Dysregulated overexpression of Sox9 induces fibroblast activation in pulmonary fibrosis
Prathibha R. Gajjala, … , Anil G. Jegga, Satish K. Madala
Prathibha R. Gajjala, … , Anil G. Jegga, Satish K. Madala
Published September 14, 2021
Citation Information: JCI Insight. 2021;6(20):e152503. https://doi.org/10.1172/jci.insight.152503.
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Research Article Pulmonology

Dysregulated overexpression of Sox9 induces fibroblast activation in pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a fatal fibrotic lung disease associated with unremitting fibroblast activation including fibroblast-to-myofibroblast transformation (FMT), migration, resistance to apoptotic clearance, and excessive deposition of extracellular matrix (ECM) proteins in the distal lung parenchyma. Aberrant activation of lung-developmental pathways is associated with severe fibrotic lung disease; however, the mechanisms through which these pathways activate fibroblasts in IPF remain unclear. Sry-box transcription factor 9 (Sox9) is a member of the high-mobility group box family of DNA-binding transcription factors that are selectively expressed by epithelial cell progenitors to modulate branching morphogenesis during lung development. We demonstrate that Sox9 is upregulated via MAPK/PI3K-dependent signaling and by the transcription factor Wilms’ tumor 1 in distal lung-resident fibroblasts in IPF. Mechanistically, using fibroblast activation assays, we demonstrate that Sox9 functions as a positive regulator of FMT, migration, survival, and ECM production. Importantly, our in vivo studies demonstrate that fibroblast-specific deletion of Sox9 is sufficient to attenuate collagen deposition and improve lung function during TGF-α–induced pulmonary fibrosis. Using a mouse model of bleomycin-induced pulmonary fibrosis, we show that myofibroblast-specific Sox9 overexpression augments fibroblast activation and pulmonary fibrosis. Thus, Sox9 functions as a profibrotic transcription factor in activating fibroblasts, illustrating the potential utility of targeting Sox9 in IPF treatment.

Authors

Prathibha R. Gajjala, Rajesh K. Kasam, Divyalakshmi Soundararajan, Debora Sinner, Steven K. Huang, Anil G. Jegga, Satish K. Madala

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Figure 2

The TGF-α/WT1 axis induces SOX9 in the distal lung fibroblasts.

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The TGF-α/WT1 axis induces SOX9 in the distal lung fibroblasts. (A) SOX9...
(A) SOX9 transcripts were measured by RT-PCR in normal lung fibroblasts treated with media or BMP2 (200 ng/mL), CTGF (50 ng/mL), TGF-α (100 ng/mL), and IGF1 (100 ng/mL) for 16 hours. (****P < 0.00005, *P < 0.05; n = 4–8/group; 1-way ANOVA.) (B) Quantification of SOX9 transcripts by RT-PCR in normal and IPF lung fibroblasts treated with media or TGF-α (100 ng/mL) for 16 hours. (****P < 0.00005, ***P < 0.0005; n = 4; 1-way ANOVA.) (C) Quantification of SOX9 transcripts by RT-PCR in IPF fibroblasts treated with vehicle, MEK inhibitor (ARRY, 0.1 μM), or PI3K inhibitor (PX-866, 0.1 μM) in the presence and absence of TGF-α (100 ng/mL) for 16 hours. (****P < 0.00005, *P < 0.05; n = 4; 1-way ANOVA.) (D) IPF fibroblasts were treated with vehicle or MEK inhibitor (ARRY, 1 μM) in the presence and absence of TGF-α (100 ng/mL) for 48 hours and immunoblotted with antibodies against SOX9 and GAPDH. SOX9 protein levels were normalized to GAPDH and are shown as fold induced change using a bar graph. (**P < 0.005; n = 3; Student’s 2-tailed t test.) (E) IPF lung fibroblasts were transiently transfected with control or WT1 siRNA for 72 hours, and SOX9 transcripts were quantified using RT-PCR. (**P < 0.005; n = 4; Student’s 2-tailed t test.) (F) Normal lung fibroblasts were transduced with control or WT1-adenoviral particles for 72 hours, and cell lysates were immunoblotted with antibodies against SOX9, WT1, and GAPDH. (G) Schemata show a human SOX9 promoter (Sox9WT1) clone or the WT1 binding site with the SOX9 promoter clone deleted (Sox9ΔWT1). (H) Luciferase activity was measured in HEK293 cells that were transiently transfected with empty, SOX9WT1, or SOX9ΔWT1 promoter plasmids and cells treated with media or TGF-α (100 ng/mL) for 16 hours (****P < 0.00005, ***P < 0.0005; n = 6; 1-way ANOVA). (I) Luciferase activity was measured in HEK293 cells cotransfected with WT1 overexpression plasmid and SOX9WT1 or SOX9ΔWT1 promoter-luciferase plasmids for 48 hours and then treated with either media or TGF-α (100 ng/mL) for another 16 hours. (****P < 0.00005; n = 6, 1-way ANOVA).