1Department of Genetics and Complex Diseases, Harvard School of Public Health, and 2Harvard Medical School Electron Microscopy Facility, Boston, Massachusetts 02115
Submitted 13 July 2004 ; accepted in final form 15 August 2004
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ABSTRACT |
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iron transport; HepG2 cells
TfR2 expression is distinct from classic TfR1 expression. It is highly expressed in the liver, with more modest mRNA levels found in some other tissues (19). The expression of TfR2 in murine liver increases during development, while TfR1 decreases (17). In contrast, TfR2 levels in the spleen are constant throughout development, while TfR1 levels increase (17). The murine TfR2 promoter can be regulated by the erythroid/liver-related transcription factors GATA, CCAAT/enhancer binding protein (C/EBP), erythroid Kruppel-like factor (EKLF), and friend of GATA-1 (FOG-1), implying tissue-specific regulation (17). The differential tissue expression of TfR1 and TfR2 during murine development and the high level of TfR2 expression in the liver suggest a tissue-specific function for TfR2 that may be distinct from TfR1 function.
Although the expression patterns of these two proteins appear to be quite different, Tf-related activities studied to date seem to be quite similar. Both bind Tf and facilitate Tf-mediated iron uptake (18, 19). However, a role for TfR2 in a process that has been referred to as "TfR1-independent Tf uptake" (15, 24, 27, 36, 37) has not been fully examined. This second Tf uptake mechanism is characterized by a pattern of biphasic internalization kinetics observed in some cell types, including liver HuH-7 cells and rat hepatocytes. While steady-state internalization of Tf typically saturates at low concentrations (<0.5 µM) (see, e.g., Refs. 1, 5), this atypical pathway displays a linear phase of uptake at ligand concentrations >0.5 µM, which appears to be independent of TfR1, because it continues when TfR1 expression is suppressed by antisense strategies or TfR1 activity is blocked by anti-TfR1 antibodies (15, 24, 36, 37). The TfR1-independent pathway in HuH-7 cells displays a slower rate of Tf recycling, an increased level of Tf degradation and a potentially unique mechanism of iron acquisition from Tf (15). Because the TfR1-independent pathway has been observed primarily in hepatic cells that express TfR2 (24, 38, 42), we considered the possibility that this receptor might be involved. Our results show that TfR2 functions in the Tf endocytic cycle are quite different from those of TfR1. Gain-of-function experiments revealed that exogenous expression of TfR2 resulted in a biphasic pattern of Tf uptake, directly demonstrating its role in a pathway previously described only in biochemical studies. Furthermore, the subcellular distribution of internalized ligand indicated that this receptor delivered Tf to the late endocytic pathway, where it accumulated in multivesicular bodies (MVB) devoid of receptors and deficient in lysosome-associated membrane protein 1 (LAMP1). Because 125I-labeled Tf did not appear to be degraded in this pathway, these coupled observations support a model in which the apparently nonsaturable linear phase of TfR1-independent Tf uptake can be explained by intracellular accrual of the ligand. The liver serves as a major depot of iron in the body, and we hypothesize that the unique mechanism of Tf import by TfR2 plays a critical role in the regulation of iron release and/or storage by this tissue.
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MATERIALS AND METHODS |
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Transfection of HeLa cells. To transiently overexpress TfR1 in HeLa cells, TfR1 cDNA was directionally subcloned into pcDNA 3.1+hygro (Invitrogen, Carlsbad, CA) using EcoRV/XbaI restriction sites (pcDNA3.1-TfR). TfR2 was exogenously expressed in HeLa cells as a FLAG-tagged fusion protein using a vector (pcDNA3-FLAG-TfR2) kindly provided by Drs. H. Kawabata and H. P. Koeffler (Division of Hematology/Oncology, Department of Medicine, University of California at Los Angeles School of Medicine, Los Angeles, CA). Briefly, subconfluent HeLa cells were washed twice in serum-free medium. Plasmidscontrol (pcDNA3.1), pcDNA3-FLAG-TfR2, or pcDNA3.1-TfR1were combined with Lipofectamine (GIBCO-BRL, Grand Island, NY) in serum-free medium, and cells were subsequently transfected according to the manufacturer's instructions. Cells were trypsinized and replated onto six-well plates. 125I-Tf uptake experiments were performed 12 days posttransfection as described above. Western blot analysis confirmed the absence or presence of exogenous FLAG-TfR2 expression or TfR1 overexpression (data not shown). To study the degradation of internalized ligand, cells transfected to express TfR1 or TfR2 were incubated with 1.5 µM 125I-Tf at 37°C for up to 6 h. Aliquots (2 µl) of the cell-conditioned medium were removed and mixed with 1 ml of 20% trichloroacetic acid (TCA)-2% casein hydrolysate for 1 h on ice. After microcentrifugation at 14,000 rpm for 5 min at 4°C, TCA-soluble radioactivity was determined using a gamma counter.
Immunoelectron microscopy. After the incubations of transfected HeLa cells were performed as described in the figure legends, cells were washed four times with (in mM) 150 NaCl, 1 CaCl2, 5 KCl, and 20 HEPES, pH 7.4, and then fixed with paraformaldehyde (4% wt/vol) and glutaraldehye (0.1% wt/vol). HepG2 cells were washed twice and incubated for 1.5 h at 37°C to deplete cells of Tf in serum-free medium containing 20 mM HEPES, pH 7.4. HepG2 cells were then incubated for 1 h at 37°C with 2 µM holotransferrin in serum-free medium containing 20 mM HEPES, pH 7.4, and 2 mg/ml BSA; washed four times in PBS; and fixed as described above. Processing for cryosectioning and immunolabeling was performed as described by Griffiths (13). Labeling for TfR2 was performed using a 1:200 dilution of M2-monoclonal anti-FLAG antisera (Sigma Chemical, St. Louis, MO) while TfR1 was detected with a 1:50 dilution of mouse anti-TfR1 (Zymed Laboratories, South San Francisco, CA). Tf was detected using 1:50 (HeLa) and 1:200 (HepG2) dilutions of rabbit anti-Tf (Rockland Immunochemicals, Gilbertsville, PA). A 1:60 dilution for Tf and a 1:100 dilution for LAMP1 (mouse anti-human LAMP1; Pharmingen, San Diego, CA) were used in colocalization studies of HeLa cells transfected with TfR2. After incubation with appropriate secondary antibody if necessary, 5 or 10 nm of protein A-gold (Dr. Jan Slot, Department of Cell Biology, University of Utrecht Medical School, Utrecht, the Netherlands) was used to detect immunoreactivity. Microscopy was performed using a Jeol 1200EX transmission electron microscope (Jeol, Peabody, MA) at 80 kV with primary magnification of x1530,000.
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RESULTS |
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DISCUSSION |
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Consistent with functional differences in the kinetics of ligand uptake, the intracellular movement of Tf taken up by TfR1 and TfR2 is also strikingly different. Our data reveal that intracellular trafficking of Tf in HeLa cells is altered by exogenous expression of TfR2 such that the ligand is directed into MVB. The fact that TfR2 does not appear in MVB suggests that Tf is released during the internalization pathway mediated by this receptor. Similar to its association with TfR1, apo-Tf is reported to strongly bind TfR2 at pH <6.5, but published studies by Kawabata et al. (19) suggest that holotransferrin dissociates from TfR2 at pH values between 6.5 and 7.0, while the iron-loaded ligand remains bound to TfR1 under these conditions. Thus it seems probable that holotransferrin could dissociate from TfR2 in early endocytic compartments to be sorted for delivery to MVB. Delivery of Tf to MVB provides an explanation for the linear phase of the biphasic uptake pattern due to the intracellular accrual of the ligand.
MVB are late endosomal compartments typically associated with the progression of membrane traffic toward the lysosome, resulting in constitutive degradation of the organelle contents (16). Perhaps best characterized for their role in EGF-EGF receptor membrane traffic, MVB lack TfR and often remain negative for LAMP1, a lysosome-associated marker, while they do contain EGF-EGF receptor complexes. Kinetic studies have shown that acid-soluble 125I release due to degradation of 125I-EGF can be detected and measured in counts per minute (cpm) as early as 30 min after internalization and transit through MVB (10). In contrast, our studies show that 125I-Tf endocytosed to MVB via TfR2 is not degraded for at least 6 h. This observation prompts the speculation that Tf is delivered to MVB as a result of its association with a saturable binding component that directs sorting of the ligand to this compartment but prevents further trafficking to lysosomes. Because of experimental limitations, we were able to study 125I-labeled Tf uptake only at concentrations up to 2 µM; therefore, it is possible that saturation of such a binding component would be achieved at even higher concentrations. In HuH7 cells, the TfR1-independent pathway for Tf uptake was recently reported to saturate to 10 µM concentration (24). In preliminary studies, we have found that recycling kinetics of 125I-labeled Tf are not altered in TfR2-expressing HeLa cells despite the accrual of ligand (Robb A and Wessling-Resnick M, unpublished observations), and this result is also consistent with saturable association of internalized ligand with some intracellular component. Besides TfR1 and TfR2, only the cubilin-megalin complex is known to bind Tf (22), so it is clear that further work is necessary to explore the molecular basis for the localization of Tf to MVB and its resistance to lysosomal proteolysis. Because TfR2 is important for iron homeostasis, we hypothesize that the differential localization of Tf in this organelle might facilitate downstream effects, perhaps via interactions with the putative MVB-targeted receptor. Interestingly, in some cell types, MVB function in regulated exocytosis such that their contents are released (20); they may also function in the biogenesis of secretory organelles that subsequently undergo regulated secretion to export their cargo (41). Tf is known to be secreted in bile, so one hypothesis is that its appearance in hepatocyte MVB might be coupled to transcytotic clearance from circulation.
Regardless of the precise mechanism, downstream consequences of the differential intracellular targeting of Tf to MVB most likely involve modulation of serum Tf concentration and/or decreased Tf stabilization or recycling. Any of these factors would contribute to the pathology of iron overload observed in patients with hemochromatosis who have mutations in TfR2 (4, 14, 26, 32). Clinical information about iron loading associated with TFR2 mutations indicates that the metal accumulates in periportal hepatocytes (30). On the basis of these observations and the pattern of Tf traffic reported in the present study, it seems rather unlikely that hepatic iron import is the major role of TfR2. In this respect, it is also interesting to note that hepatic expression of TfR2 and a newly identified iron-regulatory factor, hepcidin, are significantly correlated in a manner that is independent of body iron status (12). Hepcidin appears to be a negative regulator of iron release (11). It is possible that the delivery of Tf to MVB is associated with the regulation of hepcidin expression such that disruption of this trafficking pathway because of loss of TfR2 function leads to alterations in tissue iron storage. It is clear that further characterization of the Tf-TfR2 trafficking pathway is necessary to elucidate key elements involved in the regulation of iron homeostasis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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