WNT/β-Catenin Signaling Promotes TGF-β-Mediated Activation of Human Cardiac Fibroblasts by Enhancing IL-11 Production

Figures

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  Figure 1. Activation of canonical and non-canonical WNT pathways in human cardiac fibroblasts. Human cardiac fibroblasts were stimulated with WNT3a, WNT5a or TGF-β. Panel (A) illustrates cellular localizations of β-catenin in fibroblasts 30 min after indicated treatment. DAPI stains cell nuclei. Arrows indicate nuclear localization of β-catenin. Data are representative of three independent experiments. Bar = 50 µm. Panel (B) represents expression of canonical (AXIN2 and TCF7) and non-canonical (JUN and ATF2) WNT responsive genes 1 h after indicated treatment. Panel (C) shows quantification of AP-1 activity in AP-1-hCF reporter cardiac fibroblasts following 24 h treatment (n = 6). Graphs show means ± SEM, p values computed using one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.05 (post-hoc test vs. control).

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  Figure 2. RNAseq analysis of TGF-β-activated fibroblasts following WNT3a or WNT5a treatment. Cardiac fibroblasts were stimulated with WNT3a or WNT5a in the presence of TGF-β for 72 h. Panel (A) shows a heat map of fibrosis-related genes differentially expressed (fold change > 2 and p < 0.05) in fibroblasts treated with WNT3a/TGF-β versus TGF-β alone (n = 3). All dysregulated genes of WNT3a/TGF-β versus TGF-β are presented in the Supplementary Figure S1. Panel (B) represents a heat map of all differentially expressed genes (fold change > 2 and p < 0.05) in cardiac fibroblasts treated with WNT5a/TGF-β versus TGF-β alone (n = 3). GO categories: Cell differentiation (GO:0030154), Regulation of cellular component organization (GO:0051128), Extracellular matrix (GO:0031012), WNT signaling pathway (GO:0016055) and Muscle Contraction (GO:0006936).

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  Figure 3. Panel (A) illustrates representative immunocytochemistry staining of αSMA, F-actin and cell nuclei (DAPI) of cardiac fibroblasts stimulated with WNT3a and/or TGF-β for 72 h. Data are representative of three biological independent experiments. Bar = 100 µm. Panel (B) shows quantification of gel contraction (72 h, n = 3) and representative image of collagen gel containing cardiac fibroblasts pre-treated as indicated. Panels (C–E) describe the profibrotic effect of GSK3β inhibitor CHIR99021 (CHIR) in the presence or absence of TGF-β in cardiac fibroblasts. Expression of profibrotic genes after 72 h of stimulation is presented in panel (C) (n = 4). Panel (D) shows representative immunoblots and the respective quantifications of αSMA and fibronectin after 72 h of stimulation (all n = 4) and panel (E) indicates pro-collagen I α1 level in supernatants (n = 6) stimulated for 5 days. Bars and whiskers represent means ±SEM. Main effect of WNT3a/CHIR, TGF-β and their interaction were tested using two-way ANOVA followed by Tukey’s multiple comparisons test, when the p-value for interaction was ≤ 0.1; * p < 0.05 (post-hoc TGF-β vs. TGF-β+WNT3a or CHIR vs. TGFβ+CHIR).

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  Figure 4. WNT5a reverses profibrotic response of TGF-β-activated cardiac fibroblasts. Panel (A) shows expression of profibrotic genes (n = 8) and panel (B) illustrates representative immunoblots with the respective quantifications of αSMA (n = 4) and fibronectin (n = 4) in cardiac fibroblasts stimulated with WNT5a and/or TGF-β for 72 h. Panel (C) shows pro-collagen I α1 levels measured in supernatants of cardiac fibroblasts treated as indicated for 5 days (n = 5). Bars and whiskers represent means ±SEM. Main effect of WNT5a, TGF-β and their interaction were tested using two-way ANOVA followed by Tukey’s multiple comparisons test, when the p-value for interaction was < 0.05, * p < 0.05 (post-hoc TGF-β vs. TGF-β+WNT5a).

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  Figure 5. WNT3a enhances TAK1 activation and IL-11 production in TGF-β-activated fibroblasts. Representative immunoblots and the respective quantification of p-SMAD2/SMAD2 (A) (n = 4) and p-TAK1/TAK1 (B) (n = 4) in cardiac fibroblasts stimulated with Wnt3a and/or TGF-β for 1 h. Panel (C) indicates IL11 gene expression (24 h n = 6) and panel (D) secreted IL-11 protein level in supernatants (72 h n = 5) of cardiac fibroblast stimulated as indicated. Panel (E) shows secreted IL-11 proteins in supernatants (n = 4) of cardiac fibroblast stimulated with or without TGF-β for 24 h in the presence or absence of the WNT secretion blocker WNT-C59. Bars and whiskers represent means ±SEM. Main effect of WNT3a or WNT-C59, TGF-β and their interaction were tested using two-way ANOVA followed by Tukey’s multiple comparisons test, when the p-value for interaction was ≤ 0.1; * p < 0.05 (post-hoc TGF-β vs. TGF-β+WNT3a or TGF-β vs. TGF-β+WNT-C59).

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  Figure 6. IL-11 contributes to profibrotic response enhanced by WNT3a. Human cardiac fibroblasts were cultured with TGF-β and stimulated with WNT3a in the presence or absence of an anti-IL-11 neutralizing antibody for 72 h. Panel (A) presents mRNA levels of profibrotic genes after indicated treatment (n = 10). Panel (B) shows quantification of αSMA and fibronectin (n = 4) and the representative immunoblots. Pro-collagen I α1 levels measured in supernatants (n = 6) are presented in panel (C). Panel (D) shows quantification of gel contraction (n = 3) containing cardiac fibroblasts pre-treated as indicated. Bars and whiskers represent means ±SEM. The main effect of WNT3a-TGF-β, the anti-IL11 antibody and their interaction were tested using two-way ANOVA followed by Tukey’s multiple comparisons test, when the p-value for interaction was ≤ 0.1; * p < 0.05 (post-hoc TGF-β+WNT3a vs. TGF-β+WNT3a+anti- IL11).