Abstract
Introduction SOX9 (SRY-box transcription factor 9) controls extracellular matrix (ECM) deposition in chondrogenesis, and epithelial differentiation in alveologenesis. We hypothesised that SOX9 drives lung fibrosis progression by regulates ECM and alveolar epithelial injury and can provide downstream biomarkers for idiopathic pulmonary fibrosis (IPF).
Methods We used a Wild type (WT) time-course bleomycin model to study spatio-temporal expression of SOX9, where lungs were harvested at regular intervals (1–28 days) after injury. For functional studies we induced lung fibrosis using bleomycin in inducible global Sox9 Knockout (Sox9KO, Sox9fl/fl;ROSACreER+/−) and Control (Sox9fl/fl;ROSACreER-/−) mice. Fibrosis was assessed at 14 days, using immunohistochemistry (IHC), qPCR, Western blot and hydroxyproline assays (HPA). Bronchoalveolar lavage (BAL) was analysed by flow cytometry and proteomics. Healthy and IPF human tissue were analysed by IHC. Serum from healthy controls and IPF patients was analysed by luminex assay.
Results Using WT bleomycin time-course model we observed, using IHC, that SOX9 is upregulated in stroma during the „fibroproliferative and fibrotic period (days 7–28). SOX9 co-localised to SFTPC and recently discovered KRT17 expressing pro-fibrotic epithelium. Sox9KO reduced fibrosis severity (HPA and COL1 expression) and reduced KRT17 epithelial cell expression (IHC). Sox9 SiRNA treatment in vitro reduced MRC-5 fibroblast SOX9, α-SMA and COL1 expression. Western blot showed in vitro SOX9 expression was upregulated in TGF-β1 and PDGF-DD treated MRC-5 fibroblasts. In human IPF lung, SOX9 co-localised to SFTPC and KRT17 as well as α-SMA, PDGFR-β and COL1. Overall findings are consistent with SOX9 regulating a fibrotic phenotype in both fibrotic epithelium and fibroblasts.
K-means cluster analysis of mouse BAL proteomics identified known promising (SPP1, IGFBP-2) and novel (BM1, biomarker 1) fibrosis biomarkers. In human IPF serum (n=12) BM1, OPN and IGFBP-2 were upregulated (p<0.05) compared to controls (n=8). BM1 was significantly (p<0.05) lower in patients with stable rather than progressive IPF, indicated by lung function and radiology.
Conclusion SOX9 co-localises with, and regulates in vivo, critical components of the fibrotic niche: 1) myofibroblasts and ECM secretion; 2) recently discovered KRT17 epithelial cells that have a fibrotic gene signature. Identification of downstream known, and novel, promising predictive biomarkers highlight the translational importance of SOX9.
Methods We used a Wild type (WT) time-course bleomycin model to study spatio-temporal expression of SOX9, where lungs were harvested at regular intervals (1–28 days) after injury. For functional studies we induced lung fibrosis using bleomycin in inducible global Sox9 Knockout (Sox9KO, Sox9fl/fl;ROSACreER+/−) and Control (Sox9fl/fl;ROSACreER-/−) mice. Fibrosis was assessed at 14 days, using immunohistochemistry (IHC), qPCR, Western blot and hydroxyproline assays (HPA). Bronchoalveolar lavage (BAL) was analysed by flow cytometry and proteomics. Healthy and IPF human tissue were analysed by IHC. Serum from healthy controls and IPF patients was analysed by luminex assay.
Results Using WT bleomycin time-course model we observed, using IHC, that SOX9 is upregulated in stroma during the „fibroproliferative and fibrotic period (days 7–28). SOX9 co-localised to SFTPC and recently discovered KRT17 expressing pro-fibrotic epithelium. Sox9KO reduced fibrosis severity (HPA and COL1 expression) and reduced KRT17 epithelial cell expression (IHC). Sox9 SiRNA treatment in vitro reduced MRC-5 fibroblast SOX9, α-SMA and COL1 expression. Western blot showed in vitro SOX9 expression was upregulated in TGF-β1 and PDGF-DD treated MRC-5 fibroblasts. In human IPF lung, SOX9 co-localised to SFTPC and KRT17 as well as α-SMA, PDGFR-β and COL1. Overall findings are consistent with SOX9 regulating a fibrotic phenotype in both fibrotic epithelium and fibroblasts.
K-means cluster analysis of mouse BAL proteomics identified known promising (SPP1, IGFBP-2) and novel (BM1, biomarker 1) fibrosis biomarkers. In human IPF serum (n=12) BM1, OPN and IGFBP-2 were upregulated (p<0.05) compared to controls (n=8). BM1 was significantly (p<0.05) lower in patients with stable rather than progressive IPF, indicated by lung function and radiology.
Conclusion SOX9 co-localises with, and regulates in vivo, critical components of the fibrotic niche: 1) myofibroblasts and ECM secretion; 2) recently discovered KRT17 epithelial cells that have a fibrotic gene signature. Identification of downstream known, and novel, promising predictive biomarkers highlight the translational importance of SOX9.
| Original language | English |
|---|---|
| Pages (from-to) | A66-A67 |
| Number of pages | 2 |
| Journal | Thorax |
| Volume | 77 |
| DOIs | |
| Publication status | Published - Nov 2022 |