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 6

Overexpression of WT1 augments prosurvival gene expression in mouse lung resident fibroblasts.

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Overexpression of WT1 augments prosurvival gene expression in mouse lung...
(A) Schematic workflow for B to C, illustrating the isolation of fibroblasts from WT1OE mouse lung cultures. Fibroblasts were infected with either control or Cre-expressing adenovirus for 72 hours to induce WT1 overexpression. (B) Fibroblasts were infected with either control adenovirus (AdControl) or Cre-expressing adenovirus (AdCre) for 72 hours, and cell lysates were immunoblotted with antibodies against Wt1, Bak, Bax, αSma, or Gapdh. (C) Schematic workflow for E to I, illustrating the isolation of fibroblasts from lung cultures derived from TGF-αOEWT1fl/fl mice on doxycycline-treated food for 8 weeks. Lung-resident fibroblasts were infected with either control or Cre-expressing adenovirus for 72 hours to delete WT1. (D) RT-PCR quantification of WT1 and ECM gene transcript expression, including Col1α1, Col1α2, Col3α, Fn1, and Acta2, in WT1-deficient fibroblasts compared with control fibroblasts (n = 3/group). *P < 0.05, **P < 0.01, and ****P < 0.0001, by multiple unpaired, 2-tailed Student’s t tests. (E and F) Lung-resident fibroblasts isolated from lung cultures of TGF-αOE WT1fl/fl mice on doxycycline-treated food for 8 weeks were infected with either control or Cre-expressing adenovirus for 72 hours, and cell lysates were immunoblotted with antibodies against Wt1, Bcl2, Bcl-XL, Bax, Col1α, Eln, Fn1, αSma, or Gapdh. (G) Fibroblasts were infected with either control or Cre-expressing adenovirus for 48 hours, followed by treatment with anti-Fas for an additional 24 hours. Cells were then stained to quantify TUNEL+ apoptotic cells (yellow arrowheads). Representative confocal images are shown. Original magnification, ×20. Scale bars: 100 μm. (H) Quantification of the percentage of TUNEL+ cells (red) in total cells stained with DAPI (blue) was performed using MetaMorph image analysis (n = 3/group). *P < 0.05 and **P < 0.01, by 1-way ANOVA.