Differential Engagement Of The Integrin Binding Domain Of Fibronectin Impacts Fibroblast Behavior

Abstract

The switch between α5β1 and αvβ3 integrin engagement due to unfolding of the integrin binding domain (IBD) of fibronectin (Fn) has been theorized for a long time but was observed in vitro and murine models only recently. Fn is largely present in the extra cellular matrix (ECM) during wound healing and fibroproliferative diseases like idiopathic pulmonary fibrosis or cancer. Thus, further understanding the downstream effects of this differential integrin binding could elucidate several diseases’ mechanisms. Specifically, integrin α5β1 requires the regular, closed conformation of Fn's IBD, while αvβ3 can also bind the unfolded, open conformation. Moreover, cells can generate forces sufficient to cause such unfolding. We hypothesized that this change in Fn conformation affects lung fibroblasts by guiding integrin enrichment, pushing them towards a secretory and disease-relevant phenotype. We tested this premise by engineering Fn fragments containing the IBD site that mimicked either the open or closed conformation. Additionally, these fragments were designed to be covalently bound via thiol chemistry to a variety of substrates in order to preserve their structural integrity. Glass dishes coated with either Fn fragment were used as substrates for Single Cell Force Spectroscopy. By using fibroblasts expressing only α5β1, only αvβ3, and naïve murine lung fibroblasts we observed the need of proper α5β1 engagement to generate high adhesion forces even at short time points (<120s). Plating naïve fibroblasts on similarly functionalized dishes for up to 30 minutes enabled Mass Spec analysis of the adhesome and of the phosphorylation pathways emerging from the differential integrin engagement. Particularly, collagen XIIa and tubulin beta3 appeared to be enriched upon increased αvβ3 binding. Even on soft substrates (4kPa representing healthy lung tissue), lung fibroblasts plated on the open IBD Fn fragment engaged αvβ3 and display increased nuclear translocation of transcription factors associated with a secretory and contractile phenotype, after a 24h incubation. Fn (with Extra Domain A), Tropomyosin, Vimentin, Serpine1, and α integrins were among the genes upregulated on the open Fn fragment, compared to the closed fragment. These results were confirmed by qPCR and then bulk RNAseq assays. In conclusion, these data support that differential integrin engagement due to Fn IBD unfolding appears to affect cell phenotype in both the short and long run. Current efforts are focused on evaluating the efficacy of a novel αvβ3 inhibiting antibody on the progression of murine lung fibrosis. This work contributes to defining the link between changes in the ECM and cell behavior in the context of fibroproliferative diseases.