Nck Adaptor Proteins in Atherogenic Endothelial Activation

动脉粥样硬化易发部位血流动力学切应力和氧化应激的改变促进低水平， 慢性内皮细胞激活使这些部位易于形成斑块。在我的实验室工作 已经确定Nck信号转接子家族，包括Nck 1和Nck 2，是新的调节因子 血管内皮细胞激活。NCK1/2基因敲除或添加多肽抑制剂可钝化剪切和氧化应激- 诱导p21激活的激酶(PAK)信号转导，从而降低内皮通透性、细胞骨架 重塑和核因子-κB驱动的促炎基因表达。此外，我们还提供令人信服的 剪切和氧化应激反应中PECAM-1的磷酸化使NCK重新聚集到内皮细胞的证据 细胞-细胞连接，促进NCK依赖的PAK激活，并刺激NCK依赖的诱导 促炎症信号(NF-ICAMB)和基因表达(κ-1、VCAM1)。这些数据表明了一种模型 由此NCK募集到PECAM-1改变了本地信号环境，从而切换了PECAM-1信号 从消炎到促炎。 内皮细胞同时缺失Nck-1和Nck-2，而不是单独的亚型，会损害血管发育。 然而，一些研究已经发现了Nck 1和Nck 2的不同功能，并扰乱了流动模式 在体外和体内都不同程度地影响Nock1和Nock2的表达。虽然到目前为止还没有研究表明 在成人病理条件下检测内皮细胞Nck-1或Nck-2的功能，用Nck-1治疗 封闭多肽显著钝化多种模型系统中的白细胞募集和血管通透性 在活体内。然而，这些研究无法确定这种多肽是通过Nck起作用还是影响内皮细胞 直接起作用。我们在动脉粥样硬化易感人群中使用内皮特异性Nock1/2缺失的初步数据 APOE基因敲除小鼠表现出促炎症基因表达减少，对血流紊乱和 减少早期斑块的发展。因此，我们假设PECAM-1的磷酸化反应 切变和氧化应激促进形成不同的基于NCK的信号复合体，从而驱动 动脉粥样硬化条件下的通透性和促炎基因表达。在目标1中，我们将 通过绘制NCK结合位点来表征内皮激活过程中PECAM-1/NCK复合体的形成 在PECAM-1上，验证体内相互作用，并测定与局部氧化应激的串扰 回应。在目标2中，我们将描述基于NCK的信号复合体的组成和功能 在内皮细胞激活过程中，通过评估Nck 1/Nck 2表达变化的机制，检测其 在内皮细胞激活中的相关作用，并利用结构域映射和空间蛋白质组学来表征 血管内皮细胞激活过程中NKK1/2的相互作用。在目标3中，我们将确定内皮细胞NCK 利用携带NCK基因条件性缺失的小鼠体内信号影响内皮细胞激活 评估内皮细胞在致动脉粥样硬化条件下内皮细胞激活中的相对作用。

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.