REGULATION OF IRON REGULATORY PROTEIN-1 DURING HYPOXIA AND HYPOXIA REOXYGENATION/

Given the important relationship between O-2 and iron (Fenton chemistr y) a study was undertaken to characterize the effects of hypoxia, as w ell as subsequent reoxygenation, on the iron-regulatory proteins 1 and 2 (IRP1 and IRP2) in a rat hepatoma cell line. IRP1 and IRP2 are cyto solic RNA-binding proteins that bind RNA stem-loops located in the 5'- or 3'-untranslated regions of specific mRNAs encoding proteins that a re involved in iron homeostasis. In cells exposed to hypoxia, IRP1 RNA binding was decreased similar to 2.8-fold after a 6-h exposure to 3% O-2. Hypoxic inactivation of IRP1 was abolished when cells were pretre ated with the iron chelator desferrioxamine, indicating a role for iro n in inactivation. IRP1 inactivation was reversible since re-exposure of hypoxically-treated cells to 21% O-2 increased RNA binding activity similar to 7-fold after 21 h with an increase in activity seen as ear ly as 1-h post-reoxygenation. IRP1 protein levels were unaffected duri ng hypoxia as well as during reoxygenation. Whereas the protein synthe sis inhibitor cycloheximide did not block IRP1 inactivation during hyp oxia, it completely blocked IRP1 reactivation during subsequent reoxyg enation. Reactivation of IRP1 during reoxygenation was also partially blocked by the phosphatase inhibitor okadaic acid. Finally, reactivate d IRP1 was found to be resistant to inactivation by exogenous iron kno wn to down-regulate its activity during normoxia. These data demonstra te that IRP1 RNA binding activity is post-translationally regulated du ring hypoxia and hypoxia/reoxygenation. Regulation of IRP1 by changing oxygen tension may provide a novel mechanism for post-transcriptional ly regulating gene expression under these stresses.