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Mammalian Tissue Oxygen Levels Modulate Iron-Regulatory Protein Activities in Vivo

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Science  17 Dec 2004:
Vol. 306, Issue 5704, pp. 2087-2090
DOI: 10.1126/science.1103786

Abstract

The iron-regulatory proteins (IRPs) posttranscriptionally regulate expression of transferrin receptor, ferritin, and other iron metabolism proteins. Although both IRPs can regulate expression of the same target genes, IRP2–/– mice significantly misregulate iron metabolism and develop neurodegeneration, whereas IRP1–/– mice are spared. We found that IRP2–/– cells misregulated iron metabolism when cultured in 3 to 6% oxygen, which is comparable to physiological tissue concentrations, but not in 21% oxygen, a concentration that activated IRP1 and allowed it to substitute for IRP2. Thus, IRP2 dominates regulation of mammalian iron homeostasis because it alone registers iron concentrations and modulates its RNA-binding activity at physiological oxygen tensions.

Iron-regulatory proteins 1 and 2 (IRP1 and IRP2) bind with high affinity to RNA motifs known as iron-responsive elements (IREs) in numerous transcripts related to iron homeostasis. In ferritin H and L chains (H, heart; L, liver), IRP binding near the 5′ end of the mRNA inhibits translation. In contrast, binding of IRPs to IREs in the 3′ portion of the transferrin receptor 1 (Tf R1) mRNA stabilizes Tf R transcripts (13). Although the genes encoding IRP1 and IRP2 are highly homologous, they sense cytosolic iron levels by different mechanisms. IRP1 is a bifunctional protein; in iron-replete cells, IRP1 contains a cubane [4Fe-4S] cluster and functions as a cytosolic aconitase, interconverting citrate and isocitrate, whereas when the Fe-S cluster is absent, IRP1 apoprotein binds IREs with high affinity (4). Unlike IRP1, IRP2 undergoes iron-dependent degradation in iron-replete cells (5, 6). Thus, neither IRP binds to IREs when cells are iron-replete, and ferritin expression increases accordingly, while TfR expression decreases.

In cell lines derived from a wide variety of tissues, IRP1 generally appears to be the major contributor to total iron-regulatory activity (13). In primary cultures of wild-type (WT) mouse embryonic fibroblasts, IRE-binding activity of IRP1 increased markedly in iron-depleted cells and greatly exceeded that of IRP2 under both iron-replete and iron-depleted conditions (Fig. 1A). In addition, increased IRE-binding activity of IRP1 correlated with a marked decrease in cytosolic aconitase activity (Fig. 1B).

Fig. 1.

IRP1 is an effective iron sensor in cultured mouse embryonic fibroblasts, but not in mouse liver. (A) Embryonic fibroblasts grown for 16 hours in 21% O2 in unsupplemented medium (lane C), medium supplemented with 300 μM ferric ammonium citrate (FAC) (lane F), or 100 μM of the iron chelator deferoxamine mesilate (Dfo) (lane D) show increased IRE-binding activity of IRPs in a gel-shift assay in iron-depleted cells. IRP1 can be recruited from its non–IRE-binding form to bind IREs by addition of 2% β-mercaptoethanol (+2ME), which indirectly assesses total IRP1 levels. (B) IRP1 from iron-replete cells is an active aconitase (lanes F and C), but IRP1 in iron-depleted cells loses aconitase activity as IRP1 switches to the IRE-binding form (lane D). The mitochondrial aconitase (m-aco) and cytosolic aconitase (c-aco) bands were identified as described (9). (C) Liver lysates from WT animals on low- or high-iron diets show that IRE-binding activity of IRP2 is regulated according to iron status, whereas IRE-binding activity of IRP1 does not change. Most IRP1 is in a non–IRE-binding form that can be recruited in vitro by +2ME. (D) Cytosolic aconitase activities are equal in iron-deficient and iron-replete animals.

Despite the ability of IRP1 to significantly modulate its IRE-binding activity in cell culture according to iron status, and its ability to regulate expression of target transcripts (7, 8), animals in which IRP1 is genetically ablated display normal regulation of iron metabolism in most tissues (9). In contrast, animals that lack IRP2 express abnormally high amounts of ferritin and low amounts of TfR in multiple tissues, and they develop progressive adult-onset neurodegeneration (10). In liver lysates from iron-deficient WT animals, the IRE-binding activity of IRP2 increased in response to iron deficiency, whereas the IRE-binding activity and aconitase activity of IRP1 remained constant (Fig. 1, C and D) (9).

To understand why the regulatory activities of IRP1 and IRP2 differed in animal tissues compared to cell lines, we analyzed primary cultures of macrophages and lymphocytes from WT, IRP1–/–, and IRP2–/– animals. In both cell types, IRP1 appeared to be the predominant IRE-binding protein when cells were cultured in atmospheric oxygen (21% O2). Because mammalian tissue oxygen concentrations are closer to 3 to 6% than 21% (1114), and because IRP activities can be affected by oxygen (15), these experiments were carried out at oxygen concentrations ranging from 3 to 21% to evaluate how IRP activities vary accordingly. In macrophages, the change in oxygen concentration from 3 to 21% led to a dramatic switch in the proportion of IRE-binding activity contributed by each IRP. IRP2 was the predominant source of IRE-binding activity at 3% O2, whereas IRP1 became the major source of IRE-binding activity at 21% O2. As IRP1 activity increased at 21% O2, IRP2 levels decreased. In addition, at 3% O2, IRP1 lost most of its ability to respond to changes in iron status (Fig. 2A and fig. S1). Thus, the relative importance of each IRP in the regulation of iron metabolism varied greatly with changes in oxygen concentration.

Fig. 2.

IRP2 is an effective iron sensor in macrophages and lymphocytes grown at oxygen concentrations ranging from 3 to 21%, whereas IRP1 is a poor sensor at low oxygen concentrations. (A) The ratio of IRE-binding activities of IRP1 and IRP2 in macrophages, determined by gel retardation assay, shifts markedly when oxygen concentrations change. After 6 days of growth at 3% O2, macrophages were exposed for 48 hours to either 3 or 21% O2 and to iron treatments as described in Fig. 1A. (B) IRP2 levels are highly regulated in immunoblot assays of primary WT macrophages at both 6 and 21% O2 concentrations. After cells were incubated for 6 days at 6% O2, they were exposed for 48 hours to either 6 or 21% O2 and treated with a constant amount of 100 μM FAC and increasing amounts of Dfo. To verify even protein loading, we stained immunoblot membranes with PonceauS, and a typical band from this gel is shown. (C) IRP1 is a good iron sensor at 21% O2, but its range of regulation at 3 and 6% O2 is much lower in lymphocytes. Primary splenic lymphocytes were isolated from WT animals and immediately incubated at 3, 6, 12, and 21% O2 concentrations for 24 hours in the constant presence of 200 μM FAC and increasing amounts of Dfo. Gel-shift assays were performed to assess IRE-binding activities (fig. S1). IRP1-binding activity was quantified on a Typhoon 9200 Imager (Molecular Dynamics) with Image Quant software. (D) IRP2 levels are highly regulated in lymphocytes at multiple oxygen concentrations. Western blots of IRP2 from splenic lymphocytes that were incubated for 72 hours in the constant presence of 200 μM FAC and no Dfo or 400 μM Dfo were probed with antibody to IRP2 as described (9). Loading control is the same as in (B).

In spleen-derived lymphocytes, IRE binding activity of IRP1 also increased markedly as the oxygen concentration rose from 3 to 21% (Fig. 2C). IRP2 levels were highly regulated by iron at all oxygen concentrations examined, although IRP2 protein levels decreased gradually as the oxygen concentration increased (Fig. 2D). Thus, oxygen exposure is a critical variable that determines the potential regulatory activity of IRP1 and IRP2 and their relative importance in both macrophages and lymphocytes.

To evaluate the effect of oxygen-dependent changes of IRP activity on regulated targets, we compared ferritin and TfR expression levels in WT, IRP1–/–, and IRP2–/– primary cell cultures grown in 21 or 3 to 6% O2 concentrations. WT macrophages decreased ferritin and increased TfR expression in response to iron deficiency at both low and high oxygen concentrations (Fig. 3, A and B). In contrast, iron-deficient macrophages from IRP2–/– animals could not repress ferritin or increase TfR expression when grown at 3% O2. However, at 21% O2, iron-deficient IRP2–/– macrophages almost fully regulated ferritin and TfR expression, suggesting that the regulatory activity of IRP1 at 21% O2 allowed IRP1 to substitute for IRP2 in regulation of IRP targets.

Fig. 3.

Misregulation of ferritin and TfR occurs in IRP2–/– macrophages grown at 3% O2 but not in IRP1–/– macrophages. (A) Macrophages were grown for 8 days at either 21 or 3% O2 and, for the last 48 hours, in either unsupplemented medium (lane C), medium supplemented with 300 μM FAC (lane F), or 100 μM Dfo (lane D). Ferritin levels were detected by immunoblot as described (30). (B) Levels of TfR were detected by immunoblot in macrophages treated as in (A). (C) Ferritin and TfR biosynthesis were assessed by metabolic labeling and immunoprecipitation of ferritin and TfR in macrophages grown at 3% O2 and treated with FAC and Dfo as described in (A). (D) IRP2 levels were assessed by immunoblot in macrophages grown at 3% O2 and treated with FAC and Dfo as described in (A). Controls for (A), (B), and (D) are given in fig. S2.

Increased ferritin and decreased TfR levels were observed in IRP2–/– macrophage immunoblots grown in 3% O2 (Fig. 3, A and B) and were attributable to changes in the biosynthetic rates of ferritin and TfR grown at low oxygen concentrations (Fig. 3C). The observation that ferritin and TfR regulation were normal in IRP1–/– macrophages maintained at 3% O2 implied that IRP2 provided almost all of the regulatory activity in macrophages with minimal contribution from IRP1 at these oxygen concentrations. Levels of IRP2 did not change in IRP1–/– macrophages compared to WT at 3% O2 (Fig. 3D).

Unlike macrophages, which relied almost exclusively on IRP2 for regulation at physiologically relevant oxygen concentrations, lymphocytes appeared to depend on both IRP1 and IRP2 for normal regulation. The ability to regulate ferritin was significantly reduced in IRP2–/– lymphocytes (Fig. 4A). In IRP1–/– lymphocytes grown at 21% O2, ferritin levels were also significantly elevated compared to levels in WT controls, and in IRP1–/– lymphocytes grown at 6% O2, ferritin was slightly elevated at the lowest iron concentration points. These results revealed that IRP1 contributes a substantial fraction of IRE-binding activity in lymphocytes, even though it is minimally sensitive to changes in iron status at low oxygen concentrations (Fig. 2C), similar to its previously observed role in brown fat and kidney (9). In lymphocytes, IRP2 levels increased in IRP1–/– cells (Fig. 4B), perhaps because loss of IRP1 resulted in a slight increase in ferritin expression (Fig. 4A) that produced a state of subtle functional cytosolic iron deficiency and a concomitant decrease in the rate of iron-dependent IRP2 degradation (5, 6). The increase in IRP2 allowed cells and tissues to partially compensate for loss of IRP1, as was observed in lymphocytes (Fig. 4B) and in tissues of IRP1–/– mice (9). Thus, IRP1 contributes significantly to baseline regulation of iron metabolism in some cells, consistent with the increased severity of neurodegeneration in IRP1+/– IRP2–/– animals (16), and the embryonic lethality of IRP1–/– IRP2–/– mice (17).

Fig. 4.

Misregulation of ferritin occurs in IRP2–/– lymphocytes and to a lesser extent in IRP1–/– lymphocytes, where IRP2 levels increase in compensation for loss of IRP1. (A) Lymphocytes were harvested from animals of the different genotypes and grown for 24 hours in 21 or 6% O2 in the constant presence of 200 μM FAC and various amounts of Dfo. (B) In immunoblots on lysates from WT, IRP1–/–, and IRP2–/– lymphocytes, IRP2 levels increase substantially in IRP1–/– lymphocytes compared to WT levels. Lymphocytes were grown at 3% O2 and treated with FAC and Dfo as described in Fig. 3A. Controls for (A) and (B) are given in fig. S3.

IRP1 and IRP2 operate on a continuum in which the partial pressure of oxygen determines relative activity. IRP1 contains an oxidation-sensitive Fe-S cluster that is enzymatically assembled in cells that have sufficient iron and sulfur (18, 19). However, once the Fe-S cluster of IRP1 is assembled, it may remain stable unless it is exposed to oxidants, including oxygen, superoxide, hydrogen peroxide, nitric oxide, and peroxynitrite (4, 2023). At the ambient oxygen concentrations in tissues of healthy animals (1315), the Fe-S cluster of IRP1 appears to be stable, and IRP1 is therefore poorly suited to function as an iron sensor (9). In contrast, IRP2 has a different mechanism of iron sensing that depends on iron-dependent degradation (5, 6, 2427). Iron-dependent turnover is intact at physiologically relevant oxygen concentrations, which enables IRP2 to dominate normal regulation of iron homeostasis in mammals.

IRP1 plays a minor role in the regulation of iron metabolism by contributing a fraction of baseline IRE-binding activity in tissues. However, the observation that IRE-binding activity can be recruited by oxidative disassembly of its Fe-S cluster means that in some pathological situations, including those in which inflammation results in release of reactive oxygen species, the vast reservoir of IRP1 in the aconitase form (Fig. 1C) may be converted to the IRE-binding form (20, 21). This can lead to inappropriate repression of ferritin synthesis, increased TfR expression, and cellular iron toxicity. Increased iron content coupled with decreased ferritin has been observed in degenerating regions of the brain in Parkinson's disease (28). Thus, pathologic activation of IRP1 by reactive oxygen species may play an important role in some disease processes.

Our findings provide genetic evidence that in mammals, the tissue oxygen concentrations are a critical variable in regulating genes that are important in iron metabolism. Results obtained from tissue culture cells grown in room air may lead to conclusions that are not relevant to normal physiology. Iron- and oxygen-based chemistries affect many cellular processes, including mitochondrial function, DNA replication, and the response to hypoxia (29). Our work demonstrates that the IRP-regulatory system has evolved to tightly regulate iron metabolism at oxygen concentrations that exist in normal mammalian tissues. Even though the two IRPs are highly homologous (1), their activities are only partially redundant, and they occupy different regulatory niches. In normal physiology, tissue oxygen tension determines the contribution of each IRP to the regulation of iron homeostasis.

Supporting Online Material

www.sciencemag.org/cgi/content/full/306/5704/2087/DC1

Materials and Methods

Figs. S1 to S3

References

References and Notes

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