Regulation of Endothelial Nitric Oxide Synthase mRNA stability

描述（由申请人提供）：由内皮型一氧化氮合酶（eNOS）产生的一氧化氮（NO）通过促进血管舒张和抑制血管平滑肌细胞增殖、血小板聚集和白细胞粘附对维持血管稳态至关重要。虽然eNOS最初被认为是组成型的，后来证明，一些病理生理刺激，如剪切应力，慢性运动，和亚融合生长状态，上调eNOS表达。相反，包括肿瘤坏死因子-α、缺氧和高浓度氧化LDL的细胞因子降低eNOS水平，这可能导致与许多心血管疾病如动脉粥样硬化和心力衰竭相关的内皮功能障碍。对于许多这些刺激，eNOS mRNA稳定性的调节在控制eNOS表达中起着至关重要的作用。越来越多的证据表明，eNOS 3'非翻译区（3'-UTR）与胞浆蛋白的结合在eNOS mRNA稳定性的调节中起着关键作用。然而，这些蛋白质的身份仍有待确定。通过使用RNA亲和纯化和蛋白质质谱测序，我们已经揭示了翻译延伸因子-1 α（eEF 1A）和多聚嘧啶片段结合蛋白（PTB）特异性结合eNOS 3 '-UTR，潜在地调节人内皮细胞中eNOS mRNA的稳定性。本研究拟继续深入探讨PTB和eEF 1A 1在细胞培养和体内调控eNOS mRNA稳定性的机制。目的1研究eNOS mRNA 3 '-UTR与PTB的物理和功能相互作用，并通过RNA凝胶迁移率变动分析和紫外交联分析鉴定它们的相互作用结构域。目的2探讨eNOS mRNA 3 '-UTR核糖核蛋白复合物形成的分子调控机制。重点将放在Rho/ROCK通路和蛋白质-蛋白质相互作用在eNOS 3 '-UTR核糖核蛋白复合物形成和eNOS mRNA稳定性调节中的作用。此外，目的3将研究eNOS 3 '-UTR/eEF 1A 1/PTB复合物的破坏是否会阻止内皮细胞中TNF-1诱导的eNOS mRNA不稳定，以及eEF 1A 1和PTB是否参与兔高胆固醇血症模型中内皮功能障碍的发展。这些拟议的研究将提供更深入的了解如何eNOS表达的生理和病理条件下的调节机制。希望从拟议的研究中获得的结果将为心血管疾病的治疗带来新的治疗靶点。

DESCRIPTION (provided by applicant): Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), is critical to the maintenance of vascular homeostasis through promoting vasodilation and inhibiting vascular smooth muscle cells proliferation, platelets aggregation, and leukocyte adhesion. Although eNOS was initially considered to be constitutive, it was later demonstrated that several pathophysiological stimuli, such as shear stress, chronic exercise, and the subconfluent growth state, upregulate eNOS expression. In contrast, cytokines including tumor necrosis factor-alpha, hypoxia, and high concentrations of oxidized LDL decrease eNOS levels, which may contribute to the endothelial dysfunction associated with many cardiovascular diseases such as atherosclerosis and heart failure. For many of these stimuli, modulation of eNOS mRNA stability plays an essential role in controlling eNOS expression. Accumulating evidence indicates that the binding of cytosolic protein(s) to eNOS 3' untranslated region (3'-UTR) plays a critical role in the regulation of eNOS mRNA stability. However, the identity of these proteins remains to be identified. By using RNA affinity purification and protein sequencing by mass spectrometry, we have revealed that translation elongation factor-1 alpha (eEF1A) and polypyrimidine tract-binding protein (PTB) specifically bind to eNOS 3'-UTR, potentially regulating eNOS mRNA stability in human endothelial cells. This proposal is to continue to explore, in depth, the mechanisms by which PTB and eEF1A1 regulate eNOS mRNA stability in cell culture and in vivo. Aim 1 will characterize the physical and functional interactions of eNOS mRNA 3'-UTR with PTB and identify their interacting domains by RNA gel mobility shift assays and UV-cross linking assays. Aim 2 will investigate the molecular mechanisms regulating the eNOS mRNA 3'-UTR ribonucleoprotein complex formation. Emphasis will placed on the roles of the Rho/ROCK pathway and protein-protein interactions in the regulation of eNOS 3'-UTR ribonucleoprotein complex formation and eNOS mRNA stability. Furthermore, aim 3 will investigate whether disruption of the eNOS 3'-UTR/eEF1A1/PTB complex will prevent eNOS mRNA destabilization induced by TNF-1 in endothelial cells and whether eEF1A1 and PTB participate in the development of endothelial dysfunction in a rabbit model of hypercholesterolemia. These proposed studies will provide greater insights into the mechanisms of how eNOS expression is regulated under physiological and pathological conditions. It's hoped that the results obtained from the proposed studies will lead to novel therapeutic targets for treatment of cardiovascular diseases.