Regulation of Endothelial Nitric Oxide Synthase mRNA stability

描述（由申请人提供）：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最初被认为是组成性的，但后来证明，剪切应力、慢性运动和亚融合生长状态等几种病理生理刺激可上调eNOS的表达。相反，包括肿瘤坏死因子- α、缺氧和高浓度氧化LDL在内的细胞因子降低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表达中起着重要作用。越来越多的证据表明，胞质蛋白(s)与eNOS 3‘非翻译区（3’-UTR）的结合在eNOS mRNA稳定性的调控中起着关键作用。然而，这些蛋白质的身份仍有待确定。通过RNA亲和纯化和质谱测序，我们发现翻译延伸因子-1 α (eEF1A)和多嘧啶束结合蛋白（PTB）特异性结合eNOS 3'-UTR，可能调节人内皮细胞eNOS mRNA的稳定性。本研究将继续深入探索PTB和eEF1A1在细胞培养和体内调控eNOS mRNA稳定性的机制。目的1将描述eNOS mRNA 3'-UTR与PTB的物理和功能相互作用，并通过RNA凝胶迁移转移试验和uv交联试验鉴定它们的相互作用域。目的2将研究调节eNOS mRNA 3'-UTR核糖核蛋白复合物形成的分子机制。重点将放在Rho/ROCK途径和蛋白-蛋白相互作用在eNOS 3'-UTR核糖核蛋白复合物形成和eNOS mRNA稳定性调控中的作用。此外，目的3将研究eNOS 3'-UTR/eEF1A1/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eEF1A1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。这些拟议的研究将为eNOS在生理和病理条件下的表达调控机制提供更深入的见解。希望这些研究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. 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.