Murine Frizzled-1 Behaves as an Antagonist of the Canonical Wnt/β-Catenin Signaling
2004; Elsevier BV; Volume: 279; Issue: 7 Linguagem: Inglês
10.1074/jbc.m309233200
ISSN1083-351X
AutoresSergio Román-Román, De‐Li Shi, Véronique Stiot, Eric Haÿ, Béatrice Vayssière, Teresa García, Roland Baron, Georges Rawadi,
Tópico(s)Kruppel-like factors research
ResumoActivation of the Wnt signaling cascade provides key signals during development and in disease. Wnt signals are transduced by seven-transmembrane Frizzleds (Fzs) and the single transmembrane low density lipoprotein receptor-related proteins 5 or 6. In the course of the analysis of genes regulated by bone morphogenetic protein 2 in mesenchymal cells we found a significant induction of murine Frizzled-1 (mFz1) gene expression. Unexpectedly overexpression of mFz1 dramatically repressed the induction of alkaline phosphatase mediated by either bone morphogenetic protein 2 or Wnt3a in these cells. Moreover mFz1 overexpression significantly repressed both β-catenin translocation into the nucleus and T cell factor signaling mediated by Wnt3a. Importantly microinjection of mFz1 transcript in Xenopus embryo inhibited the ability of Wnt1 to induce the expression of the Wnt/β-catenin target gene Siamois in animal cap assay and secondary axis formation in whole embryo. By using chimeric constructs in which N- and C-terminal segments of mFz1 were replaced by the corresponding parts of Xfz3 we demonstrated that the antagonistic activity resides in the cysteine-rich domain of the N-terminal part. The antagonist activity of mFz1 could be prevented by overexpression of Gαq-(305-359), which specifically uncouples Gq-coupled receptors, suggesting that Gαq signaling contributes to the inhibition of Wnt/β-catenin pathway by mFz1. This is the first time that a Frizzled receptor has been reported to antagonize Wnt/β-catenin. Activation of the Wnt signaling cascade provides key signals during development and in disease. Wnt signals are transduced by seven-transmembrane Frizzleds (Fzs) and the single transmembrane low density lipoprotein receptor-related proteins 5 or 6. In the course of the analysis of genes regulated by bone morphogenetic protein 2 in mesenchymal cells we found a significant induction of murine Frizzled-1 (mFz1) gene expression. Unexpectedly overexpression of mFz1 dramatically repressed the induction of alkaline phosphatase mediated by either bone morphogenetic protein 2 or Wnt3a in these cells. Moreover mFz1 overexpression significantly repressed both β-catenin translocation into the nucleus and T cell factor signaling mediated by Wnt3a. Importantly microinjection of mFz1 transcript in Xenopus embryo inhibited the ability of Wnt1 to induce the expression of the Wnt/β-catenin target gene Siamois in animal cap assay and secondary axis formation in whole embryo. By using chimeric constructs in which N- and C-terminal segments of mFz1 were replaced by the corresponding parts of Xfz3 we demonstrated that the antagonistic activity resides in the cysteine-rich domain of the N-terminal part. The antagonist activity of mFz1 could be prevented by overexpression of Gαq-(305-359), which specifically uncouples Gq-coupled receptors, suggesting that Gαq signaling contributes to the inhibition of Wnt/β-catenin pathway by mFz1. This is the first time that a Frizzled receptor has been reported to antagonize Wnt/β-catenin. The transforming growth factor (TGF) 1The abbreviations used are: TGFtransforming growth factorALPalkaline phosphataseBMPbone morphogenic proteinCRDcysteinerich domainLRPlipoprotein receptor-related proteinmFz1murine Frizzled-1hFz1human Frizzled-1XfzXenopus FrizzledTCFT cell factorRTreverse transcriptaseCMconditioned mediumGFPgreen fluorescent proteinβ-catenin*constitutive active mutant form of β-cateninBAPTA/AM1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate acetoxymethyl ester.1The abbreviations used are: TGFtransforming growth factorALPalkaline phosphataseBMPbone morphogenic proteinCRDcysteinerich domainLRPlipoprotein receptor-related proteinmFz1murine Frizzled-1hFz1human Frizzled-1XfzXenopus FrizzledTCFT cell factorRTreverse transcriptaseCMconditioned mediumGFPgreen fluorescent proteinβ-catenin*constitutive active mutant form of β-cateninBAPTA/AM1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate acetoxymethyl ester.-β family plays a central role in regulating a broad range of cellular responses including cell growth and differentiation. Among members of this family, bone morphogenetic proteins (BMPs) have been shown to regulate growth and differentiation of chondroblast and osteoblast lineage cells and induce the commitment of mesenchymal cells into osteoblast/chondroblast phenotypes (1Atkinson B.L. Fantle K.S. Benedict J.J. Huffer W.E. Gutierrez-Hartmann A. J. Cell. Biochem. 1997; 65: 325-339Crossref PubMed Scopus (79) Google Scholar, 2Katagiri T. Yamaguchi A. Ikeda T. Yoshiki S. Wozney J.M. Rosen V. Wang E.A. Tanaka H. Omura S. Suda T. Biochem. Biophys. Res. Commun. 1990; 172: 295-299Crossref PubMed Scopus (451) Google Scholar, 3Wang E.A. Israel D.I. Kelly S. Luxenberg D.P. Growth Factors. 1993; 9: 57-71Crossref PubMed Scopus (450) Google Scholar). In addition, when ectopically implanted, BMPs possess the capacity to induce bone and cartilage formation (4Reddi A.H. Curr. Opin. Cell Biol. 1992; 4: 850-855Crossref PubMed Scopus (239) Google Scholar, 5Reddi A.H. 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Zhao S.C. Eustace B. Lappe M.M. Spitzer L. Zweier S. Braunschweiger K. Benchekroun Y. Hu X. Adair R. Chee L. FitzGerald M.G. Tulig C. Caruso A. Tzellas N. Bawa A. Franklin B. McGuire S. Nogues X. Gong G. Allen K.M. Anisowicz A. Morales A.J. Lomedico P.T. Recker S.M. Van Eerdewegh P. Recker R.R. Johnson M.L. Am. J. Hum. Genet. 2002; 70: 11-19Abstract Full Text Full Text PDF PubMed Scopus (1060) Google Scholar). These results clearly suggest a genetic link between LRP5, and consequently Wnt, signaling and the regeneration of bone mass. The control of osteoblast differentiation involves a complex combination of signals. However, the relationship and possible cross-talk between different signaling pathways and/or different components of these pathways remain largely unknown. In this study we report that BMP-2 up-regulates the expression of Frizzled-1 in different murine mesenchymal cell lines. Unexpectedly overexpression of Frizzled-1 counteracts the effects of BMP-2 and Wnt3a in inducing the expression of the osteoblast differentiation marker ALP, indicating that mouse Frizzled-1 (mFz1) may exert a feedback mechanism to modulate the effect of BMP-2 in murine mesenchymal cells. More importantly we have demonstrated, using a Xenopus assay, that mFz1 is capable of antagonizing Wnt/β-catenin signaling. Our data suggest that the CRD of mFz1 may be responsible for its inhibitory function. Whether mFz1 antagonizes Wnt signaling by competing with other members of the Frizzled family or with Wnt binding for LRP5 association or by both mechanisms remains to be determined. Cell Culture—C2C12 cells, kindly provided by Dr. G. Karsenty (Baylor College of Medicine, Houston, TX), and ST-2, C3H10T1/2, COS-7 and L-Wnt3a cells, obtained from ATCC, were maintained (5% CO2 at 37 °C) in Dulbecco's modified Eagle's medium supplemented with fetal calf serum at appropriate concentration. RNA Extraction, Chip Hybridizations, and Quantitative RT-PCR— Total RNA samples were obtained from three C2C12 cell cultures (BMP-2-treated, 1 μg/ml; TGF-β-treated, 2.5 ng/ml; and solvent-treated control, 10 mm HCl) by use of the RNAplus kit provided by Quantum, harvesting from each culture at six time points (4 h, 8 h, 1 day, 2 days, 3 days, and 4 days). Total RNA was also extracted from ST-2 and C3H10T1/2 cells under similar conditions. Total RNA samples from C2C12 cells were hybridized to the complete series of Affymetrix 35,000 mouse chips as described by Theilhaber et al. (23Theilhaber J. Connolly T. Roman-Roman S. Bushnell S. Jackson A. Call K. Garcia T. Baron R. Genome Res. 2002; 12: 165-176Crossref PubMed Scopus (62) Google Scholar), and data assembly and analysis were carried as described by the authors (23Theilhaber J. Connolly T. Roman-Roman S. Bushnell S. Jackson A. Call K. Garcia T. Baron R. Genome Res. 2002; 12: 165-176Crossref PubMed Scopus (62) Google Scholar). Quantitative RT-PCR was performed with TaqMan PCR reagent kits in the ABI PRISM 7700 sequence detection system (Applied Biosystems) as described by Spinella-Jaegle et al. (6Spinella-Jaegle S. Rawadi G. Kawai S. Gallea S. Faucheu C. Mollat P. Courtois B. Bergaud B. Ramez V. Blanchet A.M. Adelmant G. Baron R. Roman-Roman S. J. Cell Sci. 2001; 114: 2085-2094Crossref PubMed Google Scholar). The following (5′-3′) primer/probe set was designed using Primer-Express Version 1.0 software from Applied Biosystems: forward, 5′-GCCTACATCGCTGGCTTTCT-3′; reverse, 5′-CCCCGTCCTCTGCAAACTT-3′; probe, 5′-AGGACCGGGTGGTGTGCAACGA-3′. Wnt3a-conditioned Medium Preparation—Wnt3a-conditioned medium (Wnt3a-CM) was prepared as described by Shibamoto et al. (24Shibamoto S. Higano K. Takada R. Ito F. Takeichi M. Takada S. Genes Cells. 1998; 3: 659-670Crossref PubMed Scopus (230) Google Scholar). Briefly, to collect the conditioned medium from cultures of Wnt3a-producing L cells, these cells were seeded at a density of 6 × 106 cells in a 125-cm2 flask containing Dulbecco's modified Eagle's medium with 10% fetal calf serum. 24 h after seeding, medium was changed to Dulbecco's modified Eagle's medium with 2% fetal calf serum, and cells were cultured for 3 days. Then Wnt3a-CM was harvested, centrifuged at 1000 × g for 10 min, and filtered through a nitrocellulose membrane. The activity of Wnt3a-CM was assayed on normal L cells by examining the increase in β-catenin as described by Willert et al. (25Willert K. Shibamoto S. Nusse R. Genes Dev. 1999; 13: 1768-1773Crossref PubMed Scopus (294) Google Scholar). Wnt3a-CM was added to cells at 20% final concentration in all subsequent experiments. Measurement of Alkaline Phosphatase Activity—C2C12 cells were plated at 2 × 104/cm2 and 24 h later were transfected with the indicated constructs (1 μg total) using FuGENE 6 (Roche Applied Science). 16 h after transfection, cells were washed and cultured in media containing 2% fetal bovine serum. When indicated, cells were treated with BMP-2 (100 ng/ml) (6Spinella-Jaegle S. Rawadi G. Kawai S. Gallea S. Faucheu C. Mollat P. Courtois B. Bergaud B. Ramez V. Blanchet A.M. Adelmant G. Baron R. Roman-Roman S. J. Cell Sci. 2001; 114: 2085-2094Crossref PubMed Google Scholar) or with Wnt3a-CM. 48 h later, ALP activity was determined in cell lysates using the alkaline phosphatase Opt kit (Roche Applied Science). Cell lysates were analyzed for protein content using the micro-BCA assay kit (Pierce), and ALP activity was normalized for total protein concentration. Luciferase Assay—COS-7 cells plated in 24-well plates at 2 × 104/cm2 were transiently transfected with the indicated constructs (1 μg total) using FuGENE 6 (Roche Applied Science). 20 ng of pRL-TK (Promega, Madison, WI), which encodes a Renilla luciferase gene downstream of a minimal herpes simplex virus thymidine kinase promoter, was systematically added to the transfection mixture to assess transfection efficiency. When required, controls were carried out by replacing constructs with corresponding empty vectors. 16 h after transfection, cells were washed and cultured in media containing 2% fetal bovine serum for an additional 24 h. Cell lysates were prepared, and luciferase assays were performed with the Dual Luciferase assay kit (Promega) according to the manufacturer's instructions. 10 μl of cell lysate were assayed first for firefly luciferase and then for Renilla luciferase activity. Firefly luciferase activity was normalized to Renilla luciferase activity. Plasmids and Constructs—mFz1 and human Frizzled-1 (hFz1) were amplified by RT-PCR, and the nucleotide sequence was confirmed by DNA sequence analysis and subcloned with a Myc tag into pcDNA3.1 vector and into pTracer to co-express GFP protein (Invitrogen). Murine TCF expressing vector and the TCF-luciferase reporter construct, TOP-flash (26van de Wetering M. Cavallo R. Dooijes D. van Beest M. van Es J. Loureiro J. Ypma A. Hursh D. Jones T. Bejsovec A. Peifer M. Mortin M. Clevers H. Cell. 1997; 88: 789-799Abstract Full Text Full Text PDF PubMed Scopus (1053) Google Scholar), were obtained from Upstate Biotechnology. Human β-catenin was RT-PCR amplified, confirmed by sequencing, and subcloned into pcDNA3.1. QuikChange was used to create a constitutive active mutant form of β-catenin (β-catenin*) with the S33Y missense mutation (27Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3465) Google Scholar). Gαq-(305-359) expression vector (28Akhter S.A. Luttrell L.M. Rockman H.A. Iaccarino G. Lefkowitz R.J. Koch W.J. Science. 1998; 280: 574-577Crossref PubMed Scopus (392) Google Scholar) was kindly provided Dr. W. J. Koch (Duke University, Durham, NC). Myc-tagged Xenopus Frizzled-3 (Xfz3) was described previously (29Umbhauer M. Djiane A. Goisset C. Penzo-Mendez A. Riou J.F. Boucaut J.C. Shi D.L. EMBO J. 2000; 19: 4944-4954Crossref PubMed Google Scholar). Xenopus Wnt1 coding sequence was amplified by PCR and subcloned in pCS2 vector (30Turner D.L. Weintraub H. Genes Dev. 1994; 8: 1434-1447Crossref PubMed Scopus (947) Google Scholar). mFz1 was obtained by PCR amplification of mFz1 CRD and the downstream sequence with sense primer 5′-ATCTCCATGGCGGACCACGGCTAC-3′ and antisense primer 5′-AGTTCTAGACGGTAGTCTCCCCCT-3′ including a NcoI and XbaI site, respectively. mFz1 amplicon was cloned into pCS2 vector in-frame with the signal peptide and Myc epitopes using convenient restriction sites. Derived truncated or chimera constructs were also cloned in the same vector using convenient restriction sites in-frame with the signal peptide and Myc epitopes. mFz1 was also cloned in the same vector using convenient restriction sites. The Myc-tagged mFz1ΔC that retains seven residues after the seventh transmembrane domain was obtained by digesting the full-length mFz1 with NcoI and EcoRI. mFz1ΔN lacking the CRD was obtained by PCR amplification of the seven transmembrane and the C-terminal cytoplasmic domains using a sense and an antisense primer including a NcoI and a XbaI sites (underlined), respectively: 5′-AATCCATGGGCCAGAACACGTC-3′ and 5′-AGTTCTAGACGGTAGTCTCCCCCT-3′. The PCR product was digested by NcoI and XbaI and then subcloned into the corresponding sites of Myc-tagged Xfz3 in-frame with the signal peptide and Myc epitopes. The chimeric receptors Xfz3N/mFz1C and mFz1N/Xfz3C were generated by PCR amplification of the C-terminal cytoplasmic region of mFz1 or Xfz3 with a sense primer, 5′-ACATTCGAATGGAGGAAGTTCTAC-3′ with a BstBI site or 5′-ACTGAATTCCTGGGCCAGCTTTTTC-3′ with an EcoRI site (underlined), respectively, and the T7 primer. The PCR products were cloned into the same sites in Myc-tagged Xfz3 or mFz1, respectively, and the XbaI site. To obtain Xfz3CRD/mFz1C, the sequence corresponding to the seven transmembrane and the C-terminal cytoplasmic domains of mFz1 was amplified by PCR using sense primer 5′-AGAGGAGCTCCGCTTCTCGCGC-3′ including a SacI site (underlined) and the T7 primer and subcloned into the SacI and XbaI sites of the Myc-tagged Xfz3. Immunoblotting—Cells were transfected with expression vectors as described above, and 24 h post-transfection, cells were lysed (20 mm Tris-HCl (28Akhter S.A. Luttrell L.M. Rockman H.A. Iaccarino G. Lefkowitz R.J. Koch W.J. Science. 1998; 280: 574-577Crossref PubMed Scopus (392) Google Scholar), 150 mm NaCl, 5 mm MgCl2, 10% glycerol, 0.5% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, and 20 μg/ml leupeptin), and then the protein content of cell lysates was determined with Bio-Rad DC protein assay. 50 μg of each cell lysates were separated by SDS-PAGE. Proteins were then electrophoretically transferred to nitrocellulose membranes and probed with the indicated primary antibody. Immunoreactive bands were visualized by ECL Western blotting technique (Amersham Biosciences) using horseradish peroxidase-labeled secondary antibodies. Anti-Myc antibody was from Santa Cruz Technology, and the anti-β-catenin antibody was obtained from Upstate Biotechnology. Xenopus Embryos and Microinjections—Xenopus eggs were obtained from females injected with 500 IU of human chorionic gonadotropin (Sigma) and artificially fertilized with minced testis. Eggs were dejellied with 2% cysteine hydrochloride (pH 7.8) and kept in 0.1× modified Barth solution. Synthetic capped mRNAs were made by in vitro transcription as described previously (29Umbhauer M. Djiane A. Goisset C. Penzo-Mendez A. Riou J.F. Boucaut J.C. Shi D.L. EMBO J. 2000; 19: 4944-4954Crossref PubMed Google Scholar). Embryos were injected at the four-cell stage near the animal pole region (for ectodermal explants) or in the two ventrovegetal blastomeres (for secondary axis induction) in 0.1× modified Barth solution containing 3% Ficoll-400. Ectodermal explants were dissected at blastula stage and cultured to early gastrula stage for RT-PCR analysis. RT-PCR—For RT-PCR, RNA samples were treated with RNase-free DNase I (Roche Applied Science) and were reverse-transcribed using 200 units of SuperScript (Invitrogen). PCR and primers for the Wnt/β-catenin target gene Siamois and the housekeeping gene ornithine decarboxylase were as described previously (29Umbhauer M. Djiane A. Goisset C. Penzo-Mendez A. Riou J.F. Boucaut J.C. Shi D.L. EMBO J. 2000; 19: 4944-4954Crossref PubMed Google Scholar). Confocal Microscopy Assay—COS-7 cells are plated at 40,000 cells/well in a 6-well plate; each well contained a sterile microcoverglass, which can be removed for observation. Cells were transfected with the indicated plasmid (1 μg total) using FuGENE 6 (Roche Applied Science). Transfected cells were treated or not with Wnt3a-CM for 24 h, then fixed with 3.7% formaldehyde (Sigma) for 10 min, and washed two times with phosphate-buffered saline. Fixed cells were permeabilized by phosphate-buffered saline, 0.025% Triton X-100 (Sigma) for 5 min and blocked in phosphate-buffered saline, 3% bovine serum albumin for 15 min. After that cells were incubated overnight at 4 °C with the primary antibody, a rabbit polyclonal anti-β-catenin (Upstate Biotechnology), at 5 μg/ml in phosphate-buffered saline. After being washed two times cells were incubated for 1 h at room temperature with the secondary antibody, goat anti-rabbit conjugated to FITC (Santa Cruz Biotechnology) at a 1:100 dilution. The cells were washed tree times, mounted on a glass slide, and viewed with a confocal laser scanning microscope (LSM510, Carl Zeiss). Up-regulation of mFz1 Expression by BMP-2 in Mesenchymal Cell Lines—The myoblast cell line C2C12 displays a certain degree of plasticity in terms of differentiation, and BMP-2 treatment is known to inhibit myotube formation and converts the differentiation pathway of C2C12 cells into that of the osteoblast lineage (31Katagiri T. Yamaguchi A. Komaki M. Abe E. Takahashi N. Ikeda T. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. J. Cell Biol. 1994; 127: 1755-1766Crossref PubMed Scopus (1275) Google Scholar). To identify genes playing a role in the transdifferentiation of these pluripotent cells, we performed a genome-wide expression analysis by determining changes in expression levels of 27,000 genes with Affymetrix oligonucleotide chips (23Theilhaber J. Connolly T. Roman-Roman S. Bushnell S. Jackson A. Call K. Garcia T. Baron R. Genome Res. 2002; 12: 165-176Crossref PubMed Scopus (62) Google Scholar). Among these genes Frizzled-1 (mFz1) was found to be significantly increased in the presence of BMP-2 (Fig. 1A). This increase in the expression level of mFz1 was observed 24 h after BMP-2 treatment and was maintained until 4 days of culture. In contrast, TGF-β1, which inhibits myotube formation of C2C12 cells without inducing the osteoblastic phenotype (31Katagiri T. Yamaguchi A. Komaki M. Abe E. Takahashi N. Ikeda T. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. J. Cell Biol. 1994; 127: 1755-1766Crossref PubMed Scopus (1275) Google Scholar), did not significantly affect mFz1 expression in our experiments. The increase in expression of mFz1 in response to BMP-2, but not to TGF-β1, was confirmed by quantitative RT-PCR (TaqMan) in C2C12 cells but also in two other murine mesenchymal pluripotent cell lines, ST2 and C3H10T1/2 (Fig. 1B). Overexpression of mFz1 Represses Activity of BMP-2 and Wnt3a—BMP-2 is known to induce the osteoblast phenotype in distinct pluripotent mesenchymal cell lines including C2C12, C3H10T1/2, and ST2. To investigate the significance of the up-regulation of mFz1 by BMP-2, we examined whether mFz1 overexpression could affect the responsiveness of these cells to BMP-2. mFz1, hFz1, and Xfz3 were cloned and expressed as Myc-tagged proteins and showed comparable expression levels when transfected in C2C12 cells (Fig. 2C) or in the other cells used (data not shown). As shown in Fig. 2A, mFz1 significantly inhibits the BMP-2-induced ALP activity in C2C12. Similar data were obtained using ST-2 and C3H10T1/2 cells (data not shown), suggesting that mFz1 antagonizes the effects of BMP-2. We have recently demonstrated that BMP-2 controls ALP expression by a Wnt autocrine loop (32Rawadi G. Vayssiere B. Dunn F. Baron R. Roman-Roman S. J. Bone Miner. Res. 2003; 18: 1842-1853Crossref PubMed Scopus (657) Google Scholar). As shown in Fig. 2A, mFz1 overexpression also inhibited Wnt3a-mediated ALP activity. Moreover mFz1 was also capable of significantly decreasing Wnt3a-mediated TCF-1 activation in COS cells (Fig. 2B). In contrast, overexpression of human Frizzled-1, Xfz3 (Fig. 2, A and B), or Xfz4 (data not shown) did not decrease but rather enhanced either BMP-2 or Wnt3a activities in our assays. These observations demonstrate that mFz1 antagonizes the activity of BMP-2 and Wnt3a in different cell lines. Overexpression of mFz1 Blocks Nuclear β-Catenin Translocation in COS Cells—To investigate how mFz1 inhibits the activity of Wnt, we monitored nuclear β-catenin translocation induced by Wnt3a in the presence or absence of mFz1. COS cells were transfected with either a control vector expressing GFP or a construct that expresses both GFP and mFz1, and then cells were treated with Wnt3a-conditioned medium. Endogenous cellular β-catenin localization was determined by immunocytochemistry, and exogenous mFz1 expression was determined by GFP fluorescence. As shown in Fig. 3, in cells transfected with GFP expression vector β-catenin is located both in the cytoplasm and at the membrane but not in the nucleus (Fig. 3A). Wnt3a treatment resulted in nuclear localization of β-catenin (Fig. 3B). Importantly mFz1 transfection precluded the Wnt3a-induced β-catenin nuclear localization (Fig. 3C). As shown in Fig. 3D, cells that show β-catenin nuclear localization in response to Wnt3a was markedly lower in mFz1-transfected cells compared with control. Finally we examined the effect of mFz1 overexpression on the TCF signaling induced by β-catenin* (27Morin P
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