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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL103869-05
Application #
8587497
Study Section
Vascular Cell and Molecular Biology Study Section (VCMB)
Program Officer
Mcdonald, Cheryl
Project Start
2010-08-01
Project End
2015-11-30
Budget Start
2013-12-01
Budget End
2014-11-30
Support Year
5
Fiscal Year
2014
Total Cost
$348,750
Indirect Cost
$123,750
Name
Thomas Jefferson University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
053284659
City
Philadelphia
State
PA
Country
United States
Zip Code
19107
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Yan, Guijun; Zhu, Ni; Huang, Shengdong et al. (2015) Orphan Nuclear Receptor Nur77 Inhibits Cardiac Hypertrophic Response to Beta-Adrenergic Stimulation. Mol Cell Biol 35:3312-23
Qi, Jia; Yang, Ping; Yi, Bing et al. (2015) Heat shock protein 90 inhibition by 17-DMAG attenuates abdominal aortic aneurysm formation in mice. Am J Physiol Heart Circ Physiol 308:H841-52
Yi, Bing; Ozerova, Maria; Zhang, Guan-Xin et al. (2015) Post-Transcriptional Regulation of Endothelial Nitric Oxide Synthase Expression by Polypyrimidine Tract-Binding Protein 1. Arterioscler Thromb Vasc Biol 35:2153-60
Liu, Yan; Zhang, Jian; Yi, Bing et al. (2014) Nur77 suppresses pulmonary artery smooth muscle cell proliferation through inhibition of the STAT3/Pim-1/NFAT pathway. Am J Respir Cell Mol Biol 50:379-88
Chen, Ming; Yi, Bing; Sun, Jianxin (2014) Inhibition of cardiomyocyte hypertrophy by protein arginine methyltransferase 5. J Biol Chem 289:24325-35
Qin, Qing; Chen, Ming; Yi, Bing et al. (2014) Orphan nuclear receptor Nur77 is a novel negative regulator of endothelin-1 expression in vascular endothelial cells. J Mol Cell Cardiol 77:20-8
Huo, Yan; Yi, Bing; Chen, Ming et al. (2014) Induction of Nur77 by hyperoside inhibits vascular smooth muscle cell proliferation and neointimal formation. Biochem Pharmacol 92:590-8
Li, Pan; Liu, Yan; Yi, Bing et al. (2013) MicroRNA-638 is highly expressed in human vascular smooth muscle cells and inhibits PDGF-BB-induced cell proliferation and migration through targeting orphan nuclear receptor NOR1. Cardiovasc Res 99:185-93

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