β-Catenin mediates TGF-β-induced WNT-5A expression in airway smooth muscle cells.

<p>(A) <i>De novo</i> protein synthesis is required for TGF-β-induced WNT-5A expression. Airway smooth muscle cells were either left unstimulated (vehicle basal) or stimulated with TGF-β (2 ng/ml) in the presence or absence of the protein synthesis inhibitor cycloheximide (5 µg/ml) for 24 hours. WNT-5A mRNA induction was evaluated by qRT-PCR. Data represent mean ± SEM of 4 independent experiments. **p<0.01, ***p<0.001 compared to vehicle basal, ## p<0.01 compared to TGF-β-stimulated cells; 2-tailed Student's <i>t</i> test for paired observations. (B-D) β-Catenin silencing reduces TGF-β-induced WNT-5A expression. Airway smooth muscle cells were transfected with β-catenin-specific siRNA or a non-targeting siRNA as control. Subsequently, cells were stimulated with TGF-β (2 ng/ml) for 24 hours (mRNA; B,C) or 48 hours (protein; D). (B,C) Expression of β-catenin mRNA (B) and WNT-5A mRNA (C) was determined by qRT-PCR and expressed relative to non-targeting siRNA transfected, untreated control. Data represent mean ± SEM of 5 independent experiments. *p<0.05, **p<0.01 compared to non-targeting siRNA-transfected, untreated control, # p<0.05, ### p<0.001 compared to non-targeting siRNA-transfected, TGF-β-stimulated cells; 2-tailed Student's <i>t</i> test for paired observations. (D) Western blot analysis was performed to analyze WNT-5A and β-catenin protein expression in whole cell extracts. Equal protein loading was verified by the analysis of GAPDH. (E) Forced increase in β-catenin abundance elevates WNT-5A protein level. Cells were transfected with S33Y-β-catenin mutant or a GFP expression vector as control. Subsequently, cells were either left untreated or stimulated with TGF-β (2 ng/ml) for 48 hours. Western blot analysis was performed to determine the abundance of WNT-5A and total β-catenin at protein level. GAPDH expression assessed as loading control. (F) Canonical WNT ligand stimulation increases WNT-5A gene expression. Cells were stimulated with L-cells-derived WNT-3A conditioned medium or control conditioned medium for 24 hours. Expression of WNT-5A mRNA was evaluated by qRT-PCR and expressed relative to control conditioned medium. Data represent mean ± SEM of 5 independent experiments. **p<0.01 compared to control conditioned medium; 2-tailed Student's <i>t</i> test for paired observations.