SUMMARY The mechanisms that allow the body to sense iron levels in order to maintain iron homeostasis are unknown. Patients with the most common form of hereditary iron overload have mutations in the hereditary hemochromatosis protein, HFE. They have lower levels of hepcidin, than unaffected individuals. Hepcidin, a hepatic peptide hormone, negatively regulates iron efflux from the intestines into the blood. We report two hepatic cell lines, WIF-B cells and HepG2 cells transfected with HFE, where hepcidin expression responded to iron-loaded transferrin. The response was abolished when endogenous transferrin receptor 2 (TfR2) was suppressed or in primary hepatocytes lacking either functional TfR2 or HFE. Furthermore, transferrin-treated HepG2 cells transfected with HFE chimeras containing only the α3 and cytoplasmic domains could upregulate hepcidin expression. Since the HFE α3 domain interacts with TfR2, these results supported our finding that TfR2/HFE complex is required for transcriptional regulation of hepcidin by holo-Tf.
ZIP14 is a transmembrane metal ion transporter that is abundantly expressed in the liver, heart, and pancreas. Previous studies of HEK 293 cells and the hepatocyte cell lines AML12 and HepG2 established that ZIP14 mediates the uptake of nontransferrin-bound iron, a form of iron that appears in the plasma during pathologic iron overload. In this study we investigated the role of ZIP14 in the cellular assimilation of iron from transferrin, the circulating plasma protein that normally delivers iron to cells by receptor-mediated endocytosis. We also determined the subcellular localization of ZIP14 in HepG2 cells. We found that overexpression of ZIP14 in HEK 293T cells increased the assimilation of iron from transferrin without increasing levels of transferrin receptor 1 or the uptake of transferrin. To allow for highly specific and sensitive detection of endogenous ZIP14 in HepG2 cells, we used a targeted knock-in approach to generate a cell line expressing a FLAG-tagged ZIP14 allele. Confocal microscopic analysis of these cells detected ZIP14 at the plasma membrane and in endosomes containing internalized transferrin. HepG2 cells in which endogenous ZIP14 was suppressed by siRNA assimilated 50% less iron from transferrin compared with controls. The uptake of transferrin, however, was unaffected. We also found that ZIP14 can mediate the transport of iron at pH 6.5, the pH at which iron dissociates from transferrin within the endosome. These results suggest that endosomal ZIP14 participates in the cellular assimilation of iron from transferrin, thus identifying a potentially new role for ZIP14 in iron metabolism. Most cells acquire iron from transferrin (TF),2 a circulating plasma protein that can carry up to two ferric (Fe 3ϩ ) iron atoms. After Fe-TF binds to cell surface TF receptor 1 (TFR1), the plasma membrane invaginates into clathrin-coated pits, which internalize the Fe-TF⅐TFR1 complex into endosomes. Upon endosomal acidification, Fe 3ϩ is released and subsequently reduced to Fe 2ϩ . The liberated Fe 2ϩ is then transported across the endosomal membrane and into the cytosol (1).The assimilation of iron from TF has been best characterized in developing erythroid cells, the most avid consumers of TFbound iron (TBI). In these cells, reduction of Fe 3ϩ is catalyzed by the oxidoreductase Steap3 (2), and iron transport out of the endosome is facilitated by the transmembrane protein divalent metal transporter 1 (DMT1) (3, 4). Accordingly, mice lacking either Steap3 or DMT1 cannot incorporate sufficient iron into developing erythrocytes and become anemic (2, 3). After the erythroid marrow, the second largest consumer of TBI is the liver, accounting for 10 -20% of iron exchange with the plasma (5). Interestingly, anemic Steap3-mutant mice or DMT1-null mice are able to take up iron into the liver (6, 7), indicating that Steap3 and DMT1 are dispensable for hepatic iron uptake.Under normal conditions, Ͼ95% of plasma iron is TBI. Studies in perfused rat liver document that the liver takes up TBI, almost exclusively into he...
Both hemojuvelin (HJV) and bone morphogenic protein-6 (BMP6) are essential for hepcidin expression. Hepcidin is the key peptide hormone in iron homeostasis, and is secreted predominantly by hepatocytes. HJV expression is detected in hepatocytes, as well as in skeletal and heart muscle. HJV binds BMP6 and increases hepcidin expression presumably by acting as a BMP co-receptor. We characterized the role of hepatocyte HJV in the regulation of BMP6 and hepcidin expression. In HJV-null (Hjv Iron is an indispensable element for life. Iron homeostasis is controlled elegantly by hepcidin (1, 2). Hepcidin is a peptide hormone that is secreted predominantly by the hepatocytes in the liver. Under physiological conditions, its expression is regulated positively by bodily iron-loading (3, 4). Recent studies demonstrate that normal levels of hepatic hepcidin expression require the presence of both hemojuvelin (HJV) 2 and bone morphogenic protein-6 (BMP6) (5-7).HJV is a GPI-linked membrane protein that is encoded by the gene, HFE2, in humans (8, 9). Repulsive guidance molecule c (RGMc) is the ortholog of HJV in mice, and it is encoded by the gene, Hfe2. For simplicity, we will use Hjv for the gene and HJV for the protein in human and mice in this report. Hjv is expressed highly in skeletal and heart muscle, and at a relatively low level in hepatocytes (9, 10). Homozygous or compound heterozygous mutations of HJV in humans and disruption of both Hjv alleles (Hjv Ϫ/Ϫ ) in mice, markedly reduce hepatic hepcidin expression and cause severe iron overload (9, 11-13). Thus, HJV is a critical upstream regulator of hepcidin transcription. However, the precise role of HJV expression in different tissues in the regulation of hepcidin expression in vivo has not been addressed.Hepatic hepcidin expression is mediated via the BMP signaling cascade (14). BMP signaling is initiated upon the binding of BMP ligands to BMP receptor complexes on the cell surface. This binding triggers the phosphorylation of Smad1, Smad5, and Smad8 (Smad1/5/8) in the cytoplasm. The phosphorylated Smads (pSmad1/5/8) form heteromeric complexes with Smad4 and then translocate to the nucleus where they induce the transcription of target genes (15). Liver-specific disruption of Smad4 in mice markedly decreases hepcidin expression and causes iron accumulation in particular organs (14).Hepatocytes are the only known cell types in the body where both HJV and hepcidin are co-expressed (3,10,12,16). In hepatoma cell lines, hepcidin expression can be induced robustly by BMP2, BMP4, and BMP6 independently of HJV (10,17,18). Recent studies show that BMP6, rather than other BMP ligands, plays an essential role in iron homeostasis. BMP6 mRNA in the liver and the small intestine is positively regulated by bodily iron-loading (5, 19). Disruption of both BMP6 alleles in mice markedly suppresses hepatic hepcidin expression and causes severe iron overload (6, 7). These phenotypes resemble those reported in Hjv Ϫ/Ϫ mice (12, 13). Therefore, BMP6 and HJV are both important in the induct...
Mutations in the transmembrane glycoproteins transferrin receptor 2 (TfR2) and HFE are associated with hereditary hemochromatosis. Interactions between HFE and transferrin receptor 1 (TfR1) have been mapped to the ␣1-and ␣2-helices in HFE and to the helical domain of TfR1. Recently, TfR2 was also reported to interact with HFE in transfected mammalian cells. To test whether similar HFE residues are important for both TfR1 and TfR2 binding, a mutant form of HFE (W81AHFE) that has a ϳ5,000-fold lower affinity for TfR1 than HFE was employed. As expected, W81AHFE does not interact with TfR1. However, we found that the same mutation in HFE does not affect the TfR2/HFE interaction. This finding indicates that the TfR2/HFE and TfR1/HFE interactions are distinct. We further observed that, unlike TfR1/HFE, Tf does not compete with HFE for binding to TfR2 and that binding is independent of pH (pH 6 -7.5). TfR2-TfR1 and HFE-HLA-B7 chimeras were generated to map the domains of the TfR2/HFE interaction. TfR1 and HLA-B7 were chosen because of their similar overall structures with TfR2 and HFE, respectively. We mapped the interacting domains to the putative stalk and protease-like domains of TfR2 located between residues 104 and 250 and to the ␣3 domain of HFE, both of which differ from the TfR1/HFE interacting domains. Furthermore, we found that HFE increases TfR2 levels in hepatic cells independent of holo-Tf.Hereditary hemochromatosis (HH) 3 is a prevalent inherited iron metabolism disorder characterized by excessive iron deposition in the liver, heart, pancreas, and parathyroid and pituitary glands. The excess iron deposited in these organs is toxic, which leads to multi-organ dysfunction (1, 2). HH type 1, an autosomal recessive form of the disease, is caused by mutations in the HFE gene (3, 4), which encodes an atypical MHC class I protein. The majority of HH type 1 patients are homozygous for C282Y 4 substitution, which has a prevalence of ϳ1 per 400 individuals of northern European descent (5). Interestingly, patients with this same mutation present very different phenotypes, ranging from mild to severe (6). The different presentations of the same mutation suggest that HFE function depends on the presence of modifiers (7-10), acting directly or indirectly with HFE.The first modifier was reported to be transferrin receptor 1 (TfR1). In vitro (11) and in vivo (12) studies showed that, at slightly alkaline pH 7.5, HFE interacts with the ecto-domain of TfR1. Interestingly, both HFE and diferric-transferrin (holo-Tf) recognize overlapping regions on TfR1 (13, 14), which results in competition between HFE and holo-Tf for binding to TfR1 (15,16). At concentrations of holo-Tf (100 nM) well below the physiological level of holo-Tf in the blood (ϳ10 M), no binding of HFE to TfR1 can be detected (17). Expression of wild type HFE decreases intracellular iron levels in both the cells expressing endogenous TfR1 (16,18,19) and the cells lacking endogenous TfR1 (20). Moreover, a mutated form of HFE (W81AHFE), which has ϳ5,000-fold lowe...
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