The mechanism of iron uptake by the rat placenta

The mechanism of iron uptake from transferrin by the rat placenta in culture has been studied. Transferrin endocytosis preceded iron accumulation by the cells. Both transferrin internalisation and iron uptake were inhibited by low temperature. Transferrin endocytosis was less susceptible to the effects of metabolic inhibitors such as sodium fluoroacetate, potassium cyanide, 2,4, dinitrophenol or carbonylcyanide M-chlorophenyl hydrazone (CCCP) than was iron uptake. Iron accumulation was decreased if the cells were incubated in the presence of weak bases such as chloroquine or ammonium chloride. These results suggest that, following internalisation, the vesicles containing the transferrin and iron became acidified, and that this acidification was a necessary prerequisite for the accumulation of iron by the cell. Further, the results indicate that the intravesicular pH was maintained at the expense of metabolic energy, suggesting that a pump may be involved. The importance of the permeability properties of the vesicle membrane in the iron uptake process was investigated by incubating the cells with labelled transferrin and iron in the presence of different cation and anion ionophores. Irrespective of the normal cation that the ionophores carried, all inhibited iron uptake without altering transferrin levels. In contrast, phloridzin, a Cl- transport inhibitor, did not affect either the levels of transferrin within the cells or the amount of iron accumulated.     

Evidence that transferrin supports cell proliferation by supplying iron for DNA synthesis

Transferrin is essential for cell proliferation and it was suggested that it may trigger a proliferative response following its interaction with receptors, serving as a growth factor. However, since the only clearly defined function of transferrin is iron transport, it may merely serve as an iron donor. To further clarify this issue, we took advantage of an iron chelate, ferric salicylaldehyde isonicotinoyl hydrazone (Fe-SIH), which we developed and previously demonstrated to efficiently supply iron to cells without using physiological transferrin receptor pathway. As expected, we observed that blocking monoclonal antibodies against transferrin receptors inhibited proliferation of both Raji and murine erythroleukemia cells. This inhibited cell growth was rescued upon the addition of Fe-SIH which was also shown to deliver iron to Raji cells in the presence of blocking anti-transferrin receptor antibodies. Moreover, blocking anti-transferrin receptor antibodies inhibited [3H]thymidine incorporation into DNA and this inhibition could be overcome by added Fe-SIH. In addition, Fe-SIH slightly stimulated, while SIH (an iron chelator) significantly inhibited, DNA synthesis in phytohemagglutinin-stimulated peripheral blood lymphocytes. Taken together, these results indicate that the only function of transferrin in supporting cell proliferation is to supply cells with iron.     

Biochemistry of nonheme iron in man. I. Iron proteins and cellular iron metabolism

Total plasma iron turnover in man is about 36 mg/day. Transferrin is the iron transport protein of plasma, which can bind 2 atoms of iron per protein molecule, and which interacts with various cell types to provide them with the iron required for their metabolic and proliferative processes. All tissues contain transferrin receptors on their plasma membrane surfaces, which interact preferentially with diferric transferrin. In erythroid cells as well as certain laboratory cell lines, the removal of iron from transferrin apparently proceeds via the receptor-mediated endocytosis process. Transferrin and its receptor are recycled to the cell surface, whereas the iron remains in the cell. The mode of iron uptake in the hepatocyte, the main iron storage tissue, is less certain. The release of iron by hepatocytes, as well as by the reticuloendothelial cells, apparently proceeds nonspecifically. All tissues contain the iron storage protein ferritin, which stores iron in the ferric state, though iron must be in the ferrous state to enter and exit the ferritin molecule. Cellular cytosol also contains a small-molecular-weight ferrous iron pool, which may interact with protoporphyrin to form heme, and which apparently is the form of iron exported by hepatocytes and macrophages. In plasma, the ferrous iron is converted into the ferric form via the action of ceruloplasmin.     

Role of plasma membrane phospholipids in the uptake and release of transferrin and its iron by reticulocytes

Summary The involvement of membrane phospholipids in the utilization of transferrinbound iron by reticulocytes was investigated using [⁵⁹Fe]- and [¹²⁵I]-labelled transferrin and rabbit reticulocytes which had been incubated with phospholipas A. Transferrin and iron uptake and release were all inhibited by phospholipas A which produced a marked decrease in the relative abundance of phosphatidylcholine and phosphatidylethanolamine and equivalent increases in their lyso-compounds in the reticulocyte plasma membrane. There was a close correlation between the iron uptake rate and the rate and amount of transferrin uptake and the amount of the lysophospholipids in the membrane. Incubation of the cells with exogenous lysophosphatidylethanolamine or lysophosphatidylcholine also produced inhibition of iron and transferrin uptake. The reduced uptake produced by phospholipase A could be reversed if the lyso-compounds were removed by fatty acid-free bovine serum albumin or by reincubation in medium 199. Treatment with phospholipase A was shown to increase the amount of transferrin bound by specific receptors on the reticulocyte membrane but to inhibit the entry of transferrin into the cells.The present investigation provides evidence that the phospholipid composition of the cell membrane influences the interaction of transferrin with its receptors, the processes of endocytosis and exocytosis whereby transferrin enters and leaves the cells, and the mechanism by which iron is mobilized between its binding to transferrin and incorporation into heme. In addition, the results indicate that phosphatidylethanolamine is present in the outer half of the lipid bilayer of reticulocyte membrane.     

Transferrin receptor expression and the regulation of placental iron uptake

Placental transferrin receptors, located at the apical side of syncytiotrophoblast, mediate placental iron uptake. Regulation of transferrin receptors on the fetal-maternal exchange area could be a major determinant in the regulation of trans-placental iron transport.Transferrin receptor expression in cultured human term cytotrophoblasts is on a much lower level than in choriocarcinoma cells, with a higher proportion of receptors located on the cell surface. Differentiation of cells, either due to longer culture periods or to 8-bromo-cAMP treatment does not lead to an increase of transferrin receptor expression. In vitro, the level of expression is largely regulated by the cellular density in the culture dishes. Low cellular occupancy of the dish leads to a high level of transferrin receptors. Treatment with iron-sources results in a down regulation of transferrin receptors.Thus, though the level of transferrin receptors in cultured normal trophoblast is at a constant level, unaffected by differentiation, high levels of maternal transferrin-iron availability can lead to a decrease in placental iron uptake. This feed-back mechanism makes placental iron uptake independent of maternal iron stores.Abbreviations hCG human Chorionic Gonadotrophin - TfR Transferrin Receptor     

Receptor-mediated endocytosis: the intracellular journey of transferrin and its receptor

A variety of ligands and macromolecules enter cells by receptor-mediated endocytosis. Ligands bind to their receptors on the cell surface and ligand-receptor complexes are localized in specialized regions of the plasma membrane called coated pits. Coated pits invaginate and give rise to intracellular coated vesicles containing ligand-receptor complexes which are thus internalized. Transferrin, a major serum glycoprotein which transports iron into cells, enters cells by this pathway. It binds to its receptor on the cell surface, transferrin-receptor complexes cluster in coated pits and are internalized in coated vesicles. Coated vesicles then lose their clathrin coat and fuse with endosomes, an organelle with an internal pH of about 5-5.5. Most ligands dissociate from their receptors in endosomes and they finally end up in lysosomes where they are degraded, while their receptors remain bound to membrane structures and recycle to the cell surface. Transferrin has a different fate: in endosomes iron dissociates from transferrin but apotransferrin remains bound to its receptor because of its high affinity for the receptor at acid pH. Apotransferrin thus recycles back to the plasma membrane still bound to its receptor. When the ligand-receptor complex reaches the plasma membrane or a compartment at neutral pH, apotransferrin dissociates from its receptor with a half-life of 18 s because of its low affinity for its receptor at neutral pH. The receptor is then ready for a new cycle of internalization, while apotransferrin enters the circulation, reloads iron in the appropriate organs and is ready for a new cycle of iron transport.     

Transferrin receptors and transferrin iron uptake by cultured human blood monocytes

Transferrin receptors have been previously found on human macrophages and it has also been shown that transferrin iron is taken up by these cells. It has therefore been inferred that the uptake is receptor mediated and involves an endocytic pathway. The subject was addressed directly in the present study in which the transferrin-iron-receptor interaction was characterized in cultured human blood monocytes. Specific, saturable diferric transferrin binding was demonstrated, with a kDa of 3.6 X 10(-8) M and a calculated receptor density of 1.25-2.5 X 10(5) receptors per cell. Incubation at 4 degrees C markedly reduced transferrin binding and completely inhibited iron uptake. Chase experiments confirmed progressive cellular loading of iron, with concomitant loss of transferrin. Inhibitors of endocytic vesicle acidification (ammonium chloride and 2,4-dinitrophenol) inhibited iron unloading from endocytosed diferric transferrin, while microtubular inhibitors (colchicine and vindesine) and a microfilament inhibitor (cytochalasin B) reduced diferric transferrin uptake but had little effect on the iron unloading pathway. A similar effect was noted with a calcium ion antagonist (verapamil) and with 2 calmodulin antagonists (chlorpromazine and imipramine). These latter findings suggest the importance of cytoskeleton-membrane interactions via a calcium, calmodulin and protein kinase C mediated system. Endocytosed iron accumulated progressively as ferritin within the cultured monocytes.     

Transferrin and tooth morphogenesis: Retention of transferrin by mouse embryonic teeth in organ culture

Transferrin is the only serum protein that is required for the early morphogenesis of mouse embryonic teeth in organ culture. Transferrin is able to support tooth morphogenesis and dental cell differentiation by stimulating cell proliferation. Its role in this process is restricted exclusively to iron transport, which takes place by receptor-mediated endocytosis of iron-loaded transferrin. A lipophilic iron chelator, pyridoxal isonicotinoyl hydrazone (PIH), can replace transferrin and support tooth morphogenesis in organ culture. We studied the effects of these two iron transporters on cell proliferation in tooth germs during culture. We found that Fe-PIH and transferrin stimulate proliferation to a similar extent in early cap-stage teeth of 14-day mouse embryos, but have no effect on cell proliferation in bell-stage teeth of 16-day mouse embryos. Day-16 teeth undergo morphogenesis in unsupplemented chemically defined medium, whereas transferrin or Fe-PIH is needed for the morphogenesis of day-14 teeth. Although the need for exogenous iron-transport molecules is lost with advancing development, the level of mitotic activity is still fairly high in bell-stage teeth. The abundant binding of transferrin in areas of active cell proliferation in bell-stage teeth also suggests that transferrin is still needed and used for the transport of iron into proliferating cells. Transferrin is not degraded by the process of receptor-mediated endocytosis. After releasing iron into a cell, transferrin is returned to the extracellular space and is reused. We therefore studied whether the transferrin needed by bell-stage teeth could be adequately supplied by endogenous transferrin synthesized or stored in tissue explants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Iron metabolism in BeWo chorion carcinoma cells. Transferrin-mediated uptake and release of iron

Growing human choriocarcinoma BeWo b24 cells contain 1.5 X 10(6) functional cell surface transferrin binding sites and 2.0 X 10(6) intracellular binding sites. These cells rapidly accumulate iron at a rate of 360,000 iron atoms/min/cell. During iron uptake the transferrin and its receptor recycle at least each 19 min. The accumulated iron is released from the BeWo cells at a considerable rate. The time required to release 50% of previously accumulated iron into the extracellular medium is 30 h. This release process is cell line-specific as HeLa cells release very little if any iron. The release of iron by BeWo cells is stimulated by exogenous chelators such as apotransferrin, diethylenetriaminepenta-acetic acid, desferral, and apolactoferrin. The time required to release 50% of the previously accumulated iron into medium supplemented with chelator is 15 h. In the absence of added chelators iron is released as a low molecular weight complex, whereas in the presence of chelator the iron is found complexed to the chelator. Uptake of iron is inhibited by 250 microM primaquine or 2.5 microM monensin. However, the release of iron is not inhibited by these drugs. Intracellular iron is stored bound to ferritin. A model for the release of iron by BeWo cells and its implication for transplacental iron transport is discussed.     

Iron metabolism in K562 erythroleukemic cells

Iron delivery to K562 cells is enhanced by desferrioxamine through induction of transferrin receptors. Experiments were performed to further characterize this event with respect to iron metabolism and heme synthesis. In control cells, up to 85% of the iron taken up from iron-transferrin was incorporated into ferritin, 7% into heme, and the remainder into compartments not yet identified. In cells grown with desferrioxamine, net accumulation of intracellular desferrioxamine (14-fold) was observed and iron incorporation into ferritin and heme was inhibited by 86% and 75%, respectively. In contrast, complete inhibition of heme synthesis in cells grown with succinylacetone had no effect on transferrin binding or iron uptake. Exogenous hemin (30 microM) inhibited transferrin binding and iron uptake by 70% and heme synthesis by 90%. These effects were already evident after 2 h. Thus, although heme production could be reduced by desferrioxamine, succinylacetone, and hemin, cell iron uptake was enhanced only by the intracellular iron chelator. The effects of exogenous heme are probably unphysiologic and the greater inhibition of iron flow into heme can be explained by effects on early steps of heme synthesis. We conclude that in this cell model a chelatable intracellular iron pool rather than heme synthesis mediates regulation of iron uptake.     

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 NH₄Cl, 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 (⁵⁵Fe/⁵⁹Fe) 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.     

The effect of different iron compounds on transferrin receptor expression in term human cytotrophoblast cells

During pregnancy, the mother is faced with an increased food demand. A good example of this increased demand is iron (Fe). Fe is needed in all growing cells. During pregnancy, the Fe transport to the fetus increases enormously. This amount can easily induce an Fe deficiency in the mother. Fe suppletion is very important for her, but not for the Fe status of the fetus, which is protected against Fe toxicity as well as deficiency. The placenta seems to be autonomous in Fe uptake. Likely there is a regulation mechanism. The human placenta is hemomonochorial. The cell layer of the fetus in contact with the maternal blood is formed by syncytiotrophoblasts. Fe is transported to the placenta by transferrin. Transferrin binds to a transferrin receptor on the trophoblast membrane and is internalized via an endocytic pathway. During this cycle, Fe is released from transferrin and the transferrin-transferrin receptor complex is recycled to the membrane. Isolated trophoblast cells from term placentas form a syncytium in vitro, and transferrin receptors are expressed. Expression depends on the number of cells in culture, culture time, the amount of Fe available, and the Fe compound. By regulation of the number of transferrin receptors, trophoblasts are able to control their Fe uptake.     

Transferrin receptor 2: a new molecule in iron metabolism

Transferrin receptor 1 (TfR1) which mediates uptake of transferrin-bound iron, is essential for life in mammals. Recently, a close homologue of human transferrin receptor 1 was cloned and called transferrin receptor 2 (TfR2). A similar molecule has been identified in the mouse. Human transferrin receptor 2 is 45% identical with transferrin receptor 1 in the extracellular domain, but contains no iron responsive element in its mRNA and is apparently not regulated by intracellular iron concentration nor by interaction with HFE. Transferrin receptor 2, like transferrin receptor 1, binds transferrin in a pH-dependent manner (but with 25 times lower affinity) and delivers iron to cells. However, transferrin receptor 2 distribution differs from transferrin receptor 1, increasing in differentiating hepatocytes and decreasing in differentiating erythroblasts. Expression of both receptors is cell cycle dependent. Mutations in the human transferrin receptor 2 gene cause iron overload disease, suggesting it has a role in iron homeostasis.     

Brain iron homeostasis.

The anatomical and cellular distribution of non-haem iron, ferritin, transferrin, and the transferrin receptor have been studied in postmortem human brain and these studies, together with data on the uptake and transport of labeled iron, by the rat brain, have been used to elucidate the role of iron and other metal ions in certain neurological disorders. High levels of non-haem iron, mainly in the form of ferritin, are found in the extrapyramidal system, associated predominantly with glial cells. In contrast to non-haem iron, the density of transferrin receptors is highest in cortical and brainstem structures and appears to relate to the iron requirement of neurones for mitochondrial respiratory activity. Transferrin is synthesized within the brain by oligodendrocytes and the choroid plexus, and is present in neurones, consistent with receptor mediated uptake. The uptake of iron into the brain appears to be by a two-stage process involving initial deposition of iron in the brain capillary endothelium by serum transferrin, and subsequent transfer of iron to brain-derived transferrin and transport within the brain to sites with a high transferrin receptor density. A second, as yet unidentified mechanism, may be involved in the transfer of iron from neurones possessing transferrin receptors to sites of storage in glial cells in the extrapyramidal system. The distribution of iron and the transferrin receptor may be of relevance to iron-induced free radical formation and selective neuronal vulnerability in neurodegenerative disorders.     

An evaluation of lysosomal enzyme leakage as a factor influencing the behaviour of transformed cells.

The effect of concanavalin A on transferrin and iron uptake by reticulocytes was determined using rabbit reticulocytes and rabbit transferrin labelled with ⁵⁹Fe and ¹²⁵I and concanavalin A (ConA) labelled with ¹³¹I. In concentrations of 50–200 μg/ml ConA markedly inhibited iron uptake but did not inhibit transferrin uptake or release from the cells. ConA was itself taken up by rabbit blood cells in a manner similar to that of transferrin except that the uptake was not specific for reticulocytes but occurred also with mature erythrocytes. The inhibition of iron uptake by concanavalin and the uptake of concanavalin by the cells were both inhibited by α-methyl-d-mannoside. It is concluded that the effects observed were due to the binding of concanavalin to glycoproteins of the cell membrane, either by a direct interaction with transferrin receptors or by the production of a non-specific change in the structure of the membrane.     

Iron uptake in reticulocytes. Inhibition mediated by the ionophores monensin and nigerisin

The lipophilic carboxylic ionophores monensin and nigerisin reversibly blocked iron uptake by erythroid cells. At low concentrations of ionophores (0.25-0.5 microM), the disruption of the compartment in which iron is released affected minimally the release of iron from transferrin but effectively inhibited iron uptake. Iron released from transferrin was extruded from the cell synchronously with but not bound to transferrin. The compartment disrupted by the ionophores, and in which iron is released from transferrin, is apparently contiguous to the extracellular medium. Contiguity was assessed by determining the effect of extracellular Na+ and K+ on the activity of the ionophores. The above data fit a model of iron uptake in which iron is released from transferrin in an acidic compartment in immediate contiguity with the cell plasma membrane. Iron is then bound by its membrane acceptor and is translocated to the cytosolic side of the plasma membrane. At submicromolar concentrations, the ionophores monensin and nigerisin produce a small increase in the pH of the acidic compartment. The pH change, which is not sufficient to block the release of iron from transferrin, is enough to block the binding of released iron to its acceptor in the plasma membrane, thus producing inhibition of iron uptake.     

Growth of human tumor cell lines in transferrin-free,low-iron medium

Summary Iron is essential for tumor cell growth. Previous studies have demonstrated that apart from transferrin-bound iron uptake, mammalian cells also possess a transport system capable of efficiently obtaining iron from small molecular weight iron chelates (Sturrock et al., 1990). In the present study, we have examined the ability of tumor cells to grow in the presence of low molecular weight iron chelates of citrate. In chemically defined serum-free medium, most human tumor cell lines required either transferrin (5 μg/ml) or a higher concentration of ferric citrate (500 μM) as an iron source. However, we have also found that from 13 human cell lines tested, 4 were capable of long-term growth in transferrin-free medium with a substantially lower concentration of ferric citrate (5 μM). When grown in medium containing transferrin, both regular and low-iron dependent cell lines use transferrin-bound iron. Growth of both cell types in transferrin medium was inhibited to a certain degree by monoclonal antibody 42/6, which specifically blocks the binding of transferrin to the transferrin receptor. On the contrary, growth of low-iron dependent cell lines in transferrin-free, low-iron medium (5 μM ferric citrate) could not be inhibited by monoclonal antibody 42/6. Furthermore, no autocrine production of transferrin was observed. Low-iron dependent cell lines still remain sensitive to iron depletion as the iron(III) chelator, desferrioxamine, inhibited their growth. We conclude that low-iron dependent tumor cells in transferrin-free, low-iron medium may employ a previously unknown mechanism for uptake of non-transferrin-bound iron that allows them to efficiently use low concentrations of ferric citrate as an iron source. The results are discussed in the context of alternative iron uptake mechanisms to the well-characterized receptor-mediated endocytosis process.     

Effect of iron chelators on the transferrin receptor in K562 cells

Delivery of iron to K562 cells by diferric transferrin involves a cycle of binding to surface receptors, internalization into an acidic compartment, transfer of iron to ferritin, and release of apotransferrin from the cell. To evaluate potential feedback effects of iron on this system, we exposed cells to iron chelators and monitored the activity of the transferrin receptor. In the present study, we found that chelation of extracellular iron by the hydrophilic chelators desferrioxamine B, diethylenetriaminepentaacetic acid, or apolactoferrin enhanced the release from the cells of previously internalized 125I-transferrin. Presaturation of these compounds with iron blocked this effect. These chelators did not affect the uptake of iron from transferrin. In contrast, the hydrophobic chelator 2,2-bipyridine, which partitions into cell membranes, completely blocked iron uptake by chelating the iron during its transfer across the membrane. The 2,2-bipyridine did not, however, enhance the release of 125I-transferrin from the cells, indicating that extracellular iron chelation is the key to this effect. Desferrioxamine, unlike the other hydrophilic chelators, can enter the cell and chelate an intracellular pool of iron. This produced a parallel increase in surface and intracellular transferrin receptors, reaching 2-fold at 24 h and 3-fold at 48 h. This increase in receptor number required ongoing protein synthesis and could be blocked by cycloheximide. Diethylenetriaminepentaacetic acid or desferrioxamine presaturated with iron did not induce new transferrin receptors. The new receptors were functionally active and produced an increase in 59Fe uptake from 59Fe-transferrin. We conclude that the transferrin receptor in the K562 cell is regulated in part by chelatable iron: chelation of extracellular iron enhances the release of apotransferrin from the cell, while chelation of an intracellular iron pool results in the biosynthesis of new receptors.     

Translational control during the acute phase response. Ferritin synthesis in response to interleukin-1.

Interleukin-1 (IL-1 beta) increases the synthesis of both heavy and light (L)-ferritin subunits when added to human hepatoma cells (HepG2) grown in culture. RNase protection and Northern blot analysis with L-ferritin probes revealed that no changes in L-ferritin mRNA levels occur after cytokine stimulation. However, the induction coincides with an increased association of the L-subunit mRNA with polyribosomes. Since the recruitment of stored ferritin mRNA onto polyribosomes is seen when iron enters the cell, the effect of IL-1 beta on iron uptake was tested and was found to be unaffected by the lymphokine. Neither transferrin receptor mRNA levels nor the number of receptors displayed on the cell surface was affected by IL-1 beta. However, the action of the cytokine on ferritin translation is inhibited by the action of the intracellular iron chelator deferoxamine. These data indicate that IL-1 beta induces ferritin gene expression by translational control of its mRNA. The pathway of induction is different from iron-dependent ferritin gene expression whereas regulation requires the background presence of cellular iron.