Abstract
Cellular iron uptake and storage are coordinately controlled by binding of iron-regulatory proteins (IRP), IRP1 and IRP2, to iron-responsive elements (IREs) within the mRNAs encoding transferrin receptor (TfR) and ferritin. Under conditions of iron starvation, both IRP1 and IRP2 bind with high affinity to cognate IREs, thus stabilizing TfR and inhibiting translation of ferritin mRNAs. The IRE/IRP regulatory system receives additional input by oxidative stress in the form of H(2)O(2) that leads to rapid activation of IRP1. Here we show that treating murine B6 fibroblasts with a pulse of 100 microm H(2)O(2) for 1 h is sufficient to alter critical parameters of iron homeostasis in a time-dependent manner. First, this stimulus inhibits ferritin synthesis for at least 8 h, leading to a significant (50%) reduction of cellular ferritin content. Second, treatment with H(2)O(2) induces a approximately 4-fold increase in TfR mRNA levels within 2-6 h, and subsequent accumulation of newly synthesized protein after 4 h. This is associated with a profound increase in the cell surface expression of TfR, enhanced binding to fluorescein-tagged transferrin, and stimulation of transferrin-mediated iron uptake into cells. Under these conditions, no significant alterations are observed in the levels of mitochondrial aconitase and the Divalent Metal Transporter DMT1, although both are encoded by two as yet lesser characterized IRE-containing mRNAs. Finally, H(2)O(2)-treated cells display an increased capacity to sequester (59)Fe in ferritin, despite a reduction in the ferritin pool, which results in a rearrangement of (59)Fe intracellular distribution. Our data suggest that H(2)O(2) regulates cellular iron acquisition and intracellular iron distribution by both IRP1-dependent and -independent mechanisms.
Highlights
Cellular iron uptake and storage are coordinately controlled by binding of iron-regulatory proteins (IRP), IRP1 and IRP2, to iron-responsive elements (IREs) within the mRNAs encoding transferrin receptor (TfR) and ferritin
H2O2 Elicits a Time-dependent Stimulation of TfR and Inhibition of Ferritin Synthesis—We have shown previously that treatment of cells with micromolar concentrations of H2O2 results in rapid induction of IRP1 to bind to IREs and that IRE
B6 fibroblasts were first treated with 100 M H2O2 for 1 h and metabolically labeled with [35S]methionine/cysteine for 2 h either immediately or at different time points after treatment, and TfR and ferritin synthesis were assessed by immunoprecipitation (Fig. 1, top panel)
Summary
Transferrin; IRP1, iron regulatory protein 1; IRE, iron-responsive element; UTR, untranslated region; TfR, transferrin receptor; m-, mitochondrial and c-, cytosolic aconitase; DMT1, divalent metal transporter 1; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; DFO, desferrioxamine. Under conditions of iron starvation, IRP1 and IRP2 are activated for high affinity binding to multiple “iron-responsive elements” (IREs) in the 3Ј-untranslated region (UTR) of TfR mRNA and to a single IRE in the 5Ј-UTR of the mRNAs encoding both H- and L-ferritin chains. Failure of IRPs to bind to cognate IREs in iron-replete cells leads to degradation of TfR mRNA and synthesis of ferritin We showed that a treatment of these cells with 100 M H2O2 for 1 h inhibits ferritin synthesis, whereas longer treatments (4 – 6 h) increase TfR mRNA levels, as a result of IRP1 activation [26] These responses have not been correlated with the biological activity of TfR and ferritin, in terms of iron uptake and sequestration. We extend the previous studies and investigate the effects of H2O2 in the expression and function of several IRE-containing mRNAs, as reflected in the uptake of 59Fetransferrin and intracellular management of 59Fe
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