Regulation of eNOS Expression by NfkB, Shear Stress, and Exercise

剪切应力通过急性激活内皮型一氧化氮合酶（eNOS）酶以及上调eNOS表达增加血管一氧化氮的产生。我们的实验室已经证明，层流剪切应力激活eNOS mRNA的转录和mRNA的稳定通过不同的信号转导途径。eNOS mRNA转录的增加依赖于c-Src、RAS、MEK 1/2和ERK 1/2的激活。此外，我们已经确定了eNOS启动子内的区域，这是有助于响应剪切应力的转录。转录因子NF κ B以剪切应力依赖的方式与该区域结合，并且似乎在响应剪切的eNOS启动子活性的诱导中至关重要。NF κ B激活多种靶基因以响应血管壁中的多种刺激，这些刺激主要与动脉粥样硬化的发生有关。相比之下，NO在血管壁中具有动脉粥样硬化保护作用，并且已经显示出抑制NF κ B活化。我们认为这代表了一个经典的 负反馈环，由此剪切激活NF κ B，其刺激NO产生和eNOS表达，并且NO抑制NF κ B。在不产生NO的条件下，我们假设可能发生NF κ B的无约束激活。在这个项目中，有几个目标是为了理解这个概念。在目标1中，我们将检查负责响应剪切激活NF κ B的分子信号。在目标2中，我们将检验以下假设：响应于剪切的NO产生提供负反馈以抑制NF κ B与eNOS启动子的结合，抑制eNOS启动子活性并减少eNOS转录。为了操纵NO水平，将用NOS抑制剂L-NAME处理细胞，我们将研究来自eNOS -/-小鼠的细胞。在目标3中，我们将确定在运动训练期间NF κ B是否在体内被激活，其激活是否由NO调节，以及NF κ B激活是否介导运动期间eNOS表达的增加。在最后的目标中，我们将确定在运动过程中缺乏NO是否会促进血管炎症。这些研究将在不产生NO的eNOS -/-小鼠中进行。此外，我们有初步数据显示，运动训练对年轻健康人具有异质性影响，在约40%的受试者中，运动训练会使血流介导的血管舒张恶化。我们计划研究，以确定是否NF κ B驱动的基因产物，VCAM-1和ICAM-1，增加了这些科目的运动训练。总之，这些研究将提供新的信息，eNOS基因表达的调控和NO的作用，以防止NF κ B介导的血管炎症。

Shear stress increases vascular production of nitric oxide by acute activation of the endothelial nitric oxide synthase (eNOS) enzyme and also by upregulation of eNOS expression. Our laboratory has demonstrated that laminar shear stress activates eNOS mRNA transcription and mRNA stabilization through divergent signal transduction pathways. Increased eNOS mRNA transcription in response to shear depends on activation of c-Src, RAS, MEK1/2 and ERK1/2. Furthermore, we have identified the region within the eNOS promoter that is instrumental for transcription in response to shear stress. The transcription factor NFkappaB binds to this region in a shear stress-dependent manner, and seems to be critically important in the induction of eNOS promoter activity in response to shear. NFkappaB activates a variety of target genes in response to diverse stimuli in the vessel wall that are mostly linked to the onset of atherosclerosis. In contrast, NO has an atheroprotective effect in the vessel wall, and has been shown to inhibit NFkappaB activation. We propose that this represents a classical negative feedback loop, whereby shear activates NFkappaB which stimulates NO production and eNOS expression and that NO inhibits NFkappaB. In conditions where NO is not produced, we hypothesize that unbridled activation of NFkappaB may occur. In this project, several aims are directed toward understanding this concept. In aim 1, we will examine molecular signals responsible for activation of NFkappaB in response to shear. In aim 2, we will examine the hypothesis that NO production in response to shear provides a negative feedback to inhibit NFkappaB-binding to the eNOS promoter, inhibiting eNOS promoter activity and reducing eNOS transcription. To manipulate levels NO, cells will be treated with the NOS inhibitor L-NAME and we will study cells from eNOS -/- mice. In aim 3, we will determine if NFkappaB is activated in vivo during exercise training, if its activation is modulated by NO and if NFkappaB activation mediates the increase in eNOS expression during exercise. In a final aim, we will determine if an absence of NO promotes vascular inflammation during exercise. These studies will be performed in eNOS -/- mice that do not produce NO. In addition, we have preliminary data showing that exercise training has a heterogeneous effect in young otherwise healthy humans, and in about 40% of subjects, flow-mediated vasodilatation is worsened by exercise training. We plan studies to determine if the NFkappaB driven gene products, VCAM-1 and ICAM-1, are increased by exercise training in these subjects. Overall, these studies will provide novel information regarding the regulation of eNOS gene expression and the role of NO to prevent NFkappaB-mediated vascular inflammation.