TGF-β-activated kinase 1 (TAK1) signaling regulates TGF-β-induced WNT-5A expression in airway smooth muscle cells via Sp1 and β-catenin

WNT-5A, a key player in embryonic development and post-natal homeostasis, has been associated with a myriad of pathological conditions including malignant, fibroproliferative and inflammatory disorders. Previously, we have identified WNT-5A as a transcriptional target of TGF-β in airway smooth muscle cells and demonstrated its function as a mediator of airway remodeling. Here, we investigated the molecular mechanisms underlying TGF-β-induced WNT-5A expression. We show that TGF-β-activated kinase 1 (TAK1) is a critical mediator of WNT-5A expression as its pharmacological inhibition or siRNA-mediated silencing reduced TGF-β induction of WNT-5A. Furthermore, we show that TAK1 engages p38 and c-Jun N-terminal kinase (JNK) signaling which redundantly participates in WNT-5A induction as only simultaneous, but not individual, inhibition of p38 and JNK suppressed TGF-β-induced WNT-5A expression. Remarkably, we demonstrate a central role of β-catenin in TGF-β-induced WNT-5A expression. Regulated by TAK1, β-catenin is required for WNT-5A induction as its silencing repressed WNT-5A expression whereas a constitutively active mutant augmented basal WNT-5A abundance. Furthermore, we identify Sp1 as the transcription factor for WNT-5A and demonstrate its interaction with β-catenin. We discover that Sp1 is recruited to the WNT-5A promoter in a TGF-β-induced and TAK1-regulated manner. Collectively, our findings describe a TAK1-dependent, β-catenin- and Sp1-mediated signaling cascade activated downstream of TGF-β which regulates WNT-5A induction

TAK1 regulates total and active fraction of β-catenin in airway smooth muscle cells.

<p>(A-C) TAK1 signaling in total β-catenin regulation. Airway smooth muscle cells were either left unstimulated (vehicle basal) or stimulated with TGF-β (2 ng/ml) in the presence or absence of LL-Z1640-2 (0.5 µM), SB203580 (10 µM), SP600125 (10 µM) or the combination of SB203580 and SP600125 (10 µM each) for 24 hours. Whole cell extracts were subjected to western analysis for detection of total β-catenin protein abundance. GAPDH expression was examined as loading control. Graphs represent quantitation of band intensities for total β-catenin corrected for GAPDH as percentage of TGF-β-induced expression. Data represent mean ± SEM of 4-6 independent experiments. *p<0.05, **p<0.01 compared to vehicle basal, # p<0.05, ## p<0.01 compared to TGF-β-stimulated cells; 2-tailed Student's <i>t</i> test for paired observations. (D, E) Regulation of active β-catenin by TAK1. Airway smooth muscle cells were either left unstimulated (vehicle basal) or stimulated with TGF-β (2 ng/ml) in the presence or absence of LL-Z1640-2 (0.5 µM) for 16 or 24 hours as indicated. Whole cells extracts were subjected to western analysis for detection of active β-catenin protein abundance. Expression of GAPDH was assessed as loading control. Graphs represent quantitation of band intensities for active β-catenin corrected for loading control as percentage of TGF-β-induced expression. Data represent mean ± SEM of 5 independent experiments. *p<0.05, **p<0.01 compared to vehicle basal, # p<0.05, ## p<0.01 compared to TGF-β-stimulated cells; 2-tailed Student's <i>t</i> test for paired observations.</p

TGF-β facilitates Sp1/β-catenin interaction.

<p>Airway smooth muscle cells were stimulated with TGF-β (2 ng/ml) for 16 hours. Co-immunoprecipitation was performed as described in the Materials and Methods section. Immunocomplexes and whole cell extracts (WCE) were subjected to western analysis as indicated in the panels.</p

TAK1-activated p38/JNK signaling regulates WNT-5A induction in airway smooth muscle cells.

<p>(A) TAK1 activates p38 and JNK. Airway smooth muscle cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of LL-Z1640-2 (0.5 µM) for 30 and 60 minutes. Whole cells extracts were immunoblotted for phospho-p38 and phospho-JNK using specific antibodies. Equal protein loading was verified by the analysis of β-actin. (B–D) p38 and JNK involvement in WNT-5A expression. Airway smooth muscle cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of SB203580 (10 µM) or SP600125 (10 µM) or combination of both SB203580 and SP600125 (10 µM each) for 24 hours. RNA was isolated and WNT-5A mRNA expression was determined by qRT-PCR, corrected for 18S rRNA and expressed relative to vehicle basal. Data represent mean ± SEM of 4–6 independent experiments. **p<0.01, ***p<0.001 compared to vehicle basal, ### p<0.001 compared to TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test.</p

Sp1 is the transcription factor for TGF-β-induced WNT-5A expression in airway smooth muscle cells.

<p>(A-B) Mithramycin A attenuates WNT-5A mRNA and protein expression. (A) Cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of Mithramycin A (300 nM) for 24 hours. WNT-5A mRNA was analyzed by qRT-PCR. Data represent mean ± SEM of 4 independent experiments. **p<0.01 compared to vehicle basal, ## p<0.01 compared to TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test. (B) Cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of Mithramycin A (300 nM) for 48 hours. Whole cell extracts were prepared and WNT-5A protein abundance was evaluated by western analysis. GAPDH was assessed as loading control. (C, D) Cells were transfected with Sp1-specific or a non-targeting siRNA as control. Subsequently, cells were stimulated with TGF-β (2 ng/ml) for 24 hours and analyzed for the expression of Sp1 mRNA (C) and WNT-5A mRNA (D) by qRT-PCR. Data represent mean ± SEM of 5 independent experiments. *p<0.05, ***p<0.001 compared to non-targeting siRNA-transfected untreated control, #p<0.05, ### p<0.001 compared to non-targeting siRNA-transfected, TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test. (E) Mithramycin A attenuates TGF-β-induced extracellular matrix expression. Cells were stimulated with TGF-β (2 ng/ml) in the presence or absence of Mithramycin A (300 nM) for 24 hours. Collagen IαI and fibronectin mRNA was analyzed by qRT-PCR. Data represent mean ± SEM of 4 independent experiments. *p<0.05, **p<0.01 compared to vehicle basal, #p<0.05, ## p<0.01 compared to TGF-β-stimulated cells; 1-way ANOVA followed by Newman-Keuls multiple comparisons test. (F) Sp1 is recruited to WNT-5A promoter in response to TGF-β. Cells were left untreated or stimulated with TGF-β (2 ng/ml) for 16 hours. Chromatin was prepared and ChIP analysis was performed as described in the Materials and Methods section. PCR was carried out using primers specific for Sp1 binding region on <i>WNT-5A</i> promoter A after immunoprecipitation with anti-Sp1 or control IgG antibody. Input DNA from chromatin preparation before immunoprecipitation was amplified to ascertain the loading. Resulting PCR products were analyzed by DNA PAGE. (G) TAK1 mediates recruitment of Sp1 to <i>WNT-5A</i> promoter in response to TGF-β. Cells were left untreated or stimulated with TGF-β (2 ng/ml) in the presence or absence of LL-Z1640-2 (0.5 µM) for 16 hours. ChIP analysis was performed as described above.</p

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.</p