Aluminum exposure affects transferrin-dependent and -independent iron uptake by K562 cells

Aluminum (Al) and iron (Fe) share several physicochemical characteristics and they both bind to transferrin (Tf), entering the cell via Tf receptors (TfR). Previously, we found similar values of affinity constant for the binding of TfR to Tf carrying either Al or Fe. The competitive interaction between both metals prevented normal Fe incorporation into K562 cells and triggered the upregulation of Fe transport. In the present work we demonstrated that Al modified Fe uptake without affecting the expression of Tf receptors. Both TfR and TfR2 mRNA levels, evaluated by RT-PCR, and TfR antigenic sites, analyzed by flow cytometry, were found unchanged after Al exposure. In turn, Al did induce upregulation of non-Tf bound Fe (NTBI) uptake. This modulation was not due to intracellular Fe decrease since NTBI transport proved not to be regulated by Fe depletion. Unlike its behavior in the presence of Tf, Al was unable to compete with NTBI uptake, suggesting that both metals do not share the same alternative transport pathway. We propose that Al interference with TfR-mediated Fe incorporation might trigger the upregulation of NTBI uptake, an adaptation aimed at incorporating the essential metal required for cellular metabolism without allowing the simultaneous access of a potentially toxic metal.     

Transferrin-bound and transferrin free iron uptake by cultured rat astrocytes.

Previously we had demonstrated the presence of transferrin receptor (TfR) on the plasma membrane of cultured rat cortical astrocytes. In this study, we investigated the roles of TfR in transferrin-bound iron (Tf-Fe) as well as transferrin-free iron (Fe II) uptake by the cells. The cultured rat astrocytes were incubated with 1 microM of double-labelled transferrin (125I-Tf-59Fe) in serum- free DMEM F12 medium or 59Fe II in isotonic sucrose solution at 37 degrees C or 4 degrees C for varying times. The cellular Tf-Fe, Tf and Fe II uptake was analyzed by measuring the intracellular radioactivity with gamma counter. The result showed that Tf-Fe uptake kept increasing in a linear manner at least in the first 30-min. In contrast to Tf-Fe uptake, the internalization of Tf into the cells was rapid initially but then slowed to a plateau level after 10 min. of incubation. The addition of either NH4Cl or CH3NH2, the blockers of Tf-Fe uptake via inhibiting iron release from Tf within endosomes, decreased the cellular Tf-Fe uptake but had no significant effect on Tf uptake. Pre-treated cells with trypsin inhibited significantly the cellular uptake of Tf-Fe as well as Tf. These findings suggested that Tf-Fe transport across the membrane of astrocytes is mediated by Tf-TfR endocytosis. The results of transferrin-free iron uptake indicated that the cultured rat cortical astrocytes had the capacity to acquire Fe II. The highest uptake of Fe II occurred at pH 6.5. The Fe II uptake was time and temperature dependent, iron concentration saturable, inhibited by several divalent metal ions, such as Co2+, Zn2+, Mn2+ and Ni2+ and not significantly affected by phenylarsine oxide treatment. These characteristics of Fe II uptake by the cultured astrocytes suggested that Fe II uptake is not mediated by TfR and implied that a carrier-mediated iron transport system might be present on the membrane of the cultured cells.     

Two mechanisms of iron uptake from transferrin by melanoma cells. The effect of desferrioxamine and ferric ammonium citrate.

The effects of ferric ammonium citrate (FAC) and desferrioxamine (DFO) on iron (Fe), and transferrin (Tf) uptake have been investigated using SK-MEL-28 human melanoma cells, which express the Tf homologue, melanotransferrin, in high concentrations. Previously we demonstrated two separate Fe uptake mechanisms from Tf, viz. a specific process mediated by the transferrin receptor (TfR) and a nonspecific process (Richardson, D. R., and Baker, E. (1990) Biochim. Biophys. Acta 1053, 1-12). Cells exposed to DFO demonstrated up-regulation of the TfR with a concurrent increase in the rate of Fe uptake. Desferrioxamine also stimulated the nonspecific process of Fe uptake, resulting in a further increase in accumulation of Fe over Tf after saturation of the specific TfR. Ferric ammonium citrate had two effects. First, it resulted in down-regulation of the TfR. Second, and paradoxically, it markedly stimulated the rate of Fe uptake from Tf by the nonspecific process without increasing the rate of nonspecific Tf uptake. These data conclusively demonstrate that two entirely different mechanisms of iron uptake from Tf exist in melanoma cells and that ferric ammonium citrate may be a useful experimental tool to further characterize the specific and nonspecific mechanisms of Fe uptake from Tf.     

Transferrin receptor 2 mediates a biphasic pattern of transferrin uptake associated with ligand delivery to multivesicular bodies

The physiological role of transferrin (Tf) receptor 2 (TfR2), a homolog of the well-characterized TfR1, is unclear. Mutations in TfR2 result in hemochromatosis, indicating that this receptor has a unique role in iron metabolism. We report that HepG2 cells, which endogenously express TfR2, display a biphasic pattern of Tf uptake when presented with ligand concentrations up to 2 µM. The apparently nonsaturating pathway of Tf endocytosis resembles TfR1-independent Tf uptake, a process previously characterized in some liver cell types. Exogenous expression of TfR2 but not TfR1 induces a similar biphasic pattern of Tf uptake in HeLa cells, supporting a role for TfR2 in this process. Immunoelectron microscopy reveals that while Tf, TfR1, and TfR2 are localized in the plasma membrane and tubulovesicular endosomes, TfR2 expression is associated with the additional appearance of Tf in multivesicular bodies. These combined results imply that unlike TfR1, which recycles apo-Tf back to the cell surface after the release of iron, TfR2 promotes the intracellular deposition of ligand. Tf delivered by TfR2 does not appear to be degraded, which suggests that its delivery to this organelle may be functionally relevant to the storage of iron in overloaded states. iron transport; HepG2 cells     

Comparison of the interactions of transferrin receptor and transferrin receptor 2 with transferrin and the hereditary hemochromatosis protein HFE

The transferrin receptor (TfR) interacts with two proteins important for iron metabolism, transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. A second receptor for Tf, TfR2, was recently identified and found to be functional for iron uptake in transfected cells (Kawabata, H., Germain, R. S., Vuong, P. T., Nakamaki, T., Said, J. W., and Koeffler, H. P. (2000) J. Biol. Chem. 275, 16618-16625). TfR2 has a pattern of expression and regulation that is distinct from TfR, and mutations in TfR2 have been recognized as the cause of a non-HFE linked form of hemochromatosis (Camaschella, C., Roetto, A., Cali, A., De Gobbi, M., Garozzo, G., Carella, M., Majorano, N., Totaro, A., and Gasparini, P. (2000) Nat. Genet. 25, 14-15). To investigate the relationship between TfR, TfR2, Tf, and HFE, we performed a series of binding experiments using soluble forms of these proteins. We find no detectable binding between TfR2 and HFE by co-immunoprecipitation or using a surface plasmon resonance-based assay. The affinity of TfR2 for iron-loaded Tf was determined to be 27 nm, 25-fold lower than the affinity of TfR for Tf. These results imply that HFE regulates Tf-mediated iron uptake only from the classical TfR and that TfR2 does not compete for HFE binding in cells expressing both forms of TfR.     

Characterization of glyceraldehyde-3-phosphate dehydrogenase as a novel transferrin receptor

A majority of cells obtain of transferrin (Tf) bound iron via transferrin receptor 1 (TfR1) or by transferrin receptor 2 (TfR2) in hepatocytes. Our study establishes that cells are capable of acquiring transferrin iron by an alternate pathway via GAPDH.These findings demonstrate that upon iron depletion, GAPDH functions as a preferred receptor for transferrin rather than TfR1 in some but not all cell types. We utilized CHO-TRVb cells that do not express TfR1 or TfR2 as a model system. A knockdown of GAPDH in these cells resulted in a decrease of not only transferrin binding but also associated iron uptake. The current study also demonstrates that, unlike TfR1 and TfR2 which are localized to a specific membrane fraction, GAPDH is located in both the detergent soluble and lipid raft fractions of the cell membrane. Further, transferrin uptake by GAPDH occurs by more than one mechanism namely clathrin mediated endocytosis, lipid raft endocytosis and macropinocytosis. By determining the kinetics of this pathway it appears that GAPDH-Tf uptake is a low affinity, high capacity, recycling pathway wherein transferrin is catabolised. Our findings provide an explanation for the detailed role of GAPDH mediated transferrin uptake as an alternate route by which cells acquire iron.     

Inhibition of heme synthesis decreases transferrin receptor expression in mouse erythroleukemia cells.

The coordination of transferrin receptor (TfR) expression and heme synthesis was investigated in mouse erythroleukemia (MEL) cells of line 707 treated with heme synthesis inhibitors or in a variant line Fw genetically deficient in heme synthesis. Cells of line 707 were induced for differentiation by 5 mM hexamethylene bisacetamide (HMBA). TfR expression increased in the course of induction, as judged by increased TfR mRNA synthesis, increased cytoplasmic TfR mRNA level, and by the increased number of cellular 125I-Tf binding sites. Addition of 0.1 mM succinylacetone (SA) decreased cellular TfR to the level comparable with the uninduced cells. The decrease was reverted by the iron chelator desferrioxamine (DFO) but not by exogenous hemin. In short-term (1-2 hours) incubation, SA inhibited 59Fe incorporation from transferrin into heme, whereas total cellular 59Fe uptake was increased. A decrease in TfR mRNA synthesis was apparent after 2 hours of SA treatment. Conversely, glutathione peroxidase mRNA synthesis, previously shown to be inducible by iron, was increased by SA treatment. Cells of heme deficient line Fw did not increase the number of Tf binding sites after the induction of differentiation by 5 mM sodium butyrate. SA had no effect on TfR expression in Fw cells. The results suggest that the depletion of cellular non-heme iron due to the increase in heme synthesis maintains a high level of transferrin receptor expression in differentiating erythroid cells even after the cessation of cell division.     

Computational Structure Models of Apo and Diferric Transferrin–Transferrin Receptor Complexes

Complexation of transferrin (Tf) and its receptor (TfR) is an essential event for iron uptake by the cell. Much data has been accumulated regarding Tf-TfR complexation, such as results from mutagenesis. We created 3D structural models of apo-human Tf-TfR (apoTf-TfR) and Fe(III)₂Tf-TfR (Fe₂Tf-TfR) complexes by computational rigid body refinement. The models are consistent with published mutagenesis experiments. In our models, the C-lobes of apoTf and Fe₂Tf bind to the helical domain of TfR, and the N-lobes are sandwiched between the ectodomain of TfR and the cell membrane as previously reported. Further, the molecules of apoTf and Fe₂Tf are not forced to undergo large conformational changes upon complexation. The creation of the models led a new and important finding that a residue of TfR, R651, which is called a hot spot for Tf-TfR binding, interacts with Tf E385 when either apoTf or Fe₂Tf bind to TfR. The models rationally interpret the iron release from Fe₂Tf-TfR upon acidification, dissociation of apoTf from TfR at slightly alkaline pH, and metal specific recognition of TfR.     

The role of the transferrin receptor for the activation of human lymphocytes

The proliferative response of peripheral blood mononuclear cells (PBMC) in synthetic serum-free media depends on the presence of sufficient amounts of transferrin (Tf). In the present communication we show that the reduction of Tf concentration in culture media results in a decreased proliferation, whereas lymphokine production and the expression of activation markers (IL-2 receptor; transferrin receptor, (TfR); HLA class II) remain unchanged. To examine whether this effect is due to iron depletion we added iron chelates (ferric citrate, FeCi; ferric nitrilotriacetic acid, FeNTA) which can be internalized by cells without the requirement for Tf. The iron chelates could fully restore the proliferative response even in complete absence of Tf, suggesting that the observed inhibitory effect was indeed caused by iron depletion. Addition of a monoclonal TfR antibody, J 64, also caused a marked inhibition of proliferation of PBMC in regular serum-containing medium as well as in Tf-free synthetic medium; this effect could not be overcome by any of the tested iron chelates. Therefore, growth inhibition caused by J 64 cannot simply be attributed to iron starvation. These data suggest that J 64 may interfere with processes others than iron uptake and that the TfR might confer a necessary promoting signal for lymphocyte proliferation.     

Bronchial-associated lymphoid tissue (BALT) response to airway challenge with cigarette smoke, bovine antigen and anti-pulmonary serum.

The bronchial-associated lymphoid tissue (BALT) is a lymphoepithelial organ, related to the immune defence of the lung and to alveolar clearance, which changes size in certain states of disease. Changes in the size of BALT were quantified and compared, and Spearman's test was used to test the relation with the bronchial epithelium. A total of 180 rats were used, divided into 6 groups of 30 as follows: 1) untreated controls; 2) exposed to cigarette smoke for two months; 3) treated with anti-pulmonary serum three doses daily over five days; 4) exposed to cigarette smoke and treated with anti-pulmonary serum; 5) sensitized with bovine albumin and exposed to an environment containing this antigen for two months; 6) exposed to cigarette smoke and bovine albumin. The lungs were processed for histological study, and were stained with the PAS-Alcian blue method. The main left bronchi BALT was studied, and the following were quantified: Lymphatic area (LA), as a percentage of the lung surface occupied by BALT; the flat epithelium (FEp), as the length of bronchial epithelium anatomically related to LA, whose cells tend to adopt a flat shape; the Contact epithelium (Cep), as the length of bronchial epithelium which is in direct contact with the LA. A percentage count of bronchial cells was made in the following classifications: globet cells; globet cells stained with the PAS-Alcian blue method; flat cells; lymphoepithelium cells; columnar cells; and bronchial epithelium cells excluding the above two cell types.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ferristatin II Promotes Degradation of Transferrin Receptor-1 In Vitro and In Vivo

Previous studies have shown that the small molecule iron transport inhibitor ferristatin (NSC30611) acts by down-regulating transferrin receptor-1 (TfR1) via receptor degradation. In this investigation, we show that another small molecule, ferristatin II (NSC8679), acts in a similar manner to degrade the receptor through a nystatin-sensitive lipid raft pathway. Structural domains of the receptor necessary for interactions with the clathrin pathway do not appear to be necessary for ferristatin II induced degradation of TfR1. While TfR1 constitutively traffics through clathrin-mediated endocytosis, with or without ligand, the presence of Tf blocked ferristatin II induced degradation of TfR1. This effect of Tf was lost in a ligand binding receptor mutant G647A TfR1, suggesting that Tf binding to its receptor interferes with the drug’s activity. Rats treated with ferristatin II have lower TfR1 in liver. These effects are associated with reduced intestinal ⁵⁹Fe uptake, lower serum iron and transferrin saturation, but no change in liver non-heme iron stores. The observed hypoferremia promoted by degradation of TfR1 by ferristatin II appears to be due to induced hepcidin gene expression.     

The molecular mechanism for receptor-stimulated iron release from the plasma iron transport protein transferrin

Human transferrin receptor 1 (TfR) binds iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes where iron is released in a TfR-facilitated process. Consistent with our hypothesis that TfR binding stimulates iron release from Fe-Tf at acidic pH by stabilizing the apo-Tf conformation, a TfR mutant (W641A/F760A-TfR) that binds Fe-Tf, but not apo-Tf, cannot stimulate iron release from Fe-Tf, and less iron is released from Fe-Tf inside cells expressing W641A/F760A-TfR than cells expressing wild-type TfR (wtTfR). Electron paramagnetic resonance spectroscopy shows that binding at acidic pH to wtTfR, but not W641A/F760A-TfR, changes the Tf iron binding site > or =30 A from the TfR W641/F760 patch. Mutation of Tf histidine residues predicted to interact with the W641/F760 patch eliminates TfR-dependent acceleration of iron release. Identification of TfR and Tf residues critical for TfR-facilitated iron release, yet distant from a Tf iron binding site, demonstrates that TfR transmits long-range conformational changes and stabilizes the conformation of apo-Tf to accelerate iron release from Fe-Tf.     

Increased Iron Sequestration in Alveolar Macrophages in Chronic Obtructive Pulmonary Disease

Free iron in lung can cause the generation of reactive oxygen species, an important factor in chronic obstructive pulmonary disease (COPD) pathogenesis. Iron accumulation has been implicated in oxidative stress in other diseases, such as Alzheimer’s and Parkinson’s diseases, but little is known about iron accumulation in COPD. We sought to determine if iron content and the expression of iron transport and/or storage genes in lung differ between controls and COPD subjects, and whether changes in these correlate with airway obstruction. Explanted lung tissue was obtained from transplant donors, GOLD 2–3 COPD subjects, and GOLD 4 lung transplant recipients, and bronchoalveolar lavage (BAL) cells were obtained from non-smokers, healthy smokers, and GOLD 1–3 COPD subjects. Iron-positive cells were quantified histologically, and the expression of iron uptake (transferrin and transferrin receptor), storage (ferritin) and export (ferroportin) genes was examined by real-time RT-PCR assay. Percentage of iron-positive cells and expression levels of iron metabolism genes were examined for correlations with airflow limitation indices (forced expiratory volume in the first second (FEV₁) and the ratio between FEV₁ and forced vital capacity (FEV₁/FVC)). The alveolar macrophage was identified as the predominant iron-positive cell type in lung tissues. Futhermore, the quantity of iron deposit and the percentage of iron positive macrophages were increased with COPD and emphysema severity. The mRNA expression of iron uptake and storage genes transferrin and ferritin were significantly increased in GOLD 4 COPD lungs compared to donors (6.9 and 3.22 fold increase, respectively). In BAL cells, the mRNA expression of transferrin, transferrin receptor and ferritin correlated with airway obstruction. These results support activation of an iron sequestration mechanism by alveolar macrophages in COPD, which we postulate is a protective mechanism against iron induced oxidative stress.     

Transferrin receptor 2: evidence for ligand-induced stabilization and redirection to a recycling pathway

Transferrin receptor 2 (TfR2) is a homologue of transferrin receptor 1 (TfR1), the protein that delivers iron to cells through receptor-mediated endocytosis of diferric transferrin (Fe(2)Tf). TfR2 also binds Fe(2)Tf, but it seems to function primarily in the regulation of systemic iron homeostasis. In contrast to TfR1, the trafficking of TfR2 within the cell has not been extensively characterized. Previously, we showed that Fe(2)Tf increases TfR2 stability, suggesting that trafficking of TfR2 may be regulated by interaction with its ligand. In the present study, therefore, we sought to identify the mode of TfR2 degradation, to characterize TfR2 trafficking, and to determine how Fe(2)Tf stabilizes TfR2. Stabilization of TfR2 by bafilomycin implies that TfR2 traffics to the lysosome for degradation. Confocal microscopy reveals that treatment of cells with Fe(2)Tf increases the fraction of TfR2 localizing to recycling endosomes and decreases the fraction of TfR2 localizing to late endosomes. Mutational analysis of TfR2 shows that the mutation G679A, which blocks TfR2 binding to Fe(2)Tf, increases the rate of receptor turnover and prevents stabilization by Fe(2)Tf, indicating a direct role of Fe(2)Tf in TfR2 stabilization. The mutation Y23A in the cytoplasmic domain of TfR2 inhibits its internalization and degradation, implicating YQRV as an endocytic motif.     

Characterization of the Interaction between Diferric Transferrin and Transferrin Receptor 2 by Functional Assays and Atomic Force Microscopy

Transferrin receptor 2 (TfR2), a homologue of the classical transferrin receptor 1 (TfR1), is found in two isoforms, α and β. Like TfR1, TfR2α is a type II membrane protein, but the β form lacks transmembrane portions and therefore is likely to be an intracellular protein. To investigate the functional properties of TfR2α, we expressed the protein with FLAG tagging in transferrin-receptor-deficient Chinese hamster ovary cells. The association constant for the binding of diferric transferrin (Tf) to TfR2α is 5.6 × 10⁶ M^− 1, which is about 50 times lower than that for the binding of Tf to TfR1, with correspondingly reduced rates of iron uptake. Evidence for Tf internalization and recycling via TfR2α without degradation, as in the TfR1 pathway, was also found. The interaction of TfR2α with Tf was further investigated using atomic force microscopy, a powerful tool used for investigating the interaction between a ligand and its receptor at the single-molecule level on the living cell surface. Dynamic force microscopy reveals a difference in the interactions of Tf with TfR2α and TfR1, with Tf-TfR1 unbinding characterized by two energy barriers, while only one is present for Tf-TfR2. We speculate that this difference may reflect Tf binding to TfR2α by a single lobe, whereas two lobes of Tf participate in binding to TfR1. The difference in the binding properties of Tf to TfR1 and TfR2α may help account for the different physiological roles of the two receptors.     

Mysteries of the transferrin-transferrin receptor 1 interaction uncovered

How does the iron (Fe) binding protein, transferrin (Tf), bind to the transferrin receptor 1 (TfR1) to donate Fe to cells? In this issue of Cell, Cheng et al., describe the molecular structure of the human TfR1-Tf complex, This atomic model shows that Tf binds laterally to the TfR1 dimer and extends into the gap between the bottom of the receptor ectodomain and the membrane.     

Human MRCKα is regulated by cellular iron levels and interferes with transferrin iron uptake

Myotonic dystrophy kinase-related Cdc42-binding kinase α (MRCKα, formally known as CDC42BPA) is a serine/threonine kinase that can regulate actin/myosin assembly and activity. Recently, it has been shown that it possesses a functional iron responsive element (IRE) in the 3′-untranslated region (UTR) of its mRNA, suggesting that it may be involved in iron metabolism. Here we report that MRCKα protein expression is also regulated by iron levels; MRCKα colocalizes with transferrin (Tf)-loaded transferrin receptors (TfR), and attenuation of MRCKα expression by a short hairpin RNA silencing construct leads to a significant decrease in Tf-mediated iron uptake. Our results thus indicate that MRCKα takes part in Tf-iron uptake, probably via regulation of Tf-TfR endocytosis/endosome trafficking that is dependent on the cellular cytoskeleton. Regulation of the MRCKα activity by intracellular iron levels could thus represent another molecular feedback mechanism cells could use to finely tune iron uptake to actual needs.