Figure 6

Modulation of Wnt3a expression via a conserved TCF/LEF promoter site. (a) Phylogenetic tree of WNT3A across multiple species: human, mouse, rat, chicken, and zebrafish. The WNT3A exons were used to construct the phylogenetic tree. Evolutionary distances in the units of base substitutions per site were calculated using the maximum composite likelihood method. The phylogenetic tree was then constructed using the Neighbor-Joining method with the sum of branch lengths = 0.645. (b) Binding site analysis of aligned human, mouse, rat, chicken, and zebrafish WNT3A promoter sequences highlighting conserved β-catenin-binding TCF/LEF sites within the 5 kb region upstream of the transcriptional start site (TSS). (c) Schematic representation of our luciferase reporter of TCF/LEF-dependent WNT3A transcription (left panel). The reporter construct consists of a selected segment of the human WNT3A promoter region containing a highly conserved TCF/LEF site upstream of the Gaussia luciferase gene. We show two variants of the reporter: one containing the wild-type (WT) TCF/LEF site (Human-WT) and another where the TCF/LEF site was inactivated by mutation (Human-Mut). Upon expression of a WNT3A transcriptional reporter in human renal proximal tubule cells, the Human-Mut reporter demonstrated significantly decreased WNT3A promoter activity compared with the WT control (right panel). The data were normalized to alkaline phosphatase expression. *P < 0.05; Student’s t-test. (d) Binding site analysis of aligned human, mouse, rat, chicken, and zebrafish DRD2 promoter sequences highlighting conserved β-catenin-binding TCF/LEF sites within the 5 kb region upstream of the TSS. (e) Treatment with either LiCl (25 mM, 24 hr) or recombinant Wnt3a (100 ng/ml, 24 hr) significantly increased D2R mRNA expression. Data are expressed as mean ± SEM of 3 experiments conducted on separate experimental dates and performed in triplicate. *P < 0.05; Student’s t-test (c). *P < 0.05, ***P < 0.001; one-way ANOVA, Holm-Sidak post-hoc test (e).