Iron Carrier Transferrin Research Articles

Loss of PTPN2 Activity Alters Iron Handling Protein Expression in IBD Patients and Causes Iron Deficiency in Mice

BackgroundAnemia is the most common extraintestinal complication of inflammatory bowel disease (IBD) and is a risk factor for Crohn’s disease (CD) onset. Iron deficiency is the most common cause of anemia in IBD; however, the mechanisms involved are poorly understood. Here, we investigated the role of the IBD risk gene, protein tyrosine phosphatase non‐receptor type 2 (PTPN2), in regulating iron homeostasis.MethodsProteomic analyses were performed on serum from IBD patients genotyped for the IBD‐associated loss‐of‐function rs1893217 PTPN2variant (n=10/genotype). Constitutive Ptpn2 wild type (WT), heterozygous (Het), and knockout (KO) 3‐week‐old mice were analyzed for serum iron content, liver non‐heme iron and liver expression of the iron regulatory hormone, hepcidin (Hamp1). Protein and RNA from duodenal epithelial cells (DECs) were assayed by western blotting and qPCR. Localization of the brush border ferrous iron transporter, DMT1, in duodenal tissue was determined by immunohistochemistry (IHC).ResultsIron homeostasis genes, the iron carrier transferrin (TF) and the transferrin receptor (TFRC), were reduced (‐log p‐value = 10.7) in CD patients with the PTPN2 risk variant. Ptpn2‐KO mice had reduced i) serum iron (p<0.001; n=11); ii) serum TF saturation (p<0.01; n=9); and iii) liver non‐heme iron levels (p<0.01; n=10), vs. Ptpn2‐WT and Het mice. This indicated that PTPN2 loss decreased serum and liver iron storage. Moreover, Ptpn2‐KO mice had reduced liver expression of Hamp1 (p<0.01; n=7), likely suppressed by low serum iron. DEC gene expression of ferritin (Fth1), an intracellular iron storage molecule, was reduced (p=0.048; n=5) while Tfrc1, a mediator of basolateral cellular iron uptake, was significantly increased (p=0.0048; n=6) in Ptpn2‐KO mice. Reduced FTH1 expression (p=0.007; n=12) in DECs of Ptpn2‐KO mice was confirmed by western blot, indicating reduced intracellular iron storage. DMT1 expression was unchanged (n=6), whereas IHC showed reduced apical membrane DMT1 in duodenal epithelium of Ptpn2‐KO mice (n=6), suggesting a possible mechanism of impaired intestinal non‐heme iron uptake.ConclusionsCD patients with the PTPN2 loss‐of‐function rs1893217 SNP display alterations in serum iron handling proteins. Loss of PTPN2 activity in mice causes features of anemia including iron deficiency associated with mislocalization of the duodenal transporter DMT1. These findings identify a major role for PTPN2 in regulating iron homeostasis.

Red Blood Cell Transfusions Shape Macrophages Towards an Anti-Inflammatory Functional Phenotype and Impair the Immune Response to Infectious Cues

Reticulo-endothelial macrophages are central for the regulation of iron homeostasis, thanks to their ability to recycle red blood cell (RBC)-derived iron. Beside this role, macrophages are key innate immune cells which exhibit remarkable functional plasticity. These two roles are tightly interconnected: on the one hand, macrophage polarization dictates the expression of iron-regulated genes and determine cell iron handling; on the other, iron availability affects immune effector functions. However, it is still unclear how different iron sources affect macrophage plasticity. Here we investigated the phenotypic switching of macrophages induced by fresh RBCs compared to hemolytic RBCs, heme and different iron formulations.Repeated transfusions of fresh RBCs in mice result in macrophage iron overload and cause extensive macrophage cell death and monocyte recruitment in liver and spleen. After transfusion, macrophages acquire an M2-like anti-inflammatory phenotype, hallmarked by elevated expression of M2 markers (CD206, Arg-1, Ym1) and anti-inflammatory cytokines (IL-10, IL-4). Conversely, M1 markers (MHCII, CD86) and pro-inflammatory cytokines (IL-6, IL1b) are markedly decreased. These results indicate that transfusion-induced erythrophagocytosis likely suppresses the inflammatory response. A clear predisposition to infections has been observed in chronically transfused individuals, suggesting a detrimental impact of transfusion practice on the immune system. Therefore we analyzed whether transfusional iron overload impairs macrophage response to infectious cues. Our in vivo experiments demonstrate that transfusions shape macrophages towards an M2-like phenotype in a more pronounced way after LPS stimulation, highlighting a novel adverse anti-inflammatory effect of transfusions upon infection. Macrophages show a drastic reduction in M1 marker expression, which is reflected by decreased levels of circulating inflammatory cytokines and increased levels of anti-inflammatory cytokines. The pathophysiological relevance of our findings is currently under investigation in a model of transfused myelodysplastic (MDS) mice.Despite resulting in cell iron overload similarly to transfusions, treatment of mice with free heme and iron polarizes macrophages towards an M1-like pro-inflammatory phenotype. Interestingly, the M1 polarization potential of the different iron formulations (Fe-carboxymaltose/-dextrane/-nitrate) closely reflects their capacity to readily release free iron in the circulation. The administration of the heme scavenger hemopexin and the iron carrier transferrin or the chelator deferoxamine strongly attenuates the ability of heme and iron to induce M1 polarization. The pathophysiological relevance of heme/iron-triggered M1 polarization was confirmed in a mouse model of sickle cell disease (SCD), hallmarked by hemolytic RBCs and elevated circulating heme and iron, as well as in a model of lung cancer, characterized by hemolytic RBC extravasation and iron-loaded macrophages. Hemopexin administration to sickle mice significantly reduces the pro-inflammatory profile of macrophages, thus reducing the chronic inflammation state associated with SCD. Conversely, iron delivery in the tumor microenvironment via iron-oxide nanoparticles elicits pro-inflammatory macrophages with tumor killing properties, thus delaying tumor growth.Collectively, these results suggest that iron, in its different forms, dynamically determines macrophage polarization and function. Here we have shown that the source and route of iron acquisition have a key role in shaping macrophage phenotype, independently of the ability to produce cell iron overload. Our observations indicate that transfusions dampen macrophage immune effector functions by inducing their switching towards an anti-inflammatory phenotype, which is unlikely able to properly fight infections. The weak pro-inflammatory response of the macrophage might contribute to the increased propensity of transfused patients to develop infections, having potential implications for conditions associated with chronic transfusions (MDS, thalassemia, SCD). Importantly, these findings support the concept that macrophage plasticity can be manipulated for therapeutic purposes by applying different iron sources or scavengers/chelators according to the desired beneficial effect. DisclosuresPlatzbecker:Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Acceleron: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding.

The Lipogenic Regulator SREBP2 Induces Transferrin in Circulating Melanoma Cells and Suppresses Ferroptosis.

Circulating tumor cells (CTC) are shed by cancer into the bloodstream, where a viable subset overcomes oxidative stress to initiate metastasis. We show that single CTCs from patients with melanoma coordinately upregulate lipogenesis and iron homeostasis pathways. These are correlated with both intrinsic and acquired resistance to BRAF inhibitors across clonal cultures of BRAF-mutant CTCs. The lipogenesis regulator SREBP2 directly induces transcription of the iron carrier Transferrin (TF), reducing intracellular iron pools, reactive oxygen species, and lipid peroxidation, thereby conferring resistance to inducers of ferroptosis. Knockdown of endogenous TF impairs tumor formation by melanoma CTCs, and their tumorigenic defects are partially rescued by the lipophilic antioxidants ferrostatin-1 and vitamin E. In a prospective melanoma cohort, presence of CTCs with high lipogenic and iron metabolic RNA signatures is correlated with adverse clinical outcome, irrespective of treatment regimen. Thus, SREBP2-driven iron homeostatic pathways contribute to cancer progression, drug resistance, and metastasis. SIGNIFICANCE: Through single-cell analysis of primary and cultured melanoma CTCs, we have uncovered intrinsic cancer cell heterogeneity within lipogenic and iron homeostatic pathways that modulates resistance to BRAF inhibitors and to ferroptosis inducers. Activation of these pathways within CTCs is correlated with adverse clinical outcome, pointing to therapeutic opportunities.This article is highlighted in the In This Issue feature, p. 521.

Abstract 6073: The lipogenic regulator SREBP induces Transferrin in circulating melanoma cells, suppressing their susceptibility to ferroptosis

Abstract Circulating tumor cells (CTCs) are shed by cancers into the bloodstream, where a viable subset overcomes oxidative stress to initiate metastatic outgrowth. Clonally derived cultured CTCs from patients with BRAF-mutant melanoma reveal upregulation of lipogenesis and iron homeostasis pathways, correlated with their baseline and acquired drug resistance. In CTCs, the lipogenesis regulator SREBP directly induces transcription of the iron carrier Transferrin (TF), thereby reducing intracellular reactive oxygen species (ROS) and lipid peroxidation, and conferring resistance to BRAF inhibitors and inducers of ferroptosis. Knockdown of endogenous TF impairs tumorigenesis by melanoma CTCs; their associated soft agar clonogenic defect is rescued by the lipophilic anti-oxidants Ferrostatin-1 or Vitamin E, and by cholesterol. Single cell RNA-seq of patient-derived melanoma CTCs identifies a subset with high lipogenic, iron metabolic and proliferative signatures, correlated with adverse clinical outcome. Together, the coordinated regulation of these SREBP-driven pathways contributes to cancer progression, drug resistance and metastasis. Citation Format: Xin Hong, Whijae Roh, Ryan J. Sullivan, Keith H. Wong, Ben S. Wittner, HongShan Guo, Taronish D. Dubash, Moshe Sade-Feldman, Ben K. Wesley, Genevieve M. Boland, Dieuwke L. Marvin, Todd Bonesteel, Chenyue Lu, Elad Horwitz, François Aguet, Samuel S. Freeman, Katherine Calhoun, Michelle K. Jewett, Linda T. Nieman, Nir Hacohen, Anders M. Näär, David T. Ting, Mehmet Toner, Shannon L. Stott, Gad Getz, Shyamala Maheswaran, Daniel A. Haber. The lipogenic regulator SREBP induces Transferrin in circulating melanoma cells, suppressing their susceptibility to ferroptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6073.

Controversies on the Consequences of Iron Overload and Chelation in MDS.

Many patients with MDS are prone to develop systemic and tissue iron overload in part as a consequence of disease-immanent ineffective erythropoiesis. However, chronic red blood cell transfusions, which are part of the supportive care regimen to correct anemia, are the major source of iron overload in MDS. Increased systemic iron levels eventually lead to the saturation of the physiological systemic iron carrier transferrin and the occurrence of non-transferrin-bound iron (NTBI) together with its reactive fraction, the labile plasma iron (LPI). NTBI/LPI-mediated toxicity and tissue iron overload may exert multiple detrimental effects that contribute to the pathogenesis, complications and eventually evolution of MDS. Until recently, the evidence supporting the use of iron chelation in MDS was based on anecdotal reports, uncontrolled clinical trials or prospective registries. Despite not fully conclusive, these and more recent studies, including the TELESTO trial, unravel an overall adverse action of iron overload and therapeutic benefit of chelation, ranging from improved hematological outcome, reduced transfusion dependence and superior survival of iron-loaded MDS patients. The still limited and somehow controversial experimental and clinical data available from preclinical studies and randomized trials highlight the need for further investigation to fully elucidate the mechanisms underlying the pathological impact of iron overload-mediated toxicity as well as the effect of classic and novel iron restriction approaches in MDS. This review aims at providing an overview of the current clinical and translational debated landscape about the consequences of iron overload and chelation in the setting of MDS.

Intravenous Iron Promotes Low-Grade Inflammation in Anemic Patients By Triggering Macrophage Activation

Patients with iron deficiency anemia (IDA) who have intolerance or an unsatisfactory response to oral iron or present with chronic kidney disease often receive intravenous (iv) iron formulations to improve their anemic condition. Here we investigated whether iv iron induces inflammation in anemic patients and the underlying cellular and molecular mechanisms. We analyzed plasma samples of anemic patients before and 3 weeks after receiving a single-bolus treatment with the iv formulation iron-carboxymaltose and measured circulating inflammatory cytokines. To understand the inflammatory action of iron, we studied the effect of iron-carboxymaltose and non-transferrin bound-like iron (NTBI) source exposure both in an in vitro and in vivo model system. Anemic patients show an increase in circulating cytokine levels still 3 weeks after iron-carboxymaltose injection, compared to their steady-state condition. We found elevated IL-6, TNFα, CCL2 and INFγ. Importantly, patients with more pronounced increase in circulating inflammatory cytokines show higher serum ferritin as well as hepcidin levels, likely induced by inflammation. These observations suggest that iv iron exerts a pro-inflammatory action in anemic patients. Similarly, treatment of wild-type as well as iron-deficient mice with iron-carboxymaltose induces inflammation by triggering cytokine release in the circulation. Since iron formulations might first target macrophages to allow iron recycling, we analyzed the phenotype and production of cytokines induced by iron in these cells both in vitro and in vivo. Iron-carboxymaltose triggers a pro-inflammatory phenotypic switching of bone marrow-derived macrophages (BMDMs), hallmarked by increased expression of the M1 markers MHCII, CD86, iNOS and inflammatory cytokines (e.g. TNFα, IL-6, IL-1β) and reduced expression of the M2 markers CD206, Arg-1, Ym1 and IL-10. This effect is directly due to the presence of iron in the formulation, as exposure of BMDMs to NTBI-like iron (ferric-nitrilotriacetate) induces the same polarization shift, which is prevented by the iron carrier transferrin and the iron chelator deferoxamine. Consistently, inflammatory polarization of reticulo-endothelial (RES) macrophages of the liver and the spleen is observed in mice, either in steady-state and IDA condition, after treatment with NTBI-like iron sources (e.g. ferric-ammonium citrate, ferric-nitrate) as well as iron-carboxymaltose. RES macrophages from treated mice show iron deposition and increased expression of iron-related genes (ferroportin, ferritin, HO-1), together with high cytokine production. Transferrin significantly attenuates iron-mediated macrophage activation in mice, leading to an overall reduction of circulating inflammatory cytokines. These results indicate that iron-activated macrophages contribute to inflammatory cytokine release after administration of intravenous iron. We further confirmed that this process occurs with other iron formulations, including iron-sucrose and iron-dextran. Mechanistically, iron affects macrophage plasticity by triggering reactive oxygen species (ROS) production. Application of anti-oxidant molecules such as N-acetyl-cysteine prevents iron-mediated macrophage polarization both in BMDMs and mice. Collectively, these results indicate that iv iron exerts a clear inflammatory effect in anemic patients through the pro-inflammatory activation of macrophages. Our findings have potential implications for IDA patients who might develop a chronic low-grade inflammation in response to treatment with iv iron formulations. We suggest that inflammation in this context might have a further detrimental effect on the underlying anemia, through a multifactorial action resulting from (i) erythropoiesis suppression, due to the inhibitory action of inflammatory cytokines and (ii) hepcidin induction, which leads to further reduction in iron absorption. In addition, alteration in the inflammatory status might be responsible for commonly reported iv iron-related adverse events, including headache, dizziness, nausea, abdominal pain, constipation, diarrhea, rash and injection-site reactions. We propose that the co-administration of transferrin or anti-oxidants with iv iron in IDA patients is of benefit to counteract iron-induced inflammation, thus improving anemia more effectively and preventing adverse effects. Disclosures Vinchi: Vifor Pharma: Research Funding; Pharmanutra: Research Funding; Silence Therapeutics: Consultancy; Novartis: Research Funding. Muckenthaler:Novartis: Research Funding; Silence Therapeutics: Consultancy. Girelli:Vifor Pharma: Other: honoraria for lectures; Silence Therapeutics: Membership on an entity's Board of Directors or advisory committees; La Jolla Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy.

Transferrin Receptors TfR1 and TfR2 Bind Transferrin through Differing Mechanisms.

Hereditary hemochromatosis (HH), a disease marked by chronic iron overload from insufficient expression of the hormone hepcidin, is one of the most common genetic diseases. One form of HH (type III) results from mutations in transferrin receptor-2 (TfR2). TfR2 is postulated to be a part of signaling system that is capable of modulating hepcidin expression. However, the molecular details of TfR2's role in this system remain unclear. TfR2 is predicted to bind the iron carrier transferrin (Tf) when the iron saturation of Tf is high. To better understand the nature of these TfR-Tf interactions, a binding study with the full-length receptors was conducted. In agreement with previous studies with truncated forms of these receptors, holo-Tf binds to the TfR1 homologue significantly stronger than to TfR2. However, the binding constant for Tf-TfR2 is still far above that of physiological holo-Tf levels, inconsistent with the hypothetical model, suggesting that other factors mediate the interaction. One possible factor, apo-Tf, only weakly binds TfR2 at serum pH and thus will not be able to effectively compete with holo-Tf. Tf binding to a TfR2 chimera containing the TfR1 helical domain indicates that the differences in the helical domain account for differences in the on rate of Tf, and nonconserved inter-receptor interactions are necessary for the stabilization of the complex. Conserved residues at one possible site of stabilization, the apical arm junction, are not important for TfR1-Tf binding but are critical for the TfR2-Tf interaction. Our results highlight the differences in Tf interactions with the two TfRs.

Retinal Function and Structure in the Hypotransferrinemic Mouse

The iron carrier transferrin is expressed at remarkably high levels in normal retinas and is upregulated during retinal degeneration. The authors characterized the consequences of genetically reduced retinal transferrin production on retinal structure and function. Hypotransferrinemic (HPX⁻/⁻) mice treated with weekly intraperitoneal salvage transferrin injections were examined at 1 and 2 months of age. HPX⁻/⁻, HPX⁺/⁻, and wild-type (WT) mice were evaluated by electroretinography, ophthalmoscopy, and histology. Retinal iron content and transferrin levels were measured. RNA levels of genes involved in iron homeostasis and antioxidative response were determined by quantitative PCR. Oxidative injury was assessed by immunostaining for 4-hydroxy-2-nonenal (HNE). At 2 months, dark-adapted, mixed rod-cone response b-wave amplitudes were significantly lower in HPX⁻/⁻ mice than in WT mice (340 ± 112 μV vs. 624 ± 134 μV [mean ± SEM]; P = 0.002). Oscillatory potentials were significantly suppressed in HPX mice, and ophthalmoscopy demonstrated marked retinal pallor. Quantitative immunostaining revealed a 39% reduction of transferrin content in HPX⁻/⁻ compared with WT retinas (P = 0.01). mRNA levels of Tf, Tf receptor, and ceruloplasmin were decreased, whereas mRNA for antioxidant genes were elevated in HPX⁻/⁻ retinas. HNE staining was reduced in mice carrying the mutant HPX allele. Histologic examination demonstrated preserved retinal structure, and retinal iron content was similar across the strains. Despite the lack of wild-type retinal transferrin production and low levels of retinal transferrin protein, the retinal morphology and retinal iron content in HPX⁻/⁻ mice treated by systemic salvage transferrin injections are normal until age 2 months. However, retinal function and gene expression of some of the iron-associated genes are significantly altered.

Luminescent lanthanide complexes as analytical tools in anion sensing, pH indication and protein recognition

Although lanthanide complexes are recently used in luminescence labeling of bio-targets, this review focuses on sensing profiles of synthetic and biological lanthanide complexes. Rational design and combinatorial screening approaches toward synthetic lanthanide complexes applicable as luminescent sensing materials are described. Iron-carrying transferrin and ferritin proteins further form lanthanide complexes working as pH indicators and protein recognition reagents.

Relocation of Cell Iron. A Mode of Chelation with Application to Diseases of Regional Iron Accumulation.

Iron raises to toxic levels in mitochondria of excitable cells in some forms of neuro-degeneration with brain accumulation (NBIA), in sideroblastic anemia and in Friedreich's ataxia (FA), often leaving the cytosol iron-depleted. In anemia of chronic disease (ACD) iron is withheld by macrophages, while iron levels in extracellular fluids (e.g. plasma) are drastically reduced. Although excessive iron deposition occurring in organs of iron overloaded (IO) patients can be reduced with iron chelators, it is uncertain whether this is applicable to conditions where iron accumulates within selected tissues/cells in the absence of systemic IO.Objective. We assessed whether deferiprone (DFP), a membrane-permeant bidentate chelator in clinical use for treating systemic IO, might serve as an iron relocating agent in settings of regional iron accumulation by a. capturing labile iron accumulated in cell compartments and b. conveying the chelated iron either to other cell locations for metabolic integration or to transferrin for systemic reutilization.Methods. DFP capacity to shuttle iron intracellularly and transcellularly was assessed in macrophages (J774) and heart (H9c2) cell models using organelle-targeted fluorescent iron-sensors in conjunction with fluorescence microscopy imaging. We employed pairs of sensors targeted to different cell compartments and iron-evoked quenching and chelator-evoked dequenching as means to trace DFP mediated iron transfer. Mitochondrial iron accumulation was generated with succinylacetone or by silencing genes affecting mitochondrial iron metabolism.Results. DFP facilitated iron transfer: a. from iron-laden cell organelles to other cell compartments or to medium (and vice versa) and b. from iron loaded macrophages to pre-erythroid MEL cells (for chemically induced hemoglobin synthesis) either directly or via transfer to extracellular transferrin.Discussion The results of this study indicate that relocation of cell accumulated iron can be used as a modality of chelation for treating conditions of regional iron accumulation by applying chelators able to permeate into cell compartments but also to transfer the chelated iron to cell acceptors or to the extracellular iron carrier transferrin.

Iron homeostasis and toxicity in retinal degeneration

Iron is essential for many metabolic processes but can also cause damage. As a potent generator of hydroxyl radical, the most reactive of the free radicals, iron can cause considerable oxidative stress. Since iron is absorbed through diet but not excreted except through menstruation, total body iron levels buildup with age. Macular iron levels increase with age, in both men and women. This iron has the potential to contribute to retinal degeneration. Here we present an overview of the evidence suggesting that iron may contribute to retinal degenerations. Intraocular iron foreign bodies cause retinal degeneration. Retinal iron buildup resulting from hereditary iron homeostasis disorders aceruloplasminemia, Friedreich's ataxia, and panthothenate kinase-associated neurodegeneration cause retinal degeneration. Mice with targeted mutation of the iron exporter ceruloplasmin have age-dependent retinal iron overload and a resulting retinal degeneration with features of age-related macular degeneration (AMD). Post mortem retinas from patients with AMD have more iron and the iron carrier transferrin than age-matched controls. Over the past 10 years much has been learned about the intricate network of proteins involved in iron handling. Many of these, including transferrin, transferrin receptor, divalent metal transporter-1, ferritin, ferroportin, ceruloplasmin, hephaestin, iron-regulatory protein, and histocompatibility leukocyte antigen class I-like protein involved in iron homeostasis (HFE) have been found in the retina. Some of these proteins have been found in the cornea and lens as well. Levels of the iron carrier transferrin are high in the aqueous and vitreous humors. The functions of these proteins in other tissues, combined with studies on cultured ocular tissues, genetically engineered mice, and eye exams on patients with hereditary iron diseases provide clues regarding their ocular functions. Iron may play a role in a broad range of ocular diseases, including glaucoma, cataract, AMD, and conditions causing intraocular hemorrhage. While iron deficiency must be prevented, the therapeutic potential of limiting iron-induced ocular oxidative damage is high. Systemic, local, or topical iron chelation with an expanding repertoire of drugs has clinical potential.

The Iron Carrier Transferrin Is Upregulated in Retinas from Patients with Age-Related Macular Degeneration

Iron can cause oxidative stress, and elevated iron levels have been associated with several neurodegenerative diseases including age-related macular degeneration (AMD). Transferrin, an iron transport protein, is expressed at high levels in the retina. The purpose of this study was to assess transferrin involvement in AMD by determining the expression profile of transferrin in retinas with AMD compared with retinas without evidence of disease. Postmortem retinas were obtained from AMD and non-AMD eyes. Expression of transferrin was assessed in a microarray dataset from 33 retinas of unaffected donors and 12 retinas of patients with AMD (six with neovascular AMD and six with non-neovascular AMD). Quantitative real-time RT-PCR (QPCR) was used to confirm the microarray results. Transferrin protein expression was assessed by semiquantitative Western blot analysis and immunohistochemistry. In comparison to unaffected retinas, mean transferrin mRNA levels, as measured by microarray analysis were elevated 3.5- and 2.1-fold in non-neovascular and neovascular AMD retinas, respectively. Semiquantitative Western blot analysis demonstrated a 2.1-fold increase in transferrin protein in AMD eyes. Immunohistochemistry showed more intense and widespread transferrin label in AMD maculas, particularly in large drusen, Müller cells, and photoreceptors. These data demonstrate that transferrin expression is increased in the retinas of patients with AMD relative to those of healthy control patients of comparable age. Along with previous studies that have demonstrated elevated iron levels in AMD retinas, early onset drusen formation in a patient with retinal iron overload resulting from aceruloplasminemia, and retinal degeneration with some features of macular degeneration in the iron-overloaded retinas of ceruloplasmin/hephestin knockout mice, the present study suggests that altered iron homeostasis is associated with AMD.

Iodination significantly influences the binding of human transferrin to the transferrin receptor

The human transferrin receptor (TfR) and its ligand, the serum iron carrier transferrin, serve as a model system for endocytic receptors. Although the complete structure of the receptor’s ectodomain and a partial structure of the ligand have been published, conflicting results still exist about the magnitude of equilibrium binding constants, possibly due to different labeling techniques. In the present study, we determined the equilibrium binding constant of purified human TfR and transferrin. The results were compared to those obtained with either iodinated TfR or transferrin. Using an enzyme-linked assay for receptor–ligand interactions based on the published direct calibration ELISA technique, we determined an equilibrium constant of K d=0.22 nM for the binding of unmodified human Tf to surface-immobilized human TfR. In a reciprocal experiment using soluble receptor and surface-bound transferrin, a similar constant of K d=0.23 nM was measured. In contrast, covalent labeling of either TfR or transferrin with 125I reduced the affinity 3–5-fold to K d=0.66 nM and K d=1.01 nM, respectively. The decrease in affinity upon iodination of transferrin is contrasted by an only 1.9-fold decrease in the association rate constant, suggesting that the iodination affects rather the dissociation than the association kinetics. These results indicate that precautions should be taken when interpreting equilibrium and rate constants determined with covalently labeled components.

In Vitro Fusion of Reticulocyte Endocytic Vesicles with Liposomes

Since reticulocytes have a high demand for iron, which is required for heme biosynthesis, these cells are highly specialized in the endocytosis of the iron carrier transferrin (Tf). From the resulting endocytic vesicles (EVs), iron is released and the vesicles rapidly return to the cell membrane where they fuse, causing the release of the apotransferrin. Due to a lack of other intracellular compartments, the endocytic vesicles can be readily isolated. In this study, we have investigated the fusogenic properties of EVs, using liposomes as target membranes. Membrane fusion was monitored by a lipid mixing assay based on the relief of fluorescence self-quenching, using octadecylrhodamine B-chloride (R18). Application of this procedure was verified and solidified by analysis of the fusion event by an independent lipid mixing assay, after in situ labeling of EVs, and by determination of the mixing of aqueous contents. We demonstrate that the endocytic vesicles are particularly prone to fuse with target membranes that contain dioleoylphosphatidylethanolamine (DOPE). Relative to DOPE, bilayers composed of phosphatidylserine or phosphatidylcholine show a reduced fusion activity with EV. The specific and strong inhibition of fusion by cyclosporin A and a peptide known to interfere with the propensity of DOPE to adopt the hexagonal HII phase suggests that the mechanism of fusion involves the ability of this lipid to readily adopt non-bilayer phases. ATP, GTP, and/or cytosol are not necessary to obtain fusion. However, trypsin treatment of the endocytic vesicles inhibits fusion, indicating the involvement of (a) protein(s) in the fusion event.

Serotonin agonists increase transferrin levels via activation of 5-HT1C receptors in choroid plexus epithelium

Choroid plexus epithelial cells are enriched in mRNA for proteins such as the iron carrier transferrin, which acts as a trophic factor in the brain. Choroid plexus epithelial cells also have a high density of 5-HT1C receptors linked to activation of the phosphoinositide (PI) hydrolysis second messenger system. The present studies show that the 5-HT1C/5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) potently increases PI hydrolysis and the levels of transferrin in primary cultures of rat choroid plexus epithelial cells. These effects are blocked by the 5-HT1C/5-HT2 receptor antagonists mesulergine and mianserin, but not by the 5-HT2 receptor-selective antagonist spiperone. Similarly, mesulergine and mianserin, but not spiperone, block the increases in transferrin levels and PI hydrolysis elicited by 5-carboxamidotryptamine (5-CT), a 5-HT1 receptor-selective agonist, and by serotonin. We conclude, therefore, that 5-HT1C receptor activation in the choroid plexus leads to an increase in the production of transferrin. By promoting transferrin synthesis in the choroid plexus, 5-HT may indirectly influence brain development and differentiation.

Iron, free radicals and cancer

Free radicals, intermediates in the tissue damage caused by radiation, are formed, inter alia, in interactions catalyzed by iron, which synergizes with radiation and some cytostatics (anthracyclins) in causing cell damage. Conversely, iron chelators can counteract cell damage. Similarly, antioxidants can slow atherogenesis, caused in part by oxidative stress and free radicals. Cell damage is also prevented by physiological defense systems like superoxide dismutase, against endogenous free radicals formed by granulocytes, monocytes, etc. Iron can thus induce free radicals which cause DNA double strand breaks and oncogene activation. This is suggested by four epidemiological studies suggesting a higher cancer risk in patients with larger iron stores than in those with small iron stores. In addition to its effect on carcinogenesis, iron can also maintain the growth of malignant cells as well as growth of pathogens. Breast cancer cells, for instance, display 5-15 times more transferrin receptors than normal breast tissue. Iron-carrying transferrin is in fact a growth factor. Hyposideremia in patients with cancer or infection is not a paraphenomenon but a functioning defense mechanism ('nutritional immunity'). If this immunity is broken by iron administration, relapses of diseases like tuberculosis, brucellosis, and malaria have been described. While iron-deficiency anemia should of course be diagnosed, treated and if possible prevented, there are good reasons to avoid over-utilization of medicamental iron.

Iron metabolism of established human hematopoietic cell lines in vitro

Three malignant hematopoietic cell lines were used in studies on cellular iron metabolism. Our results show that iron-carrying transferrin became bound to specific dimeric cell surface receptors. Iron accumulated within the cell with time, whereas intact transferrin was released back to the medium. Chloroquine and NH 4Cl, known as pH-raising agents in vesicles of the lysosomal system, inhibited iron accumulation and transferrin binding in a dose-dependent manner. This suggests that the acid pH in endosomes leads to the cleavage of the iron-transferrin bonds. Transferrin degradation was not found, which leads us to suggest a process of ‘acid flushing’ for the dissociation of iron from transferrin without the involvement of endosome-lysosome fusion. Taken together, the data agree with the concept of receptor-mediated endocytosis, as described for many macromolecules. Iron was stored in ferritin in the cell types tested. Only a minor part (less than 15%) of the iron was bound in hemoglobin in the K-562 cell line. The relationship between iron stores and exogenously added iron in heme synthesis was investigated using a double labelling ( 55 Fe 59 Fe ) technique. The results showed that exogenous iron was preferentially used before the iron stored in ferritin. The results are discussed in relation to various hypotheses on cellular iron uptake and transport.