title: Reporter gene HEK 293 cells and WNT/Frizzled fusion proteins as tools to study WNT signaling pathways.
abstract: WNT/Frizzled receptor (FZD) signaling pathways are pivotal for physiological and pathophysiological processes. In humans, the complexity of WNT/FZD signaling is based on 19 WNTs, 10 FZDs and at least two (co)receptors (LRP5/6) mediating supposably four different signaling cascades. The detailed investigation of the specific function of the different initiating components is primarily hampered by the lack of most WNT proteins in a purified form. Therefore, we constructed and examined a chimeric protein of WNT3a and FZD4 as a suitable approach to overcome this obstacle for future studies of the specificity of other WNT/FZD combinations. Furthermore, we produced four different reporter HEK 293 cell lines to quantify the induced activation of the proposed signaling cascades, the β-catenin-, the NFAT-, the AP-1- and the CRE-regulated pathways. The chimera WNT3aFZD4 efficiently induced β-catenin-mediated luciferase activity. This activity was increased 40-fold compared with basal when LRP6 was stably cotransfected, proving that the chimera WNT3aFZD4 can also interact efficiently with LRP6. Our results demonstrate that the approach of using reporter gene cell lines in combination with WNT/FZD chimeras is efficient to study the β-catenin-mediated pathway and should also allow clarifying the specificity of WNT/FZD combinations in the activation of the other pathways.
authors.affiliation: Abteilung für Klinische Chemie und Klinische Biochemie, Ludwig-Maximilians-Universität, Nussbaumstrasse 20, D-80336 München, Germany.
authors.forename: Larisa
authors.initials: L
authors.lastname: Ring
authors.forename: Iris
authors.initials: I
authors.lastname: Peröbner
authors.forename: Marisa
authors.initials: M
authors.lastname: Karow
authors.forename: Marianne
authors.initials: M
authors.lastname: Jochum
authors.forename: Peter
authors.initials: P
authors.lastname: Neth
authors.forename: Alexander
authors.initials: A
authors.lastname: Faussner
mesh_terms: D001483:Base Sequence; D051157:Frizzled Receptors / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D051153:Wnt Proteins / Q000235:genetics* / Q000378:metabolism; D060449:Wnt Signaling Pathway; D060509:Wnt3A Protein / Q000235:genetics / Q000378:metabolism
chemical_list: C402930:FZD4 protein, human; D051157:Frizzled Receptors; D011993:Recombinant Fusion Proteins; D051153:Wnt Proteins; D060509:Wnt3A Protein