Wnt/β-Catenin and Estrogen Signaling Converge in Vivo

Wnt and estrogen signaling represent important regulatory pathways, each controlling a wide range of biological processes. While an increasing number of observations suggest potential convergence between these pathways, no direct evidence of their functional interaction has been reported. Using human colon and breast cancer cells, we found that estrogen receptor (ER) α- and β-catenin precipitated within the same immunocomplexes, reciprocally enhanced the transactivation of cognate reporter genes, and were reciprocally recruited to cognate response elements in the promoters of endogenous target genes. Using transgenic Drosophila that ectopically expressed human ERα alone or together with metabolically stable β-catenin/Armadillo mutants, we demonstrated genetic interaction between these signal transducers in vivo. Thus, we present here the first direct evidence of cross-talk between Wnt and estrogen signaling pathways via functional interaction between β-catenin and ERα. Wnt and estrogen signaling represent important regulatory pathways, each controlling a wide range of biological processes. While an increasing number of observations suggest potential convergence between these pathways, no direct evidence of their functional interaction has been reported. Using human colon and breast cancer cells, we found that estrogen receptor (ER) α- and β-catenin precipitated within the same immunocomplexes, reciprocally enhanced the transactivation of cognate reporter genes, and were reciprocally recruited to cognate response elements in the promoters of endogenous target genes. Using transgenic Drosophila that ectopically expressed human ERα alone or together with metabolically stable β-catenin/Armadillo mutants, we demonstrated genetic interaction between these signal transducers in vivo. Thus, we present here the first direct evidence of cross-talk between Wnt and estrogen signaling pathways via functional interaction between β-catenin and ERα. Estrogens regulate a plethora of physiological functions in the developing and adult organism and act predominantly via the activation of ERα 1The abbreviations used are: ER, estrogen receptor; ERE, estrogen response element; TCF, T cell factor; LEF, lymphoid enhancer factor; TBE, TCF/LEF binding element; CSFCS, charcoal-stripped fetal calf serum; ChIP, chromatin immunoprecipitation; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; LBD, ligand binding domain; wt, wild-type. 1The abbreviations used are: ER, estrogen receptor; ERE, estrogen response element; TCF, T cell factor; LEF, lymphoid enhancer factor; TBE, TCF/LEF binding element; CSFCS, charcoal-stripped fetal calf serum; ChIP, chromatin immunoprecipitation; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; LBD, ligand binding domain; wt, wild-type. and ERβ. Liganded ER dimers bind to promoter estrogen response elements (EREs) and regulate the transcription of target genes. This ER-mediated regulation requires the recruitment of different co-factor complexes and is associated with rearrangement of chromatin structure at EREs within target gene promoters (1Yanagisawa J. Kitagawa H. Yanagida M. Wada O. Ogawa S. Nakagomi M. Oishi H. Yamamoto Y. Nagasawa H. McMahon S.B. Cole M.D. Tora L. Takahashi N. Kato S. Mol. Cell. 2002; 9: 553-562Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 2Metivier R. Penot G. Hubner M.R. Reid G. Brand H. Kos M. Gannon F. Cell. 2003; 115: 751-763Abstract Full Text Full Text PDF PubMed Scopus (1246) Google Scholar). ER can also act as a co-factor at non-ERE sites via interaction with other DNA-bound transcriptional factor complexes, such as c-Jun/c-Fos on the AP-1 site (3Kushner P.J. Agard D.A. Greene G.L. Scanlan T.S. Shiau A.K. Uht R.M. Webb P. J. Steroid Biochem. Mol. Biol. 2000; 74: 311-317Crossref PubMed Scopus (735) Google Scholar) or c-Jun/NFκB on the tumor necrosis factor response element (4Tzagarakis-Foster C. Geleziunas R. Lomri A. An J. Dale C Leitman D.C. J. Biol. Chem. 2002; 277: 44772-44777Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The physiological significance of ERs is demonstrated by the severe abnormalities in development and function of major organs and tissues in mice with ablated ERα and/or ERβ (5Couse J.F. Korach K.S. Endocr. Rev. 1999; 20: 358-417Crossref PubMed Scopus (0) Google Scholar). Also, both positive and negative impacts of estrogens in different types of cancer have been well documented (6Creasman W.T. Gynecol. Oncol. 2002; 86: 1-9Abstract Full Text PDF PubMed Scopus (25) Google Scholar). Wnt signaling plays a critical role in numerous processes of development and in adult tissues and appears to be conserved across all animal taxa. β-Catenin is an intracellular transducer of canonical Wnt or Wnt/β-catenin signaling and, thus, has a dual function: as a transcriptional factor and, in a cadherin-bound form, as a regulator of cell adhesion and migration. Cytoplasmic or signaling β-catenin is unstable and rapidly targeted to phosphorylation-ubiquitination-coupled proteasomal degradation. Wnt signaling inhibits this degradation, resulting in the accumulation of β-catenin in the nucleus and its association with members of the T cell factor/lymphoid enhancer factor (TCF/LEF) family of transcriptional factors that leads to the activation of Wnt target genes. Mutations that increase the stability of cytoplasmic β-catenin have been implicated in numerous malignant transformations and represent a leading cause of colorectal tumorigenesis (7Akiyama T. Cytokine Growth Factor Rev. 2000; 11: 273-282Crossref PubMed Scopus (277) Google Scholar, 8Peifer M. Polakis P. Science. 2000; 287: 1606-1609Crossref PubMed Scopus (1140) Google Scholar, 9Bienz M. Clevers H. Nat. Cell Biol. 2003; 5: 179-182Crossref PubMed Scopus (129) Google Scholar). Consistent with the concept of morphogen gradients (10Tabata T. Nat. Rev. Genet. 2001; 2: 620-630Crossref PubMed Scopus (103) Google Scholar) β-catenin exerts different biological effects, such as induction of cell proliferation and apoptosis or stimulation and repression of the same target genes, in a threshold-dependent manner (11Waltzer L. Vandel L. Bienz M. EMBO J. 2001; 20: 137-145Crossref PubMed Scopus (40) Google Scholar, 12Olmeda D. Castel S. Vilaro S. Cano A. Mol. Biol. Cell. 2003; 14: 2844-2860Crossref PubMed Scopus (164) Google Scholar). Thus, slight modulation of β-catenin signaling through cross-talk with other pathways may trigger serious physiological consequences. Potential cross-talk between Wnt/β-catenin and estrogen signaling in vivo has been implicated in physiological studies on tissues as different as brain (13Cardona-Gomez P. Perez M. Avila J. Garcia-Segura L.M. Wandosell F. Mol. Cell. Neurosci. 2004; 25: 363-373Crossref PubMed Scopus (170) Google Scholar) and uterus (14Gunin A.G. Emelianov V.U. Mironkin I.U. Morozov M.P. Tolmachev A.S. Eur. J. Obstet. Gynecol. Reprod. Biol. 2004; 114: 83-91Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Furthermore, although males and females develop colorectal cancer with approximately the same frequency, its incidence rate is significantly lower in women undergoing hormone replacement therapy (15Crandall C.J. J. Womens Health Gend. Based Med. 1999; 8: 1155-1166Crossref PubMed Scopus (43) Google Scholar, 16Nelson H.D. Humphrey L.L. Nygren P. Teutsch S.M. Allan J.D. J. Am. Med. Assoc. 2002; 288: 872-881Crossref PubMed Scopus (901) Google Scholar). While these and other observations suggested the possibility of functional interaction between ER and β-catenin, previous attempts failed to detect such an interaction (13Cardona-Gomez P. Perez M. Avila J. Garcia-Segura L.M. Wandosell F. Mol. Cell. Neurosci. 2004; 25: 363-373Crossref PubMed Scopus (170) Google Scholar, 17Yang F. Yang F. Li X. Sharma M. Sasaki C.Y. Longo D.L. Lim B. Sun Z. J. Biol. Chem. 2002; 277: 11336-11344Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 18Pawlowski J.E. Ertel J.R. Allen M.P. Xu M. Butler C. Wilson E.M. Wierman M.E. J. Biol. Chem. 2002; 277: 20702-20710Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), and no direct evidence of Wnt and estrogen signaling pathway convergence has been reported. Compared with vertebrates, Wnt signaling has been far better characterized in Drosophila, in which it is not obscured by involvement of other, evolutionary more recent multiple pathways. Thus, Drosophila provides a powerful experimental system for analysis of functional interaction in vivo between Wnt signaling and other regulatory pathways, including those immerged at the later stages of evolution. Therefore, in addition to mammalian cells, to detect functional interaction between Wnt/β-catenin and estrogen signaling in vivo we used transgenic Drosophila that ectopically expressed human ERα coupled to an ERE-dependent green fluorescent protein (GFP) reporter gene alone or together with constitutively active mutants of Armadillo, a Drosophila homologue of β-catenin. Using different approaches, we obtained in this study the first evidence of physical association and transcriptional and genetic interaction in vivo between ERα and β-catenin. Immunoprecipitation and Immunoblotting—Cells grown in the presence of charcoal-stripped fetal calf serum (CSFCS) were transfected with FLAG-hERα expression vector and harvested 28–30 h post-transfection, after treatment for 3 h with vehicle (ethanol) or 10–8m 17β-estradiol (Sigma), tamoxifen (Sigma), or ICI 182,780 (Tocris). Anti-β-catenin E-5 or H-102 antibodies (Santa Cruz Biotechnology) or preimmune rabbit serum IgG (as a negative control) were used for immunoprecipitation. Western blots were visualized with anti-FLAG M2 (Sigma) or anti-ERα HC-20 (Santa Cruz Biotechnology) antibodies. Transfection and Reporter Assay—Cells grown in Opti-MEM, 5% CSFCS were transfected with 250 ng of reporter (ERE-tk-luc or tk-luc for MCF7 cells and TOPFLASH or FOPFLASH for colon cancer cells) and 1 ng of pRl (Promega) plasmid (control for transfection efficiency) together with 100 ng of empty (control) or cDNA (β-catenin S33Y for MCF7 cells and ERα for colon cancer cells) expression vector and treated for 16–20 h with vehicle or 10–8m ligand, as indicated. To nullify nonspecific effects on basal promoters, TOPFLASH and ERE-tk-luc reporter activities were normalized against FOPFLASH and tk-Luc reporter activities, respectively, from parallel experiments. Chromatin Immunoprecipitation (ChIP) Assay—Association of ERα and β-catenin with ERE in the pS2 gene promoter (19Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cell. 2000; 103: 843-852Abstract Full Text Full Text PDF PubMed Scopus (1445) Google Scholar) and TCF/LEF binding element (TBE) in the Axin2 gene promoter (20Yan D. Wiesmann M. Rohan M. Chan V. Jefferson A.B. Guo L. Sakamoto D. Caothien R.H. Fuller J.H. Reinhard C. Garcia P.D. Randazzo F.M. Escobedo J. Fantl W.J. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14973-14978Crossref PubMed Scopus (302) Google Scholar) was analyzed using the Chromatin Immunoprecipitation Kit (Upstate Biotechnology) and HC-20 or E-5 antibody, respectively. As a control for nonspecific chromatin precipitation with these antibodies, a set of primers was used to amplify a pS2 gene DNA segment that does not have ERE or TBE sequences. In addition, IgG from normal preimmune rabbit serum was used as a negative control. Histology and Immunostaining—All techniques were performed as described previously (21Takeyama K. Ito S. Yamamoto A. Tanimoto H. Furutani T. Kanuka H. Miura M. Tabata T. Kato S. Neuron. 2002; 35: 855-864Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 22Kawasaki Y. Sato R. Akiyama T. Nat. Cell Biol. 2003; 5: 211-215Crossref PubMed Scopus (179) Google Scholar). Expression of ERα and GFP in Drosophila eye discs were detected using Zeiss Confocal Laser Scanning System 510 and quantified by calculation of pixels of the corresponding signals using Adobe Photoshop 7 software facilities. TUNEL labeling was performed using the TACS2 TdT-Fluor In Situ Apoptosis Detection Kit (Trevigen). Drosophila Lines and Stocks—The UAS-ΔArm and UAS-ArmS10 mutants were obtained from the Bloomington Drosophila Stock Center. Generation and characterization of the used UAS-ERα, ERE-GFP transgenic Drosophila lines were described in Ref. 23Ito S. Takeyama K. Yamamoto A. Sawatsubashi S. Shirode Y. Kouzmenko A. Tabata T. Kato S. Genes Cells. 2004; (DOI 10.1111/j.1365-2443.2004.00777x)Google Scholar. Briefly, cDNA encoding full-length human ERα, ligand binding domain (LBD) deletion mutant ERα-(1–302), or GFP reporter under control an ERE containing promoter were recloned into the pCaSpeR vector. Transgene constructs together with pπ25.7wc transposase were microinjected into w1118 embryos using a micromanipulator (Leica). Several independent transformant lines have been generated. To target ERα expression into the eye disc, transgenic Drosophila were crossed with flies of a GMR-GAL4 line expressing GAL4 driver in the retina under control of the tissue-specific glass multimer gene promoter. Physical Association of ERα and β-Catenin—Human colon cancer HCT116 cells express metabolically stable β-catenin due to mutation at its putative phosphorylation site. These cells, however, do not express detectable ER. HCT116 cells were transfected with a FLAG-tagged human ERα expression plasmid, and endogenous β-catenin was immunoprecipitated from cell lysates following 3-h preincubation with estrogen or vehicle. IgG from normal rabbit serum was used as a control for nonspecific immunoprecipitation. Obtained immunocomplexes were subjected to Western blotting and analyzed by immunostaining with antibodies against FLAG-tag and ERα. ERα co-immunoprecipitated with β-catenin even in the absence of ligand; however, ERα-β-catenin association was markedly stimulated by estrogen (Fig. 1A). Similar results (data not shown) were obtained using SW480 human colon cancer cells, in which non-mutant β-catenin was stabilized by a loss-of-function mutation in the gene of tumor suppressor Adenomatous polyposis coli, an essential component of the β-catenin degradation machinery. Brief exposure to ligand did not affect FLAG-ERα expression in this (Fig. 1A) or further experiments. As anti-β-catenin antibodies co-precipitated a C-terminally truncated FLAG-ERα-(1–396) (Fig. 1B), it appeared that an intact LBD was not essential for the ER interaction with β-catenin. Predictably, C-terminal truncation of ERα abolished the ligand sensitivity of the interaction. We then analyzed whether ligands that inhibited the transcriptional activity of ERα would also affect its interaction with β-catenin. Immunoprecipitation of ERα with antibodies against β-catenin was significantly stimulated by the ERα partial, tamoxifen, and complete, ICI 182,780, antagonists (Fig. 1C). Transcriptional Interaction between ERα and β-Catenin— Next, we investigated whether the apparent physical association between ERα and β-catenin was consequential for transcriptional function of the proteins. Transactivation of an ERE-dependent reporter by endogenous ER was studied in human breast cancer MCF7 cells, in which the Wnt pathway is practically silent. Expression of stabilized β-catenin S33Y in these cells enhanced ligand-dependent expression of the reporter without affecting its basal activity in the absence of ligand (Fig. 2A). Expression of ERα in human colon cancer SW480 (Fig. 2B) and HCT116 (data not shown) cells enhanced the activation of the Wnt-responsive TOPFLASH reporter by endogenous β-catenin in the absence of ligand. Treatment with estrogen resulted in further moderate activation of reporter expression, while ER antagonists appeared not to affect reporter gene activity (Fig. 2B). The reciprocal activation of cognate reporters in the transfection experiments suggested that ERα and β-catenin might reciprocally recruit each other to their corresponding response elements in endogenous target gene promoters. Indeed, antibody against β-catenin precipitated ERE of the pS2 gene promoter from chromatin of β-catenin S33Y expressing MCF7 cells in an estrogen-dependent manner (Fig. 2C). Conversely, anti-ERα antibody precipitated in a ligand-dependent manner Axin2 gene promoter putative TBE from chromatin of SW480 cells transfected with an ERα expression construct, while recruitment of β-catenin to the TBE was not sensitive to the presence of estrogen (Fig. 2D). The used antibodies did not display nonspecific chromatin precipitation (Fig. 2E). Consistent with the results obtained using MCF7 cells, ERα transactivation was markedly enhanced in vivo by the stabilized Armadillo mutants ΔArm (24Tolwinski N.S. Wieschaus E. Development (Camb.). 2001; 128: 2107-2117PubMed Google Scholar) (Fig. 2F) or ArmS10 (25Pai L.M. Orsulic S. Bejsovec A. Peifer M. Development (Camb.). 1997; 124: 2255-2266PubMed Google Scholar) (data not shown) when ectopically co-expressed in the Drosophila eye disc. Genetic Interaction between ERα and β-Catenin—Constitutive activation of Armadillo in the Drosophila eye disc has been shown to induce apoptosis and consequent degeneration in the adult eye (26Greaves S. Sanson B. White P. Vincent J.P. Genetics. 1999; 153: 1753-1766PubMed Google Scholar, 27Freeman M. Bienz M. EMBO 2001; 2: PubMed Scopus Google Scholar). of β-catenin transcriptional activity by ERα in SW480 cells (Fig. 2B) and functional interaction between ERα and Armadillo (Fig. 2F) would activation of endogenous Armadillo by the ERα expression in the Drosophila eye disc leading to development of a of Wnt/β-catenin We performed TUNEL of the eye discs with expression of ERα alone or together with the constitutively active Armadillo mutant expressed ERα and ΔArm both a slight increase in apoptosis with eye discs from Drosophila of the of ERα and ΔArm resulted in a increase in cell while estrogen no on apoptosis in eye discs and those expressing ERα or ΔArm alone (data not treatment with significantly apoptosis when ERα and ΔArm were (Fig. Armadillo has a in the eye disc, due to the at this by the signaling M. Bienz M. EMBO 2001; 2: PubMed Scopus Google Scholar). This to detect in apoptosis in transgenic eye discs at this that would be to due to the of cell at the later We adult eye of flies from these transgenic lines and the (Fig. The normal Drosophila eye is by with Expression of ERα in the eye disc leads to development of to those by expression of eye and or of of ΔArm and ERα enhanced this eye while appeared not to affect the ΔArm or ERα expression (data not treatment with however, further the of eye abnormalities in the ERα and ΔArm mutants Consistent with expression of the LBD deletion mutant in the eye disc a that with the full-length ERα (data not We found that β-catenin associated with ERα even in the absence of ligand and that estrogens further enhanced this interaction. While it is that the association was at in to the of of the the association between β-catenin and C-terminally truncated ERα suggested that the ligand binding was not essential might induce a more for ERα to with β-catenin. This may be of functional significance at physiological of the proteins. β-catenin recruitment to EREs and ERα recruitment to in the promoters of endogenous target were both The stimulation of ERα-β-catenin interaction by ER and antagonists may have important for the of was that ERα with β-catenin/Armadillo in vivo in transgenic The ligand-dependent transactivation function of ERα was significantly enhanced by the of stabilized Armadillo in the eye development by targeted expression of Armadillo and ERα were of a of both enhanced the that was further by treatment with in is shown to have a activity to be by ER Wilson M.E. M. Rev. 2001; PubMed Scopus Google Scholar). Physical and transcriptional interaction between β-catenin and receptor has been previously F. Yang F. Li X. Sharma M. Sasaki C.Y. Longo D.L. Lim B. Sun Z. J. Biol. Chem. 2002; 277: 11336-11344Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 18Pawlowski J.E. Ertel J.R. Allen M.P. Xu M. Butler C. Wilson E.M. Wierman M.E. J. Biol. Chem. 2002; 277: 20702-20710Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). in experiments in these no interaction between β-catenin and other hormone including ER, has been Thus, we have shown that Wnt and estrogen signaling pathways cross-talk in vivo through functional interaction between ERα and β-catenin. This interaction may of estrogen effects in and processes in which abnormalities of Wnt/β-catenin signaling have been such as in colorectal In addition, we have a experimental system in which to factors conserved between and Drosophila that may be in regulation of cross-talk between Wnt and estrogen signaling and for the of to with this other may be transcriptional modulation appears to be the major of functional ERα-β-catenin interaction. The function of is on the recruitment of different and chromatin complexes (1Yanagisawa J. Kitagawa H. Yanagida M. Wada O. Ogawa S. Nakagomi M. Oishi H. Yamamoto Y. Nagasawa H. McMahon S.B. Cole M.D. Tora L. Takahashi N. Kato S. Mol. Cell. 2002; 9: 553-562Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 2Metivier R. Penot G. Hubner M.R. Reid G. Brand H. Kos M. Gannon F. Cell. 2003; 115: 751-763Abstract Full Text Full Text PDF PubMed Scopus (1246) Google Scholar, H. R. K. Y. Y. D. Ogawa S. K. M. A. T. Ito T. Y. Nagasawa H. T. J. Kato S. Cell. 2003; Full Text PDF PubMed Scopus Google Scholar, F. Takeyama K. T. Kitagawa H. Yamamoto Y. K. C. A. J. P. J. Y. Kato S. 2003; PubMed Scopus Google Scholar). β-Catenin has been shown to recruit such as the A. K. M.P. F. R. EMBO J. 2000; PubMed Google Scholar), and of the mammalian and chromatin complexes N. A. H. A. Bienz M. Clevers H. EMBO J. 2001; 20: PubMed Scopus Google Scholar) that also to with ERα. of and chromatin complexes may for the transcriptional of ERα-β-catenin interaction. The physiological of this interaction may also on cell and in of the recruited regulatory experiments to all ERα-β-catenin to whether the ERα-β-catenin interaction results in in the of the recruited regulatory or factors to ERα-β-catenin protein complexes