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.
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.
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