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(Circulation Research. 2008;103:591.)
© 2008 American Heart Association, Inc.

Molecular Medicine

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

Guijun Yan, Bei You, Shao-Ping Chen, James K. Liao^*, Jianxin Sun^*

From the Department of Cell Biology and Molecular Medicine (G.Y., B.Y., S.-P.C., J.S.), University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark; and Vascular Medicine Research Unit (J.K.L.), Brigham & Women’s Hospital and Harvard Medical School, Boston, Mass.

Correspondence to Jianxin Sun, PhD, Department of Cell Biology & Molecular Medicine, UMDNJ-New Jersey Medical School, 185 S Orange Ave, MGB G-653, Newark, NJ 07103. E-mail sunj1{at}umdnj.edu

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 1- 1 (eEF1A1). In HUVECs, TNF- 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-–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.

Key Words: TNF- • eNOS • mRNA • stability • translation elongation factor 1-

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