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

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)at the posttranscriptional level. To elucidate the molecular basis of TNF–mediated eNOS mRNA instability, eNOS 3 untranslated region (3 -UTR) binding proteins were purified by RNA affinity chromatography from cytosolic fractions of TNF–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. 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 11 (eEF1A1). In HUVECs, TNFrapidly 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–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–induced decrease in eNOS mRNA stability. (Circ Res. 2008;103:591-597.)