Rapid responses to oxidative stress mediated by iron regulatory protein

Reactive oxygen intermediates (ROIs), including superoxide anion (O2.-) and hydrogen peroxide (H2O2), are by-products of aerobic metabolism with potential toxicity towards cellular macromolecules, including lipids, proteins and DNA. Excess ROIs, a condition referred to as oxidative stress, is considered to be a major contributor to ageing, degenerative diseases and reperfusion injury. The reactivity of H2O2 with iron (Fenton reaction) intimately connects oxidative stress and cellular iron metabolism. We have found a novel oxidative stress response pathway in mammalian cells which links oxidative stress to the regulation of iron metabolism. Exposure of cells to H2O2 leads to reduced synthesis of the intracellular iron storage protein ferritin and stimulates transferrin receptor (TfR) mRNA expression. Both responses are post-transcriptional and result from induction of iron regulatory protein (IRP) binding to iron-responsive elements (IREs) in ferritin and TfR mRNAs. IRP induction by H2O2 appears to involve the disassembly of its cubane 4Fe-4S cluster and occurs even in the presence of the protein synthesis inhibitor cycloheximide. The induction kinetics by H2O2 far exceed those by iron starvation. The response requires cellular integrity and cannot be elicited in cell extracts. Whereas the activation of IRP by iron depletion is insensitive to okadaic acid, the rapid induction by H2O2 is blocked by this inhibitor of type I/IIa protein phosphatases. Thus okadaic acid separates the activation pathways by iron depletion and oxidative stress, suggesting the involvement of stress-induced kinase/phosphatase pathways in the latter.