Tumor necrosis factor-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)-alpha at the posttranscriptional level. To elucidate the molecular basis of TNF-alpha-mediated eNOS mRNA instability, eNOS 3' untranslated region (3'-UTR) binding proteins were purified by RNA affinity chromatography from cytosolic fractions of TNF-alpha-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-alpha. 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-alpha 1 (eEF1A1). In HUVECs, TNF-alpha 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-alpha-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-alpha-induced decrease in eNOS mRNA stability.
Yan
Guijun
G
Department of Cell Biology & Molecular Medicine, UMDNJ-New Jersey Medical School, Newark, NJ 07103, USA.
You
Bei
B
Chen
Shao-Ping
SP
Liao
James K
JK
Sun
Jianxin
J
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D015536:Down-Regulation / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D020648:Peptide Elongation Factor 1 / Q000235:genetics / Q000502:physiology*; D014176:Protein Biosynthesis / Q000502:physiology; D020871:RNA Stability / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
C094814:EEF1A1 protein, human; D020648:Peptide Elongation Factor 1; D014409:Tumor Necrosis Factor-alpha; D052250:Nitric Oxide Synthase Type III