LSC 2013 abstract - Mechanisms regulating enhanced αvβ6 expression in pulmonary fibrosis

αVβ6 integrins activate TGFβ, which plays a fundamental role in IPF pathogenesis. TGFβ increases αVβ6 and αVβ6 is upregulated in lungs of IPF patients. This suggests that dysregulation of a feedback loop may promote IPF. The aims were to investigate the mechanisms driving TGFβ-induced β6 (ITGB6) expression. We assessed β6 subunit expression by QPCR and flow cytometry. An ITGB6 promoter-reporter and truncated mutants were used to identify the regulatory region of the promoter. Transcription factor binding to the promoter was assessed by chromatin immunoprecipitation. TGFβ caused dose and time-dependent increases in αVβ6 and ITGB6, and increased promoter activity. A Smad site at -800bp was fundamental for TGFβ-induced expression. Dominant negative Smad3 inhibited ITGB6 promoter activity and αvβ6 expression. Smad3 -/- mice were protected from TGFβ-induced increases in αvβ6. Smad3 bound to the ITGB6 promoter in response to TGFβ. Smad3 binding was higher in IPF lung tissue compared with controls. Furthermore, we identified a region of the ITGB6 promoter responsible for gene repression. Glucocorticoid receptor (GR) or Elk-1 siRNA increased αvβ6 expression. Binding of the putative repressor Elk-1 to the promoter is reduced in IPF patients. To conclude, TGFβ increases αVβ6 expression by Smad3 binding to the ITGB6 promoter, which is increased in IPF. We identified Elk1 and GR as potential negative regulators of αvβ6. This highlights a feedback loop which may be dysregulated in IPF and suggests impaired repression of ITGB6 may be important.