Up-regulation of the interferon-inducible IFI16 gene by oxidative stress triggers p53 transcriptional activity in endothelial cells.
Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), induces injury of endothelium in a variety of pathophysiological conditions, such as inflammation, aging, and cancer. In our study, we characterized the signaling pathway linking oxidative stress induced by sublethal concentrations of H2O2 to p53 in primary human endothelial cells through the interferon (IFN)-inducible gene IFI16. Induction of IFI16 by H2O2 was concentration- and time-dependent (maximum at 50 microM, 6 h after treatment) and down-regulated by pretreatment with N-acetyl-L-cysteine, which acts as an antioxidant. This pathway is a general response to ROS and not specific to H2O2 treatment, as two other ROS-generating compounds, i.e., S-nitroso-N-acetylpenicillamine and tert-butyl hydroperoxide, were equally capable to induce IFI16. Moreover, IFI16 up-regulation is a result of protein accumulation, as expression of corresponding mRNA, assessed by real-time polymerase chain reaction, was not affected. To investigate the mechanism of IFI16 accumulation, cells were incubated for 6 h in the presence of H2O2 or IFN-beta, and then cycloheximide was added to inhibit further protein synthesis. The half-life of IFI16 protein was found to be significantly increased in H2O2-treated cells compared with IFN-beta-treated cells (t1/2 = 120 min vs. > 30 min in H2O2- vs. IFN-beta-treated cells, respectively). An increase of IFI16 was accompanied by interaction with p53 phosphorylated at its N terminus, as shown by immunoprecipitation experiments. Moreover, binding to IFI16 resulted in its transcriptional activation as shown by an increase in the activity of a reporter gene driven by p53-responsive sequences derived from the p21(WAF1) promoter, along with an increase in the p21 mRNA and protein levels. Altogether, these results demonstrate a novel role of IFI16 in the signal transduction pathway that leads to p53 activation by oxidative stress in endothelial cells.
Gugliesi
Francesca
F
Department of Public Health and Microbiology, University of Turin, Via Santena 9, 10126-Torino, Italy.
Mondini
Michele
M
Ravera
Raffaella
R
Robotti
Andrea
A
de Andrea
Marco
M
Gribaudo
Giorgio
G
Gariglio
Marisa
M
Landolfo
Santo
S
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004730:Endothelium, Vascular / Q000502:physiology; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D009687:Nuclear Proteins; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; C076700:IFI16 protein, human