title: Amplification of TGFβ Induced ITGB6 Gene Transcription May Promote Pulmonary Fibrosis.
abstract: Idiopathic pulmonary fibrosis (IPF) is a devastating, progressive disease with poor survival rates and limited treatment options. Upregulation of αvβ6 integrins within the alveolar epithelial cells is a characteristic feature of IPF and correlates with poor patient survival. The pro-fibrotic cytokine TGFβ1 can upregulate αvβ6 integrin expression but the molecular mechanisms driving this effect have not previously been elucidated. We confirm that stimulation with exogenous TGFβ1 increases expression of the integrin β6 subunit gene (ITGB6) and αvβ6 integrin cell surface expression in a time- and concentration-dependent manner. TGFβ1-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 αvβ6 integrins on, lung epithelial cells occurs via homeostatic αvβ6-mediated TGFβ1 activation in the absence of exogenous stimulation, and can be amplified by TGFβ1 activation. Fundamentally, we show for the first time that TGFβ1-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 TGFβ1-induced ITGB6 transcriptional activity. Using chromatin immunoprecipitation we demonstrate that TGFβ1 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 TGFβ1 over-expression model of pulmonary fibrosis we demonstrate that Smad3 is crucial for TGFβ1-induced αvβ6 integrin expression within the alveolar epithelium in vivo. Together, these data confirm that a homeostatic, autocrine loop of αvβ6 integrin activated TGFβ1-induced ITGB6 gene expression regulates epithelial basal αvβ6 integrin expression, and demonstrates that this occurs via Smad-dependent transcriptional regulation at a single Smad binding site in the promoter of the β6 subunit gene. Active TGFβ1 amplifies this pathway both in vitro and in vivo, which may promote fibrosis.
authors: Division of Respiratory Medicine-City, University of Nottingham, Nottingham, United Kingdom.
authors: Amanda L
authors: AL
authors: Tatler
authors: Division of Respiratory Medicine-City, University of Nottingham, Nottingham, United Kingdom.
authors: Amanda T
authors: AT
authors: Goodwin
authors: Division of Respiratory Medicine-City, University of Nottingham, Nottingham, United Kingdom.
authors: Olumide
authors: O
authors: Gbolahan
authors: Division of Respiratory Medicine-City, University of Nottingham, Nottingham, United Kingdom.
authors: Gauri
authors: G
authors: Saini
authors: Division of Respiratory Medicine-City, University of Nottingham, Nottingham, United Kingdom.
authors: Joanne
authors: J
authors: Porte
authors: Division of Respiratory Medicine-City, University of Nottingham, Nottingham, United Kingdom.
authors: Alison E
authors: AE
authors: John
authors: Division of Respiratory Medicine-City, University of Nottingham, Nottingham, United Kingdom.
authors: Rachel L
authors: RL
authors: Clifford
authors: Biogen, Cambridge, Massachusetts, United States of America.
authors: Shelia M
authors: SM
authors: Violette
authors: Biogen, Cambridge, Massachusetts, United States of America.
authors: Paul H
authors: PH
authors: Weinreb
authors: Department of Pathology, University of Cambridge, Cambridge, United Kingdom.
authors: Helen
authors: H
authors: Parfrey
authors: School of Medicine, University of California San Francisco, San Francisco, California, United States of America.
authors: Paul J
authors: PJ
authors: Wolters
authors: Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Canada.
authors: Jack
authors: J
authors: Gauldie
authors: Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Canada.
authors: Martin
authors: M
authors: Kolb
authors: Division of Respiratory Medicine-City, University of Nottingham, Nottingham, United Kingdom.
authors: Gisli
authors: G
authors: Jenkins
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D054990:Idiopathic Pulmonary Fibrosis / Q000378:metabolism / Q000473:pathology*; D039641:Integrin beta Chains / Q000235:genetics / Q000378:metabolism*; D016023:Integrins / Q000235:genetics / Q000378:metabolism; D008168:Lung / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D051379:Mice; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D020871:RNA Stability / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism; D051901:Smad4 Protein / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000951:Antigens, Neoplasm; D039641:Integrin beta Chains; D016023:Integrins; D051900:Smad3 Protein; D051901:Smad4 Protein; D016212:Transforming Growth Factor beta; C073878:integrin alphavbeta6; C072170:integrin beta6