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.
Highlights
Pax6 belongs to the paired box family of transcription factors, and plays an important role in the development of the central nervous system, the eyes, the nose and the pancreas
Dickkopf 3 (Dkk3) expression is upregulated in the FlpIn-3T3 Pax6and Pax6(5a) cell lines and the protein is localized in cytoplasm and nucleus
Gene expression microarray identified Dkk3 to be the gene strongest upregulated by Pax6(5a) [9]
Summary
Pax belongs to the paired box family of transcription factors, and plays an important role in the development of the central nervous system, the eyes, the nose and the pancreas (reviewed in [1]). The Pax6(5a) isoform is characterized by a 14 amino acid insertion in the Nterminal part of the paired domain This insertion changes the DNA binding specificity of the Pax paired domain from the consensus primarily recognized by a single paired domain [6], to a consensus looking like two tandem repeats, preferably bound by four Pax6(5a) paired domains simultaneously [2,7]. Both Pax isoforms are expressed together in various tissues in the eye and brain, and seem to interact functionally to stimulate transcription of target genes [8]. A link between the Wnt pathway and Pax is reported both in the brain [14], in the lens [15] and in the cornea [16]
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