Department of Physiology, University of Western Australia, Nedlands 6907, Western Australia, Australia
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ABSTRACT |
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The mechanisms of uptake of
non-transferrin-bound iron by human hepatoma cells (HuH7) were
investigated using 59Fe-citrate
and [14C]citrate. The
amount of iron associated with the cells increased linearly with time,
whereas citrate uptake reached a plateau after 45 min, resulting in a
cellular accumulation of iron over citrate. The cells displayed
high-affinity membrane binding sites for citrate with maximum binding
of 118 ± 17 pmol citrate/mg protein and a dissociation constant of
21 ± 2 µM (n = 3). Iron uptake
was saturable with a maximum uptake rate of 1.95 ± 0.43 pmol · mg
protein1 · min
1
and an apparent Michaelis constant of 1.1 ± 0.1 µM.
Nonradioactive ferric citrate and citrate inhibited
59Fe uptake to a similar degree.
This suggests that the uptake of citrate-bound iron is dependent on
either binding to specific citrate binding sites or the concentration
of unbound iron. The uptake of iron was inhibited by ferricyanide
(>100 µM) and ferrous iron chelators but stimulated by ferrocyanide
and ascorbate, suggesting that the iron is reduced from
Fe3+ to
Fe2+ and transported into the cell
by an iron carrier-mediated step.
non-transferrin-bound iron; iron overload; iron carrier; citrate binding sites
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INTRODUCTION |
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UNDER NORMAL CONDITIONS the liver acquires iron from plasma transferrin. In iron overload, plasma transferrin becomes saturated with iron, and the amount of excess iron or non-transferrin-bound iron (NTB-Fe) in the plasma increases (4, 11). Most of the NTB-Fe found in the plasma is chelated by citrate (10). Both transferrin-bound iron (Tf-Fe) and NTB-Fe are cleared by the liver (6, 7), and iron from both sources is likely to contribute to increased hepatic iron uptake in iron overload.
The uptake of Tf-Fe by hepatic cells is mediated by transferrin receptor (TR) and transferrin receptor-independent (NTR) pathways (26, 29, 30). The first of these processes involves a limited number of TRs, which are saturated at a low extracellular concentration of Tf-Fe (18, 33). Transferrin and its receptor are internalized into endocytotic vesicles, where the iron is released from transferrin in the low pH environment of the vesicle (2). The rate of iron uptake by this process is tightly controlled by intracellular iron levels via a posttranscriptional regulation of TR expression. Reduced intracellular iron levels increase the binding activity of the cytosolic iron regulatory protein to the iron-responsive element in the 3' untranslated region of TR mRNA. This stabilizes the mRNA, enhancing the rate of translation and the expression of receptor protein, and leads to increased iron uptake by the cell (13).
In vitro studies have shown that Tf-Fe taken up by the NTR pathways provides the main source of iron for hepatic cells (30). Using the human hepatoma cell line HuH7, we have shown that the uptake of Tf-Fe occurs by two NTR processes, both of which are dependent on an iron carrier-mediated step (27, 30). The first of these processes involves the internalization of transferrin. Initially, transferrin binds with low affinity to the cell surface and, similar to the TR-mediated process, the transferrin and iron are endocytosed into the cell where the iron is released from transferrin at an intracellular acidic site and the transferrin is recycled back to the cell surface (30). It is likely that an iron carrier-mediated step transfers the iron from the endocytic vesicle to the cell cytosol for utilization by the cell.
The second of the NTR-mediated processes involves the cell surface release of iron from transferrin and the subsequent transport of iron into the cell by an iron carrier. In a previous study we found that NTB-Fe inhibited the uptake of Tf-Fe released from the carrier protein at the cell surface, indicating that the uptake of iron from both sources shares a common iron-carrier mediated pathway into the cell (30). Little is known about the mechanisms involved in the uptake of Tf-Fe by this cell surface iron carrier. In this study we investigated the mechanism of uptake of NTB-Fe, which shares the iron carrier with Tf-Fe, to gain further information about the role of iron carriers in the uptake of iron by hepatic cells.
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MATERIALS AND METHODS |
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Materials. Radiochemicals iodine-125, iron-59, and carbon-14 labeled citric acid were purchased from Dupont NEN Research Products (Sydney, Australia). BSA were obtained from Sigma Chemicals (St. Louis, MO), and Pronase came from Boehringer (Sydney, Australia). Minimal essential medium (MEM), fetal bovine serum, and geneticin came from GIBCO BRL (Auckland, New Zealand). The antibiotics penicillin and streptomycin were obtained from Commonwealth Serum Laboratories (Melbourne, Australia) and Fungizone from Cytosystems (Sydney, Australia).
Cells. The human hepatoma cell line HuH7 (20) was grown in monolayer culture in MEM containing 10% fetal bovine serum, 100 µg/ml streptomycin, 100 U/ml penicillin, and 0.25 µg/ml Fungizone as described previously (30). HuH7 cells were transfected with TR antisense and sense (for control) RNA expression vectors as described previously (30) to produce a cell clone with a 50% reduction in the level of TR protein expression (TR antisense cells) compared with the control cells (TR sense cells). The vectors were constructed using a 2.4-kb Eco-RV-Xba1 fragment of the human TR cDNA pcDTR-1 (14) inserted in the antisense or sense (control) orientation into the multiple cloning site of the expression vector pRc/CMV (Invitrogen, San Diego, CA) by the method of Sasaki et al. (20).
Ferric citrate solution.
Stock solutions of 59Fe-labeled
ferric citrate (×100; usually 100 µM Fe, 10 mM citrate) were
prepared by mixing
59FeCl3
and unlabeled FeCl3 (in 0.1 M HCl)
with 100-fold molar excess of unlabeled sodium citrate for ~15 min
before the start of the experiment. The ferric citrate solution was
diluted 1:100 with MEM containing 2% BSA to give a final pH of 7.4 and
specific activity of 20-30
counts · min1 · pmol
1
Fe. Similarly, a stock solution (×100) of
14C-labeled ferric citrate was
prepared by mixing
[14C]citric acid and
unlabeled sodium citrate with
56FeCl3
(in 0.1 M HCl) and was diluted with MEM-2% BSA to give a final pH of
7.4 and specific activity of ~30 dpm/pmol citrate. Unless stated
otherwise, the final Fe and citrate concentrations in the incubation
medium were 1 and 100 µM, respectively.
Experimental procedures. HuH7 cells were grown in 35-mm cell culture dishes for 3 days until they reached ~80% confluence. Before the commencement of the uptake experiments, the cells were washed twice with MEM at 37°C to remove the cell growth medium. In most experiments the cells were then incubated with 59Fe-citrate or [14C]citrate (with and without Fe) for up to 120 min at 37°C. At the end of this incubation period, the cells were washed five times with Hanks' buffer, pH 7.4 at 4°C, and then were incubated with the proteolytic enzyme Pronase (1 mg/ml) for a further 30 min at 4°C to measure the cell membrane-bound and intracellular uptake of iron and citrate (28). The cell suspension was centrifuged at 12,000 g for 30 s, and intracellular radioactivity remained in the cell pellet and membrane-bound radioactivity was released into the cell supernatant. The cell samples containing 59Fe radioactivity were solubilized in 0.1% Triton X-100-0.1 M NaOH. Cell samples containing [14C]citrate were solubilized with 1 M NaOH and then neutralized using 1 N HCl before the addition of 5 ml scintillant (Ready Safe, Beckman, Fullerton, CA). The cell samples and the cell supernatant were counted for 59Fe in a LKB-1282 Compugamma counter (Pharmacia, Uppsala, Sweden) and 14C in a Beckman LS 6500 Liquid Scintillation Counter (Fullerton, CA).
Data analysis. In this study the term uptake refers to the amount of iron and citrate associated with the cells, whereas the uptake rate refers to the rate of accumulation of iron by the cells. 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 means ± SE. Each experiment was performed at least three times. A Student's t-test was performed to determine if the difference between sample values was statistically significant (P < 0.05).
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RESULTS |
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Iron and citrate uptake. HuH7 human hepatoma cells were incubated with 59Fe-citrate for up to 90 min. At 37°C intracellular and membrane-associated 59Fe-citrate increased with time. Approximately, 70% of the iron taken up by the cells was internalized after 90 min (Fig. 1A). The uptake of 59Fe-citrate was temperature dependent, and at 4°C nearly all the iron taken up by the cells was associated with cell membrane (Fig. 1B). At this temperature, the intracellular and membrane-associated iron were ~5 and 60%, respectively, of that observed at 37°C.
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Effects of iron and citrate concentration.
The uptake of iron by the HuH7 cells from increasing extracellular
concentrations of 59Fe-citrate was
saturable (Fig. 3). There was no
significant difference in the iron uptake by TR sense cells and TR
antisense cells, which have a 50% reduction in TR expression compared
with TR sense cells (30). The maximum iron uptake rate
(Vmax) and the
apparent Michaelis constant
(Km) for Fe
uptake by the TR sense and TR antisense cells were calculated using a
Michaelis-Menten type equation.
Vmax and Km for the TR
sense cells were 1.95 ± 0.43 pmol · mg
protein1 · min
1 and 1.1 ± 0.1 µM
(n = 3), respectively. These values
were not significantly different from
Vmax and
Km values for the
TR antisense cells, which were 2.25 ± 0.47 pmol · mg
protein
1 · min
1
and 1.0 ± 0.1 µM (n = 3),
respectively. Both intracellular and membrane uptake of iron were
saturable with ~60% of the iron internalized after 60 min of
incubation (Fig. 3B).
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Effect of unlabeled ferric citrate and iron-free citrate. The effects of unlabeled citrate and ferric citrate on the uptake of 59Fe-citrate and ferric [14C]citrate were measured by incubating the hepatoma cells with 59Fe-citrate (1 µM Fe, 100 µM citrate) in the absence and presence of increasing concentration of unlabeled citrate or ferric citrate. Both of these compounds reduced the intracellular and membrane-associated 59Fe to a similar degree with net uptake of 59Fe almost completely inhibited in the presence of 5 mM unlabeled ferric citrate or citrate only (Fig. 5, A and B). In a second series of experiments, the cells were incubated with ferric [14C]citrate in the presence of increasing concentrations of unlabeled ferric citrate or citrate only. Net uptake of ferric [14C]citrate was inhibited by both unlabeled ferric citrate and citrate to a similar degree. The intracellular and membrane-associated ferric [14C]citrate were reduced to ~50-60% of the control values when the ferric citrate or citrate concentration was 10 mM (Fig. 5, C and D).
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Effects of ferricyanide, ferrocyanide, and ascorbate. Low concentrations of potassium ferricyanide (2-20 µM) stimulated intracellular and membrane uptake of iron by about 40%. At higher concentrations (100-500 µM) intracellular uptake was almost completely inhibited, whereas membrane uptake was reduced by 20% of the control value (Fig. 6, A and B). Potassium ferrocyanide at concentrations <10 µM had little effect on intracellular and membrane-associated iron levels, but higher concentrations (100-500 µM) increased intracellular and membrane uptake to twice the control value. The reducing agent ascorbate enhanced iron net uptake markedly. The degree of stimulation increased with increasing ascorbate concentration, with cell-associated iron reaching ~600-700% of the control value at ascorbate concentrations >100 µM. Ascorbate stimulated both the intracellular and membrane uptake of iron, but the effect on intracellular uptake was far greater than on membrane-bound uptake. In the presence of 100 µM ascorbate, the intracellular uptake of iron increased 500%, whereas the membrane-bound uptake increased 300% when compared with the control value. Ferricyanide, ferrocyanide, and ascorbate had little effect on intracellular and membrane uptake of citrate (Fig. 6, C and D).
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Iron chelators. Iron chelators bathophenanthroline disulfonic acid (BPS) and 2,2'-bipyridine inhibited intracellular and membrane iron uptake in a concentration-dependent manner. At a chelator concentration of 500 µM, intracellular and membrane uptake of iron uptake was reduced by BPS and 2,2'-bipyridine to 25 and 15% of the control value, respectively (Fig. 7A). At this concentration BPS had little effect on membrane citrate uptake but there was a 20% decrease in intracellular levels compared with the control value. 2,2'-Bipyridine inhibited intracellular and membrane-associated citrate levels by ~25% at a chelator concentration of 500 µM (Fig. 7B). Other iron chelators Desferal, pyridoxal isonicotinoyl hydrazone, and diethylene triaminepentaacetic acid (250 µM) completely inhibited the uptake of iron (data not shown).
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DISCUSSION |
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In this study we have shown that human hepatoma cells (HuH7) display high-affinity and low-affinity or "nonspecific" cell membrane binding sites for citrate. Citrate binding by the high-affinity citrate binding sites was saturable with increasing concentrations of extracellular citrate and the binding of radiolabeled citrate was inhibited by an excess of unlabeled citrate. The affinity of citrate for its binding site was not dependent on the presence of iron (Figs. 4 and 5).
The amount of citrate bound to the cell surface binding sites reached a steady-state level after 45 min of incubation, and citrate was internalized into the cell by a temperature-dependent process with intracellular levels reaching steady-state levels after 60 min at 37°C. A steady state is reached when the rate of citrate entering the cell is equal to the rate of citrate leaving the cell. Citrate may leave the cell by efflux or it may be catabolized by the cells. A recent study from our laboratory (9) has shown that rat hepatocytes in primary culture also express citrate binding sites that mediate the uptake of citrate. In this study the internalized citrate was shown to be metabolized by the cells and released as CO2. This finding suggests that citrate provides a source of energy for the cells.
Iron uptake from ferric citrate by HuH7 cells was saturable, which suggests that it is mediated by an iron carrier (Fig. 3). These findings are in agreement with another study from our laboratory using primary rat hepatocytes in culture and the physiological chelator citrate, which demonstrated that NTB-Fe uptake is mediated by a membrane carrier for iron (1). Other investigators using nonphysiological chelators such as tricine (32), nitrilotriacetic acid (3, 19), and diethylene triaminepentaacetic acid (21) have obtained similar results. For two reasons it is unlikely that the uptake of ferric citrate involves TR. First, the Vmax for iron uptake from citrate by HuH7 cells transfected with TR antisense RNA expression vector, which suppressed receptor expression by ~50%, was similar to the control cells (Fig. 3A). Second, the Vmax for iron uptake from citrate by the HuH7 cells (Fig. 3A) was much greater than the Vmax that was previously found for Tf-Fe uptake by the TR-mediated pathway (30).
The uptake of iron from citrate was inhibited to a similar degree by unlabeled ferric citrate or citrate only (Fig. 5). The inhibition of radioactive iron uptake by nonradioactive ferric citrate was probably due to the reduction in specific activity of iron. However, citrate alone reduced both citrate and iron uptake. These findings can be explained two ways. First, the uptake of iron and citrate is coupled and ferric citrate binds to the citrate binding sites before delivering its iron to the cells. The second explanation is that citrate chelates most of the iron and a small amount of iron remains unbound and is transported into the cells by the iron carriers. When the concentration of chelator is increased the amount of unbound iron available for cellular transport is reduced.
Ascorbate and potassium ferrocyanide, which can reduce Fe3+ to Fe2+, enhanced membrane and intracellular uptake of iron without affecting the uptake of citrate, whereas the oxidizing reagent potassium ferricyanide at concentrations above 100 µM inhibited the membrane and intracellular uptake of iron but did not affect citrate uptake. In addition, both the membrane impermeable ferrous iron chelator BPS and permeable ferrous iron chelator 2,2'-bipyridine inhibited membrane and intracellular uptake of iron. These results suggest that the uptake of iron from citrate is dependent on the reduction of iron from Fe3+ to Fe2+ at the cell membrane. These results are consistent with the findings of Randell et al. (19), who showed that the reductant ascorbate stimulated the uptake of NTB-Fe in the form of Fe-nitrilotriacetic acid by HepG2 hepatoma cells, whereas the ferrous chelators BPS and Ferrozine, which cannot cross the cell membrane, reduced iron uptake. In other types of cells Inman et al. (12) have provided strong evidence for the role of a ferrireductase in the uptake of NTB-Fe by showing that ferricyanide competitively inhibited the uptake of Fe-nitrilotriacetic acid. Also, the transition metal cadmium inhibited both the transport of iron and the reduction of ferricyanide in K-562 erythroleukemic cells, suggesting that the reduction and transport of NTB-Fe are coupled (12).
The intracellular uptake of iron and citrate by hepatic cells may occur by several mechanisms. It is possible that the ferric citrate complex is transported into the cell by receptor-mediated endocytosis or the iron and citrate are transported into the cell separately. The following lines of evidence suggest that the second process is more likely to occur. First, using a proteolytic enzyme to distinguish between intracellular and membrane uptake, we observed there was an accumulation of iron relative to citrate at the cell membrane, as well as inside the cell. Second, iron bound to citrate is in the Fe3+ form (16); therefore reduction of iron at the cell membrane is likely to lead to the release the iron from citrate at the cell surface and subsequent transport of iron and citrate into the cells by different pathways.
Gordan and Kaplan (8) have also presented evidence to suggest that iron and citrate are taken up independently by fibroblasts and HeLa cells. They have shown that membrane impermeable ferrous chelators BPS and Ferrozine, the reducing agent ascorbate, and calcium, all affect iron uptake but have no effects on citrate uptake. However, in contrast to our findings, they did not observe saturable uptake of either iron or citrate. Hence, their results provide no evidence for iron carriers or citrate binding sites on these cells. The solubility of iron as ferric citrate is relatively low at physiological pH and it is necessary to have a large molar excess of citrate relative to iron to prevent the formation of insoluble high molecular weight ferric hydroxide complexes (22). Gordan and Kaplan used a relatively low molar excess of citrate to iron (Fe-citrate, 1:5), and it is likely that high-molecular weight ferric citrate polymers formed. By contrast, most of our experiments were performed using physiological concentrations of NTB-Fe (1 µM) and citrate (100 µM) and in the presence of this relatively large molar excess of citrate to iron, ferric citrate forms mainly low-molecular weight Fe(Cit)2 complexes with negligible amounts of high-molecular weight polymers (23). Therefore, it is likely the differences in Gordan and Kaplan and our results are due to the different forms of ferric citrate present in the cell incubation medium.
In a previous study we have shown that the uptake of NTB-Fe and Tf-Fe, by a NTR pathway involving cell surface release of iron from transferrin, is mediated by the same cell surface iron carrier (30). Therefore, it is likely that several steps in the uptake of Tf-Fe by this NTR pathway and NTB-Fe are the same. Iron is bound to transferrin in the Fe3+ form and, as for ferric citrate, it is likely that the iron from transferrin is reduced at the cell surface before it is transported into the cells by the iron carrier. This is consistent with the evidence of others that reduction occurs extracellularly because membrane impermeable ferrous iron chelators inhibited Tf-Fe uptake (5, 24, 31). It is probable that the reduction and release of iron from transferrin at the cell surface by the NTR pathway involves a ferrireductase because other studies have shown Tf-Fe uptake and ferrireductase activity are linked (15, 25). However, iron uptake from transferrin by the HuH7 also occurs by TR and NTR endocytic pathways (30) and whether the reduction and release of iron from transferrin by a ferrireductase also occur in the endosome is unknown.
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ACKNOWLEDGEMENTS |
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This study was supported by a National Health and Medical Research Council of Australia and the Raine Medical Research Foundation.
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FOOTNOTES |
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Address for reprint requests: D. Trinder, Dept. of Physiology, Nedlands 6907, Western Australia, Australia.
Received 21 November 1997; accepted in final form 17 April 1998.
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