The Hereditary Hemochromatosis Protein, HFE, Specifically Regulates Transferrin-mediated Iron Uptake in HeLa Cells*

Cindy N. RoyDagger §, David M. Penny, John N. Feder, and Caroline A. EnnsDagger parallel

From the Dagger  Department of Cell and Developmental Biology, Oregon Health Sciences University, Portland, Oregon 97201-3098 and  Progenitor Inc., Menlo Park, California 94025

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HFE is the protein product of the gene mutated in the autosomal recessive disease hereditary hemochromatosis (Feder, J. N., Gnirke, A., Thomas, W., Tsuchihashi, Z., Ruddy, D. A., Basava, A., Dormishian, F., Domingo, R. J., Ellis, M. C., Fullan, A., Hinton, L. M., Jones, N. L., Kimmel, B. E., Kronmal, G. S., Lauer, P., Lee, V. K., Loeb, D. B., Mapa, F. A., McClelland, E., Meyer, N. C., Mintier, G. A., Moeller, N., Moore, T., Morikang, E., Prasss, C. E., Quintana, L., Starnes, S. M., Schatzman, R. C., Brunke, K. J., Drayna, D. T., Risch, N. J., Bacon, B. R., and Wolff, R. R. (1996) Nat. Genet. 13, 399-408). At the cell surface, HFE complexes with transferrin receptor (TfR), increasing the dissociation constant of transferrin (Tf) for its receptor 10-fold (Gross, C. N., Irrinki, A., Feder, J. N., and Enns, C. A. (1998) J. Biol. Chem. 273, 22068-22074; Feder, J. N., Penny, D. M., Irrinki, A., Lee, V. K., Lebron, J. A., Watson, N., Tsuchihashi, Z., Sigal, E., Bjorkman, P. J., and Schatzman, R. C. (1998) Proc. Natl. Acad. Sci. U S A 95, 1472-1477). HFE does not remain at the cell surface, but traffics with TfR to Tf-positive internal compartments (Gross et al., 1998). Using a HeLa cell line in which the expression of HFE is controlled by tetracycline, we show that the expression of HFE reduces 55Fe uptake from Tf by 33% but does not affect the endocytic or exocytic rates of TfR cycling. Therefore, HFE appears to reduce cellular acquisition of iron from Tf within endocytic compartments. HFE specifically reduces iron uptake from Tf, as non-Tf-mediated iron uptake from Fe-nitrilotriacetic acid is not altered. These results explain the decreased ferritin levels seen in our HeLa cell system and demonstrate the specific control of HFE over the Tf-mediated pathway of iron uptake. These results also have implications for the understanding of cellular iron homeostasis in organs such as the liver, pancreas, heart, and spleen that are iron loaded in hereditary hemochromatotic individuals lacking functional HFE.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HFE is the protein product of the gene mutated in the autosomal recessive disease hereditary hemochromatosis (1), which was first cloned in 1996 (1). It is therefore a relatively new member of the growing group of proteins involved in iron metabolism. HFE is remarkable in that it is a nonclassical major histocompatibility complex class I type molecule, a characteristic that prevented its role in iron homeostasis from being recognized immediately. Over 80% of hereditary hemochromatosis patients have the same mutation in the alpha 3 domain of HFE that prevents its heterodimerization with beta 2 microglobulin (2). Consistent with the classification of HFE as an major histocompatibility complex class I type molecule, this C282Y mutation has been shown to prevent cell surface expression and, presumably, function of the protein (2, 3). The generation of the HFE knockout mouse supports the finding that the C282Y mutation is, indeed, a loss of function mutation because the phenotype of the knockout mouse parallels the manifestation of hereditary hemochromatosis in humans (4).

Although the gene encoding HFE was discovered by genetic mapping of individuals with a disease of iron overload, the function of the protein in iron metabolism has not been immediately appreciated. The region of the HFE molecule normally associated with peptide binding and cell surface presentation in major histocompatibility complex class I molecules is too narrow to bind peptides. HFE does not appear to bind iron either (5). HFE does, however, associate with the transferrin receptor (TfR),1 a well characterized member of the iron metabolic pathway. The TfR is a type II transmembrane protein that binds the serum protein, diferric transferrin (Tf), at the cell surface. The Tf-TfR complex is constitutively taken into the cell through clathrin-mediated endocytosis. At the low pH of the endosome, a conformational change in the TfR facilitates iron release from Tf (6-9). Apotransferrin (apo-Tf) remains bound to TfR until the complex cycles back to the cell surface. At the neutral pH of the extracellular environment, apo-Tf is quickly replaced by diferric Tf and is free to bind more iron (for further discussion, see reviews in Refs. 10 and 11).

The association of HFE with the TfR has been demonstrated in tissues and in cell culture (12, 13). Wild type HFE has been shown to associate with TfR in the placenta (13) and to have the same immunohistochemical staining pattern as TfR in the crypt cells of the intestine (14). In cultured cells, HFE traffics with TfR between the cell surface and Tf-positive perinuclear compartments (15). The association between HFE and TfR has been shown to lower the affinity of TfR for its ligand 10-fold (5, 12, 15). Other nonclassical major histocompatibility complex class I molecules have been shown to affect ligand affinity for other receptors, such as the ability of the HLA H-2 protein to associate with the insulin receptor and reduce all insulin binding sites to low affinity sites (16-20) and the ability of anti-HLA antibodies to reduce epidermal growth factor binding to its receptor (21, 22).

The physiological importance of the interaction between HFE and TfR is not immediately clear. Diferric Tf is present at concentrations of approximately 5 µM in the blood (23). The dissociation constant of Tf for the TfR-HFE complex is approximately 11 nM. Thus, the binding of diferric Tf to its receptor is saturated in the presence of HFE. These results emphasize the putative role of HFE as a regulator of iron homeostasis that acts through the TfR cycle rather than having a role as a transporter or iron-binding protein.

To investigate its influence on cellular iron metabolism, the ability of HFE to specifically regulate the Tf-mediated pathway of iron uptake was tested. We found that HFE does not alter the cycling kinetics of the TfR. Rather, HFE reduces the amount of iron assimilated from Tf by ~33% without affecting non-Tf-mediated uptake of Fe-NTA. This finding gives more insight into the role of HFE as an iron regulator through the Tf-mediated pathway. Additionally, this focuses attention on the role of Tf-iron stores as a critical part of the "sensing machinery" that regulates dietary iron uptake and the kinetics of metabolic iron recycling.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Lines

The fWTHFE/tTA HeLa cell line expressing FLAG epitope-tagged HFE (fHFE) under the tetracycline-responsive promoter has been previously described (15). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 400 µg/ml G418 (Geneticin, Calbiochem), 200 ng/ml puromycin, and with (HFE-) or without (HFE+) 2 µg/ml tetracycline.

Iodination

Human holotransferrin (Intergen, Co.) was labeled with Na[125I] (NEN Life Science Products) using lactoperoxidase as described previously (24).

125I-Tf Uptake Protocol

The rate of 125I-Tf uptake was determined as described previously (24) with the following modifications. Uptakes were performed on subconfluent HFE- and HFE+ fWTHFE/tTA HeLa cultures (~1 × 106 cells) washed two times with 2 ml of DMEM-20 mM HEPES, pH 7.4, and preincubated in the same medium for 15 min at 37 °C with 5% CO2. At the 0 min mark, 1 ml of specific (DMEM-20 mM HEPES, 2 mg/ml ovalbumin, 50 nM 125I-Tf) or nonspecific (specific medium with 1 mg/ml cold Tf) medium was added to the appropriate cells. Cells were incubated at 37 °C with 5% CO2 for 2, 4, 6, or 8 min. Externally bound Tf was stripped with an acidic buffer (0.5 N acetic acid, 0.5 M NaCl) for 3 min at 4 °C. Then, the cells were washed four times with 2 ml of 4 °C final wash buffer (150 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 5 mM KCl, 1 mM MgCl2, pH 7.4) before addition of solubilization detergent (0.1% Triton X-100, 0.1% NaOH) and counting in a gamma counter (Packard, CobraII Auto-Gamma). Surface TfR numbers were determined by counting the amount of 125I-Tf bound after incubation with 50 nM 125I-Tf for 90 min on ice at 4 °C. Following the 90-min incubation, the medium was removed, and cells were washed four times with 2 ml of 4 °C final wash buffer before solubilization and counting.

125I-Tf Efflux Protocol

The rate of Tf efflux was determined as described previously by McGraw and Maxfield (25) with the following modifications. Monolayers of subconfluent HFE- and HFE+ fWTHFE/tTA HeLa cells grown in 35-mm plates were washed three times with 2 ml of DMEM-20 mM HEPES and then preincubated in the same medium for 15 min at 37 °C and 5% CO2. The medium was removed, and cells were incubated for 2 h with specific (50 nM 125I-Tf in DMEM-20 mM HEPES and 2 mg/ml ovalbumin) or nonspecific (specific medium with 1 mg/ml unlabeled Tf) medium at 37 °C and 5% CO2. Cells were washed with 2 ml of 37 °C mild acid buffer (500 mM NaCl, 50 mM MES, pH 5.0) for 2 min and then washed two times with 2 ml of 37 °C final wash buffer with 100 µM desferoxamine (desferal; Ciba-Geigy Ltd.; Basel, Switzerland) and 3 µg/ml Tf to prevent loading and rebinding of apo-125I-Tf. 1 ml of 37 °C medium (DMEM-20 mM HEPES, 2 mg/ml ovalbumin) with 3 µg/ml Tf and 100 µM desferoxamine was added at the 0 min mark, and then at each of the appropriate time points, plates were placed on ice and the appropriate efflux, surface and internal samples were collected as follows.

Efflux-- Medium was removed by pipetting, and cells were washed one time with 1 ml of final wash, which was pooled and counted in gamma counter (Packard, CobraII Auto-Gamma).

Surface-- Cells were incubated in 1 ml of acid wash (0.2 N acetic acid, 0.5 M NaCl) for 3 min at 4 °C and then washed 1 time with 1 ml of final wash, which was pooled and counted.

Internal-- Cells were stripped and washed as per surface samples and then solubilized in 2 ml of solubilization detergent and counted.

TfR Distribution

The relative TfR distribution between cell surface (external) and internal compartments was determined as described previously (25, 26) with the following modifications. Subconfluent HFE- and HFE+ fWTHFE/tTA HeLa cells grown in 35-mm plates were either incubated for 90 min with 100 nM 125I-Tf at 37 °C with 5% CO2 for total and internal measurements or with 100 nM 125I-Tf at 4 °C for external measurements in 1 ml of specific (100 nM 125I-Tf in DMEM-20 mM HEPES) or nonspecific (specific medium with 1 mg/ml unlabeled Tf) medium at 4 °C. At the end of the incubation time, cells were placed on ice. Total and external measurements were determined by washing four times with 2 ml of final wash at 4 °C and then solubilizing and counting. The amount of internalized 125I-Tf was determined by stripping the surface for 3 min with acid wash, washing four times with 2 ml of final wash, and then solubilizing and counting in gamma counter (Packard, CobraII Auto-Gamma).

55Fe Loading of Human Apotransferrin

55FeCl3 (NEN Life Science Products) was complexed to nitrilotriacetic acid in a 1:200 ratio (Fe:NTA). Then, the 55Fe-NTA was incubated with Tf in a 2:1 ratio for 1 h in carbonate buffer (10 mM NaHCO3, 250 mM Tris-HCl). 55Fe-transferrin was separated from free 55Fe on a 20-ml G-50 Sephadex (Sigma) column. The resulting transferrin was 82% saturated with iron.

55Fe-Tf Uptake Protocol

55Fe uptake from transferrin was determined essentially as described above for 125I-Tf uptake with the following modifications. Uptakes were performed on subconfluent HFE- and HFE+ fWTHFE/tTA HeLa cells grown in 35-mm dishes. Specific medium contained 100 nM 55Fe-Tf and 2 mg/ml ovalbumin in wash medium (McCoy's 5A with 20 mM HEPES). Nonspecific medium was the same as specific with the addition of 1 mg/ml unlabeled Tf. After 45, 90, 135, or 225 min of uptake, cells were placed on ice, and externally bound Tf was stripped with an acidic buffer (0.2 N acetic acid, 500 mM NaCl, 1 mM FeCl3) for 3 min at 4 °C. Cells were solubilized in 1 ml of the same solubilization detergent described above. Lysates were mixed with 6 ml UniverSol (ICN) and counted for 10 min in a scintillation counter (Beckman LS 6000SC) with a window of 0-350 nm. TfR numbers were determined as described for the 125I-Tf uptake protocol above.

55Fe-NTA Uptake Protocol

55Fe-NTA uptake procedures were modified from a previously described protocol by Inman and Wessling-Resnick (27). A 55Fe-NTA solution was made by the addition of 55FeCl3 to NTA buffer (80 µM NTA, 20 mM HEPES-Tris) for a final concentration of 20 µM. Subconfluent fWTHFE/tTA HeLa cells grown in 35-mm dishes were washed two times with 2 ml of McCoy's 5A-20 mM HEPES at 37 °C. Wash medium was replaced with 1 ml of specific (200 nM 55Fe-NTA in wash medium) or nonspecific (specific medium with 1 mM Fe-NTA) medium. After 2, 5, 10, 15, or 30 min of uptake, cells were placed on ice, and 3 ml of 4 °C quench buffer (1 mM Fe-NTA, 25 mM HEPES, 150 mM NaCl) was added for 20 min. Finally, the cells were washed three times with 2 ml of 150 mM NaCl at 4 °C, solubilized, and counted as for 55Fe-Tf uptake protocol described above.

Iron Treatments

HFE- and HFE+ fWTHFE/tTA HeLa cells were seeded at ~1.5 × 105 cells per 35-mm dish. After 2 days, cells were treated with either 50 µM desferoxamine, 100 nM human holotransferrin (Intergen Co.), or 100 µM Fe-NTA overnight. Cells were lysed in NET-Triton (150 mM NaCl, 5 mM EDTA, and 10 mM Tris (pH 7.4) with 1% Triton X-100) after at least 12 h of treatment.

Western Immunodetection

Cell extracts from 2 × 105 cells were diluted with 4× Laemmli buffer (28) to 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol and subjected to electrophoresis on 12% SDS-polyacrylamide gels under reducing conditions. The proteins were transferred to nitrocellulose. Immunoblot analysis was performed using sheep anti-human transferrin receptor serum (described previously (24); 1:10,000 dilution), and sheep anti-human ferritin antibody (The Binding Site, Ltd., 1:100 dilution) followed by swine anti-goat secondary antibody conjugated to horseradish peroxidase (Boehringer Mannheim). Chemiluminescence (SuperSignal, Pierce) was performed per the manufacturer's directions.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

fHFE Does Not Alter Tf Endocytosis-- The influence of HFE on TfR cycling kinetics was measured to determine how HFE regulates iron levels in the cell. In previous studies, a cell line expressing fHFE under the control of the tetracycline repressible system was established (15). Induction of HFE expression by the withdrawal of tetracycline from the medium results in decreased ferritin (Ft) levels. One mechanism by which HFE could lower the amount of iron taken up into cells would be by decreasing the rate of endocytosis of the TfR. HFE co-traffics with TfR between the cell surface and Tf positive perinuclear compartments (15) and therefore might have some influence on TfR endocytic kinetics.

The rate of 125I-Tf uptake was measured to test whether or not HFE alters the endocytosis of TfR and therefore the amount of Tf-mediated iron uptake in fWTHFE/tTA HeLa cells expressing fHFE (fWTHFE/tTA HeLa cells). Uptake experiments were performed in the presence of 50 nM 125I-Tf. The association of HFE with the TfR decreases the affinity of the TfR for Tf, resulting in an increase in the Kd from 1.2 to 11 nM (15). At 50 nM Tf, essentially all the TfRs are occupied with Tf, even in the presence of HFE. Cells were incubated with 125I-Tf for 2, 4, 6, or 8 min at 37 °C with 5% CO2. The surface was stripped of bound Tf, and the amount of 125I-Tf inside the cell was counted. Both HFE- and HFE+ cells took up approximately 0.20 Tf/surface TfR/min (Fig. 1). The same rates of internalization were measured in cells treated with 100 nM 125I-Tf, confirming that the binding of Tf to its receptor was rapid and saturating (data not shown). fHFE did not significantly alter the amount of 125I-Tf uptake per TfR in fWTHFE/tTA cells and therefore did not affect the kinetics of TfR endocytosis.


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Fig. 1.   fHFE does not significantly alter Tf endocytosis. 125I-Tf uptake was measured in 7.2 × 105 HFE- (triangle , dashed line) or 7.0 × 105 HFE+ (, solid line) fWTHFE/tTA HeLa cells. The rates of 125I-Tf uptake/surface receptor/min for HFE- and HFE+ were 0.198 (r2 = 0.901) and 0.213 (r2 = 0.925), respectively. Linear regression was determined in the Cricket Graph program. These results are representative of three experiments performed with quadruplicate data points without significant variation between experiments.

fHFE Does Not Alter TfR Exocytosis-- The amount of iron taken up into cells is a function of the kinetics of TfR cycling that depends on both the endocytic and exocytic rates. Because fHFE had no effect on TfR endocytosis, the rate of 125I-Tf release was measured to test whether fHFE alters the exocytic rate of TfR in HFE- and HFE+ fWTHFE/tTA HeLa cells. Cells were loaded with 125I-Tf for 2 h at 37 °C with 5% CO2 to saturate internal and external TfR with Tf. Then, the cell surface was stripped of Tf, and fresh medium was added to the cells. The Tf released from internal cellular compartments was collected and counted at 2, 4, 6, and 8 min time points. HFE- and HFE+ fWTHFE/tTA HeLa cells did not differ significantly in their rate of TfR exocytosis (Fig. 2).


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Fig. 2.   fHFE does not significantly alter Tf exocytosis. 125I-Tf release was measured in 9.3 × 105 HFE- (triangle , dashed line) or 7.7 × 105 HFE+ (, solid line) fWTHFE/tTA HeLa cells. The rates of 125I-Tf release as a percentage of the total cell associated/125I-Tf/min for HFE- and HFE+ were 0.052 (r2 = 0.916) and 0.047 (r2 = 0.929), respectively. Linear regression was determined in the Cricket Graph program. These results are representative of three experiments performed with quadruplicate data points without significant variation between experiments.

TfR Distribution Is Not Changed in fHFE-expressing Cells-- Because the uptake and efflux kinetics of Tf did not change with the expression of fHFE, the steady state distribution of external and internal TfRs should also remain the same between cells expressing and not expressing fHFE. To test this prediction, steady state external and internal pools of 125I-Tf were measured in control cells (HFE-) and in cells expressing fHFE (HFE+). fHFE expression did not significantly alter the relative surface and internal distributions of TfR (Fig. 3). Approximately 20% of cellular TfR was found at the cell surface, whereas 80% was found in internal vesicles. The distribution of TfR between the cell surface and internal compartments was similar to that reported previously in other cell lines expressing similar amounts of TfR (29-31). Combined with the uptake and efflux data reported above, these findings support the conclusion that HFE has no effect on TfR cycling kinetics even though HFE associates and co-traffics with the TfR (15).


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Fig. 3.   fHFE does not significantly alter TfR distribution. TfR distribution was measured in 7.6 × 105 HFE- (light gray) or 6.1 × 105 HFE+ (dark gray) fWTHFE/tTA HeLa cells. The percentage of receptors on the cell surface or in internal compartments did not differ significantly between the two cell types. Both express approximately 20% of TfR on the cell surface and 80% in intracellular vesicles. These results are representative of three experiments performed with quadruplicate data points without significant variation between experiments.

fHFE Reduces Iron Uptake from Tf But Does Not Alter Non-Tf-mediated Iron Uptake-- We wanted to determine whether HFE affected the amount of Tf-mediated or non-Tf-mediated iron uptake because no effect on the cycling kinetics of the TfR was detected. HFE- and HFE+ fWTHFE/tTA HeLa cells were incubated in the presence of 100 nM 55Fe-loaded Tf for up to 240 min, and the rate of 55Fe uptake from Tf for HFE- and HFE+ cells was determined. Control cells took up 55Fe from Tf at a rate of 0.291 pmol of Fe/106 cells/min, whereas cells expressing fHFE took up 55Fe from Tf at a rate of 0.193 pmol of Fe/106 cells/min (Fig. 4). These data show that HFE-expressing cells take up approximately 33% less iron from Tf than control cells that do not express fHFE.


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Fig. 4.   fHFE significantly reduces iron uptake from Tf. 55Fe uptake from Tf was measured in 6.8 × 105 HFE- (triangle , dashed line) or 5.6 × 105 HFE+ (, solid line) fWTHFE/tTA HeLa cells. The rate of 55Fe uptake/min/106 HFE- and HFE+ cells from Tf was 0.291 (r2 = 0.939) and 0.193 (r2 = 0.923), respectively; a 33% reduction specific to HFE expression. Linear regression was determined in the Cricket Graph program. These results are representative of three experiments performed with quadruplicate data points without significant variation between experiments.

To determine whether the influences of HFE are restricted to the Tf-mediated pathway of iron uptake or if HFE additionally affects the non Tf-mediated iron uptake pathway, HFE- and HFE+ fWTHFE/tTA HeLa cells were incubated in the presence of 200 nM 55Fe-NTA for up to 30 min. No significant difference was seen in iron uptake between HFE- and HFE+ cells (Fig. 5). Combined, these results support the conclusion that the regulation of iron uptake by HFE is specific to the Tf-mediated iron uptake pathway. The effect of HFE on Tf-mediated iron uptake is therefore a direct effect on the Tf-derived iron pathway rather than an alteration of all cellular iron metabolism.


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Fig. 5.   fHFE does not significantly alter non-Tf-mediated iron uptake. 55Fe uptake from 55Fe-NTA was measured in 1.5 × 106 HFE- (triangle , dashed line) or 1.8 × 106 HFE+ (, solid line) fWTHFE/tTA HeLa cells. The rates of 55Fe uptake in pmol/million cells/min for HFE- and HFE+ were 0.275 (r2 = 0.964) and 0.255 (r2 = 0.975), respectively. Linear regression was determined in the Cricket Graph program. These results are representative of three experiments performed with quadruplicate data points without significant variation between experiments.

Ft Levels Are Responsive to Iron Treatment in fWTHFE/tTA HeLa Cells-- The response of HFE- and HFE+ fWTHFE/tTA HeLa cells to iron loading or iron chelation was observed by detection of TfR and Ft levels on Western blots. These experiments were performed to test whether HFE affects the ability of the cells to compensate for changing iron levels in the medium as predicted by the known influences of iron regulatory proteins (IRPs) on Ft and TfR expression. IRPs are the most extensively characterized mode of regulating intracellular iron concentrations. In the presence of low intracellular iron, IRPs bind to iron responsive elements in the mRNA of iron-regulated proteins. This interaction either stabilizes the mRNA by binding to the 3'-end of the transcript (as is the case for TfR mRNA), or blocks translation by binding to the 5'-end of the transcript, close to the site of translation initiation (as is the case for Ft mRNA). Thus, at low intracellular iron levels, the cell increases iron uptake by increasing TfR levels. It decreases iron storage by lowering Ft levels. When intracellular iron concentrations are high enough, the IRP no longer binds the iron responsive element. This acts to reduce iron uptake from Tf by reducing TfR levels and increasing Ft levels.

The response of cells to iron depletion and iron loading was measured in HFE- and HFE+ fWTHFE/tTA HeLa cells grown for 2 days in untreated growth medium and treated for at least 12 h with growth medium supplemented with 50 µM desferoxamine to chelate iron, human Tf, or Fe-NTA. Under control conditions, cells expressing fHFE had no detectable Ft over background, whereas those that do not express HFE showed significant amounts of Ft (Fig. 6). HFE- and HFE+ cells showed increased Ft expression following treatment with 100 nM human Tf. HFE- cells increased Ft levels only about 2-fold upon inspection of the Western blot. HFE+ cells had slightly less Ft than HFE- cells for the same treatment condition, but this was a significant increase over the undetectable Ft levels in HFE+ cells without Tf treatment. In the same way, both HFE- and HFE+ cells showed increased Ft expression following treatment with 100 µM Fe-NTA.


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Fig. 6.   Ft levels are responsive to iron treatment in fWTHFE/tTA HeLa cells. Lysates of 2 × 105 HFE- or HFE+ fWTHFE/tTA HeLa cells were run on a 12% denaturing polyacrylamide gel under reducing conditions. Proteins were transferred to nitrocellulose and detected with sheep anti-human transferrin receptor (1:10,000) and sheep anti-ferritin (1:100) antibodies and swine anti-goat horseradish peroxidase-conjugated secondary antibody (1:10,000). ~94- and 19/21-kDa bands (by chemiluminescent detection) represent TfR and Ft, respectively. The increased TfR expression and decreased Ft expression in fHFE-expressing cells is consistent with previously reported results. Both cell types increased Ft levels in the presence of an iron source and increased TfR levels in the presence of the iron chelator desferoxamine. No Ft was detectable in HFE+ cells in the presence of 50 µM desferoxamine. These results are representative of four experiments without significant variation between experiments.

TfR levels also changed, as shown in Fig. 6, as predicted by the known influence of IRPs. TfR levels were slightly elevated in fHFE-expressing cells versus HFE- cells in keeping with their iron-depleted status. Both HFE- and HFE+ cells treated with desferoxamine showed increased TfR levels above that in untreated cells. For both HFE- and HFE+ cells, TfR levels slightly decreased upon treatment with human Tf or Fe-NTA as compared with untreated cells. These results demonstrate that the outcome of the iron-responsive mechanisms used by the cell are not perturbed by HFE expression. Rather, HFE imposes an additional but separate homeostatic mechanism.

The sensitivity of the cellular IRP response to iron was also tested in the presence and absence of HFE. Even at Tf concentrations below saturation of the TfR and HFE/TfR complex, such as 5 nM, Ft levels were greater for both HFE- and HFE+ cells than without Tf treatment (Fig. 7). However, due to the difference in the Tf binding affinity and the reduced iron uptake from Tf, HFE+ cells expressed consistently smaller amounts of Ft than HFE- cells under the same Tf concentrations below 50 nM. Ft levels were also increased in HFE- and HFE+ cells when treated with Fe-NTA at concentrations as low as 50 nM. Ft levels for HFE+ cells remained lower than HFE- cells due to their initially depressed intracellular iron levels. The observation that the cells responded to Tf-derived iron at much lower concentrations than NTA-derived iron is consistent with the association constants and the kinetics of the protein components of those systems. Detection limits of the enhanced chemiluminescence and x-ray film prevent quantitative conclusions from these data; however, the qualitative results provide strong evidence that HFE does not prevent the IRP-dependent regulation of Ft and TfR expression. Rather, HFE alters the "set point" of intracellular iron levels in a Tf-mediated manner.


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Fig. 7.   Sensitivity of fWTHFE/tTA HeLa cells to iron treatment. Lysates of 4 × 105 HFE- or HFE+ fWTHFE/tTA HeLa cells treated for 12 h with the indicated amounts of Tf (A) or Fe-NTA (B) were run on a 12% denaturing polyacrylamide gel under reducing conditions. Proteins were transferred to nitrocellulose and detected with sheep anti-ferritin (1:100) antibodies and swine anti-goat horseradish peroxidase-conjugated secondary antibody (1:10,000). Sensitivity to Tf- and NTA-derived iron sources is within the concentration range consistent with the association constants of the TfR and iron transport proteins.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The mechanism by which HFE controls iron homeostasis is not yet known. We have previously reported that HFE specifically reduces Ft levels in HeLa cells expressing fHFE, showing a direct relationship between HFE expression and cellular iron homeostasis (15). HFE traffics with TfR to Tf-positive perinuclear vesicles, which are one site of cellular iron absorption (15). Regulated Tf and non-Tf-mediated iron uptake pathways have both been described for the HeLa tissue culture system (32-34) (for further discussion, see review in Ref. 35). This study shows that HFE specifically decreases the Tf-mediated pathway of iron uptake.

fHFE has no effect on the kinetics of TfR cycling in cells. If HFE were to slightly decrease the endocytic rate or increase the exocytic rate of TfR, less Tf and therefore less iron would be taken up per unit of time. At concentrations of Tf that saturate Tf binding to the HFE/TfR complex (50-100 nM), no difference in the uptake or release of 125I-Tf from HFE- and HFE+ fWTHFE/tTA HeLa cells was detected. These results were confirmed by measuring the steady state distribution of TfRs. No redistribution of TfRs was detected between the cell surface and internal compartments when fHFE is expressed at steady state.

Although HFE does not affect TfR cycling kinetics, it does regulate cellular iron homeostasis through the Tf-mediated iron uptake pathway. At saturating concentrations of 55Fe-loaded Tf (100 nM), 33% less iron was taken up by fWTHFE/tTA HeLa cells expressing fHFE. Because saturating amounts of Tf were used in these experiments, the ability of HFE to lower the TfR affinity for Tf is not the mechanism responsible for the observed decrease in cellular iron. This experiment does not differentiate between release of iron from transferrin and transport across the endosomal membrane to the cytoplasm. HFE might effect either of these steps in Tf-mediated iron transport. Because Ft levels are also reduced for HFE+ fWTHFE/tTA HeLa cells in culture, these results suggest that acquisition of iron from fetal bovine Tf in tissue culture medium takes place through the TfR. Despite the larger dissociation constant of fetal bovine Tf for the human TfR, iron is still removed from the bovine Tf but the uptake is reduced in HFE+ cells.

fWTHFE/tTA HeLa cells were incubated in the presence of 200 nM 55Fe-NTA to confirm that HFE specifically regulates Tf-mediated iron uptake. Iron presented in this form bypasses the Tf-TfR pathway and enters the cell directly through a transporter (27, 36). Candidate transporters are SFT and DCT1/Nramp2 although other, currently unidentified, cell surface iron transporters may exist as well (34, 37). No significant difference was seen in the amount of non-Tf bound iron taken up by HFE+ cells as compared with HFE- cells, suggesting that HFE acts specifically through the Tf/TfR-mediated pathway of iron uptake.

Western blots were used to determine whether cells expressing HFE can respond to bioavailable iron by increasing their Ft levels. Under control tissue culture conditions, HFE- cells have more Ft than HFE+ cells. Cells that do not express fHFE are capable of depleting Ft levels under low iron conditions, such as in the presence of the iron chelator desferoxamine. Both HFE- and HFE+ fWTHFE/tTA HeLa cells responded to iron loading via the Tf-mediated and non-Tf-mediated pathways by increasing intracellular Ft levels and decreasing TfR levels. These results emphasize that cells expressing HFE still regulate Ft levels under high and low iron conditions, presumably through the translational regulation of Ft by the IRPs. HFE has simply changed the total amount of iron that is stored within the cell.

These studies demonstrate that in a nonpolarized cell line, fHFE reduces the amount of iron taken up from the Tf-mediated iron uptake pathway. Several factors are involved in iron uptake from the endosome, any one of which could be affected by HFE. One characteristic of the endosome is its relatively low pH (between 5.5 and 6.5, depending on the cell type (9)). This low pH is responsible for a conformational change in both Tf and the TfR that facilitates the release of iron (6-9). HFE might regulate the lumenal pH of the endosome, preventing efficient removal of iron from Tf. In conflict with this hypothesis is evidence that suggests increases in endosomal pH slows recycling of TfR (38). We do not see any perturbations of TfR recycling, suggesting that the endosomal pH is close to normal.

We favor the hypothesis that HFE prevents the pH-induced conformation of the TfR that potentiates the release of iron from Tf. Aisen and co-workers (6-8) and Sipe and Murphy (9) showed the importance of the TfR-induced conformational change in Tf for the release of iron in the endosome. At pH 6.0, the association of Tf with the TfR approximately doubles the rate of loss of iron from Tf (39). If this change was prevented, then less iron would be taken up into cells. One caveat of this hypothesis is the observation that soluble TfR does not detectably bind to soluble HFE at pH 6.0 (5). For this mechanism to be responsible for the lower uptake of iron into cells, either HFE does interact with the TfR at the slightly higher pH of many endosomes or the membrane bound forms of the TfR and HFE interact with each other at endosomal pHs.

Alternatively, HFE might regulate the function of one of the known endosomal iron transporters, such as Nramp2/DCT1 or SFT (40, 41), thereby reducing iron transport across the endosomal membrane. HFE might simply use its association with TfR for endocytosis. Once in the endosome, HFE might complex with transmembrane proteins responsible for transporting, iron into the cytoplasm. HFE could modulate the transporters affinity for iron or its kinetics of iron transport across the endosomal membrane. Thus, HFE may be involved in maintaining homeostatic equilibrium of iron through its regulation of such a transporter. By modulating transporter activity in Tf-positive endosomes, HFE could facilitate appropriate flux of Tf-derived iron across the intestinal enterocyte. These three hypothesis need to be tested.

With the addition of the present findings from the fWTHFE/tTA HeLa system, we propose a new model for the role of HFE in iron regulation (Fig. 8). HFE imposes an additional regulatory step for iron uptake in cells expressing HFE. Iron responsive element-mediated iron regulation does not appear to be sufficient to regulate iron homeostasis because organisms lacking HFE or Tf but having functional IRPs eventually succumb to iron overload. We propose that HFE limits iron uptake from Tf by reducing the amount of iron released from Tf. Without HFE (as is the case in hereditary hemochromatosis patients), organs normally expressing HFE, such as the lives, clear more iron from circulating Tf, contributing to hepatic iron overload.


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Fig. 8.   Model of HFE action on cellular iron homeostasis. Diferric Tf is present at a concentration of approximately 5 µM in the blood (43) and is therefore capable of saturating all TfRs, even in the presence of HFE (arrow 1A). HFE traffics with Tf and TfR to the endosome (arrow 2A). Once the endosome has acidified, iron is released from transferrin and transported through an endosomal iron transporter, such as Nramp2/DCT1 or SFT (arrow 3A). In the presence of HFE, TfR does not potentiate the full release of iron from Tf, leaving some iron bound to Tf. Tf then cycles out of the endosome and back to the cell surface, resulting in release of ferric rather than apo-Tf (arrow 4A). TfR that is not associated with HFE (as is the case in HH patients) also binds diferric Tf preferentially at the cell surface (arrow 1B). TfR internalization is identical to that of the TfR/HFE complex (arrow 2B). Once the endosome has acidified, TfR potentiates the release of iron from Tf, which is transported across the endosomal membrane (arrow 3B). More iron is released from Tf that is bound to TfR alone than is released from Tf bound to the HFE-TfR complex. Tf then cycles out of the endosome and back to the cell surface, where apo-Tf is released (arrow 4B).

We have shown previously that HFE co-traffics with TfR from the time of its initial synthesis to its cycling through Tf-positive endosomes (15). HFE may traffic with TfR from the basolateral plasma membrane to endocytic vesicles of the enterocytes. This would be consistent with the perinuclear immunohistochemical staining of HFE (14) and TfR localization (42) in the cells of the intestinal crypts. HFE in the enterocyte endosome, where iron derived from apical dietary uptake and iron derived from bodily Tf stores meet, may play a key role in the regulation of iron transport out of the enterocyte into the blood. The mechanisms for such events remain unresolved.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK 40608.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by the Training Program in Molecular Hematology, T32-HL00781, NHLBI, National Institutes of Health.

parallel To whom correspondence should be addressed: Dept. of Cell and Developmental Biology, L215, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97201-3098. Tel.: 503-494-5845; Fax: 503-494-4253; E-mail: ennsca{at}ohsu.edu.

    ABBREVIATIONS

The abbreviations used are: Tf, transferrin; TfR, Tf receptor; fHFE, FLAG epitope-tagged HFE; Ft, ferritin; IRP, iron regulatory protein; NTA, nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis; tTA, tetracycline transactivatable; DMEM, Dulbecco's modified Eagle's medium.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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