Tumor necrosis factor-alpha downregulates endothelial nitric oxide synthase mRNA stability via translation elongation factor 1-alpha 1

Abstract

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.

Figure 1. Identification and characterization of eNOS 3'-UTR binding proteins

(A) eNOS 3'-UTR binding proteins were purified by RNA affinity chromatography from a cytosolic fraction of unstimulated and stimulated HUVECs with 20 ng/mL TNF-α for 24 hr. After extensive washing , proteins bound to the biotinylated eNOS 3'-UTR coupled to streptavidin agarose beads were eluted with SDS-PAGE sample buffer , electrophoretically separated by 12 % SDS-PAGE, and Coomassie-stained. The target protein bands at 57 kD and 52 kD were digested with trypsin followed by MALDI-TOF-MS analysis. (B) R-EMSA was performed by incubating the radiolabeled eNOS 3'-UTR with increasing concentration of GST-eEF1A1 (1μg and 5μg) and a fixed concentration of GST (5μg). Competition with specific competitor (50 molar excess) corresponding to unlabeled eNOS 3'-UTR transcript was also performed in the presence of 5μg GST-eEF1A1. (C) EMSA was performed by incubating the radiolabeled eNOS 3'-UTR with 30 μg cytosolic extracts from HUVECs transduced with AdeEF1A1 for 48 hr in the presence and absence of anti-eEF1A antibody. (D) UV Cross-linking was performed by incubating the radiolabeled eNOS 3'-UTR with increasing concentration of GST-eEF1A1 (1μg and 5μg) and a fixed concentration of GST (5μg). Competition with specific competitor (50 molar excess) corresponding to unlabeled eNOS 3'-UTR transcript was also performed in the presence of 5μg GST-eEF1A1. (E) Immunoprecipitation, followed by RT-PCR assays, showing interaction of eEF1A and eNOS mRNA 3'-UTR in HUVECs treated with or without TNF-α (20 ng/ml) for 1 hr.

Figure 2. Analysis of the eEF1A1-binding site in the human eNOS 3'-UTR

(A) Schematic representation of human eNOS 3'-UTR mutants. (B) UV cross-linking showing the binding of eEF1A1 to the different deletion mutants of human eNOS 3'-UTR.

Figure 3. Identification of the eNOS 3'-UTR binding site in eEF1A1

(A) Schematic representation of human eEF1A1 functional domains. (B) Identification of GST-fusion proteins containing different domains of eEF1A1. GST-fusion proteins were bacterially expressed and purified by glutathione-Sepharose beads. Purified proteins were separated on 12% SDS-PAGE and stained with Coomassie Blue R250. (C) UV cross-linking showing the binding of eEF1A1 domain III (D3) to the human eNOS 3'-UTR. UV cross-linking assays were performed by incubating the radiolabeled eNOS 3'-UTR with equal molar of GST fusion proteins.

Figure 4. Effect of eEF1A1 on the expression of eNOS

(A) Cos-7 cells in 100 mm plate were transfected with eNOS cDNA containing 3'-UTR in combination of either empty vector or myctagged eEF1A1 cDNAs at the concentrations indicated. 48hr after transfection, cell lysates were collected for western blot analysis. (B) Cos-7 cells and (C) BAECs in 6-well plates were cotransfected with Luc-eNOS-UTR reporter plasmid together with either pCS2-Myc-eEF1A1 expression or empty vectors. 48hr after transfection, cell lysates were assayed for luciferase activities. n=5, * p<0.05 vs AdLacZ alone.

Figure 5. Overexpression of eEF1A1 mimics the downregulation of eNOS expression by TNF-α

(A) TNF-α increases the expression of eEF1A in HUVECs. Cells were treated with TNF-α (20 ng/ml) for various time points and cell lysates were then collected for western blot analysis using anti-eEF1A and anti-α-tubulin antibodies. (B) Overexpression of Flag-tagged eEF1A1 inhibits the expression of eNOS in HUVECs. HUVECs were transduced with Ad-LacZ and AdeEF1A1 at the MOI of 100. 48 hr after transduction, cells were then treated with TNF-α (20 ng/ml) for 24 hr. The expression of eNOS was detected by western blot analysis and quantitated by densitometry. *p<0.05 vs Ad-LacZ. (C) Overexpression of eEF1A1 decreases eNOS mRNA stability. Preconfluent HUVECs were transduced with Ad-Lacz and Ad-eEF1A1 at the MOI of 100 for 48 hr. 20 μg/mL DRB was then added and eNOS mRNA half-life was determined by Northern blot analysis. A representative Northern blot is shown. (D) Combined results from 5 different experiments of eNOS mRNA half-life determination by Northern blot analysis.

Figure 6. eEF1A1 knockdown prevents TNF-α-induced destabilization of eNOS mRNA stability

(A) Western blotting showing the expression of eEF1A1 in HUVECs transfected with eEF1A1 specific small interference RNA (siRNA) and control siRNA. (B) Knockdown of eEF1A1 attenuates TNF-α induced destabilization of eNOS mRNA. HUVECs were transfected with eEF1A1 specific siRNA (eEFsiRNA) and control siRNA (CTLsiRNA). 72 hr after transfection, HUVECs were treated with TNF-α (20ng/ml) and eNOS mRNA half-life was determined by Northern blot analysis. The data are representative of 5 independent experiments. (C) Representative Western blots showing the effects of control and eEF1A1 siRNA on eEF1A and eNOS expression in the presence or absence of TNF-α (20 ng/ml). The data represents 3 independent experiments. *P<0.05 vs control siRNA alone or eEF1A siRNA + TNF-α.