The Wnt pathway destabilizes adherens junctions and promotes cell migration via β-catenin and its target gene cyclin D1

Abstract

The Wnt pathway regulates cell proliferation, mobility and differentiation. Among the many Wnt target genes is CCND1 which codes for cyclin D1. Cyclin D1, in complex with cdk4 and cdk6, regulates G1/S phase transition during cell cycle. Independently of CDK, cyclin D1 also regulates the migration of macrophages. Here we analyzed the effects of cyclin D1 on the migration of cancer cell lines using the transwell migration and scratch assays. We also tested the effect of cyclin D1 and β-catenin on E-cadherin-mediated cell-cell contacts. Our results show that the Wnt pathway promotes cellular migration via its target gene cyclin D1. Moreover we show that cyclin D1 influences the actin cytoskeleton and destabilizes adherens junctions.

Keywords: Adherens junction; Cell migration; Cyclin D1; Wnt pathway; β-Catenin.

Fig. 1

Knock-down of cyclin D1 and β-catenin lowers relative migration rate. Bars show relative migration rates after siRNA transfection. The migration rate of control cells, which were treated with control siRNA, was set as 100%. The statistical analysis was performed with the Student’s t-test. ^∗ indicates P < 0.005, ^∗∗ indicates P < 0.05.

Fig. 2

HeLa cells are impaired in their migration after treatment with siRNA against β-catenin or cyclin D1. Representative photographs of the cellular migration in the scratch assay after 6 and 13 h. Control cells were transfected with control siRNA.

Fig. 3

The actin cytoskeleton rearranges after knock-down of cyclin D1 or β-catenin. Immunofluorescences of siRNA treated and phalloidin/TRITC (red) stained HCT116 cells. Control cells were transfected with control siRNA. Nuclei are counterstained with DAPI (blue).

Fig. 4

Cyclin D1 influences polymerization of the actin cytoskeleton in colorectal cancer cells. Flow cytometry analysis of phalloidin/FITC stained and siRNA treated HCT116 and SW480 cells. Representative histograms for cyclin D1 (blue) and β-catenin siRNA (green) transfected cells are shown (A). As a control the cells were either treated with control siRNA (gray) or were left untreated and unstained (black). (B) Quantification of the relative mean fluorescence intensity (MFI).

Fig. 5

Formation of E-cadherin-mediated adherens junctions in HCT116 and SW480 cells after transfection with siRNA against cyclin D1 or β-catenin. Immunofluorescences of siRNA treated and E-cadherin/Cy3 (red) stained HCT cells and SW480 cells. As a control the cells were transfected with control siRNA. Nuclei are marked with DAPI (blue). White arrows show E-cadherin-mediated cellular contacts.