Fibronectin-dependent integrin signaling drives EphA2 expression in vascular smooth muscle cells

Abstract

Vascular smooth muscle cells undergo a phenotypic shift to a "synthetic" phenotype during atherosclerosis characterized by downregulation of contractile markers and augmented proliferation, migration, and extracellular matrix deposition. While absent in contractile smooth muscle cells, the receptor tyrosine kinase EphA2 shows enhanced expression in synthetic vascular smooth muscle in vitro and in atherosclerotic plaques in vivo. EphA2 deletion in atheroprone ApoE knockout mice reduces plaque size, fibrous tissue, and smooth muscle content. However, the mechanisms regulating smooth muscle EphA2 expression remain unknown. Although serum strongly induces EphA2 expression, individual growth factors and insulin all failed to stimulate EphA2 expression in smooth muscle cells. In contrast, adhesion to fibronectin stimulated the expression of EphA2, while blunting serum-induced fibronectin deposition attenuated EphA2 expression, suggesting a critical role for fibronectin signaling. Fibronectin binds to a subset of extracellular matrix-binding integrins, and blocking fibronectin-integrin interactions or inhibiting specific fibronectin-binding integrins both attenuated EphA2 expression. Furthermore, pharmacological inhibition of fibronectin-binding integrins significantly reduced EphA2 expression in atherosclerotic plaques. RNA sequencing analysis of fibronectin-associated gene expression pointed to NF-κB as a likely transcription factor mediating fibronectin-responsive genes. Adhesion to fibronectin enhanced NF-κB activation in smooth muscle cells and inhibiting NF-κB blunted EphA2 expression associated with fibronectin. In addition, chromatin immunoprecipitation showed that NF-κB directly interacts with the EphA2 promoter, and mutating this site blunts fibronectin-dependent EphA2 promoter activity. Together these data identify a novel role for fibronectin-dependent integrin signaling in the induction of smooth muscle EphA2 expression.NEW & NOTEWORTHY Here, we demonstrate a novel interplay between cell-cell and cell-matrix adhesions, showing that fibronectin-dependent integrin signaling promotes NF-κB activation and interaction with the EphA2 promoter to drive smooth muscle EphA2 expression, whereas integrin inhibition attenuates EphA2 expression in atherosclerotic plaques in vivo. Although this relationship has clear implications on smooth muscle fibroproliferative remodeling in atherosclerosis, the matrix-specific regulation of EphA2 expression may impact a variety of pathological conditions.

Keywords: EphA2; NF-κB; fibronectin; integrins; vascular smooth muscle.