Given the important relationship between O2 and
iron (Fenton chemistry) a study was undertaken to characterize the effects of
hypoxia, as well as subsequent reoxygenation, on the
iron-regulatory proteins 1 and 2 (IRP1 and IRP2) in a rat
hepatoma cell line. IRP1 and IRP2 are cytosolic
RNA-binding proteins that bind
RNA stem-loops located in the 5'- or 3'-untranslated regions of specific mRNAs encoding
proteins that are involved in
iron homeostasis. In cells exposed to
hypoxia, IRP1
RNA binding was decreased approximately 2. 8-fold after a 6-h exposure to 3% O2. Hypoxic inactivation of IRP1 was abolished when cells were pretreated with the
iron chelator desferrioxamine, indicating a role for
iron in inactivation. IRP1 inactivation was reversible since re-exposure of hypoxically-treated cells to 21% O2 increased
RNA binding activity approximately 7-fold after 21 h with an increase in activity seen as early as 1-h post-reoxygenation.
IRP1 protein levels were unaffected during
hypoxia as well as during reoxygenation. Whereas the
protein synthesis inhibitor cycloheximide did not block IRP1 inactivation during
hypoxia, it completely blocked IRP1 reactivation during subsequent reoxygenation. Reactivation of IRP1 during reoxygenation was also partially blocked by the
phosphatase inhibitor
okadaic acid. Finally, reactivated IRP1 was found to be resistant to inactivation by exogenous
iron known to down-regulate its activity during normoxia. These data demonstrate that IRP1
RNA binding activity is post-translationally regulated during
hypoxia and
hypoxia/reoxygenation. Regulation of IRP1 by changing
oxygen tension may provide a novel mechanism for post-transcriptionally regulating gene expression under these stresses.