Histochem Cell Biol (2007) 127:221–226 DOI 10.1007/s00418-006-0229-7

S H O RT C OM M UN IC A TI O N

Localization of the iron-regulatory proteins hemojuvelin and transferrin receptor 2 to the basolateral membrane domain of hepatocytes Uta Merle · Franziska Theilig · Evelyn Fein · Sven Gehrke · Birgit Kallinowski · Hans-Dieter Riedel · Sebastian Bachmann · Wolfgang Stremmel · Hasan Kulaksiz

Accepted: 24 July 2006 / Published online: 25 August 2006 © Springer-Verlag 2006

Abstract The newly discovered proteins hemojuvelin (Hjv) and transferrin receptor type 2 (TfR2) are involved in iron metabolism. Mutations in the Hjv and TfR2 gene cause hemochromatosis. We investigated the expression and cellular localization of Hjv and TfR2 in rat and human liver. The expression of Hjv and TfR2 was shown on mRNA and protein level by RT–PCR and immunoblot experiments. Their cellular localization was studied by immunoXuorescence with antibodies raised against Hjv and TfR2. Hjv and TfR2 are present in human and rat liver and in primary human hepatocytes. Antisera raised against Hjv identiWed immunoreactive proteins with an apparent size of 44 and 46 kDa in immunoblot experiments of rat and human liver extracts, which are in accordance with the putative membrane-bound and cleaved soluble forms

Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at http://dx.doi.org/10.1007/s00418-006-0229-7. Kulaksiz and Stremmel contributed equally to this work. U. Merle · E. Fein · S. Gehrke · B. Kallinowski · H.-D. Riedel · W. Stremmel · H. Kulaksiz Department of Gastroenterology, University Hospital, Heidelberg, Germany F. Theilig · S. Bachmann Institute of Anatomy, Charité, Humboldt University, Berlin, Germany H. Kulaksiz (&) Department of Internal Medicine IV, University Hospital Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany e-mail: [email protected]

of this protein, respectively. TfR2 was detected as a 105 kDa protein corresponding to the predicted size of glycosylated TfR2 monomers. In immunoXuorescence experiments, Hjv and TfR2 were found in rat liver only in hepatocytes. At the subcellular level, both proteins were predominantly localized to the basolateral membrane domain of hepatocytes. The localization of Hjv and TfR2 at the same membrane domain renders a functional interaction of these two proteins in iron homeostasis possible. Keywords Hemochromatosis · Hemojuvelin · TfR2 · Cellular localization

Introduction Hereditary hemochromatosis (HH) represents a group of genetic disorders that manifest as iron deposition in a variety of organs such as the liver, pancreas, heart, and skin. If untreated, liver cirrhosis, heart failure, and diabetes can develop. Most often HH is associated with mutations in the HFE gene (Feder et al. 1996). However, recent studies show that mutations in several other genes also produce an HH phenotype, including hepcidin [hepatic antimicrobial peptide (HAMP)] (Roetto et al. 2003), hemojuvelin (HFE2/Hjv) (Papanikolaou et al. 2004), and transferrin receptor 2 (TfR2) (Camaschella et al. 2000). The hemojuvelin gene was Wrst identiWed by Papanikolaou et al. (2004) and Hjv gene mutations were found in association with juvenile hemochromatosis. Hemojuvelin is transcribed into a full-length messenger RNA with Wve hypothetical splicing isoforms encoding a protein of 200, 313, and 426 amino acids. Hemojuvelin

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contains a C-terminal putative transmembrane domain characteristic of a glycosylphosphatidylinositol-linked membrane anchor (GPI-anchor). By northern blot analysis of human tissues, Papanikolaou et al. (2004) found hemojuvelin transcript expression predominantly in the liver, heart, and skeletal muscle. Currently it is under discussion if hemojuvelin modulates expression of hepcidin by an interaction with an as yet unknown protein, most likely a transmembrane receptor like TfR2 (Lin et al. 2005). The TfR2 protein is a 105-kDa membrane glycoprotein that can interact with transferrin (Kawabata et al. 1999). The fact that mutations in TfR2 cause HH indicates that TfR2 has an important, but unknown role in iron homeostasis. As humans with TfR2-related HH, TfR2 mutant mice present a phenotype resembling Hfe-associated iron overload (Kawabata et al. 2005). Individuals with hemojuvelin and TfR2 mutations as well as hemojuvelin and TfR2 mutant mice demonstrate an in vivo down-regulation of hepatic hepcidin expression despite iron overload (Huang et al. 2005; Kawabata et al. 2005; Nemeth et al. 2005). It can be speculated that all four genes that are associated with diVerent types of primary iron overload including HFE, TfR2, hemojuvelin, and hepcidin are functionally linked. In this liver-speciWc iron-sensing pathway, hepcidin most likely represents the central regulator lying downstream of HFE, TfR2, and hemojuvelin. To test the hypothesis that in this iron-sensing pathway controlling iron absorption in mammals hemojuvelin and TfR2 interact functionally, we raised antibodies against hemojuvelin and TfR2 and examined the expression and cellular localization of these proteins in the liver by real-time RT–PCR, immunoblot, and immunoXuorescence experiments.

Materials and methods Tissues and tissue preparation Human liver samples were obtained after hemi-hepatectomy in adult patients with liver metastases. The ethics committee of University Hospital Heidelberg approved the study, and experiments were undertaken in accordance with the Helsinki Declaration of 1975. All patients had given written consent for the use of the tissue for research purposes. Rats were anaesthetized and subsequently sacriWced by cervical dislocation. Tissue specimens from liver were resected, for immunoXuorescence analysis embedded in OCT (Tissue Tek; Miles Inc., Germany), and subsequently frozen in liquid nitrogen for further analysis. Hep3B

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and HepG2 human hepatoma cells were obtained from the German Collection of Microorganisms and Cell Culture (Braunschweig, Germany) and were grown at 37°C with 5% CO2 in Dulbecco’s ModiWed Eagle Medium (DMEM; Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (FBS). Peptide synthesis, immunization procedure, and antibodies From the published human hemojuvelin and transferrin receptor 2 sequence (GenBank accession numbers NP_660320.3 and NP_003218.2), the peptides hemojuvelin-(80–98) and transferrin receptor 2-(14–36) (Camaschella et al. 2000; Papanikolaou et al. 2004) were synthesized as C-terminal amides using a standard Fmoc (N-alpha-(9-Xuorenylmethyloxycarbonyl)) protocol (Kulaksiz et al. 2002a, b). Peptides were coupled to keyhole limpet hemocyanin using m-maleimidobenzoylN-hydroxy-succinimide ester, and SPF rabbits (Charles River-IVa Credo, Wilmington, MA, USA) were immunized with each peptide conjugate (Eurogentec, Seraing, Belgium). After testing the titre by ELISA, two antisera against hemojuvelin-(80–98) [EG(1)-Hjv and EG(2)-Hjv] and two antisera against TfR2-(14–36) [EG(1)-TfR2, EG(2)-TfR2] were used in the present study. The peptides were produced and used for generation of the antisera displayed no homology to any hitherto reported proteins except Hjv and TfR2 as conWrmed by the BLAST P2 search. Quantitative RT–PCR Total RNA was isolated from rat and human liver tissue and human hepatoma cells Hep3B and HepG2 using the RNAeasy Mini Kit (Qiagen, Hilden, Germany) including DNAse digestion according to manufacturer’s instructions. As previously described (Gehrke et al. 2003; Herrmann et al. 2004), real-time quantiWcation of rat and human hemojuvelin, TfR2, and actin mRNA transcripts was performed with a two-step RT–PCR using the LightCycler system and the Relative QuantiWcation Software Version 1.0 (Roche Diagnostics, Mannheim, Germany). QuantiWcation of human hemojuvelin mRNA transcripts was performed using the sense primer hsHJV-Q502 (5⬘TCT TAG CTC CAC TCC TTT CTG G) and the antisense primer hsHJV-Q301 (5⬘-GAT AAT TTC AAC CCT GGA CTG C). These primers were designed to amplify hemojuvelin transcripts 1–5. Rat hemojuvelin primers (rnHJV-Q501, 5⬘-TGC CAG AAG GCT GTG TAA GG; and rnHJV-Q302, 5⬘-TCT AAA TCC GTC AAG AAG ACT CG) were generated based on the published sequence for RGMc, which diVers from hemo-

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juvelin only in nomenclature (GenBank accession number BC089203). QuantiWcation of human TfR2 mRNA transcripts was performed using the sense primer hsTfR2-Q501 (5⬘-TGG CCC TCG AGG CCA AGA ATT CGG CAC) and the antisense primer hsTFR2Q301 (5⬘-ATA AGC TTG CGG CCG CTT A). QuantiWcation of rat TfR2 mRNA transcripts was performed using the predicted sequence for rat TfR2 (GenBank accession number XM_222022) with the sense primer rnTfR2-Q501 (5⬘-TCC TTT CTC CCT CTT TGA GTG G) and the antisense primer rnTFR2-Q301 (5⬘-GAT ATG GGG AAC TTT GCT CCT G). Normalization to actin mRNA levels was performed as previously described (Gehrke et al. 2003; Herrmann et al. 2004). Calibrators for human hemojuvelin and TfR2 were generated from EST clones (human: IMAGE: 122292 and IMAGE: 201221 from RZPD, Berlin, Germany). Due to the homology with the mouse sequences for hemojuvelin and TfR2 calibrators for rat hemojuvelin and TfR2 were generated from mouse EST clones (IMAGE: 1886105 and IMAGE: 4196597 from RZPD, Berlin, Germany). Immunoblot analysis Immunoblot experiments were performed on SDS-polyacrylamide gels. Proteins from human and rat liver, as well as from human Hep3B and HepG2 cells were extracted according to the published protocols. (Kulaksiz et al. 2002a, b, 2004) Following electrophoresis, proteins were transferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) by semidry blotting. After blocking with 2% BSA in PBS the membranes were incubated overnight with the hemojuvelin and TfR2 antisera diluted 1:1,000 and 1:2,000 respectively. After washing in Tris-buVered saline containing 10 mM Tris–HCl (pH 8.0), 150 mM NaCl and 0.05% Tween 20, immunoreactive proteins were visualized after incubation with horseradish oxidase-conjugated goat anti-rabbit antibody (diluted 1:4,000; Sigma, Germany) using ECL immunoblotting detection reagents (Amersham Pharmacia, Uppsala, Sweden). Molecular mass marker used was PageRuler (Fermentas, St. LeonRoth, Germany). The immunoreaction on the immunoblot was speciWcally blocked after preincubation of the antisera with the corresponding peptide immunogens. Cross-reactivity with the second goat anti-rabbit antibody was excluded by appropriate controls. ImmunoXuorescence analysis Four micrometer thick serial cryostat sections were used. After blocking with 5% skim milk in PBS (pH 7.4) for 1 h at room temperature, sections were

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incubated with primary antibody (1:1,000, diluted in PBS) for 2 h at room temperature and then overnight at 4°C, followed by incubation with Xuorochrome-coupled secondary antibody. Immunostaining was visualized on a Leica Xuorescence microscope equipped with a high pressure mercury lamp and the appropriate Wlter sets. Images were acquired with a digital camera and processed with MetaVue software (Universal Imaging; Visitron, Puchheim, Germany). Appropriate controls have been carried out to support the speciWc immunoreactivity of antibodies for immunoXuorescence staining (Kulaksiz et al. 2002a, b, 2004).

Results and discussion Since mutations in the recently discovered proteins hemojuvelin and transferrin receptor 2 can cause hemochromatosis, both proteins are supposed to be important regulators of iron homeostasis. However, the exact function of hemojuvelin and TfR2 especially in the liver as one central organ of iron metabolism is still unclear. Although TfR2 is a homologue of transferrin receptor 1 (TfR1), the primary molecule responsible for the uptake of transferrin bound iron into cells, the aYnity of TfR2 to Fe-transferrin is 25–30 times lower than that of TfR1 (Kawabata et al. 2000). Such a low aYnity of TfR2 to transferrin suggests the presence of other ligands for TfR2 and especially a functional interaction of hemojuvelin with TfR2 is currently under discussion (Lin et al. 2005). For this reason it is of central interest to know the cellular and subcellular localization of these proteins in the liver. Expression of hemojuvelin and TfR2 on mRNA level was analyzed by highly sensitive real-time RT– PCR. Both proteins were expressed in rat and human liver and human hepatoma cells (Electronic Supplemental Material 1). This is in accordance with previous reports on detection of hepatic expression of hemojuvelin (Papanikolaou et al. 2004) and TfR2 (Kawabata et al. 1999; Fleming et al. 2000) by northern blot analysis. Of note, the expression level for both proteins was lower in the hepatoma cells than in the liver and no hemojuvelin mRNA expression was detectable in HepG2 cells. The lower expression level found in hepatoma cells is most likely due to the altered overall expression proWle commonly observed in tumor cell lines when compared to primary cells. To verify the presence of the translated proteins in the liver, we raised speciWc antisera against hemojuvelin and TfR2 and used them in immunoblot and immunoXuorescence experiments. Immunoblot analysis conWrmed

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To analyze the cellular and subcellular localization of hemojuvelin and TfR2, immunoXuorescence microscopy was performed with the speciWc antisera tested in immunoblot experiments. In rat liver, both proteins were speciWcally localized to the hepatocytes with no signal detectable in KupVer cells (Fig. 2). At the subcellular level, hemojuvelin immunoreactivity was restricted to the basolateral (sinusoidal) membrane domain of hepatocytes. In serial sections, TfR2 immunoreactivity was detected at the same membrane domain of hepatocytes. Control experiments with preimmuneserum of the antisera used for immunoXuorescence analysis revealed no immunostaining conWrming the speciWcity of the immunoreactions (Fig. 2). Hemojuvelin and TfR2 were both found at the basolateral membrane domain where a direct contact with blood and sensing of inXammatory or iron-homeostasis related signals is possible. This is the Wrst report on the cellular and subcellular localization of Hjv in the liver. Just recently, Zhang et al. (2005) reported on the distribution of hemojuvelin in HJV-overexpressing HEK293 cells analyzed by immunoXuorescence analysis and detected Hjv in accordance to our results evenly distributed on the plasma membrane. Concordantly to

expression of hemojuvelin and TfR2 in rat and human liver at the protein level (Fig. 1). With antisera raised against human hemojuvelin-(80–98), an immunoreactive band of »46 kDa was identiWed in rat and human liver. Interestingly, the speciWc hemojuvelin antisera also identiWed a second protein at »44 kDa in the same tissues. The apparent molecular masses of these proteins are in accordance with the molecular masses detected in hemojuvelin-overexpressing cells (Lin et al. 2005) and most likely correspond to a cell-associated isoform (»46 kDa) and a cleaved, soluble isoform (»44 kDa) of hemojuvelin. Notably, in contrast to liver tissue, in HepG2 and Hep3B cells the cell-associated form was predominant with no signal of the cleaved form. In immunoblot analyses of rat and human liver tissue and HepG2 cells, TfR2 was identiWed as a protein with a molecular weight of 105 kDa (Fig. 1). No immunoreactivity was found in Hep3B cells. The apparent molecular mass of this immunoreactive protein is in accordance with the predicted size of glycosylated TfR2 (Kawabata et al. 2000). Of note, the immunoreactions on the western blots were completely blocked after preincubation of the antisera with the corresponding peptide immunogens conWrming the speciWcity of the immunoreactions (Fig. 1).

kDa

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4

kDa

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B

4

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Fig. 1 a Western blot analysis of Hjv in extracts of HepG2 (lane 1), Hep3B (lane 2), rat liver (lane 3), and human liver (lane 4) with antibody EG(1)-Hjv. b Western blot analysis of TfR2 in extracts of HepG2 (lane 1), Hep3B (lane 2), rat liver (lane 3), and human liver (lane 4) with antibody EG(1)-TfR2. c Western blot analysis of Hjv in extracts of human liver with antiserum EG(1)-Hjv (lane 1) and in extracts of human liver after preadsorption of the anti-

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kDa

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D

serum EG(1)-Hjv with Hjv peptide immunogen (lane 2). d Western blot analysis of TfR2 in extracts of human liver with antiserum EG(1)-TfR2 (lane 1) and in extracts of human liver after preadsorption of the antiserum EG(1)-TfR2 with TfR2 peptide immunogen (lane 2). For all western blot analyses equal amount of protein (50
g) were loaded per lane

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Fig. 2 Cellular localization of immunoreactive TfR2 (a, c) and hemojuvelin (b, d) in serial sections of rat liver using polyclonal antisera. a EG(2)-TfR2, b EG(2)-Hjv, c EG(1)-TfR2, d EG(1)-Hjv. a, b MagniWcation £200; c, d MagniWcation £330. Note the strong immunostaining at the basolateral membrane domain of hepatocytes (arrowheads) with TfR (e) and Hjv (f) antisera. MagniWcation £400

our results for TfR2, Wallace et al. (2005) reported of a predominant basolateral localization of TfR2 in hepatocytes. As we reported previously, pro-hepcidin localizes to the basolateral membrane domain too (Kulaksiz et al. 2004). We hypothesize a functional interaction of hepcidin with hemojuvelin and TfR2 in this functional membrane domain in the context of iron-metabolism regulation. Interestingly, the hepatic lobules were homogeneous with respect to hemojuvelin and TfR2 distribution. This immunoreactivity pattern distributed throughout the liver is consistent with their proposed function. Hemojuvelin and TfR2 may act as iron-sensors in blood. Activation of TfR2 and hemojuvelin could then cause a cascade signaling to hepcidin localized in the same membrane domain of hepatocytes. Interestingly, in contrast to distribution of hemojuvelin and TfR2 in portal lobules, pro-hepcidin expression is mainly localized in periportal zones (Kulaksiz et al. 2004). Hepcidin immunoreactivity decreases from the periportal zones toward the central veins. This zonation within the portal lobules may

have a functional signiWcance as in iron-overloading iron-staining also shows the same distribution pattern with iron deposits located predominantly periportal (Whittaker et al. 1996). Due to results of our immunoXuorescence analysis with a regional overlap of the three iron-regulatory proteins hemojuvelin, TfR2, and hepcidin, a functional interaction of these proteins is highly possible. In conclusion, we speculate that cellassociated hemojuvelin functionally interacts with TfR2 at the basolateral membrane domain to regulate iron homeostasis by signaling to hepcidin. Acknowledgments The authors thank Sabine Tuma and Karin Bents for expert technical support. The study was supported by Deutsche Forschungsgemeinschaft (KU 1253/5-1).

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