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Arterioscler Thromb Vasc Biol. 2015 Oct;35(10):2153-60. doi: 10.1161/ATVBAHA.115.305750. Epub 2015 Aug 20.

Post-Transcriptional Regulation of Endothelial Nitric Oxide Synthase Expression by Polypyrimidine Tract-Binding Protein 1.

Author information

1
From the Center for Translational Medicine, Department of Medicine, Thomas Jefferson University, Philadelphia, PA (B.Y., M.O., J.S.); Reproductive Medicine Center, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China (G.Y.); and Department of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, China (G.Z., S.H., J.S.).
2
From the Center for Translational Medicine, Department of Medicine, Thomas Jefferson University, Philadelphia, PA (B.Y., M.O., J.S.); Reproductive Medicine Center, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China (G.Y.); and Department of Cardiothoracic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, China (G.Z., S.H., J.S.). jianxin.sun@jefferson.edu.

Abstract

OBJECTIVE:

Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular function and its expression is regulated at post-transcriptional levels through a yet unknown mechanism. The purpose of this study is to elucidate the post-transcriptional factors regulating eNOS expression and function in endothelium.

APPROACHES AND RESULTS:

To elucidate the molecular basis of tumor necrosis factor (TNF)-α-mediated eNOS mRNA instability, biotinylated eNOS 3'-untranslational region (UTR) was used to purify its associated proteins by RNA affinity chromatography from cytosolic fractions of TNF-α-stimulated human umbilical vein endothelial cells (HUVECs). We identified 2 cytosolic proteins, with molecular weight of 52 and 57 kDa, which specifically bind to eNOS 3'-UTR in response to TNF-α stimulation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis identified the 57-kDa protein as polypyrimidine tract-binding protein 1 (PTB1). RNA gel mobility shift and UV cross-linking assays demonstrated that PTB1 binds to a UCUU-rich sequence in eNOS 3'-UTR, and the C-terminal half of PTB1 is critical to this interaction. Importantly, PTB1 overexpression leads to decreased activity of luciferase gene fused with eNOS 3'-UTR as well as reduced eNOS expression and activity in human ECs. In HUVECs, we show that TNF-α markedly increased PTB1 expression, whereas adenovirus-mediated PTB1 overexpression decreased eNOS mRNA stability and reduced protein expression and endothelium-dependent relaxation. Furthermore, knockdown of PTB1 substantially attenuated TNF-α-induced destabilization of eNOS transcript and downregulation of eNOS expression.

CONCLUSIONS:

These results indicate that PTB1 is essential for regulating eNOS expression at post-transcriptional levels and suggest a novel therapeutic target for treatment of vascular diseases associated with inflammatory endothelial dysfunction.

KEYWORDS:

RNA stability; endothelium; luciferase; tumor necrosis factor-α; vascular diseases

PMID:
26293469
PMCID:
PMC6027644
DOI:
10.1161/ATVBAHA.115.305750
[Indexed for MEDLINE]
Free PMC Article

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