The Fibronectin Domain ED-A Is Crucial for Myofibroblastic Phenotype Induction by Transforming Growth Factor-β1

TLDR

TGFβ1 promotes myofibroblast differentiation by inducing α‑smooth muscle actin, modulating adhesive receptors, and upregulating extracellular matrix components such as the wound‑healing isoform ED‑A fibronectin. The authors show that blocking ED‑A fibronectin with an anti‑ED‑A antibody or soluble ED‑A domain inhibits TGFβ1‑induced α‑smooth muscle actin and collagen I expression, indicating that ED‑A FN is required for TGFβ1 signaling to the myofibroblast phenotype. The study demonstrates that ED‑A fibronectin deposition precedes and correlates with α‑smooth muscle actin expression, and that blocking ED‑A (with antibody or soluble domain) prevents TGFβ1‑induced α‑smooth muscle actin and collagen I while leaving FN assembly intact, proving that polymerized ED‑A FN is essential for TGFβ1‑driven myofibroblast differentiation via an ECM‑mediated outside‑in signaling mechanism.

Abstract

Transforming growth factor-beta1 (TGFbeta1), a major promoter of myofibroblast differentiation, induces alpha-smooth muscle (sn) actin, modulates the expression of adhesive receptors, and enhances the synthesis of extracellular matrix (ECM) molecules including ED-A fibronectin (FN), an isoform de novo expressed during wound healing and fibrotic changes. We report here that ED-A FN deposition precedes alpha-SM actin expression by fibroblasts during granulation tissue evolution in vivo and after TGFbeta1 stimulation in vitro. Moreover, there is a correlation between in vitro expression of alpha-SM actin and ED-A FN in different fibroblastic populations. Seeding fibroblasts on ED-A FN does not elicit per se alpha-SM actin expression; however, incubation of fibroblasts with the anti-ED-A monoclonal antibody IST-9 specifically blocks the TGFbeta1-triggered enhancement of alpha-SM actin and collagen type I, but not that of plasminogen activator inhibitor-1 mRNA. Interestingly, the same inhibiting action is exerted by the soluble recombinant domain ED-A, but neither of these inhibitory agents alter FN matrix assembly. Our findings indicate that ED-A-containing polymerized FN is necessary for the induction of the myofibroblastic phenotype by TGFbeta1 and identify a hitherto unknown mechanism of cytokine-determined gene stimulation based on the generation of an ECM-derived permissive outside in signaling, under the control of the cytokine itself.