Targeted inhibition of transferrin-mediated iron uptake in Hep G2 hepatoma cells

We have used a model system consisting of two human hepatoma cell lines, Hep G2, representing well differentiated normal hepatocytes, and PLC/PRF/5, representing poorly differentiated malignant hepatocytes, to demonstrate that the differential presence of asialoglycoprotein receptor activity in these cell lines can be used to influence transferrin-mediated iron uptake. We based our experiments on the following facts: Hep G2 cells possess receptors that bind, internalize, and degrade galactose-terminal (asialo-)glycoproteins; PLC/PRF/5 cells have barely detectable asialoglycoprotein receptor activity; both cell lines possess active transferrin-mediated iron uptake; transferrin releases iron during acidification of intracellular vesicular compartments; primary amines, e.g. primaquine, inhibit acidification and iron release from transferrin. When added to culture medium, [55Fe]transferrin delivered 55Fe well to both cell lines. As expected, in the presence of [55Fe]transferrin, free primaquine caused a concentration-dependent decrease in 55Fe uptake in both cell lines. To create a targetable conjugate, primaquine was covalently coupled to asialofetuin to form asialofetuin-primaquine. When PLC/PRF/5 (asialoglycoprotein receptor (-)) cells were preincubated with this conjugate, transferrin-mediated 55Fe uptake was unaffected. However, transferrin-mediated 55Fe uptake by Hep G2 (asialoglycoprotein receptor (+)) cells under identical conditions was specifically decreased by 55% compared to control cells incubated without the conjugate.     

Specificity of hepatic iron uptake from plasma transferrin in the rat.

1. The role of specific interaction between transferrin and its receptors in iron uptake by the liver in vivo was investigated using 59Fe-125I-labelled transferrins from several animal species, and adult and 15-day rats. Transferrin-free hepatic uptake of 59Fe was measured 2 or 0.5 hr after intravenous injection of the transferrins. 2. Rat, rabbit and human transferrins gave high and approximately equal levels of hepatic iron uptake while transferrins from a marsupial (Sentonix brachyurus), lizard, crocodile, toad and fish gave very low uptake values. Chicken ovotransferrin resulted in higher uptake than with any other species of transferrin. 3. Iron uptake by the femurs (as a sample of bone marrow erythroid tissue) and, in another group of 19-day pregnant animals by the placentas and fetuses, was also measured, for comparison with the liver results. The pattern of uptake from the different transferrins was found to be similar to that of iron uptake by the liver except that with femurs, placentas and fetuses ovotransferrin gave low values comparable to those of the other non-mammalian species. 4. It is concluded that iron uptake by the liver from plasma transferrin in vivo is largely or completely dependent on specific transferrin-receptor interaction. The high hepatic uptake of iron from ovotransferrin was probably mediated by the asialoglycoprotein receptors on hepatocytes.     

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 iron interactions with cultured hepatocellular carcinoma cells (PLC/PRF/5)

Hepatocellular carcinoma cells of the PLC/PRF/5 cell line had 1.9 x 10(5) transferrin receptors per tumor cell with a Kd of 1.5 x 10(-8) M. At high concentrations of transferrin the binding was not saturable. Transferrin internalization by hepatoma cells was shown by time and temperature-dependent binding studies and by pronase experiments. Transferrin recycling was confirmed by the demonstration of a progressive increase in the cellular molar ratios of iron to transferrin and by chase experiments. Ammonium chloride interfered with iron unloading. The vinca alkaloid vincristine inhibited iron and transferrin uptake. The hepatocarcinoma cells appeared to lack asialoglycoprotein receptors and therefore internalized partially desialated transferrin by the regular route. Iron uptake from transferrin was markedly inhibited by the hydrophobic ferrous chelator 2,2' bipyridine but was relatively unaffected by the hydrophilic ferric chelator desferroxamine. The implication that ferrous iron was involved in postendocytic transvesicular membrane iron transport was supported by a study in which hepatoma cells were shown to take up large amounts of ferrous iron suspended in 270 mM sucrose at pH 5.5. The interaction at this pH between surface labeled hepatoma cell extracts and ferrous iron on a Sephacryl S-300 column suggested that the postendocytic transvesicular transport of iron through the membrane was in part protein mediated. The endocytosed iron in hepatoma cells was found in association with ferritin (33%), transferrin (31%) and a low molecular weight fraction (21%).     

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.     

The role of calcium in transferrin and iron uptake by reticulocytes

The possible role of calcium in the uptake of transferrin and iron by rabbit reticulocytes was investigated by altering cellular calcium levels through the use of the chelating agents EDTA and $\text{ethyleneglycol-bis}\text{-(3-}\text{aminoethylether}\text{)-N,N′-}\text{tetraacetic}$ acid (EGTA) and the ionophores, A23187 and X537A. Incubation of reticuloyctes with EDTA or EGTA at 4°C had no effect on transferrin and iron uptake but incubation at 37°C resulted in an irreversible inhibition associated with decreased adsorption of transferrin to the cells and evidence of inactivation or loss of the transferrin receptors. Transferrin and iron uptake were also inhibited when the cells were incubated with A23187 or X537A. In the case of A23187 the action was primarily exerted on the temperature-sensitive stage of transferrin uptake and was associated with loss of cellular K⁺ and decrease in cell size. The effect was greater when Ca²⁺ was added to the incubation medium than its absence. X537A produced relatively greater inhibition of iron uptake than of transferrin uptake, associated with a reduction in cellular ATP concentratio. The action of X537A was unaffected by the presence of Ca²⁺ in the incubation medium.The results obtained with EDTA and EGTA indicate that cell membrane Ca²⁺ is required for the integrity or binding of transferrin receptors to the reticulocyte membrane. No evidence was obtained from the experiments with ionophores that an increase of cellular Ca²⁺ affects transferrin and iron uptake directly. The inhibition caused by A23187 was mainly due to a reduction in cell size resulting from increased membrane permeability to K⁺ and that caused by X537A appeared to result from an inhibition of energy metabolism and ATP production.     

Hepatocytes and reticulocytes have different mechanisms for the uptake of iron from transferrin

The uptake of iron from transferrin by isolated rat hepatocytes and rat reticulocytes has been compared. The results show the following. 1) Reticulocytes and hepatocytes express plasma membrane NADH:ferricyanide oxidoreductase activity. The activity, expressed per 10(6) cells, is approximately 60-fold higher in the hepatocyte than in the reticulocyte. 2) Hepatocyte plasma membrane NADH:ferricyanide oxidoreductase activity and uptake of iron from transferrin are stimulated by low oxygen concentration and inhibited by iodoacetate. In reticulocytes, similar changes are seen in NADH:ferricyanide oxidoreductase activity, but not on iron uptake. 3) Ferricyanide inhibits the uptake of iron from transferrin by hepatocytes, but has no effect on iron uptake by reticulocytes. 4) Perturbants of endocytosis and endosomal acidification have no inhibitory effect on hepatocyte iron uptake, but inhibit reticulocyte iron uptake. 5) Hydrophilic iron chelators effectively inhibit hepatocyte iron uptake, but have no effect on reticulocyte iron uptake. Hydrophobic iron chelators generally inhibit both hepatocyte and reticulocyte iron uptake. 6) Divalent metal cations with ionic radii similar to or less than the ferrous iron ion are effective inhibitors of hepatocyte iron uptake with no effect on reticulocyte iron uptake. The results are compatible with hepatocyte uptake of iron from transferrin by a reductive process at the cell surface and reticulocyte iron uptake by receptor-mediated endocytosis.     

Uptake of iron from transferrin by isolated hepatocytes

Isolated rat hepatocytes containing 0.56-1.79 micrograms iron/10(6) cells and with an intracellular ATP concentration of 3-4 mM, accumulate iron from transferrin linearly with time for at least 3 h. At 37 degrees C the rate of uptake amounts to 0.3-0.7 pmol/mg cell protein per min. The uptake reaches a saturation level of 21-40 pmol/mg cell protein per h at 2.2 microM iron. At 5 degrees C the uptake does not increase over the time of incubation. Uptake of iron, but not binding of transferrin is increased 4-5-fold at oxygen concentrations 10-20 microM. At oxygen concentrations beyond these limits iron uptake is decreased. Iron taken up at low oxygen concentrations can be chelated by bathophenanthroline and bathophenanthroline disulphonate , but only if the chelators are present during the uptake experiments. The results suggest that iron uptake from transferrin by hepatocytes in suspension involves reductive removal of iron.     

Transferrin binding and iron uptake in mouse hepatocytes

Methods were developed for obtaining highly viable mouse hepatocytes in single cell suspension and for maintaining the hepatocytes in adherent static culture. The characteristics of transferrin binding and iron uptake into these hepatocytes was investigated. (1) After attachment to culture dishes for 18–24 h hepatocytes displayed an accelerating rate of iron uptake with time. Immediately after isolation mouse hepatocytes in suspension exhibited a linear iron uptake rate of 1.14·10⁵molecules/cell per min in 5 μM transferrin. Iron uptake also increased with increasing transferrin concentration both in suspension and adherent culture. Pinocytosis measured in isolated hepatocytes could account only for 10–20% of the total iron uptake. Iron uptake was completely inhibited at 4°C. (2) A transferrin binding component which saturated at 0.5 μM diferric transferrin was detected. The number of specific, saturable diferric transferrin binding sites on mouse hepatocytes was 4.4·10⁴±1.9·10⁴ for cells in suspension and 6.6·10⁴±2.3·10⁴ for adherent cultured cells. The apparent association constants were 1.23·10⁷ 1·mol^?1 and 3.4·10⁶ 1·mol^?1 for suspension and cultured cells respectively. (3) Mouse hepatocytes also displayed a large component of non-saturable transferrin binding sites. This binding increased linearly with transferrin concentration and appeared to contribute to iron uptake in mouse hepatocytes. Assuming that only saturable transferrin binding sites donate iron, the rate of iron uptake is about 2.5 molecules iron/receptor per min at 5 μM transferrin in both suspension and adherent cells and increases to 4 molecules iron/receptor per min at 10 μM transferrin in adherent cultured cells. These rates are considerably greater than the 0.5 molcules/receptor per min observed at 0.5 μM transferrin, the concentration at which the specific transferrin binding sites are fully occupied. The data suggest that either the non-saturable binding component donates some iron or that this component stimulates the saturable component to increase the rate of iron uptake. (4) During incubations at 4°C the majority of the transferrin bound to both saturable and nonsaturable binding sites lost one or more iron atoms. Incubations including 2 mM α,α′-dipyridyl (an Fe¹¹ chelator) decreased the cell associated ⁵⁹Fe at both 4 and 37°C while completely inhibiting iron uptake within 2–3 min of exposure at 37°C. These observations suggest that most if not all iron is loosened from transferrin upon interaction of transferrin with the hepatocyte membrane. There is also greater sensitivity of ⁵⁹Fe uptake compared to transferrin binding to pronase digestion, suggesting that an iron acceptor moiety on the cell surface is available to proteolysis.     

Effect of nicotine on transferrin binding and iron uptake by cultured rat placenta

The effect of nicotine on transferrin and iron transport in placental cells has been studied. Nicotine inhibits iron uptake but has little effect on the steady-state levels of transferrin. The effect is temperature and concentration dependent and is not reversible. At a concentration of 15 mM nicotine inhibited transferrin endocytosis by 40%, while iron uptake was decreased by nearly 60%. Nicotine exerted a similar effect on reticulocytes, but other amines, either tertiary or quaternary, had little or no effect on either iron uptake or steady-state intracellular transferrin levels. The results suggest that nicotine acts by blocking uptake, probably by acting as a weak base inhibiting iron release from transferrin, and inhibiting exocytosis with a resultant block of endocytosis. The concentrations required to exert an effect are too high to implicate inhibition of iron transport in the effects of smoking on pregnancy.     

The effect of lysosomotrophic bases and inhibitors of transglutaminase on iron uptake by immature erythroid cells

The mechanism by which weak bases block iron uptake by immature erythroid cells was investigated using rabbit and rat reticulocytes and erythroblasts from the fetal rat liver. A large variety of bases was found to inhibit iron uptake but to have a much smaller or no effect on transferrin uptake by the cells. Quinacrine and chloroquine were active at the lowest concentrations. Dansylcadaverine, an inhibitor of transglutaminase, was also active at low concentration. However, the results do not indicate a role for transglutaminase in the iron uptake process. Instead they show that the major effect of the bases is to inhibit iron release from transferrin molecules on or within the cells. The possible mechanism of this effect was investigated by measurement of intracellular ATP levels, intracellular pH and by morphological studies utilizing fluorescent and electron microscopy. The bases caused little change in ATP levels, but elevated intracellular pH, probably due to accumulation within intracellular vesicles, which were shown to accumulate fluorescent weak bases, to swell under the action of the bases and to be the site of intracellular localization of transferrin. It is concluded that the bases tested in this work inhibit iron release from transferrin in intracellular vesicles by increasing their pH rather than by blocking transglutaminase and thereby restricting endocytosis. Reduction of transferrin uptake by the cells when it occurs is probably due to inhibition of recycling of transferrin receptors to the outer cell membrane.     

Non-transferrin donors of iron for heme synthesis in immature erythroid cells

The mechanism of iron uptake from several iron-containing compounds by transferrin-depleted rabbit reticulocytes and mouse spleen erythroid cells was investigated. Iron complexes of DL-penicillamine, citrate and six different aroyl hydrazones may be utilized by immature erythroid cells for hemoglobin synthesis, although less efficiently than iron from transferrin. HTF-14, a monoclonal antibody against human transferrin, reacts with rabbit transferrin and inhibits iron uptake and heme synthesis by rabbit reticulocytes. HTF-14 had no significant effect on iron uptake and heme synthesis when non-transferrin donors of iron were examined. Ammonium chloride (NH4Cl) increases intracellular pH and blocks the release or utilization of iron from the internalized transferrin. NH4Cl only slightly affected iron incorporation and heme synthesis from non-transferrin donors of iron. Hemin inhibited transferrin iron uptake and heme synthesis, but had a much lesser effect on iron incorporation and heme synthesis from non-transferrin donors of iron. These results allow us to conclude that transferrin-depleted reticulocytes take up iron from all of the examined non-transferrin iron donors without the involvement of the transferrin/transferrin receptor pathway.     

Transferrin and iron in cultured chick embryonic neurons: a comparison between human and chick transferrins

Transferrin was not required for the short-term survival of cultured chick retinal neurons. Both human and chick transferrin failed to enhance the in vitro survival of 8- or 11-day embryonic chick retinal neurons when cultured in a defined medium. Furthermore, maintenance of neurons in the presence of chick transferrin antibody did not alter in vitro survival. Retinal neurons, however, could bind and internalize human or chick transferrin when assayed for by fluorescence immunohistochemical techniques. Binding and internalization of chick transferrin appeared to be greater than human transferrin. Iron uptake was measured in cultures maintained in the absence of transferrin. After incubation with 59FeCl3, iron uptake was 3.5 +/- 1.1 fmoles/cell. The presence of chick transferrin antibody did not significantly alter the amount of iron uptake occurring in this assay. In a comparison of human and chick transferrin mediated iron uptake, chick transferrin was 50% more effective than human transferrin in transporting iron. This study demonstrates that cultured embryonic retinal neurons are not dependent on transferrin for survival or iron uptake, although they actively bind and internalize transferrin. Results also demonstrate that whereas cultured chick retinal neurons can bind and utilize human transferrin, they do so with less efficiency than chick transferrin.     

Effect of aluminium on iron uptake and transferrin-receptor expression by human erythroleukaemia K562 cells.

Incubation of human erythroleukaemia K562 cells with Al-transferrin inhibited iron uptake from 59Fe-transferrin by about 80%. The inhibition was greater than that produced by a similar quantity of Fe-transferrin. Preincubation of cells for 6 h with either Al-transferrin or Fe-transferrin diminished the number of surface transferrin receptors by about 40% compared with cells preincubated with apo-transferrin. Al-transferrin did not compete significantly with Fe-transferrin for transferrin receptors and, when cells were preincubated for 15 min instead of 6 h, the inhibitory effect of Al-transferrin on receptor expression was lost. Both forms of transferrin also decreased the level of transferrin receptor mRNA by about 50%, suggesting a common regulatory mechanism. Aluminium citrate had no effect on iron uptake or transferrin-receptor expression. AlCl3 also had no effect on transferrin-receptor expression, but at high concentration it caused an increase in iron uptake by an unknown, possibly non-specific, mechanism. Neither Al-transferrin nor AlCl3 caused a significant change in cell proliferation. It is proposed that aluminium, when bound to transferrin, inhibits iron uptake partly by down-regulating transferrin-receptor expression and partly by interfering with intracellular release of iron from transferrin.     

Over-expression of wild-type and mutant HFE in a human melanocytic cell line reveals an intracellular bridge between MHC class I pathway and transferrin iron uptake

Hereditary hemochromatosis (HH) is a frequent recessive disorder of iron metabolism characterised by systemic iron overload. In Northern Europe, more than 90% of HH patients are homozygous for a mis-sense mutation (C282Y) in the HFE1 gene product. The HFE protein is the heavy chain of a MHC class I-related molecule and associates with beta2 microglobulin and the transferrin receptor. Its precise roles in iron metabolism and in the pathophysiology of HH are still unclear. In order to identify the cellular processing of HFE, an important step towards the understanding of the function of the protein, we stably over-expressed the wild type and mutated forms fused to the Green Fluorescent Protein in a melanocytic MHC class I expressing cell line, the Mel Juso cell line. In wild type and mutant clones, the fusion proteins were not detected at the cell surface but only in the cytoplasm. Their sub-cellular localisation was determined by co-labelling of cells with organite-specific antibodies and confocal microscopy. HFE-GFP followed initially HLA class I intracellular processing but co-localised with transferrin in early endosomes without recycling at the cell surface. The C282Y-GFP fusion protein followed a different folding pathway to exit endoplasmic reticulum. Over-expression of the wild-type protein lead to a decrease in diferric transferrin uptake. Our model will be of use in the elucidation of the functional interaction between intracellular HFE and iron transporters transferrin/transferrin receptor complexes and Slc11A2 (also named N-Ramp2 or DMT1) in different endosomal compartments.     

Staphylococcus aureus siderophore-mediated iron-acquisition system plays a dominant and essential role in the utilization of transferrin-bound iron

Staphylococcus aureus is known to be capable of utilizing transferrin-bound iron, via both siderophore- and transferrin-binding protein (named IsdA)-mediated iron-acquisition systems. This study was designed in order to determine which iron-acquisition system plays the essential or dominant role with respect to the acquisition of iron from human transferrin, in the growth of S. aureus. Holotransferrin (HT) and partially iron-saturated transferrin (PT), but not apotransferrin (AT), were found to stimulate the growth of S. aureus. S. aureus consumed most of the transferrin-bound iron during the exponential growth phase. Extracellular proteases were not, however, involved in the liberation of iron from transferrin. Transferrin-binding to the washed whole cells via IsdA was not observed during the culture. The expression of IsdA was observed only in the deferrated media with AT, but not in the media supplemented with PT or HT. In contrast, siderophores were definitely produced in the deferrated media with PT and HT, as well as in the media supplemented with AT. The siderophores proved to have the ability to remove iron directly from transferrin, but the washed whole cells expressing IsdA did not. In the bioassay, the growth of S. aureus on transferrin-bound iron was stimulated by the siderophores alone. These results demonstrate that the siderophore-mediated iron-acquisition system plays a dominant and essential role in the uptake of iron from transferrin, whereas the IsdA-mediated iron-acquisition system may play only an ancillary role in the uptake of iron from transferrin.     

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.     

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.     

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.     

Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. Effect of lysosomotropic agents

Growing HepG2 cells contain 50,000 functional surface transferrin-binding sites (Ciechanover, A., Schwartz, A.L., and Lodish, H.F. (1983) Cell 32,267-275) and 100,000 intracellular sites. At saturating concentrations of [59Fe]transferrin, and under conditions in which protein synthesis is blocked, iron uptake is linear for several hours at a rate of 9,500 transferrin molecules/cell/min. Thus, each receptor must recycle a ligand, on the average, each 15.8 min. Surface-bound transferrin is rapidly endocytosed (t1/2 = 3.5 min). All of the iron remains within the cell, while the apotransferrin is rapidly (t1/2 = 5.0 min) secreted into the medium. Previously, we showed (Dautry-Varsat, A., Ciechanover, A., and Lodish, H.F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2258-2262) that exposure of a ferrotransferrin-receptor complex to medium of pH less than 5.0 results in dissociation of iron, but that apotransferrin remains bound to its receptor. If the pH is raised to 7.0, such as would occur when an acidic intracellular vesicle fuses with the plasma membrane, apotransferrin is very rapidly dissociated (t1/2 = 17 s at 37 degrees C) from its receptor. Taken together, these results indicate that transferrin remains bound to its receptor throughout the endocytic cycle. In the present study, we have directly measured all the kinetic parameters involved in the transferrin receptor cycle. They are similar to those of the asialoglycoprotein receptor in the same cell line, and can be described by a simple kinetic model. In the presence of lysosomotropic agents, ferrotransferrin binds to its surface receptor and is internalized normally. However, iron is not dissociated from transferrin, and ferrotransferrin recycles back to the cell surface and is secreted into the medium. We conclude that the low pH in endocytic vesicles is essential for the dissociation of iron from transferrin and its delivery to the cell, but is not required for recycling of transferrin, and presumably of its receptor.