Crosstalk mechanisms in lung fibrosis: Investigation of the role of integrin αVβ6, Galectin-3 and EGFR in TGF-β activation

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disease with high morbidity. Transforming Growth Factor-β (TGF-β) and integrin αVβ6 are key drivers of IPF, inducing epithelial-to-mesenchymal transition (EMT), myofibroblast activation and pathological matrix remodelling. Integrin αVβ6 binds and applies mechanical force on latent TGF-β to induce activation and release of the potent cytokine. Galectin-3, EGFR, MUC-1 are highly expressed in IPF and regulate a range of pro-fibrotic functions. The aim of this study was to examine whether galectin-3, EGFR, MUC-1 and integrin αVβ6 are functionally integrated and to determine whether and how they regulate TGF-β activation to promote IPF progression. Following initial optimisation and selection of appropriate cellular systems, imaging and biochemical analyses demonstrated subcellular co-localisation and co-immunoprecipitation of galectin-3 with αVβ6 and EGFR in lung epithelial cells. Moreover, surface plasmon resonance (SPR) revealed galectin-3 binds directly to αVβ6, EGFR and MUC-1 in a carbohydrate-dependent manner. Exogenous galectin-3 promotes co-clustering of EGFR and integrin αVβ6 in caveolin-rich, vesicular-like structures and increases αVβ6 protein levels, most likely by modulating its degradation. Additionally, galectin-3 inhibition suppresses TGF-β-induced αVβ6 expression and αVβ6-mediated force application on latent TGF-β and fibronectin and reduces TGF-β activation. Similarly, EGFR inhibition by gefitinib reduces the αVβ6-mediated force dependent TGF-β activation. MUC-1 inhibition with GO-201, a peptide blocking the MUC-1 oligomerisation, suppressed the protein levels of αVβ6 and decreased co-localisation of Galectin-3 with integrin αVβ6. GO-201 treatment also reduced the cell surface levels of EGFR and αVβ6 in lung epithelial cells. These data indicate a functional relationship between EGFR, galectin-3, MUC-1 and αVβ6. Most importantly, we demonstrate a novel regulator of integrin αVβ6, galectin-3, which binds αVβ6 to control its trafficking and proteostasis. A feed-forward model is proposed, whereby galectin-3 promotes TGF-β-dependent αVβ6 expression, which further promotes αVβ6-mediated TGF-β activation, to nucleate and propagate fibrotic foci formation. The mechanism by which galectin-3 amplifies αVβ6-mediated TGF-β activation may ultimately serve as a novel therapeutic target or prognostic indicator in IPF.