ABSTRACT
Background Hemodynamic shear stress critically regulates endothelial activation and atherogenesis by affecting cytoskeletal dynamics and endothelial gene expression. The Nck adaptor proteins (Nck1 and Nck2) regulate cytoskeletal remodeling pathways and play redundant roles during development. While a cell permeable Nck-binding peptide reduces shear-induced inflammation, the roles of Nck1 and Nck2 in atherosclerosis remain unknown.
Methods and Results Herein, we show that Nck1 deficiency (siRNA/shRNA knockdown, genetic knockout), but not Nck2, decreases basal and shear stress-induced proinflammatory signaling (NF-κB phosphorylation and nuclear translocation) and ICAM-1/VCAM-1 expression. In contrast, neither Nck1 nor Nck2 were required for flow-induced Akt and ERK1/2 activation, and only Nck2 was required for laminar flow-induced cytoskeletal alignment. Using the partial carotid ligation model of disturbed flow, we found that Nck1 knockout mice showed significantly reduced proinflammatory gene expression and macrophage infiltration that was not further diminished upon Nck2 deletion. Consistent with these findings, Nck1 knockout mice showed significantly diminished diet-induced atherosclerosis, associated with reduced plasma cytokine levels and diminished macrophage content. To define the mechanisms of differential Nck1 and Nck2 signaling in endothelial activation, we performed domain swap experiments mixing SH2 and SH3 domains between Nck1 and Nck2. These Nck1/Nck2 chimeras define a critical role for the Nck1 SH2 domain (phosphotyrosine binding) but a redundant role for Nck1/2 SH3 domains (proline rich binding) in rescuing shear stress-induced endothelial activation in Nck1/2 DKO cells. Using domain point mutations, we confirmed the vital role for Nck1’s SH2 domain and identify the first Nck SH3 domain (DY pocket containing domain) in meditating NF-κB activation and endothelial inflammation. Pre-treatment of endothelial cells with the small molecule Nck1 SH3.1 inhibitor confirmed the critical role of this domain in flow-induced NF-κB activation and ICAM-1/ VCAM-1 expression.
Conclusions Taken together, our data reveal a hitherto unknown link between Nck1 signaling in endothelial cell activation and atherosclerosis development, highlighting the potential for targeting Nck1 to control atherogenic inflammation.