Tumor Necrosis Factor-α Downregulates Endothelial Nitric Oxide Synthase mRNA Stability via Translation Elongation Factor 1-α 1

• Published in: Circulation research Vol. 103; no. 6; pp. 591 - 597
• Main Authors: Yan, Guijun, You, Bei, Chen, Shao-Ping, Liao, James K, Sun, Jianxin
• Format: Journal Article
• Language: English
• Published: United States American Heart Association, Inc 12.09.2008
• Subjects: Amino Acid Sequence; Animals; Cattle; Cells, Cultured; Cercopithecus aethiops; COS Cells; Down-Regulation - physiology; Humans; Molecular Sequence Data; Nitric Oxide Synthase Type III - antagonists & inhibitors; Nitric Oxide Synthase Type III - genetics; Nitric Oxide Synthase Type III - metabolism; Peptide Elongation Factor 1 - genetics; Peptide Elongation Factor 1 - physiology; Protein Biosynthesis - physiology; RNA Stability - genetics; Tumor Necrosis Factor-alpha - physiology
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/CIRCRESAHA.108.173963

Endothelium-derived nitric oxide (NO) is an important regulator of vascular function. NO is produced by endothelial NO synthase (eNOS), whose expression is downregulated by tumor necrosis factor (TNF)-α at the posttranscriptional level. To elucidate the molecular basis of TNF-α–mediated eNOS mRNA instability, eNOS 3′ untranslated region (3′-UTR) binding proteins were purified by RNA affinity chromatography from cytosolic fractions of TNF-α–stimulated human umbilical vein endothelial cells (HUVECs). The formation of 3′-UTR ribonucleoprotein complexes, with molecular weight of 52 and 57 kDa, was increased by TNF-α. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis of the 52-kDa protein identified 3 peptides that comprise the peptide sequence of translation elongation factor 1-α 1 (eEF1A1). In HUVECs, TNF-α rapidly increased eEF1A1 expression, which is maximal after 1 hour and persists for up to 48 hours. RNA gel mobility-shift and UV cross-linking assays indicated that recombinant glutathione S-transferase–eEF1A1 fusion protein specifically binds to a UC-rich sequence in the 3′-UTR of eNOS mRNA. In addition, the domain III of eEF1A1 mediates the binding of eNOS 3′-UTR in eEF1A1. Overexpression of eEF1A1 markedly attenuated the expression of eNOS and luciferase gene fused with eNOS 3′-UTR in both COS-7 cells and bovine aortic endothelial cells (BAECs). Furthermore, adenovirus-mediated overexpression of eEF1A1 increased eNOS mRNA instability, whereas knockdown of eEF1A1 substantially attenuated TNF-α–induced destabilization of eNOS mRNA and downregulation of eNOS expression in HUVECs. These results indicate that eEF1A1 is a novel eNOS 3′-UTR binding protein that plays a critical role in mediating TNF-α–induced decrease in eNOS mRNA stability.