Selective role of Nck1 in atherogenic inflammation and plaque formation
Mabruka Alfaidi, … , James G. Traylor, A. Wayne Orr
Mabruka Alfaidi, … , James G. Traylor, A. Wayne Orr
Published May 19, 2020
Citation Information: J Clin Invest. 2020;130(8):4331-4347. https://doi.org/10.1172/JCI135552.
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Research Article Cell biology Vascular biology Article has an altmetric score of 9

Selective role of Nck1 in atherogenic inflammation and plaque formation

Abstract

Although the Canakinumab Anti-Inflammatory Thrombosis Outcomes Study (CANTOS) established the role of treating inflammation in atherosclerosis, our understanding of endothelial activation at atherosclerosis-prone sites remains limited. Disturbed flow at atheroprone regions primes plaque inflammation by enhancing endothelial NF-κB signaling. Herein, we demonstrate a role for the Nck adaptor proteins in disturbed flow–induced endothelial activation. Although highly similar, only Nck1 deletion, but not Nck2 deletion, limited flow-induced NF-κB activation and proinflammatory gene expression. Nck1-knockout mice showed reduced endothelial activation and inflammation in both models, disturbed flow– and high fat diet–induced atherosclerosis, whereas Nck2 deletion did not. Bone marrow chimeras confirmed that vascular Nck1, but not hematopoietic Nck1, mediated this effect. Domain-swap experiments and point mutations identified the Nck1 SH2 domain and the first SH3 domain as critical for flow-induced endothelial activation. We further characterized Nck1’s proinflammatory role by identifying interleukin 1 type I receptor kinase-1 (IRAK-1) as a Nck1-selective binding partner, demonstrating that IRAK-1 activation by disturbed flow required Nck1 in vitro and in vivo, showing endothelial Nck1 and IRAK-1 staining in early human atherosclerosis, and demonstrating that disturbed flow–induced endothelial activation required IRAK-1. Taken together, our data reveal a hitherto unknown link between Nck1 and IRAK-1 in atherogenic inflammation.

Authors

Mabruka Alfaidi, Christina H. Acosta, Dongdong Wang, James G. Traylor, A. Wayne Orr

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Figure 1

Nck1/2 deletion ameliorates shear stress–induced NF-κB activation.

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Nck1/2 deletion ameliorates shear stress–induced NF-κB activation. (A) H...
(A) Human aortic endothelial cells (HAECs) were transfected with siRNA specific for Nck1/2, and transfection efficiency was assessed using Western blotting. (B and C) HAECs were subjected to acute shear stress for the indicated times, and NF-κB activation was assessed by detection of p65 serine 536 phosphorylation using Western blotting. Densitometric analysis was performed using ImageJ. (D and E) p65 nuclear translocation was measured after 45 minutes of shear stress in Nck1/2 siRNA–treated and mock control cells. Images were analyzed using NIS Elements software. Scale bar: 50 μm. Data are presented as the mean ± SEM, n = 4. *P < 0.05, **P < 0.01, ****P < 0.0001 by 2-way ANOVA followed by Bonferroni’s post hoc test. HPF, high-power field. (F and G) HAECs were transfected with Nck1/2 siRNA, and oscillatory shear stress–induced (OSS-induced; ±5 dynes/cm2 with 1 dyne/cm2 forward flow) proinflammatory gene expression (VCAM-1, ICAM-1) and proinflammatory signaling (p-NF-κB Ser536) were assessed by Western blotting. n = 4 in G. *P < 0.05 by Kruskal-Wallis test. (H) HAECs were treated as in F, and mRNA expression was assessed by qRT-PCR (n = 4). ***P < 0.001, ****P < 0.0001 by 2-way ANOVA followed by Bonferroni’s post hoc test.