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低水平， 慢性内皮活化，使这些部位易于形成斑块。从我的实验室工作 已经确定了Nck家族的信号转导衔接子，包括Nck 1和Nck 2，作为新的调节剂， 内皮激活Nck 1/2敲低或添加肽抑制剂减弱剪切和氧化应激- 诱导p21激活激酶（PAK）信号传导，从而降低内皮通透性、细胞骨架 重塑和NF-κ B驱动的促炎基因表达。此外，我们还提供令人信服的 PECAM-1磷酸化对剪切和氧化应激的反应将Nck募集到内皮细胞的证据 细胞-细胞连接，促进Nck依赖性PAK激活，并刺激Nck依赖性诱导 促炎信号传导（NF-κB）和基因表达（ICAM-1、VCAM-1）。这些数据表明 由此Nck向PECAM-1的募集改变了局部信号环境， 从抗炎到促炎 内皮细胞缺失Nck 1和Nck 2，而不是个别亚型，损害血管发育。 然而，几项研究发现Nck 1和Nck 2具有不同的功能， 在体外和体内均不同地影响Nck 1和Nck 2表达。虽然迄今为止还没有研究表明 在成人病理条件下检查内皮Nck 1或Nck 2功能，用Nck- 阻断肽在多模型系统中显著减弱白细胞募集和血管通透性 in vivo.然而，这些研究不能确定这种肽是否通过Nck起作用或影响内皮细胞， 功能直接。我们利用动脉粥样硬化易感人群中内皮特异性Nck 1/2缺失的初步数据 ApoE基因敲除小鼠显示出对干扰的血流的反应中促炎基因表达减少， 减少早期斑块的发展。因此，我们假设PECAM-1磷酸化反应于 剪切和氧化应激促进了不同的NCK信号复合物的形成， 动脉粥样硬化条件下的渗透性和促炎基因表达。在目标1中，我们 通过绘制Nck结合位点来表征内皮激活期间PECAM-1/Nck复合物的形成 在PECAM-1上，验证体内的相互作用，并确定在这种情况下与局部氧化应激的串扰。 反应在目标2中，我们将描述NCK信号复合物的组成和功能 通过评估Nck 1/Nck 2表达改变的机制，检测其在内皮细胞活化过程中的作用， 在内皮细胞活化中的相对作用，并利用结构域作图和空间蛋白质组学来表征 内皮细胞活化过程中Nck 1/2相互作用体。在目标3中，我们将确定内皮Nck是否 信号转导影响体内内皮细胞活化，使用含有Nck基因条件性缺失的小鼠， 内皮细胞，以评估它们在致动脉粥样硬化条件下的内皮活化中的相对作用。

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.