Keywords:
IRE (iron-responsive element), IRP (iron-regulatory protein), LIPS (labile iron pool), mRNA (messenger RNA), NO (nitrogen monoxide), TfR (transferrin receptor), UTR (untranslated region)Over the last 10 years, great interest has been focused on the effects of hypoxia and NO on cellular iron metabolism. This can be attributed mainly to the discovery of iron-regulatory protein-1 (IRP-1), which is a [4Fe-4S] cluster–containing RNA-binding molecule (see references 1 and 2 for reviews). This protein regulates intracellular iron metabolism and is sensitive to iron levels and intracellular redox status.
1
, 2
Indeed, NO, oxidant stress, and hypoxia are known to regulate IRP-1 RNA–binding activity.1
, 2
The discovery of a second IRP, known as IRP-2, that is also sensitive to iron and free radicals adds further complexity to this intricate regulatory mechanism.1
, 2
Both IRPs bind to IREs in the UTRs of mRNAs that encode molecules involved in iron metabolism.
1
These include TfR-1, which is involved in iron uptake from transferrin and the iron storage protein ferritin.1
, 2
Both IRPs regulate the levels of the LIP, a poorly characterized intermediate form of iron that exists in equilibrium between different compartments.1
, 2
This iron is thought to be present as Fe(II) and Fe(III), and the equilibrium between these two states can be markedly altered by intracellular redox potential. Both IRPs sense iron levels in the LIP and control the synthesis of ferritin and TfR-1 by binding to IREs in the 5′ or 3′-UTRs of their mRNAs.1
, 2
When intracellular iron levels are low in the LIP, IRPs bind to IREs stabilizing TfR-1 mRNA and blocking translation of ferritin mRNA, leading to increased iron uptake and decreased iron storage, respectively. In contrast, high levels of iron in the LIP prevent RNA-binding of IRP-1 as a result of the formation of an [4Fe4S] cluster within the protein. In addition, high iron levels result in the proteasome-mediated degradation of IRP-2.
1
, 2
With the use of fibroblasts, hepatoma cells, and macrophages in culture, NO has been shown to increase IRP RNA–binding activity, resulting in increased TfR-1 expression and reduced ferritin translation.
1
, 2
, 3
, 4
, 5
The mechanism behind this increase in IRP RNA–binding activity may be a result of the NO-mediated depletion of cellular iron or the ability of NO to directly disassemble the [4Fe-4S] cluster of IRP-1 to increase RNA-binding activity.1
, 2
However, these studies conflict with other investigations involving macrophages showing that inflammation or incubation with interferonγ/lipopolysaccharide leads to marked NO production that stimulates ferritin synthesis and decreases TfR-1 mRNA levels.6
, 7
, 8
, 9
, 10
This effect was caused by NO as inhibitors of nitric oxide synthase prevented the response.10
Moreover, the increase in ferritin and decrease in TfR-1 expression were mediated by IRP-2, evidenced by a marked decrease in its RNA–binding activity.8
, 9
, 10
The findings of recent studies
9
, 10
have suggested that the different effects of NO described above are a result of the various redox-related states of this molecule generated under different conditions, eg, the nitrosonium ion [NO+] and nitric oxide [NO·]. For instance, the effect of NO in mediating increased IRP RNA–binding activity may be mediated by NO·; as it avidly binds iron and could result in iron release from IRP-1 and the cell.10
Exposure of macrophages to inflammatory cytokines may generate NO+, which then S-nitrosylates IRP-2 and results in its degradation, leading to increased ferritin translation.10
However, further studies are required to confirm this hypothesis, as there is no direct evidence that IRP-2 is S-nitrosylated by NO+.The effects of hypoxia on iron metabolism can also be at least partly linked to the changes in RNA–binding activity of the IRPs. Indeed, IRP-1 RNA–binding activity has been shown to decrease in macrophages and hepatoma cells during hypoxia,
11
, 12
and these conditions are known to increase ferritin expression.13
Theoretically, under hypoxic conditions, the Fe(II) state is favored in preference to Fe(III), which could result in the release of iron from ferritin or other compartments. The increased amount of Fe(II) could result in two effects: (1) It may be incorporated into the [4Fe-4S] cluster of IRP-1 and decrease RNA-binding activity, and (2) it could result in increased IRP-2 degradation.It is notable that oxidant stress has been shown to have both transcriptional and post-transcriptional effects on ferritin expression.
2
Indeed, oxidant stress can directly increase the transcription of the ferritin gene.14
For example, this occurs after exposure to agents which decrease glutathione levels that acts as a defense against free radicals.15
Further, oxidant stress can inactivate IRP-1 by oxidizing critical sulfydryl groups of cysteine residues that are important for mRNA binding.16
On the other hand, incubation of cells with hydrogen peroxide can activate IRP-1 RNA–binding by inducing disassembly of the [4Fe-4S] cluster that consequently increases IRP-IRE binding and prevents ferritin translation.17
Such a response is not protective in terms of oxidant stress and it could result in decreased levels of ferritin, which is necessary to store catalytically-active iron.18
In this issue of the journal, Smith et al
19
publish their findings on the effects of NO and hypoxia on the ferritin content of alveolar cells. This work was done in an attempt to understand why ferritin levels are enhanced in the lungs of patients with respiratory disorders. In this study,19
two cell types were assessed — namely, a lung cancer–derived epithelial cell line (A549) and primary cultures of alveolar macrophages. Hypoxia resulted in increased ferritin levels in both cell types, and this was well correlated with the findings of previous studies.11
, 12
In contrast, the effects of NO on each cell type was different, increasing ferritin content in A549 cells but having no appreciable effect on total ferritin levels in alveolar macrophages.19
In addition, in contrast to A549 cells, ferritin release was found to occur in macrophages at NO levels that did not cause marked cytotoxicity.19
The increase in ferritin levels after exposure of A549 cells to NO defies its “classic” effect at increasing IRP RNA–binding activity that depresses ferritin translation.
1
, 2
, 3
, 4
, 5
One possible important clue to elucidating the mechanism of this effect is the finding that the GSH precursor N-acetylcysteine reduced hypoxia-inducible ferritin accumulation in both cell types and the NO-inducible ferritin expression in A549 cells.19
These results suggest that the increase in ferritin levels in the presence of NO or hypoxia causes glutathione depletion as a result of oxidative stress. This could have led to a decrease in IRP RNA–binding activity, direct transcriptional up-regulation of ferritin, or both. It is of interest that the macrophages did not respond to NO in the same way as A549 cells and this discrepancy could be related to differences in the ability of these cell types to handle oxidative stress. Indeed, macrophages are “professional killer cells” with an impressive oxidative arsenal that may lead to efficient mechanisms with which to deal with collateral insults on their own biochemistry.In the investigation by Smith et al,
19
the effects of NO and hypoxia were focused on examination of iron and ferritin levels. Obviously, these studies provide solid groundwork for further experiments assessing the precise molecular mechanisms involved. Indeed, evaluating the effect of NO and hypoxia on IRP-IRE–binding activity and ferritin gene transcription in both cell types could be useful in further understanding the changes in ferritin observed in respiratory disorders.References
- Molecular control of vertebrate iron metabolism.Proc Natl Acad Sci U S A. 1996; 93: 8175-8182
- Regulation of ferritin genes and protein.Blood. 2002; 99: 3505-3516
- Biosynthesis of nitric oxide activates iron regulatory factor in macrophages.EMBO J. 1993; 12: 3643-3649
- Translational regulation via ironresponsive elements by the nitric oxide NO-synthase pathway.EMBO J. 1993; 12: 3651-3657
- Effects of nitrogen monoxide species on cellular proliferation and transferrin and iron uptake by erythroleukemia (K562) cells.Blood. 1995; 86: 3211-3219
- Ferritin synthesis in inflammation. I. Pathogenesis of impaired iron release.Br J Haematol. 1977; 37: 7-16
- Ferritin synthesis in inflammation. II. Mechanisms of increased ferritin synthesis.Br J Haematol. 1981; 49: 361-370
- Nitric oxide–mediated induction of ferritin synthesis in J774 macrophages by inflammatory cytokines.Blood. 1998; 91: 1059-1061
- Effects of interferon-γ and lipopolysaccharide on macrophage iron metabolism are mediated by nitric oxide-induced degradation of iron regulatory protein 2.J Biol Chem. 2000; 275: 6220-6226
- Nitrogen monoxide–mediated control of ferritin synthesis.Proc Natl Acad Sci U S A. 2002; 99: 12214-12219
- Regulation of iron regulatory protein 1 during hypoxia and hypoxia/reoxygenation.J Biol Chem. 1998; 273: 7588-7593
- Effects of hyperoxia and iron on iron regulatory protein-1 activity and the ferritin synthesis in mouse peritoneal macrophages.Biochim Biophys Acta. 2001; 1544: 370-377
- Hypoxia alters iron homeostasis and induces ferritin synthesis in oligodendrocytes.J Neurochem. 1995; 64: 2458-2464
- Coordinate transcriptional and translational regulation of ferritin in response to oxidative stress.Mol Cell Biol. 2000; 20: 5818-5827
- Induction of ferritin synthesis by oxidative stress.J Biol Chem. 1995; 270: 700-703
- Superoxide and hydrogen peroxide–dependent inhibition of iron regulatory protein activity.FASEB J. 1996; 10: 1326-1335
- Activation of iron regulatory protein-1 by oxidative stress in vitro.Proc Natl Acad Sci U S A. 1998; 95: 10559-10563
- Nitric oxide signaling to iron-regulatory protein.Proc Natl Acad Sci U S A. 1995; 92: 1267-1271
Smith JL, O’Brien-Ladner AR, Kaiser CR, Wesselius LJ. Effects of hypoxia and nitric oxide on ferritin content of alveolar cells. J Lab Clin Med 2003;141:309-17
Article info
Identification
Copyright
© 2003 Elsevier Science Inc. Published by Elsevier Inc. All rights reserved.
