Wilms tumor 1 impairs apoptotic clearance of fibroblasts in distal fibrotic lung lesions
Harshavardhana H. Ediga, Chanukya P. Vemulapalli, Vishwaraj Sontake, Pradeep K. Patel, Hikaru Miyazaki, Dimitry Popov, Martin B. Jensen, Anil G. Jegga, Steven K. Huang, Christoph Englert, Andreas Schedl, Nishant Gupta, Francis X. McCormack, Satish K. Madala
Harshavardhana H. Ediga, Chanukya P. Vemulapalli, Vishwaraj Sontake, Pradeep K. Patel, Hikaru Miyazaki, Dimitry Popov, Martin B. Jensen, Anil G. Jegga, Steven K. Huang, Christoph Englert, Andreas Schedl, Nishant Gupta, Francis X. McCormack, Satish K. Madala
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Research Article Inflammation Pulmonology

Wilms tumor 1 impairs apoptotic clearance of fibroblasts in distal fibrotic lung lesions

Abstract

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease characterized by impaired fibroblast clearance and excessive extracellular matrix (ECM) protein production. Wilms tumor 1 (WT1), a transcription factor, is selectively upregulated in IPF fibroblasts. However, the mechanisms by which WT1 contributes to fibroblast accumulation and ECM production remain unknown. Here, we investigated the heterogeneity of WT1-expressing mesenchymal cells using single-nucleus RNA-Seq of distal lung tissues from patients with IPF and control donors. WT1 was selectively upregulated in a subset of IPF fibroblasts that coexpressed several prosurvival and ECM genes. The results of both loss-of-function and gain-of-function studies were consistent with a role for WT1 as a positive regulator of prosurvival genes to impair apoptotic clearance and promote ECM production. Fibroblast-specific overexpression of WT1 augmented fibroproliferation, myofibroblast accumulation, and ECM production during bleomycin-induced pulmonary fibrosis in young and aged mice. Together, these findings suggest that targeting WT1 is a promising strategy for attenuating fibroblast expansion and ECM production during fibrogenesis.

Authors

Harshavardhana H. Ediga, Chanukya P. Vemulapalli, Vishwaraj Sontake, Pradeep K. Patel, Hikaru Miyazaki, Dimitry Popov, Martin B. Jensen, Anil G. Jegga, Steven K. Huang, Christoph Englert, Andreas Schedl, Nishant Gupta, Francis X. McCormack, Satish K. Madala

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Figure 3

WT1 is upregulated in IPF distal lung mesenchymal cells.

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WT1 is upregulated in IPF distal lung mesenchymal cells. (A) Immunostain...
(A) Immunostaining was performed with the anti-WT1 antibody on lung sections from control and individuals with IPF. Representative images of pleural and parenchymal regions were obtained at low (original magnification, ×20; scale bar: 200 μm) and high (original magnification, ×40; scale bar: 100 μm) magnification. Arrowheads highlight WT1 staining on pleural mesothelial cells and spindle-shaped fibroblasts in the distal fibrotic lesions of lung parenchyma. (B) Representative fluorescence confocal RNA-ISH images showing WT1 (red) and ACTA2 (green) expression in control and IPF lung tissues. White arrowheads illustrate the double-positive cells. Scale bars: 20 μm. (C) Quantification of WT1+ cells normalized to total lung cells in lung section images from IPF and control lungs (n = 4/group). **P < 0.01, by 2-tailed Student’s t test. (D) Quantification of mesenchymal cells double-positive for WT1 and ACTA2 among total lung cells in lung section images from IPF and control lungs (n = 4/group). ***P < 0.001, by 2-tailed Student’s t test. (E) Primary lung-resident fibroblasts isolated from lung cultures of control and IPF lungs were immunoblotted with antibodies against WT1 and GAPDH. (F) WT1 protein levels were normalized to GAPDH and are shown as fold change (n = 3/group). *P < 0.05, by 2-tailed Student’s t test. (G) WT1 transcripts were measured by RT-PCR in normal lung fibroblasts treated with media, TGF-α (50 ng/mL), or TGF-β1 (20 ng/mL) for 16 hours (n = 4/group). **P < 0.01, by 1-way ANOVA.