Hepcidin Regulation by HFE and TFR2: Is It Enough to Give a Hepatocyte a Complex?

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Gao J, Chen J, Kramer M, et al. (Department of Cell and Developmental Biology, Oregon Health & Science University, Portland, OR; others). Interaction of the hereditary hemochromatosis protein HFE with Transferrin Receptor 2 is required for transferrin-induced hepcidin expression. Cell Metabolism 2009;9:217–227.

The regulation of iron homeostasis is controlled to a large extent by the iron-regulatory hormone hepcidin. Hepcidin is secreted from the liver in situations of iron overload and acts to reduce levels of iron in the circulation by reducing iron release from cells by binding to the iron exporter ferroportin, inducing its internalization and degradation. Hepcidin expression in the liver is regulated by a number of factors, including iron, inflammatory cytokines, bone morphogenetic proteins (BMPs) and hypoxia. In all forms of hereditary hemochromatosis (HH), there is reduced expression of hepcidin in relation to iron stores. The role of the hepatocyte and HH-associated proteins in the regulation of hepcidin and in turn iron homeostasis is becoming clearer. For example hemojuvelin, the protein mutated in most forms of juvenile hemochromatosis is a BMP co-receptor and enhances BMP-induced regulation of hepcidin (Nat Genet 2006;38:531–539). The role of 2 other important HH-associated proteins—HFE and TFR2—in iron metabolism has recently been the subject of intensive research. The genetic basis of the most common form of HH, mutations in the HFE gene, was identified in 1996 (Nat Genet 1996;13:399–340). The aberrant localization of the most common mutant form (the C282Y mutation) of this major histocompatibility complex class I-like molecule in the endoplasmic reticulum suggested that its regulatory role lay in its expression on the cell surface. Not long after the HFE gene was identified, it was shown that the encoded protein interacted with the transferrin receptor (TFR1), thus linking its function with iron metabolism (Proc Natl Acad Sci U S A 1998;5:1472–1477). When the TFR1 homolog transferrin receptor 2 (TFR2) was identified and implicated in another form of HH (Nat Genet 2000;25:14–15), an interaction between HFE and TFR2 was postulated but could not be demonstrated using soluble forms of the 2 proteins (J Biol Chem 2000;275:38135–38138). In the last 3 years a number of in vitro studies have suggested that HFE and TFR2 do in fact bind and that this interaction is distinct from the HFE–TFR1 interaction (J Biol Chem 2006;281:28494–28498; J Biol Chem 2007;282:36862–36870; Arch Biochem Biophys 2008;474:193–197). It was postulated that HFE and TFR2 form an iron-sensing complex responsible for regulating hepcidin in response to iron loaded transferrin (holo-Tf).

In this collaborative study by Gao et al (Cell Metabolism 2009;9:217–227), the regulation of hepcidin by HFE and TFR2 was investigated further. One of the main barriers to studying the regulation of hepcidin by holo-Tf has been the lack of an appropriate hepatocyte cell line responsive to holo-Tf. In this study, the authors identified a rat hepatoma/human fibroblast hybrid cell line WIF-B that responded to holo-Tf by up-regulating hepcidin mRNA expression. A comparative analysis of the expression of key iron-related genes in WIF-B cells, the human hepatoma cell line HepG2, and isolated rat hepatocytes was used to determine the basis for the differences in hepcidin responsiveness to holo-Tf. They found that HFE and, to a lesser extent, TFR2 were expressed at higher levels in WIF-B cells than in HepG2 cells. HepG2 cells engineered to express HFE, through the use of a tetracycline-inducible HFE plasmid, were then found to be responsive to holo-Tf and up-regulated hepcidin promoter activity as measured by luciferase reporter assay and mRNA expression as measured by qualitative reverse transcriptase polymerase chain reaction (qRT-PCR).

Interestingly W81A-HFE, a mutant that does not bind to TFR1 had the same effect as wild-type HFE, suggesting that the interaction between HFE and TFR1 is not required for holo-Tf–induced up-regulation of hepcidin. Knockdown of TFR2 in HepG2 cells expressing HFE abolished holo-Tf–mediated regulation of hepcidin transcription, suggesting that TFR2 is also required for the holo-Tf–mediated regulation of hepcidin. Immunoprecipitation experiments showed that in the absence of holo-Tf, HFE can be found in a complex with TFR1, but when holo-Tf is present, HFE dissociates from TFR1 and is found in complex with TFR2 and Tf. It was proposed that this Tf/TFR2/HFE complex is responsible for increasing hepcidin expression. Further support for the role of HFE and TFR2 in holo-Tf–mediated hepcidin regulation came from the analysis of mouse primary hepatocytes isolated from Hfe^−/−, Tfr2^245x/245x mutant and control mice. Although these mice were on different backgrounds, they were compared with their respective wild-type control strains. Hepatocytes isolated from both control strains of mice responded to holo-Tf by up-regulating hepcidin mRNA expression. However, in both the Hfe^−/− and Tfr2^245x/245x mutant hepatocytes holo-Tf had no effect on hepcidin expression.

Finally, the authors performed some domain swapping experiments to determine the domains of HFE necessary to regulate hepcidin. Chimeric HFE/HLA-B7 molecules were systematically generated and their responsiveness to holo-Tf was measured using the hepcidin-promoter luciferase reporter assay. HLA-B7 was chosen to generate chimeras because of its similar domain structure to the atypical major histocompatibility complex class I molecule HFE. The results of this analysis showed that only the α3 domain of HFE was necessary for binding to TFR2, but both the α3 and cytoplasmic domains were required for hepcidin regulation.

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Comment 

This well-designed in vitro study analyzed the binding and functional significance of HFE and TFR2 on hepcidin regulation. Several studies have now demonstrated that HFE and TFR2 interact in vitro (J Biol Chem 2006;281:28494–28498; J Biol Chem 2007;282:36862–36870; Arch Biochem Biophys 2008;474:193–197), but this is the first demonstration of the functional consequence of this complex on hepcidin regulation. An important point brought up in this paper is that the liver cell lines commonly used to study the regulation of iron metabolism and hepcidin regulation are inadequate. HepG2 cells, a commonly used human liver cell line, required the addition of HFE to become responsive to holo-Tf. The authors analyzed the expression of iron-related genes by qRT-PCR in 3 cell lines. These were derived from human (HepG2), rat (isolated hepatocytes), and human/rat hybrid (WIF-B); therefore, a direct comparison of expression by qRT-PCR across species (as shown in Figure 1B) is not strictly appropriate. Nevertheless, the expression of HFE in HepG2 cells did render them responsive to holo-Tf. The experiments where HFE is over-expressed and TFR2 is knocked down in HepG2 cells are convincing and suggest that both HFE and TFR2 are required for holo-Tf–mediated expression of hepcidin. The domain swapping experiments suggest that HFE binds to TFR2 through its α3 domain and that the cytoplasmic domain of HFE is important for signaling. However, this study does not explain some of the observations made in HH patients and animal models of HH. If a complex between HFE and TFR2 were absolutely required for the regulation of hepcidin by holo-Tf, then the absence of one of these molecules would be predicted to lead to the same iron overload phenotype. At first glance, this would seem to be the case. However, a number of patients with TFR2-HH have now been reported with earlier onset and more severe iron loading than patients with HFE-HH (Haematologica 2004;89:359–360; Br J Haematol 2004;125:674–678; Haematologica 2008;93:309–310). Most important, the absence of both HFE and TFR2 would be predicted to lead to the same level of iron loading; this also is not the case. In the only report of patients with a combination of HFE and TFR2 mutations, the iron overload in the subjects was severe and resembled juvenile hemochromatosis, suggesting that the loss of HFE and TFR2 have a compounding affect on hepcidin regulation and iron homeostasis (Gastroenterology 2005;128:470–479). Animal models where both Hfe and Tfr2 are disrupted also have more iron loading and dysregulation of hepcidin, suggesting that these 2 molecules work in parallel to regulate hepcidin (unpublished observations). These in vivo studies suggest that an interaction between HFE and TFR2 is not absolutely necessary for the regulation of hepcidin by iron and that HFE and TFR2, when alone, do exert some hepcidin regulatory ability. Indeed patients with HFE-HH and Hfe^−/− mice do retain the ability to regulate hepcidin, but basal expression levels are reduced, leading to inappropriately low hepcidin for a given iron load (Digestion 2005;72:25–32; Am J Physiol Gastrointest Liver Physiol 2006;291:G229–237; Blood 2007;110:4096–4100). Together these studies suggest that the ability of holo-Tf to regulate hepcidin and ultimately iron homeostasis must involve more than just a simple complex between HFE and TFR2. Future research including a demonstration that HFE and TFR2 interact in vivo are required to fully understand how HFE and TFR2 regulate hepcidin and iron homeostasis.