Abstract
The proto-oncogene Wnt-1 plays an essential role in fetal brain development and causes hyperplasia and tumorigenesis when activated ectopically in the mouse mammary gland. When expressed in certain mammary epithelial cell lines, the gene causes morphological transformation and excess cell proliferation at confluence. Like other members of the mammalian Wnt family, Wnt-1 encodes secretory glycoproteins which have been detected in association with the extracellular matrix or cell surface but which have not previously been found in a soluble or biologically active form. We show here that conditioned medium harvested from a mammary cell line expressing Wnt-1 contains soluble Wnt-1 protein and induces mitogenesis and transformation of mammary target cells. By immunodepletion of medium containing epitope-tagged Wnt-1, we show that at least 60% of this activity is specifically dependent on Wnt-1 protein. These results provide the first demonstration that a mammalian Wnt protein can act as a diffusible extracellular signaling factor.
Full Text
The Full Text of this article is available as a PDF (793.3 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Blasband A., Schryver B., Papkoff J. The biochemical properties and transforming potential of human Wnt-2 are similar to Wnt-1. Oncogene. 1992 Jan;7(1):153–161. [PubMed] [Google Scholar]
- Bradbury J. M., Niemeyer C. C., Dale T. C., Edwards P. A. Alterations of the growth characteristics of the fibroblast cell line C3H 10T1/2 by members of the Wnt gene family. Oncogene. 1994 Sep;9(9):2597–2603. [PubMed] [Google Scholar]
- Bradley R. S., Brown A. M. The proto-oncogene int-1 encodes a secreted protein associated with the extracellular matrix. EMBO J. 1990 May;9(5):1569–1575. doi: 10.1002/j.1460-2075.1990.tb08276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bradley R. S., Cowin P., Brown A. M. Expression of Wnt-1 in PC12 cells results in modulation of plakoglobin and E-cadherin and increased cellular adhesion. J Cell Biol. 1993 Dec;123(6 Pt 2):1857–1865. doi: 10.1083/jcb.123.6.1857. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brown A. M., Papkoff J., Fung Y. K., Shackleford G. M., Varmus H. E. Identification of protein products encoded by the proto-oncogene int-1. Mol Cell Biol. 1987 Nov;7(11):3971–3977. doi: 10.1128/mcb.7.11.3971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brown A. M., Wildin R. S., Prendergast T. J., Varmus H. E. A retrovirus vector expressing the putative mammary oncogene int-1 causes partial transformation of a mammary epithelial cell line. Cell. 1986 Sep 26;46(7):1001–1009. doi: 10.1016/0092-8674(86)90699-9. [DOI] [PubMed] [Google Scholar]
- Couso J. P., Bate M., Martínez-Arias A. A wingless-dependent polar coordinate system in Drosophila imaginal discs. Science. 1993 Jan 22;259(5094):484–489. doi: 10.1126/science.8424170. [DOI] [PubMed] [Google Scholar]
- Dickinson M. E., Krumlauf R., McMahon A. P. Evidence for a mitogenic effect of Wnt-1 in the developing mammalian central nervous system. Development. 1994 Jun;120(6):1453–1471. doi: 10.1242/dev.120.6.1453. [DOI] [PubMed] [Google Scholar]
- Gavin B. J., McMahon A. P. Differential regulation of the Wnt gene family during pregnancy and lactation suggests a role in postnatal development of the mammary gland. Mol Cell Biol. 1992 May;12(5):2418–2423. doi: 10.1128/mcb.12.5.2418. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gavin B. J., McMahon J. A., McMahon A. P. Expression of multiple novel Wnt-1/int-1-related genes during fetal and adult mouse development. Genes Dev. 1990 Dec;4(12B):2319–2332. doi: 10.1101/gad.4.12b.2319. [DOI] [PubMed] [Google Scholar]
- González F., Swales L., Bejsovec A., Skaer H., Martinez Arias A. Secretion and movement of wingless protein in the epidermis of the Drosophila embryo. Mech Dev. 1991 Aug;35(1):43–54. doi: 10.1016/0925-4773(91)90040-d. [DOI] [PubMed] [Google Scholar]
- Herzlinger D., Qiao J., Cohen D., Ramakrishna N., Brown A. M. Induction of kidney epithelial morphogenesis by cells expressing Wnt-1. Dev Biol. 1994 Dec;166(2):815–818. doi: 10.1006/dbio.1994.1360. [DOI] [PubMed] [Google Scholar]
- Hinck L., Nelson W. J., Papkoff J. Wnt-1 modulates cell-cell adhesion in mammalian cells by stabilizing beta-catenin binding to the cell adhesion protein cadherin. J Cell Biol. 1994 Mar;124(5):729–741. doi: 10.1083/jcb.124.5.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jue S. F., Bradley R. S., Rudnicki J. A., Varmus H. E., Brown A. M. The mouse Wnt-1 gene can act via a paracrine mechanism in transformation of mammary epithelial cells. Mol Cell Biol. 1992 Jan;12(1):321–328. doi: 10.1128/mcb.12.1.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kirschmeier P. T., Housey G. M., Johnson M. D., Perkins A. S., Weinstein I. B. Construction and characterization of a retroviral vector demonstrating efficient expression of cloned cDNA sequences. DNA. 1988 Apr;7(3):219–225. doi: 10.1089/dna.1988.7.219. [DOI] [PubMed] [Google Scholar]
- Kitajewski J., Mason J. O., Varmus H. E. Interaction of Wnt-1 proteins with the binding protein BiP. Mol Cell Biol. 1992 Feb;12(2):784–790. doi: 10.1128/mcb.12.2.784. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klingensmith J., Nusse R. Signaling by wingless in Drosophila. Dev Biol. 1994 Dec;166(2):396–414. doi: 10.1006/dbio.1994.1325. [DOI] [PubMed] [Google Scholar]
- Krauss S., Korzh V., Fjose A., Johansen T. Expression of four zebrafish wnt-related genes during embryogenesis. Development. 1992 Sep;116(1):249–259. doi: 10.1242/dev.116.1.249. [DOI] [PubMed] [Google Scholar]
- Markowitz D., Goff S., Bank A. A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol. 1988 Apr;62(4):1120–1124. doi: 10.1128/jvi.62.4.1120-1124.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mason J. O., Kitajewski J., Varmus H. E. Mutational analysis of mouse Wnt-1 identifies two temperature-sensitive alleles and attributes of Wnt-1 protein essential for transformation of a mammary cell line. Mol Biol Cell. 1992 May;3(5):521–533. doi: 10.1091/mbc.3.5.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Massagué J. Receptors for the TGF-beta family. Cell. 1992 Jun 26;69(7):1067–1070. doi: 10.1016/0092-8674(92)90627-o. [DOI] [PubMed] [Google Scholar]
- McMahon A. P., Bradley A. The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell. 1990 Sep 21;62(6):1073–1085. doi: 10.1016/0092-8674(90)90385-r. [DOI] [PubMed] [Google Scholar]
- Moon R. T., DeMarais A., Olson D. J. Responses to Wnt signals in vertebrate embryos may involve changes in cell adhesion and cell movement. J Cell Sci Suppl. 1993;17:183–188. doi: 10.1242/jcs.1993.supplement_17.26. [DOI] [PubMed] [Google Scholar]
- Moon R. T. In pursuit of the functions of the Wnt family of developmental regulators: insights from Xenopus laevis. Bioessays. 1993 Feb;15(2):91–97. doi: 10.1002/bies.950150204. [DOI] [PubMed] [Google Scholar]
- Nusse R. Insertional mutagenesis in mouse mammary tumorigenesis. Curr Top Microbiol Immunol. 1991;171:43–65. doi: 10.1007/978-3-642-76524-7_3. [DOI] [PubMed] [Google Scholar]
- Nusse R., Varmus H. E. Wnt genes. Cell. 1992 Jun 26;69(7):1073–1087. doi: 10.1016/0092-8674(92)90630-u. [DOI] [PubMed] [Google Scholar]
- Nusse R., van Ooyen A., Cox D., Fung Y. K., Varmus H. Mode of proviral activation of a putative mammary oncogene (int-1) on mouse chromosome 15. Nature. 1984 Jan 12;307(5947):131–136. doi: 10.1038/307131a0. [DOI] [PubMed] [Google Scholar]
- Papkoff J., Brown A. M., Varmus H. E. The int-1 proto-oncogene products are glycoproteins that appear to enter the secretory pathway. Mol Cell Biol. 1987 Nov;7(11):3978–3984. doi: 10.1128/mcb.7.11.3978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Papkoff J., Schryver B. Secreted int-1 protein is associated with the cell surface. Mol Cell Biol. 1990 Jun;10(6):2723–2730. doi: 10.1128/mcb.10.6.2723. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Parkin N. T., Kitajewski J., Varmus H. E. Activity of Wnt-1 as a transmembrane protein. Genes Dev. 1993 Nov;7(11):2181–2193. doi: 10.1101/gad.7.11.2181. [DOI] [PubMed] [Google Scholar]
- Parr B. A., McMahon A. P. Wnt genes and vertebrate development. Curr Opin Genet Dev. 1994 Aug;4(4):523–528. doi: 10.1016/0959-437x(94)90067-d. [DOI] [PubMed] [Google Scholar]
- Ramakrishna N. R., Brown A. M. Wingless, the Drosophila homolog of the proto-oncogene Wnt-1, can transform mouse mammary epithelial cells. Dev Suppl. 1993:95–103. [PubMed] [Google Scholar]
- Rijsewijk F., Schuermann M., Wagenaar E., Parren P., Weigel D., Nusse R. The Drosophila homolog of the mouse mammary oncogene int-1 is identical to the segment polarity gene wingless. Cell. 1987 Aug 14;50(4):649–657. doi: 10.1016/0092-8674(87)90038-9. [DOI] [PubMed] [Google Scholar]
- Rijsewijk F., van Deemter L., Wagenaar E., Sonnenberg A., Nusse R. Transfection of the int-1 mammary oncogene in cuboidal RAC mammary cell line results in morphological transformation and tumorigenicity. EMBO J. 1987 Jan;6(1):127–131. doi: 10.1002/j.1460-2075.1987.tb04729.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shackleford G. M., Shivakumar S., Shiue L., Mason J., Kenyon C., Varmus H. E. Two wnt genes in Caenorhabditis elegans. Oncogene. 1993 Jul;8(7):1857–1864. [PubMed] [Google Scholar]
- Shackleford G. M., Varmus H. E. Expression of the proto-oncogene int-1 is restricted to postmeiotic male germ cells and the neural tube of mid-gestational embryos. Cell. 1987 Jul 3;50(1):89–95. doi: 10.1016/0092-8674(87)90665-9. [DOI] [PubMed] [Google Scholar]
- Sidow A. Diversification of the Wnt gene family on the ancestral lineage of vertebrates. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5098–5102. doi: 10.1073/pnas.89.11.5098. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smolich B. D., McMahon J. A., McMahon A. P., Papkoff J. Wnt family proteins are secreted and associated with the cell surface. Mol Biol Cell. 1993 Dec;4(12):1267–1275. doi: 10.1091/mbc.4.12.1267. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sonnenberg A., van Balen P., Hilgers J., Schuuring E., Nusse R. Oncogene expression during progression of mouse mammary tumor cells; activity of a proviral enhancer and the resulting expression of int-2 is influenced by the state of differentiation. EMBO J. 1987 Jan;6(1):121–125. doi: 10.1002/j.1460-2075.1987.tb04728.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stark K., Vainio S., Vassileva G., McMahon A. P. Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4. Nature. 1994 Dec 15;372(6507):679–683. doi: 10.1038/372679a0. [DOI] [PubMed] [Google Scholar]
- Struhl G., Basler K. Organizing activity of wingless protein in Drosophila. Cell. 1993 Feb 26;72(4):527–540. doi: 10.1016/0092-8674(93)90072-x. [DOI] [PubMed] [Google Scholar]
- Takada S., Stark K. L., Shea M. J., Vassileva G., McMahon J. A., McMahon A. P. Wnt-3a regulates somite and tailbud formation in the mouse embryo. Genes Dev. 1994 Jan;8(2):174–189. doi: 10.1101/gad.8.2.174. [DOI] [PubMed] [Google Scholar]
- Thomas K. R., Capecchi M. R. Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development. Nature. 1990 Aug 30;346(6287):847–850. doi: 10.1038/346847a0. [DOI] [PubMed] [Google Scholar]
- Tsukamoto A. S., Grosschedl R., Guzman R. C., Parslow T., Varmus H. E. Expression of the int-1 gene in transgenic mice is associated with mammary gland hyperplasia and adenocarcinomas in male and female mice. Cell. 1988 Nov 18;55(4):619–625. doi: 10.1016/0092-8674(88)90220-6. [DOI] [PubMed] [Google Scholar]
- Vaidya A. B., Lasfargues E. Y., Sheffield J. B., Coutinho W. G. Murine mammary tumor virus (MuMTV) infection of an epithelial cell line established from C57BL/6 mouse mammary glands. Virology. 1978 Oct 1;90(1):12–22. doi: 10.1016/0042-6822(78)90328-8. [DOI] [PubMed] [Google Scholar]
- Weber-Hall S. J., Phippard D. J., Niemeyer C. C., Dale T. C. Developmental and hormonal regulation of Wnt gene expression in the mouse mammary gland. Differentiation. 1994 Sep;57(3):205–214. doi: 10.1046/j.1432-0436.1994.5730205.x. [DOI] [PubMed] [Google Scholar]
- Wilkinson D. G., Bailes J. A., McMahon A. P. Expression of the proto-oncogene int-1 is restricted to specific neural cells in the developing mouse embryo. Cell. 1987 Jul 3;50(1):79–88. doi: 10.1016/0092-8674(87)90664-7. [DOI] [PubMed] [Google Scholar]
- Wilson I. A., Niman H. L., Houghten R. A., Cherenson A. R., Connolly M. L., Lerner R. A. The structure of an antigenic determinant in a protein. Cell. 1984 Jul;37(3):767–778. doi: 10.1016/0092-8674(84)90412-4. [DOI] [PubMed] [Google Scholar]
- Wolda S. L., Moon R. T. Cloning and developmental expression in Xenopus laevis of seven additional members of the Wnt family. Oncogene. 1992 Oct;7(10):1941–1947. [PubMed] [Google Scholar]
- Yayon A., Klagsbrun M., Esko J. D., Leder P., Ornitz D. M. Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell. 1991 Feb 22;64(4):841–848. doi: 10.1016/0092-8674(91)90512-w. [DOI] [PubMed] [Google Scholar]
- Zákány J., Duboule D. Correlation of expression of Wnt-1 in developing limbs with abnormalities in growth and skeletal patterning. Nature. 1993 Apr 8;362(6420):546–549. doi: 10.1038/362546a0. [DOI] [PubMed] [Google Scholar]
- van Leeuwen F., Samos C. H., Nusse R. Biological activity of soluble wingless protein in cultured Drosophila imaginal disc cells. Nature. 1994 Mar 24;368(6469):342–344. doi: 10.1038/368342a0. [DOI] [PubMed] [Google Scholar]
- van den Heuvel M., Harryman-Samos C., Klingensmith J., Perrimon N., Nusse R. Mutations in the segment polarity genes wingless and porcupine impair secretion of the wingless protein. EMBO J. 1993 Dec 15;12(13):5293–5302. doi: 10.1002/j.1460-2075.1993.tb06225.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- van den Heuvel M., Nusse R., Johnston P., Lawrence P. A. Distribution of the wingless gene product in Drosophila embryos: a protein involved in cell-cell communication. Cell. 1989 Nov 17;59(4):739–749. doi: 10.1016/0092-8674(89)90020-2. [DOI] [PubMed] [Google Scholar]