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 UniversityMedical Center, Nashville, Tennessee 37232; and Laboratory Animal Center (Y.T.), ChongqingUniversity of Medical Sciences, Chongqing 400016, People’s Republic of China

Major biological effects of estrogen in the uterusare thought to be primarily mediated by nuclearestrogen receptors, ER� and ER�. We show herethat estrogen in an ER-independent manner rapidlyup-regulates the expression of Wnt4 and Wnt5a ofthe Wnt family and frizzled-2 of the Wnt receptorfamily in the mouse uterus. One of the mechanismsby which Wnts mediate canonical signaling in-volves stabilization of intracellular �-catenin. Weobserved that estrogen treatment prompts nuclearlocalization of active �-catenin in the uterine epi-thelium. We also found that adenovirus mediated in

vivo delivery of SFRP-2, a Wnt antagonist, down-regulates estrogen-dependent �-catenin activitywithout affecting some of the early effects (waterimbibition and angiogenic markers) and inhibitsuterine epithelial cell growth, suggesting thatcanonical Wnt signaling is critical to estrogen-induced uterine growth. Our present results pro-vide evidence for a novel role of estrogen thattargets early Wnt/�-catenin signaling in an ER-independent manner to regulate the late uterinegrowth response that is ER dependent. (MolecularEndocrinology 18: 3035–3049, 2004)

DIVERSE BIOLOGICAL EFFECTS of estrogen areprimarily mediated via activation of its nuclear

estrogen receptors ER� and ER�, which are ligand-inducible transcription factors (1, 2). However, in-creased uterine expression of specific genes after es-trogen exposure in mice missing the ER� gene or inwhich both ER� and ER� functions have been si-lenced by an ER antagonist ICI 182,780 (ICI) sug-gested an alternative pathway for specific estrogenfunctions. For example, we have previously shownthat that uterine expression of secreted frizzled-relatedprotein-2 (SFRP-2), a negative regulator of Wnt sig-naling, is rapidly down-regulated by estrogen in wild-type or ER�(�/�) mice in the presence of ICI or aprotein synthesis inhibitor cycloheximide (3). The re-sult suggested that a rapid onset of Wnt signalingplays a role in mediating estrogen actions in the mouseuterus.

The Wnt family of genes encodes a large group ofhighly conserved secreted glycoproteins. They playcrucial role in embryonic developmental processes(4–6) and are also involved in tumorigenesis (6–8). To

be effective in autocrine or paracrine signaling, Wntproteins must associate with their extracellular surfacereceptors frizzled (Fz) to mediate intracellular signaltransduction pathways (9). Fzs constitute a large fam-ily of seven transmembrane G protein-coupled recep-tors and possess an extracellular cysteine-rich domain(CRD) for Wnt binding (10, 11). Another family of pro-teins, termed secreted Fz-related proteins (SFRPs)produced by different genes, is structurally similar toFzs with respect to CRD, but lacks the seven trans-membrane and the intracellular signaling domains (12,13). SFRPs primarily exert inhibitory effects on Wntsignaling by competing with Wnt ligands for the Fzreceptor and forming a nonfunctional complex withFzs in a dominant-negative manner (14–16).

Wnt functions are executed at least via three intra-cellular signaling pathways in the context of cell types(17–19). Most widely studied, the canonical Wnt sig-naling pathway involves regulation of �-catenin. Theactivation of Wnt signaling stabilizes intracellular�-catenin by inhibiting serine-threonine kinase activityof GSK3� (glycogen synthase kinase 3�). In the ab-sence of Wnt signaling, GSK3� binds to axin (a bridg-ing molecule), adenomatous polyposis coli and�-catenin complex, leading to �-catenin phosphoryla-tion and its degradation by ubiquitination. The activeintracellular �-catenin is considered to be the stabi-lized dephosphorylated from (20–22), which translo-cates to the nucleus and forms complex with lymphoidenhancer factor (Lef)/T-cell factor (Tcf) family of tran-scription factors to stimulate transcription of Wnt tar-get genes (23, 24). Two other noncanonical Wnt sig-naling pathways include Wnt/Ca2� and Wnt/ras

Abbreviations: �ABC, Active �-catenin; BrdU, bromode-oxyuridine; CMV, cytomegalovirus; CRD, cysteine-rich do-main; ER, estrogen receptor; Fz, frizzled; GFP, green fluores-cent protein; GSK3�, glycogen synthase kinase 3�; ICI, ICI182,780; Lef, lymphoid enhancer factor; LF, lactoferrin; MOI,multiplication of infection; nts, nucleotides; rpL7, ribosomalprotein L-7; SFRP-2, secreted Fz-related protein-2; Tcf, T-cell factor; VEGF, vascular endothelial growth factor; VEGFR-2/Flk1, VEGF receptor-2.

Molecular Endocrinology is published monthly by TheEndocrine Society (http://www.endo-society.org), theforemost professional society serving the endocrinecommunity.

0888-8809/04/$15.00/0 Molecular Endocrinology 18(12):3035–3049Printed in U.S.A. Copyright © 2004 by The Endocrine Society

doi: 10.1210/me.2004-0259

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homolog gene family, member A/c-Jun NH2-terminalkinase pathways that primarily affect actin cytoskele-ton and planar polarity of cells (17, 19, 25). Activationof each signaling pathway depends on the type ofligands and receptors involved because Wnt proteinspossess preferential binding to specific receptors (26).Moreover, distinct Fzs are also known to exhibit dif-ferential activation of these various signaling pathways(27–29), although the pathway specificity appears tobe developmentally regulated.

It is well known that Wnt signaling plays rolesin epithelial-mesenchymal interactions and cellularorganization during embryonic and postembryonic de-velopment that involve cell proliferation and differen-tiation, cell fate specification and cell-to-cell commu-nication (4–6). Wnt signaling is also consideredimportant for female reproductive functions (30, 31)and has been described as a target for endocrinedisruptors (32). Our present study shows for the firsttime that several genes of the Wnt signaling pathwayare regulated by estrogen in the mouse uterus in anearly responsive manner without involving classicalERs. Furthermore, we show here that �-catenin-mediated rapid canonical Wnt signaling is activated inuterine epithelial cells under the direction of estrogenin both wild-type and ER�(�/�) mice. Most impor-tantly, in vivo delivery of adenovirally expressedSFRP-2 (a Wnt antagonist) in ovariectomized wild-type mice markedly inhibits estrogen-dependent uter-ine growth and �-catenin activity. Collectively, thesestudies demonstrate that estrogen-dependent controlof Wnt/�-catenin signaling that is mediated in a non-classical manner regulates late uterine growth re-sponse that is ER dependent.

RESULTS

Estrogen Regulates Early Wnt Signaling in theMouse Uterus in the Absence of ER Function

Our previous studies demonstrated that estrogen reg-ulates the expression of several early responsivegenes in the ovariectomized uteri of both wild-typeand ER�(�/�) mice (3). One of those genes, SFRP-2,an antagonist of Wnt signaling, was strongly sup-pressed after estrogen stimulation, suggesting estro-gen’s influence in regulating early Wnt signaling in theuterus. This observation led us to examine whether theWnt and Fz family members are regulated by estrogenin the mouse uterus in the presence or absence ofER�. Our initial uterine analysis by RT-PCR using ei-ther types of mice showed that expression of Wnt4,Wnt5a, and Fz2 genes was predominately up-regu-lated due to an injection of estradiol-17� (E2) by 6 h,whereas that of Wnt1, Wnt3, Wnt7a, Wnt7b, Fz4, Fz6,or Fz7 genes was below the level of detection orremained unaffected by this treatment (data notshown). Although a previous study (30) showed thatuterine expression of Wnt4, Wnt5a, and Wnt7a genes

was regulated during various stages of the cycle inmice; however, our analysis in ovariectomized miceindicates that the expression of Wnt7a may not beregulated by estrogen during its early phase of action.Thus, our subsequent studies focused on the regula-tion of uterine Wnt4, Wnt5a, and Fz2 genes byestrogen.

To examine whether estrogen influences the ex-pression of Wnt4, Wnt5a, and Fz2, ovariectomizedwild-type mice received a sc injection of E2 (100 ng/mouse) and killed at 1, 2, 6, 12, and 24 h. Mice injectedwith oil (vehicle) served as controls. Total RNA sam-ples obtained from whole uteri were analyzed byNorthern blot hybridization. Analysis of our data showsthat, although the mRNA levels of these genes werelow in oil-treated samples, an injection of E2 causedrapid induction of these mRNAs by 1 h with a peakresponse at 2 h and maintained through 6 h (Fig. 1A).The levels of Wnt4 and Fz2 mRNAs showed somedecline by 12 and 24 h, whereas that of Wnt5a re-mained unaffected. The maximal inductive responseby E2 of Wnt4, Wnt5a, and Fz2 genes was about 5-, 3-,and 6-fold, respectively, as compared with the corre-sponding oil-injected groups.

We next asked whether estrogen-regulated re-sponses, as observed above, were mediated throughthe participation of ER� and/or ER�. To address thisquestion, ovariectomized wild-type mice were givenan injection of oil, E2, ICI or ICI plus E2 as describedin Materials and Methods, and uterine tissues werecollected at 6 h after the last injection. Total uterineRNA samples were analyzed by Northern blotting.As shown in Fig. 1B, the results show that uterinelevels of Wnt4, Wnt5a, and Fz2 expression were lowafter an injection of oil or ICI. However, an injectionof E2 caused up-regulation of the mRNA levels ofthese genes. Notably, the treatment of ICI before E2

injection failed to attenuate estrogen-induced ex-pression of these genes. To evaluate the efficacyof ICI on Wnt signaling target genes and to ruleout the role of ER in these responses, we analyzedthe expression of lactoferrin (LF), a highly ER-dependent estrogen-responsive gene in the mouseuterus (33) (Fig. 1B). Our results show that estrogen-induced expression of LF in uteri of wild-type miceis suppressed by a coinjection ICI with estrogen.The results suggest that ICI used in this study isindeed effective in antagonizing the expression ofER-dependent target genes. In contrast, ICI’s failureto influence the expression of estrogen-regulatedWnt signaling genes provides strong evidence thatthe effects of estrogen on these genes are not me-diated via ER-� and/or ER-�.

To provide further genetic evidence that estro-genic responses were mediated via a non-ER mech-anism, we analyzed uterine expression of Wnt4,Wnt5a, and Fz2 in ER�(�/�) mice and comparedwith that in the wild-type littermates using a quan-titative RT-PCR as previously reported (3, 33, 34).We used this technique because of the limited avail-

3036 Mol Endocrinol, December 2004, 18(12):3035–3049 Hou et al. • Estrogen Mediates Uterine Growth via Wnt Signaling

ability of uterine RNA samples from ER�(�/�) mice.Injection schedules were same as described abovefor wild-type mice. Irrespective of the treatments,our results show that the relative levels of mRNAsfor Wnt4, Wnt5a, and Fz2 genes in the wild-typeuteri are higher as compared with those in ER�(�/�)mice (Fig. 1C). However, an injection of E2 increasedthe mRNA copy numbers of Wnt4, Wnt5a, and Fz2genes by approximately 4-, 3-, and 4-fold, respec-tively, compared with the corresponding controls (oiltreatment). The ICI compound again failed to antag-onize these estrogenic responses in either wild-typeor mutant mice. Collectively, these results suggestthat the regulation of uterine expression for Wnt4,Wnt5a, and Fz2 genes by E2 occurs in an early

responsive manner without involving ERs in themouse uterus.

Estrogen Regulates Uterine Wnt4, Wnt5a, andFz2 Expression in a Cell-Specific Manner

To examine cell-specific uterine expression of Wnt4,Wnt5a, and Fz2 genes by estrogen, in situ hybridiza-tion was performed on frozen uterine sections ob-tained from ovariectomized wild-type or ER�(�/�)mice after receiving estrogen alone or estrogen plusantiestrogen for 6 h as described in Materials andMethods. The results show that uterine cell-specificaccumulation of Wnt4 (Fig. 2A), Wnt5a (Fig. 2B), andFz2 (Fig. 2C) mRNAs was low in both the ovariecto-

Fig. 1. Analysis of Uterine Gene ExpressionA, 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 oilwere killed after 6 h and served as controls. Total RNA samples (6 �g) 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. Adultovariectomized mice were given an injection of oil, E2 (100 ng/mouse), ICI (500 �g/mouse) or ICI 30 min before an injection of E2

and killed at 6 h after the last injection. Total RNA samples (6 �g) 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�(�/�) mice in response to estrogen and/or ICI as described inFig. 1B. Quantitative RT-PCR for gene-specific mRNAs was performed on total RNA samples as described (34). *, Values arestatistically different (P � 0.05, ANOVA followed by Newman-Keul’s multiple range test).

Hou et al. • Estrogen Mediates Uterine Growth via Wnt Signaling Mol Endocrinol, December 2004, 18(12):3035–3049 3037

Fig. 2. In Situ Hybridization of Wnt4, Wnt5a, and Fz2 in Wild-Type or ER�(�/�) MiceWnt4 (A), Wnt5a (B), and Fz2 (C). Ovariectomized wild-type or ER�(�/�) mice were treated with oil, E2, ICI, or E2 plus ICI as

described in Fig. 1B. Frozen sections (10 �m), fixed in paraformaldehyde, were mounted onto glass slides, prehybridized andhybridized with 35S-labeled sense or antisense riboprobes for 4 h at 45 C. Ribonuclease A-resistant hybrids were detected after5–7 d of autoradiography using Kodak NTB-2 liquid emulsion (Eastman Kodak, Rochester, NY). Sections were poststained lightlywith hematoxylin and eosin. Representative dark-field photomicrographs of uterine sections are shown. The reddish brown grains

3038 Mol Endocrinol, December 2004, 18(12):3035–3049 Hou et al. • Estrogen Mediates Uterine Growth via Wnt Signaling

mized wild-type and ER� mutant mice after an injec-tion of oil or ICI. However, an injection of E2 causeddifferential cell-specific up-regulation of these genesin wild-type or ER�(�/�) mice. For example, a distinctaccumulation of Wnt4 mRNA was predominantlynoted in the luminal and glandular epithelium and alsoin the subluminal stroma (Fig. 2A). In contrast, Wnt5amRNA was primarily detected in luminal and glandularepithelial cells, although some patchy weak signalswere also noted in the stroma (Fig. 2B). In contrast,the expression of Fz2 was evident in the epitheliumand throughout the stroma of both wild-type andER�(�/�) mice (Fig. 2C). An injection of ICI before E2

injection failed to alter estrogen-induced expressionpattern of these genes either in wild-type or mutantmice (Fig. 2, A–C). Hybridization with the correspond-ing sense cRNA probes did not show any positivesignals (data not shown).

Wnt4, Wnt5a, and SFRP-2 Proteins AreExpressed by E2 in the Mouse Uterus

To determine whether Wnt4 and Wnt5a mRNAs in-duced by estrogen in the uterus are effectively trans-lated, immunohistochemistry was used to localize cell-specific accumulation of Wnt4 and Wnt5a proteins inuterine sections obtained from ovariectomized wild-type or ER�(�/�) mice at 6 h of oil or E2 injection (Fig.3, A and B, respectively). Although weak immunostain-ing was observed for either of these proteins in theuterine epithelium of oil-treated mice, strong stainingwas noted for Wnt4 in the epithelium and in the sub-luminal stroma in both wild-type and ER�(�/�) miceafter an injection of E2. Wnt5a showed a similar patternof signal, but at a reduced intensity.

We have previously shown that SFRP-2 is stronglyexpressed in the ovariectomized mouse uterine stroma,and this expression is rapidly suppressed by an injec-tion of E2 (3). Because SFRP-2 antagonizes Wnt sig-naling, we wanted to examine whether SFRP-2 proteinis indeed suppressed during the time of E2 mediatedup-regulation of Wnt4 and Wnt5a. As shown by West-ern blot analysis (Fig. 3C), a distinct protein band (�30kDa) for SFRP-2 was detected in ovariectomized uter-ine tissue extracts after an injection of oil at 6 h. Incontrast, the intensity of this band was remarkablyweak in uterine samples after an injection of E2 eitherat 6 or 24 h (Fig. 3C). This band is specific for SFRP-2because a similar blot incubated in parallel with theprimary antibody preneutralized by 250-fold molar ex-cess of antigenic peptide (sc-7426P, Santa Cruz Bio-technology, Inc.) failed to show such a band (data notshown). These results correlate well with our mRNAdata and suggest that estrogen-stimulated up-regula-

tion of Wnt4 and Wnt5a occurs in the uterus during thetime when uterine SFRP-2 expression is remainedsuppressed by E2.

E2 Activates Canonical Wnt Signaling in theMouse Uterus

Because Wnt signaling mediated by canonical path-way regulates intracellular concentration of �-catenin,we examined whether estrogen can modulate the lev-els of �-catenin in the uterus. Western blot analysisusing an anti-�-catenin antibody was performed toexamine the levels of total �-catenin in uterine extractsobtained from ovariectomized wild-type or ER�(�/�)mice after an injection of oil or E2. The results showthat an accumulation of �-catenin was induced by E2

at 6 and 24 h both in the wild-type (Fig. 4A) orER�(�/�) (data not shown) mice as compared with alow basal level after the oil injection (control).

We also examined cell-specific distribution of total�-catenin by immunohistochemistry on formalin-fixedparaffin-embedded uterine sections of both wild-typeand ER�(�/�) mice under similar conditions. Wefound that total �-catenin was predominantly localizedin the uterine epithelium, apparently in the cell junc-tion, after an injection of oil (Fig. 4B). The intensity ofthe signal was significantly enhanced by E2 treatmentat 6 h in both wild-type and ER�(�/�) mice (Fig. 4B);low levels of accumulation were also evident in stromalcells in the subluminal region irrespective of the treat-ment. Similar results were also obtained after E2 injec-tion by 24 h (data not shown). Collectively, theseresults suggest that E2 is capable of stimulating epi-thelial cell-specific accumulation of �-catenin in a sus-tained manner without requiring ER.

It is well established that the canonical Wnt signalingstabilizes intracellular �-catenin in a dephosphorylatedform. In cells that do not receive Wnt signaling, free�-catenin is targeted for proteasomal degradation viaits N-terminal Ser-37/Thr-41 phosphorylation byGSK3� (20). The canonical Wnt signaling inhibitsGSK3� activity, resulting in the accumulation of active�-catenin that is targeted to the nucleus to form nu-clear complex with Tcf/Lef transcription factors (21,22). We used an antibody specific to active �-catenin(�ABC) to further examine the activation of Wnt sig-naling in the mouse uterus after an injection of E2 inboth ovariectomized wild-type and ER�(�/�) mice(Fig. 4C). The results of immunostaining revealed thatactive �-catenin is exclusively localized in uterine lu-minal and glandular epithelial cell nuclei of both wild-type and ER�(�/�) mice by 6 h (Fig. 4C) and retainedfor 24 h (data not shown) after exposure to E2. Incontrast, we did not see any such nuclear accumula-

indicat the sites of mRNA accumulation. This color shade is the result of lateral light scattering from the eosin staining underdark-field microscopy. Sectioned hybridized with the sense probes served as negative controls (data not shown). Bars, 100 �M.le, Luminal epithelium; s, stroma; and myo, myometrium. These experiments were repeated three times with three to four micein each group and similar results were obtained.

Hou et al. • Estrogen Mediates Uterine Growth via Wnt Signaling Mol Endocrinol, December 2004, 18(12):3035–3049 3039

tion after the oil injection (Fig. 4C). Collectively, theseresults provide clear evidence that the canonical Wntsignaling becomes active in the uterus by estrogen notinvolving ER�.

Adenoviral Expression of SFRP-2 SuppressesEstrogen-Dependent Uterine Regulationof �-Catenin

Because SFRP-2 acts as a negative regulator of Wntsignaling, we examined whether adenovirally ex-pressed SFRP-2 influences estrogen-dependent ca-nonical Wnt signaling in the uterus. The recombinantadenovirus particles carrying SFRP-2 and the empty

vector (control) under the direction of a cytomega-lovirus (CMV) promoter were made. These con-structs were also equipped with a green fluorescentprotein (GFP) expression system under a separateCMV promoter. In our initial experiments, we verifiedwhether these virus particles appropriately regulatethe expression of genes in uterine cells in vitro.Western blot analyses show that the adenoviral par-ticles carrying the SFRP-2 gene indeed expressedSFRP-2 in primary uterine stromal cell cultures,whereas the control virus particles failed to expressthis protein under similar cell culture conditions(Fig. 5A). The expression of GFP by either type ofvirus particles suggests that virus-driven expression

Fig. 3. Immunolocalization of Wnt4 (A) and Wnt5a (B) in Uteri of Ovariectomized Wild-Type and ER�(�/�) Mice after Exposureto Oil or E2.

Injection schedules and the dose of various agents were same as described in Fig. 1. Paraformaldehyde-fixed paraffin-embedded sections (6 �m) were used. The sections were incubated with primary antibody at 1:500 dilution in PBS for 17 h at 4C. Representative photomicrographs of uterine sections are shown. Red deposits indicate positive immunostaining. Theseexperiments were repeated three times with three to four mice in each group and similar results were obtained. No immuno-staining was noted when sections were incubated with preimmune serum instead of primary antibody (data not shown). Bars, 100�m. le, Luminal epithelium; ge, glandular epithelium; s, stroma; and myo, myometrium. C, Western blot analysis of SFRP-2 inwild-type uterine tissue extracts. Adult ovariectomized wild-type mice were given a single injection of E2 (100 ng/mouse) and killedat times indicated. Mice injected with oil were killed after 6 h and served as controls. Uterine tissue extracts (80 �g 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 notshown).

3040 Mol Endocrinol, December 2004, 18(12):3035–3049 Hou et al. • Estrogen Mediates Uterine Growth via Wnt Signaling

of the transgene is appropriately regulated in uterinecells in vitro (Fig. 5A).

We next examined whether these virus-driven par-ticles reach the uterine target and produce the expres-sion of the transgene after in vivo delivery via a tailvein. Because the SFRP-2 transgene, which we usedin generating the adenovirus particles, is a mouse-specific gene, it is not possible to distinguish the ex-pression levels of the SFRP-2 transgene vs. the nativeSFRP-2 gene in the uterus. To circumvent this prob-lem, we analyzed the expression of virus-driven GFP.Analysis of uterine proteins by immunoprecipitationand Western blotting showed that the viral-driven GFPis expressed after the delivery of virus particles (Fig.5B), providing evidence that in vivo administration ofthese particles can reach the uterine target and drive

gene expression. The results of our in vitro analysiswould suggest that both the transgenes are regulatedin a similar manner in the uterus in vivo. To furtherexamine the effectiveness of this approach, estrogen-dependent uterine �-catenin expression was exam-ined after in vivo delivery of these virus particles. Theresults show that estrogen-induced up-regulation oftotal �-catenin at 24 h is strongly suppressed by viral-driven SFRP-2, whereas the control virus fails to showany such effects (Fig. 5C). Under similar conditions,we also analyzed uterine cell-specific expression ofGFP by direct visualization of GFP fluorescence inuterine sections. As shown in Fig. 5D, uterine epithelialand stromal cells exhibited the expression of GFP afteradministration of the SFRP-2 or control (data notshown) virus particles in mice. This provides evidence

Fig. 4. Analysis of Uterine Expression of �-CateninA, Western blot analysis of total �-catenin (�BC) in wild-type uterine tissue extracts. Injection schedules and Western blotting

experiments are same as described in Fig. 3C. Primary antibody preneutralized by 250-fold molar excess of the antigenic peptidedid not detect any bands (data not shown). Immunolocalization of total �-catenin (�BC) (B) and �ABC (C) in uteri of ovariectomizedwild-type and ER�(�/�) mice after exposure to oil or E2 as described in Fig. 1B. Red deposits indicate positive immunoreactivity.These experiments were repeated three times with three to four mice in each group, and similar results were obtained. Noimmunostaining was noted when similar sections were incubated with preimmune serum instead of primary antibody (data notshown). Bars, 100 �m. le, Luminal epithelium; ge, glandular epithelium; s, stroma. Note: Distinct nuclear localization of active�-catenin is clearly detected in uterine epithelial cells for either type of mice after the treatment of estrogen. Pictures are shownby high magnification within the insets. Bars, 40 �m.

Hou et al. • Estrogen Mediates Uterine Growth via Wnt Signaling Mol Endocrinol, December 2004, 18(12):3035–3049 3041

that the virus-mediated SFRP-2 transgene expressionin the uterus is available to act in a dominant-negativemanner for Wnt signaling in vivo. Overall, these resultssuggest that E2-induced canonical Wnt signaling in theuterus is neutralized by the SFRP-2 virus particles.

Inhibition of Canonical Wnt Signaling AbrogatesEstrogen-Dependent Uterine Growth Response

Uterine estrogenic effects are traditionally consideredbiphasic: the early response that occurs within 6 h ischaracterized by increased water imbibitions andmacromolecular uptake, whereas the late responseoccurs between 16–30 h and is defined by increasedepithelial cell DNA synthesis and hyperplasia (35, 36).

Because forced expression of SFRP-2 negatively im-pacts Wnt/�-catenin signaling in the mouse uterus, wenext asked whether similar expression of SFRP-2would interfere with estrogenic responses in the uterus.As shown in Fig. 6, A–D, in vivo delivery of viral-drivenSFRP-2 demonstrated a striking suppression of estro-gen-dependent late phase of uterine epithelial cellDNA synthesis and growth, whereas the control virusfailed to evoke such a response. In contrast, estrogen-induced uterine water imbibition and vascular perme-ability genes VEGF (vascular endothelial growth factor)and Flk1 (VEGF receptor-2) (37) that occur during theearly phase were not affected by similar treatments(Fig. 6, B and E). Overall, these results suggest thatcanonical Wnt signaling that occurs during the early

Fig. 5. Analysis of Adenovirus-Mediated ExpressionA, 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 GFPexpression in the uterus. SFRP-2 or control virus particles were injected in ovariectomized mice through a tail vein. They wererested for 7 d to achieve optimum infection. Uterine tissue extracts were analyzed for viral-driven expression of GFP byimmunoprecipitation followed by Western blotting using a GFP antibody. Primary uterine stromal cell extracts expressing GFPwere used as a positive control. C, Western blot analysis of �-catenin in uterine tissue extracts. Virus (SFRP-2 or control) infectedmice, rested for 7 d, were given a single injection of E2 and killed 24 h later. Another group of noninfected mice was similarlytreated with E2 injection. Uterine tissue extracts were analyzed for the expression of �-catenin and actin proteins. D, Directvisualization of GFP fluorescence in uterine tissue sections. Treatments were same as described in Fig. 5C. Frozen uterine tissuesections (10 �m) were covered with mounting solution and then subjected to capture photomicrographs under fluorescencemicroscope for GFP (right panels) or under the phase for bright-fields (left panels). Bars, 40 �m. le, Luminal epithelium; s, stroma.

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estrogenic response without involving ER is crucial tolate estrogenic uterine growth response.

DISCUSSION

The uterus is a primary target of estrogen for variousfunctions during the reproductive cycle and preg-nancy. In mice, estrogen induces uterine epithelial cellproliferation and is essential for the maintenance ofnormal epithelial morphogenesis, cytodifferentiation,and secretory activity (38). The general consensus isthat estrogen exerts its effects by modifying gene ex-pression through activation of its nuclear receptor ER�in the uterus (39–41). Here we have shown that estro-gen in an ER-independent manner up-regulates ca-nonical Wnt signaling in the uterus, suggesting a novelrole for estrogen action in the target tissue (Fig. 7). It isa long-sought question how the biphasic estrogenicresponses in the uterus are molecularly linked. Ourpresent observation that the canonical Wnt signalingpathway is activated very early in response to estro-gen and is crucial to late estrogenic growth providesstrong evidence that Wnt/�-catenin signaling is a linkbetween the two phases. Our study also provides thefirst evidence how an ER-independent early responsepathway is integrated into an ER-dependent lategrowth response pathway.

Wnt signaling is known to participate in epithelial-mesenchymal communication (30, 42–47). Severalmembers of the Wnt gene family are expressed in theuterus (30) and mammary glands (45–47). Expressionof specific Wnt genes in these tissues is restricted toeither epithelial, stromal, or both cell types, and theirexpression levels fluctuate with changing hormonelevels during the reproductive cycle and pregnancy.Although genetic studies in mice involving severalWnts have established that Wnt signaling is crucial toembryonic developmental processes (4–6), the biolog-ical significance of Wnt signaling in adult tissue ho-meostasis remains poorly understood. It is believedthat estrogen-regulated epithelial cell-specific func-tions are directed by signals emanating from the stro-mal cell compartment (48). However, the nature of theregulatory signals still remains largely unknown. Ourprevious observation of heightened expression ofSFRP-2 in stromal cells when the uterus is quiescentafter ovariectomy (3), together with our present obser-vation of suppressed SFRP-2 expression with up-reg-ulation of Wnt4, Wnt5a, and Fz2 in both the epithelialand stromal cells by estrogen (Fig. 3), suggests thatWnt signaling is operative in both cell types.

�-Catenin binds to the cytoplasmic domain of E-cadherin to form a linkage with the actin cytoskeletonand regulates its function at the adherence junctions(49). The increased accumulation of total �-catenin atthe epithelial cell junctions after estrogen stimulationsuggests that this protein is involved in the mainte-nance of adherence junctions in these cells (Fig. 4B). In

contrast, estrogen-stimulated distinct nuclear accu-mulation of active �-catenin in epithelial cells suggeststhe operation of a canonical Wnt signaling (Fig. 4C).The absence of active �-catenin in stromal cells sug-gests that these cells are not involved in canonical Wntsignaling. Wnts can also execute signal transduction ina noncanonical manner using Wnt/Ca2� or Wnt/rashomolog gene family, member A/c-Jun NH2-terminalkinase pathway (17, 19, 25). It is possible that thesealternative Wnt signaling pathways are operative instromal cells.

It is generally believed that Wnts are secreted cell-signaling molecules (50–52). There is evidence thatWnts bind to Bip (also known as glucose-regulatedprotein 78 kDa, GRP78), a resident protein in the en-doplasmic reticulum. This interaction is thought to berequired for preventing secretion of improperly foldedpolypeptides and for efficient secretion of Wnts (53,54). We have previously shown that estrogen alsoup-regulates the expression of Bip in a non-ER-medi-ated manner in uterine epithelial and stromal cells (3).We speculate that a direct interaction of Bip with Wntligands occurs in the mouse uterus after estrogenstimulation to regulate Wnt secretion.

Our present study presents strong evidence thatuterine epithelial cell-specific activation of canonicalWnt signaling by estrogen is critical to epithelial cellgrowth in response to estrogen. Our studies show thatthe virus-driven expression of GFP and thus SFRP-2 isdetectable in uterine epithelial and stromal cells. How-ever, we cannot be absolutely certain that the expres-sion of the SFRP-2 transgene in the uterus directlyaffected the regulation of uterine �-catenin expressionbecause it is not possible to distinguish the uterineexpression of mouse-specific SFRP-2 transgene fromthe native gene. It is also possible that other nonuter-ine targets are involved, but our experiments using theovariectomized mouse model with supplementation ofsteroid hormones excludes at least the involvement ofovaries. Nonetheless, evidence for nuclear transloca-tion of �-catenin in the regulation of gene transcriptionand cellular proliferation (23, 24) is consistent with ourpresent findings of nuclear translocation of �-cateninin uterine epithelial cells and their proliferative re-sponse by estrogen. Previous studies that used recip-rocal tissue recombination experiments between theuterine epithelium and stroma from the wild-type andER�(�/�) mice suggest that estrogen-mediated epi-thelial cell growth is positively controlled by stromalER� (55). However, our findings of ER-independentWnt signaling in uterine epithelial cells suggest anothermechanism that does not appear to involve stromabecause regulation of �-catenin by estrogen is notdifferent in uteri of wild-type and ER�(�/�) mice.There is also evidence to suggest that Wnt4, Wnt5a,and Fz2 do not necessarily involve canonical Wnt sig-naling (23–25). However, our results demonstratingestrogen-dependent up-regulation of these genes inconjunction with the stabilization of �-catenin in theuterus in an early time-dependent manner suggest

Hou et al. • Estrogen Mediates Uterine Growth via Wnt Signaling Mol Endocrinol, December 2004, 18(12):3035–3049 3043

Fig. 6. Analysis of Estrogen-Dependent Uterine Effects in Ovariectomized Mice after Administration of AdenovirusesA, 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 wetweights in similarly treated mice. Normal estrogenic responses (E2, 100 ng/mouse) were determined in parallel without any viralinfection. Oil-injected mice served as controls. Uterine wet weights were recorded at 6 and 24 h after E2 injection. The numberof mice used in each group is indicated within the bars. Values with asterisks are statistically different (P � 0.01, ANOVA followed

3044 Mol Endocrinol, December 2004, 18(12):3035–3049 Hou et al. • Estrogen Mediates Uterine Growth via Wnt Signaling

that the activation of canonical Wnt signaling in theuterus is a function of time.

The mechanism of ER-independent regulation ofuterine genes is currently unknown. The answer to thisquestion awaits the cloning and identification of agene(s) encoding functional membrane ER(s). Al-though the physiological implication of up-regulationof Wnt4, Wnt5a, and Fz2 by estrogen, but down-regulation of SFRP-2 in the uterus, follows a temporalrelationship and is to potentiate Wnt signaling, themolecular mechanism by which this is achieved is not

currently known. However, it is to be noted thatSFRP-2 is regulated by estrogen without involving anyintermediary protein synthesis (3). This suggests post-translational mechanism, i.e. phosphorylation and/ordephosphorylation could be involved.

The downstream effects of canonical Wnt signalinglead to the association of �-catenin with its effectorproteins Lef/Tcf family member of transcription factorsbefore their interaction on specific target gene pro-moters for gene transcription. We have preliminaryresults to show that Tcf3 and Tcf4 are expressed in the

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

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

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 positiveimmunostaining. Bars, 100 �m. le, Luminal epithelium. D, Quantitation of BrdU-positive cells in the luminal epithelium as shownin C. Approximately 500 cells were counted in each group. E, Analysis of early estrogenic effects at 6 h. Uterine sections wereanalyzed histologically (hematoxylin and eosin staining), and by in situ hybridization of VEGF and Flk-1. Bars, 100 �m. le, Luminalepithelium; myo, myometrium; s, stroma.

Hou et al. • Estrogen Mediates Uterine Growth via Wnt Signaling Mol Endocrinol, December 2004, 18(12):3035–3049 3045

mouse uterus after estrogen stimulation (Cox, S., andS. K. Das, unpublished data). ER-dependent transcrip-tional activation is not only influenced by specific li-gand binding to its receptor, but also by the recruit-ment of coregulatory proteins at the level of chromatininteraction. The manifestation of estrogen-regulatedlate growth response in the uterus is believed to be theresult of ER�-dependent gene transcriptional activities(56). Changes in gene expression are generallyachieved by the convergence of signaling pathwayson transcription factors, governing their transactiva-tion potential in the context of specific target genes.We speculate that estrogen-regulated �-catenin/tran-scription factor complex mutually interacts with ER toachieve combinatorial or distinct functions at the levelof DNA transcription machinery. Both Tcf and ER rec-ognize discrete nucleotide sequences in the promoterof their target genes. There is also evidence that thesetwo nuclear transcription factors interact directly eitheron the DNA or in the absence of DNA, and the resultingcomplex interaction could be antagonistic or stimula-tory depending on the target gene promoter activity(57). It is possible that an interaction between thesetwo transcription factors in response to estrogen setsup a dialogue to induce the late estrogenic growthresponse. In conclusion, our present findings providenew insights into a complex system that impactsestrogen-dependent regulation of uterine growth re-sponse. The activation of ER-independent Wnt/�-catenin signaling by estrogen in uterine epithelial cellsduring the early phase significantly contributes to theER-dependent late growth response.

MATERIALS AND METHODS

Animals and Injections

Wild-type and ER�(�/�) mice of the same genetic back-ground (C57BL/6J/129/J) were produced by crossing of het-erozygous females and males [Lubahn et al., (64)]. In allstudies, wild-type littermates were analyzed in parallel withER�(�/�) mice. They were housed in the institutional animalcare facility according to the National Institutes of Health andinstitutional guidelines for the care and use of laboratoryanimals. Genotyping was performed by PCR analysis of tailDNA. In general, 8- to 10-wk-old adult mice were ovariecto-mized and rested for 2 wk before they received any injections(3, 34). Mice were injected with oil (0.1 ml) or estradiol-17�(E2, 100 ng/mouse) or ICI 182,780 (ICI, 500 �g/mouse), apotent antiestrogen for both ER� and ER� or the same doseof ICI 30 min before E2 injection. They were killed at differenttimes as indicated after the last injection. The test agentswere dissolved in sesame oil and injected (0.1 ml/mouse) sc.

Probes and Hybridizations

cRNA probes were generated from mouse-specific cDNAclones. The clones of Wnt4 and Wnt5a in pGEM7Zf(�) vec-tors (58) or Fz2 in a pBluescript vector (10) were used. cDNAclones for VEGF, Flk-1, LF, and ribosomal protein L-7 (rpL7)have previously been described (33, 34, 37). Northern or insitu hybridization technique was same as previously de-

scribed (59, 60). Hybridized bands on Northern blots wereanalyzed by densitometric scanning. In situ hybridizationslides were post-stained with hematoxylin and eosin. Seriallysectioned slides hybridized with the sense probe were servedas negative controls.

Quantification of RNAs by Competitive RT-PCR

This was performed as previously described (34). Competi-tive templates used for this study were generated by intro-ducing a nonspecific DNA fragment into or by deleting acDNA fragment from the mouse-specific target gene. Spe-cifically, a 185 bp blunt-ended fragment (Ssp1), obtainedfrom pGEM7Zf (�) vector, was inserted into cDNA clones forWnt5a at the SmaI site or Fz2 at the StuI site. For Wnt4, aSmaI fragment (438 bp) was deleted from the original cloneobtained from Andrew McMahon (58) and subsequently li-gated at the ends to generate the competitor template. Thefollowing primers were used for RT-PCR: 5�-ATT GTC CCCCAA 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 accessionno. AF363723); 5�-CGA GGA GTG CCA ATA CCA GT-3�(270–289 nts, sense) and 5�-GCC GTC AAT GGC TTT AGATG-3� (941–960 nts, antisense) for Wnt4 mRNA (GenBankaccession no. M89797). The internal primers 5�-CCT TGAGAA AGT CCT GCC AG-3� (764–783 nts, antisense) forWnt5a, 5�-CGA TGA GCG TCA TGA GGT AT-3� (670–689, ntsantisense) for Fz2, and 5�-CTC ACA GAA GTC CGG GCTAG-3� (869–888, nts antisense) for Wnt4 were used for South-ern blot hybridization of the RT-PCR amplified products.

Antibodies

The affinity purified goat polyclonal antibodies for Wnt4 (cat-alog no. AF475) and Wnt5a (catalog no. AF645), raisedagainst E. coli-derived recombinant mouse peptides, werepurchased from R&D Systems, Inc. (Minneapolis, MN). Theaffinity purified goat polyclonal antibodies generated againstmouse-specific synthetic peptide located at the carboxy ter-minus of �-catenin (�BC) (catalog no. sc-1496) or of SFRP2(catalog no. sc-7426) or of actin (catalog no. sc-1615) werepurchased from Santa Cruz Biotechnology, Inc. (Santa Cruz,CA). An antibody specific to GFP (catalog no. A-11122) wasprocured from Molecular Probes (Eugene, OR). Anti-�ABC,clone 8E7, mouse monoclonal IgG1k was bought from Up-state (Lake Placid, NY).

Recombinant Adenoviral Plasmids

The replication-defective adenoviral vectors were generatedas previously described by us (63). The full-length codingregion of mouse SFRP-2 cDNA was generated by RT-PCRusing ovariectomized mouse uterine total RNA. Primers car-rying the linkers for XhoI at 5�-ends were used for RT-PCRas follows: 5�-GGCTCGAGATGCCGCGGGGCCCTGCCTC-3� (sense) and 5�-GGCTCGAGCTAGCATTGCAGCTTGCG-GA-3� (antisense). The amplified DNA fragment was clonedinto XhoI site, in a direction of the sense orientation withrespect to a CMV promoter in a shuttle vector, pAdTrack-CMV. The selected clone was sequenced to confirm its iden-tity. Both the SFRP-2 plasmid and the empty shuttle plasmidpossess an additional CMV promoter which drives GFP ex-pression independently. Plasmid DNAs were linearized withPmeI and subsequently cotransfected with pAdEasy-1 forrecombination into E. coli BJ5183. The recombinant plasmidclones, harboring either SFRP-2 DNA or none (empty control)were confirmed by restriction cutting using PacI and bysequencing.

3046 Mol Endocrinol, December 2004, 18(12):3035–3049 Hou et al. • Estrogen Mediates Uterine Growth via Wnt Signaling

Preparation of Adenovirus Particles

Virus packaging was carried out into 293 cells as described(61). Virus particles were purified by CsCl density gradientcentrifugation and stored at �70 C.

Immunohistochemical Staining

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

Immunoprecipitation and Western Blotting

These procedures followed the protocol as previously de-scribed (62) with some modification for immunoprecipitationstudies. In brief, the protein extracts (500 �g) were incubatedin a buffer [0.1% Triton X-100, 20 mM HEPES (pH 7.5), 150mM NaCl, 10% glycerol] containing 1 �g primary antibodyconjugated with protein A-Sepharose beads (catalog no. 17-0780-01, Pharmacia Sweden) for overnight at 4 C with agentle shaking. The beads were washed three times with thesame buffer and the bound proteins were eluted by boilingthe beads in 1 � sodium dodecyl sulfate sample buffer for 5min. After centrifugation at 10,000 � g for 5 min, superna-tants were separated by 10% SDS-PAGE, transferred ontoImmuno-Blot polyvinylidene difluoride (PVDF) membrane(catalog no. 162-0177, Bio-Rad), and Western blotted asdescribed before (62).

Adenoviral Infection of Mouse Uterine Stromal Cellsin Vitro

To evaluate the appropriate regulation of adenovirus-drivenexpression of genes, we first analyzed their effects in an invitro system. Primary culture of mouse uterine stromal cellswas grown as previously described by us (63). Cells at 70–80% confluence (in six-well dishes) were subjected to ad-enoviral infection at 10 multiplication of infection (MOI). Theextent of infection was monitored by GFP expression in thesecells using a fluorescence microscopy. For SFRP-2 and GFPexpression studies, cells were collected after 48 h of infectionand proteins were extracted for analysis by Western blottingas described above.

In Vivo Delivery of Adenovirus in Mice

To assess adenovirus-mediated gene expression in vivo,adult ovariectomized mice were inoculated with virus parti-cles iv through a tail vein. Approximately 100 �l of viralsolution in saline containing 1 � 1011 virus particles wasinjected per mouse. After inoculation, they were rested for 7 dto achieve optimum infection. The analysis of viral-drivenexpression of proteins was performed in uterine tissue ex-tracts by immunoprecipitation and Western blotting for GFPas described above. To examine the effects of E2 on uterinebiphasic responses in the adenovirus-infected mice, uterinewet weights were measured at 6 and 24 h after injections ofE2 (100 ng/mouse) or oil (control) to analyze early and lateestrogenic effects, respectively. The early effects were furtherassessed by gene expression studies for VEGF and Flk-1,known permeability regulators (37), whereas the late effectswere analyzed by bromodeoxyuridine (BrdU) incorporationinto DNA. For studies with BrdU, mice were injected sc withBrdU (50 mg/kg body weight) 2 h before they were killed.Formaldehyde-fixed paraffin-embedded tissue sections werestained for BrdU incorporation using biotinylated antibodyaccording to the manufacturer’s instruction (catalog no. 93-3943; Zymed Laboratories Inc., San Francisco, CA).

Acknowledgments

We thank Bert Vogelstein (Johns Hopkins University, Bal-timore, MD) for providing reagents to generate recombinantadenoviral clones.

Received June 28, 2004. Accepted August 31, 2004.Address all correspondence and requests for reprints to:

Dr. Sanjoy Das, Division of Reproductive and DevelopmentalBiology, Department of Pediatrics, D-4105 Medical CenterNorth, Vanderbilt University Medical Center, 1161 21st Ave-nue South, Nashville, Tennessee 37232-2678. E-mail: sanjoy.das@vanderbilt.edu.

This work was supported in part by National Institutes ofHealth Grants (HD12304 and HD33994 to S.K.De., andES07814 and HD37830 to S.K.Da.).

REFERENCES

1. Tsai MJ, O’Malley BW 1994 Molecular mechanisms ofaction of steroid/thyroid receptor superfamily members.Annu Rev Biochem 63:451–486

2. Beato M, Herrlich P, Schutz G 1995 Steroid hormonereceptors: many actors in search of a plot. Cell 83:851–857

3. Das SK, Tan J, Raja S, Halder J, Paria BC, Dey SK 2000Estrogen targets genes involved in protein processing,calcium homeostasis, and Wnt signaling in the mouseuterus independent of estrogen receptor-� and -�. J BiolChem 275:28834–28842

4. Cadigan KM, Nusse R 1997 Wnt signaling: a commontheme in animal development. Genes Dev 11:3286–3305

5. Wodarz A, Nusse R 1998 Mechanisms of Wnt signaling indevelopment. Annu Rev Cell Dev Biol 14:59–88

6. Smalley MJ, Dale TC 1999 Wnt signalling in mammaliandevelopment and cancer. Cancer Metastasis Rev 18:215–230

7. Tsukamoto AS, Grosschedl R, Guzman RC, Parslow T,Varmus HE 1988 Expression of the int-1 gene in trans-genic mice is associated with mammary gland hyperpla-sia and adenocarcinomas in male and female mice. Cell55:619–625

8. Lustig B, Behrens J 2003 The Wnt signaling pathway andits role in tumor development. J Cancer Res Clin Oncol129:199–221

9. Dale TC 1998 Signal transduction by the Wnt family ofligands. Biochem J 329:209–223

10. Wang Y, Macke JP, Abella BS, Andreasson K, Worley P,Gilbert DJ, Copeland NG, Jenkins NA, Nathans J 1996 Alarge family of putative transmembrane receptors homol-ogous to the product of the Drosophila tissue polaritygene frizzled. J Biol Chem 271:4468–4476

11. Liu X, Liu T, Slusarski DC, Yang-Snyder J, Malbon CC,Moon RT, Wang H 1999 Activation of a frizzled-2/�-adrenergic receptor chimera promotes Wnt signaling anddifferentiation of mouse F9 teratocarcinoma cells via G�oand G�t. Proc Natl Acad Sci USA 96:14383–14388

12. Finch PW, He X, Kelley MJ, Uren A, Schaudies RP,Popescu NC, Rudikoff S, Aaronson SA, Varmus HE, Ru-bin JS 1997 Purification and molecular cloning of a se-creted, Frizzled-related antagonist of Wnt action. ProcNatl Acad Sci USA 94:6770–6775

13. Rattner A, Hsieh JC, Smallwood PM, Gilbert DJ, Cope-land NG, Jenkins NA, Nathans J 1997 A family of se-creted proteins contains homology to the cysteine-richligand-binding domain of frizzled receptors. Proc NatlAcad Sci USA 94:2859–2863

14. Bafico A, Gazit A, Pramila T, Finch PW, Yaniv A, Aaron-son SA 1999 Interaction of frizzled related protein (FRP)

Hou et al. • Estrogen Mediates Uterine Growth via Wnt Signaling Mol Endocrinol, December 2004, 18(12):3035–3049 3047

with Wnt ligands and the frizzled receptor suggests al-ternative mechanisms for FRP inhibition of Wnt signaling.J Biol Chem 274:16180–16187

15. Jones SE, Jomary C 2002 Secreted Frizzled-relatedproteins: searching for relationships and patterns. Bioes-says 24:811–820

16. Kawano Y, Kypta R 2003 Secreted antagonists of theWnt signalling pathway. J Cell Sci 116:2627–2634

17. Kuhl M, Sheldahl LC, Park M, Miller JR, Moon RT 2000The Wnt/Ca2� pathway: a new vertebrate Wnt signalingpathway takes shape. Trends Genet 16:279–283

18. Liu T, DeCostanzo AJ, Liu X, Wang Hy, Hallagan S, MoonRT, Malbon CC 2001 G protein signaling from activatedrat frizzled-1 to the �-catenin-Lef-Tcf pathway. Science292:1718–1722

19. Pandur P, Lasche M, Eisenberg LM, Kuhl M 2002 Wnt-11activation of a non-canonical Wnt signalling pathway isrequired for cardiogenesis. Nature 418:636–641

20. van Noort M, Meeldijk J, van der Zee R, Destree O,Clevers H 2002 Wnt signaling controls the phosphoryla-tion status of �-catenin. J Biol Chem 277:17901–17905

21. Staal FJ, Noort Mv M, Strous GJ, Clevers HC 2002 Wntsignals are transmitted through N-terminally dephospho-rylated �-catenin. EMBO Rep 3:63–68

22. Bienz M, Clevers H 2003 Armadillo/�-catenin signals inthe nucleus–proof beyond a reasonable doubt? Nat CellBiol 5:179–182

23. Willert K, Nusse R 1998 �-Catenin: a key mediator of Wntsignaling. Curr Opin Genet Dev 8:95–102

24. van Noort M, Clevers H 2002 TCF transcription factors,mediators of Wnt-signaling in development and cancer.Dev Biol 244:1–8

25. Huelsken J, Birchmeier W 2001 New aspects of Wntsignaling pathways in higher vertebrates. Curr OpinGenet Dev 11:547–553

26. He X, Saint-Jeannet JP, Wang Y, Nathans J, Dawid I,Varmus H 1997 A member of the Frizzled protein familymediating axis induction by Wnt-5A. Science 275:1652–1654

27. Slusarski DC, Corces VG, Moon RT 1997 Interaction ofWnt and a Frizzled homologue triggers G-protein-linkedphosphatidylinositol signalling. Nature 390:410–413

28. Hartmann C, Tabin CJ 2000 Dual roles of Wnt signalingduring chondrogenesis in the chicken limb. Development127:3141–3159

29. Medina A, Reintsch W, Steinbeisser H 2000 Xenopusfrizzled 7 can act in canonical and non-canonical Wntsignaling pathways: implications on early patterning andmorphogenesis. Mech Dev 92:227–237

30. Miller C, Pavlova A, Sassoon DA 1998 Differential ex-pression patterns of Wnt genes in the murine femalereproductive tract during development and the estrouscycle. Mech Dev 76:91–99

31. Daikoku T, Song H, Guo Y, Riesewijk A, Mosselman S,Das SK, Dey SK 2004 Uterine Msx-1 and Wnt4 signalingbecomes aberrant in mice with the loss of leukemiainhibitory factor or Hoxa-10: evidence for a novel cyto-kine-homeobox-Wnt signaling in implantation. Mol En-docrinol 18:1238–1250

32. Sassoon D 1999 Wnt genes and endocrine disruption ofthe female reproductive tract: a genetic approach. MolCell Endocrinol 158:1–5

33. Das SK, Taylor JA, Korach KS, Paria BC, Dey SK, LubahnDB 1997 Estrogenic responses in estrogen receptor-�deficient mice reveal a distinct estrogen signaling path-way. Proc Natl Acad Sci USA 94:12786–12791

34. Das SK, Das N, Wang J, Lim H, Schryver B, PlowmanGD, Dey SK 1997 Expression of �cellulin and epiregulingenes in the mouse uterus temporally by the blastocystsolely at the site of its apposition is coincident with the“window” of implantation. Dev Biol 190:178–190

35. Kirkland JL, Gardner RM, Ireland JS, Stancel GM 1977The effect of hypophysectomy on the uterine response toestradiol. Endocrinology 101:403–410

36. Harris J, Gorski J 1978 Evidence for a discontinuousrequirement for estrogen in stimulation of deoxyribonu-cleic acid synthesis in the immature rat uterus. Endocri-nology 103:240–245

37. Ma W, Tan J, Matsumoto H, Robert B, Abrahamson DR,Das SK, Dey SK 2001 Adult tissue angiogenesis: evi-dence for negative regulation by estrogen in the uterus.Mol Endocrinol 15:1983–1992

38. Dey SK 1996 In: Adashi E, Rock JA, Rosenwaks Z, eds.Reproductive endocrinology, surgery, and technology.New York: Lippincott-Raven; 421–434

39. Kumar V, Green S, Stack G, Berry M, Jin JR, Chambon P1987 Functional domains of the human estrogen recep-tor. Cell 51:941–951

40. Tan J, Paria BC, Dey SK, Das SK 1999 Differential uterineexpression of estrogen and progesterone receptors cor-relates with uterine preparation for implantation and de-cidualization in the mouse. Endocrinology 140:5310–5321

41. Carson-Jurica MA, Schrader WT, O’Malley BW 1990 Ste-roid receptor family: structure and functions. Endocr Rev11:201–220

42. Mericskay M, Kitajewski J, Sassoon D 2004 Wnt5a isrequired for proper epithelial-mesenchymal interactionsin the uterus. Development 131:2061–2072

43. Kispert A, Vainio S, McMahon AP 1998 Wnt-4 is a mes-enchymal signal for epithelial transformation of meta-nephric mesenchyme in the developing kidney. Develop-ment 125:4225–4234

44. Miller C, Sassoon DA 1998 Wnt-7a maintains appropriateuterine patterning during the development of the mousefemale reproductive tract. Development 125:3201–3211

45. Gavin BJ, McMahon AP 1992 Differential regulation ofthe Wnt gene family during pregnancy and lactation sug-gests a role in postnatal development of the mammarygland. Mol Cell Biol 12:2418–2423

46. Weber-Hall SJ, Phippard DJ, Niemeyer CC, Dale TC1994 Developmental and hormonal regulation of Wntgene expression in the mouse mammary gland. Differ-entiation 57:205–214

47. Bradbury JM, Edwards PA, Niemeyer CC, Dale TC 1995Wnt-4 expression induces a pregnancy-like growth pat-tern in reconstituted mammary glands in virgin mice. DevBiol 170:553–563

48. Cunha GR, Chung LW, Shannon JM, Taguchi O, Fujii H1983 Hormone-induced morphogenesis and growth: roleof mesenchymal-epithelial interactions. Recent ProgHorm Res 39:559–598

49. Gumbiner BM 1996 Cell adhesion: the molecular basisof tissue architecture and morphogenesis. Cell 84:345–357

50. Papkoff J, Brown AM, Varmus HE 1987 The int-1 proto-oncogene products are glycoproteins that appear to en-ter the secretory pathway. Mol Cell Biol 7:3978–3984

51. Brown AM, Papkoff J, Fung YK, Shackleford GM,Varmus HE 1987 Identification of protein products en-coded by the proto-oncogene int-1. Mol Cell Biol7:3971–977

52. Papkoff J 1989 Inducible overexpression and secretionof int-1 protein. Mol Cell Biol 9:3377–3384

53. Burrus LW, McMahon AP 1995 Biochemical analysis ofmurine Wnt proteins reveals both shared and distinctproperties. Exp Cell Res 220:363–373

54. Kitajewski J, Mason JO, Varmus HE 1992 Interaction ofWnt-1 proteins with the binding protein BiP. Mol Cell Biol12:784–790

55. Cooke PS, Buchanan DL, Young P, Setiawan T, Brody J,Korach KS, Taylor J, Lubahn DB, Cunha GR 1997 Stro-mal estrogen receptors mediate mitogenic effects of es-tradiol on uterine epithelium. Proc Natl Acad Sci USA94:6535–6540

3048 Mol Endocrinol, December 2004, 18(12):3035–3049 Hou et al. • Estrogen Mediates Uterine Growth via Wnt Signaling

56. Couse JF, Korach KS 1999 Estrogen receptor null mice:what have we learned and where will they lead us? En-docr Rev 20:358–417

57. El-Tanani M, Fernig DG, Barraclough R, Green C,Rudland P 2001 Differential modulation of transcriptionalactivity of estrogen receptors by direct protein-protein-interactions with the T cell factor family of transcriptionfactors. J Biol Chem 276:41675–41682

58. Gavin BJ, McMahon JA, McMahon AP 1990 Expressionof multiple novel Wnt-1/int-1-related genes during fetaland adult mouse development. Genes Dev 4:2319–2332

59. Das SK, Wang XN, Paria BC, Damm D, Abraham JA,Klagsbrun M, Andrews GK, Dey SK 1994 Heparin-bind-ing EGF-like growth factor gene is induced in the mouseuterus temporally by the blastocyst solely at the site of itsapposition: a possible ligand for interaction with blasto-cyst EGF-receptor in implantation. Development 120:1071–1083

60. Das SK, Chakraborty I, Paria BC, Wang XN, Plowman G,Dey SK 1995 Amphiregulin is an implantation-specific

and progesterone-regulated gene in the mouse uterus.Mol Endocrinol 9:691–705

61. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW,Vogelstein B 1998 A simplified system for generatingrecombinant adenoviruses. Proc Natl Acad Sci USA95:2509–2514

62. Tan J, Raja S, Davis MK, Tawfik O, Dey SK, Das SK 2002Evidence for coordinated interaction of cyclin D3 withp21 and cdk6 in directing the development of uterinestromal cell decidualization and polyploidy during im-plantation. Mech Dev 111:99–113

63. Tan Y, Li M, Cox S, Davis MK, Tawfik O, Paria BC, DasSK 2004 HB-EGF directs stromal cell polyploidy anddecidualization via cyclin D3 during implantation. DevBiol 265:181–195

64. Lubahn DB, Moyer JS, Golding TS, Couse JF, KorachKS, Smithies O 1993 Alteration of reproductive functionbut not prenatal sexual development after insertionaldisruption of the mouse estrogen receptor gene. ProcNatl Acad Sci USA 90:11162-11166

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