Abstract
The Wnt and Hedgehog signalling pathways serve key roles in diverse developmental processes. However, the molecular associations between these two signalling pathways remains unclear. Previous transcriptome studies on human foreskin fibroblasts have indicated that Wnt signalling activation induces the expression of key Hedgehog signalling genes, including smoothened, frizzled class receptor (Smo) and GLI family zinc finger 1 (Gli1). Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results revealed that Wnt3a treatment induced the expression of the key Hedgehog signalling genes, including Smo, patched (PTCH), Gli1, Gli2 and Gli3. In addition, western blot analyses demonstrated that Wnt3a treatment resulted in the accumulation of cellular Smo and Gli proteins. Furthermore, promoter sequence analysis revealed that the putative β-catenin/T-cell factor (TCF)-4 complex binding motifs (T/AC/GAAAG) were located within 1.5 kb of the Smo and Gli1 promoters. Results of the chromatin immunoprecipitation experiments and yeast-one hybrid assays revealed that TCF4 directly binds to the Smo and Gli1 promoters, with two binding sites for Smo and a single binding site for Gli1. Further analysis showed that the β-catenin/TCF4 complex binds to the Smo and Gli1 promoters. To investigate the functions of TCF4 and β-catenin in transcriptional regulation of Smo and Gli1, TCF4 and β-catenin were transiently expressed in fibroblast cells. RT-qPCR results demonstrated that overexpression of TCF4 and β-catenin induced the expression of Smo and Gli1. In addition, small interfering RNA-mediated suppression of β-catenin resulted in the downregulation of Smo and Gli1 expression levels, even under Wnt3a treatment. Suppression of β-catenin and Gli1 expression inhibited cell proliferation. Taken together, the results of the present study suggested that the β-catenin/TCF4 complex directly activates Smo and Gli1 by binding to their promoters, which in turn controls cell proliferation in human fibroblasts.