­Dysregulation of the sensory and regulatory pathways controlling cellular iron metabolism in unilateral obstructive nephropathy

2021 ◽  
Author(s):  
James A Votava ◽  
Shannon Reese ◽  
Kathryn M Deck ◽  
Christopher P Nizzi ◽  
Sheila Anderson ◽  
...  
Keyword(s):  
Iron Metabolism ◽  
Kidney Failure ◽  
Rna Binding ◽  
Binding Activity ◽  
Iron Storage ◽  
Iron Regulatory Proteins ◽  
Regulatory Pathways ◽  
Genetic Ablation ◽  
Cellular Iron ◽  
The Impact

Chronic kidney disease (CKD) involves disturbances in iron metabolism including anemia caused by insufficient erythropoietin (EPO) production. However, underlying mechanisms responsible for the dysregulation of cellular iron metabolism are incompletely defined. Using the unilateral ureteral obstruction (UUO) model in Irp1+/+ and Irp1-/- mice we asked if iron regulatory proteins (IRP), the central regulators of cellular iron metabolism and also suppressors of EPO production, contribute to the etiology of anemia in kidney failure. We identified a significant reduction in IRP protein level and RNA binding activity that associated with a loss of the iron uptake protein transferrin receptor 1, increased expression of the iron storage protein subunits H- and L-ferritin, and a low but overall variable level of stainable iron in the obstructed kidney. This reduction in IRP RNA binding activity and ferritin RNA levels suggests the concomitant rise in ferritin expression and iron content in kidney failure is IRP-dependent. In contrast, the reduction in Epo mRNA level in the obstructed kidney was not rescued by genetic ablation of IRP1 suggesting disruption of normal HIF-2a regulation. Furthermore, reduced expression of some HIFa target genes in UUO occurred in the face of increased expression of HIFa proteins and the prolyl hydroxylases (PHD) 2 and PHD1, the latter of which is not known to be HIFa mediated. Our results suggest that the IRP system drives changes in cellular iron metabolism that are associated with kidney failure in UUO but that the impact of IRP on EPO production is overridden by disrupted hypoxia signaling.

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

Blood ◽  
2005 ◽  
Vol 105 (5) ◽  
pp. 2161-2167 ◽  
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.

Effects of Mitochondrial Ferritin Expression on Tumor Iron Metabolism and Tumor Growth in Nude Mice Xenografts.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3582-3582
Author(s):  
Guangjun Nie ◽  
Guohua Chen ◽  
Alex Sheftel ◽  
Kostas Pantopoulos ◽  
Prem Ponka
Keyword(s):  
Tumor Growth ◽  
Nude Mice ◽  
Iron Metabolism ◽  
Rna Binding ◽  
Heme Oxygenase 1 ◽  
Iron Starvation ◽  
Iron Storage ◽  
Iron Regulatory Proteins ◽  
Tumor Xenografts ◽  
Mitochondrial Iron

Abstract Mitochondrial ferritin (MtFt) is a mitochondrial iron storage protein, whose function and regulation is largely unknown. Our previous results have shown that MtFt markedly affects intracellular iron distribution and homeostasis in mammalian cells (Blood105: 2161–2167, 2005). Using tumor xenografts, we examined the effects of expression MtFt on tumor iron metabolism and growth. H1299 parental or MtFt overexpressing cells were implanted into nude mice. As compared to control tumor xenografts, the expression of MtFt dramatically reduced the implanted tumor growth. A cytosolic iron starvation phenotype in MtFt expressing tumors was revealed by increased RNA-binding activity of iron regulatory proteins (IRPs) and, concomitantly, both an increase in transferrin receptor levels and a decrease in cytosolic ferritin. MtFt overexpression also led to a decrease in both total cellular heme content and heme oxygenase-1 levels. In addition, the expression of MtFt in tumors was associated with a decrease in aconitase activity and lower frataxin protein levels. Mitochondrial iron deposition in MtFt expressing tumors was directly observed by transmission electron microscopy. The pattern of iron accumulation in MtFt overexpressing tumor cells is remarkably similar to that observed in the mitochondria of sideroblastic anemia patients. In conclusion, our study shows that MtFt expression significantly affected tumor iron homeostasis by shunting iron into mitochondria; iron scarcity resulted in partial defects in heme and iron-sulfur cluster syntheses. It is likely that deprivation of iron in the cytosol is the cause of the significant inhibition of xenograft tumor growth.

Iron regulatory protein-1 and -2: transcriptome-wide definition of binding mRNAs and shaping of the cellular proteome by iron regulatory proteins

Blood ◽  
2011 ◽  
Vol 118 (22) ◽  
pp. e168-e179 ◽  
Author(s):  
Mayka Sanchez ◽  
Bruno Galy ◽  
Bjoern Schwanhaeusser ◽  
Jonathon Blake ◽  
Tomi Bähr-Ivacevic ◽  
...  
Keyword(s):  
Iron Metabolism ◽  
Rna Binding ◽  
Isotope Labeling ◽  
Rna Binding Proteins ◽  
Regulatory Protein ◽  
Regulatory Proteins ◽  
Iron Regulatory Proteins ◽  
Ribonucleoprotein Complexes ◽  
Cellular Iron ◽  
Irp1 And Irp2

Abstract Iron regulatory proteins (IRPs) 1 and 2 are RNA-binding proteins that control cellular iron metabolism by binding to conserved RNA motifs called iron-responsive elements (IREs). The currently known IRP-binding mRNAs encode proteins involved in iron uptake, storage, and release as well as heme synthesis. To systematically define the IRE/IRP regulatory network on a transcriptome-wide scale, IRP1/IRE and IRP2/IRE messenger ribonucleoprotein complexes were immunoselected, and the mRNA composition was determined using microarrays. We identify 35 novel mRNAs that bind both IRP1 and IRP2, and we also report for the first time cellular mRNAs with exclusive specificity for IRP1 or IRP2. To further explore cellular iron metabolism at a system-wide level, we undertook proteomic analysis by pulsed stable isotope labeling by amino acids in cell culture in an iron-modulated mouse hepatic cell line and in bone marrow-derived macrophages from IRP1- and IRP2-deficient mice. This work investigates cellular iron metabolism in unprecedented depth and defines a wide network of mRNAs and proteins with iron-dependent regulation, IRP-dependent regulation, or both.

scholarly journals Oxalomalate, a competitive inhibitor of aconitase, modulates the RNA-binding activity of iron-regulatory proteins

2000 ◽  
Vol 348 (2) ◽  
pp. 315-320 ◽  
Author(s):  
Michela FESTA ◽  
Alfredo COLONNA ◽  
Concetta PIETROPAOLO ◽  
Alfredo RUFFO
Keyword(s):  
Iron Metabolism ◽  
Rna Binding ◽  
Competitive Inhibitor ◽  
Binding Activity ◽  
Regulatory Proteins ◽  
Mobility Shift ◽  
Prolonged Incubation ◽  
Iron Regulatory Proteins

We investigated the effect of oxalomalate (OMA, α-hydroxy-β-oxalosuccinic acid), a competitive inhibitor of aconitase, on the RNA-binding activity of the iron-regulatory proteins (IRP1 and IRP2) that control the post-transcriptional expression of various proteins involved in iron metabolism. The RNA-binding activity of IRP was evaluated by electrophoretic mobility-shift assay of cell lysates from 3T3-L1 mouse fibroblasts, SH-SY5Y human cells and mouse livers incubated in vitro with OMA, with and without 2-mercaptoethanol (2-ME). Analogous experiments were performed in vivo by prolonged incubation (72 h) of 3T3-L1 cells with OMA, and by injecting young mice with equimolar concentrations of oxaloacetate and glyoxylate, which are the precursors of OMA synthesis. OMA remarkably decreased the binding activity of IRP1 and, when present, of IRP2, in all samples analysed. In addition, the recovery of IRP1 by 2-ME in the presence of OMA was constantly lower versus control values. These findings suggest that the severe decrease in IRP1 RNA-binding activity depends on: (i) linking of OMA to the active site of aconitase, which prevents the switch to IRP1 and explains resistance to the reducing agents, and (ii) possible interaction of OMA with some functional amino acid residues in IRP that are responsible for binding to the specific mRNA sequences involved in the regulation of iron metabolism.

Abstract 293: Mammalian Target of Rapamycin and Tristetraprolin Regulate Iron Homeostasis in Cardiac Cells

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Marina Bayeva ◽  
Arineh Khechaduri ◽  
Hossein Ardehali
Keyword(s):  
Energy Metabolism ◽  
Iron Homeostasis ◽  
Heart Function ◽  
Iron Regulation ◽  
Mrna Levels ◽  
Iron Regulatory Proteins ◽  
Regulatory Pathways ◽  
Genetic Knockout ◽  
Cellular Iron ◽  
Vital Functions

Introduction: Iron is essential for normal heart function, and disruption of iron homeostasis can lead to cardiomyopathy. However, our understanding of iron regulation on a cellular level is incomplete, with a single model involving iron regulatory proteins (IRP) described to date. Here, we report the existence of a parallel iron regulatory pathway by energy sensor mTOR and inflammatory mediator trsitetraprolin (TTP). Results: To examine the role of energy metabolism in the regulation of cellular iron, we used rapamycin to inhibit mTOR pathway in H9c2 cardiac myoblasts and mouse embryonic fibroblasts (MEFs). Rapamycin treatment significantly elevated cellular iron content through a coordinated reduction in iron import (transferrin receptor, TfR1) and iron export (ferroportin, Fpn1), leading to deceleration of iron flux and net iron accumulation. We found that the primary action of rapamycin was to reduce TfR1 through destabilization of its mRNA. Surprisingly, this effect was not mediated by IRP1/2, the “classical” sensors of cellular iron levels, as TfR1 mRNA levels were significantly reduced by rapamycin even in cells with the genetic knockout of IRP1 and IRP2. In yeast, a tandem zinc finger (TZF) protein Cth2 was found to conserve cellular iron in states of deficiency by preferentially degrading mRNA of non-essential iron-containing proteins thus reducing iron requirements and liberating iron for vital functions. We found that the mammalian TZF protein TTP, an established mediator of inflammation, was greatly induced by iron deficiency, enhanced degradation of iron-containing proteins, and complemented Cth2 deletion in yeast, thus establishing TTP as the functional homolog of Cth2 in mammalian iron regulation. Finally, TTP levels were increased by rapamycin in IRP1/2-independent manner, and genetic knockout of TTP in MEFs significantly reversed the effects of rapamycin on TfR1 mRNA levels and stability. These findings establish TTP as the mediator of iron-regulatory effects of mTOR and provide a novel link between energy metabolism, inflammation and iron regulatory pathways. Conclusions: We identified a novel pathway of cellular iron regulation by mTOR and TTP, which complements the “classical” IRP1/2 model.

Iron Regulatory Proteins in Systemic Iron Metabolism and Erythropoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-22-SCI-22
Author(s):  
Matthias W. Hentze
Keyword(s):  
Iron Metabolism ◽  
Iron Homeostasis ◽  
Mutant Mouse ◽  
Conflicts Of Interest ◽  
Regulatory Proteins ◽  
Iron Regulatory Proteins ◽  
Cellular Iron ◽  
Regulatory Systems ◽  
Biology I ◽  
Mouse Lines

Abstract Abstract SCI-22 Imbalances of iron homeostasis account for some of the most common human diseases. Pathologies can result from both iron deficiency or overload. The hepcidin/ferroportin and the IRE/IRP regulatory systems balance systemic and cellular iron metabolism, respectively, and understanding their points of intersection and crosstalk represents a major challenge in iron biology. I will discuss an emerging picture from studies with different mutant mouse lines according to which the “cellular” IRE/IRP system determines “set points” via its targets (including ferroportin and HIF2α). These are then subject to modulation via hepcidin in response to systemic cues. Disclosures: No relevant conflicts of interest to declare.

The Potential of Iron Chelators of the Pyridoxal Isonicotinoyl Hydrazone Class as Effective Antiproliferative Agents III: The Effect of the Ligands on Molecular Targets Involved in Proliferation

Blood ◽  
1999 ◽  
Vol 94 (2) ◽  
pp. 781-792 ◽  
Author(s):  
G. Darnell ◽  
D.R. Richardson
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Rna Binding ◽  
Neuroblastoma Cells ◽  
Molecular Targets ◽  
Binding Activity ◽  
Time Dependent ◽  
Mrna Levels ◽  
Iron Regulatory Proteins ◽  
Pyridoxal Isonicotinoyl Hydrazone

We have identified specific iron (Fe) chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class that are far more effective ligands than desferrioxamine (DFO; Richardson et al, Blood 86:4295, 1995; Richardson and Milnes, Blood 89:3025, 1997). In the present study, we have compared the effect of DFO and one of the most active chelators (2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone; 311) on molecular targets involved in proliferation. This was performed to further understand the mechanisms involved in the antitumor activity of Fe chelators. Ligand 311 was far more active than DFO at increasing Fe release from SK-N-MC neuroepithelioma and BE-2 neuroblastoma cells and preventing Fe uptake from transferrin. Like DFO, 311 increased the RNA-binding activity of the iron-regulatory proteins (IRPs). However, despite the far greater Fe chelation efficacy of 311 compared with DFO, a similar increase in IRP-RNA binding activity occurred after 2 to 4 hours of incubation with either chelator, and the binding activity was not inhibited by cycloheximide. These results suggest that, irrespective of the Fe chelation efficacy of a ligand, an increase IRP-RNA binding activity occurred via a time-dependent step that did not require protein synthesis. Further studies examined the effect of 311 and DFO on the expression of p53-transactivated genes that are crucial for cell cycle control and DNA repair, namely WAF1,GADD45, and mdm-2. Incubation of 3 different cell lines with DFO or 311 caused a pronounced concentration- and time-dependent increase in the expression of WAF1 and GADD45 mRNA, but not mdm-2 mRNA. In accordance with the distinct differences in Fe chelation efficacy and antiproliferative activity of DFO and 311, much higher concentrations of DFO (150 μmol/L) than 311 (2.5 to 5 μmol/L) were required to markedly increase GADD45 and WAF1 mRNA levels. The increase in GADD45 and WAF1 mRNA expression was seen only after 20 hours of incubation with the chelators and was reversible after removal of the ligands. In contrast to the chelators, the Fe(III) complexes of DFO and 311 had no effect on increasing GADD45 and WAF1 mRNA levels, suggesting that Fe chelation was required. Finally, the increase in GADD45 and WAF1 mRNAs appeared to occur by a p53-independent pathway in SK-N-MC and K562 cells, because these cell lines lack functional p53. Our results suggest that GADD45 and WAF1 may play important roles in the cell cycle arrest observed after exposure to these chelators.

Effects of iron regulatory protein regulation on iron homeostasis during hypoxia

Blood ◽  
2003 ◽  
Vol 102 (9) ◽  
pp. 3404-3411 ◽  
Author(s):  
Brian D. Schneider ◽  
Elizabeth A. Leibold
Keyword(s):  
Iron Uptake ◽  
Iron Homeostasis ◽  
Rna Binding ◽  
Late Phase ◽  
Regulatory Protein ◽  
Binding Activity ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Iron Regulatory Proteins ◽  
Ft Synthesis

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.

Modulation of IRP Binding Activity by Oxygen in Primary Erythroid Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2662-2662
Author(s):  
Matthias Schranzhofer ◽  
Manfred Schifrer ◽  
Prem Ponka ◽  
Ernst W. Muellner
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Activity ◽  
Ammonium Citrate ◽  
Ferric Ammonium Citrate ◽  
Erythroid Cells ◽  
Stem Loop ◽  
Iron Regulatory Proteins ◽  
Protein Levels ◽  
Irp1 And Irp2

Abstract Iron regulatory proteins 1 and 2 (IRP1 and IRP2) are cytoplasmic RNA-binding proteins that target specific stem-loop RNA structures known as iron responsive elements (IRE). Binding of IRPs to IREs inhibits translation of ferritin mRNA and stabilizes transferrin receptor (TfR) mRNA. Various factors have been reported to regulate binding activity of IRPs, such as iron, phosphorylation, nitric oxide and hypoxia. While there is a consistent agreement on the negative effect of iron on the interaction between IRPs and IREs, reports regarding the influence of hypoxia on the IRE-binding activity of IRPs vary in a species and cell specific manner. It was the aim of this work to study the effect of hypoxic (3% oxygen) and normoxic (20% oxygen) conditions on IRP binding activity in primary erythroid cells. The cells were induced for differentiation and incubated under physiological, low (Desferrioxamine) and high (ferric ammonium citrate) iron conditions. Binding activity of IRPs and protein levels of ferritin and TfR as well as cell proliferation and differentiation parameters were determined to analyze the regulation of iron metabolism during terminal differentiation. The data show, that in developing red blood cells binding activities of IRP1 and IRP2 are reduced at 3% oxygen. This reduction correlates with increased ferritin protein levels and decreased TfR protein levels. Moreover, incubation under hypoxia strongly decreased cell expansion and reduces hemoglobinization. These results suggest that terminal erythroid differentiation in the bone marrow might occur under normoxic rather than hypoxic conditions.

Analysis of Ferritins in Lymphoblastoid Cell Lines and in the Lens of Subjects With Hereditary Hyperferritinemia-Cataract Syndrome

Blood ◽  
1998 ◽  
Vol 91 (11) ◽  
pp. 4180-4187 ◽  
Author(s):  
Sonia Levi ◽  
Domenico Girelli ◽  
Federica Perrone ◽  
Marcella Pasti ◽  
Carole Beaumont ◽  
...  
Keyword(s):  
Cell Lines ◽  
Iron Metabolism ◽  
Antioxidant Properties ◽  
Water Soluble ◽  
Lymphoblastoid Cell Lines ◽  
Cataract Formation ◽  
Iron Regulatory Proteins ◽  
Control Subjects ◽  
Cellular Iron ◽  
Chain Synthesis

Hereditary hyperferritinemia-cataract syndrome (HHCS) is an autosomal and dominant disease caused by heterogeneous mutations in the iron responsive element (IRE) of the 5′ untranslated flanking region of ferritin L-chain mRNA, which reduce the binding to the trans iron regulatory proteins and make L-chain synthesis constitutively upregulated. In the several families identified so far, the serum and tissue L-ferritin levels are fivefold to 20-fold higher than in nonaffected control subjects, iron metabolism is apparently normal, and the only relevant clinical symptom is early onset, bilateral cataract. Some pathogenetic aspects of HHCS remain obscure, with particular reference to the isoferritins produced by HHCS cells, as well as the mechanism of cataract formation. We analyzed lymphoblastoid cell lines obtained from two nonaffected control subjects and from HHCS patients carrying the substitution A40G (Paris-1), G41C (Verona-1), and the deletion of the residues 10-38 (Verona-2) in the IRE structure. Enzyme-linked immunosorbent assays specific for the H- and L-type ferritins showed that L-ferritin levels were up to 20-fold higher in HHCS than in control cells and were not affected by iron supplementation or chelation. Sequential immunoprecipitation experiments of metabolically-labeled cells with specific antibodies indicated that in HHCS cells about half of the L-chain was assembled in L-chain homopolymers, which did not incorporate iron, and the other half was assembled in isoferritins with a high proportion of L-chain. In control cells, all ferritin was assembled in functional heteropolymers with equivalent proportion of H- and L-chains. Cellular and ferritin iron uptake was slightly higher in HHCS than control cells. In addition, we analyzed the lens recovered from cataract surgery of a HHCS patient. We found it to contain about 10-fold more L-ferritin than control lens. The ferritin was fully soluble with a low iron content. It was purified and partially characterized. Our data indicate that: (1) in HHCS cells a large proportion of L-ferritin accumulates as nonfunctional L-chain 24 homopolymers; (2) the concomitant fivefold to 10-fold expansion of ferritin heteropolymers, with a shift to L-chain–rich isoferritins, does not have major effects on cellular iron metabolism; (3) L-chain accumulation occurs also in the lens, where it may induce cataract formation by altering the delicate equilibrium between other water-soluble proteins (ie, crystallins) and/or the antioxidant properties.

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