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.

Keywords: Extracellular matrix; Fibrosis; Pulmonology; Respiration.

Figure 1. Postnatal WT1-positive mesothelial cell contributions to myofibroblasts in pulmonary fibrosis.

(A) The dramatic decrease in the expression of WT1 in developing mouse lung at E14.5 and expression levels were normalized to HPRT mRNA (n = 3–5/gestational age). Data are presented as mean ± SEM. Statistical significance was calculated using 1-way ANOVA with Sidak’s multiple comparison for multiple comparisons. **P < 0.005, ***P < 0.0005. (B) Immunostaining shows the presence of WT1 protein in mesothelial cells (pleural surface) that coexpress calretinin (red) but not in myofibroblasts (green) of αSMA^YFP mice embryos at E15.5. Scale bar: 50 μm. (C) WT1 staining (white) is detected in a subset of mesothelial cells positive for calretinin (red) in WT mice embryos at E15.5. Scale bar: 50 μm. (D) Schematic diagram of treatments with tamoxifen and Dox. Control or TGFα/WT1^CreERT2/mTmG mice were induced with tamoxifen, and 1 week later, pulmonary fibrosis was induced by administering Dox in food for 4 or 6 weeks. (E) Immunofluorescence images show progressive accumulation of WT1-derived cells residing in subpleura but not adventitia in TGFα/WT1^CreERT2/mTmG mice compared with control WT1^CreERT2/mTmG mice on Dox for 4 or 6 weeks. Scale bar: 50 μm. (F) Immunostaining shows the presence of αSMA protein in WT1-positive mesothelium-derived mesenchymal cells of TGFα/WT1^CreERT2/mTmG mice on Dox for 4 weeks. Note the presence of 2 distinct mesenchymal cells in subpleural fibrotic lesions that are marked by αSMA protein in GFP-positive cells (WT1-positive mesothelial cells) and GFP-negative (WT1-positive mesenchymal cells) cells. Scale bar: 50 μm.

Figure 2. WT1-lineage cells are the major source of mesenchymal cells in subpleural fibrotic lesions in vivo.

(A) Schematic diagram of treatments with tamoxifen and Dox. For pretamoxifen treatments (Pre-Tam), control, or TGFα/WT1^CreERT2/mTmG mice were induced with tamoxifen, and 1 week later, pulmonary fibrosis was induced by administering Dox in food for 4 weeks. For Pre- and posttamoxifen treatments (Pre/Post-Tam), TGFα/WT1^CreERT2/mTmG mice were treated with tamoxifen before and after pulmonary fibrosis was induced by Dox in food for 4 weeks. (B) Representative FACS plots show increased accumulation of WT1-drived myofibroblasts in Pre-/Post-Tam–treated TGFα/WT1^CreERT2/mTmG mice compared with Pre-Tam–treated TGFα/WT1^CreERT2/mTmG mice or control mice on Dox for 4 weeks. (C) Quantification of GFP-positive myofibroblasts in lung cultures of tamoxifen-treated TGFα/WT1^CreERT2/mTmG mice or control mice on Dox for 4 weeks. (D) Immunofluorescence images show increased accumulation of WT1-derived mesenchymal cells residing in subpleura but not adventitia in tamoxifen-treated TGFα/WT1^CreERT2/mTmG mice compared with control mice on Dox for 4 weeks. Scale bar: 50 μm.

Figure 3. WT1 is a positive regulator of fibroblast-to-myofibroblast transformation.

(A) Human non-IPF fibroblasts were transduced with either control adenovirusor WT1 adenovirus for 24 hours. Transcripts of WT1 and αSMA were quantified using RT-PCR (n = 6). Results are cumulative, from 2 independent experiments with similar results. (B) Human non-IPF fibroblasts were transduced with either control adenovirusor WT1 adenovirus for 72 hours. Protein lysates were immunoblotted for αSMA and β-actin. αSMA quantification was performed by normalizing to the endogenous β-actin control. Results are representative of 2 independent experiments with similar results (n = 3). (C) Fibroblasts of nontransgenic mice on Dox for 4 weeks were transduced with either control lentivirus or WT1 lentivirus (10 MOI) for 24 hours. Transcripts of WT1 and αSMA were quantified using RT-PCR (n = 3). Results are representative of 3 independent experiments with similar results. (D) Fibroblasts of αSMA^CreERT2/mTmG mice were transduced with either control lentivirus or WT1 lentivirus for 72 hours in the presence of 4-hydroxy tamoxifen. Immunofluorescence images were obtained at an original magnification of ×20. Scale bar: 100 μm. The number of αSMA-positive (GFP-positive) myofibroblasts were quantified in images, and the data shown are cumulative of 2 independent experiments with similar results (n = 4). (E) Fibroblasts from IPF primary lung cultures were transiently transfected with either control or WT1-specific siRNA for 72 hours, and αSMA gene expression was analyzed by RT-PCR. Results are representative of 2 independent experiments with similar results (n = 4). (F) IMR-90 cells were transduced with WT1 adenovirus (100 MOI) for 72 hours. Cell lysates were prepared, and the ChIP assay was performed with anti-WT1 antibody or normal rabbit IgG as a negative control using αSMA gene promoter–specific PCR primers. Nonimmunoprecipitated DNA is represented as input DNA (product size, 140 bp). (G) Primary lung-resident fibroblasts were isolated from lung cultures of TGFα mice placed on Dox for 8 weeks. Cell lysates were prepared, and the ChIP assay was performed with anti-WT1 antibody or normal rabbit IgG as a negative control using αSMA gene promoter–specific PCR primers. Nonimmunoprecipitated DNA is represented as input DNA (product size, 104 bp). Data are representative of 2 independent experiments. Data are presented as mean ± SEM. Unpaired Student t test, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.

Figure 4. WT1 is a positive regulator of fibroproliferation.

Primary lung-resident fibroblasts were isolated from the lung cultures of IPF, non-IPF, WT, or TGFα transgenic mice by negative selection using ant-CD45 magnetic beads. (A) Human non-IPF fibroblasts were transduced with either control adeno or WT1 adenovirus for 72 hours. Proliferation was assessed by immunoblotting WT1 and proliferating cell nuclear antigen (PCNA). PCNA quantification was performed by normalizing to the endogenous β-actin control. Results are representative of 2 independent experiments with similar results (n = 3). (B) Human IPF fibroblasts were transfected with either control or WT1 siRNA for 72 hours, and proliferation was assessed by BrdU incorporation. Results are cumulative of 2 independent experiments with similar results (n = 4). (C) Fibroblasts of nontransgenic mice on Dox for 4 weeks were transduced with either control lenti or WT1 lentivirus for 72 hours. Proliferation was assessed by BrdU incorporation assay. Results are representative of 2 independent experiments with similar results (n = 6). (D) Fibroblasts of TGFα transgenic mice on Dox for 4 weeks were transiently transfected with either control or WT1 siRNA for 72 hours, and proliferation was assessed by BrdU incorporation. Results are cumulative of 2 independent experiments with similar results (n = 4). (E) WT1-driven proliferative genes including Grem1, Runx1, Wnt4, Stat3, Prrx1, Igf1, Ccnb1, and E2f8 were quantified in fibroblasts of TGFα transgenic mice on Dox for 4 weeks transfected with either control or WT1 siRNA for 48 hours. Results are representative of 3 independent experiments with similar results (n = 3). Data are presented as mean ± SEM. Unpaired Student t test *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.

Figure 5. The partial loss of WT1 attenuates pulmonary fibrosis in vivo.

CCSP/rtTA and TetO/TGFα transgenes were bred into WT1^CreERT2/– knock-in mice (WT1^+/–) to generate TGFα transgenic mice with WT or mutant WT1 allele, and all groups of mice were administered Dox for 4 weeks. In bleomycin model, WT1^+/+ and WT1^+/– mice were treated with bleomycin (6 units per kg body weight) in 50 μl of saline solution intradermally (i.d.) for 5 days per week for a total of 4 weeks. (A) Masson’s trichrome–stained lung sections of TGFα/WT1^+/+ and TGFα/WT1^+/– mice on Dox for 4 weeks (n = 4–5/group). Image magnification, ×5. Scale bar: 400 μm. (B) TGFα transcripts were measured in WT (WT1^+/+ and TGFα/WT1^+/+) and WT1 mutant (WT1^+/– and TGFα/WT1^+/) mice on Dox for 4 weeks (n = 4–5/group). (C) WT1 transcripts were measured in WT (WT1^+/+ and TGFα/WT1^+/+) and WT1 mutant (WT1^+/– and TGFα/WT1^+/–) mice on Dox for 4 weeks (n = 4–5/group). (D) The total lung hydroxyproline levels in WT (WT1^+/+ and TGFα/WT1^+/+) and WT1 mutant (WT1^+/– and TGFα/WT1^+/–) mice on Dox for 4 weeks (n = 4–5/group). (E) The lung compliance was measured using FlexiVent in WT (WT1^+/+ and TGFα/WT1^+/+) and WT1 mutant (WT1^+/– and TGFα/WT1^+/–) mice on Dox for 4 weeks (n = 4–5/group). (F) Masson’s trichrome–stained lung sections of WT1^+/+ and WT1^+/– mice treated with bleomycin for 4 weeks (n = 6/group). Image magnification, ×5. Scale bar: 400 μm. (G) WT1 transcripts were measured in the total RNA isolated from the lungs of WT1^+/+ and WT1^+/– mice treated with bleomycin for 4 weeks (n = 6/group). (H and I) Col1α and Col5α transcripts were measured in the total RNA isolated from the lungs of WT1^+/+ and WT1^+/– mice treated with bleomycin for 4 weeks (n = 6/group). (J) The lung compliance was measured using FlexiVent in WT1^+/+ and WT1^+/– mice treated with bleomycin for 4 weeks (n = 6/group). Results are representative of 2 independent experiments with similar results. Data are presented as mean ± SEM. Statistical significance was calculated using 1-way ANOVA with Sidak’s multiple comparison test or unpaired Student t test for comparison between 2 groups. *P < 0.05, **P < 0.005, ****P < 0.0005.

Figure 6. Mechanisms of WT1-driven fibroproliferation and myofibroblast transformation in vivo.

(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/WT1^CreERT2/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 αSMA^CreERT2/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.

Figure 7. Schematic representation of WT1-driven fibroblast activation in pulmonary fibrosis.

In the peripheral areas of the lung, WT1-positive mesothelial cells and mesenchymal cells undergo myofibroblast transformation and activate lung-resident fibroblasts via direct and indirect mechanisms in the pathogenesis of pulmonary fibrosis.