EphA2 signaling within integrin adhesions regulates fibrillar adhesion elongation and fibronectin deposition.

The multifunctional glycoprotein fibronectin influences several crucial cellular processes and contributes to multiple pathologies. While a link exists between fibronectin-associated pathologies and the receptor tyrosine kinase EphA2, the mechanism by which EphA2 promotes fibronectin matrix remodeling remains unknown. We previously demonstrated that EphA2 deletion reduces smooth muscle fibronectin deposition and blunts fibronectin deposition in atherosclerosis without influencing fibronectin expression. We now show that EphA2 expression is required for contractility-dependent elongation of tensin- and α5ß1 integrin-rich fibrillar adhesions that drive fibronectin fibrillogenesis. Mechanistically, EphA2 localizes to integrin adhesions where focal adhesion kinase mediates ligand-independent Y772 phosphorylation, and mutation of this site significantly blunts fibrillar adhesion length. EphA2 deficiency decreases smooth muscle cell contractility by enhancing p190RhoGAP activation and reducing RhoA activity, whereas stimulating RhoA signaling in EphA2 deficient cells rescues fibrillar adhesion elongation. Together, these data identify EphA2 as a novel regulator of fibrillar adhesion elongation and provide the first data identifying a role for EphA2 signaling in integrin adhesions.

Integrin affinity modulation critically regulates atherogenic endothelial activation in vitro and in vivo.

While vital to platelet and leukocyte adhesion, the role of integrin affinity modulation in adherent cells remains controversial. In endothelial cells, atheroprone hemodynamics and oxidized lipoproteins drive an increase in the high affinity conformation of α5ß1 integrins in endothelial cells in vitro, and α5ß1 integrin inhibitors reduce proinflammatory endothelial activation to these stimuli in vitro and in vivo. However, the importance of α5ß1 integrin affinity modulation to endothelial phenotype remains unknown. We now show that endothelial cells (talin1 L325R) unable to induce high affinity integrins initially adhere and spread but show significant defects in nascent adhesion formation. In contrast, overall focal adhesion number, area, and composition in stably adherent cells are similar between talin1 wildtype and talin1 L325R endothelial cells. However, talin1 L325R endothelial cells fail to induce high affinity α5ß1 integrins, fibronectin deposition, and proinflammatory responses to atheroprone hemodynamics and oxidized lipoproteins. Inducing the high affinity conformation of α5ß1 integrins in talin1 L325R endothelial cells suggest that NF-κB activation and maximal fibronectin deposition require both integrin activation and other integrin-independent signaling. In endothelial-specific talin1 L325R mice, atheroprone hemodynamics fail to promote inflammation and macrophage recruitment, demonstrating a vital role for integrin activation in regulating endothelial phenotype.