Distribution Of Transferrin Receptors Research Articles

Apicobasal transferrin receptor localization and trafficking in brain capillary endothelial cells

The detailed mechanisms by which the transferrin receptor (TfR) and associated ligands traffic across brain capillary endothelial cells (BECs) of the CNS-protective blood–brain barrier constitute an important knowledge gap within maintenance and regulation of brain iron homeostasis. This knowledge gap also presents a major obstacle in research aiming to develop strategies for efficient receptor-mediated drug delivery to the brain. While TfR-mediated trafficking from blood to brain have been widely studied, investigation of TfR-mediated trafficking from brain to blood has been limited. In this study we investigated TfR distribution on the apical and basal plasma membranes of BECs using expansion microscopy, enabling sufficient resolution to separate the cellular plasma membranes of these morphological flat cells, and verifying both apical and basal TfR membrane domain localization. Using immunofluorescence-based transcellular transport studies, we delineated endosomal sorting of TfR endocytosed from the apical and basal membrane, respectively, as well as bi-directional TfR transcellular transport capability. The findings indicate different intracellular sorting mechanisms of TfR, depending on the apicobasal trafficking direction across the BBB, with the highest transcytosis capacity in the brain-to-blood direction. These results are of high importance for the current understanding of brain iron homeostasis. Also, the high level of TfR trafficking from the basal to apical membrane of BECs potentially explains the low transcytosis which are observed for the TfR-targeted therapeutics to the brain parenchyma.

Heterogeneity of Transferrin receptor expression, distribution and transferrin uptake in breast cancer cells in 2D and 3D spheroid cultures

Alterations in the expression of proteins involved in iron homeostasis have been detected in cancer cells, and their levels appear to correlate inversely with patient survival. Thus, elucidating the mechanisms underlying defective iron transport is crucial to understand the faulty iron homeostasis that is typical of cancer cells. Rapidly proliferating cancer cells show increased expression of proteins that support iron import, ex. transferrin receptor (TfR), as well as decreased levels of proteins involved in iron export, ex. ferroportin. TfR (CD71 or p90) delivers iron into cells via the binding of iron‐loaded transferrin (holo‐Tf) and subsequent endocytosis and recycling of TfR‐Tf complexes. Tf is a commonly used ligand for receptor mediated drug delivery and molecular imaging of breast tumors. However, TfR expression and Tf uptake tumor heterogeneity (Figure 1) is a crucial issue that limits our understanding of the role of defective iron transport in tumor progression as well as the use of Tf in targeted drug delivery and tumor imaging. Our long‐term goal is to develop novel imaging methodologies and biochemical approaches to establish, quantitate and modify TfR expression and Tf uptake heterogeneity using tumor xenografts and 3D cancer spheroids (Figure 1). Herein, novel approaches to modulate Tf uptake will be tested for their effect on the heterogeneity of Tf delivery across solid tumors. Previously, increasing the serum concentration of iron‐deleted Tf (apo‐Tf) has been shown to alter Tf saturation as well as the intracellular distribution and level of TfR expression in mice models of anemia, e.g. β‐Thalassemia. We currently hypothesize that increasing concentration of apo‐Tf will lead to a re‐distribution of TfR to the surface of the cells, as well as altered trafficking of iron in cancer cells. We will test the effect of apo‐Tf in TfR expression and distribution in human breast cancer 2D cell cultures, 3D cancer spheroids and tumor xenografts. Firstly, Tf uptake plate reader assays have shown that 24h incubation with apo‐Tf modulates Tf uptake differently in non‐cancerous breast epithelial cells (MCF10A) and in human breast cancer cells (MDA‐MB‐231 and T47D cells). Immunofluorescence imaging of non‐cancerous vs. breast cancer cells has shown that apo‐Tf treatment induces significant changes in the intracellular distribution of TfR and early endosomal marker EEA1. These results suggest that treatment with apo‐Tf acts via alteration of TfR‐Tf endocytic trafficking pathway. Similar experiments will be performed to determine how incubation with apo‐Tf affects TfR expression and Tf uptake heterogeneity in 3D cancer spheroids. Ability to potentially alter TfR trafficking in breast cancer cells is highly significant not only to alter iron homeostasis as a way to reduce cancer progression but also to optimize Tf‐mediated drug delivery approaches. Funding source: This work was supported by the National Institute of Health (NIH) R01 BRG CA207725.Support or Funding InformationFunding source: This work was supported by the National Institute of Health (NIH) R01 BRG CA207725.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Iron transport kinetics through blood-brain barrier endothelial cells

BackgroundTransferrin and its receptors play an important role during the uptake and transcytosis of iron through blood-brain barrier (BBB) endothelial cells (ECs) to maintain iron homeostasis in BBB endothelium and brain. Any disruptions in the cell environment may change the distribution of transferrin receptors on the cell surface, which eventually alter the homeostasis and initiate neurodegenerative disorders. In this paper, we developed a comprehensive mathematical model that considers the necessary kinetics for holo-transferrin internalization and acidification, apo-transferrin recycling, and exocytosis of free iron and transferrin-bound iron through basolateral side of BBB ECs. MethodsOrdinary differential equations are formulated based on the first order reaction kinetics to model the iron transport considering their interactions with transferrin and transferrin receptors. Unknown kinetics rate constants are determined from experimental data by applying a non-linear optimization technique. ResultsUsing the estimated kinetic rate constants, the presented model can effectively reproduce the experimental data of iron transports through BBB ECs for many in-vitro studies. Model results also suggest that the BBB ECs can regulate the extent of the two possible iron transport pathways (free and transferrin-bound iron) by controlling the receptor expression, internalization of holo-transferrin-receptor complexes and acidification of holo-transferrin inside the cell endosomes. ConclusionThe comprehensive mathematical model described here can predict the iron transport through BBB ECs considering various possible routes from blood side to brain side. The model can also predict the transferrin and iron transport behavior in iron-enriched and iron-depleted cells, which has not been addressed in previous work.

EGFR regulates iron homeostasis to promote cancer growth through redistribution of transferrin receptor 1

Dysregulation in iron metabolism can lead to a wide range of diseases including cancer. Iron-regulatory proteins (IRPs) and iron responsive elements (IREs) have been established as post-transcriptional regulators of intracellular iron homeostasis. The roles of other pathways involved in this process, however, remain largely unknown. Here we report that epidermal growth factor receptor (EGFR), an oncogenic driver, binds to and regulates the subcellular distribution of transferrin receptor 1(TfR1) through its tyrosine kinase activity and thus is required for cellular iron import. Inactivation of EGFR reduces the cell surface TfR1 expression, which leads to decreased iron import due to impaired TfR1-mediated iron uptake. This damaged iron assimilation results in cell cycle arrest and growth inhibition, which can be partially rescued by non-Tf-bound iron supplements. Evaluation of non-small cell lung cancer samples reveals a positive correlation between EGFR activation and membrane TfR1 expression. Our findings uncover a new role of EGFR in modulating cellular iron homeostasis through redistribution of TfR1, which is essential for cancer development and progression.

Transferrin Receptor Levels are increased by Interleukin 1β in a Human Kidney Proximal Tubular Cell line

BackgroundIron is required for several essential cellular processes such as DNA synthesis and energy metabolism, but when present in excess can cause cellular injury. Although kidney iron levels and urinary excretion of iron and iron transport proteins such as transferrin are increased in patients with kidney disease, it remains unclear how iron is handled by the kidneys during inflammation. We hypothesize that inflammation increases renal tubular iron uptake mechanisms, which in turn may promote cellular injury.Methods and ResultsTo understand how iron is handled by proximal tubule cells during inflammation, we turned to HK‐2 cells, a human proximal tubular cell line, and investigated the levels and distribution of transferrin receptor and divalent metal transporter 1 (DMT‐1) in response to interleukin 1β, a pro‐inflammatory cytokine. Transferrin receptor allows the uptake of iron after binding and internalization of iron‐loaded transferrin while DMT‐1 carries divalent metals like iron, zinc and copper inside the cells. Our data with HK‐2 cells indicated that transferrin receptor levels doubled upon treatment with 0.1 ng/mL interleukin 1β for 24 hours (P<0.005 by one‐way ANOVA, followed by Dunnett's test). Immunofluorescent staining of HK‐2 cells showed no apparent change in transferrin receptor distribution. In contrast, DMT‐1 levels in HK‐2 cells remained unchanged upon treatment with interleukin 1β for 24 hours at concentrations as high as 50 ng/mL. Increased intracellular levels of iron may lead to mitochondrial oxidative stress, which in turn may impact ATP production. After treating HK‐2 cells with 1 ng/mL interleukin 1β for 24 hours, intracellular ATP levels were measured and indicated no significant difference with untreated cells.ConclusionsThese data suggest that under inflammatory conditions, the uptake mechanism for the key iron transport protein transferrin is increased in the proximal tubule. This could have important implications in the setting of glomerular injury, which permits greater amounts of transferrin to be filtered, resulting in increased exposure of proximal tubular cells to transferrin and thus iron uptake.

A novel model for brain iron uptake: introducing the concept of regulation.

Neurologic disorders such as Alzheimer's, Parkinson's disease, and Restless Legs Syndrome involve a loss of brain iron homeostasis. Moreover, iron deficiency is the most prevalent nutritional concern worldwide with many associated cognitive and neural ramifications. Therefore, understanding the mechanisms by which iron enters the brain and how those processes are regulated addresses significant global health issues. The existing paradigm assumes that the endothelial cells (ECs) forming the blood-brain barrier (BBB) serve as a simple conduit for transport of transferrin-bound iron. This concept is a significant oversimplification, at minimum failing to account for the iron needs of the ECs. Using an in vivo model of brain iron deficiency, the Belgrade rat, we show the distribution of transferrin receptors in brain microvasculature is altered in luminal, intracellular, and abluminal membranes dependent on brain iron status. We used a cell culture model of the BBB to show the presence of factors that influence iron release in non-human primate cerebrospinal fluid and conditioned media from astrocytes; specifically apo-transferrin and hepcidin were found to increase and decrease iron release, respectively. These data have been integrated into an interactive model where BBB ECs are central in the regulation of cerebral iron metabolism.

Heterogenous distribution of ferroportin-containing neurons in mouse brain

Iron is crucial for a variety of cellular functions in neuronal cells. Neuronal iron uptake is reflected in a robust and consistent expression of transferrin receptors and divalent metal transporter 1 (DMT 1). Conversely, the mechanisms by which neurons neutralize and possibly excrete iron are less clear. Studies indicate that neurons express ferroportin which could reflect a mechanism for iron export. We mapped the distribution of ferroportin in the adult mouse brain using an antibody prepared from a peptide representing amino acid sequences 223-303 of mouse ferroportin. The antibody specifically detected ferroportin in brain homogenates, whereas homogenates of cultured endothelial cells were devoid of immunoreactivity. In brain sections, ferroportin was confined to neuronal cell bodies and peripheral processes of cerebral cortex, hippocampus, thalamus, brain stem, and cerebellum. In brain stem ferroportin-labeling was particularly high in neurons of cranial nerve nuclei and reticular formation. Ferroportin was hardly detectable in striatum, pallidum, or hypothalamus. Among non-neuronal cells, ferroportin was detected in oligodendrocytes and choroid plexus epithelial cells. A comparison with previous studies on the distribution of transferrin receptors in neurons shows that many neuronal pools coincide with those expressing ferroportin. The data therefore indicate that neuronal iron homeostasis consists of a delicate balance between transferrin receptor-mediated uptake of iron-transferrin and ferroportin-related iron excretion. The findings also suggest a particular high turnover of iron in neuronal regions, such as habenula, hippocampus, reticular formation and cerebellum, as several neurons in these regions exhibit a prominent co-expression of transferrin receptors and ferroportin.

Impairment of protein trafficking upon overexpression and mutation of optineurin.

BackgroundGlaucoma is a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and axons. Optineurin is one of the candidate genes identified so far. A mutation of Glu50 to Lys (E50K) has been reported to be associated with a more progressive and severe disease. Optineurin, known to interact with Rab8, myosin VI and transferrin receptor (TfR), was speculated to have a role in protein trafficking. Here we determined whether, and how optineurin overexpression and E50K mutation affect the internalization of transferrin (Tf), widely used as a marker for receptor-mediated endocytosis.Methodology/Principal FindingsHuman retinal pigment epithelial (RPE) and rat RGC5 cells transfected to overexpress wild type optineurin were incubated with Texas Red-Tf to evaluate Tf uptake. Granular structures or dots referred to as foci formed in perinuclear regions after transfection. An impairment of the Tf uptake was in addition observed in transfected cells. Compared to overexpression of the wild type, E50K mutation yielded an increased foci formation and a more pronounced defect in Tf uptake. Co-transfection with TfR, but not Rab8 or myosin VI, construct rescued the optineurin inhibitory effect, suggesting that TfR was the factor involved in the trafficking phenotype. Forced expression of both wild type and E50K optineurin rendered TfR to colocalize with the foci. Surface biotinylation experiments showed that the surface level of TfR was also reduced, leading presumably to an impeded Tf uptake. A non-consequential Leu157 to Ala (L157A) mutation that displayed much reduced foci formation and TfR binding had normal TfR distribution, normal surface TfR level and normal Tf internalization.Conclusions/SignificanceThe present study demonstrates that overexpression of wild type optineurin results in impairment of the Tf uptake in RPE and RGC5 cells. The phenotype is related to the optineurin interaction with TfR. Our results further indicate that E50K induces more dramatic effects than the wild type optineurin, and is thus a gain-of-function mutation. The defective protein trafficking may be one of the underlying bases why glaucoma pathology develops in patients with E50K mutation.

Cellular distributions of molecules with altered expression specific to the tumor promotion process from the early stage in a rat two-stage hepatocarcinogenesis model

A global gene expression profiling specific to the early process of tumor promotion by fenbendazole (FB) or phenobarbital (PB) in a rat two-stage hepatocarcinogenesis model revealed 33 genes to show altered expression in common with both chemicals. The immunohistochemical distribution of transferrin receptor (Tfrc), nuclear receptor subfamily 0, group B, member 2 (Nr0b2) and minichromosome maintenance deficient 6 (MCM6), included in the altered expression profile, were therefore examined in FB- and PB-induced proliferative lesions at both early and late stages of tumor promotion. In addition, immunoexpression of transforming growth factor beta receptor (TGFbetaR) I, TGFbetaRII, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and phosphorylated phosphatase and tensin homolog deleted on chromosome 10 (pPTEN) was also examined. In the early stage, most hepatocellular foci positive for glutathione S-transferase placental form (GST-P) showed co-expression of TGFbetaRI and lack of PTEN and pPTEN, some GST-P-positive foci co-expressing Tfrc and Nr0b2. In the late stage, selective expression of TGFbetaRI, but not TGFbetaRII, was also observed in many adenomas and carcinomas consistently expressing GST-P. Nr0b2 was variably expressed in the proliferative lesions, irrespective of the carcinogenic stage. Like the GST-P-positive foci, adenomas and carcinomas consistently lacked PTEN and pPTEN. Expression of Tfrc and MCM6 was increased in parallel with the carcinogenic stage. In conclusion, loss of PTEN and dysregulation of transforming growth factor beta signaling can be considered to be involved in rat hepatocarcinogenesis from early stages. Selective expression of Tfrc in proliferative lesions suggests an involvement of changes in iron homeostasis during the process of tumor promotion/progression driven by FB or PB.

Functional analysis of individual cells and microenvironment of breast cancer‐draining lymph nodes

The development of distant metastases is the major cause of death in breast cancer (BC). In many BC cases, metastases are present in patients with no metastasis-positive lymph nodes (LN). Hence, there is a need to improve prognosis by a better prediction of the nodal status and tumor spread. The current study is designed to develop and utilize new functional characteristics of the cells and microenvironment of BC-draining LN, which may help to improve the estimation of LN metastatic involvement. Innovative devices and methodologies were developed for collecting, transferring, and analyzing LN at an individual-cell resolution. Using these devices, a suspension of living cells were prepared from the LN and processed for various assays, including immunophenotypic analysis, activation status, and invasion activity. The functional profile of tumor-activated LN cells showed an increase in the intracellular enzymatic reaction rate, accompanied by a homogeneous distribution of transferrin receptor as well as by a significant increase in matrix metalloproteinase proteolytic activity. Moreover, the proportion of cells exhibiting such a profile was significantly higher in tumor-containing LN than in tumor-free LN. Thus, the live and postfixation features of LN cells and their microenvironment, correlated with the functional status of the LN, may serve to improve their predictive value in breast cancer examination.

Distribution of transferrin receptor of the 1st type (TfR1) in rat convoluted seminiferous tubules at different periods of postnatal ontogenesis

Distribution of transferrin receptor of the 1st type (TfR1) in rat convoluted seminiferous tubules at different periods of postnatal ontogenesis

Altered iron metabolism, transferrin receptor 1 and ferritin in patients with colon cancer

In this study, the level and distribution of transferrin receptor 1 (TfR1) and ferritin in colorectal carcinoma and in normal colon epithelium has been determined relative to the tumor stage and iron status of patients using immunohistochemical staining methods. While the majority of carcinoma patients were anemic, no relationship between the level of colon tissue ferritin and TfR1 and the systemic parameters of iron metabolism was evident. Furthermore, no association between ferritin content and the grade of colorectal carcinoma was observed. However, a relationship between the expression of TfR1 and the grade of colorectal carcinoma was observed. In this case high expression of TfR1 was found in colorectal carcinoma samples of Dukes A or B grade, and well differentiated colorectal carcinoma cells. In comparison, weak or no expression of TfR1 was observed in carcinoma samples of Dukes C or D grade with poorly differentiated cells and in carcinoma samples that had lymph node infiltration and distant metastasis.

Receptor sérico de transferrina en niños sanos. Valor diagnóstico en la anemia infecciosa y en la ferropénica

The serum transferrin receptor (TfR) presents certain advantages over other parameters of cellular iron status because it does not vary in acute or chronic diseases. To establish reference ranges of TfR in healthy children for our laboratory, to define the distribution of this variable in children with acute illness and in those with iron deficiency, and to evaluate the diagnostic yield of TfR, the transferrin-receptor/ferritin ratio (TfR/F) and the transferrin-receptor-ferritin index (TfR-F) in distinguishing ferropenic from infectious anemia. A descriptive, cross-sectional analysis was conducted in 132 children aged from 6 months to 16 years for a period of 18 months. The subjects were classified in three groups: healthy children, children with acute illness, and children with iron deficiency. The distribution of TfR and its diagnostic yield were evaluated. Of the 132 subjects, 30 were excluded because they lacked one or more of the parameters under analysis and 19 were excluded because they showed a thalassemic trait. In the 30 healthy children, the mean TfR concentration was 1.2 mg/l (SD 0.36) and the median was 1.02 (IQR 0.7-1.7). In the 32 children with acute illness, with or without anemia, TfR values were similar to those found in healthy children (p > 0.05). TfR values were higher in children with iron deficiency (21 patients; mean TfR value: 1.67 mg/l SD 0:98) than in healthy children but this difference was not statistically significant (p 0.08). The highest TfR values were found in the group with ferropenic anemia (mean TfR value: 2.13 mg/l SD 1.14) with a statistically significant difference between healthy children (p 0.04) and those with iron deficiency without anemia (p 0.01). The TfR/F ratio showed an optimal diagnostic yield in distinguishing ferropenic from acute disease anemia. If this ratio is higher than 80.7 ferropenia can be suspected as the cause of the anemia with a global value of the test of 100 % (95 % CI: 75.91-99.42). TfR could be useful in evaluating intracellular iron status in children. Acute disease does not alter TfR values and, in combination with ferritin, TfR offers an optimal diagnostical yield in distinguishing ferropenic from acute illness anemia.

The annexin 2/S100A10 complex controls the distribution of transferrin receptor-containing recycling endosomes.

The Ca2+- and lipid-binding protein annexin 2, which resides in a tight heterotetrameric complex with the S100 protein S100A10 (p11), has been implicated in the structural organization and dynamics of endosomal membranes. To elucidate the function of annexin 2 and S100A10 in endosome organization and trafficking, we used RNA-mediated interference to specifically suppress annexin 2 and S100A10 expression. Down-regulation of both proteins perturbed the distribution of transferrin receptor- and rab11-positive recycling endosomes but did not affect uptake into sorting endosomes. The phenotype was highly specific and could be rescued by reexpression of the N-terminal annexin 2 domain or S100A10 in annexin 2- or S100A10-depleted cells, respectively. Whole-mount immunoelectron microscopy of the aberrantly localized recycling endosomes in annexin 2/S100A10 down-regulated cells revealed extensively bent tubules and an increased number of endosome-associated clathrin-positive buds. Despite these morphological alterations, the kinetics of transferrin uptake and recycling was not affected to a significant extent, indicating that the proper positioning of recycling endosomes is not a rate-limiting step in transferrin recycling. The phenotype generated by this transient loss-of-protein approach shows for the first time that the annexin 2/S100A10 complex functions in the intracellular positioning of recycling endosomes and that both subunits are required for this activity.

Transcytosis of GCSF-transferrin across rat alveolar epithelial cell monolayers.

The purpose of this study was to use primary cultured rat alveolar epithelial cell monolayers to examine the potential of using transferrin receptor (TfR)-mediated transcytosis for noninvasive systemic protein drug delivery via the pulmonary route. Freshly isolated rat type II pneumocytes were plated onto tissue culture-treated polycarbonate 12-mm Transwells. AEC monolayers (> or = 2500 omega(cm2)) were treated with keratinocyte growth factor (10 ng/mL) for maintenance of type II cell-like characteristics. Filgrastim (GCSF)-Tf conjugates were prepared using the linkers SPDP and DPDPB. TfR-specific binding and uptake were determined using 125I-Tf and 59Fe-Tf treatment, respectively. Apical-to-basolateral (A-to-B) transferrin receptor (TfR)-mediated transcytosis was determined by dosing the apical compartment with 1.5 microg/mL of 125I-Tf or 125I-GCSF-Tf. Nonspecific TfR-independent transport of 125I-Tf and 125I-GCSF-Tf was determined in parallel by including 150 microg/mL of nonradiolabeled Tf. Basolateral samples (500 microL) were taken at 2, 4, and 6 h post-dosing, subjected to 15% trichloroacetic acid precipitation, and assayed in a Packard gamma counter. TfR-specific transport was determined as the difference between total and nonspecifc transport. The effects of brefeldin-A (BFA) on TfR distribution and (A-to-B) transport of 125I-Tf, 125I-GCSF and 125I-GCSF-Tf was studied by including the agent in the apical fluid at 1 microg/mL. BFA treatment resulted in a small significant reduction in TfR at the basolateral surface of type II cell-like monolayers, while it had no effect on TfR distribution in type I cell-like monolayers. In contrast, BFA treatment significantly altered the endocytosis of TfR, reducing the basolateral uptake of 59Fe-Tf while greatly increasing the apical uptake of 59Fe-Tf. BFA treatment, however, did not affect the TfR-specific uptake of 59Fe-Tf in type I cell-like monolayers. TfR-specific apical-to-basolateral transcytosis of 125I-Tf and 126I-GCSF-Tf conjugates was significantly enhanced in the presence of BFA in type II cell-like monolayers, whereas it had no effect on apical-to-basolateral transport of 125I-GCSF. BFA-enhanced transport of 125I-GCSF-Tf was approximately 3-fold higher than that of 125I-GCSF in the presence or absence of BFA. Moreover, 125I-GCSF transport in the presence of BFA was not significantly different from non-specific 125I-GCSF-Tf transport. Chromatographic analyses and bio-assays revealed that GCSF-Tf was not degraded during transport via TfR-specific processes, and that GCSF retained biologic activity when liberated from the conjugate via dithiothreitol reduction. This study suggests the possibility of using TfR-mediated transcytosis for systemic delivery of therapeutic proteins via the alveolar epithelium.

Iron transport in a lymphoid cell line with the hemochromatosis C282Y mutation

AbstractThe gene for hemochromatosis (HFE) is expressed in a variety of cells, including those not thought to be affected by this disease. The impact of HFE on iron transport was examined in B-lymphoid cell lines developed from a patient with hemochromatosis with the HFE C282Y mutation (C282Y cells) and an individual with the wild-type HFE gene (WT cells). Whereas both cell lines expressed HFE protein, C282Y cells displayed less HFE protein at the cell surface. Transferrin receptor (TfR) number was 2- to 3-fold greater in WT cells than in C282Y cells, while TfR affinity for transferrin (Tf) was slightly lower in C282Y cells. TfR distribution between intracellular and cell-surface compartments was similar in both cell lines. Iron uptake per cell was greater in WT cells but was not increased proportional to TfR number. When considered relative to cell-surface TfR number, however, iron uptake and Tf internalization were actually greater in C282Y cells. Surprisingly, Tf-independent iron uptake was also significantly greater in C282Y cells than in WT cells. The ferritin content of C282Y cells was approximately 40% that of WT cells. Exposure of cells to pro-oxidant conditions in culture led to a greater inhibition of proliferation in C282Y cells than in WT cells. Our results indicate that in this B-lymphoid cell line, the HFE C282Y mutation affects both Tf-dependent and -independent iron uptake and enhances cell sensitivity to oxidative stress. The role of HFE in iron uptake by B cells may extend beyond its known interaction with the TfR.

Characterization and distribution of ferritin binding sites in the adult mouse brain.

Studies on iron uptake into the brain have traditionally focused on transport by transferrin. However, transferrin receptors are not found in all brain regions and are especially low in white matter tracts where high iron concentrations have been reported. Several lines of research suggest that a receptor for ferritin, the intracellular storage protein for iron, may exist. We present, herein, evidence for ferritin binding sites in the brains of adult mice. Autoradiographic studies using 125I-recombinant human ferritin demonstrate that ferritin binding sites in brain are predominantly in white matter. Saturation binding analyses revealed a single class of binding sites with a dissociation constant (K(D)) of 4.65 x 10(-9) M and a binding site density (Bmax of 17.9 fmol bound/microg of protein. Binding of radiolabeled ferritin can be competitively displaced by an excess of ferritin but not transferrin. Ferritin has previously been shown to affect cellular proliferation, protect cells from oxidative damage, and deliver iron. The significance of a cellular ferritin receptor is that ferritin is capable of delivering 2,000 times more iron per mole of protein than transferrin. The distribution of ferritin binding sites in brain vis-à-vis transferrin receptor distribution suggests distinct methods for iron delivery between gray and white matter.

Characterization of Transferrin Receptor in an Immortalized Cell Line of Rat Brain Endothelial Cells, RBE4

The content and distribution of transferrin receptors in an immortalized cell line, RBE4, derived from rat cerebral capillary endothelial cells was investigated using the monoclonal antibody MRC OX-26 (OX-26 mAb) specific for the rat transferrin receptor. An ELISA assay was developed with which the OX-26 mAb can be determined quantiatively. The detection limit of the assay was 10 pg or 0.07 fmol of murine antibody. With this technique accurate measurement of native antibody is now possible without the need for isotope labeling (iodination). Immunostaining of confluent monolayers of RBE4 cells using an antibody directed against the tight junction associated protein ZO-1 was indicative for structural intactness of RBE4 cell mono-layers. OX-26 immunostaining demonstrated localization of the transferrin receptor at the plasma membrane and/or in the cytosol. Binding studies showed saturation of OX-26 mAb binding. The antibody binding analysis gave a dissociation constant (KD) of 17.1 ± 1.2 nmol/l. The total amount of transferrin receptors present per cell was 70,800 ± 17,000. Our results indicate that receptor binding of OX-26 mAb can be studied using an in vitro cell culture model of rat brain mircrovessel endothelium in conjunction with an ELISA technique for detection of native antibody. This approach will be used to investigate mechanisms of transendothelial transport of OX-26 in vitro.

Apotransferrin and Holotransferrin Undergo Different Endocytic Cycles in Intestinal Epithelia (Caco-2) Cells

Previous studies have demonstrated that diferric transferrin and apotransferrin compete for the binding to basolateral transferrin receptors and that transferrin-mediated iron uptake by Caco-2 cells is inhibited by apotransferrin to a larger extent than that predicted solely by receptor competition. This inhibition can have important implications in determining the net exchange of iron between intestinal cells and the basolateral milieu. Accordingly, we further characterized the endocytic cycles of apotransferrin and diferric transferrin in Caco-2 cells. We found that after internalization both apotransferrin and diferric transferrin recycled to the cell exterior, but that apotransferrin had a protracted endocytic cycle. Confocal microscopy studies revealed a different cellular distribution of apotransferrin and diferric transferrin; both were found in a compartment close to the basal membrane, but apotransferrin reached as well regions closer to the apical membrane. Moreover, the intracellular distribution of transferrin receptors was influenced by the iron load of transferrin; cells incubated with apotransferrin presented a more apical distribution of transferrin receptors than cells incubated with diferric transferrin. These results indicate for the first time that the endocytic cycle of transferrin receptors in intestinal epithelial cells is determined by the iron content of transferrin. They explain also the marked inhibitory effect of apotransferrin on transferrin-mediated iron uptake by Caco-2 cells, since incubation of cells with apotransferrin resulted in the actual sequestration of the receptor in the cell interior.

Autoradiography of transferrin receptors in the human brain

Transferrin is the major protein concerned with iron transport in the serum and may provide a route by which iron enters the brain. This study was designed to show the optimal binding conditions for in vitro transferrin receptor autoradiography and to show the regional distribution of transferrin receptors in the human brain. Optimal binding conditions were: 120 min incubation with 5.0 nM 125I-transferrin at 37°C. Transferrin receptors degrade quickly even with storage at −70°C, therefore binding studies should be performed within 7 days post mortem. Transferrin receptors were widely distributed in the human brain, with high density in the neocortex, moderate densities in the putamen and caudate nuclei, and very low densities in the globus pallidus and substantia nigra. Therefore transferrin receptor density shows a mismatch with the known distribution of iron in the human brain. The presence and distribution of transferrin receptors in the human brain are important because they may provide a route to deliver lipophobic substances across the blood-brain barrier by binding them to antibodies raised against transferrin receptors.