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 (H ₂O₂), 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 μM, 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 H₂O ₂ 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 H ₂O₂ or IFN-β, 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-β-treated cells (t1/2 = 120 min vs. >30 min in H ₂O₂- vs. IFN-β-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.