The role of Hfe in transferrin-bound iron uptake by hepatocytes

Abstract

HFE-related hereditary hemochromatosis results in hepatic iron overload. Hepatocytes acquire transferrin-bound iron via transferrin receptor (Tfr) 1 and Tfr1-independent pathways (possibly Tfr2-mediated). In this study, the role of Hfe in the regulation of hepatic transferrin-bound iron uptake by these pathways was investigated using Hfe knockout mice. Iron and transferrin uptake by hepatocytes from Hfe knockout, non-iron-loaded and iron-loaded wild-type mice were measured after incubation with 50 nM 125 I-Tf-59 Fe (Tfr1 pathway) and 5 M 125 I-Tf-59 Fe (Tfr1-independent or putative Tfr2 pathway). Tfr1 and Tfr2 messenger RNA (mRNA) and protein expression were measured by real-time polymerase chain reaction and western blotting, respectively. Tfr1-mediated iron and transferrin uptake by Hfe knockout hepatocytes were increased by 40% to 70% compared with iron-loaded wild-type hepatocytes with similar iron levels and Tfr1 expression. Iron and transferrin uptake by the Tfr1-independent pathway was approximately 100-fold greater than by the Tfr1 pathway and was not affected by the absence of Hfe. Diferric transferrin increased hepatocyte Tfr2 protein expression, resulting in a small increase in transferrin but not iron uptake by the Tfr1-independent pathway. Conclusion: Tfr1-mediated iron uptake is regulated by Hfe in hepatocytes. The Tfr1-independent pathway exhibited a much greater capacity for iron uptake than the Tfr1 pathway but it was not regulated by Hfe. A homozygous C282Y mutation in the HFE gene results in hereditary hemochromatosis and is characterized by increased iron absorption and transferrin saturation that leads to hepatic iron overload. The hemochromatosis protein, HFE, is closely associated with transferrin receptor 1 (TFR1) and competes with transferrin for the same binding sites on TFR1. 1 Hepatocytes acquire transferrin-bound iron via TFR1 by a high-affinity endocytic process. 2 A second transferrin receptor has been identified and was named transferrin receptor 2 (TFR2). Like TFR1, it transports transferrin-bound iron by a receptor-mediated endocytic process, 3 although the affinity of this pathway is up to 30-fold lower than that of TFR1. 4 In iron overload, transferrin saturation and diferric transferrin levels are increased, resulting in an up-regulation of TFR2 protein expression, 5,6 suggesting that hepatocyte iron loading may involve TFR2. It has been postulated that TFR2 may mediate the TFR1-independent uptake process described previously in hepatic cells. [7][8][9] In addition to its role in iron transport, TFR2 also has a role in the regulation of cellular iron metabolism because mutations in TFR2 cause hepatic iron overload. 10,11 TFR2 is predominantly expressed in hepatocytes 12 and has recently been shown to interact with HFE. 13 Unlike TFR1, TFR2 is not regulated by intracellular iron levels because there are no iron-responsive elements in the untranslated region of its messenger RNA (mRNA). 12 Hepcidin is synthesized by hepatocytes and is a key regulator of iron homeostasis. 14 It ...

The role of transferrin receptor 1 and 2 in transferrin-bound iron uptake in human hepatoma cells

“…Taken together, these observations make it clear that TFR1 is the dominant TFR involved in iron uptake in hepatoma cells. Furthermore, we showed that cells treated with both TFR1 and TFR2 siRNA did not exhibit impaired iron uptake at a high transferrin concentration, providing support for the existence of a TFR-independent pathway in hepatoma cells which involves the low-affinity endocytosis of transferrin as described previously in hepatocytes (9).…”

“…Human apotransferrin was labeled with 57 CoCl2 (Perkin Elmer) as detailed above for unlabeled Co2Tf. TFR1-mediated uptake of Fe2Tf was measured at a transferrin concentration of 50 nM, and TFR1-independent uptake (putative TFR2 pathway) was measured at a transferrin concentration of 5 M, as described previously (9,28). Uptake of Co 2Tf was measured at the same concentrations as Fe2Tf.…”

“…In liver, TFR1 expression is low and is reduced even further with iron loading, whereas basal liver TFR2 expression is normally high and increases with rising transferrin saturation (21,35). The high-affinity TFR1-mediated pathway in hepatocytes and HuH7 human hepatoma cells saturates at low transferrin concentrations between 50 and 100 nM in vitro (9,40). A TFR1-independent uptake pathway for transferrin-bound iron (TBI) has been described in hepatic cells as a low-affinity, highercapacity process (19,28,39,40).…”

ZRT/IRT-like Protein 14 (ZIP14) Promotes the Cellular Assimilation of Iron from Transferrin

“…Hepatocytes and hepatoma cells are known to take up Fe-TF by several mechanisms including a high affinity, low capacity TFR1-mediated endocytic pathway and a low affinity, high capacity TFR1-independent pathway (28 -31). In primary mouse hepatocytes and HuH7 hepatoma cells, the TFR1-mediated pathway saturates at low TF concentrations between 50 and 100 nM (31,32). Here, we document that in HepG2 cells incubated with 100 nM TF, knockdown of endogenous ZIP14-FLAG resulted in 45% less assimilation of iron from TF compared with controls.…”

Enhanced erythropoiesis in Hfe-KO mice indicates a role for Hfe in the modulation of erythroid iron homeostasis

“…Several studies showed that Hfe competes with Tf for the same binding sites on Tfrc, thereby impairing iron uptake. [16][17][18][19] Yet, despite all of the studies describing interaction of Hfe with Tfrc, [19][20][21] few investigated their association within the erythroid compartment, where the latter is known to be essential. 22 Earlier studies reported that Hfe could not be detected in nucleated erythroid cells 23 and that liver-specific ablation of Hfe mirrors most of the HH phenotype, indicating that it plays a pivotal role in this organ.…”