Probing the structure of the regulatory region of human transferrin receptor messenger RNA and its interaction with iron regulatory protein-1.

A portion of the 3'UTR of the human transferrin receptor mRNA mediates iron-dependent regulation of mRNA stability. The minimal RNA regulatory region contains three conserved hairpins, so-called iron responsive elements (IREs), that are recognized specifically by iron regulatory proteins (IRPs). The structure of this regulatory region and its complex with IRP-1 was probed using a combination of enzymes and chemicals. The data support the existence of an intrinsic IRE loop structure that is constrained by an internal C-G base pair. This particular structure is one of the determinants required for optimal IRP binding. IRP-1 covers one helical turn of the IRE and protects conserved residues in each of the three IREs: the bulged cytosine and nucleotides in the hairpin loops. Two essential IRP-phosphate contacts were identified by ethylation interference. Three-dimensional modeling of one IRE reveals that IRP-1 contacts several bases and the ribose-phosphate backbone located on one face in the deep groove, but contacts also exist with the shallow groove. A conformational change of the IRE loop mediated by IRP-1 binding was visualized by Pb2+-catalyzed hydrolysis. This effect is dependent on the loop structure and on the nature of the closing base pair. Within the regulatory region of transferrin receptor mRNA, IRP-1 induces reactivity changes in a U-rich hairpin loop that requires the presence of the stem-loop structure located just downstream the endonucleolytic cleavage site identified by Binder et al. (Binder R et al. 1994, EMBO J 13:1969-1980). These results provide indications of the mechanism by which IRP-1 stabilizes the transferrin receptor mRNA under iron depletion conditions.     

Regulation of ferritin and transferrin receptor mRNAs

Iron regulates the synthesis of two proteins critical for iron metabolism, ferritin and the transferrin receptor, through novel mRNA/protein interactions. The mRNA regulatory sequence (iron-responsive element (IRE)) occurs in the 5'-untranslated region of all ferritin mRNAs and is repeated as five variations in the 3'-untranslated region of transferrin receptor mRNA. When iron is in excess, ferritin synthesis and iron storage increase. At the same time, transferrin receptor synthesis and iron uptake decrease. Location of the common IRE regulatory sequence in different noncoding regions of the two mRNAs may explain how iron can have opposite metabolic effects; when the IRE is in the 5'-untranslated region of ferritin mRNA, translation is enhanced by excess iron whereas the presence of the IREs in the 3'-untranslated region of the transferrin receptor mRNA leads to iron-dependent degradation. How and where iron actually acts is not yet known. A soluble 90-kDa regulatory protein which has been recently purified to homogeneity from liver and red cells specifically blocks translation of ferritin mRNA and binds IRE sequences but does not appear to be an iron-binding protein. The protein is the first specific eukaryotic mRNA regulator identified and confirms predictions 20 years old. Concerted regulation by iron of ferritin and transferrin receptor mRNAs may also define a more general strategy for using common mRNA sequences to coordinate the synthesis of metabolically related proteins.     

Identification and characterization of the iron regulatory element in the ferritin gene of a plant (soybean)

Iron increases ferritin synthesis, targeting plant DNA and animal mRNA. The ferritin promoter in plants has not been identified, in contrast to the ferritin promoter and mRNA iron-responsive element (IRE) in animals. The soybean leaf, a natural tissue for ferritin expression, and DNA, with promoter deletions and luciferase or glucuronidase reporters, delivered with particle bombardment, were used to show that an 86-base pair fragment (iron regulatory element (FRE)) controlled iron-mediated derepression of the ferritin gene. Mutagenesis with linkers of random sequence detected two subdomains separated by 21 base pairs. FRE has no detectable homology to the animal IRE or to known promoters in DNA and bound a trans-acting factor in leaf cell extracts. FRE/factor binding was abrogated by increased tissue iron, in analogy to mRNA (IRE)/iron regulatory protein in animals. Maximum ferritin derepression was obtained with 50 microm iron citrate (1:10) or 500 microm iron citrate (1:1) but Fe-EDTA was ineffective, although the leaf iron concentration was increased; manganese, zinc, and copper had no effect. The basis for different responses in ferritin expression to different iron complexes, as well as the significance of using DNA but not mRNA as an iron regulatory target in plants, remain unknown.     

Alterations in the interaction between iron regulatory proteins and their iron responsive element in normal and Alzheimer's diseased brains.

Iron regulatory proteins (IRPs) are cytoplasmic mRNA binding proteins involved in intracellular regulation of iron homeostasis. IRPs regulate expression of ferritin and transferrin receptor at the mRNA level by interacting with a conserved RNA structure termed the iron-responsive element (IRE). This concordant regulation of transferrin receptors and ferritin is designed so a cell can obtain iron when it is needed, and sequester iron when it is in excess. However, we have reported that iron accumulates in the brain in Alzheimer's disease without a concomitant increase in ferritin. An increase in iron without proper sequestration can increase the vulnerability of cells to oxidative stress. Oxidative stress is a component of many neurological diseases including Alzheimer's. We hypothesized that alterations in the IRP/IRE interaction could be the site at which iron mismanagement occurs in the Alzheimer's brains. In this report we demonstrate that in normal human brain extracts, the IRP is detected as a double IRE/IRP complex by RNA band shift assay, but in 2 of 6 Alzheimer's brain (AD) extracts examined a single IRE/IRP complex was obtained. Furthermore, the mobility of the single IRE/IRP complex in Alzheimer's brain extracts is decreased relative to the double IRE/IRP complex. Western blot and RNA band super shift assay demonstrate that IRP1 is involved in the formation of the single IRE/IRP complex. In vitro analyses suggest that the stability of the doublet complex and single AD complex are different. The single complex from the AD brain are more stable. A more stable IRE/IRP complex in the AD brain could increase stability of the transferrin receptor mRNA and inhibit ferritin synthesis. At the cellular level, the outcome of this alteration in the molecular regulatory mechanism would be increased iron accumulation without an increase in ferritin; identical to the observation we reported in AD brains. The appearance of the single IRE/IRP complex in Alzheimer's brain extracts is associated with relatively high endogenous ribonuclease activity. We propose that elevated RNase activity is one mechanism by which the iron regulatory system becomes dysfunctional.     

Correlation between the expression of divalent metal transporter 1 and the content of hypoxia-inducible factor-1 in hypoxic HepG2 cells

Transferrin and transferrin receptor are two key proteins of iron metabolism that have been identified to be hypoxia-inducible genes. Divalent metal transporter 1 (DMT1) is also a key transporter of iron under physiological conditions. In addition, in the 5' regulatory region of human DMT1 (between -412 and -570), there are two motifs (CCAAAGTGCTGGG) that are similar to hypoxia-inducible factor-1 (HIF-1) binding sites. It was therefore speculated that DMT1 might also be a hypoxia-inducible gene. We investigated the effects of hypoxia and hypoxia/re-oxygenation on the expression of DMT1 and the content of HIF-1alpha in HepG2 cells. As we expected, a very similar tendency in the responses of the expression of HIF-1alpha, DMT1+IRE (iron response element) and DMT1-IRE proteins to chemical (CoCl(2)) or physical hypoxia was observed. A highly significant correlation was found between the expression of DMT1 proteins and the contents of HIF-1 in hypoxic cells. After the cells were exposed to hypoxia and subsequent normoxia, no HIF-1alpha could be detected and a significant decrease in DMT1+IRE expression (P<0.05), but not in DMT1-IRE protein (versus the hypoxia group), was observed. The findings implied that the HIF-1 pathway might have a role in the regulation of DMT1+IRE expression during hypoxia.     

Iron uptake and Nramp2/DMT1/DCT1 in human bronchial epithelial cells

The capacity of natural resistance-associated macrophage protein-2 [Nramp2; also called divalent metal transporter-1 (DMT1) and divalent cation transporter-1 (DCT1)] to transport iron and its ubiquitous expression make it a likely candidate for transferrin-independent uptake of iron in peripheral tissues. We tested the hypothesis that non-transferrin-bound iron uptake by airway epithelial cells is associated with Nramp2/DMT1/DCT1 and that exposure to iron can increase Nramp2/DMT1/DCT1 mRNA and protein expression and transport of this metal. Exposure of BEAS-2B cells to ferric ammonium citrate (FAC) resulted in a decrease in Fe(3+) concentration in the supernatant that was dependent on time and initial iron concentration. In the presence of internalized calcein, FAC quenched the fluorescent signal, indicating intracellular transport of the metal. The Nramp2/DMT1/DCT1 mRNA isoform without an iron-response element (IRE) increased with exposure of BEAS-2B cells to FAC. RT-PCR demonstrated no change in the mRNA for the isoform with an IRE. Similarly, Western blot analysis for the isoform without an IRE confirmed an increased expression of this protein after FAC exposure, whereas the isoform with an IRE exhibited no change. Finally, immunohistochemistry revealed an increase in the isoform without an IRE in the rat lung epithelium after instillation of FAC. Comparable to mRNA and protein increases, iron transport was elevated after pretreatment of BEAS-2B cells with iron-containing compounds. We conclude that airway epithelial cells increase mRNA and expression of the Nramp2/DMT1/DCT1 without an IRE after exposure to iron. The increase results in an elevated transport of iron and its probable detoxification by these cells.     

A novel iron responsive element in the 3'UTR of human MRCKalpha

Human untranslated region (UTR) databases were searched to identify novel proteins potentially regulated by an iron responsive element (IRE), and found two candidates-cell cycle phosphatase Cdc14A variant 1 and myotonic dystrophy kinase-related Cdc42-binding kinase alpha (MRCKalpha), both possessing a putative IRE in their 3'UTR. In further experiments, we focused on MRCKalpha. Biochemical analyses of the MRCKalpha IRE revealed that it was functional and mediated the response to iron level in the same way as transferrin receptor 1 IREs (TfR) did. Similarly to TfR mRNA, MRCKalpha mRNA is stabilized, when iron supply is low, while it is destabilized under iron-rich conditions. The expression of MRCKalpha mRNA was found to be ubiquitous; the highest levels were noted in testes, the lowest in skeletal muscle. The level of MRCKalpha mRNA in various tissues strongly positively correlates with the level of TfR mRNA, indicating its possible role in the transferrin iron uptake pathway.     

Self-association and ligand-induced conformational changes of iron regulatory proteins 1 and 2

Iron regulatory proteins (IRPs) regulate iron metabolism in mammalian cells. We used biophysical techniques to examine the solution properties of apo-IRP1 and apo-IRP2 and the interaction with their RNA ligand, the iron regulatory element (IRE). Sedimentation velocity and equilibrium experiments have shown that apo-IRP1 exists as an equilibrium mixture of monomers and dimers in solution, with an equilibrium dissociation constant in the low micromolar range and slow kinetic exchange between the two forms. However, only monomeric IRP1 is observed in complex with IRE. In contrast, IRP2 exists as monomer in both the apo-IRP2 form and in the IRP2/IRE complex. For both IRPs, sedimentation velocity and dynamic light-scattering experiments show a decrease of the Stokes radius upon binding of IRE. This conformational change was also observed by circular dichroism. Studies with an RNA molecule complementary to IRE indicate that, although specific base interactions can increase the stability of the protein/RNA complex, they are not essential for inducing this conformational change. The dynamic change of the IRP between different oligomeric and conformational states induced by interaction with IRE may play a role in the iron regulatory functions of IRPs.     

Overexpression of the ferritin iron-responsive element decreases the labile iron pool and abolishes the regulation of iron absorption by intestinal epithelial (Caco-2) cells

Mammalian cells regulate iron levels tightly through the activity of iron-regulatory proteins (IRPs) that bind to RNA motifs called iron-responsive elements (IREs). When cells become iron-depleted, IRPs bind to IREs present in the mRNAs of ferritin and the transferrin receptor, resulting in diminished translation of the ferritin mRNA and increased translation of the transferrin receptor mRNA. Likewise, intestinal epithelial cells regulate iron absorption by a process that also depends on the intracellular levels of iron. Although intestinal epithelial cells have an active IRE/IRP system, it has not been proven that this system is involved in the regulation of iron absorption in these cells. In this study, we characterized the effect of overexpression of the ferritin IRE on iron absorption by Caco-2 cells, a model of intestinal epithelial cells. Cells overexpressing ferritin IRE had increased levels of ferritin, whereas the levels of the transferrin receptor were decreased. Iron absorption in IRE-transfected cells was deregulated: iron uptake from the apical medium was increased, but the capacity to retain this newly incorporated iron diminished. Cells overexpressing IRE were not able to control iron absorption as a function of intracellular iron, because both iron-deficient cells as well as iron-loaded cells absorbed similarly high levels of iron. The labile iron pool of IRE-transfected cell was extremely low. Likewise, the reduction of the labile iron pool in control cells resulted in cells having increased iron absorption. These results indicate that cells overexpressing IRE do not regulate iron absorption, an effect associated with decreased levels of the regulatory iron pool.     

Ligand-induced structural alterations in human iron regulatory protein-1 revealed by protein footprinting.

Human iron regulatory protein-1 (IRP-1) is a bifunctional protein that regulates iron metabolism by binding to mRNAs encoding proteins involved in iron uptake, storage, and utilization. Intracellular iron accumulation regulates IRP-1 function by promoting the assembly of an iron-sulfur cluster, conferring aconitase activity to IRP-1, and hindering RNA binding. Using protein footprinting, we have studied the structure of the two functional forms of IRP-1 and have mapped the surface of the iron-responsive element (IRE) binding site. Binding of the ferritin IRE or of the minimal regulatory region of transferrin receptor mRNA induced strong protections against proteolysis in the region spanning amino acids 80 to 187, which are located in the putative cleft thought to be involved in RNA binding. In addition, IRE-induced protections were also found in the C-terminal domain at Arg-721 and Arg-728. These data implicate a bipartite IRE binding site located in the putative cleft of IRP-1. The aconitase form of IRP-1 adopts a more compact structure because strong reductions of cleavage were detected in two defined areas encompassing residues 149 to 187 and 721 to 735. Thus both ligands of apo-IRP-1, the IRE and the 4Fe-4S cluster, induce distinct but overlapping alterations in protease accessibility. These data provide evidences for structural changes in IRP-1 upon cluster formation that affect the accessibility of residues constituting the RNA binding site.     

TNF, IFN-gamma, and endotoxin increase expression of DMT1 in bronchial epithelial cells

Regulation of the metal transport protein divalent metal transporter-1 (DMT1) may contribute to the uptake and detoxification of iron by cells resident in the respiratory tract. Inflammation has been associated with an increased availability of this metal resulting in an oxidative stress. Because proinflammatory cytokines and LPS have been demonstrated to affect an elevated expression of DMT1 in a macrophage cell line, we tested the hypothesis that tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and LPS increase DMT1 expression in airway epithelial cells. We used RT-PCR to detect mRNA for both -IRE DMT1 and +IRE DMT1 in BEAS-2B cells. Treatment with TNF-alpha, IFN-gamma, or LPS increased both forms. Western blot analysis also demonstrated an increase in the expression of both isoforms of DMT1 after these treatments. Twenty-four hours after exposure of an animal model to TNF-alpha, IFN-gamma, or LPS, a significant increase in pulmonary expression of -IRE DMT1 was seen by immunohistochemistry; the level of +IRE DMT1 was too low in the lung to be visualized using this methodology. Finally, iron transport into BEAS-2B cells was increased after inclusion of TNF-alpha, IFN-gamma, or LPS in the media. We conclude that proinflammatory cytokines and LPS increase mRNA and protein expression of DMT1 in airway cells in vitro and in vivo. Furthermore, both -IRE and +IRE isoforms are elevated after exposures. Increased expression of this protein appears to be included in a coordinated response of the cell and tissue where the function might be to diminish availability of metal.