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

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cells, Cultured
  • Down-Regulation
  • Endothelium, Vascular / metabolism
  • Gene Expression Regulation*
  • Human Umbilical Vein Endothelial Cells
  • Humans
  • Nitric Oxide Synthase Type III / genetics*
  • Polypyrimidine Tract-Binding Protein / metabolism*
  • Protein Binding
  • RNA Stability / genetics
  • RNA, Messenger / metabolism
  • Sensitivity and Specificity
  • Transcription Factors / metabolism
  • Tumor Necrosis Factor-alpha / metabolism*


  • RNA, Messenger
  • TNF protein, human
  • Transcription Factors
  • Tumor Necrosis Factor-alpha
  • Polypyrimidine Tract-Binding Protein
  • NOS3 protein, human
  • Nitric Oxide Synthase Type III