Nitric Oxide and Oxidative Stress (H₂O₂) Control Mammalian Iron Metabolism by Different Pathways

Abstract

Several cellular mRNAs are regulated posttranscriptionally by iron-responsive elements (IREs) and the cytosolic IRE-binding proteins IRP-1 and IRP-2. Three different signals are known to elicit IRP-1 activity and thus regulate IRE-containing mRNAs: iron deficiency, nitric oxide (NO), and the reactive oxygen intermediate hydrogen peroxide (H2O2). In this report, we characterize the pathways for IRP-1 regulation by NO and H2O2 and examine their effects on IRP-2. We show that the responses of IRP-1 and IRP-2 to NO remarkably resemble those elicited by iron deficiency: IRP-1 induction by NO and by iron deficiency is slow and posttranslational, while IRP-2 induction by these inductive signals is slow and requires de novo protein synthesis. In contrast, H2O2 induces a rapid posttranslational activation which is limited to IRP-1. Removal of the inductive signal H2O2 after < or = 15 min of treatment (induction phase) permits a complete IRP-1 activation within 60 min (execution phase) which is sustained for several hours. This contrasts with the IRP-1 activation pathway by NO and iron depletion, in which NO-releasing drugs or iron chelators need to be present during the entire activation phase. Finally, we demonstrate that biologically synthesized NO regulates the expression of IRE-containing mRNAs in target cells by passive diffusion and that oxidative stress endogenously generated by pharmacological modulation of the mitochondrial respiratory chain activates IRP-1, underscoring the physiological significance of NO and reactive oxygen intermediates as regulators of cellular iron metabolism. We discuss models to explain the activation pathways of IRP-1 and IRP-2. In particular, we suggest the possibility that NO affects iron availability rather than the iron-sulfur cluster of IRP-1.

Redox control of iron regulatory protein 2 stability

“…Our findings seemingly contradict previous conclusions that H 2 O 2 promotes rapid induction of IRP1 without affecting IRP2 activity [18]. Early studies did not always take into account the notorious sensitivity of IRP2 to post-lysis degradation in tissue and cell extracts [30].…”

“…Human H1299 lung cancer cells [16] and mouse Ltk-fibroblasts [18] were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, and 0.1 ng/ml streptomycin. RAW 264.7 macrophages (ATCC number CRL-2278™) were cultured in RPMI-1640 medium including supplements.…”

“…2B), despite the fact that extracellular H 2 O 2 is rapidly metabolized by cultured cells [21,22]. We chose this time point to avoid possible interference with an early transient H 2 O 2 -mediated activation of IRP1 [18,21], and assess the implications of IRP2 stabilization on IRE-binding activity and expression of IRE-containing mRNAs. We then evaluated the effects of H 2 O 2 -mediated stabilization of IRP2 in the expression of two known IRP downstream targets, ferritin and transferrin receptor 1 (TfR1).…”

Dinitrosyliron complexes and the mechanism(s) of cellular protein nitrosothiol formation from nitric oxide

“…Additionally, we have used •NO donor rather than •NO from endogenous formation. It may be, for example, that relatively long-term •NO exposure during up-regulation of inducible NOS results in •NO-induced modifications in protein expression and/or iron homeostatic responses to modifications in the CIP (39). Finally, our proposal that •NO affects nitrosation via DNIC formation and subsequent transnitrosation to thiols is based on indirect evidence (kinetics, O 2 independence, CIP dependence) and the direct correlation between RSNO and DNIC with different experimental manipulations.…”

Translational Regulation of Human Neuronal Nitric-oxide Synthase by an Alternatively Spliced 5′-Untranslated Region Leader Exon

“…We initially favored an IRE/IRP hypothesis for the regulation of nNOS given the known effects of NO in augmenting IRP binding to IRE sequences (62)(63)(64). This would incorporate a translational feedback inhibition loop into the regulation of select human nNOS mRNAs.…”