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Transferrin receptor-independent uptake of differic transferrin by human hepatoma cells with antisense inhibition of receptor expression
Transferrin Receptor–Independent Uptake of Differic Transferrin by Human Hepatoma Cells With Antisense Inhibition of Receptor Expression DEBORAH TRINDER, OLGA ZAK,
The hepatic uptake of transferrin-bound iron by a nontransferrin receptor (NTR)–mediated process was investigated using the human hepatoma cell line HuH7. Because HuH7 cells also acquire iron from transferrin by a receptor (TR)-mediated process, TR expression was inhibited by transfecting the cells with a plasmid containing human TR complementary DNA in antisense orientation relative to a human cytomegalovirus promoter/ enhancer element. Cell clones were obtained that expressed a 50% to 60% reduction in cell surface TR, leading to a corresponding decrease in transferrin and iron uptake compared with wild-type cells. Uptake of transferrin by a second process was nonsaturable and not inhibited by a 100-fold excess of unlabeled transferrin. The amounts of transferrin taken up by the wild-type and antisense cells by this process were similar, showing that it did not involve TR. The proteolytic enzyme Pronase reduced the uptake of transferrin, suggesting that the NTR-mediated process entailed the nonsaturable binding of transferrin to plasma membrane proteins. This process, like the TR-mediated one, involved the internalization and recycling of transferrin, leading to accumulation of iron with time. Iron uptake mediated by NTR process was saturable and displaced by 100-fold excess unlabeled transferrin and reduced by weak bases and metabolic inhibitors. Therefore, the NTR-mediated process entailed transferrin adsorption to membranebound proteins, internalization, and release of iron from transferrin by a pH-dependent step followed by the intracellular transport of iron into ferritin and heme by a saturable carrier-mediated mechanism. (HEPATOLOGY 1996;23:1512-1520.) Abbreviations: TR, transferrin receptor; NTR, nontransferrin receptor; MEM, minimal essential medium; FBS, fetal bovine serum; Vmax , maximum rate of iron uptake; Km , equilibrium constant. From the Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461. Received July 13, 1995; accepted December 29, 1995. Supported by a National Health and Medical Research Council of Australia CJ Martin Fellowship to D.T. and grants from the National Institutes of Health (DK15056 and DK37927). Dr. Trinder is now at the Department of Physiology, University of Western Australia, Nedlands 6907, Western Australia, Australia. Address reprint requests to: Deborah Trinder, Ph.D., Department of Physiology, University of Western Australia, Nedlands 6907, Western Australia, Australia. Copyright q 1996 by the American Association for the Study of Liver Diseases. 0270-9139/96/2306-0031$3.00/0
AND
PHILIP AISEN
Iron is required for the synthesis of many heme and non-heme proteins and enzymes necessary for cellular growth and metabolism. Most cells take up iron from serum transferrin by a transferrin receptor (TR)-mediated process. Hepatocytes, in addition to their own need for iron, are also an important site of iron storage. They acquire iron by many pathways, including TRmediated uptake of iron from transferrin,1 iron taken up by from desialylated transferrin through the asialoglycoprotein receptor,2 iron carried by ferritin,3,4 and transferrin-iron secured by a non–transferrin receptormediated process5 (NTR). The TR-mediated uptake of transferrin-iron has been studied extensively, particularly in the developing erthyroid cell.6 The receptormediated process involves the binding of transferrin to a cell surface TR, internalization of the transferrin-TR complex into an endocytotic vesicle, the release of iron from transferrin, and transport into the cell cytosol and the recycling of the apotransferrin-TR complex to the cell surface, where the apotransferrin is released.7 By contrast, the other processes of iron delivery, especially the TR-independent uptake of transferrin-iron, have not been well characterized. In an earlier study from this laboratory, data were presented indicating that the uptake of iron from transferrin by isolated hepatocytes was dependent on the extracellular transferrin concentration.1 This indicated that, in addition to the uptake of transferrin-iron through a limited number of TR, there was a second nonsaturating pathway delivering iron to the hepatocyte. This process involves the low-affinity, nonspecific uptake of transferrin8,9 which is independent of TR.10 There is an accumulation of iron over transferrin by the cell by this process, and in vitro, it provides a major source of iron for the liver cell,5 although the physiological importance of this process remains unclear.11 One of the major hindrances to the elucidation of the second process of NTR-mediated uptake of transferriniron has been to differentiate it from the TR-mediated process. To overcome this problem, we have transfected a human hepatoma cell line HuH712 with a fragment of the human TR complementary DNA13 in the antisense direction to suppress TR expression.14 HuH7 cells, such as hepatocytes, were able to take up transferrin-iron by the NTR process providing a model to examine he-
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patic iron uptake by receptor-dependent and -independent routes. MATERIALS AND METHODS Human transferrin, Pronase, and horse spleen ferritin were purchased from Boehringer Mannheim (Indianapolis, IN). Bovine serum albumin was obtained from Sigma Chemicals (St. Louis, MO). Restriction enzymes were obtained from New England Biolabs (Beverley, MA). Lipofectin, Geneticin, trypsin-ethylenediaminetetraacetic acid (EDTA) and minimal essential medium (MEM) were purchased from Gibco (Bethesda, MD). 125I and 59Fe were received from New England Nuclear/Dupont (Boston, MA). Fetal bovine serum (FBS) was obtained from Gemini (Berkeley, CA). HuH7 hepatoma cells were a generous gift from Dr. Richard Stockert. The wild-type cells were grown in monolayer culture on plastic culture flasks in MEM containing 10% heat-inactivated FBS and 1 mg/mL horse spleen ferritin to ensure the cells are not deprived of iron. TR antisense and sense cells were grown in the above media containing 200 mg/mL Geneticin. The cells were removed from the flasks by incubation with 0.05% trypsin and 0.53 mmol/L EDTA for 5 minutes at 377C. The cell suspension was centrifuged, and the pellet was resuspended in media. The cells were counted in a hemocytometer and subcultured into new cell culture flasks or cell culture plates for experiments. TR antisense RNA expression vectors were constructed using a 2.4-kb EcoRV-Xba1 fragment of human TR complementary DNA pcDTR-1.13 This fragment contains 58 bp of the 5* untranslated region, the entire coding sequence, and 49 bp of the 3* untranslated region and was inserted in the antisense or sense (control) orientation into the Xba1 multiple cloning site of the expression vector pRc/CMV (Invitrogen, CA) as described previously.14 The recombinant plasmids were grown in DHa5 Escherichia coli and purified by alkaline lysis and equilibrium centrifugation using cesium chloride.15 Orientation of the insert (sense or antisense) was determined by digestion with the restriction enzyme Nde I. HuH7 cells were grown in 60-mm plates until they reached 50% confluence (106 cells). The cells were transfected with 5 to 10 mg antisense or sense TR vector, pRc/CMV/TR using Lipofectin (30 mg) for 24 hours according to the manufacture’s instructions. The cell medium was then replaced with MEM/ 10% FBS containing 1 mg/mL horse spleen ferritin. After 48 hours, the cells were replated at a 1:10 dilution, and stable transfectants with high number of copies of the TR antisense gene were selected for neomycin resistance by growing the cells in the presence of a high concentration of Geneticin (1,600 mg/mL). After 4 weeks, the Geneticin concentration was reduced to 200 mg/mL, and the transfected cells were cloned by limiting dilution. The cell clones were screened for reduced transferrin receptor expression by measuring the binding of 2.5 mg/mL 125I-transferrin to TR at 47C compared with the wild-type cells after experimental procedures described below. Iron was removed from transferrin as described previously16 and labeled with 59Fe, using citrate as an iron donor to give a specific activity of approximately 2,000 cpm/ng transferrin-iron. Diferric transferrin was labeled with 125I using the Enzymobead method following the manufacturer’s instructions (Bio-Rad, Hercules, CA). The labeled protein was then passed through 10-DG desalting column (Bio-Rad, CA) to remove unbound radioactivity. Specific activity was ap-
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proximately 80 to 100 cpm/ng transferrin with more than 99% 125I protein-bound. HuH7 wild-type and transferrin receptor sense and antisense cells were plated at a density of 2 1 105 cells per 35mm six-well plate and grown for 3 days in MEM/10% FBS with horse spleen ferritin until they reach approximately 80% confluence. To remove endogenous transferrin and FBS, the cells were preincubated two times for 15 minutes at 377C with MEM. Cells were then incubated with MEM containing 2% bovine serum albumin and 125I-transferrin or 59Fe-transferrin. At the end of the incubation, the cells were washed five times with Hank’s buffer (pH 7.4) at 47C and solubilized for gamma counting using 0.1% Triton/0.1 N NaOH. Protein concentration was determined by the method of Bradford17 with bovine serum albumin as a standard. Protein content was 276 { 3 (n Å 5) pg/cell for wild-type cells, 240 { 11 (n Å 3) pg/cell for TR sense cells, and 204 { 12 (n Å 3) pg/cell for TR antisense cells. Nonheme iron levels were measured by growing wildtype, TR sense, and antisense cells in MEM/10% FBS containing 1 mg/mL horse spleen ferritin. The cells (approximately 10 1 106) were washed five times with Hank’s buffer (pH 7.4) and then removed from the cell culture flasks using 0.05% trypsin and 0.53 mmol/L EDTA as described. The cells were resuspended in 2.8 mol/L nitric acid, and the nonheme iron levels were determined as described previously18 using a Varian Spectra AA-640Z atomic absorption spectrophotometer equipped with a graphite furnace atomizer. Intracellular and membrane-bound radioactivity were determined using the enzyme Pronase as described previously.5 The washed cells were incubated for 30 minutes with 1 mg/ mL pronase at 47C and centrifuged at 12,000g for 30 seconds. The cell pellet contained the intracellular radioactivity, and the supernatant contained the membrane-bound radioactivity. NTR-mediated uptake of transferrin and iron was measured in the presence of a 100-fold excess of unlabeled human transferrin. TR-mediated uptake of transferrin and iron was calculated as the difference between uptake measured in the absence and presence of 100-fold excess of unlabeled human transferrin. Cells were preincubated with the weak bases chloroquine, NH4Cl, and methylamine and the metabolic inhibitors KCN and NaN3 for 15 minutes at 377C before the addition of 60 nmol/L or 1.25 mmol/L 59Fe-transferrin for a further 60 minutes at 377C, and then cells were treated as described. To determine the amount of 59Fe incorporated into ferritin and heme, the cells were incubated with 59Fe-transferrin for 2 hours at 377C or 47C and washed five times with Hank’s buffer (pH 7.4). The cells were then solubilized with 1 mL of lysis buffer containing 1% Triton/1% deoxycholate and 25 mmol/L HEPES (pH 7.4) at 47C and centrifuged at 12,000g for 5 minutes to remove insoluble materials. The incorporation of 59 Fe into ferritin was measured by incubating the supernatant with rabbit anti-human serum ferritin antibody (Boehringer Mannheim) for 1 hour at 377C or overnight at 47C. The antibody-antigen complex was precipitated using protein-A– positive cells (Boehringer Mannheim), and the precipitate was washed three times with lysis buffer at 47C and counted for gamma radiation. To determine 59Fe incorporated into heme, the cells were incubated with 59Fe-transferrin and solubilized as described. The cell supernatant was acidified with 1N HCl to pH 2, and the heme was extracted with an equal volume of ice-cold cyclohexanone.19
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TABLE 1. Binding Parameters for Transferrin With HuH7 Cells at 47C Cell Type
Number of TransferrinBinding Sites per Cell
Association Constant M01 (11008)
Wild-type Sense Antisense
210,000 187,500 98,500
2.0 1.0 1.6
NOTE. The results are the mean values obtained from two separate experiments.
Cellular degradation of transferrin was determined by measuring the amount of protein-free 125I inside the cells or released to the incubation media by trichloroacetic acid precipitation. One milliliter of cell suspension or media containing 2% bovine serum albumin as a protein carrier was incubated with trichloroacetic acid to give a final concentration of 10% for 15 minutes at 47C. The samples were centrifuged, and the pellet and supernatant were counted for gamma radiation. Unless otherwise stated, the results are the means of duplicate or triplicate determinations made within each experiment, and the SEM has been included where appropriate. Results were analyzed by one-way ANOVA using a Fisher post hoc comparison test as appropriate. Significant differences were obtained when P õ .05. All experiments were performed at least twice, and the data presented are representative of a typical experiment. RESULTS Transferrin Binding by HuH7 Cells. For permanent inhibition of TR expression, human hepatoma cells HuH7 were transfected with TR antisense expression vector pRc/CMV/TR (sense vector used as control). Stable clones were selected by neomycin resistance and screened for reduced TR expression by measuring 125Itransferrin binding to TR at 47C. Six stable hepatoma cell clones that had a 50% to 60% reduction in transferrin binding were obtained. The TR antisense clone AS/C9 was selected for further studies. The number of transferrin binding sites per cell expressed by HuH7 wild-type cells and the TR sense and antisense cell clones was determined by Scatchard analysis of 125I-transferrin binding at 47C (Table 1). The level of expression of transferrin binding sites by the TR antisense cells was reduced to 47% when compared with the wild-type, whereas the TR sense S/H2 expressed 89% of the control level. Furthermore, if the number of TR was expressed per gram of protein, there was 7.6 1 1014 TR/g protein for the wild-type cells, 7.8 1 1014 TR/g protein for the sense cells, and 4.8 1 1014 TR/g protein for the antisense cells. Therefore, the number of TR/g protein expressed by the antisense cells was reduced to approximately 60% compared with the wild-type and sense cells. The TR on all cell types were saturated at an extracellular transferrin concentration of 60 nmol/L. Transferrin and Iron Uptake With Time. The uptake of 59Fe from transferrin by the wild-type, sense, and
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antisense cells at 377C increased with time. The rate of iron uptake by AS/C9 antisense cells was reduced to approximately 45% of the wild-type cells, and uptake by S/H2 sense cells was approximately 90% of the control (Fig. 1A). The rate of accumulation of transferrin at 377C was initially rapid, but decreased markedly after 15 minutes until a steady-state level was reached after 60 minutes’ incubation (Fig. 1B). The initial rate of transferrin uptake over the first 5 minutes by the antisense cells was 40% of the wild-type cells, with values of 12,000 molecules/cell/min and 30,000 molecules/cell/min, respectively. The steady-state level of cell-bound transferrin in the antisense cells was 44% of the wild-type cells (Fig. 1B). There was a progressive increase in the iron to transferrin molar ratio with time, reaching 12.6 after 120 minutes for the wild-type cells and 12.8 for the antisense cells. The cellular nonheme iron level was 20.1 { 0.7 ng Fe/106 cells (73 { 2 mg Fe/g protein, n Å 3) for the wildtype cells, 18.2 { 0.6 ng Fe/106 cells (76 { 2 mg Fe/g protein, n Å 3) and 17.6 { 1.4 ng Fe/106 cells (86 { 7 mg Fe/g protein, n Å 3) for the antisense cells. There was no significant difference in the iron concentration, expressed either per cell or per gram of protein, between the three cell types (P ú .05). Two Mechanisms of Transferrin Uptake. To determine if the wild-type and antisense cells could acquire iron by NTR-mediated as well as by TR-mediated processes, the cells were incubated with transferrin in the concentration range 2.5 to 120 nmol/L at 377C. The total transferrin taken up by the cells at 377C reflects binding to the cell surface and internalization of transferrin into the cell. Cell-associated transferrin increased rapidly as the transferrin concentration was initially increased, but leveled off as the TR saturated at a concentration of 60 nmol/L (Fig. 2A). Transferrin uptake reached a maximum level of 5.7 1 105 molecules/cell by the wild-type cells and 2.7 1 105 molecules/ cell by the antisense cells. Using transferrin uptake at 377C as an indication of the total number of binding sites and binding at 47C as a measure of cell surface binding sites, approximately 35% of the receptors were found on the cell surface. The distribution of TR was similar for both cell types. In a second experiment, the extracellular transferrin concentration was increased to approximately 2 mmol/ L. Plots of the cell-associated transferrin that were initially curvilinear became linear, suggesting the presence of a second nonsaturable process of transferrin uptake (Fig. 2B). When the uptake of 125I-transferrin was measured in the presence of a 100-fold molar excess of unlabeled transferrin at each concentration point, it increased at a constant rate of approximately 330 molecules/cell/nmol/L transferrin. This rate was similar to the rate of increase of total transferrin uptake when the concentration was increased above 150 nmol/L for both the wild-type (360 molecules/cell/nmol/ L transferrin) and antisense cells (345 molecules/cell/ nmol/L transferrin). In the presence of unlabeled trans-
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FIG. 1. (A) Uptake of iron from diferric transferrin at 377C by HuH7 wild-type cells (●), sense cells (s), and antisense cells (m). Transferrin concentration was 60 nmol/L. The results are the means of duplicate determinations. (B) Transferrin uptake by HuH7 wildtype cells (●) and antisense cells (m) at 377C. Transferrin concentration was 60 nmol/L. The results are the means of duplicate measurements.
FIG. 2. Effect of transferrin concentration on transferrin uptake by wild-type and antisense cells at 377C. Total uptake 125I-transferrin uptake by wild (●) and antisense cells (m) and NTR uptake by wild (s) and antisense cells (n). The results are means of duplicate determinations.
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FIG. 3. Effect of transferrin concentration on iron accumulation from diferric transferrin by wild and antisense cells at 377C. Total iron uptake by wild (●) and antisense cells (m). The results are means of duplicate determinations.
ferrin, the amount of cell-associated transferrin was similar for the wild and antisense cells. These results indicate that the nonsaturating process does not involve high-affinity TR and, at 1.25 mmol/L, it accounted for approximately 40% and 60% of total transferrin accumulation by the wild-type and antisense cells, respectively. Two Processes of Iron Uptake. Iron uptake from diferric transferrin was measured over a transferrin concentration range of 0.025 to 2.5 mmol/L. The amount of iron taken up by the wild and antisense cells increased rapidly at low diferric transferrin concentrations, but the rate of increase declined with transferrin concentrations above 0.1 mmol/L (Fig. 3A) consistent with a TR-mediated pathway of iron uptake. At higher transferrin concentrations, the uptake of iron continued to increase, suggesting the presence of an additional iron uptake process (Fig. 3B). When the uptake of 59Fetransferrin was measured in the presence of a 100-fold molar excess of unlabeled transferrin at each concentration point, iron uptake was greatly reduced, and at a transferrin concentration of 1.25 mmol/L, iron uptake by the wild and antisense cells was only 10% to 15% of that observed in the absence of unlabeled transferrin-iron. The maximum rate of iron uptake (Vmax ) and equilibrium constant (Km) were calculated using nonlinear least squares fitting of the iron uptake data to a Michaelis Menten–type equation. The best fit was obtained when two saturable processes of iron uptake by the wild and antisense cells were assumed.
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Vi Å
V1m 1 [F] V2m 1 [F] / Km1 / [F] Km2 / [F]
where Vi is the velocity of iron uptake at transferrin concentration [F], V1m and V2m are the maximal rates for the first and second processes of iron uptake, and Km1 Km2 are the equilibrium constants for the two processes. For the first process of iron uptake, Vmax for antisense cells (Vmax 40,000 atoms/cell/min or 1.9 1 1014 atoms/g protein/min; Km 22 nmol/L) was 50% to 65% of the wild-type cells (Vmax 79,000 atoms/cell/min or 2.9 1 1014 atoms/g protein/min; Km 13 nmol/L), depending on whether the results were expressed per cell or per gram of protein. These results are consistent with a TR-mediated process. For the second process, the Vmax for the antisense cells was (Vmax 67,000 atoms/cell/min or 3.2 1 1014 atoms/g protein/min; Km 2.0 mmol/L) was approximately 90% to 115% of the wild-type cells (Vmax 77,000 atoms/cell/min or 2.8 1 1014 atoms/g protein/ min; Km was 1.5 mmol/L). This suggests that a similar low-affinity mechanism was involved in both cell types. In Fig. 4, an Eadie-Hofstee plot20 of iron uptake illustrates that there were two components of iron uptake for both the wild-type and the antisense cells. Internalization of Transferrin and Iron. A series of experiments was performed to compare iron uptake by the TR- and NTR-mediated processes. The antisense cells were incubated with a low concentration of transferrin (60 nmol/L) where the uptake of transferrin and iron occurs by the TR-mediated process and at a higher transferrin concentration (1.25 mmol/L) where uptake
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FIG. 4. Data shown in Fig. 3 are presented as an Eadie Hofstee plot of iron uptake by wild (●) and antisense cells (m).
occurs predominantly by the NTR-mediated process (Fig. 2B). The difference in transferrin and iron uptake at these two concentrations was used as a measure of uptake by NTR-mediated process. Iron uptake at 377C is shown in Fig. 5A. The rate of total, membrane-bound,
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and intracellular uptake with time increased approximately twofold on increasing the transferrin concentration from 60 nmol/L to 1.25 mmol/L. Iron uptake at 47C, which reflects iron bound to the cell membrane, also increased approximately twofold and accounted for 12% of total uptake at both transferrin concentrations. After 2 hours’ incubation with 60 nmol/L and 1.25 mmol/L 59Fe-transferrin at 377C, 43% and 41% of the iron was incorporated into ferritin, respectively (Table 2) and less than 5% into heme. When the incubation temperature was reduced to 47C, only 5% to 10% of iron taken up by the cells at both transferrin concentrations was incorporated into ferritin. Transferrin uptake from 60 nmol/L and 1.25 mmol/L diferric transferrin is shown in Fig. 5B. Transferrin uptake at both transferrin concentrations reached a steady-state level after 60 minutes’ incubation with 50% and 42% of the transferrin internalized at 60 nmol/ L and 1.25 mmol/L, respectively. The steady-state level of total, intracellular, and membrane-bound associated transferrin increased approximately two to three times when the transferrin concentration was raised from 60 nmol/L to 1.25 mmol/L. There was a net accumulation of iron by the cell, and after 120 minutes’ incubation, the intracellular iron-to-transferrin molar ratio reached approximately 20 at both transferrin concentrations. More than 90% of 125I-transferrin taken up by the cells was precipitated by 10% trichloroacetic acid, indicating that there was little catabolism of transferrin by the cells. These findings suggest that the
FIG. 5. Iron (A) and transferrin (B) uptake by antisense cells. The cells were incubated with 59Fe-transferrin or 125I-transferrin for 0 to 120 minutes and then reincubated with 1 mg/mL Pronase for 30 minutes at 47C to separate surface-bound and intracellular iron. At a transferrin concentration of 60 nmol/L, total (s), intracellular (●) uptake and 1.25 mmol/L total (n) and intracellular (m) uptake. Fig. 5A results are expressed as mean { SEM, where n Å 3, and Fig. 5B results are means of duplicate estimations.
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TABLE 2. Effects of Inhibitors on Iron Uptake by HuH7 Cells Wild-Type Inhibitor
Chloroquine (200 mmol/L) NH4Cl (20 mmol/L) Methylamine (30 mmol/L) KCN (1 mmol/L) NaN3 (1 mmol/L) Ferritin
Antisense
0.06 mmol/L (% control)
1.25 mmol/L (% control)
36 (3) 41 (3) 50 (3)
31 (3) 32 (3) 34 (3)
45 (3)
39 (3)
0.06 mmol/L (% control)
32 32 33 75 87 43
(3) (6) (3) (3) (3) (3)
1.25 mmol/L (% control)
28 28 30 80 104 41
(3) (6) (3) (3) (3) (3)
59
NOTE. The cells were incubated with Fe-transferrin (0.06 mmol/L and 1.25 mmol/L) in the presence and absence of inhibitors for 60 minutes. Iron uptake (with inhibitor) is expressed as percentage of control value (without inhibitor). Ferritin was measured by immunoprecipitation as described in Materials and Methods. The amount of iron incorporated into ferritin is expressed as a percentage of total iron uptake. The number of estimations are shown in parentheses.
NTR-mediated process, like the TR-mediated process, involved the endocytosis and recycling of transferrin. The weak bases chloroquine, NH4Cl, and methylamine inhibited the uptake of iron by the antisense cells by approximately 70% at both transferrin concentrations. Uptake by the wild-type cells was also reduced, and at the higher transferrin concentration the effect was similar to the antisense cells; however, at the lower concentration, the percentage inhibition was slightly reduced. The metabolic inhibitor KCN inhibited iron uptake by approximately 20% at both transferrin concentrations, with NaN3 having a lesser effect (Table 2). DISCUSSION
HuH7 hepatoma cells acquired iron from transferrin by two processes. The first pathway involved high-affinity TR-mediated endocytosis of transferrin, and the second was a low-affinity process not involving TR receptors. The high-affinity process played an important role in the uptake of iron at low extracellular transferrin concentrations, and the low-affinity process became more predominant at higher transferrin concentrations. This study provided the first evidence of TR-independent uptake of transferrin-iron by hepatoma cells and indicated that the uptake of transferriniron by hepatoma cells entailed similar mechanisms to those found in hepatocytes.5,10 An antisense TR vector was introduced into the hepatoma cells to reduce TR expression by interfering with TR gene translation.14 This resulted in the decline of the number of cell surface TR and a reduction in the uptake of transferrin and iron uptake by the TR antisense cells. A reduction in the initial rate and steadystate levels of transferrin accumulation was consistent with the decline in the number of TR, demonstrating that the inhibition of iron uptake was completely attributable to the reduction in the level of receptor expression, and there was no alteration to the recycling time of transferrin through the cell. This was confirmed by similar iron-to-transferrin molar ratio for both the wild-type and antisense cells, indicating that the effi-
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ciency of iron accumulation was consistent for both cell types. There was no difference in the nonheme iron levels of the wild-type, TR sense, and antisense cells grown in the presence of horse spleen ferritin, suggesting that the alteration to TR expression was independent of the iron status of the cells. There was a difference in the protein content per cell for the wild-type cells and TR sense and antisense cell clones. However, it is unlikely that the differences in TR expression were caused by a generalized alteration in cellular protein synthesis because, irrespective of whether the results are expressed per cell or per gram of protein, there was a significant reduction in the number of TR and the rate of iron uptake by the antisense cells compared with the wild-type or sense cells. In a control experiment, wild-type cells were transfected with pRc/CMV vector only. Southern and Northern blot analysis showed that there was neither rearrangement or amplification of the endogenous TR gene or changes in the TR messenger RNA levels caused by transfection itself (Sasaki K et al, Unpublished observation). The number of TR and rate of iron uptake was similar for the wild-type and TR sense control cells (Table 1 and Fig. 1) with inhibition of TR expression specific to cells transfected with antisense TR. TR were not involved in the uptake of transferriniron by the second process because transfection of the HuH7 cells with the TR antisense vector had no effect on transferrin or iron uptake by this process. Transferrin uptake by both cell types was nonsaturable and not inhibited by excess of unlabeled transferrin, characteristics that are inconsistent with a receptor-mediated process. This is in agreement with findings from another study using Chinese hamster ovary cells, which possessed no detectable transferrin binding sites but were still able to accumulate iron by an NTR-mediated process.21 Similarly, an antibody to the TR directly inhibited the TR-mediated uptake of transferrin and iron by rat hepatocytes, but there was no effect on the second process.10 If transferrin uptake by the second process does not involve TR, then how is the iron delivered to the cell?
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The uptake of transferrin at 377C reflects total cellular accumulation of transferrin, and the use of Pronase to remove transferrin bound to the cell surface showed approximately 40% of the transferrin to be inside the cell. Uptake and internalization of transferrin by the NTR process reached a steady state and acid precipitation of the transferrin indicated that there was very little catabolism of the protein. Therefore, like the TRmediated process, transferrin taken up by NTR-mediated process was internalized into the cell, releasing its iron before being recycled to the extracellular medium. This was also corroborated by an intracellular iron-totransferrin molar ratio of approximately 20 for the NTR-mediated process (Fig. 5A and B). The possibility that fluid-phase endocytosis accounted for the NTR uptake must be considered. Uptake of extracellular macromolecules by fluid-phase endocytosis generally leads to targeting to the lysosomes, where they are degraded.22 In this study, however, we have observed transferrin being recycled rather than degraded. A computer model23 adapted to transferrin cell interactions24 indicated that fluid-phase endocytosis, involving the recycling of fluid from the endocytotic vesicles to the extracellular medium, would lead to the recycling of transferrin and the accumulation of iron sufficient to account for NTR uptake by hepatocytes. Although the recycling of transferrin by fluid-phase endocytosis may therefore play a role in the delivery of iron to the hepatoma cells, our results point to involvement of adsorptive endocytosis in the uptake of transferrin. Inhibition of transferrin and iron uptake by the proteolytic enzyme Pronase suggests that NTR-mediated mechanism entails low-affinity binding or adsorption of transferrin to plasma membrane proteins, nonsaturated at the transferrin concentrations used in this study, leading to the endocytosis and recycling of transferrin and the accumulation of iron by the cell. In the search for a transferrin membrane-binding component that may be involved in the NTR-mediated process, there is evidence that proteoglycans isolated from fibroblasts,25 liver endothelium cells,26 and whole liver27 can bind transferrin. Interaction of transferrin with proteoglycans appeared to be dependent on the pH and the iron content of the transferrin. Release of iron from transferrin was promoted particularly from the N-terminal lobe of the protein.27,28 The proteoglycan-transferrin–binding activity was also sensitive to treatment with trypsin,25 which is consistent with our observations that proteolytic enzymes can reduce transferrin and iron uptake by NTR process. However, there is no direct evidence of the involvement of proteoglycans in the cellular uptake of transferrin-iron. Interestingly, in contrast to transferrin accumulation, most of the iron taken up by the NTR- and TRmediated processes was saturated with increasing amounts of diferric transferrin and was inhibited by an excess of unlabeled diferric transferrin. The inhibition of TR-mediated iron uptake by unlabeled diferric transferrin was caused by the competition between the
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labeled and unlabeled transferrin for the TR-binding sites. However, this does not account for the inhibition of iron uptake by the NTR-mediated process, and it is likely that the labeled and unlabeled iron were competing at another stage in the iron uptake pathway that involved a second saturable iron carrier-mediated step. Similarly, it has been reported that melanoma cells acquire iron by two saturable mechanisms. The first process was TR mediated, and the second was TR independent. The Vmax and Km for iron uptake by the TRindependent pathway were similar to our results in hepatoma cells.29,30 Irrespective of the pathway of transferrin-iron entering into the cell, either by the TR or NTR process, weak bases, metabolic inhibitors, and reduced incubation temperature each reduced iron uptake. The low pH of the endocytotic vesicle has been demonstrated to be an important factor in the release of iron from transferrin by the TR process.31 The weak bases increase the pH of the transferrin-iron containing endosomes inhibiting iron release.32,33 The results obtained in this study demonstrate that the uptake of iron by the NTR process, like the TR process, entails an energy-dependent step with the pH-dependent release of iron from transferrin. Once the iron is released from transferrin, it is transferred into the cytosol and incorporated into ferritin for storage or heme synthesis. In the hepatoma cells, intracellular incorporation of iron into ferritin and heme was similar for iron taken up from transferrin by both processes, indicating that the intracellular pathways taken by iron were the same. The exact mechanism involved in the transport of iron across the vesicle membrane remains unclear. Numerous studies have now been done using rat liver,34 erythroid, and nonerythroid cells,35-37 demonstrating the presence of nontransferrin bound iron transport systems for conveying iron complexes across membrane barriers by carrier-mediated processes. It is therefore possible that the second rate-limiting step in the uptake of iron by the HuH7 cells entails the binding of iron to a carrier that transports iron across the endosome membrane to the cytosol for heme synthesis or incorporation into storage ferritin. Acknowledgment: The authors thank Professor Evan Morgan for his comments on the manuscript. REFERENCES 1. Young SP, Aisen P. The interaction of transferrin with isolated hepatocytes. Biochim Biophys Acta 1980;633:145-153. 2. Rudolph JR, Regoeczi E. Interaction of rat asialotransferrin with adult rat hepatocyte: its relevance for iron uptake and protein degradation. J Cell Physiol 1988;135:539-544. 3. Sibille J-C, Kondo H, Aisen P. Interactions between isolated hepatocytes and kupffer cells in iron metabolism: a possible role for ferritin as an iron carrier protein. HEPATOLOGY 1988;8:296301. 4. Osterloh K, Aisen P. Pathways in the binding and uptake of ferritin by hepatocytes. Biochim Biophys Acta 1989;1011:40-45. 5. Trinder D, Morgan E, Baker E. The mechanism of iron uptake by fetal rat hepatocytes in culture. HEPATOLOGY 1986;6:852-858.
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6. Iacopetta B, Morgan EH. The kinetics of transferrin endocytosis and iron uptake from transferrin in rabbit reticulocytes. J Biol Chem 1983;258:9108-9115. 7. Morgan EH. Transferrin, biochemistry, physiology and clinical significance. Mol Aspects Med 1981;4:1-123. 8. Cole ES, Glass J. Transferrin binding and iron uptake in mouse hepatocytes. Biochim Biophys Acta 1983;762:102-110. 9. Page M, Baker E, Morgan EH. Transferrin and iron uptake by rat hepatocytes in culture. Am J Physiol 1984;246:G26-G33. 10. 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;943:440-446. 11. Morgan EH. Specificity of hepatic iron uptake from plasma transferrin in the rat. Comp Biochem Physiol 1991;99A:91-95. 12. 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. 13. Ku`hn LC, McCelland A, Ruddle FH. Gene transfer, expression, and molecular cloning of the human transferrin receptor gene. Cell 1984;37:95-103. 14. 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. 15. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning, A Laboratory Manual. Ed 2. New York: Cold Spring Habor Press, 1989. 16. Zak O, Aisen P. Evidence for functional differences between the two sites of rabbit transferrin: effects of serum and carbon dioxide. Biochim Biophys Acta 1990;1052:24-28. 17. Bradford M. A rapid method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-254. 18. Trinder D, Batey RG, Morgan EH, Baker E. Effect of cellular iron concentration on iron uptake by hepatocytes. Am J Physiol 1990;259:G611-G617. 19. Teale FWJ. Cleavage of the haem-protein link by acid methylethylketone. Biochim Biophys Acta 1959;35:543. 20. Hoftsee BHJ. Non-inverted versus inverted plots in enzyme kinetics. Nature 1959;184:1296-1298. 21. 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. 22. Steinman RM, Mellman IS, Muller WA, Cohn ZA. Endocytosis and the recycling of plasma membrane. J Cell Biol 1983;96:127.
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23. Blomhoff R, Nenseter MS, Green MH, Berg T. A multicompartmental model of fluid-phase endocytosis in rabbit liver parenchymal cells. Biochem J 1989;262:605-610. 24. Bakoy OE, Thorstensen K. The process of cellular uptake of iron from transferrin: a computer simulation program. Eur J Biochem 1994;222:105-112. 25. Fransson L-A, Carlstedt I, Costar L, Malmstrom A. Binding of transferrin to the core protein of fibroblast proteoheparan sulfate. Proc Natl Acad Sci 1984;81:5657-5661. 26. Omoto E, Minguell JJ, Tavassoli MJ. Proteoglycan synthesis by cultured liver endothelium: the role of membrane-associated heparan sulfate in transferrin binding. Exp Cell Res 1990;187: 85-89. 27. Hu W-L, Regoeczi E. Hepatic heparan proteoglycan and the recycling of transferrin. Biochem Cell Biol 1992;70:535-538. 28. Regoeczi E, Chindemi PA, Hu W-L. Interaction of transferrin and its iron-binding fragments with heparin. Biochem J 1994; 299:819-823. 29. Richardson DR, Baker E. The uptake of iron and transferrin by the human malignant melanoma cell. Biochim Biophys Acta 1990;1053:1-12. 30. Richardson DR, Baker E. Two saturable mechanisms of iron uptake from transferrin in human melanoma cells: the effects of transferrin concentration, chelators, and metabolic probes on transferrin and iron uptake. J Cell Physiol 1994;161:160-168. 31. Klausner RD, Ashwell G, van Renswoude J, Hardford JB, Bridges KR. Binding of apotransferrin to K562 cells: explanation of transferrin cycle. Proc Natl Acad Sci 1983;80:2263-2266. 32. Morgan EH. Inhibition of reticulocyte iron uptake by NH4Cl and CH3NH2 . Biochim Biophys Acta 1981;642:119-134. 33. Ciechanover A, Schwartz AL, Dautry-Varsat A, Lodish HF. Kinetics of internalisation and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. J Biol Chem 1983;258:9681-9689. 34. Wright TL, Brissot P, Ma W-L, Weisiger RA. Characterization of non-transferrin-bound iron clearance by rat liver. J Biol Chem 1986;261:10909-10914. 35. Morgan EH. Membrane transport of non-transferrin-bound iron by reticulocytes. Biochim Biophys Acta 1988;943:428-439. 36. Sturrock A, Alexander J, Lamb J, Craven CM, Kaplan J. Characterization of a transferrin-independent uptake system for iron in Hela cells. J Biol Chem 1990;265:3139-3145. 37. Nunez MT, Escobar A, Ahumada A, Gonzalez-Sepulveda M. Sealed reticulocyte ghosts: an experimental model for the study of Fe2/ transport. J Biol Chem 1992;267:11490-11494.
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The hepatic uptake of transferrin-bound iron by a nontransferrin receptor (NTR)–mediated process was investigated using the human hepatoma cell line HuH7. Because HuH7 cells also acquire iron from transferrin by a receptor (TR)-mediated process, TR expression was inhibited by transfecting the cells with a plasmid containing human TR complementary DNA in antisense orientation relative to a human cytomegalovirus promoter/ enhancer element. Cell clones were obtained that expressed a 50% to 60% reduction in cell surface TR, leading to a corresponding decrease in transferrin and iron uptake compared with wild-type cells. Uptake of transferrin by a second process was nonsaturable and not inhibited by a 100-fold excess of unlabeled transferrin. The amounts of transferrin taken up by the wild-type and antisense cells by this process were similar, showing that it did not involve TR. The proteolytic enzyme Pronase reduced the uptake of transferrin, suggesting that the NTR-mediated process entailed the nonsaturable binding of transferrin to plasma membrane proteins. This process, like the TR-mediated one, involved the internalization and recycling of transferrin, leading to accumulation of iron with time. Iron uptake mediated by NTR process was saturable and displaced by 100-fold excess unlabeled transferrin and reduced by weak bases and metabolic inhibitors. Therefore, the NTR-mediated process entailed transferrin adsorption to membranebound proteins, internalization, and release of iron from transferrin by a pH-dependent step followed by the intracellular transport of iron into ferritin and heme by a saturable carrier-mediated mechanism. (HEPATOLOGY 1996;23:1512-1520.) Abbreviations: TR, transferrin receptor; NTR, nontransferrin receptor; MEM, minimal essential medium; FBS, fetal bovine serum; Vmax , maximum rate of iron uptake; Km , equilibrium constant. From the Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461. Received July 13, 1995; accepted December 29, 1995. Supported by a National Health and Medical Research Council of Australia CJ Martin Fellowship to D.T. and grants from the National Institutes of Health (DK15056 and DK37927). Dr. Trinder is now at the Department of Physiology, University of Western Australia, Nedlands 6907, Western Australia, Australia. Address reprint requests to: Deborah Trinder, Ph.D., Department of Physiology, University of Western Australia, Nedlands 6907, Western Australia, Australia. Copyright q 1996 by the American Association for the Study of Liver Diseases. 0270-9139/96/2306-0031$3.00/0
AND
PHILIP AISEN
Iron is required for the synthesis of many heme and non-heme proteins and enzymes necessary for cellular growth and metabolism. Most cells take up iron from serum transferrin by a transferrin receptor (TR)-mediated process. Hepatocytes, in addition to their own need for iron, are also an important site of iron storage. They acquire iron by many pathways, including TRmediated uptake of iron from transferrin,1 iron taken up by from desialylated transferrin through the asialoglycoprotein receptor,2 iron carried by ferritin,3,4 and transferrin-iron secured by a non–transferrin receptormediated process5 (NTR). The TR-mediated uptake of transferrin-iron has been studied extensively, particularly in the developing erthyroid cell.6 The receptormediated process involves the binding of transferrin to a cell surface TR, internalization of the transferrin-TR complex into an endocytotic vesicle, the release of iron from transferrin, and transport into the cell cytosol and the recycling of the apotransferrin-TR complex to the cell surface, where the apotransferrin is released.7 By contrast, the other processes of iron delivery, especially the TR-independent uptake of transferrin-iron, have not been well characterized. In an earlier study from this laboratory, data were presented indicating that the uptake of iron from transferrin by isolated hepatocytes was dependent on the extracellular transferrin concentration.1 This indicated that, in addition to the uptake of transferrin-iron through a limited number of TR, there was a second nonsaturating pathway delivering iron to the hepatocyte. This process involves the low-affinity, nonspecific uptake of transferrin8,9 which is independent of TR.10 There is an accumulation of iron over transferrin by the cell by this process, and in vitro, it provides a major source of iron for the liver cell,5 although the physiological importance of this process remains unclear.11 One of the major hindrances to the elucidation of the second process of NTR-mediated uptake of transferriniron has been to differentiate it from the TR-mediated process. To overcome this problem, we have transfected a human hepatoma cell line HuH712 with a fragment of the human TR complementary DNA13 in the antisense direction to suppress TR expression.14 HuH7 cells, such as hepatocytes, were able to take up transferrin-iron by the NTR process providing a model to examine he-
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patic iron uptake by receptor-dependent and -independent routes. MATERIALS AND METHODS Human transferrin, Pronase, and horse spleen ferritin were purchased from Boehringer Mannheim (Indianapolis, IN). Bovine serum albumin was obtained from Sigma Chemicals (St. Louis, MO). Restriction enzymes were obtained from New England Biolabs (Beverley, MA). Lipofectin, Geneticin, trypsin-ethylenediaminetetraacetic acid (EDTA) and minimal essential medium (MEM) were purchased from Gibco (Bethesda, MD). 125I and 59Fe were received from New England Nuclear/Dupont (Boston, MA). Fetal bovine serum (FBS) was obtained from Gemini (Berkeley, CA). HuH7 hepatoma cells were a generous gift from Dr. Richard Stockert. The wild-type cells were grown in monolayer culture on plastic culture flasks in MEM containing 10% heat-inactivated FBS and 1 mg/mL horse spleen ferritin to ensure the cells are not deprived of iron. TR antisense and sense cells were grown in the above media containing 200 mg/mL Geneticin. The cells were removed from the flasks by incubation with 0.05% trypsin and 0.53 mmol/L EDTA for 5 minutes at 377C. The cell suspension was centrifuged, and the pellet was resuspended in media. The cells were counted in a hemocytometer and subcultured into new cell culture flasks or cell culture plates for experiments. TR antisense RNA expression vectors were constructed using a 2.4-kb EcoRV-Xba1 fragment of human TR complementary DNA pcDTR-1.13 This fragment contains 58 bp of the 5* untranslated region, the entire coding sequence, and 49 bp of the 3* untranslated region and was inserted in the antisense or sense (control) orientation into the Xba1 multiple cloning site of the expression vector pRc/CMV (Invitrogen, CA) as described previously.14 The recombinant plasmids were grown in DHa5 Escherichia coli and purified by alkaline lysis and equilibrium centrifugation using cesium chloride.15 Orientation of the insert (sense or antisense) was determined by digestion with the restriction enzyme Nde I. HuH7 cells were grown in 60-mm plates until they reached 50% confluence (106 cells). The cells were transfected with 5 to 10 mg antisense or sense TR vector, pRc/CMV/TR using Lipofectin (30 mg) for 24 hours according to the manufacture’s instructions. The cell medium was then replaced with MEM/ 10% FBS containing 1 mg/mL horse spleen ferritin. After 48 hours, the cells were replated at a 1:10 dilution, and stable transfectants with high number of copies of the TR antisense gene were selected for neomycin resistance by growing the cells in the presence of a high concentration of Geneticin (1,600 mg/mL). After 4 weeks, the Geneticin concentration was reduced to 200 mg/mL, and the transfected cells were cloned by limiting dilution. The cell clones were screened for reduced transferrin receptor expression by measuring the binding of 2.5 mg/mL 125I-transferrin to TR at 47C compared with the wild-type cells after experimental procedures described below. Iron was removed from transferrin as described previously16 and labeled with 59Fe, using citrate as an iron donor to give a specific activity of approximately 2,000 cpm/ng transferrin-iron. Diferric transferrin was labeled with 125I using the Enzymobead method following the manufacturer’s instructions (Bio-Rad, Hercules, CA). The labeled protein was then passed through 10-DG desalting column (Bio-Rad, CA) to remove unbound radioactivity. Specific activity was ap-
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proximately 80 to 100 cpm/ng transferrin with more than 99% 125I protein-bound. HuH7 wild-type and transferrin receptor sense and antisense cells were plated at a density of 2 1 105 cells per 35mm six-well plate and grown for 3 days in MEM/10% FBS with horse spleen ferritin until they reach approximately 80% confluence. To remove endogenous transferrin and FBS, the cells were preincubated two times for 15 minutes at 377C with MEM. Cells were then incubated with MEM containing 2% bovine serum albumin and 125I-transferrin or 59Fe-transferrin. At the end of the incubation, the cells were washed five times with Hank’s buffer (pH 7.4) at 47C and solubilized for gamma counting using 0.1% Triton/0.1 N NaOH. Protein concentration was determined by the method of Bradford17 with bovine serum albumin as a standard. Protein content was 276 { 3 (n Å 5) pg/cell for wild-type cells, 240 { 11 (n Å 3) pg/cell for TR sense cells, and 204 { 12 (n Å 3) pg/cell for TR antisense cells. Nonheme iron levels were measured by growing wildtype, TR sense, and antisense cells in MEM/10% FBS containing 1 mg/mL horse spleen ferritin. The cells (approximately 10 1 106) were washed five times with Hank’s buffer (pH 7.4) and then removed from the cell culture flasks using 0.05% trypsin and 0.53 mmol/L EDTA as described. The cells were resuspended in 2.8 mol/L nitric acid, and the nonheme iron levels were determined as described previously18 using a Varian Spectra AA-640Z atomic absorption spectrophotometer equipped with a graphite furnace atomizer. Intracellular and membrane-bound radioactivity were determined using the enzyme Pronase as described previously.5 The washed cells were incubated for 30 minutes with 1 mg/ mL pronase at 47C and centrifuged at 12,000g for 30 seconds. The cell pellet contained the intracellular radioactivity, and the supernatant contained the membrane-bound radioactivity. NTR-mediated uptake of transferrin and iron was measured in the presence of a 100-fold excess of unlabeled human transferrin. TR-mediated uptake of transferrin and iron was calculated as the difference between uptake measured in the absence and presence of 100-fold excess of unlabeled human transferrin. Cells were preincubated with the weak bases chloroquine, NH4Cl, and methylamine and the metabolic inhibitors KCN and NaN3 for 15 minutes at 377C before the addition of 60 nmol/L or 1.25 mmol/L 59Fe-transferrin for a further 60 minutes at 377C, and then cells were treated as described. To determine the amount of 59Fe incorporated into ferritin and heme, the cells were incubated with 59Fe-transferrin for 2 hours at 377C or 47C and washed five times with Hank’s buffer (pH 7.4). The cells were then solubilized with 1 mL of lysis buffer containing 1% Triton/1% deoxycholate and 25 mmol/L HEPES (pH 7.4) at 47C and centrifuged at 12,000g for 5 minutes to remove insoluble materials. The incorporation of 59 Fe into ferritin was measured by incubating the supernatant with rabbit anti-human serum ferritin antibody (Boehringer Mannheim) for 1 hour at 377C or overnight at 47C. The antibody-antigen complex was precipitated using protein-A– positive cells (Boehringer Mannheim), and the precipitate was washed three times with lysis buffer at 47C and counted for gamma radiation. To determine 59Fe incorporated into heme, the cells were incubated with 59Fe-transferrin and solubilized as described. The cell supernatant was acidified with 1N HCl to pH 2, and the heme was extracted with an equal volume of ice-cold cyclohexanone.19
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TABLE 1. Binding Parameters for Transferrin With HuH7 Cells at 47C Cell Type
Number of TransferrinBinding Sites per Cell
Association Constant M01 (11008)
Wild-type Sense Antisense
210,000 187,500 98,500
2.0 1.0 1.6
NOTE. The results are the mean values obtained from two separate experiments.
Cellular degradation of transferrin was determined by measuring the amount of protein-free 125I inside the cells or released to the incubation media by trichloroacetic acid precipitation. One milliliter of cell suspension or media containing 2% bovine serum albumin as a protein carrier was incubated with trichloroacetic acid to give a final concentration of 10% for 15 minutes at 47C. The samples were centrifuged, and the pellet and supernatant were counted for gamma radiation. Unless otherwise stated, the results are the means of duplicate or triplicate determinations made within each experiment, and the SEM has been included where appropriate. Results were analyzed by one-way ANOVA using a Fisher post hoc comparison test as appropriate. Significant differences were obtained when P õ .05. All experiments were performed at least twice, and the data presented are representative of a typical experiment. RESULTS Transferrin Binding by HuH7 Cells. For permanent inhibition of TR expression, human hepatoma cells HuH7 were transfected with TR antisense expression vector pRc/CMV/TR (sense vector used as control). Stable clones were selected by neomycin resistance and screened for reduced TR expression by measuring 125Itransferrin binding to TR at 47C. Six stable hepatoma cell clones that had a 50% to 60% reduction in transferrin binding were obtained. The TR antisense clone AS/C9 was selected for further studies. The number of transferrin binding sites per cell expressed by HuH7 wild-type cells and the TR sense and antisense cell clones was determined by Scatchard analysis of 125I-transferrin binding at 47C (Table 1). The level of expression of transferrin binding sites by the TR antisense cells was reduced to 47% when compared with the wild-type, whereas the TR sense S/H2 expressed 89% of the control level. Furthermore, if the number of TR was expressed per gram of protein, there was 7.6 1 1014 TR/g protein for the wild-type cells, 7.8 1 1014 TR/g protein for the sense cells, and 4.8 1 1014 TR/g protein for the antisense cells. Therefore, the number of TR/g protein expressed by the antisense cells was reduced to approximately 60% compared with the wild-type and sense cells. The TR on all cell types were saturated at an extracellular transferrin concentration of 60 nmol/L. Transferrin and Iron Uptake With Time. The uptake of 59Fe from transferrin by the wild-type, sense, and
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antisense cells at 377C increased with time. The rate of iron uptake by AS/C9 antisense cells was reduced to approximately 45% of the wild-type cells, and uptake by S/H2 sense cells was approximately 90% of the control (Fig. 1A). The rate of accumulation of transferrin at 377C was initially rapid, but decreased markedly after 15 minutes until a steady-state level was reached after 60 minutes’ incubation (Fig. 1B). The initial rate of transferrin uptake over the first 5 minutes by the antisense cells was 40% of the wild-type cells, with values of 12,000 molecules/cell/min and 30,000 molecules/cell/min, respectively. The steady-state level of cell-bound transferrin in the antisense cells was 44% of the wild-type cells (Fig. 1B). There was a progressive increase in the iron to transferrin molar ratio with time, reaching 12.6 after 120 minutes for the wild-type cells and 12.8 for the antisense cells. The cellular nonheme iron level was 20.1 { 0.7 ng Fe/106 cells (73 { 2 mg Fe/g protein, n Å 3) for the wildtype cells, 18.2 { 0.6 ng Fe/106 cells (76 { 2 mg Fe/g protein, n Å 3) and 17.6 { 1.4 ng Fe/106 cells (86 { 7 mg Fe/g protein, n Å 3) for the antisense cells. There was no significant difference in the iron concentration, expressed either per cell or per gram of protein, between the three cell types (P ú .05). Two Mechanisms of Transferrin Uptake. To determine if the wild-type and antisense cells could acquire iron by NTR-mediated as well as by TR-mediated processes, the cells were incubated with transferrin in the concentration range 2.5 to 120 nmol/L at 377C. The total transferrin taken up by the cells at 377C reflects binding to the cell surface and internalization of transferrin into the cell. Cell-associated transferrin increased rapidly as the transferrin concentration was initially increased, but leveled off as the TR saturated at a concentration of 60 nmol/L (Fig. 2A). Transferrin uptake reached a maximum level of 5.7 1 105 molecules/cell by the wild-type cells and 2.7 1 105 molecules/ cell by the antisense cells. Using transferrin uptake at 377C as an indication of the total number of binding sites and binding at 47C as a measure of cell surface binding sites, approximately 35% of the receptors were found on the cell surface. The distribution of TR was similar for both cell types. In a second experiment, the extracellular transferrin concentration was increased to approximately 2 mmol/ L. Plots of the cell-associated transferrin that were initially curvilinear became linear, suggesting the presence of a second nonsaturable process of transferrin uptake (Fig. 2B). When the uptake of 125I-transferrin was measured in the presence of a 100-fold molar excess of unlabeled transferrin at each concentration point, it increased at a constant rate of approximately 330 molecules/cell/nmol/L transferrin. This rate was similar to the rate of increase of total transferrin uptake when the concentration was increased above 150 nmol/L for both the wild-type (360 molecules/cell/nmol/ L transferrin) and antisense cells (345 molecules/cell/ nmol/L transferrin). In the presence of unlabeled trans-
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FIG. 1. (A) Uptake of iron from diferric transferrin at 377C by HuH7 wild-type cells (●), sense cells (s), and antisense cells (m). Transferrin concentration was 60 nmol/L. The results are the means of duplicate determinations. (B) Transferrin uptake by HuH7 wildtype cells (●) and antisense cells (m) at 377C. Transferrin concentration was 60 nmol/L. The results are the means of duplicate measurements.
FIG. 2. Effect of transferrin concentration on transferrin uptake by wild-type and antisense cells at 377C. Total uptake 125I-transferrin uptake by wild (●) and antisense cells (m) and NTR uptake by wild (s) and antisense cells (n). The results are means of duplicate determinations.
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FIG. 3. Effect of transferrin concentration on iron accumulation from diferric transferrin by wild and antisense cells at 377C. Total iron uptake by wild (●) and antisense cells (m). The results are means of duplicate determinations.
ferrin, the amount of cell-associated transferrin was similar for the wild and antisense cells. These results indicate that the nonsaturating process does not involve high-affinity TR and, at 1.25 mmol/L, it accounted for approximately 40% and 60% of total transferrin accumulation by the wild-type and antisense cells, respectively. Two Processes of Iron Uptake. Iron uptake from diferric transferrin was measured over a transferrin concentration range of 0.025 to 2.5 mmol/L. The amount of iron taken up by the wild and antisense cells increased rapidly at low diferric transferrin concentrations, but the rate of increase declined with transferrin concentrations above 0.1 mmol/L (Fig. 3A) consistent with a TR-mediated pathway of iron uptake. At higher transferrin concentrations, the uptake of iron continued to increase, suggesting the presence of an additional iron uptake process (Fig. 3B). When the uptake of 59Fetransferrin was measured in the presence of a 100-fold molar excess of unlabeled transferrin at each concentration point, iron uptake was greatly reduced, and at a transferrin concentration of 1.25 mmol/L, iron uptake by the wild and antisense cells was only 10% to 15% of that observed in the absence of unlabeled transferrin-iron. The maximum rate of iron uptake (Vmax ) and equilibrium constant (Km) were calculated using nonlinear least squares fitting of the iron uptake data to a Michaelis Menten–type equation. The best fit was obtained when two saturable processes of iron uptake by the wild and antisense cells were assumed.
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Vi Å
V1m 1 [F] V2m 1 [F] / Km1 / [F] Km2 / [F]
where Vi is the velocity of iron uptake at transferrin concentration [F], V1m and V2m are the maximal rates for the first and second processes of iron uptake, and Km1 Km2 are the equilibrium constants for the two processes. For the first process of iron uptake, Vmax for antisense cells (Vmax 40,000 atoms/cell/min or 1.9 1 1014 atoms/g protein/min; Km 22 nmol/L) was 50% to 65% of the wild-type cells (Vmax 79,000 atoms/cell/min or 2.9 1 1014 atoms/g protein/min; Km 13 nmol/L), depending on whether the results were expressed per cell or per gram of protein. These results are consistent with a TR-mediated process. For the second process, the Vmax for the antisense cells was (Vmax 67,000 atoms/cell/min or 3.2 1 1014 atoms/g protein/min; Km 2.0 mmol/L) was approximately 90% to 115% of the wild-type cells (Vmax 77,000 atoms/cell/min or 2.8 1 1014 atoms/g protein/ min; Km was 1.5 mmol/L). This suggests that a similar low-affinity mechanism was involved in both cell types. In Fig. 4, an Eadie-Hofstee plot20 of iron uptake illustrates that there were two components of iron uptake for both the wild-type and the antisense cells. Internalization of Transferrin and Iron. A series of experiments was performed to compare iron uptake by the TR- and NTR-mediated processes. The antisense cells were incubated with a low concentration of transferrin (60 nmol/L) where the uptake of transferrin and iron occurs by the TR-mediated process and at a higher transferrin concentration (1.25 mmol/L) where uptake
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FIG. 4. Data shown in Fig. 3 are presented as an Eadie Hofstee plot of iron uptake by wild (●) and antisense cells (m).
occurs predominantly by the NTR-mediated process (Fig. 2B). The difference in transferrin and iron uptake at these two concentrations was used as a measure of uptake by NTR-mediated process. Iron uptake at 377C is shown in Fig. 5A. The rate of total, membrane-bound,
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and intracellular uptake with time increased approximately twofold on increasing the transferrin concentration from 60 nmol/L to 1.25 mmol/L. Iron uptake at 47C, which reflects iron bound to the cell membrane, also increased approximately twofold and accounted for 12% of total uptake at both transferrin concentrations. After 2 hours’ incubation with 60 nmol/L and 1.25 mmol/L 59Fe-transferrin at 377C, 43% and 41% of the iron was incorporated into ferritin, respectively (Table 2) and less than 5% into heme. When the incubation temperature was reduced to 47C, only 5% to 10% of iron taken up by the cells at both transferrin concentrations was incorporated into ferritin. Transferrin uptake from 60 nmol/L and 1.25 mmol/L diferric transferrin is shown in Fig. 5B. Transferrin uptake at both transferrin concentrations reached a steady-state level after 60 minutes’ incubation with 50% and 42% of the transferrin internalized at 60 nmol/ L and 1.25 mmol/L, respectively. The steady-state level of total, intracellular, and membrane-bound associated transferrin increased approximately two to three times when the transferrin concentration was raised from 60 nmol/L to 1.25 mmol/L. There was a net accumulation of iron by the cell, and after 120 minutes’ incubation, the intracellular iron-to-transferrin molar ratio reached approximately 20 at both transferrin concentrations. More than 90% of 125I-transferrin taken up by the cells was precipitated by 10% trichloroacetic acid, indicating that there was little catabolism of transferrin by the cells. These findings suggest that the
FIG. 5. Iron (A) and transferrin (B) uptake by antisense cells. The cells were incubated with 59Fe-transferrin or 125I-transferrin for 0 to 120 minutes and then reincubated with 1 mg/mL Pronase for 30 minutes at 47C to separate surface-bound and intracellular iron. At a transferrin concentration of 60 nmol/L, total (s), intracellular (●) uptake and 1.25 mmol/L total (n) and intracellular (m) uptake. Fig. 5A results are expressed as mean { SEM, where n Å 3, and Fig. 5B results are means of duplicate estimations.
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TABLE 2. Effects of Inhibitors on Iron Uptake by HuH7 Cells Wild-Type Inhibitor
Chloroquine (200 mmol/L) NH4Cl (20 mmol/L) Methylamine (30 mmol/L) KCN (1 mmol/L) NaN3 (1 mmol/L) Ferritin
Antisense
0.06 mmol/L (% control)
1.25 mmol/L (% control)
36 (3) 41 (3) 50 (3)
31 (3) 32 (3) 34 (3)
45 (3)
39 (3)
0.06 mmol/L (% control)
32 32 33 75 87 43
(3) (6) (3) (3) (3) (3)
1.25 mmol/L (% control)
28 28 30 80 104 41
(3) (6) (3) (3) (3) (3)
59
NOTE. The cells were incubated with Fe-transferrin (0.06 mmol/L and 1.25 mmol/L) in the presence and absence of inhibitors for 60 minutes. Iron uptake (with inhibitor) is expressed as percentage of control value (without inhibitor). Ferritin was measured by immunoprecipitation as described in Materials and Methods. The amount of iron incorporated into ferritin is expressed as a percentage of total iron uptake. The number of estimations are shown in parentheses.
NTR-mediated process, like the TR-mediated process, involved the endocytosis and recycling of transferrin. The weak bases chloroquine, NH4Cl, and methylamine inhibited the uptake of iron by the antisense cells by approximately 70% at both transferrin concentrations. Uptake by the wild-type cells was also reduced, and at the higher transferrin concentration the effect was similar to the antisense cells; however, at the lower concentration, the percentage inhibition was slightly reduced. The metabolic inhibitor KCN inhibited iron uptake by approximately 20% at both transferrin concentrations, with NaN3 having a lesser effect (Table 2). DISCUSSION
HuH7 hepatoma cells acquired iron from transferrin by two processes. The first pathway involved high-affinity TR-mediated endocytosis of transferrin, and the second was a low-affinity process not involving TR receptors. The high-affinity process played an important role in the uptake of iron at low extracellular transferrin concentrations, and the low-affinity process became more predominant at higher transferrin concentrations. This study provided the first evidence of TR-independent uptake of transferrin-iron by hepatoma cells and indicated that the uptake of transferriniron by hepatoma cells entailed similar mechanisms to those found in hepatocytes.5,10 An antisense TR vector was introduced into the hepatoma cells to reduce TR expression by interfering with TR gene translation.14 This resulted in the decline of the number of cell surface TR and a reduction in the uptake of transferrin and iron uptake by the TR antisense cells. A reduction in the initial rate and steadystate levels of transferrin accumulation was consistent with the decline in the number of TR, demonstrating that the inhibition of iron uptake was completely attributable to the reduction in the level of receptor expression, and there was no alteration to the recycling time of transferrin through the cell. This was confirmed by similar iron-to-transferrin molar ratio for both the wild-type and antisense cells, indicating that the effi-
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ciency of iron accumulation was consistent for both cell types. There was no difference in the nonheme iron levels of the wild-type, TR sense, and antisense cells grown in the presence of horse spleen ferritin, suggesting that the alteration to TR expression was independent of the iron status of the cells. There was a difference in the protein content per cell for the wild-type cells and TR sense and antisense cell clones. However, it is unlikely that the differences in TR expression were caused by a generalized alteration in cellular protein synthesis because, irrespective of whether the results are expressed per cell or per gram of protein, there was a significant reduction in the number of TR and the rate of iron uptake by the antisense cells compared with the wild-type or sense cells. In a control experiment, wild-type cells were transfected with pRc/CMV vector only. Southern and Northern blot analysis showed that there was neither rearrangement or amplification of the endogenous TR gene or changes in the TR messenger RNA levels caused by transfection itself (Sasaki K et al, Unpublished observation). The number of TR and rate of iron uptake was similar for the wild-type and TR sense control cells (Table 1 and Fig. 1) with inhibition of TR expression specific to cells transfected with antisense TR. TR were not involved in the uptake of transferriniron by the second process because transfection of the HuH7 cells with the TR antisense vector had no effect on transferrin or iron uptake by this process. Transferrin uptake by both cell types was nonsaturable and not inhibited by excess of unlabeled transferrin, characteristics that are inconsistent with a receptor-mediated process. This is in agreement with findings from another study using Chinese hamster ovary cells, which possessed no detectable transferrin binding sites but were still able to accumulate iron by an NTR-mediated process.21 Similarly, an antibody to the TR directly inhibited the TR-mediated uptake of transferrin and iron by rat hepatocytes, but there was no effect on the second process.10 If transferrin uptake by the second process does not involve TR, then how is the iron delivered to the cell?
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The uptake of transferrin at 377C reflects total cellular accumulation of transferrin, and the use of Pronase to remove transferrin bound to the cell surface showed approximately 40% of the transferrin to be inside the cell. Uptake and internalization of transferrin by the NTR process reached a steady state and acid precipitation of the transferrin indicated that there was very little catabolism of the protein. Therefore, like the TRmediated process, transferrin taken up by NTR-mediated process was internalized into the cell, releasing its iron before being recycled to the extracellular medium. This was also corroborated by an intracellular iron-totransferrin molar ratio of approximately 20 for the NTR-mediated process (Fig. 5A and B). The possibility that fluid-phase endocytosis accounted for the NTR uptake must be considered. Uptake of extracellular macromolecules by fluid-phase endocytosis generally leads to targeting to the lysosomes, where they are degraded.22 In this study, however, we have observed transferrin being recycled rather than degraded. A computer model23 adapted to transferrin cell interactions24 indicated that fluid-phase endocytosis, involving the recycling of fluid from the endocytotic vesicles to the extracellular medium, would lead to the recycling of transferrin and the accumulation of iron sufficient to account for NTR uptake by hepatocytes. Although the recycling of transferrin by fluid-phase endocytosis may therefore play a role in the delivery of iron to the hepatoma cells, our results point to involvement of adsorptive endocytosis in the uptake of transferrin. Inhibition of transferrin and iron uptake by the proteolytic enzyme Pronase suggests that NTR-mediated mechanism entails low-affinity binding or adsorption of transferrin to plasma membrane proteins, nonsaturated at the transferrin concentrations used in this study, leading to the endocytosis and recycling of transferrin and the accumulation of iron by the cell. In the search for a transferrin membrane-binding component that may be involved in the NTR-mediated process, there is evidence that proteoglycans isolated from fibroblasts,25 liver endothelium cells,26 and whole liver27 can bind transferrin. Interaction of transferrin with proteoglycans appeared to be dependent on the pH and the iron content of the transferrin. Release of iron from transferrin was promoted particularly from the N-terminal lobe of the protein.27,28 The proteoglycan-transferrin–binding activity was also sensitive to treatment with trypsin,25 which is consistent with our observations that proteolytic enzymes can reduce transferrin and iron uptake by NTR process. However, there is no direct evidence of the involvement of proteoglycans in the cellular uptake of transferrin-iron. Interestingly, in contrast to transferrin accumulation, most of the iron taken up by the NTR- and TRmediated processes was saturated with increasing amounts of diferric transferrin and was inhibited by an excess of unlabeled diferric transferrin. The inhibition of TR-mediated iron uptake by unlabeled diferric transferrin was caused by the competition between the
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labeled and unlabeled transferrin for the TR-binding sites. However, this does not account for the inhibition of iron uptake by the NTR-mediated process, and it is likely that the labeled and unlabeled iron were competing at another stage in the iron uptake pathway that involved a second saturable iron carrier-mediated step. Similarly, it has been reported that melanoma cells acquire iron by two saturable mechanisms. The first process was TR mediated, and the second was TR independent. The Vmax and Km for iron uptake by the TRindependent pathway were similar to our results in hepatoma cells.29,30 Irrespective of the pathway of transferrin-iron entering into the cell, either by the TR or NTR process, weak bases, metabolic inhibitors, and reduced incubation temperature each reduced iron uptake. The low pH of the endocytotic vesicle has been demonstrated to be an important factor in the release of iron from transferrin by the TR process.31 The weak bases increase the pH of the transferrin-iron containing endosomes inhibiting iron release.32,33 The results obtained in this study demonstrate that the uptake of iron by the NTR process, like the TR process, entails an energy-dependent step with the pH-dependent release of iron from transferrin. Once the iron is released from transferrin, it is transferred into the cytosol and incorporated into ferritin for storage or heme synthesis. In the hepatoma cells, intracellular incorporation of iron into ferritin and heme was similar for iron taken up from transferrin by both processes, indicating that the intracellular pathways taken by iron were the same. The exact mechanism involved in the transport of iron across the vesicle membrane remains unclear. Numerous studies have now been done using rat liver,34 erythroid, and nonerythroid cells,35-37 demonstrating the presence of nontransferrin bound iron transport systems for conveying iron complexes across membrane barriers by carrier-mediated processes. It is therefore possible that the second rate-limiting step in the uptake of iron by the HuH7 cells entails the binding of iron to a carrier that transports iron across the endosome membrane to the cytosol for heme synthesis or incorporation into storage ferritin. Acknowledgment: The authors thank Professor Evan Morgan for his comments on the manuscript. REFERENCES 1. Young SP, Aisen P. The interaction of transferrin with isolated hepatocytes. Biochim Biophys Acta 1980;633:145-153. 2. Rudolph JR, Regoeczi E. Interaction of rat asialotransferrin with adult rat hepatocyte: its relevance for iron uptake and protein degradation. J Cell Physiol 1988;135:539-544. 3. Sibille J-C, Kondo H, Aisen P. Interactions between isolated hepatocytes and kupffer cells in iron metabolism: a possible role for ferritin as an iron carrier protein. HEPATOLOGY 1988;8:296301. 4. Osterloh K, Aisen P. Pathways in the binding and uptake of ferritin by hepatocytes. Biochim Biophys Acta 1989;1011:40-45. 5. Trinder D, Morgan E, Baker E. The mechanism of iron uptake by fetal rat hepatocytes in culture. HEPATOLOGY 1986;6:852-858.
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6. Iacopetta B, Morgan EH. The kinetics of transferrin endocytosis and iron uptake from transferrin in rabbit reticulocytes. J Biol Chem 1983;258:9108-9115. 7. Morgan EH. Transferrin, biochemistry, physiology and clinical significance. Mol Aspects Med 1981;4:1-123. 8. Cole ES, Glass J. Transferrin binding and iron uptake in mouse hepatocytes. Biochim Biophys Acta 1983;762:102-110. 9. Page M, Baker E, Morgan EH. Transferrin and iron uptake by rat hepatocytes in culture. Am J Physiol 1984;246:G26-G33. 10. 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;943:440-446. 11. Morgan EH. Specificity of hepatic iron uptake from plasma transferrin in the rat. Comp Biochem Physiol 1991;99A:91-95. 12. 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. 13. Ku`hn LC, McCelland A, Ruddle FH. Gene transfer, expression, and molecular cloning of the human transferrin receptor gene. Cell 1984;37:95-103. 14. 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. 15. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning, A Laboratory Manual. Ed 2. New York: Cold Spring Habor Press, 1989. 16. Zak O, Aisen P. Evidence for functional differences between the two sites of rabbit transferrin: effects of serum and carbon dioxide. Biochim Biophys Acta 1990;1052:24-28. 17. Bradford M. A rapid method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-254. 18. Trinder D, Batey RG, Morgan EH, Baker E. Effect of cellular iron concentration on iron uptake by hepatocytes. Am J Physiol 1990;259:G611-G617. 19. Teale FWJ. Cleavage of the haem-protein link by acid methylethylketone. Biochim Biophys Acta 1959;35:543. 20. Hoftsee BHJ. Non-inverted versus inverted plots in enzyme kinetics. Nature 1959;184:1296-1298. 21. 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. 22. Steinman RM, Mellman IS, Muller WA, Cohn ZA. Endocytosis and the recycling of plasma membrane. J Cell Biol 1983;96:127.
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23. Blomhoff R, Nenseter MS, Green MH, Berg T. A multicompartmental model of fluid-phase endocytosis in rabbit liver parenchymal cells. Biochem J 1989;262:605-610. 24. Bakoy OE, Thorstensen K. The process of cellular uptake of iron from transferrin: a computer simulation program. Eur J Biochem 1994;222:105-112. 25. Fransson L-A, Carlstedt I, Costar L, Malmstrom A. Binding of transferrin to the core protein of fibroblast proteoheparan sulfate. Proc Natl Acad Sci 1984;81:5657-5661. 26. Omoto E, Minguell JJ, Tavassoli MJ. Proteoglycan synthesis by cultured liver endothelium: the role of membrane-associated heparan sulfate in transferrin binding. Exp Cell Res 1990;187: 85-89. 27. Hu W-L, Regoeczi E. Hepatic heparan proteoglycan and the recycling of transferrin. Biochem Cell Biol 1992;70:535-538. 28. Regoeczi E, Chindemi PA, Hu W-L. Interaction of transferrin and its iron-binding fragments with heparin. Biochem J 1994; 299:819-823. 29. Richardson DR, Baker E. The uptake of iron and transferrin by the human malignant melanoma cell. Biochim Biophys Acta 1990;1053:1-12. 30. Richardson DR, Baker E. Two saturable mechanisms of iron uptake from transferrin in human melanoma cells: the effects of transferrin concentration, chelators, and metabolic probes on transferrin and iron uptake. J Cell Physiol 1994;161:160-168. 31. Klausner RD, Ashwell G, van Renswoude J, Hardford JB, Bridges KR. Binding of apotransferrin to K562 cells: explanation of transferrin cycle. Proc Natl Acad Sci 1983;80:2263-2266. 32. Morgan EH. Inhibition of reticulocyte iron uptake by NH4Cl and CH3NH2 . Biochim Biophys Acta 1981;642:119-134. 33. Ciechanover A, Schwartz AL, Dautry-Varsat A, Lodish HF. Kinetics of internalisation and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. J Biol Chem 1983;258:9681-9689. 34. Wright TL, Brissot P, Ma W-L, Weisiger RA. Characterization of non-transferrin-bound iron clearance by rat liver. J Biol Chem 1986;261:10909-10914. 35. Morgan EH. Membrane transport of non-transferrin-bound iron by reticulocytes. Biochim Biophys Acta 1988;943:428-439. 36. Sturrock A, Alexander J, Lamb J, Craven CM, Kaplan J. Characterization of a transferrin-independent uptake system for iron in Hela cells. J Biol Chem 1990;265:3139-3145. 37. Nunez MT, Escobar A, Ahumada A, Gonzalez-Sepulveda M. Sealed reticulocyte ghosts: an experimental model for the study of Fe2/ transport. J Biol Chem 1992;267:11490-11494.
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