The ED-A domain enhances the capacity of fibronectin to store latent TGF-β binding protein-1 in the fibroblast matrix

Abstract

Dysregulated secretion and extracellular activation of TGF-β1 stimulates myofibroblasts to accumulate disordered and stiff extracellular matrix (ECM) in fibrosis. Fibronectin immobilizes latent TGF-β binding protein-1 (LTBP-1) and thus stores TGF-β1 in the ECM. Because the ED-A fibronectin splice variant is prominently expressed in fibrosis and supports myofibroblast activation, we investigated whether ED-A promotes LTBP-1-fibronectin interactions. Using stiffness-tuneable substrates for human dermal fibroblast cultures, we showed that high ECM stiffness promotes expression and co-localization of LTBP-1 and ED-A fibronectin. When rescuing fibronectin-depleted fibroblasts with specific fibronectin splice variants, LTBP-1 bound more efficiently to ED-A fibronectin than to ED-B fibronectin and fibronectin lacking splice domains. Function blocking of the ED-A domain using antibodies and competitive peptides resulted in reduced LTBP-1 binding to ED-A fibronectin, reduced LTBP-1 incorporation into the fibroblast ECM, and reduced TGF-β1 activation. Similar results were obtained by blocking the heparin-binding stretch FNIII12-13-14 (HepII), adjacent to the ED-A domain in fibronectin. Collectively, our results suggest that the ED-A domain enhances association of the latent TGF-β1 by promoting weak direct binding to LTBP-1 and by enhancing heparin-mediated protein interactions through HepII in fibronectin.