Wilms’ tumor 1 drives fibroproliferation and myofibroblast transformation in severe fibrotic lung disease
Vishwaraj Sontake, Rajesh K. Kasam, Debora Sinner, Thomas R. Korfhagen, Geereddy B. Reddy, Eric S. White, Anil G. Jegga, Satish K. Madala
Vishwaraj Sontake, Rajesh K. Kasam, Debora Sinner, Thomas R. Korfhagen, Geereddy B. Reddy, Eric S. White, Anil G. Jegga, Satish K. Madala
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Research Article Pulmonology

Wilms’ tumor 1 drives fibroproliferation and myofibroblast transformation in severe fibrotic lung disease

Abstract

Wilms’ tumor 1 (WT1) is a critical transcriptional regulator of mesothelial cells during lung development but is downregulated in postnatal stages and adult lungs. We recently showed that WT1 is upregulated in both mesothelial cells and mesenchymal cells in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a fatal fibrotic lung disease. Although WT1-positive cell accumulation leading to severe fibrotic lung disease has been studied, the role of WT1 in fibroblast activation and pulmonary fibrosis remains elusive. Here, we show that WT1 functions as a positive regulator of fibroblast activation, including fibroproliferation, myofibroblast transformation, and extracellular matrix (ECM) production. Chromatin immunoprecipitation experiments indicate that WT1 binds directly to the promoter DNA sequence of α-smooth muscle actin (αSMA) to induce myofibroblast transformation. In support, the genetic lineage tracing identifies WT1 as a key driver of mesothelial-to-myofibroblast and fibroblast-to-myofibroblast transformation. Importantly, the partial loss of WT1 was sufficient to attenuate myofibroblast accumulation and pulmonary fibrosis in vivo. Further, our coculture studies show that WT1 upregulation leads to non–cell autonomous effects on neighboring cells. Thus, our data uncovered a pathogenic role of WT1 in IPF by promoting fibroblast activation in the peripheral areas of the lung and as a target for therapeutic intervention.

Authors

Vishwaraj Sontake, Rajesh K. Kasam, Debora Sinner, Thomas R. Korfhagen, Geereddy B. Reddy, Eric S. White, Anil G. Jegga, Satish K. Madala

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Figure 6

Mechanisms of WT1-driven fibroproliferation and myofibroblast transformation in vivo.

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Mechanisms of WT1-driven fibroproliferation and myofibroblast transforma...
(A) Immunostainings show Ki67-positive cells residing in subpleura and adventitia were reduced in TGFα/WT1+/– mice compared with TGFα/WT1+/+ mice on Dox for 4 weeks. Scale bar: 50 μm. (B) Immunostainings show αSMA-positive cells residing in subpleura and adventitia were reduced in TGFα/WT1+/– mice compared with TGFα/WT1+/+ mice on Dox for 4 weeks. Scale bar: 50 μm. (C) Immunostainings show αSMA-positive cells residing in subpleura and adventitia were reduced in WT1+/– mice compared with WT1+/+ mice treated with bleomycin for 4 weeks. Scale bar: 200 μm. (D) Primary lung resident fibroblasts were isolated TGFα/WT1CreERT2/mTmG mice on Dox for 4 weeks and treated with either control or WT1 siRNA in presence of 4-hydroxy tamoxifen for 72 hours. Cells were immunostained for PCNA and immunofluorescence images were collected at original magnification ×10 (Scale bar: 50 μm). (E-G) The number of PCNA-positive cells that coexpresses WT1 (green) in total DAPI-positive cells were quantified using Metamorph image analysis software and were indicated as PCNA+/total cells, WT1+/PCNA+ cells/total cells, or WT1–/PCNA+/total cells. (H) Fibroblast cocultures experiments were performed using lung-resident fibroblasts isolated from control mice (bottom chamber) or TGFα transgenic mice on Dox for 6 weeks (top chambers). Cells in the bottom chambers were immunostained for PCNA. Images were collected at original magnification ×10. Total DAPI-positive and PCNA-positive cells were quantified using ND2 analysis software. (I) Control mice fibroblasts were cultured for 48 hours in the presence of conditioned media obtained from control or WT1 siRNA transfected fibrotic fibroblasts for 72 hours. After 48 hours, cells were immunostained for PCNA. Images were collected at original magnification ×10. Total DAPI-positive and PCNA-positive cells were quantified using ND2 analysis software. (J) Fibroblast cocultures experiments were performed using lung-resident fibroblasts isolated from the lung cultures of control αSMACreERT2/mTmG mice (bottom chamber) or TGFα transgenic mice on Dox for 6 weeks (top chambers). RNA was isolated from the cell lysates of bottom chamber, and quantification of αSMA gene expression was performed. All data are representative of 2 independent experiments with similar results (n = 3–4/group). All data in the figure are presented as mean ± SEM. Statistical significance was calculated using unpaired 2-tailed Student’s t test for comparison between 2 groups. *P < 0.05, **P < 0.005, ***P < 0.0005.