Amplification of TGFβ Induced ITGB6 Gene Transcription May Promote Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a devastating, progressive disease with poor survival rates and limited treatment options. Upregulation of alpha v beta 6 integrins within the alveolar epithelial cells is a characteristic feature of IPF and correlates with poor patient survival. The profibrotic cytokine TGF beta 1 can upregulate alpha v beta 6 integrin expression but the molecular mechanisms driving this effect have not previously been elucidated. We confirm that stimulation with exogenous TGF beta 1 increases expression of the integrin beta 6 subunit gene (ITGB6) and alpha v beta 6 integrin cell surface expression in a time-and concentration-dependent manner. TGF beta 1-induced ITGB6 expression occurs via transcriptional activation of the ITGB6 gene, but does not result from effects on ITGB6 mRNA stability. Basal expression of ITGB6 in, and alpha v beta 6 integrins on, lung epithelial cells occurs via homeostatic alpha v beta 6-mediated TGF beta 1 activation in the absence of exogenous stimulation, and can be amplified by TGF beta 1 activation. Fundamentally, we show for the first time that TGF beta 1-induced ITGB6 expression occurs via canonical Smad signalling since dominant negative constructs directed against Smad3 and 4 inhibit ITGB6 transcriptional activity. Furthermore, disruption of a Smad binding site at -798 in the ITGB6 promoter abolishes TGF beta 1-induced ITGB6 transcriptional activity. Using chromatin immunoprecipitation we demonstrate that TGF beta 1 stimulation of lung epithelial cells results in direct binding of Smad3, and Smad4, to the ITGB6 gene promoter within this region. Finally, using an adenoviral TGF beta 1 over-expression model of pulmonary fibrosis we demonstrate that Smad3 is crucial for TGF beta 1-induced alpha v beta 6 integrin expression within the alveolar epithelium in vivo. Together, these data confirm that a homeostatic, autocrine loop of alpha v beta 6 integrin activated TGF beta 1-induced ITGB6 gene expression regulates epithelial basal alpha v beta 6 integrin expression, and demonstrates that this occurs via Smad-dependent transcriptional regulation at a single Smad binding site in the promoter of the beta 6 subunit gene. Active TGF beta 1 amplifies this pathway both in vitro and in vivo, which may promote fibrosis.