Effects of iron regulatory protein regulation on iron homeostasis during hypoxia

AbstractIron regulatory proteins (IRP1 and IRP2) are RNA-binding proteins that affect the translation and stabilization of specific mRNAs by binding to stem-loop structures known as iron responsive elements (IREs). IREs are found in the 5′-untranslated region (UTR) of ferritin (Ft) and mitochondrial aconitase (m-Aco) mRNAs, and in the 3′-UTR of transferrin receptor (TfR) and divalent metal transporter-1 (DMT1) mRNAs. Our previous studies show that besides iron, IRPs are regulated by hypoxia. Here we describe the consequences of IRP regulation and show that iron homeostasis is regulated in 2 phases during hypoxia: an early phase where IRP1 RNA-binding activity decreases and iron uptake and Ft synthesis increase, and a late phase where IRP2 RNA-binding activity increases and iron uptake and Ft synthesis decrease. The increase in iron uptake is independent of DMT1 and TfR, suggesting an unknown transporter. Unlike Ft, m-Aco is not regulated during hypoxia. During the late phase of hypoxia, IRP2 RNA-binding activity increases, becoming the dominant regulator responsible for decreasing Ft synthesis. During reoxygenation (ReO2), Ft protein increases concomitant with a decrease in IRP2 RNA-binding activity. The data suggest that the differential regulation of IRPs during hypoxia may be important for cellular adaptation to low oxygen tension.

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Overexpression of mitochondrial ferritin causes cytosolic iron depletion and changes cellular iron homeostasis

Blood ◽

10.1182/blood-2004-07-2722 ◽

2005 ◽

Vol 105 (5) ◽

pp. 2161-2167 ◽

Cited By ~ 97

Author(s):

Guangjun Nie ◽

Alex D. Sheftel ◽

Sangwon F. Kim ◽

Prem Ponka

Keyword(s):

Iron Homeostasis ◽

Rna Binding ◽

Binding Activity ◽

Intracellular Iron ◽

Free Radical Damage ◽

Iron Regulatory Proteins ◽

Cellular Iron ◽

Cellular Iron Uptake ◽

A Cell ◽

Cellular Iron Homeostasis

AbstractCytosolic ferritin sequesters and stores iron and, consequently, protects cells against iron-mediated free radical damage. However, the function of the newly discovered mitochondrial ferritin (MtFt) is unknown. To examine the role of MtFt in cellular iron metabolism, we established a cell line that stably overexpresses mouse MtFt under the control of a tetracycline-responsive promoter. The overexpression of MtFt caused a dose-dependent iron deficiency in the cytosol that was revealed by increased RNA-binding activity of iron regulatory proteins (IRPs) along with an increase in transferrin receptor levels and decrease in cytosolic ferritin. Consequently, the induction of MtFt resulted in a dramatic increase in cellular iron uptake from transferrin, most of which was incorporated into MtFt. The induction of MtFt caused a shift of iron from cytosolic ferritin to MtFt. In addition, iron inserted into MtFt was less available for chelation than that in cytosolic ferritin and the expression of MtFt was associated with decreased mitochondrial and cytosolic aconitase activities, the latter being consistent with the increase in IRP-binding activity. In conclusion, our results indicate that overexpression of MtFt causes a dramatic change in intracellular iron homeostasis and that shunting iron to MtFt likely limits its availability for active iron proteins.

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HFE Downregulates Iron Uptake From Transferrin and Induces Iron-Regulatory Protein Activity in Stably Transfected Cells

Blood ◽

10.1182/blood.v94.11.3915.423k27_3915_3921 ◽

1999 ◽

Vol 94 (11) ◽

pp. 3915-3921 ◽

Cited By ~ 2

Author(s):

H.D. Riedel ◽

M.U. Muckenthaler ◽

S.G. Gehrke ◽

I. Mohr ◽

K. Brennan ◽

...

Keyword(s):

Transferrin Receptor ◽

Iron Uptake ◽

Iron Homeostasis ◽

Hereditary Hemochromatosis ◽

Regulatory Protein ◽

Intracellular Iron ◽

Iron Storage ◽

Iron Regulatory Proteins ◽

Cellular Iron

Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder of iron metabolism. More than 80% of HH patients are homozygous for a point mutation in a major histocompatibility complex (MHC) class I type protein (HFE), which results in a lack of HFE expression on the cell surface. A previously identified interaction of HFE and the transferrin receptor suggests a possible regulatory role of HFE in cellular iron absorption. Using an HeLa cell line stably transfected with HFE under the control of a tetracycline-sensitive promoter, we investigated the effect of HFE expression on cellular iron uptake. We demonstrate that the overproduction of HFE results in decreased iron uptake from diferric transferrin. Moreover, HFE expression activates the key regulators of intracellular iron homeostasis, the iron-regulatory proteins (IRPs), implying that HFE can affect the intracellular “labile iron pool.” The increase in IRP activity is accompanied by the downregulation of the iron-storage protein, ferritin, and an upregulation of transferrin receptor levels. These findings are discussed in the context of the pathophysiology of HH and a possible role of iron-responsive element (IRE)-containing mRNAs.

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Cysteine Oxidation Regulates the RNA-Binding Activity of Iron Regulatory Protein 2

Molecular and Cellular Biology ◽

10.1128/mcb.00004-09 ◽

2009 ◽

Vol 29 (8) ◽

pp. 2219-2229 ◽

Cited By ~ 35

Author(s):

Kimberly B. Zumbrennen ◽

Michelle L. Wallander ◽

S. Joshua Romney ◽

Elizabeth A. Leibold

Keyword(s):

Oxidative Stress ◽

Transferrin Receptor ◽

Iron Homeostasis ◽

Rna Binding ◽

Regulatory Protein ◽

Binding Activity ◽

Structural Basis ◽

Iron Regulatory Protein ◽

Transferrin Receptor 1 ◽

Binding Cleft

ABSTRACT Iron regulatory protein 2 (IRP2) is an RNA-binding protein that regulates the posttranscriptional expression of proteins required for iron homeostasis such as ferritin and transferrin receptor 1. IRP2 RNA-binding activity is primarily regulated by iron-mediated proteasomal degradation, but studies have suggested that IRP2 RNA binding is also regulated by thiol oxidation. We generated a model of IRP2 bound to RNA and found that two cysteines (C512 and C516) are predicted to lie in the RNA-binding cleft. Site-directed mutagenesis and thiol modification show that, while IRP2 C512 and C516 do not directly interact with RNA, both cysteines are located within the RNA-binding cleft and must be unmodified/reduced for IRP2-RNA interactions. Oxidative stress induced by cellular glucose deprivation reduces the RNA-binding activity of IRP2 but not IRP2-C512S or IRP2-C516S, consistent with the formation of a disulfide bond between IRP2 C512 and C516 during oxidative stress. Decreased IRP2 RNA binding is correlated with reduced transferrin receptor 1 mRNA abundance. These studies provide insight into the structural basis for IRP2-RNA interactions and reveal an iron-independent mechanism for regulating iron homeostasis through the redox regulation of IRP2 cysteines.

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The iron-responsive element (IRE)/iron-regulatory protein 1 (IRP1)–cytosolic aconitase iron-regulatory switch does not operate in plants

Biochemical Journal ◽

10.1042/bj20061874 ◽

2007 ◽

Vol 405 (3) ◽

pp. 523-531 ◽

Cited By ~ 45

Author(s):

Nicolas Arnaud ◽

Karl Ravet ◽

Andrea Borlotti ◽

Brigitte Touraine ◽

Jossia Boucherez ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Regulatory Protein ◽

Binding Activity ◽

Loss Of Function ◽

Structural Element ◽

Iron Regulatory Proteins ◽

Cis Acting ◽

Model Plant Arabidopsis Thaliana ◽

Responsive Element

Animal cytosolic ACO (aconitase) and bacteria ACO are able to switch to RNA-binding proteins [IRPs (iron-regulatory proteins)], thereby playing a key role in the regulation of iron homoeostasis. In the model plant Arabidopsis thaliana, we have identified three IRP1 homologues, named ACO1–3. To determine whether or not they may encode functional IRP proteins and regulate iron homoeostasis in plants, we have isolated loss-of-function mutants in the three genes. The aco1-1 and aco3-1 mutants show a clear decrease in cytosolic ACO activity. However, none of the mutants is affected in respect of the accumulation of the ferritin transcript or protein in response to iron excess. cis-acting elements potentially able to bind to the IRP have been searched for in silico in the Arabidopsis genome. They appear to be very rare sequences, found in the 5′-UTR (5′-untranslated region) or 3′-UTR of a few genes unrelated to iron metabolism. They are therefore unlikely to play a functional role in the regulation of iron homoeostasis. Taken together, our results demonstrate that, in plants, the cytosolic ACO is not converted into an IRP and does not regulate iron homoeostasis. In contrast with animals, the RNA binding activity of plant ACO, if any, would be more likely to be attributable to a structural element, rather than to a canonical sequence.

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Retinoic Acid Modulates Hepatic Iron Homeostasis in Rats by Attenuating the RNA-Binding Activity of Iron Regulatory Proteins

Journal of Nutrition ◽

10.1093/jn/137.12.2686 ◽

2007 ◽

Vol 137 (12) ◽

pp. 2686-2690 ◽

Cited By ~ 11

Author(s):

Stacy E. Schroeder ◽

Manju B. Reddy ◽

Kevin L. Schalinske

Keyword(s):

Retinoic Acid ◽

Iron Homeostasis ◽

Rna Binding ◽

Binding Activity ◽

Regulatory Proteins ◽

Hepatic Iron ◽

Iron Regulatory Proteins

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HFE Downregulates Iron Uptake From Transferrin and Induces Iron-Regulatory Protein Activity in Stably Transfected Cells

Blood ◽

10.1182/blood.v94.11.3915 ◽

1999 ◽

Vol 94 (11) ◽

pp. 3915-3921 ◽

Cited By ~ 66

Author(s):

H.D. Riedel ◽

M.U. Muckenthaler ◽

S.G. Gehrke ◽

I. Mohr ◽

K. Brennan ◽

...

Keyword(s):

Transferrin Receptor ◽

Iron Uptake ◽

Iron Homeostasis ◽

Hereditary Hemochromatosis ◽

Regulatory Protein ◽

Intracellular Iron ◽

Iron Storage ◽

Iron Regulatory Proteins ◽

Cellular Iron

Abstract Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder of iron metabolism. More than 80% of HH patients are homozygous for a point mutation in a major histocompatibility complex (MHC) class I type protein (HFE), which results in a lack of HFE expression on the cell surface. A previously identified interaction of HFE and the transferrin receptor suggests a possible regulatory role of HFE in cellular iron absorption. Using an HeLa cell line stably transfected with HFE under the control of a tetracycline-sensitive promoter, we investigated the effect of HFE expression on cellular iron uptake. We demonstrate that the overproduction of HFE results in decreased iron uptake from diferric transferrin. Moreover, HFE expression activates the key regulators of intracellular iron homeostasis, the iron-regulatory proteins (IRPs), implying that HFE can affect the intracellular “labile iron pool.” The increase in IRP activity is accompanied by the downregulation of the iron-storage protein, ferritin, and an upregulation of transferrin receptor levels. These findings are discussed in the context of the pathophysiology of HH and a possible role of iron-responsive element (IRE)-containing mRNAs.

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Unexpected Anthracycline-Mediated Alterations in Iron-Regulatory Protein-RNA-Binding Activity: The Iron and Copper Complexes of Anthracyclines Decrease RNA-Binding Activity

Molecular Pharmacology ◽

10.1124/mol.62.4.888 ◽

2002 ◽

Vol 62 (4) ◽

pp. 888-900 ◽

Cited By ~ 37

Author(s):

Juliana C. Kwok ◽

Des R. Richardson

Keyword(s):

Copper Complexes ◽

Rna Binding ◽

Regulatory Protein ◽

Binding Activity ◽

Iron Regulatory Protein

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Copper repletion enhances apical iron uptake and transepithelial iron transport by Caco-2 cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00414.2001 ◽

2002 ◽

Vol 282 (3) ◽

pp. G527-G533 ◽

Cited By ~ 38

Author(s):

Okhee Han ◽

Marianne Wessling-Resnick

Keyword(s):

Iron Uptake ◽

Iron Transport ◽

Cell Monolayer ◽

Transepithelial Transport ◽

Apical Surface ◽

Divalent Metal Transporter 1 ◽

Divalent Metal Transporter ◽

Metal Transporter ◽

Copper Status ◽

Copper Supplementation

The influence of copper status on Caco-2 cell apical iron uptake and transepithelial transport was examined. Cells grown for 7–8 days in media supplemented with 1 μM CuCl2had 10-fold higher cellular levels of copper compared with control. Copper supplementation did not affect the integrity of differentiated Caco-2 cell monolayers grown on microporous membranes. Copper-repleted cells displayed increased uptake of iron as well as increased transport of iron across the cell monolayer. Northern blot analysis revealed that expression of the apical iron transporter divalent metal transporter-1 (DMT1), the basolateral transporter ferroportin-1 (Fpn1), and the putative ferroxidase hephaestin (Heph) was upregulated by copper supplementation, whereas the recently identified ferrireductase duodenal cytochrome b (Dcytb) was not. These results suggest that DMT1, Fpn1, and Heph are involved in the iron uptake process modulated by copper status. Although a clear role for Dcytb was not identified, an apical surface ferrireductase was modulated by copper status, suggesting that its function also contributes to the enhanced iron uptake by copper-repleted cells. A model is proposed wherein copper promotes iron depletion of intestinal Caco-2 cells, creating a deficiency state that induces upregulation of iron transport factors.

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