Translational control of Nrf2 protein in activation of antioxidant response by oxidants

Nf-E2 related factor-2 (Nrf2) is a basic leucine zipper transcription factor that binds and activates the antioxidant response element (ARE) in the promoters of many antioxidant and detoxification genes. We found that H ₂O₂ treatment caused a rapid increase in endogenous Nrf2 protein level in rat cardiomyocytes. Semiquantitative or real-time reverse transcription-polymerase chain reaction failed to show an increase of Nrf2 mRNA level by H₂O₂ treatment. Measurements of Nrf2 protein stability excluded the possibility of Nrf2 protein stabilization. Although inhibiting protein synthesis with cycloheximide prevented H₂O ₂ from elevating Nrf2 protein level, RNA synthesis inhibition with actinomycin D failed to do so. Measurements of new protein synthesis with [ ³⁵S]methionine incorporation confirmed that H₂O ₂ increased the translation of Nrf2 protein. Inhibitors of phosphoinositide 3-kinase were able to abolish the induction of Nrf2 protein by H₂O₂. Although H₂O₂ increased phosphorylation of p70 S6 kinase, rapamycin failed to inhibit H ₂O₂ from elevating Nrf2 protein. H₂O ₂ also induced phosphorylation of eukaryotic translation initiation factor (eIF) 4E and eIF2α within 30 and 10 min, respectively. Inhibiting eIF4E with small interfering siRNA or increasing eIF2α phosphorylation with salubrinal did not affect Nrf2 elevation by H₂O₂. Our data present a novel phenomenon of quick onset of the antioxidant/ detoxification response via increased translation of Nrf2 by oxidants. The mechanism underlying such stress-induced de novo protein translation may involve multiple components of translational machinery.