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Inhibition of uptake of transferrin-bound iron by human hepatoma cells by nontransferrin-bound iron
Inhibition of Uptake of Transferrin-Bound Iron by Human Hepatoma Cells by Nontransferrin-Bound Iron DEBORAH TRINDER
The liver acquires iron from transferrin by transferrin receptor-mediated (TR) and transferrin receptor-independent pathways (NTR) and from nontransferrin-bound iron (NTBFe). Iron uptake by the NTR processes involves an iron-carrier mediated step. Experiments, using human hepatoma cells (HuH7) transfected with TR antisense (sense for control) RNA expression vectors to suppress TR expression, were performed to examine the effect of unlabeled NTB-Fe as iron citrate on the uptake of 59Fe-125I-transferrin. This was to determine if the uptake of transferrin-bound iron (Tf-Fe) and NTBFe uptake is mediated by a common iron-carrier. Iron citrate inhibited the uptake of 59Fe-transferrin (2.5 mmol/L Fe) in a concentration-dependent manner with a maximum effect when the citrate-iron:Tf-Fe molar ratio was 10:1. Transferrin uptake was not affected. At a lower Tf-Fe concentration of (0.125 mmol/L) when uptake of iron is TR-mediated, a 10fold molar excess of iron citrate had no effect on Tf-Fe uptake by HuH7 TR antisense and sense cells. However, at a higher Tf-Fe concentration (2.5 mmol/L), when uptake occurs mainly by the NTR-mediated process, there was a 40% reduction in the membrane-bound and intracellular uptake of iron. Iron citrate did not affect the maximum rate (Vmax ) of Tf-Fe uptake but the Michaelis-Menten constant (Km ) for Tf-Fe uptake by the NTR-mediated process was increased, indicating there was competitive inhibition of Tf-Fe uptake by iron citrate. These results suggest that the uptake of NTB-Fe and Tf-Fe by the NTR- mediated process occurs by the same cellular pathway, using a common iron-carrier. (HEPATOLOGY 1997; 26:691-698.) Most cells acquire iron from transferrin by receptor-mediated endocytosis.1 The expression of receptors and the subsequent uptake of iron is regulated by the intracellular iron level that affects the affinity of the cytosolic iron regulatory protein for the iron regulatory element in the 3* untranslated region of the transferrin receptor (TR) messenger RNA, alter-
Abbreviations: TR, transferrin receptor; Tf-Fe, transferrin-bound iron; NTR, transferrin receptor-independent; NTB-Fe, nontransferrin-bound iron; MEM, minimal essential media; Vmax , maximum velocity; Km , Michaelis-Menten constant. From the Department of Physiology, University of Western Australia, Nedlands, 6907, Western Australia, Australia. Received December 28, 1996; accepted April 18, 1997. D.T. was supported by National Health and Medical Research Council of Australia (Canberra, ACT) CJ Martin Fellowship. Address reprint requests to: Dr. D. Trinder, Department of Physiology, University of Western Australia, Nedlands, 6097, Western Australia, Australia. Fax: 618-93801025. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2603-0023$3.00/0
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EVAN MORGAN
ing its stability and the rate of translation of the messenger RNA.2,3 In addition, a number of cell types, including hepatocytes,4 hepatoma cells,5 melanoma cells,6 and Chinese hamster ovary cells7 have been shown to be able to take up transferrin-bound iron (Tf-Fe) by another pathway as well as by the receptor-mediated process. Several studies have shown that this process is not mediated by TRs. First, when hepatocytes were incubated with a TR antibody that blocks transferrin binding to the TR, the cells still took up some iron, indicating that a transferrin receptor-independent (NTR) pathway was involved.8 Second, the N-terminal halftransferrin molecule does not bind to TR but can donate iron to hepatocytes.9,10 Furthermore, human hepatoma cells (HuH7) transfected with a TR antisense RNA expression vector to suppress TR expression5 and Chinese hamster ovary cells selected by resistance to cholera toxin conjugated to transferrin7 can still accumulate iron, confirming the presence of an NTR-mediated pathway. Recently, we have shown that the uptake of Tf-Fe by the NTR-mediated process is saturable, indicating the involvement of an iron carrier-mediated process.5 However, the nature and location of this carrier is unknown. Iron can be transported in the plasma in a nontransferrinbound (NTB-Fe) form as well as in its complex with transferrin. The plasma concentration of NTB-Fe is normally extremely low but is much higher in individuals with iron overload diseases such as thalassemia11 and hemochromatosis.12 NTB-Fe may be largely bound to citrate13 and is rapidly cleared by the liver.14 The uptake of NTB-Fe by liver cells occurs by a saturable, temperature-dependent process and is inhibited by other metal ions, indicating that an iron carriermediated pathway is involved.15 The aim of this study was to investigate the role of iron carriers in hepatic iron uptake. The experiments, using human HuH7 hepatoma cells transfected with TR antisense (sense for control) RNA expression vectors to reduce TR expression, were designed to show if the transport of Tf-Fe and NTB-Fe is mediated by the same iron carrier. Competitive uptake studies using 59Fe-125I-transferrin and unlabeled NTB-Fe as iron citrate showed that the uptake of Tf-Fe by the NTR-mediated process was inhibited by NTB-Fe, indicating there is a common pathway for the uptake of Tf-Fe and NTBFe in hepatoma cells. MATERIALS AND METHODS Minimal essential medium (MEM), Geneticin, and fetal bovine serum were purchased from Gibco BRL (Auckland, New Zealand). Antibiotics, streptomycin, and penicillin were obtained from Commonwealth Serum Laboratories (Melbourne, Australia). Pronase was bought from Boehringer (Sydney, Australia). Horse spleen ferri-
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tin, bovine serum albumin and human transferrin, apotransferrin, and desferrioxamine were purchased from Sigma Chemicals (St. Louis, Mo), and 59Fe and 125I from Dupont NEN Research Products (Sydney, Australia). HuH7 hepatoma cells16 were grown in monolayer culture in 75cm2 plastic cell culture flasks in the MEM containing 10% fetal bovine serum, 100 mg/mL streptomycin and 100 U/mL penicillin as described previously.5 HuH7 TR sense and antisense cells were grown in the above medium supplemented with 200 mg/mL Geneticin. TR sense and antisense cells were obtained by transfecting HuH7 wild-type cells with TR antisense and sense RNA expression vectors.5 The vectors were constructed as described previously17 using a 2.4-kb EcoRV-Xba1 fragment of the human TR complementary DNA pcDTR-118 inserted in the antisense or sense (control) orientation into the multiple cloning site of the expression vector pRc/ CMV (Invitrogen, San Diego, CA). Human apotransferrin was saturated with iron using 56FeSO4 and 59 FeCl3 mixed with nitrilotriacetatic acid (Fe:nitrilotriacetatic acid; 1:10) to give a specific activity of approximately 2,000 cpm/ng TfFe. Diferric transferrin was labeled with 125I using iodine monochloride.19 The protein was passed through an Amberlite CG-400 anion exchange column (Fluka Chemika, Buchs, Switzerland) followed by dialysis against 0.9% NaCl-containing 20 mmol/L NaHCO3 until more than 99% of the 125I was protein-bound and any NTB-Fe was removed. The specific activity of the 125I-transferrin was approximately 30 cpm/ng protein. HuH7 sense and antisense TR cells were plated out in growth media in 6-well plates at a density of 2 1 105 cells per 35 mm well. After 3 days when the cells had reached 70% to 80% confluence they were used for transferrin and iron uptake studies. First, the cells were preincubated two times with MEM for 15 minutes at 377C to remove fetal bovine serum and endogenous transferrin. The cells were then incubated with MEM containing 2% bovine serum albumin and 59Fe-125I-transferrin in the absence or presence of either iron citrate or citrate only, for up to 2 hours at 377C. In most experiments the diferric transferrin concentration was 0.125 or 2.5 mmol/L Fe (0.06 or 1.25 mmol/L transferrin). The effect of NTB-Fe on 59Fe-125I-transferrin uptake was determined by adding unlabeled iron to the incubation media in a 1-20 molar excess as citrate-iron. The iron citrate solution was prepared by adding FeCl3 (in 0.1N HCl) to at least a 100-fold molar excess of sodium citrate. In some wells the iron chelating effect of citrate was determined by adding an equivalent concentration of citrate, only, to the incubation media containing 59Fe-125I-transferrin. After incubation with 59Fe-125Itransferrin the cells were washed five times with Hanks’ buffer at 47C and solubilized with 0.1% Triton containing 0.1N NaOH. Cell samples were counted for gamma radiation using an LKB 1282 Compugamma counter (Uppsala, Sweden). The protein concentration was determined using a BCA protein assay (Pierce, IL). The protein concentration of TR sense cells is 240 pg/cell and TR antisense cells 204 pg/cell, as reported previously.5 The endocytosis of transferrin and iron was measured by incubating the cells with 59Fe-125I-transferrin for up to 120 minutes at 377C. The intracellular and membrane-bound radioactivity were distinguished by two techniques, the first using a proteolytic enzyme Pronase and the second using an acid buffer wash. In most experiments the Pronase method as described previously was used to remove cell membrane-bound protein.20 The washed cells were incubated with 1 mg/mL Pronase for 30 minutes at 47C and then the cell suspension was centrifuged at 12,000g for 30 seconds. Intracellular radioactivity remained associated with the cell pellet and membrane-bound radioactivity was released into the cell supernatant. The acid-wash method21 entailed incubating washed cells with a citrate buffer containing 25 mmol/L citric acid, 25 mmol/L sodium citrate, 150 mmol/L sucrose, and 10 mmol/L desferrioxamine (pH 4.6) for 2 minutes to release iron from the cell surface followed by two washes with a MEM containing 50 mmol/L desferrioxamine and
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1.25 mmol/L unlabeled diferric transferrin (pH7.4) to release the iron-depleted transferrin from the cell surface. All wash solutions were collected and pooled to determine the total membrane-bound radioactivity. The intracellular radioactivity remained associated with the cells. To determine the rate of transfer of iron from transferrin to citrate, 59Fe-125I-transferrin (2.5 mmol/L Fe) was mixed with unlabeled iron citrate (25 mmol/L Fe) for up to 24 hours at 377C in the cell incubation medium containing MEM and 2% bovine serum albumin. The protein-bound 59Fe and low molecular weight 59Fecitrate were separated using an Amicon Centricon-30 concentrator (Amicon, Beverly, MA) (molecular weight cutoff 30,000). The solution was centrifuged at 5,000g for 30 minutes in the filtration unit and the supernatant containing the Tf-Fe and the filtrate containing iron citrate were counted for gamma radioactivity. All determinations were made at least in duplicate within each experiment. If duplicate determinations were made all data are represented in the figures. If three or more determinations were made the results are expressed as mean { SEM. A t test or one-way ANOVA using a Fisher post-hoc comparison was performed to determine if the difference between sample values was statistically significant (P õ .05). All experiments was performed at least twice. RESULTS Effect of Iron Citrate on the Uptake of Transferrin and Iron. At low transferrin concentration (0.125 mmol/L Fe), when the uptake of iron and transferrin occurs mainly by the TRmediated process,5 iron citrate (1.25 mmol/L Fe) or citrate (125 mmol/L) had no significant effect on Tf-Fe uptake by the HuH7 TR sense or antisense cells. However, at higher transferrin concentrations (2.5 mmol/L Fe) when transferrin and iron uptake occurs mainly by the NTR-mediated pathways,5 iron citrate (25 mmol/L Fe) significantly inhibited TfFe uptake by both the TR sense and antisense cells, whereas citrate (2.5 mmol/L) alone had no effect (Fig. 1A and 1B). The amount of inhibition of Tf-Fe uptake by iron citrate was similar for both cell types even though Tf-Fe uptake was higher in the TR sense cells than the TR antisense cells, because of the presence of a larger number of TR.5 Iron uptake was reduced by 97 { 14 1 10/5 atoms/cell/120 minutes (n Å 3) for the TR sense cells and 88 { 15 1 10/5 atoms Fe/cell/120 minutes (n Å 3) for the TR antisense cells. Iron citrate significantly inhibited both the intracellular and cell surface uptake of Tf-Fe and the degree of inhibition was similar for both cell types. Membrane and intracellular uptake was reduced by 20 { 4 and 76 { 9 1 10/5 atoms/ cell/minutes (n Å 3), respectively, for the sense cells and 14 { 5 and 74 { 10 1 10/5 atoms/cell/minute (n Å 3), respectively, for the antisense cells. In contrast, there was no significant effect of iron citrate or citrate on the uptake of transferrin by the TR sense or antisense cells at either extracellular transferrin concentration (Fig. 1C and 1D). These results suggest that iron citrate inhibits the uptake of Tf-Fe by the NTR-mediated pathway but not by TR-mediated processes. As the effects of iron citrate on iron and transferrin uptake were similar for both types of cells, further experiments were performed using the TR antisense cells only. TR expression in these cells is suppressed allowing easier examination of the effects of iron citrate on the NTR-mediated uptake process. At a Tf-Fe concentration of 2.5 mmol/L, iron uptake was progressively inhibited by increasing concentrations of iron citrate, reaching a maximum level of inhibition at 10-fold molar excess of citrate-iron to Tf-Fe. Tf-Fe uptake was not
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FIG. 1. Effect of iron citrate on (A and B) iron and (C and D) transferrin uptake by (A and C) HuH7 transferrin receptor sense and (B and D) antisense cells. Uptake was measured by incubating the cells with 59Fe-125I-transferrin (0.125 mmol/L or 2.5 mmol/L Fe) in the absence (control) or presence of iron citrate (25 mmol/L Fe, 2.5 mmol/L citrate) or citrate only (2.5 mmol/L) for 2 hours. The figure shows ( ) internalized and (h) membrane uptake. The results are mean { SEM of triplicate determinations.
affected by increasing concentration of citrate alone. At the lower Tf-Fe concentration (0.125 mmol/L), increasing concentrations iron citrate or citrate only had no effect on TfFe uptake (Fig. 2A). As TR saturate at a transferrin concentration of approximately 0.06 mmol/L (0.12 mmol/L Fe),5 the uptake of TfFe by the NTR-mediated process can be determined by the difference between the Tf-Fe uptake at the Tf-Fe concentration of 0.125 mmol/L and 2.5 mmol/L. There was a significant decline in this value for Tf-Fe uptake by the NTR-mediated process with increasing iron citrate concentrations, decreasing to 40% of the control value at 10-fold molar excess of citrate-iron to Tf-Fe (Fig. 2B). The uptake of transferrin at either the high or low concentration was not affected by increasing concentrations of iron citrate or citrate (Fig. 2C).
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Exchange of Iron Between Transferrin and Citrate. To determine if the inhibition of uptake Tf-Fe by iron citrate was because of the extracellular exchange of iron from transferrin to citrate the rate of transfer of 59Fe from transferrin to citrate was measured over a 24-hour period as outlined in Materials and Methods using a 30-kd molecular weight cutoff membrane. The results are shown in Fig. 3. The rate of transfer of 59 Fe from transferrin to citrate was õ1.5% per hour. Similar results were obtained when gel filtration (Sephadex G-25; Pharmacia Biotech, Uppsala, Sweden) was used to separate transferrin-bound and low molecular weight 59Fe (data not shown). Intracellular and Membrane-Bound Iron Uptake. Intracellular and membrane uptake of iron was measured using the proteolytic enzyme Pronase20 or an acid wash technique.21 Simi-
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lar results were obtained using both methods. The intracellular and membrane uptake of iron increased with time and transferrin concentration and after 60-minute incubation, approximately 70% of the total iron uptake was internalized. At a Tf-Fe concentration of 2.5 mmol/L, the addition of iron citrate reduced both the rate of intracellular and membrane uptake of Tf-Fe to approximately 60% of the control value obtained in the absence of iron citrate. However, the rate of Tf-Fe uptake was still greater than that observed at a Tf-Fe concentration of 0.125 mmol/L (Fig. 4A and 4B). The intracellular and membrane uptake of transferrin increased rapidly in the first 15 minutes of incubation, reaching steady state levels after 30 minutes, with approximately 30% to 40% of the total transferrin inside the cell. Both the initial rate and steady-state level of intracellular and membrane uptake of transferrin increased with transferrin concentration. Iron citrate had no effect on intracellular uptake of transferrin but there was a small increase in membrane uptake at the higher transferrin concentration (Fig. 4C and 4D). Rates of Iron and Transferrin Internalization. The results in Fig. 4 indicate that iron citrate inhibited the uptake of TfFe by the cell membrane, leading to the reduced intracellular uptake of iron. To confirm this finding the rate of iron internalization was measured over a 60-minute period, whereas the rate of transferrin endocytosis was measured over the first 5 minutes of incubation when intracellular accumulation occurs at a constant rate (Table 1). At the low transferrin concentration, where uptake is TR-mediated, the iron to transferrin molar ratio was approximately 2. As each molecule of transferrin binds two atoms of iron this indicates that all of the iron was entering the cell bound to transferrin. When the transferrin concentration was increased, the rates of transferrin and iron internalization increased and the iron to transferrin molar ratio increased to 3.3, indicating that some of the iron was released from transferrin at the cell membrane before entering the cell. When iron citrate (25 mmol/L Fe) was added to 59Fe-125I-transferrin (1.25 mmol/L transferrin), the rate of transferrin internalization was not affected but 59Fe uptake was reduced by 40% and the iron to transferrin molar ratio was reduced to 2.0. The data obtained in these experiments indicate that iron citrate inhibited the uptake of iron released from transferrin at the cell membrane by the NTR mechanism. Inhibition of Iron Uptake. TR antisense cells were incubated with increasing concentrations of 59Fe-transferrin (0.025-10 mmol/L Fe) for 60 minutes in the presence or absence of iron citrate (25 mmol/L Fe) or citrate (2.5 mmol/L). The maximum rate of iron uptake (Vmax ) and the Michaelis-Menten constant (Km ) were calculated using nonlinear least squares fitting of the iron uptake to a Michaelis-Menten type equation as described before.5 The best fit was obtained when two saturable processes for iron uptake were assumed, consistent with TR- and NTR-mediated processes (Table 2). In the presence of iron citrate, the Vmax and Km for the TR-mediated process remained unchanged. For the NTR-mediated process, the Vmax was the same but the Km was increased by 2.5fold compared with the control in the absence of iron citrate, indicating there was competitive inhibition of Tf-Fe uptake by iron citrate. DISCUSSION
In this study the mechanisms of NTR-mediated uptake of iron were investigated using Tf-Fe and NTB-Fe. NTB-Fe in
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FIG. 2. Effect of iron citrate concentration on iron (A and B) and transferrin (C) uptake. TR antisense cells were incubated with 59Fe-125I-transferrin (0.125 mmol/L Fe; ●, s) and (2.5 mmol/L Fe; j, h) in presence of increasing concentrations iron citrate (●, j) or citrate (s, h) for 2 hours. The figure shows the total uptake of iron from transferrin at two Tf-Fe concentrations (A) and difference between these values (B) in the presence of iron citrate (m) or citrate (n). The results are means { SEM of triplicate determinations.
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FIG. 3. The release of 59Fe from transferrin (2.5 mmol/L Fe) in (h) absence or (j) presence of iron citrate (25 mmol/L Fe, 2.5 mmol/L citrate) during incubation at 377C for up to 24 hours in the cell incubation medium.
the form of iron citrate inhibited Tf-Fe uptake, suggesting that iron from both sources was transported into the human hepatoma cells (HuH7) by a common pathway. The evidence suggests that NTB-Fe did not affect the uptake of iron by the TR-mediated process but inhibited the NTR-mediated uptake of Tf-Fe. At a low extracellular transferrin concentration where uptake of iron and transferrin by the TR sense and antisense cells occurs by the TR-mediated pathway,5 iron citrate did not inhibit iron uptake. However, at higher transferrin concentrations where the uptake of transferrin and iron by the TR sense and antisense cells occurs mainly by NTR-mediated processes,5 NTB-Fe had an inhibitory effect on iron uptake. Also, at a higher transferrin concentration the amount of inhibition of Tf-Fe uptake by NTB-Fe was the same for both the TR sense and antisense cells. It was shown in an earlier study that, while TR antisense cells express approximately 50% as many TR as sense cells, the amount of iron taken by the NTR-mediated pathway is the same.5 Thus, similar effects of NTB-Fe on Tf-Fe uptake by both cell types supports the view that only the NTR-mediated process was impaired. Also the similar effect of NTB-Fe on the uptake of Tf-Fe by both types of cells at both low and high transferrin concentrations indicates that the process of transfection with TR antisense RNA does not affect the common pathway for the uptake of Tf-Fe and NTB-Fe. NTB-Fe inhibited both the intracellular and membrane uptake of iron but had no effect on transferrin uptake. Also, for both the TR sense and antisense cells, the degree of inhibition of intracellular and membrane-bound iron uptake was similar. These findings suggest that NTB-Fe inhibited the uptake of Tf-Fe at the cell surface, leading to a subsequent reduction in intracellular uptake of iron. At low transferrin concentrations, the rate of internalization of iron was twice the rate of transferrin endocytosis. As each molecule of transferrin binds two atoms of iron this finding is consistent with a TR-mediated process where all the iron enters the cell
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bound to transferrin.1 At the higher transferrin concentration, the rates of transferrin and iron internalization increased, indicating that additional transferrin and iron were entering the cell by NTR-mediated pathways. However, the ratio of the rate of iron to transferrin internalization was greater than 2. Therefore, extra iron had entered the cell not bound to transferrin. In the presence of NTB-Fe, the rates of iron internalization were reduced to 2, showing that under these conditions all iron was entering the cell bound to transferrin. Taken together the effects of NTB-Fe on the kinetics of intracellular and membrane uptake of iron suggest that NTB-Fe inhibited the uptake of iron released from transferrin at the cell surface and the subsequent internalization of this iron by the cell. The uptake of Tf-Fe by the NTR-mediated process is saturable and involves an iron carrier-mediated step.5 NTB-Fe did not affect the Vmax of iron uptake by the NTR-mediated process but the Km was more than doubled. This indicates that the NTB-Fe competitively inhibited the uptake of Tf-Fe, probably by preventing the binding of iron released from transferrin to a limited number of iron carriers. Little information about the effects of NTB-Fe on the uptake of Tf-Fe has been reported previously. Several workers observed inhibitory effects of NTB-Fe but the mechanisms were not elucidated. Thorstensen22 showed that ferrous ion and other metal ions such as cobalt, copper, and zinc inhibited the uptake of Tf-Fe by hepatocytes. However, a much higher concentration of the Fe2/ (500 mmol/L) was required to reduce the uptake of Tf-Fe (1 mmol/L) by half than in the present study. We have23 also observed in reticulocytes a reduction in Tf-Fe uptake by several metals of which zinc and Fe2/ were the most effective. There is also recent experimental evidence to suggest that the reverse effect occurs, that is, diferric transferrin inhibits NTB-Fe uptake. Scheiber and Goldenberg24 showed that diferric transferrin completely inhibited the uptake of NTB-Fe presented to the cells in the form of iron-diethylenetriamine pentaacetate, by hepatocytes. However, apotransferrin can also reduce the uptake of iron-diethylenetriamine pentaacetate, suggesting that the protein, probably through its iron-binding properties, rather than iron alone has an inhibitory effect on the NTB-Fe uptake. For several reasons it is unlikely that the effects of iron citrate on Tf-Fe uptake were caused by the transfer of 59Fe from transferrin to citrate or the exchange of 59Fe from transferrin with 56Fe from citrate in the extracellular medium. The rate of release of 59Fe from transferrin to citrate measured in a cell-free system was õ1.5% per hour (Fig. 3), which is insufficient to account for the reduction in Tf-Fe uptake. Also, iron citrate suppresses the uptake of Tf-Fe by the NTR pathway only and has no effect on the TR-mediated uptake of iron. Therefore, this inhibitory effect cannot be accounted for by a reduction in the specific activity of 59Fe-transferrin as this would lead to a reduction in 59Fe uptake by both processes. Iron-depleted transferrin is generated by the cellular uptake of iron from transferrin and the subsequent recycling of apotransferrin to the extracellular medium. However, this could account for õ1% of the total diferric transferrin in the extracellular medium, and saturating this low level of apotransferrin with 56Fe would not have a significant effect on the 59Fe uptake. Furthermore, any 59Fe transferred from transferrin to citrate is unlikely to reduce the uptake of 59Fe
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FIG. 4. Uptake of iron and transferrin from 59Fe-125I-transferrin with time at 377C. The figures show (A) intracellular iron, (C) transferrin, (B) membrane-bound iron, and (D) transferrin uptake from (s) 0.125 mmol/L and 2.5 mmol/L Tf-Fe (h) in the absence and (j) presence of iron citrate (25 mmol/L Fe, 2.5 mmol/L citrate).
because iron citrate can also donate iron to the hepatoma cells (Trinder D, Morgan E, unpublished observation, December 1996). The inhibitory effects of NTB-Fe on Tf-Fe uptake provide new insights into the mechanisms involved in the NTR-mediated uptake of Tf-Fe by hepatoma cells. The results provide
evidence that there are at least two NTR-mediated pathways, the first involving the cell surface release of iron from transferrin and the second mediated by the endocytosis of transferrin. At a transferrin concentration greater than that required to saturate the TR, the rate of iron internalization from transferrin was greater than that of the protein itself
TABLE 1. Rates of Iron and Transferrin Internalization by HuH7 Antisense Cells Diferric Transferrin Concentration (mmol/L)
Rate of Iron Internalization (atoms/cell/min 11003)
Rates of Transferrin Internalization (molecules/cell/min 11003)
Iron:Transferrin Molar Ratio
1.25 1.25 / (25 mmol/L iron citrate) 0.06
105 { 14 (4) 66 { 12 (4)* 40 { 2 (3)†
33 { 4 (4) 33 { 4 (4) 19 { 1 (3)†
3.3 { 0.4 (4) 2.0 { 0.2 (4)* 2.1 { 0.1 (3)*
NOTE. The cells were incubated with 59Fe-125I-transferrin (0.06 and 1.25 mmol/L) in the absence (control) and presence of iron citrate (25 mmol/L Fe) at 377C. The rate of iron internalization was measured over 60 minutes of incubation and transferrin internalization was measured over the first 5 minutes of incubation as described in the text. Results are expressed as the mean { SEM. The number of estimations is shown in parenthesis. *P õ .05 compared with 1.25 mmol/L diferric transferrin. †P õ .01 compared with 1.25 nmol/L diferric transferrin.
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TABLE 2. Maximum Velocity (Vmax) and Michaelis-Menten Constant (Km) for Iron Uptake by HuH7 Antisense Cells Incubation Conditions
Vmax1 (atoms/cell/min 11003)
Kml (nmol/L)
Vmax2 (atoms/cell/min 1 1003)
Km2 (nmol/L)
Tf-Fe Tf-Fe / iron citrate (25 mmol/L) Tf-Fe / citrate (2.5 mmol/L)
45 { 7 33 { 5 37 { 5
38 { 12 31 { 11 29 { 4
226 { 33 265 { 36 196 { 33
5,012 { 1,060 12,484 { 2,030* 3,967 { 333
NOTE. The cells were incubated with increasing concentrations of 59Fe-labeled transferrin in the absence or presence of iron citrate (25 mmol/L) or citrate alone (2.5 mmol/L). Vmax1 and Km1 for the TR-mediated process and Vmax2 and Km2 for the NTR-mediated process were determined as described in the text. Results are expressed as the mean { SEM, where the number of determinations is 3. *P õ .01 compared with diferric transferrin.
and only the excess iron uptake was inhibited by NTB-Fe. This inhibitory effect of NTB-Fe on Tf-Fe uptake shows that some iron was released from transferrin at the cell membrane and was transported into the cell independently of transferrin by an iron carrier-mediated step that was shared with NTBFe. However, uptake of iron by this pathway accounted for only approximately 60% of the total iron taken up by the NTR-mediated pathways and the other 40% was dependent on transferrin endocytosis by the NTR-mediated process (Fig. 4). These conclusions are in agreement with evidence provided by several other investigators that membrane impermeable chelators can partially inhibit the uptake of Tf-Fe by hepatocytes, indicating that iron can be released from transferrin and chelated at an extracellular site.22,25,26 It has been proposed that a plasma membrane reduced nicotinamide adenine dinucleotide:ferric reductase redox system may be involved in the reduction and release of iron from transferrin at the cell surface.27,28 Although the equilibrium constant for the reduction of diferric transferrin by reduced nicotinamide adenine dinucelotide is unfavorable29 other factors such as changes in pH at the cell membrane or conformational changes to the transferrin molecule or iron acceptors may be present in biological systems allowing the reduction of Tf-Fe to proceed. As mentioned above, a second pathway of iron uptake by NTR-mediated process was not affected by NTB-Fe and does not appear to involve the release of iron from transferrin at the cell surface. Instead the transport of iron by this process involves the endocytosis of transferrin. These conclusions are in agreement with those of our earlier studies using HuH7 hepatoma TR sense and antisense cells.5 Then it was found that Tf-Fe uptake could occur by an NTR-mediated pathway that involved the endocytosis and recycling of transferrin and the release of iron from transferrin at an intracellular, acidic site. HuH7 hepatoma cells have many characteristics of primary hepatocytes. First, these cells have been shown to retain many differentiated functions of primary hepatocyte cells including the synthesis of plasma proteins and liver-specific enzymes.16 Second, results from this and our previous study5 indicate that the iron uptake processes by the HuH7 cells are similar to those described for primary hepatocytes.4,8,28 In fact, HuH7 cells are the only hepatoma cell line, like hepatocytes, that has been shown to take up transferrinbound iron by transferrin-independent pathways and therefore, it provides a good model for hepatic iron transport. It is most likely that the effects of NTB-Fe on Tf-Fe uptake by hepatocytes will be similar to those observed for hepatoma cells. In vivo, the plasma transferrin concentration is higher than
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that required to saturate the TR and Tf-Fe is probably delivered to hepatic cells primarily by NTR-mediated processes via the iron carrier-dependent pathway. Therefore, the iron carrier that is shared by Tf-Fe and NTB-Fe plays a central role in the uptake of iron by hepatic cells. In iron overloaded states associated with the diseases hemochromatosis and thalassemia, both Tf-Fe and NTB-Fe are present in high concentrations in the plasma and the iron from both sources is probably cleared by the liver by the common iron carriermediated pathway. The uptake of iron by the carrier-mediated pathway is rate limiting and therefore, regulation of this process is likely to be of major importance in the control of iron hepatic transport in both normal and iron overloaded states. We have previously shown that HuH7 hepatoma cells can take up Tf-Fe by TR and NTR pathways. Iron uptake by the NTR mechanisms is mediated by an iron carrier-mediated step.5 In the present study it was shown that NTB-Fe partially inhibited the uptake of Tf-Fe by the NTR-mediated process but had no effect on the TR-mediated process. NTB-Fe competitively inhibited the uptake of iron, released from transferrin at the cell membrane, by an iron carrier-mediated NTR pathway. NTB-Fe did not affect the uptake of Tf-Fe by NTR pathway, which is dependent on transferrin endocytosis. These results show Tf-Fe is taken up by two NTR pathways, one involves the endocytosis of transferrin and the other involves the release of iron from transferrin at the cell surface and the subsequent transport of iron into the cell by an iron carrier-mediated pathway that is shared with NTB-Fe. REFERENCES 1. Baker E, Morgan EH. Iron transport. In: Brock JH, Halliday JJ, Pippard M, Powell L, eds. Iron Metabolism in Health and Disease. London: Saunders, 1994:63-95. 2. Owen D, Kuhn LC. Noncoding 3* sequences of the transferrin receptor gene are required for mRNA regulation by iron. EMBO J 1987;6:12871293. 3. Klausner RD, Rouault TA, Harford JB. Regulating the fate of mRNA: The control of cellular iron metabolism. Cell 1993;72:19-28. 4. Page MA, Morgan EH, Baker E. Transferrin binding and iron uptake by rat hepatocytes in culture. Am J Physiol 1984;246:G26-G33. 5. Trinder D, Zak O, Aisen P. Transferrin receptor independent uptake of diferric transferrin by human hepatoma cells with antisense inhibition of receptor expression. HEPATOLOGY 1996;23:1512-1520. 6. Richardson D, Baker E. Two mechanisms of iron uptake from transferrin by melanoma cells. The effect of desferrioxamine and ferric ammonium citrate. J Biol Chem 1992;267:13972-13979. 7. Chan RYY, Ponka P, Schulman HM. Transferrin-receptor-independent but iron-dependent proliferation of variant Chinese hamster ovary cells. Exp Cell Res 1992;202:326-336. 8. Trinder D, Morgan E, Baker E. The effects of an antibody to the rat transferrin receptor and of serum albumin on the uptake of diferric transferrin by rat hepatocytes. Biochim Biophys Acta 1988;943:440446.
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9. Zak O, Trinder D, Aisen P. Primary receptor-recognition site of human transferrin is in the C-terminal lobe. J Biol Chem 1994;269:7110-7114. 10. Thorstensen K, Trinder D, Zak O, Aisen P. Uptake of iron from Nterminal half-transferrin by isolated rat hepatocytes: evidence of transferrin receptor-independent iron uptake. Eur J Biochem 1995;232:129133. 11. Hershko C, Graham G, Bates GW, Rachmilewitz EA. Non specific serum iron in thalassemia: An abnormal serum iron fraction of potential toxicity. Br J Haematol 1978;50:255-263. 12. Batey RG, Lai Chung Fong P, Shamir S, Sherlock S. A non-transferrin bound serum iron in idiopathic hemochromatosis. Dig Dis Sci 1980; 25:340-346. 13. Grootveld M, Bell JD, Halliwell B, Aruoma OI, Bomford A, Sadlier PJ. Non-transferrin bound iron in plasma or serum from patients with idiopathic hemochromatosis. J Biol Chem 1989;264:4417-4422. 14. Craven CM, Alexander J, Eldridge JP, Kushner S, Bernstein S, Kaplan J. Tissue distribution and clearance kinetics of non-transferrin-bound iron in the hypotransferrinemic mouse: a rodent model for hemochromatosis. Proc Natl Acad Sci U S A 1987;84:3457-3461. 15. Wright TL, Brissot P, Ma W-L, Weisiger RA. Characterization of nontransferrin-bound iron clearance by rat liver. J Biol Chem 1986;261: 10909-10914. 16. Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J. Growth of human hepatoma cell line with differentiated functions in chemically defined medium. Cancer Res 1982;42:3858-3863. 17. Sasaki K, Zak O, Aisen P. Antisense suppression of transferrin receptor gene expression in a human hepatoma cell (HuH-7) line. Am J Hematol 1993;42:74-80. 18. Kuhn LC, McCelland A, Ruddle FH. Gene transfer, expression, and molecular cloning of the human transferrin receptor gene. Cell 1984; 37:95-103.
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19. Macfarlane AS. In vivo behavior of 131-fibrinogen. J Clin Invest 1963; 42:346-361. 20. Trinder D, Morgan E, Baker E. The mechanism of iron uptake by fetal rat hepatocytes in culture. HEPATOLOGY 1986;6:852-858. 21. Ghosh RN, Maxfield FR. Evidence for nonvectorial, retrograde transferrin trafficking in the early endosomes of HepG2 cells. J Cell Biol 1995;128:549-561. 22. Thorstensen K. Hepatocytes and reticulocytes have different mechanisms for the uptake of iron from transferrin. J Biol Chem 1988;263: 16837-16841. 23. Morgan EH. Membrane transport of non-transferrin iron by reticulocytes. Biochim Biophys Acta 1988;943:428-439. 24. Scheiber B, Goldenberg H. Uptake of iron by isolated rat hepatocytes from a hydrophilic impermeant ferric chelate, Fe(III)-DPTA. Archive Biochem Biophys 1996;326:185-192. 25. Cole ES, Glass J. Transferrin binding and iron uptake in mouse hepatocytes. Biochim Biophys Acta 1983;762:102-110. 26. Wong A. ‘‘The effect of iron chelators on hepatocyte iron metabolism.’’ 1995: PhD Thesis. University of Western Australia: Perth, Western Australia. 27. Low H, Sun IL, Navas P, Grebing C, Crane FL, Morre DJ. Transplasmalemma electron transport from cells is part of a diferric transferrin reductase system. Biochim Biophys Res Commun 1986;139:1117-1123. 28. Thorstensen K, Romslo I. Uptake of iron from transferrin by isolated rat hepatocytes: a redox-mediated plasma membrane process. J Biol Chem 1988;263:8844-8850 29. Thorstensen K, Aisen P. Release of iron from diferric transferrin in the presence of rat liver plasma membranes: no evidence of a plasma membrane diferric transferrin reductase. Biochim Biophys Acta 1990; 1052:29-55.
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The liver acquires iron from transferrin by transferrin receptor-mediated (TR) and transferrin receptor-independent pathways (NTR) and from nontransferrin-bound iron (NTBFe). Iron uptake by the NTR processes involves an iron-carrier mediated step. Experiments, using human hepatoma cells (HuH7) transfected with TR antisense (sense for control) RNA expression vectors to suppress TR expression, were performed to examine the effect of unlabeled NTB-Fe as iron citrate on the uptake of 59Fe-125I-transferrin. This was to determine if the uptake of transferrin-bound iron (Tf-Fe) and NTBFe uptake is mediated by a common iron-carrier. Iron citrate inhibited the uptake of 59Fe-transferrin (2.5 mmol/L Fe) in a concentration-dependent manner with a maximum effect when the citrate-iron:Tf-Fe molar ratio was 10:1. Transferrin uptake was not affected. At a lower Tf-Fe concentration of (0.125 mmol/L) when uptake of iron is TR-mediated, a 10fold molar excess of iron citrate had no effect on Tf-Fe uptake by HuH7 TR antisense and sense cells. However, at a higher Tf-Fe concentration (2.5 mmol/L), when uptake occurs mainly by the NTR-mediated process, there was a 40% reduction in the membrane-bound and intracellular uptake of iron. Iron citrate did not affect the maximum rate (Vmax ) of Tf-Fe uptake but the Michaelis-Menten constant (Km ) for Tf-Fe uptake by the NTR-mediated process was increased, indicating there was competitive inhibition of Tf-Fe uptake by iron citrate. These results suggest that the uptake of NTB-Fe and Tf-Fe by the NTR- mediated process occurs by the same cellular pathway, using a common iron-carrier. (HEPATOLOGY 1997; 26:691-698.) Most cells acquire iron from transferrin by receptor-mediated endocytosis.1 The expression of receptors and the subsequent uptake of iron is regulated by the intracellular iron level that affects the affinity of the cytosolic iron regulatory protein for the iron regulatory element in the 3* untranslated region of the transferrin receptor (TR) messenger RNA, alter-
Abbreviations: TR, transferrin receptor; Tf-Fe, transferrin-bound iron; NTR, transferrin receptor-independent; NTB-Fe, nontransferrin-bound iron; MEM, minimal essential media; Vmax , maximum velocity; Km , Michaelis-Menten constant. From the Department of Physiology, University of Western Australia, Nedlands, 6907, Western Australia, Australia. Received December 28, 1996; accepted April 18, 1997. D.T. was supported by National Health and Medical Research Council of Australia (Canberra, ACT) CJ Martin Fellowship. Address reprint requests to: Dr. D. Trinder, Department of Physiology, University of Western Australia, Nedlands, 6097, Western Australia, Australia. Fax: 618-93801025. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2603-0023$3.00/0
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ing its stability and the rate of translation of the messenger RNA.2,3 In addition, a number of cell types, including hepatocytes,4 hepatoma cells,5 melanoma cells,6 and Chinese hamster ovary cells7 have been shown to be able to take up transferrin-bound iron (Tf-Fe) by another pathway as well as by the receptor-mediated process. Several studies have shown that this process is not mediated by TRs. First, when hepatocytes were incubated with a TR antibody that blocks transferrin binding to the TR, the cells still took up some iron, indicating that a transferrin receptor-independent (NTR) pathway was involved.8 Second, the N-terminal halftransferrin molecule does not bind to TR but can donate iron to hepatocytes.9,10 Furthermore, human hepatoma cells (HuH7) transfected with a TR antisense RNA expression vector to suppress TR expression5 and Chinese hamster ovary cells selected by resistance to cholera toxin conjugated to transferrin7 can still accumulate iron, confirming the presence of an NTR-mediated pathway. Recently, we have shown that the uptake of Tf-Fe by the NTR-mediated process is saturable, indicating the involvement of an iron carrier-mediated process.5 However, the nature and location of this carrier is unknown. Iron can be transported in the plasma in a nontransferrinbound (NTB-Fe) form as well as in its complex with transferrin. The plasma concentration of NTB-Fe is normally extremely low but is much higher in individuals with iron overload diseases such as thalassemia11 and hemochromatosis.12 NTB-Fe may be largely bound to citrate13 and is rapidly cleared by the liver.14 The uptake of NTB-Fe by liver cells occurs by a saturable, temperature-dependent process and is inhibited by other metal ions, indicating that an iron carriermediated pathway is involved.15 The aim of this study was to investigate the role of iron carriers in hepatic iron uptake. The experiments, using human HuH7 hepatoma cells transfected with TR antisense (sense for control) RNA expression vectors to reduce TR expression, were designed to show if the transport of Tf-Fe and NTB-Fe is mediated by the same iron carrier. Competitive uptake studies using 59Fe-125I-transferrin and unlabeled NTB-Fe as iron citrate showed that the uptake of Tf-Fe by the NTR-mediated process was inhibited by NTB-Fe, indicating there is a common pathway for the uptake of Tf-Fe and NTBFe in hepatoma cells. MATERIALS AND METHODS Minimal essential medium (MEM), Geneticin, and fetal bovine serum were purchased from Gibco BRL (Auckland, New Zealand). Antibiotics, streptomycin, and penicillin were obtained from Commonwealth Serum Laboratories (Melbourne, Australia). Pronase was bought from Boehringer (Sydney, Australia). Horse spleen ferri-
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tin, bovine serum albumin and human transferrin, apotransferrin, and desferrioxamine were purchased from Sigma Chemicals (St. Louis, Mo), and 59Fe and 125I from Dupont NEN Research Products (Sydney, Australia). HuH7 hepatoma cells16 were grown in monolayer culture in 75cm2 plastic cell culture flasks in the MEM containing 10% fetal bovine serum, 100 mg/mL streptomycin and 100 U/mL penicillin as described previously.5 HuH7 TR sense and antisense cells were grown in the above medium supplemented with 200 mg/mL Geneticin. TR sense and antisense cells were obtained by transfecting HuH7 wild-type cells with TR antisense and sense RNA expression vectors.5 The vectors were constructed as described previously17 using a 2.4-kb EcoRV-Xba1 fragment of the human TR complementary DNA pcDTR-118 inserted in the antisense or sense (control) orientation into the multiple cloning site of the expression vector pRc/ CMV (Invitrogen, San Diego, CA). Human apotransferrin was saturated with iron using 56FeSO4 and 59 FeCl3 mixed with nitrilotriacetatic acid (Fe:nitrilotriacetatic acid; 1:10) to give a specific activity of approximately 2,000 cpm/ng TfFe. Diferric transferrin was labeled with 125I using iodine monochloride.19 The protein was passed through an Amberlite CG-400 anion exchange column (Fluka Chemika, Buchs, Switzerland) followed by dialysis against 0.9% NaCl-containing 20 mmol/L NaHCO3 until more than 99% of the 125I was protein-bound and any NTB-Fe was removed. The specific activity of the 125I-transferrin was approximately 30 cpm/ng protein. HuH7 sense and antisense TR cells were plated out in growth media in 6-well plates at a density of 2 1 105 cells per 35 mm well. After 3 days when the cells had reached 70% to 80% confluence they were used for transferrin and iron uptake studies. First, the cells were preincubated two times with MEM for 15 minutes at 377C to remove fetal bovine serum and endogenous transferrin. The cells were then incubated with MEM containing 2% bovine serum albumin and 59Fe-125I-transferrin in the absence or presence of either iron citrate or citrate only, for up to 2 hours at 377C. In most experiments the diferric transferrin concentration was 0.125 or 2.5 mmol/L Fe (0.06 or 1.25 mmol/L transferrin). The effect of NTB-Fe on 59Fe-125I-transferrin uptake was determined by adding unlabeled iron to the incubation media in a 1-20 molar excess as citrate-iron. The iron citrate solution was prepared by adding FeCl3 (in 0.1N HCl) to at least a 100-fold molar excess of sodium citrate. In some wells the iron chelating effect of citrate was determined by adding an equivalent concentration of citrate, only, to the incubation media containing 59Fe-125I-transferrin. After incubation with 59Fe-125Itransferrin the cells were washed five times with Hanks’ buffer at 47C and solubilized with 0.1% Triton containing 0.1N NaOH. Cell samples were counted for gamma radiation using an LKB 1282 Compugamma counter (Uppsala, Sweden). The protein concentration was determined using a BCA protein assay (Pierce, IL). The protein concentration of TR sense cells is 240 pg/cell and TR antisense cells 204 pg/cell, as reported previously.5 The endocytosis of transferrin and iron was measured by incubating the cells with 59Fe-125I-transferrin for up to 120 minutes at 377C. The intracellular and membrane-bound radioactivity were distinguished by two techniques, the first using a proteolytic enzyme Pronase and the second using an acid buffer wash. In most experiments the Pronase method as described previously was used to remove cell membrane-bound protein.20 The washed cells were incubated with 1 mg/mL Pronase for 30 minutes at 47C and then the cell suspension was centrifuged at 12,000g for 30 seconds. Intracellular radioactivity remained associated with the cell pellet and membrane-bound radioactivity was released into the cell supernatant. The acid-wash method21 entailed incubating washed cells with a citrate buffer containing 25 mmol/L citric acid, 25 mmol/L sodium citrate, 150 mmol/L sucrose, and 10 mmol/L desferrioxamine (pH 4.6) for 2 minutes to release iron from the cell surface followed by two washes with a MEM containing 50 mmol/L desferrioxamine and
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1.25 mmol/L unlabeled diferric transferrin (pH7.4) to release the iron-depleted transferrin from the cell surface. All wash solutions were collected and pooled to determine the total membrane-bound radioactivity. The intracellular radioactivity remained associated with the cells. To determine the rate of transfer of iron from transferrin to citrate, 59Fe-125I-transferrin (2.5 mmol/L Fe) was mixed with unlabeled iron citrate (25 mmol/L Fe) for up to 24 hours at 377C in the cell incubation medium containing MEM and 2% bovine serum albumin. The protein-bound 59Fe and low molecular weight 59Fecitrate were separated using an Amicon Centricon-30 concentrator (Amicon, Beverly, MA) (molecular weight cutoff 30,000). The solution was centrifuged at 5,000g for 30 minutes in the filtration unit and the supernatant containing the Tf-Fe and the filtrate containing iron citrate were counted for gamma radioactivity. All determinations were made at least in duplicate within each experiment. If duplicate determinations were made all data are represented in the figures. If three or more determinations were made the results are expressed as mean { SEM. A t test or one-way ANOVA using a Fisher post-hoc comparison was performed to determine if the difference between sample values was statistically significant (P õ .05). All experiments was performed at least twice. RESULTS Effect of Iron Citrate on the Uptake of Transferrin and Iron. At low transferrin concentration (0.125 mmol/L Fe), when the uptake of iron and transferrin occurs mainly by the TRmediated process,5 iron citrate (1.25 mmol/L Fe) or citrate (125 mmol/L) had no significant effect on Tf-Fe uptake by the HuH7 TR sense or antisense cells. However, at higher transferrin concentrations (2.5 mmol/L Fe) when transferrin and iron uptake occurs mainly by the NTR-mediated pathways,5 iron citrate (25 mmol/L Fe) significantly inhibited TfFe uptake by both the TR sense and antisense cells, whereas citrate (2.5 mmol/L) alone had no effect (Fig. 1A and 1B). The amount of inhibition of Tf-Fe uptake by iron citrate was similar for both cell types even though Tf-Fe uptake was higher in the TR sense cells than the TR antisense cells, because of the presence of a larger number of TR.5 Iron uptake was reduced by 97 { 14 1 10/5 atoms/cell/120 minutes (n Å 3) for the TR sense cells and 88 { 15 1 10/5 atoms Fe/cell/120 minutes (n Å 3) for the TR antisense cells. Iron citrate significantly inhibited both the intracellular and cell surface uptake of Tf-Fe and the degree of inhibition was similar for both cell types. Membrane and intracellular uptake was reduced by 20 { 4 and 76 { 9 1 10/5 atoms/ cell/minutes (n Å 3), respectively, for the sense cells and 14 { 5 and 74 { 10 1 10/5 atoms/cell/minute (n Å 3), respectively, for the antisense cells. In contrast, there was no significant effect of iron citrate or citrate on the uptake of transferrin by the TR sense or antisense cells at either extracellular transferrin concentration (Fig. 1C and 1D). These results suggest that iron citrate inhibits the uptake of Tf-Fe by the NTR-mediated pathway but not by TR-mediated processes. As the effects of iron citrate on iron and transferrin uptake were similar for both types of cells, further experiments were performed using the TR antisense cells only. TR expression in these cells is suppressed allowing easier examination of the effects of iron citrate on the NTR-mediated uptake process. At a Tf-Fe concentration of 2.5 mmol/L, iron uptake was progressively inhibited by increasing concentrations of iron citrate, reaching a maximum level of inhibition at 10-fold molar excess of citrate-iron to Tf-Fe. Tf-Fe uptake was not
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FIG. 1. Effect of iron citrate on (A and B) iron and (C and D) transferrin uptake by (A and C) HuH7 transferrin receptor sense and (B and D) antisense cells. Uptake was measured by incubating the cells with 59Fe-125I-transferrin (0.125 mmol/L or 2.5 mmol/L Fe) in the absence (control) or presence of iron citrate (25 mmol/L Fe, 2.5 mmol/L citrate) or citrate only (2.5 mmol/L) for 2 hours. The figure shows ( ) internalized and (h) membrane uptake. The results are mean { SEM of triplicate determinations.
affected by increasing concentration of citrate alone. At the lower Tf-Fe concentration (0.125 mmol/L), increasing concentrations iron citrate or citrate only had no effect on TfFe uptake (Fig. 2A). As TR saturate at a transferrin concentration of approximately 0.06 mmol/L (0.12 mmol/L Fe),5 the uptake of TfFe by the NTR-mediated process can be determined by the difference between the Tf-Fe uptake at the Tf-Fe concentration of 0.125 mmol/L and 2.5 mmol/L. There was a significant decline in this value for Tf-Fe uptake by the NTR-mediated process with increasing iron citrate concentrations, decreasing to 40% of the control value at 10-fold molar excess of citrate-iron to Tf-Fe (Fig. 2B). The uptake of transferrin at either the high or low concentration was not affected by increasing concentrations of iron citrate or citrate (Fig. 2C).
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Exchange of Iron Between Transferrin and Citrate. To determine if the inhibition of uptake Tf-Fe by iron citrate was because of the extracellular exchange of iron from transferrin to citrate the rate of transfer of 59Fe from transferrin to citrate was measured over a 24-hour period as outlined in Materials and Methods using a 30-kd molecular weight cutoff membrane. The results are shown in Fig. 3. The rate of transfer of 59 Fe from transferrin to citrate was õ1.5% per hour. Similar results were obtained when gel filtration (Sephadex G-25; Pharmacia Biotech, Uppsala, Sweden) was used to separate transferrin-bound and low molecular weight 59Fe (data not shown). Intracellular and Membrane-Bound Iron Uptake. Intracellular and membrane uptake of iron was measured using the proteolytic enzyme Pronase20 or an acid wash technique.21 Simi-
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lar results were obtained using both methods. The intracellular and membrane uptake of iron increased with time and transferrin concentration and after 60-minute incubation, approximately 70% of the total iron uptake was internalized. At a Tf-Fe concentration of 2.5 mmol/L, the addition of iron citrate reduced both the rate of intracellular and membrane uptake of Tf-Fe to approximately 60% of the control value obtained in the absence of iron citrate. However, the rate of Tf-Fe uptake was still greater than that observed at a Tf-Fe concentration of 0.125 mmol/L (Fig. 4A and 4B). The intracellular and membrane uptake of transferrin increased rapidly in the first 15 minutes of incubation, reaching steady state levels after 30 minutes, with approximately 30% to 40% of the total transferrin inside the cell. Both the initial rate and steady-state level of intracellular and membrane uptake of transferrin increased with transferrin concentration. Iron citrate had no effect on intracellular uptake of transferrin but there was a small increase in membrane uptake at the higher transferrin concentration (Fig. 4C and 4D). Rates of Iron and Transferrin Internalization. The results in Fig. 4 indicate that iron citrate inhibited the uptake of TfFe by the cell membrane, leading to the reduced intracellular uptake of iron. To confirm this finding the rate of iron internalization was measured over a 60-minute period, whereas the rate of transferrin endocytosis was measured over the first 5 minutes of incubation when intracellular accumulation occurs at a constant rate (Table 1). At the low transferrin concentration, where uptake is TR-mediated, the iron to transferrin molar ratio was approximately 2. As each molecule of transferrin binds two atoms of iron this indicates that all of the iron was entering the cell bound to transferrin. When the transferrin concentration was increased, the rates of transferrin and iron internalization increased and the iron to transferrin molar ratio increased to 3.3, indicating that some of the iron was released from transferrin at the cell membrane before entering the cell. When iron citrate (25 mmol/L Fe) was added to 59Fe-125I-transferrin (1.25 mmol/L transferrin), the rate of transferrin internalization was not affected but 59Fe uptake was reduced by 40% and the iron to transferrin molar ratio was reduced to 2.0. The data obtained in these experiments indicate that iron citrate inhibited the uptake of iron released from transferrin at the cell membrane by the NTR mechanism. Inhibition of Iron Uptake. TR antisense cells were incubated with increasing concentrations of 59Fe-transferrin (0.025-10 mmol/L Fe) for 60 minutes in the presence or absence of iron citrate (25 mmol/L Fe) or citrate (2.5 mmol/L). The maximum rate of iron uptake (Vmax ) and the Michaelis-Menten constant (Km ) were calculated using nonlinear least squares fitting of the iron uptake to a Michaelis-Menten type equation as described before.5 The best fit was obtained when two saturable processes for iron uptake were assumed, consistent with TR- and NTR-mediated processes (Table 2). In the presence of iron citrate, the Vmax and Km for the TR-mediated process remained unchanged. For the NTR-mediated process, the Vmax was the same but the Km was increased by 2.5fold compared with the control in the absence of iron citrate, indicating there was competitive inhibition of Tf-Fe uptake by iron citrate. DISCUSSION
In this study the mechanisms of NTR-mediated uptake of iron were investigated using Tf-Fe and NTB-Fe. NTB-Fe in
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FIG. 2. Effect of iron citrate concentration on iron (A and B) and transferrin (C) uptake. TR antisense cells were incubated with 59Fe-125I-transferrin (0.125 mmol/L Fe; ●, s) and (2.5 mmol/L Fe; j, h) in presence of increasing concentrations iron citrate (●, j) or citrate (s, h) for 2 hours. The figure shows the total uptake of iron from transferrin at two Tf-Fe concentrations (A) and difference between these values (B) in the presence of iron citrate (m) or citrate (n). The results are means { SEM of triplicate determinations.
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FIG. 3. The release of 59Fe from transferrin (2.5 mmol/L Fe) in (h) absence or (j) presence of iron citrate (25 mmol/L Fe, 2.5 mmol/L citrate) during incubation at 377C for up to 24 hours in the cell incubation medium.
the form of iron citrate inhibited Tf-Fe uptake, suggesting that iron from both sources was transported into the human hepatoma cells (HuH7) by a common pathway. The evidence suggests that NTB-Fe did not affect the uptake of iron by the TR-mediated process but inhibited the NTR-mediated uptake of Tf-Fe. At a low extracellular transferrin concentration where uptake of iron and transferrin by the TR sense and antisense cells occurs by the TR-mediated pathway,5 iron citrate did not inhibit iron uptake. However, at higher transferrin concentrations where the uptake of transferrin and iron by the TR sense and antisense cells occurs mainly by NTR-mediated processes,5 NTB-Fe had an inhibitory effect on iron uptake. Also, at a higher transferrin concentration the amount of inhibition of Tf-Fe uptake by NTB-Fe was the same for both the TR sense and antisense cells. It was shown in an earlier study that, while TR antisense cells express approximately 50% as many TR as sense cells, the amount of iron taken by the NTR-mediated pathway is the same.5 Thus, similar effects of NTB-Fe on Tf-Fe uptake by both cell types supports the view that only the NTR-mediated process was impaired. Also the similar effect of NTB-Fe on the uptake of Tf-Fe by both types of cells at both low and high transferrin concentrations indicates that the process of transfection with TR antisense RNA does not affect the common pathway for the uptake of Tf-Fe and NTB-Fe. NTB-Fe inhibited both the intracellular and membrane uptake of iron but had no effect on transferrin uptake. Also, for both the TR sense and antisense cells, the degree of inhibition of intracellular and membrane-bound iron uptake was similar. These findings suggest that NTB-Fe inhibited the uptake of Tf-Fe at the cell surface, leading to a subsequent reduction in intracellular uptake of iron. At low transferrin concentrations, the rate of internalization of iron was twice the rate of transferrin endocytosis. As each molecule of transferrin binds two atoms of iron this finding is consistent with a TR-mediated process where all the iron enters the cell
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bound to transferrin.1 At the higher transferrin concentration, the rates of transferrin and iron internalization increased, indicating that additional transferrin and iron were entering the cell by NTR-mediated pathways. However, the ratio of the rate of iron to transferrin internalization was greater than 2. Therefore, extra iron had entered the cell not bound to transferrin. In the presence of NTB-Fe, the rates of iron internalization were reduced to 2, showing that under these conditions all iron was entering the cell bound to transferrin. Taken together the effects of NTB-Fe on the kinetics of intracellular and membrane uptake of iron suggest that NTB-Fe inhibited the uptake of iron released from transferrin at the cell surface and the subsequent internalization of this iron by the cell. The uptake of Tf-Fe by the NTR-mediated process is saturable and involves an iron carrier-mediated step.5 NTB-Fe did not affect the Vmax of iron uptake by the NTR-mediated process but the Km was more than doubled. This indicates that the NTB-Fe competitively inhibited the uptake of Tf-Fe, probably by preventing the binding of iron released from transferrin to a limited number of iron carriers. Little information about the effects of NTB-Fe on the uptake of Tf-Fe has been reported previously. Several workers observed inhibitory effects of NTB-Fe but the mechanisms were not elucidated. Thorstensen22 showed that ferrous ion and other metal ions such as cobalt, copper, and zinc inhibited the uptake of Tf-Fe by hepatocytes. However, a much higher concentration of the Fe2/ (500 mmol/L) was required to reduce the uptake of Tf-Fe (1 mmol/L) by half than in the present study. We have23 also observed in reticulocytes a reduction in Tf-Fe uptake by several metals of which zinc and Fe2/ were the most effective. There is also recent experimental evidence to suggest that the reverse effect occurs, that is, diferric transferrin inhibits NTB-Fe uptake. Scheiber and Goldenberg24 showed that diferric transferrin completely inhibited the uptake of NTB-Fe presented to the cells in the form of iron-diethylenetriamine pentaacetate, by hepatocytes. However, apotransferrin can also reduce the uptake of iron-diethylenetriamine pentaacetate, suggesting that the protein, probably through its iron-binding properties, rather than iron alone has an inhibitory effect on the NTB-Fe uptake. For several reasons it is unlikely that the effects of iron citrate on Tf-Fe uptake were caused by the transfer of 59Fe from transferrin to citrate or the exchange of 59Fe from transferrin with 56Fe from citrate in the extracellular medium. The rate of release of 59Fe from transferrin to citrate measured in a cell-free system was õ1.5% per hour (Fig. 3), which is insufficient to account for the reduction in Tf-Fe uptake. Also, iron citrate suppresses the uptake of Tf-Fe by the NTR pathway only and has no effect on the TR-mediated uptake of iron. Therefore, this inhibitory effect cannot be accounted for by a reduction in the specific activity of 59Fe-transferrin as this would lead to a reduction in 59Fe uptake by both processes. Iron-depleted transferrin is generated by the cellular uptake of iron from transferrin and the subsequent recycling of apotransferrin to the extracellular medium. However, this could account for õ1% of the total diferric transferrin in the extracellular medium, and saturating this low level of apotransferrin with 56Fe would not have a significant effect on the 59Fe uptake. Furthermore, any 59Fe transferred from transferrin to citrate is unlikely to reduce the uptake of 59Fe
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FIG. 4. Uptake of iron and transferrin from 59Fe-125I-transferrin with time at 377C. The figures show (A) intracellular iron, (C) transferrin, (B) membrane-bound iron, and (D) transferrin uptake from (s) 0.125 mmol/L and 2.5 mmol/L Tf-Fe (h) in the absence and (j) presence of iron citrate (25 mmol/L Fe, 2.5 mmol/L citrate).
because iron citrate can also donate iron to the hepatoma cells (Trinder D, Morgan E, unpublished observation, December 1996). The inhibitory effects of NTB-Fe on Tf-Fe uptake provide new insights into the mechanisms involved in the NTR-mediated uptake of Tf-Fe by hepatoma cells. The results provide
evidence that there are at least two NTR-mediated pathways, the first involving the cell surface release of iron from transferrin and the second mediated by the endocytosis of transferrin. At a transferrin concentration greater than that required to saturate the TR, the rate of iron internalization from transferrin was greater than that of the protein itself
TABLE 1. Rates of Iron and Transferrin Internalization by HuH7 Antisense Cells Diferric Transferrin Concentration (mmol/L)
Rate of Iron Internalization (atoms/cell/min 11003)
Rates of Transferrin Internalization (molecules/cell/min 11003)
Iron:Transferrin Molar Ratio
1.25 1.25 / (25 mmol/L iron citrate) 0.06
105 { 14 (4) 66 { 12 (4)* 40 { 2 (3)†
33 { 4 (4) 33 { 4 (4) 19 { 1 (3)†
3.3 { 0.4 (4) 2.0 { 0.2 (4)* 2.1 { 0.1 (3)*
NOTE. The cells were incubated with 59Fe-125I-transferrin (0.06 and 1.25 mmol/L) in the absence (control) and presence of iron citrate (25 mmol/L Fe) at 377C. The rate of iron internalization was measured over 60 minutes of incubation and transferrin internalization was measured over the first 5 minutes of incubation as described in the text. Results are expressed as the mean { SEM. The number of estimations is shown in parenthesis. *P õ .05 compared with 1.25 mmol/L diferric transferrin. †P õ .01 compared with 1.25 nmol/L diferric transferrin.
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TABLE 2. Maximum Velocity (Vmax) and Michaelis-Menten Constant (Km) for Iron Uptake by HuH7 Antisense Cells Incubation Conditions
Vmax1 (atoms/cell/min 11003)
Kml (nmol/L)
Vmax2 (atoms/cell/min 1 1003)
Km2 (nmol/L)
Tf-Fe Tf-Fe / iron citrate (25 mmol/L) Tf-Fe / citrate (2.5 mmol/L)
45 { 7 33 { 5 37 { 5
38 { 12 31 { 11 29 { 4
226 { 33 265 { 36 196 { 33
5,012 { 1,060 12,484 { 2,030* 3,967 { 333
NOTE. The cells were incubated with increasing concentrations of 59Fe-labeled transferrin in the absence or presence of iron citrate (25 mmol/L) or citrate alone (2.5 mmol/L). Vmax1 and Km1 for the TR-mediated process and Vmax2 and Km2 for the NTR-mediated process were determined as described in the text. Results are expressed as the mean { SEM, where the number of determinations is 3. *P õ .01 compared with diferric transferrin.
and only the excess iron uptake was inhibited by NTB-Fe. This inhibitory effect of NTB-Fe on Tf-Fe uptake shows that some iron was released from transferrin at the cell membrane and was transported into the cell independently of transferrin by an iron carrier-mediated step that was shared with NTBFe. However, uptake of iron by this pathway accounted for only approximately 60% of the total iron taken up by the NTR-mediated pathways and the other 40% was dependent on transferrin endocytosis by the NTR-mediated process (Fig. 4). These conclusions are in agreement with evidence provided by several other investigators that membrane impermeable chelators can partially inhibit the uptake of Tf-Fe by hepatocytes, indicating that iron can be released from transferrin and chelated at an extracellular site.22,25,26 It has been proposed that a plasma membrane reduced nicotinamide adenine dinucleotide:ferric reductase redox system may be involved in the reduction and release of iron from transferrin at the cell surface.27,28 Although the equilibrium constant for the reduction of diferric transferrin by reduced nicotinamide adenine dinucelotide is unfavorable29 other factors such as changes in pH at the cell membrane or conformational changes to the transferrin molecule or iron acceptors may be present in biological systems allowing the reduction of Tf-Fe to proceed. As mentioned above, a second pathway of iron uptake by NTR-mediated process was not affected by NTB-Fe and does not appear to involve the release of iron from transferrin at the cell surface. Instead the transport of iron by this process involves the endocytosis of transferrin. These conclusions are in agreement with those of our earlier studies using HuH7 hepatoma TR sense and antisense cells.5 Then it was found that Tf-Fe uptake could occur by an NTR-mediated pathway that involved the endocytosis and recycling of transferrin and the release of iron from transferrin at an intracellular, acidic site. HuH7 hepatoma cells have many characteristics of primary hepatocytes. First, these cells have been shown to retain many differentiated functions of primary hepatocyte cells including the synthesis of plasma proteins and liver-specific enzymes.16 Second, results from this and our previous study5 indicate that the iron uptake processes by the HuH7 cells are similar to those described for primary hepatocytes.4,8,28 In fact, HuH7 cells are the only hepatoma cell line, like hepatocytes, that has been shown to take up transferrinbound iron by transferrin-independent pathways and therefore, it provides a good model for hepatic iron transport. It is most likely that the effects of NTB-Fe on Tf-Fe uptake by hepatocytes will be similar to those observed for hepatoma cells. In vivo, the plasma transferrin concentration is higher than
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that required to saturate the TR and Tf-Fe is probably delivered to hepatic cells primarily by NTR-mediated processes via the iron carrier-dependent pathway. Therefore, the iron carrier that is shared by Tf-Fe and NTB-Fe plays a central role in the uptake of iron by hepatic cells. In iron overloaded states associated with the diseases hemochromatosis and thalassemia, both Tf-Fe and NTB-Fe are present in high concentrations in the plasma and the iron from both sources is probably cleared by the liver by the common iron carriermediated pathway. The uptake of iron by the carrier-mediated pathway is rate limiting and therefore, regulation of this process is likely to be of major importance in the control of iron hepatic transport in both normal and iron overloaded states. We have previously shown that HuH7 hepatoma cells can take up Tf-Fe by TR and NTR pathways. Iron uptake by the NTR mechanisms is mediated by an iron carrier-mediated step.5 In the present study it was shown that NTB-Fe partially inhibited the uptake of Tf-Fe by the NTR-mediated process but had no effect on the TR-mediated process. NTB-Fe competitively inhibited the uptake of iron, released from transferrin at the cell membrane, by an iron carrier-mediated NTR pathway. NTB-Fe did not affect the uptake of Tf-Fe by NTR pathway, which is dependent on transferrin endocytosis. These results show Tf-Fe is taken up by two NTR pathways, one involves the endocytosis of transferrin and the other involves the release of iron from transferrin at the cell surface and the subsequent transport of iron into the cell by an iron carrier-mediated pathway that is shared with NTB-Fe. REFERENCES 1. Baker E, Morgan EH. Iron transport. In: Brock JH, Halliday JJ, Pippard M, Powell L, eds. Iron Metabolism in Health and Disease. London: Saunders, 1994:63-95. 2. Owen D, Kuhn LC. Noncoding 3* sequences of the transferrin receptor gene are required for mRNA regulation by iron. EMBO J 1987;6:12871293. 3. Klausner RD, Rouault TA, Harford JB. Regulating the fate of mRNA: The control of cellular iron metabolism. Cell 1993;72:19-28. 4. Page MA, Morgan EH, Baker E. Transferrin binding and iron uptake by rat hepatocytes in culture. Am J Physiol 1984;246:G26-G33. 5. Trinder D, Zak O, Aisen P. Transferrin receptor independent uptake of diferric transferrin by human hepatoma cells with antisense inhibition of receptor expression. HEPATOLOGY 1996;23:1512-1520. 6. Richardson D, Baker E. Two mechanisms of iron uptake from transferrin by melanoma cells. The effect of desferrioxamine and ferric ammonium citrate. J Biol Chem 1992;267:13972-13979. 7. Chan RYY, Ponka P, Schulman HM. Transferrin-receptor-independent but iron-dependent proliferation of variant Chinese hamster ovary cells. Exp Cell Res 1992;202:326-336. 8. Trinder D, Morgan E, Baker E. The effects of an antibody to the rat transferrin receptor and of serum albumin on the uptake of diferric transferrin by rat hepatocytes. Biochim Biophys Acta 1988;943:440446.
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9. Zak O, Trinder D, Aisen P. Primary receptor-recognition site of human transferrin is in the C-terminal lobe. J Biol Chem 1994;269:7110-7114. 10. Thorstensen K, Trinder D, Zak O, Aisen P. Uptake of iron from Nterminal half-transferrin by isolated rat hepatocytes: evidence of transferrin receptor-independent iron uptake. Eur J Biochem 1995;232:129133. 11. Hershko C, Graham G, Bates GW, Rachmilewitz EA. Non specific serum iron in thalassemia: An abnormal serum iron fraction of potential toxicity. Br J Haematol 1978;50:255-263. 12. Batey RG, Lai Chung Fong P, Shamir S, Sherlock S. A non-transferrin bound serum iron in idiopathic hemochromatosis. Dig Dis Sci 1980; 25:340-346. 13. Grootveld M, Bell JD, Halliwell B, Aruoma OI, Bomford A, Sadlier PJ. Non-transferrin bound iron in plasma or serum from patients with idiopathic hemochromatosis. J Biol Chem 1989;264:4417-4422. 14. Craven CM, Alexander J, Eldridge JP, Kushner S, Bernstein S, Kaplan J. Tissue distribution and clearance kinetics of non-transferrin-bound iron in the hypotransferrinemic mouse: a rodent model for hemochromatosis. Proc Natl Acad Sci U S A 1987;84:3457-3461. 15. Wright TL, Brissot P, Ma W-L, Weisiger RA. Characterization of nontransferrin-bound iron clearance by rat liver. J Biol Chem 1986;261: 10909-10914. 16. Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J. Growth of human hepatoma cell line with differentiated functions in chemically defined medium. Cancer Res 1982;42:3858-3863. 17. Sasaki K, Zak O, Aisen P. Antisense suppression of transferrin receptor gene expression in a human hepatoma cell (HuH-7) line. Am J Hematol 1993;42:74-80. 18. Kuhn LC, McCelland A, Ruddle FH. Gene transfer, expression, and molecular cloning of the human transferrin receptor gene. Cell 1984; 37:95-103.
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19. Macfarlane AS. In vivo behavior of 131-fibrinogen. J Clin Invest 1963; 42:346-361. 20. Trinder D, Morgan E, Baker E. The mechanism of iron uptake by fetal rat hepatocytes in culture. HEPATOLOGY 1986;6:852-858. 21. Ghosh RN, Maxfield FR. Evidence for nonvectorial, retrograde transferrin trafficking in the early endosomes of HepG2 cells. J Cell Biol 1995;128:549-561. 22. Thorstensen K. Hepatocytes and reticulocytes have different mechanisms for the uptake of iron from transferrin. J Biol Chem 1988;263: 16837-16841. 23. Morgan EH. Membrane transport of non-transferrin iron by reticulocytes. Biochim Biophys Acta 1988;943:428-439. 24. Scheiber B, Goldenberg H. Uptake of iron by isolated rat hepatocytes from a hydrophilic impermeant ferric chelate, Fe(III)-DPTA. Archive Biochem Biophys 1996;326:185-192. 25. Cole ES, Glass J. Transferrin binding and iron uptake in mouse hepatocytes. Biochim Biophys Acta 1983;762:102-110. 26. Wong A. ‘‘The effect of iron chelators on hepatocyte iron metabolism.’’ 1995: PhD Thesis. University of Western Australia: Perth, Western Australia. 27. Low H, Sun IL, Navas P, Grebing C, Crane FL, Morre DJ. Transplasmalemma electron transport from cells is part of a diferric transferrin reductase system. Biochim Biophys Res Commun 1986;139:1117-1123. 28. Thorstensen K, Romslo I. Uptake of iron from transferrin by isolated rat hepatocytes: a redox-mediated plasma membrane process. J Biol Chem 1988;263:8844-8850 29. Thorstensen K, Aisen P. Release of iron from diferric transferrin in the presence of rat liver plasma membranes: no evidence of a plasma membrane diferric transferrin reductase. Biochim Biophys Acta 1990; 1052:29-55.
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