A Force-mediated Conformational Change in Fibronectin's Integrin Binding Domain is Associated with Fibrotic Remodeling and Drives Fibroblast Activation
Moretti, Leandro, Biomedical Engineering - School of Engineering and Applied Science, University of Virginia
Barker, Tom, EN-Biomed Engr Dept, University of Virginia
Fibronectin (Fn) is a major component of the extracellular matrix during homeostatic conditions, wound healing, and is enriched in diseases like pulmonary fibrosis and cancer. Fn provides binding for growth factors and cells’ physical tethers, integrins. It has been theorized that small, cell-generated forces unfold Fn’s integrin binding domain (IBD). Fibroblasts, the effectors of fibrosis, express integrins αvβ3 and α5β1, that is susceptible to the IBD switching between the regular and unfolded state. Specifically, integrin α5β1 requires the physiological, folded (closed) conformation of Fn’s IBD, while αvβ3 can also bind the unfolded, open conformation. Since increased αvβ3 signaling has been associated with fibrosis, further understanding the downstream effects of this differential integrin binding could elucidate several unresolved diseases’ mechanisms. We hypothesized that this change in Fn conformation affects lung fibroblasts by guiding integrin enrichment, pushing them towards a secretory and pro-fibrotic phenotype.
What follows is the recollection of a multi-pronged attempt to first find the Integrin Switch (IntSw) in mouse models of lung fibrosis in vivo. I rediscovered an antibody that can target the Fn IBD, with increased specificity towards its unfolded, pathologically associated conformation where it outcompetes integrin αvβ3. The antibody was labelled with VivoTag dyes and used to first monitor lung fibrosis development in live mice that received intratracheal bleomycin after 2 weeks. I also developed a novel triple-transgenic mouse line in order to test whether affecting the IntSw by conditionally knocking out the other integrin involved, α5β1, aggravates lung fibrosis.
In my single cell force spectroscopy experiments I observed that α5β1 engagement was needed to generate high adhesion forces even at short time points (<120s), in accordance with previous literature. Luminex analysis highlighted increased activity in Rac, Akt pathways over Ras signaling for fibroblasts seeded on the open IBD conformation fragment. My mass spectroscopy work indicated that pro-fibrotic pathways and, particularly, collagen XIIa, Integrin-Linked Kinase, Rap1B, and tubulin beta3 appeared to be enriched upon increased αvβ3 binding at 30’.
After culturing for 24h on soft substrates (5 kPa representing healthy lung tissue), lung fibroblasts plated on the open IBD Fn fragment predominantly engaged αvβ3 and displayed increased nuclear translocation of transcription factors associated with a secretory and contractile phenotype. More than 9000 genes with significantly different expression were discovered by bulk RNAseq assays with fibroblasts seeded the on the open Fn fragment, compared to the closed fragment.
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. This work contributes to understanding the link between changes in the ECM and cell behavior in the context of fibroproliferative diseases.
PHD (Doctor of Philosophy)
fibrosis, integrin, fibroblast, fibronectin