TNF-alpha Downregulates Endothelial Nitric Oxide Synthase mRNA Stability via Translation Elongation Factor 1-alpha 1

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 post-transcriptional 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 (RNP) complexes, with molecular weight of 52 and 57 kDa, was increased by TNF-α. MALDI-TOF-MS analysis of the 52 kDa protein identified three peptides that comprise the peptide sequence of translation elongation factor 1-alpha 1(eEF1A1). In HUVECs, TNF-α rapidly increased eEF1A1 expression, which is maximal after 1 hr and persists for up to 48 hr. RNA gel mobility shift and UV cross-linking assays indicated that recombinant GST-eEF1A1 fusion protein specifically binds to an 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, while 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-αinduced decrease in eNOS mRNA stability.