title: Post-Transcriptional Regulation of Endothelial Nitric Oxide Synthase Expression by Polypyrimidine Tract-Binding Protein 1.
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.
  
  
  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.
authors: 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.).
authors: Bing
authors: B
authors: Yi
authors: 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.).
authors: Maria
authors: M
authors: Ozerova
authors: 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.).
authors: Guan-Xin
authors: GX
authors: Zhang
authors: 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.).
authors: Guijun
authors: G
authors: Yan
authors: 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.).
authors: Shengdong
authors: S
authors: Huang
authors: 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.
authors: Jianxin
authors: J
authors: Sun
mesh_terms: D002478:Cells, Cultured; D015536:Down-Regulation; D004730:Endothelium, Vascular / Q000378:metabolism; D005786:Gene Expression Regulation; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D052250:Nitric Oxide Synthase Type III / Q000235:genetics*; D038941:Polypyrimidine Tract-Binding Protein / Q000378:metabolism*; D011485:Protein Binding; D020871:RNA Stability / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
chemical_list: D012333:RNA, Messenger; C439524:TNF protein, human; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D038941:Polypyrimidine Tract-Binding Protein; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III
keywords: RNA stability; endothelium; luciferase; tumor necrosis factor-α; vascular diseases