Endothelial nitric oxide synthase (eNOS) is a key enzyme involved in the regulation of vascular tone and altered eNOS activity leads to endothelial dysfunction and atherosclerosis. Post-transcriptional modulation of eNOS mRNA stability is a major determinant of eNOS expression in vascular endothelium. The molecular mechanisms regulating eNOS mRNA stability remain poorly understood and represent an important gap in knowledge. This competitive renewal application proposes to investigate the molecular mechanisms by which eNOS mRNA stability is regulated. Preliminary studies suggest that the binding of Polypyrimidine Tract Binding Protein 1 (PTB1) to the eNOS mRNA 3?-UTR leads to decreased eNOS mRNA stability and expression. Moreover, we found that PTB1 specifically interacts with the mammalian Ste20-like kinase 1 (Mst1), an upstream serine/threonine-specific protein kinase of the Hippo pathway, which leads to PTB1 phosphorylation and decreased eNOS mRNA stability and protein expression in vascular endothelial cells. Our preliminary data further demonstrate that Mst1 kinase is activated in the atherosclerotic lesions and blocking Mst1 activation by overexpression of dominant negative protein (DN-Mst1) (K59R) in vascular endothelial cells markedly increased eNOS mRNA stability and expression, implicating a functional significance of Mst1 kinase in regulating endothelial functions. We therefore hypothesize that hyperlipidemia-induced activation of Mst1 promotes the binding of PTB1 to eNOS 3?-UTR, which in turn downregulates eNOS expression and causes endothelial dysfunction in the atherogenesis. Accordingly, we propose three comprehensive specific aims to delineate how the regulation of PTB1 by Mst1 kinase affects eNOS mRNA stability and expression, and the extent to which this contributes to the pathogenesis of endothelial dysfunction and vascular disease.
Specific aim 1 will examine the functional significance of Mst1 in regulating eNOS mRNA stability by PTB1. We will identify the Mst1 specific phosphorylation site(s) on PTB1 and then determine whether phosphorylation of PTB1 by Mst1 alters the binding affinity of PTB1 to the eNOS 3?-UTR, leading to decreased eNOS mRNA stability and protein expression in vascular endothelial cells.
Specific aim 2 will determine whether PTB1 induces eNOS mRNA instability by inhibiting 3?-polyadenylation of eNOS mRNA and/or by increasing the binding of microRNAs to the eNOS 3?-UTR.
Specific aim 3 will determine whether the Mst1/PTB1 pathway regulates endothelial dysfunction in vivo. By using our newly generated endothelial specific DN-Mst1 transgenic and Mst1 knockout mice, we will investigate whether eNOS expression, endothelium-dependent vascular relaxation, and atherosclerosis are altered by specifically blocking Mst1 activation in vascular endothelial cells. We envision these will establish the importance of Mst1 as a therapeutic target for improving endothelial function and decreasing cardiovascular disease.
Endothelial dysfunction is a major problem associated with many human diseases, including atherosclerosis, hypertension, heart failure, diabetes, and chronic renal failure, etc. Our studies aim to understand the key factors that cause this problem. Understanding these processes will allow development of new therapies to treat the problem in the future.
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