Increased Transferrin Receptor Research Articles

Abstract 4493: Ironing out breast cancer: investigation of a novel iron chelator

Abstract Introduction: Breast cancer is the second leading cause of cancer-related death in women. Current breast cancer therapy is hindered by dose-limiting toxicities, demonstrating the need for less toxic alternatives. As cancer cells have an increased requirement for iron, we hypothesized that iron withdrawal using a highly selective iron chelator (DIBI) developed by Chelation Partners Incorporated (CPI) would enhance breast cancer cell killing by chemotherapeutic drugs and ionizing radiation. Results: DIBI inhibited the growth of a panel of breast cancer cell lines while similar doses had limited effects on the growth of normal fibroblasts. In breast cancer cells, DIBI-mediated iron chelation increased transferrin receptor 1 mRNA expression and decreased the expression of ferroportin1 mRNA, which is indicative of decreased intracellular iron stores. Iron citrate reversed the inhibitory effect of DIBI, confirming the iron-specific activity of the compound. On the basis of iron-binding capacity, a comparison between DIBI and two conventional iron chelators demonstrated that DIBI was more effective at inhibiting breast cancer cell proliferation. DIBI-treated breast cancer cells showed a dose-dependent reduction in rounds of cell division, as well as arrest in the S-phase of the cell cycle. Higher doses of DIBI also induced apoptosis in breast cancer cells. In addition, DIBI treatment of breast cancer cells induced double-stranded DNA breaks, which are likely important for its anticancer activity. Pretreatment of breast cancer cells with DIBI enhanced the grow-inhibitory effects of ionizing radiation and conventional chemotherapeutic agents, suggesting that DIBI may increase the effectiveness of current breast cancer therapies. Combination treatment with cisplatin and DIBI demonstrated that DIBI enhanced both the cytotoxic and cytostatic anticancer effects of cisplatin. Conclusion: These findings suggest that selective sequestration of iron from the tumor microenvironment may prevent or diminish tumor progression. In the future, treating patients with this novel iron chelator in combination with conventional therapies may increase the effectiveness of current breast cancer treatments. Citation Format: Anna Greenshields, David Hoskin, Melanie Coombs, Taryn Grant. Ironing out breast cancer: investigation of a novel iron chelator. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4493. doi:10.1158/1538-7445.AM2015-4493

Nitric oxide induces hypoxia ischemic injury in the neonatal brain via the disruption of neuronal iron metabolism

We have recently shown that increased hydrogen peroxide (H2O2) generation is involved in hypoxia–ischemia (HI)-mediated neonatal brain injury. H2O2 can react with free iron to form the hydroxyl radical, through Fenton Chemistry. Thus, the objective of this study was to determine if there was a role for the hydroxyl radical in neonatal HI brain injury and to elucidate the underlying mechanisms. Our data demonstrate that HI increases the deposition of free iron and hydroxyl radical formation, in both P7 hippocampal slice cultures exposed to oxygen–glucose deprivation (OGD), and the neonatal rat exposed to HI. Both these processes were found to be nitric oxide (NO) dependent. Further analysis demonstrated that the NO-dependent increase in iron deposition was mediated through increased transferrin receptor expression and a decrease in ferritin expression. This was correlated with a reduction in aconitase activity. Both NO inhibition and iron scavenging, using deferoxamine administration, reduced hydroxyl radical levels and neuronal cell death. In conclusion, our results suggest that increased NO generation leads to neuronal cell death during neonatal HI, at least in part, by altering iron homeostasis and hydroxyl radical generation.

IRP2 Regulates Breast Tumor Growth

Experimental and epidemiologic evidence suggests that dysregulation of proteins involved in iron metabolism plays a critical role in cancer. The mechanisms by which cancer cells alter homeostatic iron regulation are just beginning to be understood. Here, we demonstrate that iron regulatory protein 2 (IRP2) plays a key role in iron accumulation in breast cancer. Although both IRP1 and IRP2 are overexpressed in breast cancer, the overexpression of IRP2, but not IRP1, is associated with decreased ferritin H and increased transferrin receptor 1 (TfR1). Knockdown of IRP2 in triple-negative MDA-MB-231 human breast cancer cells increases ferritin H expression and decreases TfR1 expression, resulting in a decrease in the labile iron pool. Further, IRP2 knockdown reduces growth of MDA-MB-231 cells in the mouse mammary fat pad. Gene expression microarray profiles of patients with breast cancer demonstrate that increased IRP2 expression is associated with high-grade cancer. Increased IRP2 expression is observed in luminal A, luminal B, and basal breast cancer subtypes, but not in breast tumors of the ERBB2 molecular subtype. These results suggest that dysregulation of IRP2 is an early nodal point underlying altered iron metabolism in breast cancer and may contribute to poor outcome of some patients with breast cancer.

M98K-OPTN induces transferrin receptor degradation and RAB12-mediated autophagic death in retinal ganglion cells

Mutations in the autophagy receptor OPTN/optineurin are associated with the pathogenesis of glaucoma and amyotrophic lateral sclerosis, but the underlying molecular basis is poorly understood. The OPTN variant, M98K has been described as a risk factor for normal tension glaucoma in some ethnic groups. Here, we examined the consequence of the M98K mutation in affecting cellular functions of OPTN. Overexpression of M98K-OPTN induced death of retinal ganglion cells (RGC-5 cell line), but not of other neuronal and non-neuronal cells. Enhanced levels of the autophagy marker, LC3-II, a post-translationally modified form of LC3, in M98K-OPTN-expressing cells and the inability of an LC3-binding-defective M98K variant of OPTN to induce cell death, suggested that autophagy contributes to cell death. Knockdown of Atg5 reduced M98K-induced death of RGC-5 cells, further supporting the involvement of autophagy. Overexpression of M98K-OPTN enhanced autophagosome formation and potentiated the delivery of transferrin receptor to autophagosomes for degradation resulting in reduced cellular transferrin receptor levels. Coexpression of transferrin receptor or supplementation of media with an iron donor reduced M98K-induced cell death. OPTN complexes with RAB12, a GTPase involved in vesicle trafficking, and M98K variant shows enhanced colocalization with RAB12. Knockdown of Rab12 increased transferrin receptor level and reduced M98K-induced cell death. RAB12 is present in autophagosomes and knockdown of Rab12 resulted in reduced formation of autolysosomes during starvation-induced autophagy, implicating a role for RAB12 in autophagy. These results also show that transferrin receptor degradation and autophagy play a crucial role in RGC-5 cell death induced by M98K variant of OPTN.

Deferasirox (ICL670A) effectively inhibits oesophageal cancer growth in vitro and in vivo

Growing evidence implicates iron in the aetiology of gastrointestinal cancer. Furthermore, studies demonstrate that iron chelators possess potent anti-tumour activity, although whether iron chelators show activity against oesophageal cancer is not known. The effect of the iron chelators, deferoxamine (DFO) and deferasirox, on cellular iron metabolism, viability and proliferation was assessed in two oesophageal adenocarcinoma cell lines, OE33 and OE19, and the squamous oesophageal cell line, OE21. A murine xenograft model was employed to assess the effect of deferasirox on oesophageal tumour burden. The ability of chelators to overcome chemoresistance and to enhance the efficacy of standard chemotherapeutic agents (cisplatin, fluorouracil and epirubicin) was also assessed. Deferasirox and DFO effectively inhibited cellular iron acquisition and promoted intracellular iron mobilization. The resulting reduction in cellular iron levels was reflected by increased transferrin receptor 1 expression and reduced cellular viability and proliferation. Treating oesophageal tumour cell lines with an iron chelator in addition to a standard chemotherapeutic agent resulted in a reduction in cellular viability and proliferation compared with the chemotherapeutic agent alone. Both DFO and deferasirox were able to overcome cisplatin resistance. Furthermore, in human xenograft models, deferasirox was able to significantly suppress tumour growth, which was associated with decreased tumour iron levels. The clinically established iron chelators, DFO and deferasirox, effectively deplete iron from oesophageal tumour cells, resulting in growth suppression. These data provide a platform for assessing the utility of these chelators in the treatment of oesophageal cancer patients.

The armadillo protein p0071 is involved in Rab11-dependent recycling

p0071 is an intercellular junction protein of the p120 catenin family. We have identified Rab11a as a novel interaction partner of p0071. p0071 interacted preferentially with active Rab11a. Knockdown experiments revealed an interdependent regulation of both proteins. On the one hand, p0071 depletion induced a perinuclear accumulation of Rab11, suggesting a role of p0071 in the anterograde transport of Rab11 from the pericentrosomal region to the plasma membrane but not in retrograde transport. p0071 as well as Rab11 depletion increased transferrin receptor recycling indicating that p0071-induced Rab11 mislocalization interfered with Rab11 function and shifted recycling from the slow Rab11-dependent pathway to the fast Rab4-dependent pathway. When p0071 or Rab11 depletion was combined with a Rab4 knockdown the effect was reversed. On the other hand, Rab11a depletion increased p0071 recycling to cell contacts thereby identifying p0071 as a Rab11 cargo protein. This correlated with increased intercellular adhesion. Thus, we propose that p0071 has a key role in regulating recycling through the Rab11-dependent perinuclear recycling compartment, and links the regulation of adherens junctions to recycling to allow dynamic modulation of intercellular adhesion.

Iron Deprivation Impairs Proliferation of CALM-AF10 leukemia Cells in Vitro and in Vivo

Abstract 1386 Background:The CALM-AF10 fusion protein arises from the t(10;11) chromosomal translocation and is found in 8–10% of T-cell acute lymphoblastic leukemias and a smaller percentage of acute myeloid leukemias; these hematopoietic malignancies are associated with a poor clinical outcome. The precise contributions of the Clathrin Assembly Lymphoid Myeloid leukemia (CALM) protein to leukemogenesis remain uncertain. CALM plays a role in clathrin-dependent endocytosis, which mediates the entry of growth factor receptors and nutrients into cells and is essential for the internalization of iron-bound transferrin. We have previously shown that Calm-deficient (Calm−/−) mouse fibroblasts derived from fit1 mice are iron deficient, and are more sensitive to treatment with iron chelators. Objective:Since CALM-AF10 leukemia cells are haploinsufficient for CALM, we hypothesize that reduced levels of CALM in CALM-AF10 leukemia cells cause a relative iron deficiency, and result in enhanced sensitivity to iron deprivation. Design/Methods:Fibroblasts and fetal liver hematopoietic progenitors (HP) were derived from Calm−/−, Calm+/− or Calm+/+ E14 embryos. The proliferation rates of non-immortalized fibroblasts were compared in the presence and absence of supplemental iron (ferric ammonium citrate (FAC)) or treatment with an iron chelator (deferoxamine (DFO)). Surface transferrin receptor expression was quantified by flow cytometry. Primary HP cells were cultured in the presence or absence of DFO and viable cell numbers were determined. To examine the anti-leukemic effect of iron deprivation in vivo, C57BL/6J-Tyrc-2J mice were fed a low-iron diet, transplanted with CALM-AF10-transduced Calm+/− leukemia cells and leukemia latency was determined. Results:Heterozygous Calm+/− fibroblasts exhibit CALM protein levels that are intermediate between their wildtype (Calm+/+) and deficient (Calm−/−) counterparts. Calm+/− cells display a slower rate of proliferation in vitro (50% reduction of viable cells) compared to their wildtype fibroblasts, and this growth deficiency can be corrected by iron supplementation with 50 mM FAC. The presence of reduced intracellular iron levels in Calm+/− fibroblasts was manifested by increased transferrin receptor expression relative to wildtype cells. In vitro, Calm+/− HP cells showed greater sensitivity to iron chelation by DFO (5 mM) than Calm+/+ controls: the relative number of viable cells in the presence of DFO was 20% lower in Calm+/− compared to Calm+/− HP cells. To assess the impact of iron deprivation in vivo, bone marrow cells from Calm+/−CALM-AF10 leukemic mice were injected into secondary recipient mice preconditioned by having received a low-iron diet for 8 weeks (n=9). The onset of leukemia in transplanted mice maintained on the iron-deficient diet was considerably delayed (median survival 93 days versus 63 days, p=0.013) relative to age- and gender-matched control mice fed a normal diet (n=9). Conclusions:We have shown that reduced CALM expression impairs iron import and consequently limits the rate of cell proliferation. Both in vitro and in vivo results suggest that CALM haploinsufficient CALM-AF10 leukemias are particularly sensitive to iron deprivation. This raises the possibility that iron chelation may be a previously unappreciated treatment option for patients with aggressive CALM-rearranged leukemias. We are currently studying the impact of iron chelation in murine CALM-AF10 in vivo leukemia models. Disclosures:No relevant conflicts of interest to declare.

A Mechanistic Study of Proteasome Inhibition-Induced Iron Misregulation in Dopamine Neuron Degeneration

Ubiquitin proteasome system (UPS) impairment and iron misregulation have been implicated in dopamine (DA) neuron degeneration in Parkinson’s disease. As previously shown, proteasome inhibition in a rodent model can cause nigral neuron degeneration accompanied by iron accumulation. To investigate the involvement of iron in DA neuron degeneration, we generated an in vitro model by applying proteasome inhibitor lactacystin in DAergic cell line MES23.5 culture. We found that lactacystin caused marked increase in labile iron, reactive oxygen species and ubiquitin-conjugated protein aggregation prior to cell injury. These effects were attenuated by iron chelators or antioxidants. Furthermore, we demonstrated that the iron regulatory protein (IRP)/iron response element system contributed to UPS impairment-mediated DA neuron injury. We documented that IRP2 disruption resulted in an increase in transferrin receptor 1 (TfR1), a decrease in ferritin heavy chain (H-Frt), and eventually cell death. These findings provide insight into the mechanistic interplay between UPS impairment and iron misregulation and suggest that the disturbances in IRP2, TfR1 and H-Frt may contribute to DA neuron degeneration.

The Oral Iron Chelator Deferiprone Protects against Iron Overload–Induced Retinal Degeneration

Iron-induced oxidative stress may exacerbate age-related macular degeneration (AMD). Ceruloplasmin/Hephaestin double-knockout (DKO) mice with age-dependent retinal iron accumulation and some features of AMD were used to test retinal protection by the oral iron chelator deferiprone (DFP). Cultured retinal pigment epithelial (ARPE-19) cells and mice were treated with DFP. Transferrin receptor mRNA (Tfrc), an indicator of iron levels, was quantified by qPCR. In mice, retinal oxidative stress was assessed by mass spectrometry, and degeneration by histology and electroretinography. DFP at 60 μM decreased labile iron in ARPE-19 cells, increasing Tfrc and protecting 70% of cells against a lethal dose of H(2)O(2). DFP 1 mg/mL in drinking water increased retinal Tfrc mRNA 2.7-fold after 11 days and also increased transferrin receptor protein. In DKOs, DFP over 8 months decreased retinal iron levels to 72% of untreated mice, diminished retinal oxidative stress to 70% of the untreated level, and markedly ameliorated retinal degeneration. DFP was not retina toxic in wild-type (WT) or DKO mice, as assessed by histology and electroretinography. Oral DFP was not toxic to the mouse retina. It diminished retinal iron levels and oxidative stress and protected DKO mice against iron overload-induced retinal degeneration. Further testing of DFP for retinal disease involving oxidative stress is warranted.

TfR1 and Ferrochelatase Expression During Erythroid Differentiation Induced by 5-Aza-Cdr In Both MEL Cells and BFU-E-Derived Erythroblasts Correlates with c-Myc Function

AbstractAbstract 4248During erythroid differentiation, heme biosynthesis increases dramatically and this is accompanied by a marked increase in transferrin receptor 1 (TfR1) expression and iron uptake from transferrin. It has long been known that 5-aza-2′deoxycytidine (5-aza-CdR), a demethylating agent, is able to induce murine erythroid leukemia (MEL) cell differentiation. However, the detailed mechanism underlying this induction remains elusive. In the current study, we report that 5-aza-CdR induces erythroid differentiation in MEL cells and BFU-E-derived erythroblasts and that this is associated with the nuclear translocation of the transcription factor c-Myc and its binding to Max to form functional complex. As markers of erythroid differentiation, the expression levels of TfR1, ferrochelatase and ALAS2 were analyzed by quantitative real-time PCR and western blotting. TfR1 and ferrochelatase expression were increased 5-fold following 5-aza-CdR treatment. To determine whether these changes in gene expression reflected differential methylation, we analyzed 1.5 kb up- and downstream from the transcription start site (TS) of these genes. Although many potential methylation sites (CpG islands) were found in both the TfR1 and ferrochelatase genes, bisulfite sequencing revealed that few sites (< 5%) were methylated in these promoters in untreated MEL cells and that there was no difference following 5-aza-CdR treatment. This observation suggests that the increased expression of TfR1 and ferrochelatase is not directly mediated by 5-aza-CdR-induced DNA demethylation. EKLF and GATA1 are key transcription regulators in erythroid differentiation, and the iron regulatory proteins (IRPs) are important for the post-transcriptional regulation of TfR1 mRNA levels. Bioinformatic analysis showed that there were many potential methylation sites in the EKLF and IRP2 promoters, however, bisulfite sequencing showed that there was no effect of 5-aza-CdR treatment at these sites in MEL cells. Since c-Myc has been reported to stimulate TfR1 expression, we then investigated the role of c-Myc in erythroid differentiation. Levels of c-Myc mRNA and protein were not increased by 5-aza-CdR, however, we did observe a dramatic increase in the nuclear fraction of c-Myc. Furthermore, co-immunopricipitation studies showed that the amount of the Myc-Max complex, which is the form in which c-Myc activates downstream gene expression, increased in the nucleus after 5-aza-CdR treatment. Interestingly, using chromatin-immunoprecipitation we also observed very strong binding of c-Myc to the CACGTG sequence upstream of the TfR1 transcription start site following 5-aza-CdR treatment. Analogous studies using primary mouse erythroblasts derived from BFU-Es gave similar results, with 5-aza-CdR treatment leading to increased TfR1 and ferrochelatase expression, c-Myc translocation to the nucleus and increased c-Myc binding activity to TfR1 promoter. Taken together, these studies point to an important role for c-Myc in the regulation of TfR1 during erythroid differentiation induced by demethylation. Precisely how demethylation is linked to alterations in c-Myc activity is under investigating. Disclosures:No relevant conflicts of interest to declare.

Insulin-stimulated Phosphorylation of the Rab GTPase-activating Protein TBC1D1 Regulates GLUT4 Translocation

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular locations to the plasma membrane in adipose and muscle cells. Prior studies have shown that Akt phosphorylation of the Rab GTPase-activating protein, AS160 (160-kDa Akt substrate; also known as TBC1D4), triggers GLUT4 translocation, most likely by suppressing its Rab GTPase-activating protein activity. However, the regulation of a very similar protein, TBC1D1 (TBC domain family, member 1), which is mainly found in muscle, in insulin-stimulated GLUT4 translocation has been unclear. In the present study, we have identified likely Akt sites of insulin-stimulated phosphorylation of TBC1D1 in C2C12 myotubes. We show that a mutant of TBC1D1, in which several Akt sites have been converted to alanine, is considerably more inhibitory to insulin-stimulated GLUT4 translocation than wild-type TBC1D1. This result thus indicates that similar to AS160, Akt phosphorylation of TBC1D1 enables GLUT4 translocation. We also show that in addition to Akt activation, activation of the AMP-dependent protein kinase partially relieves the inhibition of GLUT4 translocation by TBC1D1. Finally, we show that the R125W variant of TBC1D1, which has been genetically associated with obesity, is equally inhibitory to insulin-stimulated GLUT4 translocation, as is wild-type TBC1D1, and that healthy and type 2 diabetic individuals express approximately the same level of TBC1D1 in biopsies of vastus lateralis muscle. In conclusion, phosphorylation of TBC1D1 is required for GLUT4 translocation. Thus, the regulation of TBC1D1 resembles that of its paralog, AS160.

A Disruption in Iron-Sulfur Center BiogenesisviaInhibition of Mitochondrial Dithiol Glutaredoxin 2 May Contribute to Mitochondrial and Cellular Iron Dysregulation in Mammalian Glutathione-Depleted Dopaminergic Cells: Implications for Parkinson's Disease

Parkinson's disease (PD) is characterized by early glutathione depletion in the substantia nigra (SN). Among its various functions in the cell, glutathione acts as a substrate for the mitochondrial enzyme glutaredoxin 2 (Grx2). Grx2 is involved in glutathionylation of protein cysteine sulfhydryl residues in the mitochondria. Although monothiol glutathione-dependent oxidoreductases (Grxs) have previously been demonstrated to be involved in iron-sulfur (Fe-S) center biogenesis, including that in yeast, here we report data suggesting the involvement of mitochondrial Grx2, a dithiol Grx, in iron-sulfur biogenesis in a mammalian dopaminergic cell line. Given that mitochondrial dysfunction and increased cellular iron levels are two important hallmarks of PD, this suggests a novel potential mechanism by which glutathione depletion may affect these processes in dopaminergic neurons. We report that depletion of glutathione as substrate results in a dose-dependent Grx2 inhibition and decreased iron incorporation into a mitochondrial complex I (CI) and aconitase (m-aconitase). Mitochondrial Grx2 inhibition through siRNA results in a corresponding decrease in CI and m-aconitase activities. It also results in significant increases in iron-regulatory protein (IRP) binding, likely as a consequence of conversion of Fe-S-containing cellular aconitase to its non-Fe-S-containing IRP1 form. This is accompanied by increased transferrin receptor, decreased ferritin, and subsequent increases in mitochondrial iron levels. This suggests that glutathione depletion may affect important pathologic cellular events associated with PD through its effects on Grx2 activity and mitochondrial Fe-S biogenesis.

The CCL7-CCL2-CCR2 Axis Regulates IL-4 Production in Lungs and Fungal Immunity

Expression of the chemokine receptor CCR2 can be detrimental or beneficial for infection resolution. Herein, we examined whether CCR2 was requisite for control of infection by the dimorphic fungus Histoplasma capsulatum. H. capsulatum-infected CCR2(-/-) mice manifested defects in inflammatory cell recruitment, increased IL-4, and progressive infection. Increased IL-4 in CCR2(-/-) mice primarily contributed to decreased host resistance as demonstrated by the ability of IL-4-neutralized CCR2(-/-) mice to resolve infection without altering inflammatory cell recruitment. Surprisingly, numerous alveolar macrophages and dendritic cells contributed to IL-4 production in CCR2(-/-) mice. IL-4-mediated impairment of immunity in CCR2(-/-) mice was associated with increased arginase-1 and YM1 transcription and increased transferrin receptor expression by phagocytic cells. Immunity in mice lacking the CCR2 ligand CCL2 was not impaired despite decreased inflammatory cell recruitment. Neutralization of the CCR2 ligand CCL7 in CCL2(-/-) mice, but not wild type, resulted in increased IL-4 and fungal burden. Thus, CCL7 in combination with CCL2 limits IL-4 generation and exerts control of host resistance. Furthermore, increased phagocyte-derived IL-4 in CCR2(-/-) mice is associated with the presence of alternatively activated phagocytic cells.

The Role of Heme Oxygenase 1 in Erythroid Differentiation.

Heme is a complex of iron with protoporphyrin IX that is essential for the function of all aerobic cells. However, if left unguarded, non-protein-bound heme promotes free radical formation, resulting in cell damage and tissue injury. The highest amounts of organismal heme (75–80%) are present in circulating red blood cells (RBC) whose precursors synthesize heme with rates that are at least 1 order of magnitude higher than those in the liver (on the per cell basis), which is the second most active heme producer in the body. The only physiological mechanism of heme degradation is performed by heme oxygenases (HO1 and HO2), which catalyze the rate-limiting step in the oxidative degradation of heme and are, therefore, involved in the control of cellular heme levels. Red blood cells contain the majority of heme destined for catabolism; this process takes place in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Although the heme-inducible HO isoform, HO1, has been extensively studied in hepatocytes and many other non-erythroid cells, virtually nothing is known about the expression of HO1 in developing RBC. Similarly, it is unknown whether HO-1 plays any role in erythroid cell development under physiological or pathophysiological conditions. In this study we have shown that HO1 protein is expressed in uninduced murine erythroleukemic (MEL) cells and that its levels, somewhat surprisingly, do not decrease during DMSO-induced erythroid differentiation. Moreover, we demonstrated that heme significantly induces HO1 in both uninduced and induced MEL cells. Additionally, we investigated the effect of overexpressed HO1 on heme and iron metabolism in stably transfected MEL cells (MEL-HO1) and their non-transfected counterparts. Compared to wild type cells, DMSO-treated MEL-HO1 cells displayed a reduction in heme stability (measured by the incorporation of 59Fe into heme) in addition to impairment of erythroid differentiation. Moreover, although wild type and transfected cells expressed similar levels of transferrin receptors in the uninduced state, MEL-HO1 cells, as compared to wild type MEL cells, showed only a small increase in transferrin receptors upon treatment with DMSO. Finally, we measured apoptosis using annexin-V and observed an increase in the number of apoptotic cells in HO1 transfectants, but not in wild type MEL cells. These results suggest that an as yet unknown mechanism exists to protect heme against endogenous HO1 action during physiological erythroid differentiation. In addition, our results showing that high levels of HO1 in erythroid cells cause heme catabolism and a defect in erythroid differentiation raise the possibility that HO1 could play a role in some pathophysiological conditions such as unbalanced globin synthesis in thalassemias.

Human Nbp35 Is Essential for both Cytosolic Iron-Sulfur Protein Assembly and Iron Homeostasis

The maturation of cytosolic iron-sulfur (Fe/S) proteins in mammalian cells requires components of the mitochondrial iron-sulfur cluster assembly and export machineries. Little is known about the cytosolic components that may facilitate the assembly process. Here, we identified the cytosolic soluble P-loop NTPase termed huNbp35 (also known as Nubp1) as an Fe/S protein, and we defined its role in the maturation of Fe/S proteins in HeLa cells. Depletion of huNbp35 by RNA interference decreased cell growth considerably, indicating its essential function. The deficiency in huNbp35 was associated with an impaired maturation of the cytosolic Fe/S proteins glutamine phosphoribosylpyrophosphate amidotransferase and iron regulatory protein 1 (IRP1), while mitochondrial Fe/S proteins remained intact. Consequently, huNbp35 is specifically involved in the formation of extramitochondrial Fe/S proteins. The impaired maturation of IRP1 upon huNbp35 depletion had profound consequences for cellular iron metabolism, leading to decreased cellular H-ferritin, increased transferrin receptor levels, and higher transferrin uptake. These properties clearly distinguished huNbp35 from its yeast counterpart Nbp35, which is essential for cytosolic-nuclear Fe/S protein assembly but plays no role in iron regulation. huNbp35 formed a complex with its close homologue huCfd1 (also known as Nubp2) in vivo, suggesting the existence of a heteromeric P-loop NTPase complex that is required for both cytosolic Fe/S protein assembly and cellular iron homeostasis.

Changes in iron-regulatory proteins in the aged rodent neural retina

Iron accumulation is associated with age-related neurodegenerations and may contribute to age-related increased susceptibility of neurons to damage. We compared young and old rodent retinas to assess iron homeostasis during normal aging and the effects of increased iron on the susceptibility of retinal neurons to degeneration. Retinal iron was significantly increased with age. Quantitative RT-PCR showed that transferrin and ferritin genes were upregulated in the aged retina. At the protein level, we found decreased transferrin, and increased transferrin receptor, ferritin, ferroportin, and ceruloplasmin in the aged retina. These results support an increased steady state of iron with age in the retina. We tested susceptibility of retinal neurons with increased intracellular iron to damage in vitro. Exposure of RGC-5 cells to increased iron potentiated the neurotoxicity induced by paraquat, glutamate, and TNFα. Our results demonstrate that iron homeostasis in the retina is altered with age and suggest that iron accumulation, due to altered levels of iron-regulatory proteins in the aged retina, could be a susceptibility factor in age-related retinal diseases.

Zinc Deficiency-induced Iron Accumulation, a Consequence of Alterations in Iron Regulatory Protein-binding Activity, Iron Transporters, and Iron Storage Proteins

One consequence of zinc deficiency is an elevation in cell and tissue iron concentrations. To examine the mechanism(s) underlying this phenomenon, Swiss 3T3 cells were cultured in zinc-deficient (D, 0.5 microM zinc), zinc-supplemented (S, 50 microM zinc), or control (C, 4 microM zinc) media. After 24 h of culture, cells in the D group were characterized by a 50% decrease in intracellular zinc and a 35% increase in intracellular iron relative to cells in the S and C groups. The increase in cellular iron was associated with increased transferrin receptor 1 protein and mRNA levels and increased ferritin light chain expression. The divalent metal transporter 1(+)iron-responsive element isoform mRNA was decreased during zinc deficiency-induced iron accumulation. Examination of zinc-deficient cells revealed increased binding of iron regulatory protein 2 (IRP2) and decreased binding of IRP1 to a consensus iron-responsive element. The increased IRP2-binding activity in zinc-deficient cells coincided with an increased level of IRP2 protein. The accumulation of IRP2 protein was independent of zinc deficiency-induced intracellular nitric oxide production but was attenuated by the addition of the antioxidant N-acetylcysteine or ascorbate to the D medium. These data support the concept that zinc deficiency can result in alterations in iron transporter, storage, and regulatory proteins, which facilitate iron accumulation.

Roles of hormone replacement therapy and iron in proliferation of breast epithelial cells with different estrogen and progesterone receptor status

Estrogen and iron play critical roles in a female body development and were investigated in the present study in relation to in vitro cell proliferation. Prempro™, a hormone replacement therapy drug, and 17 β-estradiol (E2) were shown to increase cell proliferations in estrogen receptor positive (ER +) cells independent of progesterone receptor (PR) status. For example, increased cell proliferation was observed in ER +/PR + human breast cancer MCF-7, its matching non-cancerous human breast epithelial MCF-12A, and ER +/PR + murine mammary cancer MXT + cells, but not in ER −/PR − MDA-MB-231, its matching non-cancerous MCF-10A, and MXT − (ER −/PR +) cells. By mimicking post-menopausal conditions of high estrogen in local breast tissue and increased iron levels due to cessation of menstrual periods, E2 and iron were shown to exert synergistic effects on proliferation of MCF-7 cells and significantly increased Ki67 and proliferating cell nuclear antigen. Western blotting of E2-treated ER + but not ER − cells showed that E2 also increased transferrin receptor (TfR). Further studies are needed to assess the mitogenic effects of iron and estrogen in normal post-menopausal breast.

Manganese targets m‐aconitase and activates iron regulatory protein 2 in AF5 GABAergic cells

Studies suggest that disturbances of amino acid metabolism and cellular iron regulation are important mechanisms underlying manganese (Mn) neurotoxicity, although the targets underlying these disturbances are poorly defined. Using the AF5 neural-derived cell line, which displays GABAergic properties, we showed that Mn significantly increased glutamate release to 174%-214% of that of the control and that the effects of Mn exposure on the metabolism of glutamate, glutamine, alanine, and GABA resembled the effects of fluorocitrate, an inhibitor of aconitase, but not the effects of other toxicants including paraquat, rotenone, or 3-nitropropionic acid. Consistent with this, Mn inhibited aconitase activity in AF5 cells, resulting in a 90% increase in intracellular citrate; an in vitro assay revealed that m-aconitase was significantly more sensitive to inhibition by Mn than was c-aconitase. RNA mobility shift assay and Western blot showed that Mn treatment caused c-aconitase to be converted to iron regulatory protein 1 (IRP1) and increased the abundance of IRP2, leading to reduced H-ferritin expression, increased transferrin receptor expression, and increased uptake of transferrin. To determine the relative contributions of IRP1 and IRP2 in mediating the effects of Mn on iron homeostasis, we exposed transgenic fibroblasts lacking either c-aconitase/IRP1 or IRP2 to Mn. Manganese exposure minimally altered ferritin levels in cells possessing only c-aconitase/IRP1, whereas cells possessing only IRP2 showed a robust decrease in ferritin, indicating a dominant role of IRP2 in Mn-induced alteration of iron homeostasis. Together, these results demonstrate that m-aconitase is an important target of Mn and thatMn-induced alteration of iron homeostasis is mediated predominantly through IRP2.

Tissue-specific expression of ferritin H regulates cellular iron homoeostasis in vivo

Ferritin is a ubiquitously distributed iron-binding protein. Cell culture studies have demonstrated that ferritin plays a role in maintenance of iron homoeostasis and in the protection against cytokine- and oxidant-induced stress. To test whether FerH (ferritin H) can regulate tissue iron homoeostasis in vivo, we prepared transgenic mice that conditionally express FerH and EGFP (enhanced green fluorescent protein) from a bicistronic tetracycline-inducible promoter. Two transgenic models were explored. In the first, the FerH and EGFP transgenes were controlled by the tTA(CMV) (Tet-OFF) (where tTA and CMV are tet transactivator protein and cytomegalovirus respectively). In skeletal muscle of mice bearing the FerH/EGFP and tTA(CMV) transgenes, FerH expression was increased 6.0+/-1.1-fold (mean+/-S.D.) compared with controls. In the second model, the FerH/EGFP transgenes were controlled by an optimized Tet-ON transactivator, rtTA2(S)-S2(LAP) (where rtTA is reverse tTA and LAP is liver activator protein), resulting in expression predominantly in the kidney and liver. In mice expressing these transgenes, doxycycline induced FerH in the kidney by 14.2+/-4.8-fold (mean+/-S.D.). Notably, increases in ferritin in overexpressers versus control littermates were accompanied by an elevation of IRP (iron regulatory protein) activity of 2.3+/-0.9-fold (mean+/-S.D.), concurrent with a 4.5+/-2.1-fold (mean+/-S.D.) increase in transferrin receptor, indicating that overexpression of FerH is sufficient to elicit a phenotype of iron depletion. These results demonstrate that FerH not only responds to changes in tissue iron (its classic role), but can actively regulate overall tissue iron balance.