title: Pax6 regulates the expression of Dkk3 in murine and human cell lines, and altered responses to Wnt signaling are shown in FlpIn-3T3 cells stably expressing either the Pax6 or the Pax6(5a) isoform.
abstract: Pax6 is a transcription factor important for early embryo development. It is expressed in several cancer cell lines and tumors. In glioblastoma, PAX6 has been shown to function as a tumor suppressor. Dickkopf 3 (Dkk3) is well established as a tumor suppressor in several tumor types, but not much is known about the regulation of its expression. We have previously found that Pax6 and Pax6(5a) increase the expression of the Dkk3 gene in two stably transfected mouse fibroblast cell lines. In this study the molecular mechanism behind this regulation is looked at. Western blot and reverse transcriptase quantitative PCR (RT-qPCR) confirmed higher level of Dkk3 expression in both Pax6 and Pax6(5a) expressing cell lines compared to the control cell line. By the use of bioinformatics and electrophoretic mobility shift assay (EMSA) we have mapped a functional Pax6 binding site in the mouse Dkk3 promoter. The minimal Dkk3 promoter fragment required for transcriptional activation by Pax6 and Pax6(5a) was a 200 bp region just upstream of the transcriptional start site. Mutation of the evolutionary conserved binding site in this region abrogated transcriptional activation and binding of Pax6/Pax6(5a) to the mouse Dkk3 promoter. Since the identified Pax6 binding site in this promoter is conserved, RT-qPCR and Western blot were used to look for regulation of Dkk3/REIC expression in human cell lines. Six of eight cell lines tested showed changes in Dkk3/REIC expression after PAX6 siRNA knockdown. Interestingly, we observed that the Pax6/Pax6(5a) expressing mouse fibroblast cell lines were less responsive to canonical Wnt pathway stimulation than the control cell line when TOP/FOP activity and the levels of active β-catenin and GSK3-β Ser9 phosphorylation were measured after LiCl stimulation.
authors.affiliation: Research Group of Pharmacology, Department of Pharmacy, UiT - The Artic University of Norway, Tromsoe, Norway.
authors.forename: Siri
authors.initials: S
authors.lastname: Forsdahl
authors.affiliation: Research Group of Pharmacology, Department of Pharmacy, UiT - The Artic University of Norway, Tromsoe, Norway; Norwegian Translational Cancer Research Center, Department of Medical Biology, UiT - The Arctic University of Norway, Tromsoe, Norway.
authors.forename: Yury
authors.initials: Y
authors.lastname: Kiselev
authors.affiliation: Research Group of Pharmacology, Department of Pharmacy, UiT - The Artic University of Norway, Tromsoe, Norway.
authors.forename: Rune
authors.initials: R
authors.lastname: Hogseth
authors.affiliation: Research Group of Pharmacology, Department of Pharmacy, UiT - The Artic University of Norway, Tromsoe, Norway.
authors.forename: Janne E
authors.initials: JE
authors.lastname: Mjelle
authors.affiliation: Research Group of Pharmacology, Department of Pharmacy, UiT - The Artic University of Norway, Tromsoe, Norway.
authors.forename: Ingvild
authors.initials: I
authors.lastname: Mikkola
mesh_terms: D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D018925:Chemokines; D005136:Eye Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D055785:Gene Knockdown Techniques; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D000071841:PAX6 Transcription Factor; D051761:Paired Box Transcription Factors / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D020033:Protein Isoforms / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D060449:Wnt Signaling Pathway / Q000502:physiology*; D051176:beta Catenin / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018925:Chemokines; C498624:DKK3 protein, human; D005136:Eye Proteins; D018398:Homeodomain Proteins; D036341:Intercellular Signaling Peptides and Proteins; D000071841:PAX6 Transcription Factor; C000605730:PAX6 protein, human; D051761:Paired Box Transcription Factors; C000605732:Pax6 protein, mouse; D020033:Protein Isoforms; D012097:Repressor Proteins; D051176:beta Catenin; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3