An MRTF-A–ZEB1–IRF9 axis contributes to fibroblast–myofibroblast transition and renal fibrosis

Figures

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  Fig. 1. Myofibroblast-specific MRTF-A deletion attenuates renal fibrosis in mice. MRTF-Af/f mice and MRTF-AMFKO mice were subjected to the UUO procedure. A Scheme of the experimental protocol. B Plasma creatinine levels. C Plasma BUN levels. D, E Pro-fibrogenic gene expression levels were examined by qPCR and Western blotting. F Picrosirius red and Masson’s trichrome staining. N = 4-8 mice for each group.

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  Fig. 2. RNA-seq reveals Zeb1 as a target of MRTF-A. A-F Primary murine renal fibroblasts were transduced with lentivirus carrying an MRTF-A expression vector (ΔN200) or empty vector (EV). RNA-seq was performed as described in the Methods. PCA plot (A). Volcano plot (B). GO analysis (C). KEGG analysis (D). GSEA (E). Heatmap of differentially expressed genes (F). G, H Primary murine renal fibroblasts were transduced with lentivirus carrying an MRTF-A expression vector (ΔN200) or empty vector (EV). Zeb1 expression was examined by qPCR and Western blotting. I, J Primary murine renal fibroblasts were isolated from MRTF-Af/f mice and transduced with adenovirus carrying Cre or GFP followed by treatment with TGF-β (2 ng/ml) for 24 h. Zeb1 expression was examined by qPCR and Western blot. K Primary murine renal fibroblasts were transduced with MRTF-A ΔN200 followed by transfection with the indicated siRNAs. Myofibroblast marker genes were examined by qPCR.

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  Fig. 3. MRTF-A interacts with TEAD1 to regulate Zeb1 transcription. A Truncated Zeb1 promoter–luciferase constructs were transfected into HEK293 cells with or without MRTF-A. Luciferase activities were normalized by protein concentration and GFP fluorescence. B Primary renal fibroblasts were treated with TGF-β (2 ng/ml) and harvested at the indicated time points. ChIP assays were performed with anti-MRTF-A or IgG. C C57/B6 mice were subjected to the UUO procedure or the sham procedure as described in the Methods. ChIP assays were performed with kidney tissues. D Wild-type and mutated Zeb1 promoter-luciferase constructs were transfected into HEK293 cells with or without MRTF-A. Luciferase activities were normalized by protein concentration and GFP fluorescence. E Primary renal fibroblasts were treated with TGF-β (2 ng/ml) for 24 h. Re-ChIP assays were performed with the indicated antibodies. F–H Primary renal fibroblasts were transfected with siRNA targeting TEAD1 or scrambled siRNA (SCR) followed by treatment with TGF-β (2 ng/ml) for 24 h. Zeb1 expression was examined by qPCR and Western blot. ChIP assays were performed with the indicated antibodies.

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  Fig. 4. Zeb1 regulates FMyTin vitro and renal fibrosis in vivo. A–D Primary murine renal fibroblasts were transfected with siRNA targeting Zeb1 or scrambled siRNA (SCR) followed by treatment with TGF-β (2 ng/ml) for 24 h. Myofibroblast marker gene expression was examined by qPCR (A). Cell proliferation and migration were examined by EdU incorporation (B) and Transwell assays (C), respectively. D Collagen contraction assay. E–I C57/B6 mice were injected via tail vein AAV2 carrying shRNA targeting Zeb1 (shZeb1) or control shRNA (shC) followed by the UUO procedure to induce renal fibrosis. The mice were sacrificed 2w after the surgery. (E) Scheme of the experimental protocol. F Plasma creatinine levels. G Plasma BUN levels. H Pro-fibrogenic gene expression levels were examined by qPCR. I Picrosirius red and Masson’s trichrome staining. N = 6-8 mice for each group.

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  Fig. 5. IRF9 is a novel target of Zeb1. A–E Primary murine renal fibroblasts were transfected with siRNA targeting Zeb1 or scrambled siRNA (SCR) followed by treatment with TGF-β (2 ng/ml) for 24 h. RNA-seq was performed as described in the Methods. PCA plot (A). Volcano plot (B). GO analysis (C). GSEA (D). Heatmap of differentially expressed genes (E). F Primary murine renal fibroblasts were transfected with siRNA targeting Zeb1 or scrambled siRNA (SCR) followed by treatment with TGF-β (2 ng/ml) for 24 h. IRF9 expression was examined by qPCR. G–J Primary murine renal fibroblasts were transfected with the indicated siRNAs followed by treatment with TGF-β (2 ng/ml) for 24 h. Myofibroblast marker gene expression was examined by qPCR (G). Cell proliferation and migration were examined by EdU incorporation (H) and Transwell assays (I), respectively. J Collagen contraction assay.

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  Fig. 6. IRF9 is a novel regulator of TGF-β-induced fibroblast–myofibroblast transition. A-D Primary murine renal fibroblasts were transduced with adenovirus carrying an IRF9 vector (Ad-IRF9) or an empty vector (Ad-EV) followed by treatment with TGF-β (2 ng/ml) for 24 h. Myofibroblast marker gene expression was examined by qPCR (A). Cell proliferation and migration were examined by EdU incorporation (B) and Transwell assays (C), respectively. (d) Collagen contraction assay. E–I Primary murine renal fibroblasts were transduced with adenovirus carrying an IRF9 vector (Ad-IRF9) or an empty vector (Ad-EV)followed by treatment with TGF-β (2 ng/ml) for 24 h. RNA-seq was performed as described in the Methods. PCA plot (E). Volcano plot (F). GO analysis (G). GSEA (H). Heatmap of differentially expressed genes (I). J A schematic model.