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 4

SOX9 is a positive regulator of migration, transformation, and survival of IPF fibroblasts.

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SOX9 is a positive regulator of migration, transformation, and survival ...
(A) Quantification of migration of IPF fibroblasts treated with either control or SOX9 siRNA for 72 hours. (****P < 0.00005, **P < 0.005; n = 12; 2-way ANOVA.) (B) Quantification of DPYSL3 gene transcripts in the total transcripts of IPF compared with normal lungs (*P < 0.05; n = 4; Student’s 2-tailed t test). (C) Quantification of DPYSL3 gene transcripts in IPF fibroblasts treated with either control or SOX9 siRNA for 72 hours (***P < 0.0005; n = 4; Student’s 2-tailed t test). (D) Fibroblasts isolated from distal lung cultures of αSMACreERT RosamTmG mice were transduced with either control or SOX9-overexpressing lentivirus in the presence of 4-hydroxy-tamoxifen for 72 hours. Representative confocal images were obtained at 60× original magnification. Scale bar: 100 μm (n = 4/group). (E) The number of ACTA2-positive (GFP or green color) cells in the total cells (DAPI or blue color) was quantified using MetaMorph image analysis software and presented as the percentage of ACTA2-positive cells in total fibroblasts (**P < 0.005; n = 4; Student’s 2-tailed t test). (F) IPF fibroblasts were treated with either control or SOX9 siRNA for 72 hours and cultured with collagen gels to measure the changes in collagen gel contraction after 6 hours (****P < 0.00005; n = 6; Student’s 2-tailed t test). (G) IPF fibroblasts were treated with either control or SOX9 siRNA for 48 hours, followed by anti-Fas treatment for another 24 hours, and cells stained to quantify the total TUNEL-positive cells. Representative confocal images were obtained at 20× original magnification. Scale bar: 200 μm. (H) The numbers of TUNEL-positive (red color) cells and the total cells (DAPI or blue color) were quantified using Elements image analysis software and are presented as the percentage of TUNEL-positive cells in total cells. (**P < 0.005, *P < 0.05; n = 3/group; 1-way ANOVA). (I) Quantification of antiapoptotic gene transcripts, BCL-XL and BCL-2L2, in IPF fibroblasts treated with either control or SOX9-specific siRNA for 72 hours (***P < 0.0005, **P < 0.005; n = 4; Student’s 2-tailed t test).