in the ovariectomized uteri of both wild-type and ER{alpha}(–/–) mice (3). One of those genes, SFRP-2, an antagonist of Wnt signaling, was strongly suppressed after estrogen stimulation, suggesting estrogen’s influence in regulating early Wnt signaling in the uterus. This observation led us to examine whether the Wnt and Fz family members are regulated by estrogen in the mouse uterus in the presence or absence of ER{alpha}. Our initial uterine analysis by RT-PCR using either types of mice showed that expression of Wnt4, Wnt5a, and Fz2 genes was predominately up-regulated due to an injection of estradiol-17ß (E2) by 6 h, whereas that of Wnt1, Wnt3, Wnt7a, Wnt7b, Fz4, Fz6, or Fz7 genes was below the level of detection or remained unaffected by this treatment (data not shown). Although a previous study (30) showed that uterine expression of Wnt4, Wnt5a, and Wnt7a genes was regulated during various stages of the cycle in mice; however, our analysis in ovariectomized mice indicates that the expression of Wnt7a may not be regulated by estrogen during its early phase of action. Thus, our subsequent studies focused on the regulation of uterine Wnt4, Wnt5a, and Fz2 genes by estrogen.

To examine whether estrogen influences the expression of Wnt4, Wnt5a, and Fz2, ovariectomized wild-type mice received a sc injection of E2 (100 ng/mouse) and killed at 1, 2, 6, 12, and 24 h. Mice injected with oil (vehicle) served as controls. Total RNA samples obtained from whole uteri were analyzed by Northern blot hybridization. Analysis of our data shows that, although the mRNA levels of these genes were low in oil-treated samples, an injection of E2 caused rapid induction of these mRNAs by 1 h with a peak response at 2 h and maintained through 6 h (Fig. 1AGo). The levels of Wnt4 and Fz2 mRNAs showed some decline by 12 and 24 h, whereas that of Wnt5a remained unaffected. The maximal inductive response by E2 of Wnt4, Wnt5a, and Fz2 genes was about 5-, 3-, and 6-fold, respectively, as compared with the corresponding oil-injected groups.



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  		Fig. 1. Analysis of Uterine Gene Expression

A, Temporal effects of E2 on uterine mRNA expression for Wnt4, Wnt5a, Fz 2, and rPL7 in ovariectomized wild-type mice. Adult ovariectomized mice were given a single injection of E2 (100 ng/mouse) and killed at the times indicated. Mice injected with oil were killed after 6 h and served as controls. Total RNA samples (6 µg) were analyzed by Northern hybridization. B, Estrogen-dependent uterine expression of Wnt4, Wnt5a, Fz2, LF, and rPL7 are not responsive to ICI in ovariectomized wild-type mice. Adult ovariectomized mice were given an injection of oil, E2 (100 ng/mouse), ICI (500 µg/mouse) or ICI 30 min before an injection of E2 and killed at 6 h after the last injection. Total RNA samples (6 µg) were analyzed by Northern hybridization. These experiments (A and B) were repeated twice with independent RNA samples and similar results were obtained. C, Effects of E2 on uterine Wnt4, Wnt5a, and Fz2 mRNA levels in ovariectomized wild-type or ER{alpha}(–/–) mice in response to estrogen and/or ICI as described in Fig. 1BGo. Quantitative RT-PCR for gene-specific mRNAs was performed on total RNA samples as described (34 ). *, Values are statistically different (P < 0.05, ANOVA followed by Newman-Keul’s multiple range test).

 
We next asked whether estrogen-regulated responses, as observed above, were mediated through the participation of ER{alpha} and/or ERß. To address this question, ovariectomized wild-type mice were given an injection of oil, E2, ICI or ICI plus E2 as described in Materials and Methods, and uterine tissues were collected at 6 h after the last injection. Total uterine RNA samples were analyzed by Northern blotting. As shown in Fig. 1BGo, the results show that uterine levels of Wnt4, Wnt5a, and Fz2 expression were low after an injection of oil or ICI. However, an injection of E2 caused up-regulation of the mRNA levels of these genes. Notably, the treatment of ICI before E2 injection failed to attenuate estrogen-induced expression of these genes. To evaluate the efficacy of ICI on Wnt signaling target genes and to rule out the role of ER in these responses, we analyzed the expression of lactoferrin (LF), a highly ER-dependent estrogen-responsive gene in the mouse uterus (33) (Fig. 1BGo). Our results show that estrogen-induced expression of LF in uteri of wild-type mice is suppressed by a coinjection ICI with estrogen. The results suggest that ICI used in this study is indeed effective in antagonizing the expression of ER-dependent target genes. In contrast, ICI’s failure to influence the expression of estrogen-regulated Wnt signaling genes provides strong evidence that the effects of estrogen on these genes are not mediated via ER-{alpha} and/or ER-ß.

To provide further genetic evidence that estrogenic responses were mediated via a non-ER mechanism, we analyzed uterine expression of Wnt4, Wnt5a, and Fz2 in ER{alpha}(–/–) mice and compared with that in the wild-type littermates using a quantitative RT-PCR as previously reported (3, 33, 34). We used this technique because of the limited availability of uterine RNA samples from ER{alpha}(–/–) mice. Injection schedules were same as described above for wild-type mice. Irrespective of the treatments, our results show that the relative levels of mRNAs for Wnt4, Wnt5a, and Fz2 genes in the wild-type uteri are higher as compared with those in ER{alpha}(–/–) mice (Fig. 1CGo). However, an injection of E2 increased the mRNA copy numbers of Wnt4, Wnt5a, and Fz2 genes by approximately 4-, 3-, and 4-fold, respectively, compared with the corresponding controls (oil treatment). The ICI compound again failed to antagonize these estrogenic responses in either wild-type or mutant mice. Collectively, these results suggest that the regulation of uterine expression for Wnt4, Wnt5a, and Fz2 genes by E2 occurs in an early responsive manner without involving ERs in the mouse uterus.

Estrogen Regulates Uterine Wnt4, Wnt5a, and Fz2 Expression in a Cell-Specific Manner
To examine cell-specific uterine expression of Wnt4, Wnt5a, and Fz2 genes by estrogen, in situ hybridization was performed on frozen uterine sections obtained from ovariectomized wild-type or ER{alpha}(–/–) mice after receiving estrogen alone or estrogen plus antiestrogen for 6 h as described in Materials and Methods. The results show that uterine cell-specific accumulation of Wnt4 (Fig. 2AGo), Wnt5a (Fig. 2BGo), and Fz2 (Fig. 2CGo) mRNAs was low in both the ovariectomized wild-type and ER{alpha} mutant mice after an injection of oil or ICI. However, an injection of E2 caused differential cell-specific up-regulation of these genes in wild-type or ER{alpha}(–/–) mice. For example, a distinct accumulation of Wnt4 mRNA was predominantly noted in the luminal and glandular epithelium and also in the subluminal stroma (Fig. 2AGo). In contrast, Wnt5a mRNA was primarily detected in luminal and glandular epithelial cells, although some patchy weak signals were also noted in the stroma (Fig. 2BGo). In contrast, the expression of Fz2 was evident in the epithelium and throughout the stroma of both wild-type and ER{alpha}(–/–) mice (Fig. 2CGo). An injection of ICI before E2 injection failed to alter estrogen-induced expression pattern of these genes either in wild-type or mutant mice (Fig. 2Go, A–C). Hybridization with the corresponding sense cRNA probes did not show any positive signals (data not shown).



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  		Fig. 2. In Situ Hybridization of Wnt4, Wnt5a, and Fz2 in Wild-Type or ER{alpha}(–/–) Mice

Wnt4 (A), Wnt5a (B), and Fz2 (C). Ovariectomized wild-type or ER{alpha}(–/–) mice were treated with oil, E2, ICI, or E2 plus ICI as described in Fig. 1BGo. Frozen sections (10 µm), fixed in paraformaldehyde, were mounted onto glass slides, prehybridized and hybridized with 35S-labeled sense or antisense riboprobes for 4 h at 45 C. Ribonuclease A-resistant hybrids were detected after 5–7 d of autoradiography using Kodak NTB-2 liquid emulsion (Eastman Kodak, Rochester, NY). Sections were poststained lightly with hematoxylin and eosin. Representative dark-field photomicrographs of uterine sections are shown. The reddish brown grains indicat the sites of mRNA accumulation. This color shade is the result of lateral light scattering from the eosin staining under dark-field microscopy. Sectioned hybridized with the sense probes served as negative controls (data not shown). Bars, 100 µM. le, Luminal epithelium; s, stroma; and myo, myometrium. These experiments were repeated three times with three to four mice in each group and similar results were obtained.

 
Wnt4, Wnt5a, and SFRP-2 Proteins Are Expressed by E2 in the Mouse Uterus
To determine whether Wnt4 and Wnt5a mRNAs induced by estrogen in the uterus are effectively translated, immunohistochemistry was used to localize cell-specific accumulation of Wnt4 and Wnt5a proteins in uterine sections obtained from ovariectomized wild-type or ER{alpha}(–/–) mice at 6 h of oil or E2 injection (Fig. 3Go, A and B, respectively). Although weak immunostaining was observed for either of these proteins in the uterine epithelium of oil-treated mice, strong staining was noted for Wnt4 in the epithelium and in the subluminal stroma in both wild-type and ER{alpha}(–/–) mice after an injection of E2. Wnt5a showed a similar pattern of signal, but at a reduced intensity.



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  		Fig. 3. Immunolocalization of Wnt4 (A) and Wnt5a (B) in Uteri of Ovariectomized Wild-Type and ER{alpha}(–/–) Mice after Exposure to Oil or E2.

Injection schedules and the dose of various agents were same as described in Fig. 1Go. Paraformaldehyde-fixed paraffin-embedded sections (6 µm) were used. The sections were incubated with primary antibody at 1:500 dilution in PBS for 17 h at 4 C. Representative photomicrographs of uterine sections are shown. Red deposits indicate positive immunostaining. These experiments were repeated three times with three to four mice in each group and similar results were obtained. No immunostaining was noted when sections were incubated with preimmune serum instead of primary antibody (data not shown). Bars, 100 µm. le, Luminal epithelium; ge, glandular epithelium; s, stroma; and myo, myometrium. C, Western blot analysis of SFRP-2 in wild-type uterine tissue extracts. Adult ovariectomized wild-type mice were given a single injection of E2 (100 ng/mouse) and killed at times indicated. Mice injected with oil were killed after 6 h and served as controls. Uterine tissue extracts (80 µg in each lane) were fractionated by 10% SDS-PAGE. Immunoblotting was performed using a primary antibody specific to mouse SRRP-2. Primary antibody preneutralized by 250-fold molar excess of antigenic peptide did not detect SFRP-2 specific band (data not shown).

 
We have previously shown that SFRP-2 is strongly expressed in the ovariectomized mouse uterine stroma, and this expression is rapidly suppressed by an injection of E2 (3). Because SFRP-2 antagonizes Wnt signaling, we wanted to examine whether SFRP-2 protein is indeed suppressed during the time of E2 mediated up-regulation of Wnt4 and Wnt5a. As shown by Western blot analysis (Fig. 3CGo), a distinct protein band (~30 kDa) for SFRP-2 was detected in ovariectomized uterine tissue extracts after an injection of oil at 6 h. In contrast, the intensity of this band was remarkably weak in uterine samples after an injection of E2 either at 6 or 24 h (Fig. 3CGo). This band is specific for SFRP-2 because a similar blot incubated in parallel with the primary antibody preneutralized by 250-fold molar excess of antigenic peptide (sc-7426P, Santa Cruz Biotechnology, Inc.) failed to show such a band (data not shown). These results correlate well with our mRNA data and suggest that estrogen-stimulated up-regulation of Wnt4 and Wnt5a occurs in the uterus during the time when uterine SFRP-2 expression is remained suppressed by E2.

E2 Activates Canonical Wnt Signaling in the Mouse Uterus
Because Wnt signaling mediated by canonical pathway regulates intracellular concentration of ß-catenin, we examined whether estrogen can modulate the levels of ß-catenin in the uterus. Western blot analysis using an anti-ß-catenin antibody was performed to examine the levels of total ß-catenin in uterine extracts obtained from ovariectomized wild-type or ER{alpha}(–/–) mice after an injection of oil or E2. The results show that an accumulation of ß-catenin was induced by E2 at 6 and 24 h both in the wild-type (Fig. 4AGo) or ER{alpha}(–/–) (data not shown) mice as compared with a low basal level after the oil injection (control).



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  		Fig. 4. Analysis of Uterine Expression of ß-Catenin

A, Western blot analysis of total ß-catenin ({alpha}BC) in wild-type uterine tissue extracts. Injection schedules and Western blotting experiments are same as described in Fig. 3CGo. Primary antibody preneutralized by 250-fold molar excess of the antigenic peptide did not detect any bands (data not shown). Immunolocalization of total ß-catenin ({alpha}BC) (B) and {alpha}ABC (C) in uteri of ovariectomized wild-type and ER{alpha}(–/–) mice after exposure to oil or E2 as described in Fig. 1BGo. Red deposits indicate positive immunoreactivity. These experiments were repeated three times with three to four mice in each group, and similar results were obtained. No immunostaining was noted when similar sections were incubated with preimmune serum instead of primary antibody (data not shown). Bars, 100 µm. le, Luminal epithelium; ge, glandular epithelium; s, stroma. Note: Distinct nuclear localization of active ß-catenin is clearly detected in uterine epithelial cells for either type of mice after the treatment of estrogen. Pictures are shown by high magnification within the insets. Bars, 40 µm.

 
We also examined cell-specific distribution of total ß-catenin by immunohistochemistry on formalin-fixed paraffin-embedded uterine sections of both wild-type and ER{alpha}(–/–) mice under similar conditions. We found that total ß-catenin was predominantly localized in the uterine epithelium, apparently in the cell junction, after an injection of oil (Fig. 4BGo). The intensity of the signal was significantly enhanced by E2 treatment at 6 h in both wild-type and ER{alpha}(–/–) mice (Fig. 4BGo); low levels of accumulation were also evident in stromal cells in the subluminal region irrespective of the treatment. Similar results were also obtained after E2 injection by 24 h (data not shown). Collectively, these results suggest that E2 is capable of stimulating epithelial cell-specific accumulation of ß-catenin in a sustained manner without requiring ER.

It is well established that the canonical Wnt signaling stabilizes intracellular ß-catenin in a dephosphorylated form. In cells that do not receive Wnt signaling, free ß-catenin is targeted for proteasomal degradation via its N-terminal Ser-37/Thr-41 phosphorylation by GSK3ß (20). The canonical Wnt signaling inhibits GSK3ß activity, resulting in the accumulation of active ß-catenin that is targeted to the nucleus to form nuclear complex with Tcf/Lef transcription factors (21, 22). We used an antibody specific to active ß-catenin ({alpha}ABC) to further examine the activation of Wnt signaling in the mouse uterus after an injection of E2 in both ovariectomized wild-type and ER{alpha}(–/–) mice (Fig. 4CGo). The results of immunostaining revealed that active ß-catenin is exclusively localized in uterine luminal and glandular epithelial cell nuclei of both wild-type and ER{alpha}(–/–) mice by 6 h (Fig. 4CGo) and retained for 24 h (data not shown) after exposure to E2. In contrast, we did not see any such nuclear accumulation after the oil injection (Fig. 4CGo). Collectively, these results provide clear evidence that the canonical Wnt signaling becomes active in the uterus by estrogen not involving ER{alpha}.

Adenoviral Expression of SFRP-2 Suppresses Estrogen-Dependent Uterine Regulation of ß-Catenin
Because SFRP-2 acts as a negative regulator of Wnt signaling, we examined whether adenovirally expressed SFRP-2 influences estrogen-dependent canonical Wnt signaling in the uterus. The recombinant adenovirus particles carrying SFRP-2 and the empty vector (control) under the direction of a cytomegalovirus (CMV) promoter were made. These constructs were also equipped with a green fluorescent protein (GFP) expression system under a separate CMV promoter. In our initial experiments, we verified whether these virus particles appropriately regulate the expression of genes in uterine cells in vitro. Western blot analyses show that the adenoviral particles carrying the SFRP-2 gene indeed expressed SFRP-2 in primary uterine stromal cell cultures, whereas the control virus particles failed to express this protein under similar cell culture conditions (Fig. 5A). The expression of GFP by either type of virus particles suggests that virus-driven expression of the transgene is appropriately regulated in uterine cells in vitro (Fig. 5AGo).



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  		Fig. 5. Analysis of Adenovirus-Mediated Expression

A, Western blot analysis of primary culture of mouse uterine stromal cells. Cells were infected with virus particles carrying SFRP-2 or control virus particles for 48 h and cell extracts were analyzed. B, Immunoprecipitation and Western blotting for GFP expression in the uterus. SFRP-2 or control virus particles were injected in ovariectomized mice through a tail vein. They were rested for 7 d to achieve optimum infection. Uterine tissue extracts were analyzed for viral-driven expression of GFP by immunoprecipitation followed by Western blotting using a GFP antibody. Primary uterine stromal cell extracts expressing GFP were used as a positive control. C, Western blot analysis of ß-catenin in uterine tissue extracts. Virus (SFRP-2 or control) infected mice, rested for 7 d, were given a single injection of E2 and killed 24 h later. Another group of noninfected mice was similarly treated with E2 injection. Uterine tissue extracts were analyzed for the expression of ß-catenin and actin proteins. D, Direct visualization of GFP fluorescence in uterine tissue sections. Treatments were same as described in Fig. 5CGo. Frozen uterine tissue sections (10 µm) were covered with mounting solution and then subjected to capture photomicrographs under fluorescence microscope for GFP (right panels) or under the phase for bright-fields (left panels). Bars, 40 µm. le, Luminal epithelium; s, stroma.

 
We next examined whether these virus-driven particles reach the uterine target and produce the expression of the transgene after in vivo delivery via a tail vein. Because the SFRP-2 transgene, which we used in generating the adenovirus particles, is a mouse-specific gene, it is not possible to distinguish the expression levels of the SFRP-2 transgene vs. the native SFRP-2 gene in the uterus. To circumvent this problem, we analyzed the expression of virus-driven GFP. Analysis of uterine proteins by immunoprecipitation and Western blotting showed that the viral-driven GFP is expressed after the delivery of virus particles (Fig. 5BGo), providing evidence that in vivo administration of these particles can reach the uterine target and drive gene expression. The results of our in vitro analysis would suggest that both the transgenes are regulated in a similar manner in the uterus in vivo. To further examine the effectiveness of this approach, estrogen-dependent uterine ß-catenin expression was examined after in vivo delivery of these virus particles. The results show that estrogen-induced up-regulation of total ß-catenin at 24 h is strongly suppressed by viral-driven SFRP-2, whereas the control virus fails to show any such effects (Fig. 5CGo). Under similar conditions, we also analyzed uterine cell-specific expression of GFP by direct visualization of GFP fluorescence in uterine sections. As shown in Fig. 5DGo, uterine epithelial and stromal cells exhibited the expression of GFP after administration of the SFRP-2 or control (data not shown) virus particles in mice. This provides evidence that the virus-mediated SFRP-2 transgene expression in the uterus is available to act in a dominant-negative manner for Wnt signaling in vivo. Overall, these results suggest that E2-induced canonical Wnt signaling in the uterus is neutralized by the SFRP-2 virus particles.

Inhibition of Canonical Wnt Signaling Abrogates Estrogen-Dependent Uterine Growth Response
Uterine estrogenic effects are traditionally considered biphasic: the early response that occurs within 6 h is characterized by increased water imbibitions and macromolecular uptake, whereas the late response occurs between 16–30 h and is defined by increased epithelial cell DNA synthesis and hyperplasia (35, 36). Because forced expression of SFRP-2 negatively impacts Wnt/ß-catenin signaling in the mouse uterus, we next asked whether similar expression of SFRP-2 would interfere with estrogenic responses in the uterus. As shown in Fig. 6Go, A–D, in vivo delivery of viral-driven SFRP-2 demonstrated a striking suppression of estrogen-dependent late phase of uterine epithelial cell DNA synthesis and growth, whereas the control virus failed to evoke such a response. In contrast, estrogen-induced uterine water imbibition and vascular permeability genes VEGF (vascular endothelial growth factor) and Flk1 (VEGF receptor-2) (37) that occur during the early phase were not affected by similar treatments (Fig. 6Go, B and E). Overall, these results suggest that canonical Wnt signaling that occurs during the early estrogenic response without involving ER is crucial to late estrogenic uterine growth response.



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  		Fig. 6. Analysis of Estrogen-Dependent Uterine Effects in Ovariectomized Mice after Administration of Adenoviruses

A, Morphological appearances of two representative uteri from ovariectomized mice are shown after infection with virus-driven SFRP-2 or control virus for 7 d and followed by injection with E2 (100 ng/mouse) for another 24 h. B, Analysis of uterine wet weights in similarly treated mice. Normal estrogenic responses (E2, 100 ng/mouse) were determined in parallel without any viral infection. Oil-injected mice served as controls. Uterine wet weights were recorded at 6 and 24 h after E2 injection. The number of mice used in each group is indicated within the bars. Values with asterisks are statistically different (P < 0.01, ANOVA followed by Newman-Keul’s multiple range test) as compared with their corresponding control groups. C, Analysis of cell proliferation. Uterine cross-sections were examined by BrdU immunostaining. Reddish-brown nuclear deposits indicate the sites of positive immunostaining. Bars, 100 µm. le, Luminal epithelium. D, Quantitation of BrdU-positive cells in the luminal epithelium as shown in C. Approximately 500 cells were counted in each group. E, Analysis of early estrogenic effects at 6 h. Uterine sections were analyzed histologically (hematoxylin and eosin staining), and by in situ hybridization of VEGF and Flk-1. Bars, 100 µm. le, Luminal epithelium; myo, myometrium; s, stroma.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The uterus is a primary target of estrogen for various functions during the reproductive cycle and pregnancy. In mice, estrogen induces uterine epithelial cell proliferation and is essential for the maintenance of normal epithelial morphogenesis, cytodifferentiation, and secretory activity (38). The general consensus is that estrogen exerts its effects by modifying gene expression through activation of its nuclear receptor ER{alpha} in the uterus (39, 40, 41). Here we have shown that estrogen in an ER-independent manner up-regulates canonical Wnt signaling in the uterus, suggesting a novel role for estrogen action in the target tissue (Fig. 7Go). It is a long-sought question how the biphasic estrogenic responses in the uterus are molecularly linked. Our present observation that the canonical Wnt signaling pathway is activated very early in response to estrogen and is crucial to late estrogenic growth provides strong evidence that Wnt/ß-catenin signaling is a link between the two phases. Our study also provides the first evidence how an ER-independent early response pathway is integrated into an ER-dependent late growth response pathway.



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  		Fig. 7. A Proposed Model Showing a Role for Estrogen-Induced Early Wnt/ß-Catenin Signaling that Leads to Late Uterine Growth Response

Estrogen, without involving ER{alpha}/ß, induces early onset of uterine expression of Wnt4/Wnt5a/Fz2/ß-catenin genes and nuclear translocation of ß-catenin, leading to the activation of the canonical Wnt signaling in uterine epithelial cells. The nuclear protein complex, formed by the active ß-catenin and the effector protein (TCF/LEF) acts directly or together with liganded-ER{alpha} to control differential gene transcription functions, which in turn impact cellular growth response.

 
Wnt signaling is known to participate in epithelial-mesenchymal communication (30, 42, 43, 44, 45, 46, 47). Several members of the Wnt gene family are expressed in the uterus (30) and mammary glands (45, 46, 47). Expression of specific Wnt genes in these tissues is restricted to either epithelial, stromal, or both cell types, and their expression levels fluctuate with changing hormone levels during the reproductive cycle and pregnancy. Although genetic studies in mice involving several Wnts have established that Wnt signaling is crucial to embryonic developmental processes (4, 5, 6), the biological significance of Wnt signaling in adult tissue homeostasis remains poorly understood. It is believed that estrogen-regulated epithelial cell-specific functions are directed by signals emanating from the stromal cell compartment (48). However, the nature of the regulatory signals still remains largely unknown. Our previous observation of heightened expression of SFRP-2 in stromal cells when the uterus is quiescent after ovariectomy (3), together with our present observation of suppressed SFRP-2 expression with up-regulation of Wnt4, Wnt5a, and Fz2 in both the epithelial and stromal cells by estrogen (Fig. 3Go), suggests that Wnt signaling is operative in both cell types.

ß-Catenin binds to the cytoplasmic domain of E-cadherin to form a linkage with the actin cytoskeleton and regulates its function at the adherence junctions (49). The increased accumulation of total ß-catenin at the epithelial cell junctions after estrogen stimulation suggests that this protein is involved in the maintenance of adherence junctions in these cells (Fig. 4BGo). In contrast, estrogen-stimulated distinct nuclear accumulation of active ß-catenin in epithelial cells suggests the operation of a canonical Wnt signaling (Fig. 4CGo). The absence of active ß-catenin in stromal cells suggests that these cells are not involved in canonical Wnt signaling. Wnts can also execute signal transduction in a noncanonical manner using Wnt/Ca2+ or Wnt/ras homolog gene family, member A/c-Jun NH2-terminal kinase pathway (17, 19, 25). It is possible that these alternative Wnt signaling pathways are operative in stromal cells.

It is generally believed that Wnts are secreted cell-signaling molecules (50, 51, 52). There is evidence that Wnts bind to Bip (also known as glucose-regulated protein 78 kDa, GRP78), a resident protein in the endoplasmic reticulum. This interaction is thought to be required for preventing secretion of improperly folded polypeptides and for efficient secretion of Wnts (53, 54). We have previously shown that estrogen also up-regulates the expression of Bip in a non-ER-mediated manner in uterine epithelial and stromal cells (3). We speculate that a direct interaction of Bip with Wnt ligands occurs in the mouse uterus after estrogen stimulation to regulate Wnt secretion.

Our present study presents strong evidence that uterine epithelial cell-specific activation of canonical Wnt signaling by estrogen is critical to epithelial cell growth in response to estrogen. Our studies show that the virus-driven expression of GFP and thus SFRP-2 is detectable in uterine epithelial and stromal cells. However, we cannot be absolutely certain that the expression of the SFRP-2 transgene in the uterus directly affected the regulation of uterine ß-catenin expression because it is not possible to distinguish the uterine expression of mouse-specific SFRP-2 transgene from the native gene. It is also possible that other nonuterine targets are involved, but our experiments using the ovariectomized mouse model with supplementation of steroid hormones excludes at least the involvement of ovaries. Nonetheless, evidence for nuclear translocation of ß-catenin in the regulation of gene transcription and cellular proliferation (23, 24) is consistent with our present findings of nuclear translocation of ß-catenin in uterine epithelial cells and their proliferative response by estrogen. Previous studies that used reciprocal tissue recombination experiments between the uterine epithelium and stroma from the wild-type and ER{alpha}(–/–) mice suggest that estrogen-mediated epithelial cell growth is positively controlled by stromal ER{alpha} (55). However, our findings of ER-independent Wnt signaling in uterine epithelial cells suggest another mechanism that does not appear to involve stroma because regulation of ß-catenin by estrogen is not different in uteri of wild-type and ER{alpha}(–/–) mice. There is also evidence to suggest that Wnt4, Wnt5a, and Fz2 do not necessarily involve canonical Wnt signaling (23, 24, 25). However, our results demonstrating estrogen-dependent up-regulation of these genes in conjunction with the stabilization of ß-catenin in the uterus in an early time-dependent manner suggest that the activation of canonical Wnt signaling in the uterus is a function of time.

The mechanism of ER-independent regulation of uterine genes is currently unknown. The answer to this question awaits the cloning and identification of a gene(s) encoding functional membrane ER(s). Although the physiological implication of up-regulation of Wnt4, Wnt5a, and Fz2 by estrogen, but down-regulation of SFRP-2 in the uterus, follows a temporal relationship and is to potentiate Wnt signaling, the molecular mechanism by which this is achieved is not currently known. However, it is to be noted that SFRP-2 is regulated by estrogen without involving any intermediary protein synthesis (3). This suggests posttranslational mechanism, i.e. phosphorylation and/or dephosphorylation could be involved.

The downstream effects of canonical Wnt signaling lead to the association of ß-catenin with its effector proteins Lef/Tcf family member of transcription factors before their interaction on specific target gene promoters for gene transcription. We have preliminary results to show that Tcf3 and Tcf4 are expressed in the mouse uterus after estrogen stimulation (Cox, S., and S. K. Das, unpublished data). ER-dependent transcriptional activation is not only influenced by specific ligand binding to its receptor, but also by the recruitment of coregulatory proteins at the level of chromatin interaction. The manifestation of estrogen-regulated late growth response in the uterus is believed to be the result of ER{alpha}-dependent gene transcriptional activities (56). Changes in gene expression are generally achieved by the convergence of signaling pathways on transcription factors, governing their transactivation potential in the context of specific target genes. We speculate that estrogen-regulated ß-catenin/transcription factor complex mutually interacts with ER to achieve combinatorial or distinct functions at the level of DNA transcription machinery. Both Tcf and ER recognize discrete nucleotide sequences in the promoter of their target genes. There is also evidence that these two nuclear transcription factors interact directly either on the DNA or in the absence of DNA, and the resulting complex interaction could be antagonistic or stimulatory depending on the target gene promoter activity (57). It is possible that an interaction between these two transcription factors in response to estrogen sets up a dialogue to induce the late estrogenic growth response. In conclusion, our present findings provide new insights into a complex system that impacts estrogen-dependent regulation of uterine growth response. The activation of ER-independent Wnt/ß-catenin signaling by estrogen in uterine epithelial cells during the early phase significantly contributes to the ER-dependent late growth response.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals and Injections
Wild-type and ER{alpha}(–/–) mice of the same genetic background (C57BL/6J/129/J) were produced by crossing of heterozygous females and males [Lubahn et al., (64)]. In all studies, wild-type littermates were analyzed in parallel with ER{alpha}(–/–) mice. They were housed in the institutional animal care facility according to the National Institutes of Health and institutional guidelines for the care and use of laboratory animals. Genotyping was performed by PCR analysis of tail DNA. In general, 8- to 10-wk-old adult mice were ovariectomized and rested for 2 wk before they received any injections (3, 34). Mice were injected with oil (0.1 ml) or estradiol-17ß (E2, 100 ng/mouse) or ICI 182,780 (ICI, 500 µg/mouse), a potent antiestrogen for both ER{alpha} and ERß or the same dose of ICI 30 min before E2 injection. They were killed at different times as indicated after the last injection. The test agents were dissolved in sesame oil and injected (0.1 ml/mouse) sc.

Probes and Hybridizations
cRNA probes were generated from mouse-specific cDNA clones. The clones of Wnt4 and Wnt5a in pGEM7Zf(+) vectors (58) or Fz2 in a pBluescript vector (10) were used. cDNA clones for VEGF, Flk-1, LF, and ribosomal protein L-7 (rpL7) have previously been described (33, 34, 37). Northern or in situ hybridization technique was same as previously described (59, 60). Hybridized bands on Northern blots were analyzed by densitometric scanning. In situ hybridization slides were post-stained with hematoxylin and eosin. Serially sectioned slides hybridized with the sense probe were served as negative controls.

Quantification of RNAs by Competitive RT-PCR
This was performed as previously described (34). Competitive templates used for this study were generated by introducing a nonspecific DNA fragment into or by deleting a cDNA fragment from the mouse-specific target gene. Specifically, a 185 bp blunt-ended fragment (Ssp1), obtained from pGEM7Zf (+) vector, was inserted into cDNA clones for Wnt5a at the SmaI site or Fz2 at the StuI site. For Wnt4, a SmaI fragment (438 bp) was deleted from the original clone obtained from Andrew McMahon (58) and subsequently ligated at the ends to generate the competitor template. The following primers were used for RT-PCR: 5'-ATT GTC CCC CAA GGC TTA AC-3' [454–473 nucleotides (nts), sense] and 5'-CTG TGC TGC AGT TCC ATC TC-3' (883–902 nts, antisense) for Wnt5a mRNA (GenBank accession no. NM009524); 5'-CAA GAC GGA GAA GCT GGA GA-3' (448–467 nts, sense) and 5'-AGA ACT TCC TCC ACG AGT GC-3' (733–752 nts, antisense) for Fz2 mRNA (GenBank accession no. AF363723); 5'-CGA GGA GTG CCA ATA CCA GT-3' (270–289 nts, sense) and 5'-GCC GTC AAT GGC TTT AGA TG-3' (941–960 nts, antisense) for Wnt4 mRNA (GenBank accession no. M89797). The internal primers 5'-CCT TGA GAA AGT CCT GCC AG-3' (764–783 nts, antisense) for Wnt5a, 5'-CGA TGA GCG TCA TGA GGT AT-3' (670–689, nts antisense) for Fz2, and 5'-CTC ACA GAA GTC CGG GCT AG-3' (869–888, nts antisense) for Wnt4 were used for Southern blot hybridization of the RT-PCR amplified products.

Antibodies
The affinity purified goat polyclonal antibodies for Wnt4 (catalog no. AF475) and Wnt5a (catalog no. AF645), raised against E. coli-derived recombinant mouse peptides, were purchased from R&D Systems, Inc. (Minneapolis, MN). The affinity purified goat polyclonal antibodies generated against mouse-specific synthetic peptide located at the carboxy terminus of ß-catenin ({alpha}BC) (catalog no. sc-1496) or of SFRP2 (catalog no. sc-7426) or of actin (catalog no. sc-1615) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). An antibody specific to GFP (catalog no. A-11122) was procured from Molecular Probes (Eugene, OR). Anti-{alpha}ABC, clone 8E7, mouse monoclonal IgG1k was bought from Upstate (Lake Placid, NY).

Recombinant Adenoviral Plasmids
The replication-defective adenoviral vectors were generated as previously described by us (63). The full-length coding region of mouse SFRP-2 cDNA was generated by RT-PCR using ovariectomized mouse uterine total RNA. Primers carrying the linkers for XhoI at 5'-ends were used for RT-PCR as follows: 5'-GGCTCGAGATGCCGCGGGGCCCTGCCTC-3' (sense) and 5'-GGCTCGAGCTAGCATTGCAGCTTGCGGA-3' (antisense). The amplified DNA fragment was cloned into XhoI site, in a direction of the sense orientation with respect to a CMV promoter in a shuttle vector, pAdTrack-CMV. The selected clone was sequenced to confirm its identity. Both the SFRP-2 plasmid and the empty shuttle plasmid possess an additional CMV promoter which drives GFP expression independently. Plasmid DNAs were linearized with PmeI and subsequently cotransfected with pAdEasy-1 for recombination into E. coli BJ5183. The recombinant plasmid clones, harboring either SFRP-2 DNA or none (empty control) were confirmed by restriction cutting using PacI and by sequencing.

Preparation of Adenovirus Particles
Virus packaging was carried out into 293 cells as described (61). Virus particles were purified by CsCl density gradient centrifugation and stored at –70 C.

Immunohistochemical Staining
This technique was essentially same as previously described (62). For negative controls, serial sections were incubated with the preimmune serum instead of the primary antibody or preneutralized primary antibody after incubation with 250-fold molar excess of the antigenic peptide.

Immunoprecipitation and Western Blotting
These procedures followed the protocol as previously described (62) with some modification for immunoprecipitation studies. In brief, the protein extracts (500 µg) were incubated in a buffer [0.1% Triton X-100, 20 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol] containing 1 µg primary antibody conjugated with protein A-Sepharose beads (catalog no. 17-0780-01, Pharmacia Sweden) for overnight at 4 C with a gentle shaking. The beads were washed three times with the same buffer and the bound proteins were eluted by boiling the beads in 1 x sodium dodecyl sulfate sample buffer for 5 min. After centrifugation at 10,000 x g for 5 min, supernatants were separated by 10% SDS-PAGE, transferred onto Immuno-Blot polyvinylidene difluoride (PVDF) membrane (catalog no. 162-0177, Bio-Rad), and Western blotted as described before (62).

Adenoviral Infection of Mouse Uterine Stromal Cells in Vitro
To evaluate the appropriate regulation of adenovirus-driven expression of genes, we first analyzed their effects in an in vitro system. Primary culture of mouse uterine stromal cells was grown as previously described by us (63). Cells at 70–80% confluence (in six-well dishes) were subjected to adenoviral infection at 10 multiplication of infection (MOI). The extent of infection was monitored by GFP expression in these cells using a fluorescence microscopy. For SFRP-2 and GFP expression studies, cells were collected after 48 h of infection and proteins were extracted for analysis by Western blotting as described above.

In Vivo Delivery of Adenovirus in Mice
To assess adenovirus-mediated gene expression in vivo, adult ovariectomized mice were inoculated with virus particles iv through a tail vein. Approximately 100 µl of viral solution in saline containing 1 x 1011 virus particles was injected per mouse. After inoculation, they were rested for 7 d to achieve optimum infection. The analysis of viral-driven expression of proteins was performed in uterine tissue extracts by immunoprecipitation and Western blotting for GFP as described above. To examine the effects of E2 on uterine biphasic responses in the adenovirus-infected mice, uterine wet weights were measured at 6 and 24 h after injections of E2 (100 ng/mouse) or oil (control) to analyze early and late estrogenic effects, respectively. The early effects were further assessed by gene expression studies for VEGF and Flk-1, known permeability regulators (37), whereas the late effects were analyzed by bromodeoxyuridine (BrdU) incorporation into DNA. For studies with BrdU, mice were injected sc with BrdU (50 mg/kg body weight) 2 h before they were killed. Formaldehyde-fixed paraffin-embedded tissue sections were stained for BrdU incorporation using biotinylated antibody according to the manufacturer’s instruction (catalog no. 93-3943; Zymed Laboratories Inc., San Francisco, CA).


   ACKNOWLEDGMENTS
 
We thank Bert Vogelstein (Johns Hopkins University, Baltimore, MD) for providing reagents to generate recombinant adenoviral clones.


   FOOTNOTES
 
This work was supported in part by National Institutes of Health Grants (HD12304 and HD33994 to S.K.De., and ES07814 and HD37830 to S.K.Da.).

Abbreviations: {alpha}ABC, Active ß-catenin; BrdU, bromodeoxyuridine; CMV, cytomegalovirus; CRD, cysteine-rich domain; ER, estrogen receptor; Fz, frizzled; GFP, green fluorescent protein; GSK3ß, glycogen synthase kinase 3ß; ICI, ICI 182,780; Lef, lymphoid enhancer factor; LF, lactoferrin; MOI, multiplication of infection; nts, nucleotides; rpL7, ribosomal protein L-7; SFRP-2, secreted Fz-related protein-2; Tcf, T-cell factor; VEGF, vascular endothelial growth factor; VEGFR-2/Flk1, VEGF receptor-2.

Received for publication June 28, 2004. Accepted for publication August 31, 2004.


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