Regulation of Endothelial Nitric Oxide Synthase mRNA stability

说明书(申请人提供)：一氧化氮(NO)，由内皮型一氧化氮合酶(ENOS)产生，通过促进血管扩张和抑制血管平滑肌细胞增殖、血小板聚集和白细胞黏附，对维持血管稳态至关重要。虽然eNOS最初被认为是结构性的，但后来证明，几种病理生理刺激，如切应力、慢性运动和亚融合生长状态，上调了eNOS的表达。相反，包括肿瘤坏死因子-α、缺氧和高浓度氧化低密度脂蛋白在内的细胞因子会降低eNOS水平，这可能是许多心血管疾病(如动脉粥样硬化和心力衰竭)导致的内皮功能障碍的原因之一。对于这些刺激中的许多，enos mRNA稳定性的调节在控制enos表达方面起着至关重要的作用。越来越多的证据表明，胞浆蛋白(S)与eNOS 3‘非翻译区(3’-UTR)的结合在调节eNOS基因稳定性中起着关键作用。然而，这些蛋白质的特性仍有待鉴定。通过RNA亲和纯化和蛋白质测序，我们发现翻译延长因子-1α(EEF1a)和多嘧啶结合蛋白(PTB)与eNOS 3‘-UTR特异性结合，可能调节人内皮细胞enos mRNA的稳定性。本研究旨在继续深入探讨PTB和eEF1A1在细胞培养和体内调节enos基因稳定性的机制。目的1研究内皮型一氧化氮合酶基因3‘-非编码区与PTB的物理和功能相互作用，并通过RNA凝胶迁移率分析和紫外光交联实验鉴定其相互作用结构域。目的研究内皮型一氧化氮合酶基因3‘-非编码区核糖核蛋白复合体形成的分子机制。重点是Rho/ROCK途径和蛋白质-蛋白质相互作用在调节eNOS 3‘-UTR核糖核蛋白复合体形成和enos mRNA稳定性中的作用。此外，Aim 3将研究eNOS3‘-UTR/eEF1A1/PTB复合体的破坏是否可以防止由肿瘤坏死因子-1诱导的内皮细胞内eNOS mRNA的不稳定，以及eEF1A1和PTB是否参与了高胆固醇血症兔模型的内皮功能障碍的发展。这些拟议的研究将为eNOS在生理和病理条件下的表达调控机制提供更深入的了解。希望从拟议的研究中获得的结果将导致治疗心血管疾病的新的治疗靶点。 与公共健康相关：拟议的研究将揭示控制内皮细胞内皮型一氧化氮合酶(ENOS)mRNA衰退和蛋白表达的基本原理。由于血管内皮型一氧化氮合酶表达失调会导致许多人类疾病(如高血压、冠状动脉疾病、慢性心力衰竭、外周动脉疾病、糖尿病和慢性肾功能衰竭等)所致的内皮功能障碍的发生和发展，因此这项研究将很好地为这些疾病的分子机制提供重要的见解，从而促进新型治疗药物的开发。

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. PUBLIC HEALTH RELEVANCE: The proposed studies will reveal fundamental principles that govern endothelial nitric oxide synthase (eNOS) mRNA decay and protein expression in endothelial cells. Since vascular dysregulation of eNOS expression occurs and contributes to the development of endothelial dysfunction associated with many human diseases (e.g., hypertension, coronary artery disease, chronic heart failure, peripheral artery disease, diabetes, and chronic renal failure, etc.), the proposed study may well provide significant insights into the molecular mechanisms underlying these conditions and thus facilitate the development of novel therapeutic agents.