Regulation of hepatic transferrin, transferrin receptor and ferritin genes in human siderosis

Although many studies have examined the regulation of transferrin, transferrin receptor and ferritin subunit gene expression in experimental systems, no molecular biological data in humans have been documented to date. In this study we simultaneously analyzed the hepatic content of transferrin, transferrin receptor and heavy and light ferritin subunit messenger RNAs in tissue samples obtained from subjects with normal iron balance and patients with primary or secondary iron overload. Steady-state levels of transferrin messenger RNA were not depressed by iron overload. On the contrary, they were increased (p less than 0.001) in patients with severe hepatic siderosis (liver iron content greater than 200 mumol/gm dry wt) as compared with the control group. This indicates that, as already suggested by our previous data in experimental siderosis, iron maintains the ability to induce transferrin gene activity even when cellular iron content is significantly increased. Transferrin receptor gene expression was found to respond in the same manner to any cause of iron-tissue load, regardless of the cause. In fact, a lower signal for transferrin receptor messenger RNA was consistently detected in iron-overloaded patients vs. control subjects, particularly in patients with thalassemia major and idiopathic hemochromatosis (p less than 0.001). Ferritin light-subunit messenger RNA accumulation was significantly increased in those patients with severe siderosis (idiopathic hemochromatosis and thalassemia major = liver iron between 200 and 600 mumol/gm dry wt). The fact that no significant change in hepatic ferritin heavy-subunit gene expression was detected in iron-loaded patients confirms preferential production of light-subunit--enriched ferritins in long-term iron overload.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical evidence for a lack of ferritin in duodenal absorptive epithelial cells in idiopathic hemochromatosis

Patients with idiopathic hemochromatosis exhibit an unexplained increase in intestinal iron absorption. The aim of this work was to study immunohistochemical H- and L-ferritin distribution in duodenal mucosal cells of patients with idiopathic hemochromatosis, and of subjects with various degrees of iron loading. Biopsy sections of gastrointestinal mucosa from 24 patients with idiopathic hemochromatosis, 10 patients with secondary iron overload, 6 normal subjects, and 13 iron-deficient subjects were analyzed with monoclonal antibodies for the presence of immunohistochemical H and L ferritin types, and with Perls' stain for hemosiderin. Ferritin content of duodenal homogenates was evaluated in 5 cases. The absorptive duodenal cells were found to contain ferritin, mostly of the L type, in apical granules; these ferritin granules were present in all normal, iron-deficient, and iron-over-loaded subjects, but were absent in 21 (87%) of the patients with established idiopathic hemochromatosis. In cells other than those of the duodenal epithelium, such as lamina propria or antral mucosa, ferritin and hemosiderin contents were related to iron loading and no difference was evident between primary and secondary iron overload. These findings indicate that (a) idiopathic hemochromatosis is associated with an altered ferritin expression in the duodenal absorptive epithelial cells, (b) this alteration cannot be detected by analysis of duodenal homogenates, (c) idiopathic hemochromatosis does not affect ferritin accumulation in the other cell types analyzed, and (d) ferritin in absorptive duodenal cells may have a regulatory role in iron absorption.     

Heterotypic interactions between transferrin receptor and transferrin receptor 2

Cellular iron uptake in most tissues occurs via endocytosis of diferric transferrin (Tf) bound to the transferrin receptor (TfR). Recently, a second transferrin receptor, transferrin receptor 2 (TfR2), has been identified and shown to play a critical role in iron metabolism. TfR2 is capable of Tf-mediated iron uptake and mutations in this gene result in a rare form of hereditary hemochromatosis unrelated to the hereditary hemochromatosis protein, HFE. Unlike TfR, TfR2 expression is not controlled by cellular iron concentrations and little information is currently available regarding the role of TfR2 in cellular iron homeostasis. To investigate the relationship between TfR and TfR2, we performed a series of in vivo and in vitro experiments using antibodies generated to each receptor. Western blots demonstrate that TfR2 protein is expressed strongest in erythroid/myeloid cell lines. Metabolic labeling studies indicate that TfR2 protein levels are approximately 20-fold lower than TfR in these cells. TfR and TfR2 have similar cellular localizations in K562 cells and coimmunoprecipitate to only a very limited extent. Western analysis of the receptors under nonreducing conditions reveals that they can form heterodimers.     

Differential expression of transferrin receptor in duodenal mucosa in iron overload. Evidence for a site-specific defect in genetic hemochromatosis

In genetic hemochromatosis, metabolic studies have demonstrated inappropriately increased iron absorption by cells of the duodenal mucosa. It is not clear whether this reflects an intrinsic abnormality of iron homeostasis at this site or is a consequence of a more generalized defect in cellular iron metabolism particularly involving the liver. We have previously used the expression of iron-related proteins as markers of iron homeostasis and have demonstrated normal regulation of the transferrin receptor and ferritin in the liver in this condition. In the present study we used immunohistochemical techniques to study transferrin-receptor expression in the gastrointestinal epithelium in normal subjects and patients with iron overload. In untreated genetic hemochromatosis and normal subjects, villus epithelial cells expressed receptor in the basolateral, subnuclear region. In contrast, in patients with secondary iron overload, receptor staining was absent in villus epithelial cells. The cells in the duodenal crypts showed intense staining for the transferrin receptor in all subjects investigated, a finding consistent with the known behavior of this receptor in proliferating cells. Given that body iron stores in both types of iron overload were comparable, these findings indicating a failure of down-regulation of the villus enterocyte transferrin receptor in genetic hemochromatosis may reflect the presence of a regulatory defect associated with the inability to control iron absorption in this condition.     

The hereditary hemochromatosis protein, HFE, lowers intracellular iron levels independently of transferrin receptor 1 in TRVb cells

Hereditary hemochromatosis (HH) is an autosomal recessive disease that leads to parenchymal iron accumulation. The most common form of HH is caused by a single amino acid substitution in the HH protein, HFE, but the mechanism by which HFE regulates iron homeostasis is not known. In the absence of transferrin (Tf), HFE interacts with transferrin receptor 1 (TfR1) and the 2 proteins co-internalize, and in vitro studies have shown that HFE and Tf compete for TfR1 binding. Using a cell line lacking endogenous transferrin receptors (TRVb cells) transfected with different forms of HFE and TfR1, we demonstrate that even at low concentrations Tf competes effectively with HFE for binding to TfR1 on living cells. Transfection of TRVb cells or the derivative line TRVb1 (which stably expresses human TfR1) with HFE resulted in lower ferritin levels and decreased Fe2+ uptake. These data indicate that HFE can regulate intracellular iron storage independently of its interaction with TfR1. Earlier studies found that in HeLa cells, HFE expression lowers Tf-mediated iron uptake; here we show that HFE lowers non-Tf-bound iron in TRVb cells and add to a growing body of evidence that HFE may play different roles in different cell types.     

Regulation of transferrin receptor 2 protein levels by transferrin

Transferrin receptor 2 (TfR2) plays a critical role in iron homeostasis because patients carrying disabling mutations in the TFR2 gene suffer from hemochromatosis. In this study, iron-responsive regulation of TfR2 at the protein level was examined in vitro and in vivo. HepG2 cell TfR2 protein levels were up-regulated after exposure to holotransferrin (holoTf) in a time- and dose-responsive manner. ApoTf or high-iron treatment with non-Tf-bound iron failed to elicit similar effects, suggesting that TfR2 regulation reflects interactions of the iron-bound ligand. Hepatic TfR2 protein levels also reflected an adaptive response to changing iron status in vivo. Liver TfR2 protein levels were down- and up-regulated in rats fed an iron-deficient and a high-iron diet, respectively. TfR2 was also up-regulated in Hfe(-/-) mice, an animal model that displays liver iron loading. In contrast, TfR2 levels were reduced in hypotransferrinemic mice despite liver iron overload, supporting the idea that regulation of the receptor is dependent on Tf. This idea is confirmed by up-regulation of TfR2 in beta-thalassemic mice, which, like hypotransferrinemic mice, are anemic and incur iron loading, but have functional Tf. Based on these combined results, we hypothesize that TfR2 acts as a sensor of iron status such that receptor levels reflect Tf saturation.     

Duodenal ferritin content and structure: relationship with body iron stores in man

The relationship between serum ferritin and duodenal ferritin was examined in normal subjects and in patients with iron deficiency, secondary iron overload, or idiopathic hemochromatosis (IHC). A positive correlation between serum ferritin and duodenal ferritin concentrations was found in all groups. In the iron-overload conditions, duodenal ferritin concentration was lower at all levels of serum ferritin in comparison with normal and iron-deficient subjects. Patients with secondary iron overload did not differ from those with IHC, which indicates that any decrease in duodenal ferritin concentration was secondary to the excess body iron stores. Purified duodenal ferritin from normal subjects and patients with iron-overload conditions showed the same two distinct isoferritins by isoelectric focusing. After the oral administration of iron, two additional isoferritins were detected. These resembled the major isoferritins of liver.     

Mechanisms of HFE-induced regulation of iron homeostasis: Insights from the W81A HFE mutation

The mechanisms by which the hereditary hemochromatosis protein, HFE, decreases transferrin-mediated iron uptake were examined. Coimmunoprecipitation studies using solubilized cell extracts demonstrated that transferrin (Tf) competed with HFE for binding to the transferrin receptor (TfR) similar to previous in vitro studies using soluble truncated forms of HFE and the TfR. At concentrations of Tf approaching those found in the blood, no differences in Tf binding to cells were detected, which is consistent with the lower binding constant of HFE for TfR versus Tf. However, cells expressing HFE still showed a decrease in Tf-mediated iron uptake at concentrations of Tf sufficient to dissociate HFE from the TfR. These results indicate that the association of HFE with TfR is not essential for its ability to lower intracellular iron stores. To test the effect of HFE on lowering intracellular iron levels independently of its association with TfR, a mutated HFE (fW81AHFE) that shows greatly reduced affinity for the TfR was transfected into tetracycline-controlled transactivator HeLa cells. HeLa cells expressing fW81AHFE behaved in a similar manner to cells expressing wild-type HFE with respect to decreased intracellular iron levels measured by iron regulatory protein gel-shift assays and ferritin levels. The results indicate that HFE can lower intracellular iron levels independently of its interaction with the TfR.     

Quantitation of ferritin iron in plasma, an explanation for non- transferrin iron

In 33 patients with thalassemia and idiopathic hemochromatosis, plasma ferritin protein levels ranged from 36 to 5,850 micrograms/L. The iron content of this ferritin as determined by immunoprecipitation ranged from undetectable amounts to 507 micrograms/L. The mean iron content of ferritin protein in those and other subjects with plasma ferritin concentrations of over 1,000 was 6.8% +/- 2.7%. Plasma transferrin was usually saturated with iron in patients with measurable ferritin iron, but exceptions occurred. In studies using electrophoretic separation, it was shown that some ferritin iron moved to transferrin during in vitro incubation, whereas exchange in the opposite direction was extremely limited. Because some plasma ferritin iron was measured by the standard colorimetric plasma iron determination, these observations (a) indicate that plasma ferritin contains a significant amount of iron (b) indicate that a significant proportion of nontransferrin iron in individuals with nontransferrin iron as detected by standard plasma iron and total iron-binding capacity measurements is due to the presence of ferritin, and (c) suggest that large amounts of ferritin iron may affect the saturation of plasma transferrin.     

Serum transferrin receptor in hereditary hemochromatosis and African siderosis

The present investigation evaluated the serum transferrin receptor concentration in subjects with nontransfusional iron overload who were identified in two separate studies on the basis of a serum ferritin level above 400 μg/L. Subjects with preciinical hereditary hemochromatosis were evaluated in the first study and those with the African form of iron overload in the second. in the first study, hereditary hemochromatosis was identified in 14 white men on the basis of a persistent elevation in transferrin saturation above 55%. The serum receptor concentration was elevated above the upper cut-off of 8.5 mg/L in two of the subjects, but the mean receptor of 6.1 ± 1.4 mg/L (mean ± 2 SE) did not differ significantly from the normal mean for this assay of 5.6 ± 0.3 mg/L. In the same study, 60 control subjects with secondary iron overload were identified on the basis of a serum ferritin persistently above 400 μg/L, with a normal serum C-reactive protein concentration but with a transferrin saturation <55%. Three of these subjects had an elevated serum receptor concentration but the mean value of 5.5 ± 0.4 mg/L did not differ from normals nor from subjects with hemochromatosis. In the second study, 49 black Africans with iron overload were divided into those with or without an elevated transferrin saturation. The mean serum receptor concentration of 5.0 ± 0.8 mg/L and 4.5 ± 0.4 mg/L, respectively, did not differ statistically. It was concluded that there is no evidence of generalized dysreguiation of the transferrin receptor in hemochromatosis or African siderosis. © 1994 Wiley-Liss, Inc.     

Duodenal expression of a putative stimulator of Fe transport and transferrin receptor in anemia and hemochromatosis

BACKGROUND & AIMS: Stimulator of Fe Transport (SFT) and transferrin receptor (TfR) are proteins involved in iron transport. This study evaluated iron metabolism protein expression in duodenal biopsy specimens from controls and patients with abnormal iron metabolism. METHODS: Twelve controls, 8 patients with iron deficiency anemia, 7 with HFE-related hemochromatosis, and 6 with non-HFE-related iron overload were studied. Immunohistochemistry was performed on duodenal biopsy specimens with anti-TfR and anti-SFT antibodies which recognize a putative stimulator of Fe transport of ~80 kilodaltons. RESULTS: In controls, the putative stimulator of Fe transport was expressed in the middle and distal part of the villi in the subapical cytoplasmatic region. Its expression increased in anemics and, to a lesser degree, in HFE-related hemochromatotics, whereas it was reduced in patients with non-HFE-related iron overload. TfR expression showed a crypt-to-tip gradient in controls, but not in anemics, in whom it was uniformly overexpressed. TfR expression was intermediate in HFE-related hemochromatotics and similar to controls in non-HFE-related iron overload. CONCLUSIONS: Expression of the putative stimulator of Fe transport and TfR increases in iron deficiency. Increased expression of both proteins is present only in HFE-related hemochromatotics suggesting that other factors may be involved in determining non-HFE-related iron overload phenotype.     

Mutation analysis of the transferrin receptor-2 gene in patients with iron overload

Three mutations in the transferrin receptor-2 gene have recently been identified in four Sicilian families with iron overload who had a normal hemochromatosis gene, HFE (C. Camaschella, personal communication). To determine the extent to which mutations in the transferrin receptor-2 gene occur in other populations with iron overload, we have completely sequenced this gene in 17 whites, 10 Asians, and 8 African Americans with iron overload and a C282C/C282C HFE genotype, as well as 4 subjects without iron overload and homozygous for the mutant HFE C282Y genotype, 5 patients with iron overload and homozygous for the mutant HFE C282Y genotype, and 5 normal individuals. None of the individuals exhibited the Sicilian mutations, Y250X in exon 6, M172K in exon 4, and E60X in exon 2. One iron-overloaded individual of Asian descent exhibited a I238M mutation which was subsequently found to be a polymorphism present in the Asian population at a frequency of 0.0192. The presence of the I238M mutation was not associated with an increase in ferritin or transferrin saturation levels. Three silent polymorphisms were also identified, nt 1770 (D590D) and nt 1851 (A617A) and a polymorphism at nt 2255 in the 3' UTR. Thus, mutations in the transferrin receptor-2 gene were not responsible for the iron overload seen in our subjects.     

Unsaturated iron binding capacity and transferrin saturation are equally reliable in detection of HFE hemochromatosis

OBJECTIVE: Unsaturated iron binding capacity (UIBC) has been proposed as an inexpensive alternative to transferrin saturation for detection of hereditary hemochromatosis. The aim of this study was to compare, in a hospital referral clinic, the reliability of transferrin saturation and UIBC for detection of subjects who have inherited HFE (HLA-asociated iron overload) genotypes predisposing to iron overload. METHODS: Serum transferrin saturation, UIBC, and ferritin were tested in 110 consecutive subjects. Optimum thresholds were determined from receiver operating characteristic curves. RESULTS: Of 110 subjects, 44 carried significant HFE mutations (C282Y/C282Y or C282Y/H63D). In genetically predisposed subjects with biochemical expression, the optimum threshold for transferrin saturation was 43%, giving a sensitivity of 0.88 and specificity 0.95. For UIBC, the optimum threshold was 143 microg/dL (25.6 micromol/L), giving a sensitivity of 0.91 and specificity of 0.95. In patients referred with a family history or clinical suspicion of hemochromatosis, transferrin saturation and UIBC were highly reliable predictors of genotype. In patients referred for investigation of abnormal liver enzymes without a known family history of hemochromatosis, a normal transferrin saturation or normal UIBC was highly reliable in excluding hemochromatosis. CONCLUSIONS: Transferrin saturation and UIBC have equal reliability in ability to predict hemochromatosis. UIBC should be considered as an alternative to transferrin saturation in detection of hemochromatosis.     

Analyses for binding of the transferrin family of proteins to the transferrin receptor 2

Transferrin receptor 2 alpha (TfR2 alpha), the major product of the TfR2 gene, is the second receptor for transferrin (Tf), which can mediate cellular iron uptake in vitro. Homozygous mutations of TfR2 cause haemochromatosis, suggesting that TfR2 alpha may not be a simple iron transporter, but a regulator of iron by identifying iron-Tf. In this study, we analysed the ligand specificity of TfR2 alpha using human transferrin receptor 1 (TfR1) and TfR2 alpha-stably transfected and expressing cells and flow-cytometric techniques. We showed that human TfR2 alpha interacted with both human and bovine Tf, whereas human TfR1 interacted only with human Tf. Neither human TfR1 nor TfR2 alpha interacted with either lactoferrin or melanotransferrin. In addition, by creating point mutations in human TfR2 alpha, the RGD sequence in the extracellular domain of TfR2 alpha was shown to be crucial for Tf-binding. Furthermore, we demonstrated that mutated TfR2 alpha (Y250X), which has been reported in patients with hereditary haemochromatosis, also lost its ability to interact with both human and bovine Tf. Although human TfR1 and TfR2 alpha share an essential structure (RGD) for ligand-binding, they have clearly different ligand specificities, which may be related to the differences in their roles in iron metabolism.     

A novel mutation of transferrin receptor 2 in a Taiwanese woman with type 3 hemochromatosis

Hereditary hemochromatosis (HH) is very rare in Asia. Here, we describe a Taiwanese woman presenting with fully developed characteristics of HH including bronze skin, DM, decreased MRI T2 signal intensity over liver and pituitary gland. Biochemistry of iron profile indicated a severe status of iron overload by serum iron: 194 microg/dL, serum ferritin: 6640 microg/L, transferrin saturation: 92.8%. By measuring the hepatic iron index 8.48 (>1.9) of her liver biopsy tissue, the diagnosis of HH was established. Diagnosis of non-HFE HH was carried out since the whole HFE genome was sequenced but failed to localize any genetic alterations. The whole genome of transferrin receptor 2 (TfR2) was sequenced and a novel mutation of 13528 G-->A (Arg 481 His) in exon 11 was detected. Therefore, type 3 hemochromatosis was confirmed. The distinct clinical features, extremely high iron index and impressive iron staining in her liver biopsy tissue may represent an aggravated iron deposition in the liver caused by this novel mutation. Our finding implicates functional importance of histidine in exchange of arginine at amino acid 481 of transferrin receptor 2 in iron homeostasis. This case reminds physicians in Asia to keep in mind that hemochromatosis could be a rare cause of DM.     

Stoichiometries of transferrin receptors 1 and 2 in human liver

Mutations in either the hereditary hemochromatosis protein, HFE, or transferrin receptor 2, TfR2, result in a similarly severe form of the most common type of iron overload disease called hereditary hemochromatosis. Models of the interactions between HFE, TfR1, and TfR2 imply that these proteins are present in different molar concentrations in the liver, where they control expression of the iron regulatory hormone, hepcidin, in response to body iron loading. The aim of this study was to determine in vivo levels of mRNA by quantitative RT-PCR and concentrations of these proteins by quantitative immunoblotting in human liver tissues. The level of TfR2 mRNA was 21- and 63-fold higher than that of TfR1 and HFE, respectively. Molar concentration of TfR2 protein was the highest and determined to be 1.95 nmol/g protein in whole cell lysates and 10.89 nmol/g protein in microsomal membranes. Molar concentration of TfR1 protein was 4.5- and 6.1-fold lower than that of TfR2 in whole cell lysates and membranes, respectively. The level of HFE protein was below 0.53 nmol/g of total protein. HFE is thus present in substoichiometric concentrations with respect to both TfR1 and TfR2 in human liver tissue. This finding supports a model, in which availability of HFE is limiting for formation of complexes with TfR1 or TfR2.