Nck Adaptor Proteins in Atherogenic Endothelial Activation

动脉粥样硬化易发部位的血流动力学剪切应力和氧化应激的改变促进了

Alterations in hemodynamic shear stress and oxidant stress at atherosclerosis-prone sites promote a low level, chronic endothelial activation that predisposes these sites to plaque development. Work from my laboratory has identified the Nck family of signaling adaptors, including both Nck1 and Nck2, as novel regulators of endothelial activation. Nck1/2 knockdown or addition of a peptide inhibitor blunts shear and oxidant stress- induced p21 activated kinase (PAK) signaling, thereby reducing endothelial permeability, cytoskeletal remodeling, and NF-κB-driven proinflammatory gene expression. Additionally, we provide compelling evidence that PECAM-1 phosphorylation in response to shear and oxidant stress recruits Nck to endothelial cell-cell junctions, promotes Nck-dependent PAK activation, and stimulates Nck-dependent induction of proinflammatory signaling (NF-κB) and gene expression (ICAM-1, VCAM-1). These data suggest a model whereby Nck recruitment to PECAM-1 alters the local signaling milieu thereby switching PECAM-1 signaling from anti-inflammatory to pro-inflammatory. Endothelial deletion of both Nck1 and Nck2, but not individual isoforms, impairs vascular development. However, several studies have found distinct functions for Nck1 and Nck2, and disturbed flow patterns differentially affect Nck1 and Nck2 expression both in vitro and in vivo. While no studies to date have examined endothelial Nck1 or Nck2 function during pathological conditions in adults, treatment with a Nck- blocking peptide significantly blunts leukocyte recruitment and vascular permeability in multiple model systems in vivo. However, these studies cannot determine whether this peptide acts through Nck or affects endothelial function directly. Our preliminary data utilizing endothelial-specific Nck1/2 deletion in atherosclerosis-prone ApoE knockout mice show reduced proinflammatory gene expression in response to disturbed flow and reduced early plaque development. Therefore, we hypothesize that PECAM-1 phosphorylation in response to shear and oxidant stress facilitates the formation of distinct Nck-based signaling complexes that drive permeability and proinflammatory gene expression under atherogenic conditions. In Aim 1, we will characterize PECAM-1/Nck complex formation during endothelial activation by mapping the Nck-binding sites on PECAM-1, verifying the interaction in vivo, and determining the crosstalk with local oxidant stress in this response. In Aim 2, we will characterize the composition and function of Nck-based signaling complexes during endothelial cell activation by assessing the mechanisms of altered Nck1/Nck2 expression, testing their relative roles in endothelial cell activation, and utilizing domain mapping and spatial proteomics to characterize the Nck1/2 interactome during endothelial activation. In Aim 3, we will determine whether endothelial Nck signaling affects endothelial activation in vivo utilizing mice containing conditional deletion of the Nck genes in endothelial cells to assess their relative roles in endothelial activation under atherogenic conditions.