Wnt-1 Induces Growth, Cytosolic β-Catenin, and Tcf/Lef Transcriptional Activation in Rat-1 Fibroblasts

ABSTRACT Genetic evidence suggests that regulation of β-catenin and regulation of Tcf/Lef family transcription factors are downstream events of the Wnt signal transduction pathway. However, a direct link between Wnt activity and Tcf/Lef transcriptional activation has yet to be established. In this study, we show that Wnt-1 induces a growth response in a cultured mammalian cell line, Rat-1 fibroblasts. Wnt-1 induces serum-independent cellular proliferation of Rat-1 fibroblasts and changes in morphology. Rat-1 cells stably expressing Wnt-1 (Rat-1/Wnt-1) show a constitutive up-regulation of cytosolic β-catenin, while membrane-associated β-catenin remains unaffected. Induction of cytosolic β-catenin in Rat-1/Wnt-1 cells is correlated with activation of a Tcf-responsive transcriptional element. We thus provide evidence that Wnt-1 induces Tcf/Lef transcriptional activation in a mammalian system. Expression of a mutant β-catenin (β-CatS37A) in Rat-1 cells does not result in a proliferative response or a detectable change in the cytosolic β-catenin protein level. However, β-CatS37A expression in Rat-1 cells results in strong Tcf/Lef transcriptional activation, comparable to that seen in Wnt-1-expressing cells. These results suggest that Wnt-1 induction of cytosolic β-catenin may have functions in addition to Tcf/Lef transcriptional activation.

[1]  R. Krumlauf,et al.  Evidence for a mitogenic effect of Wnt-1 in the developing mammalian central nervous system. , 1994, Development.

[2]  K. Kinzler,et al.  Association of the APC tumor suppressor protein with catenins. , 1993, Science.

[3]  J. Papkoff,et al.  Wnt-1 modulates cell-cell adhesion in mammalian cells by stabilizing beta-catenin binding to the cell adhesion protein cadherin , 1994, The Journal of cell biology.

[4]  C W Turck,et al.  A homolog of the armadillo protein in Drosophila (plakoglobin) associated with E-cadherin. , 1991, Science.

[5]  R. Moon,et al.  Signal transduction through beta-catenin and specification of cell fate during embryogenesis. , 1996, Genes & development.

[6]  D. Pinkel,et al.  Deficiency of p53 accelerates mammary tumorigenesis in Wnt-1 transgenic mice and promotes chromosomal instability. , 1995, Genes & development.

[7]  Paul Polakis,et al.  Stabilization of β-Catenin by Genetic Defects in Melanoma Cell Lines , 1997, Science.

[8]  Hans Clevers,et al.  Armadillo Coactivates Transcription Driven by the Product of the Drosophila Segment Polarity Gene dTCF , 1997, Cell.

[9]  Michael Kühl,et al.  Functional interaction of β-catenin with the transcription factor LEF-1 , 1996, Nature.

[10]  R. Nusse,et al.  A Versatile Transcriptional Effector of Wingless Signaling , 1997, Cell.

[11]  P. Robbins,et al.  Stabilization of beta-catenin by genetic defects in melanoma cell lines. , 1997, Science.

[12]  Hans Clevers,et al.  XTcf-3 Transcription Factor Mediates β-Catenin-Induced Axis Formation in Xenopus Embryos , 1996, Cell.

[13]  F. Graham,et al.  An efficient and flexible system for construction of adenovirus vectors with insertions or deletions in early regions 1 and 3. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[14]  M. Peifer.  Beta-catenin as oncogene: the smoking gun. , 1997, Science.

[15]  M. Kitamura,et al.  Construction of adenovirus vectors through Cre-lox recombination , 1997, Journal of virology.

[16]  Gerard I. Evan,et al.  Induction of apoptosis in fibroblasts by c-myc protein , 1992, Cell.

[17]  R. Moon,et al.  Activities of the Wnt-1 class of secreted signaling factors are antagonized by the Wnt-5A class and by a dominant negative cadherin in early Xenopus development , 1996, The Journal of cell biology.

[18]  M. Peifer,et al.  Phosphorylation of the Drosophila adherens junction protein Armadillo: roles for wingless signal and zeste-white 3 kinase. , 1994, Developmental biology.

[19]  H. Varmus,et al.  Expression of the int-1 gene in transgenic mice is associated with mammary gland hyperplasia and adenocarcinomas in male and female mice , 1988, Cell.

[20]  F. Masiarz,et al.  Association of the APC gene product with beta-catenin. , 1993, Science.

[21]  Anthony M. C. Brown,et al.  A retrovirus vector expressing the putative mammary oncogene int-1 causes partial transformation of a mammary epithelial cell line , 1986, Cell.

[22]  Jörg Stappert,et al.  β‐catenin is a target for the ubiquitin–proteasome pathway , 1997 .

[23]  Jeremy Nathans,et al.  A new member of the frizzled family from Drosophila functions as a Wingless receptor , 1996, Nature.

[24]  G. Nolan,et al.  Production of high-titer helper-free retroviruses by transient transfection. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[25]  E. Wieschaus,et al.  A Drosophila homolog of the tumor suppressor gene adenomatous polyposis coli down-regulates beta-catenin but its zygotic expression is not essential for the regulation of Armadillo. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[26]  E. Wieschaus,et al.  腺腫性結腸ポリープ癌抑制遺伝子のショウジョウバエ相同体はβ‐カテニンをダウンレギュレートするが,その接合体発現はアルマジロ蛋白質の調節に必須ではない , 1997 .

[27]  A. Brown,et al.  Transformation by Wnt family proteins correlates with regulation of beta-catenin. , 1997, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[28]  Hans Clevers,et al.  An HMG-box-containing T-cell factor required for thymocyte differentiation , 1995, Nature.

[29]  R. Montesano,et al.  Notch4 and Wnt-1 proteins function to regulate branching morphogenesis of mammary epithelial cells in an opposing fashion. , 1998, Developmental biology.

[30]  P. Polakis,et al.  Regulation of intracellular beta-catenin levels by the adenomatous polyposis coli (APC) tumor-suppressor protein. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[31]  D. Olson,et al.  Regulated expression of Wnt family members during proliferation of C57mg mammary cells. , 1994, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[32]  A. Brown,et al.  Induction of kidney epithelial morphogenesis by cells expressing Wnt-1. , 1994, Developmental biology.

[33]  Paul Polakis,et al.  Binding of GSK3β to the APC-β-Catenin Complex and Regulation of Complex Assembly , 1996, Science.

[34]  N. Perrimon.  Serpentine Proteins Slither into the Wingless and Hedgehog Fields , 1996, Cell.

[35]  Robert Kay,et al.  Expression cloning of oncogenes by retroviral transfer of cDNA libraries , 1995, Molecular and cellular biology.

[36]  I Fariñas,et al.  Development of several organs that require inductive epithelial-mesenchymal interactions is impaired in LEF-1-deficient mice. , 1994, Genes & development.

[37]  K. Kinzler,et al.  Constitutive Transcriptional Activation by a β-Catenin-Tcf Complex in APC−/− Colon Carcinoma , 1997, Science.

[38]  Mark Peifer,et al.  β-Catenin as Oncogene--The Smoking Gun , 1997, Science.

[39]  A. Sonnenberg,et al.  Transfection of the int‐1 mammary oncogene in cuboidal RAC mammary cell line results in morphological transformation and tumorigenicity. , 1987, The EMBO journal.

[40]  F. Luyten,et al.  The cysteine-rich frizzled domain of Frzb-1 is required and sufficient for modulation of Wnt signaling. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[41]  R. Moon,et al.  Responses to Wnt signals in vertebrate embryos may involve changes in cell adhesion and cell movement , 1993, Journal of Cell Science.

[42]  Konrad Basler,et al.  pangolinencodes a Lef-1 homologue that acts downstream of Armadillo to transduce the Wingless signal in Drosophila , 1997, Nature.

[43]  Roel Nusse,et al.  Wnt genes , 1992, Cell.

[44]  B. Herrmann,et al.  Nuclear localization of β-catenin by interaction with transcription factor LEF-1 , 1996, Mechanisms of Development.

[45]  A. Brown,et al.  Expression of Wnt-1 in PC12 cells results in modulation of plakoglobin and E-cadherin and increased cellular adhesion , 1993, The Journal of cell biology.

[46]  D. Crowe.  Retinoic acid mediates post-transcriptional regulation of keratin 19 mRNA levels. , 1993, Journal of cell science.

[47]  P. McCrea,et al.  Embryonic axis induction by the armadillo repeat domain of beta- catenin: evidence for intracellular signaling , 1995, The Journal of cell biology.

[48]  B. Gumbiner,et al.  Cell Adhesion: The Molecular Basis of Tissue Architecture and Morphogenesis , 1996, Cell.

[49]  R. Moon,et al.  Identification of distinct classes and functional domains of Wnts through expression of wild-type and chimeric proteins in Xenopus embryos , 1995, Molecular and cellular biology.

[50]  P. Polakis,et al.  Wnt-1 regulates free pools of catenins and stabilizes APC-catenin complexes , 1996, Molecular and cellular biology.

[51]  Mariann Bienz,et al.  LEF-1, a Nuclear Factor Coordinating Signaling Inputs from wingless and decapentaplegic , 1997, Cell.

[52]  R Grosschedl,et al.  Functional interaction of beta-catenin with the transcription factor LEF-1. , 1996, Nature.

[53]  P. Edwards,et al.  Alterations of the growth characteristics of the fibroblast cell line C3H 10T1/2 by members of the Wnt gene family. , 1994, Oncogene.

[54]  Detlef Weigel,et al.  The Drosophila homology of the mouse mammary oncogene int-1 is identical to the segment polarity gene wingless , 1987, Cell.

[55]  A. McMahon,et al.  Differential transformation of mammary epithelial cells by Wnt genes , 1994, Molecular and cellular biology.

[56]  B. Gumbiner,et al.  beta-Catenin has Wnt-like activity and mimics the Nieuwkoop signaling center in Xenopus dorsal-ventral patterning. , 1995, Developmental biology.