Physiologic systemic iron metabolism in mice deficient for duodenal Hfe

Because there is no known physiologic pathway for iron excretion, intestinal iron absorption must be tightly controlled to maintain iron balance in the organism. Dietary iron is primarily absorbed in the duodenum, where it is reduced to its ferrous form (Fe²⁺), most likely by the activity of the intestinal brush border ferric reductase Dcytb (Cybrd1), to be transported across the intestinal membrane by the apical permease DMT1 (DCT1, Nramp2, Slc11a2). Alternatively, enterocytes acquire iron by taking up dietary heme, possibly via the newly identified transporter HCP.¹  Absorbed iron is then transferred across the basolateral membrane by the iron export protein ferroportin (Ireg1, MTP1, and Slc11a3). During export, ferrous iron is oxidized by the ferroxidase hephaestin to bind to transferrin for transport in the blood. In healthy subjects the intestinal iron transport machinery is adjusted in conditions of systemic iron deficiency and iron overload to maintain adequate iron supply.^2,3  However, patients with hereditary hemochromatosis (HH) show progressive iron overload as a consequence of increased intestinal iron absorption. The majority of these patients are homozygous for a missense mutation (C282Y) in the HFE gene, which codes for an atypical MHC class I molecule.⁴  Mice homozygous for a null allele of Hfe^5–7  or the orthologous disease mutation (Hfe^845A/845A)⁸  develop iron overload that recapitulates human HH.

In the small intestine, Hfe is found in duodenal crypt cells,⁹  as well as within the extreme apical cytoplasm of intestinal villus enterocytes,¹⁰  and its expression appears to be regulated in response to iron overload.¹¹  Moreover, transgenic overexpression of Hfe in mature duodenal enterocytes produced hepatic iron overload in the mouse, suggesting that Hfe can operate within these cells to augment iron absorption.¹²  Whether expression of Hfe at physiologic levels affects iron absorption is still unknown. Based on the findings that (1) Hfe protein is expressed in duodenal crypt cells where it can interact with the coexpressed transferrin receptor 1 (TfR1)⁹  and (2) Hfe competes with transferrin for binding to TfR1 to modulate cellular iron uptake,^13–15  it has been hypothesized that crypt stem cells could use the Hfe-TfR1 complex to sense systemic iron availability (ie, the amount of iron bound to plasma transferrin) and subsequently program the expression of iron transporters in the absorptive progeny cells of the villi.¹⁶ 

Both in patients and mice, HFE deficiency correlates with inappropriately low basal expression of the hepatically expressed iron-hormone hepcidin (LEAP1, Hamp)^17–20  that fails to be adjusted appropriately in response to elevated hepatic iron levels in mice.^18,19  Hepcidin is a β-defensin–like polypeptide whose expression is normally increased in response to secondary iron overload and which inhibits iron release from duodenal enterocytes (and also macrophages and hepatocytes) by inducing the lysosomal degradation of the iron exporter ferroportin.²¹  Thus, an alternative model for HFE function suggests that the inappropriately low-hepcidin levels in HH trigger increased iron absorption by elevated expression of ferroportin.

To differentiate between the 2 models for HFE-mediated HH, and to study the in vivo function of Hfe in the duodenum, we used the Cre/loxP technology and established a mouse line with selective and complete Hfe deficiency in intestinal crypt and villus enterocytes. We show that duodenal Hfe is dispensable for physiologic iron homeostasis under steady state conditions, excluding one of the previously favored models for HH.

The generation of mice carrying a null (Hfe^−/−) or a “floxed” (Hfe^flox) Hfe allele was described previously.⁷  In Hfe^flox mice, exons 3 to 5 are flanked with 2 loxP sites; Cre-mediated excision of these exons has been shown to generate an Hfe null allele.⁷  Selective Hfe ablation in enterocytes of the intestine was achieved by crossing Hfe^flox mice with a transgenic line (B6SJL-Tg(Vil-Cre)997Gum; Jackson Laboratory, Bar Harbor, ME) expressing the Cre recombinase under the control of promoter elements of the murine Villin gene to obtain Hfe^VillinCre mice.²² 

Mice were genotyped by polymerase chain reaction (PCR) analysis of genomic DNA extracted from the tail tip using the DNeasy kit (Qiagen, Hilden, Germany). PCR conditions were as follows: 95°C for 5 minutes, (95°C for 1 minute, 63°C for 1 minute, 72°C for 1 minute) for 30 cycles. Primers used to detect the full-length Hfe^flox allele were F and R1. Primers used to detect the truncated allele were F and R2. Primers used to detect the Cre transgene were Cre3 and Cre5. Primer locations are indicated in Figure 1A and primer sequences are given in Table 1PCR products were resolved on 1.5% agarose gels and visualized by ethidium-bromide staining.

Hfe^−/− and Hfe^flox mice on a mixed genetic background (C57BL/6 × 129P2/OlaHsd⁷ ) were back-crossed to the C57BL/6 genetic background. Hfe^VillinCre mice of both sexes were analyzed at 18 to 22 weeks of age. Wild-type age- and sex-matched C57BL/6 mice were used as controls throughout the study. All mice were born and housed in the SPF animal facility of the EMBL. They were maintained on a standard mouse diet containing 225 mg/kg iron (Teklad 2018 S; Harlan Winkelmann, Borchen, Germany) on average and a constant dark-light cycle and allowed continuous access to food. Mice were killed by CO₂ inhalation. Heparinized blood was collected by cardiac puncture. Longitudinal sections of the proximal duodenum were collected first, then other tissues were collected. All mouse breeding and animal experiments were approved by and in compliance with the guidelines of the Institutional Animal Care and Use Committee of the EMBL.

The duodenum was dissected from each mouse, opened longitudinally, placed on a piece of aluminum foil, and immediately frozen in liquid nitrogen. Samples were stored at −80°C until further use. Microdissection was carried out on 5-μm thin duodenal tissue sections cut on 1 mm polyethylenenapthalate membrane on glass slides (P.A.L.M. Laser Microscopy System, Zeiss Axiovert 135 microscope, 337 nm laser, intensity 1012 W/m²; Zeiss, Jena, Germany). DNA was isolated by lysing the captured cells overnight at 37°C in a buffer containing 0.04% Proteinase K, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% Tween-20. PCR analysis was performed under the conditions described for genotyping.

Nonheme iron content of liver and duodenal tissues was measured by a slight modification of the method described by Torrence and Bothwell.²³  Briefly, tissues were dried (72 hours at 42°C) and digested in 10% TCA/10% HCl (48 hours at 65°C). Tissue debris was pelleted by centrifugation (15 700g for 5 minutes). Twenty microliters of 10 × diluted supernatant was added to 100 μL chromogenic solution (6 M Na acetate/0.01% bathophenanthroline-disulfonic acid/0.1% thioglycolic acid) in a 96-well plate and incubated for 10 minutes at room temperature. Absorbance (535 nm) was measured in a photometer (SpectramaxPlus; Molecular Devices, Munich, Germany). Iron content was calculated using serial dilution of an iron-standard (Iron atomic absorption standard solution; Sigma-Aldrich, Deisenhofen, Germany); values are expressed as micrograms of iron per gram of dry tissue.

Serum samples have been analyzed by the clinical-chemical laboratory of the German Mouse Clinic²⁴  using the AU400-System (Olympus Diagnostica, Hamburg, Germany) and adapted reagents (Olympus Diagnostica) according to the manufacturer's instructions to determine serum iron (OSR6186) and ferritin (OSR6150) concentrations as well as the UIBC (OSR6124).

Total RNA was isolated using Trizol reagent, and 2 μg total RNA was reverse transcribed using the Revertaid H Minus M-MuLV Reverse Transcriptase (Fermentas, Hanover, MD) following the manufacturer's instructions. Quantitative real-time PCR was carried out in 25-μL reaction volume using SYBR Green I dye on ABI Prism 7500 (Applied Biosystems, Applera Deutschland, Weiterstadt, Germany). Primers were designed to specifically amplify hepcidin, Dcytb, DMT1, Ireg1, and Gapdh sequences. Primer sequences are indicated in Table 1. Amplification conditions were as follows: 50°C for 2 minutes, 95°C for 10 minutes (95°C for 15 seconds, 60°C for 15 seconds) for 45 cycles. The mRNA abundance of each transcript was calculated relative to the expression of the housekeeping gene Gapdh (glycraldehyde-3-phosphate-dehydrogenase). Data have been analyzed using the modified REST program,²⁵  which allows analyzing the expression of a target gene standardized by a nonregulated reference gene. The mathematical model used is based on the correction for exact PCR efficiencies and the mean crossing point deviation between sample group(s) and control group(s).

To assess whether Hfe expression in the intestine contributes to the maintenance of murine iron homeostasis, we used the Cre/loxP technology²²  to establish a mouse line that lacks Hfe selectively in the small intestine but not in other tissues. We used a previously generated mouse line carrying a floxed Hfe allele (Hfe^flox), in which exons 3 to 5 are flanked with loxP sites⁷  (Figure 1A). Global Cre-mediated excision of exons 3 to 5 in this line generates an Hfe-null allele and recapitulates the phenotype seen in HH.⁷  The Hfe^flox mouse line was crossed with transgenic mice expressing the Cre recombinase under the control of the murine Villin 1 promoter²⁶  to generate Hfe^VillinCre mice (Figure 1A). Hfe^VillinCre mice are viable, fertile, normal in size, and do not display any overt physical or behavioral abnormality (data not shown).

Tissue specificity of Hfe inactivation by the Villin promoter-driven Cre transgene was determined by genomic PCR analysis as schematically illustrated in Figure 1A. Recombination of the Hfe^flox allele is observed along the entire small intestine in Hfe^VillinCre mice, including the proximal section of the duodenum where most of the iron absorption takes place (Figure 1B). Messenger RNA analysis using primers specific for the wild-type and recombined Hfe alleles yielded comparable results (data not shown). As expected, no recombination of the Hfe^flox allele is detected in any of the other organs tested (Figure 1C). However, both recombined and non-recombined alleles are detected when analyzing genomic DNA prepared from whole intestinal sections of the Hfe^VillinCre mice (Figure 1B-C). This indicates either that the recombination of the Hfe^flox allele in enterocytes is incomplete or that DNA from connective tissue and smooth muscle cells not expressing the Cre recombinase contributes to the signal of the intact Hfe^flox allele (Figure 1B-C). Importantly, only the non-recombined or the fully recombined allele is detected in intestinal samples from Hfe^flox or Hfe^−/− mice, respectively (Figure 1C).

Because Hfe was suggested to function in crypt and/or villi enterocytes,^9,10  it is important to determine the efficiency of Cre-mediated recombination in both cell types. Therefore, crypt and villus enterocytes were isolated by laser capture microdissection (Figure 2A-D), and recombination efficiency was assessed by genomic PCR analysis in these samples. For ease of handling, larger tissue regions containing mainly tip (Figure 2A) and crypt enterocytes (Figure 2B) were isolated from mice lacking the Cre recombinase transgene (Cre⁻), whereas pure tip (p.t.; Figure 2C) and pure crypt (p.c.; Figure 2D) enterocytes were isolated from mice carrying the Cre transgene (Cre⁺). DNA analysis of these preparations shows that the full-length Hfe^flox allele present in Cre⁻ mice is absent in both crypt and tip enterocytes of Cre⁺ mice where, importantly, only the recombined allele is detected (Figure 2E). This demonstrates that recombination of the Hfe^flox allele in mice carrying the Cre transgene is highly efficient and specific in crypt and villus enterocytes of the intestine.

To test whether Hfe expression in intestinal enterocytes is required to maintain normal systemic iron homeostasis, we analyzed the liver iron content of Hfe^VillinCre mice (18-22 weeks of age) expressing the Cre transgene (Cre⁺) in comparison to Cre⁻ littermates. As a positive control for liver iron loading, we used mice derived from the same original line with constitutive Hfe deficiency (Hfe^−/−) (together with age- and sex-matched wild-type mice) whose generation and analysis has been described previously.⁷  As expected, both Hfe^−/− males and females accumulate significantly more iron in the liver than C57BL/6 control mice (Table 2). Hfe^−/− females also accumulate significantly more iron in the liver compared with Hfe^−/− males (1.8-fold; P = .001). By contrast, mice with complete and selective Hfe deficiency in enterocytes display no hepatic iron accumulation, associated with unchanged serum iron levels, unsaturated iron-binding capacity (UIBC), transferrin saturation, and serum ferritin values. As expected, the UIBC is reduced in Hfe^−/− male and female mice compared with respective wild-type controls (Table 2).

Previous work showed increased hepcidin mRNA expression in response to secondary iron overload, but a failure to increase hepcidin in response to the primary iron overload caused by Hfe deficiency.^7,10  Consistently, Hfe^−/− mice lack a significant increase in hepcidin mRNA expression (Figure 3) despite increased hepatic iron levels (Table 2). Importantly, and in agreement with the other systemic iron homeostasis parameters, Hfe^VillinCre Cre⁺ and Cre⁻ mice show comparable hepcidin mRNA expression (Figure 3). However, hepcidin mRNA levels are 2-fold higher in female mice compared with males independent of the genotype (Figure 3), correlating with elevated liver iron content in female mice (Table 2).

Taken together, these data show that duodenal Hfe expression is dispensable for normal iron homeostasis and liver iron loading under steady state conditions.

To examine whether enterocytic Hfe deficiency exerts local effects by modulating the expression of duodenal iron transporters, we assayed the mRNA expression of the brush border ferric reductase Dcytb (Cybrd1), the transmembrane permease DMT1 and the iron export protein ferroportin in the duodenum of C57BL/6, Hfe^−/−, and Hfe^VillinCre mice (Cre⁺ and Cre⁻) by quantitative real-time RT-PCR (Figure 4).

As noted before,⁷  we find a significant increase in Dcytb mRNA expression in Hfe^−/− compared with C57BL/6 wild-type mice, with a more pronounced augmentation in males (3.74-fold; P = .001) than in females (1.4-fold; P = .03). In contrast, Dcytb mRNA expression remains unchanged both in male and female Hfe^VillinCre (Cre⁺) mice compared with Cre⁻ littermates. These data strongly suggest that elevated Dcytb mRNA expression in Hfe^−/− mice is not a primary effect of intestinal Hfe deficiency, but rather a consequence of the systemic changes in iron homeostasis associated with constitutive HFE deficiency. DMT-1 and ferroportin mRNA expression also remains unaltered in sex- and age-matched C57BL/6, Hfe^−/−, and Hfe^VillinCre mice (Figure 4B-C). Together with an unchanged duodenal iron content (data not shown), our data suggest that duodenal Hfe plays no direct role in the local regulation of iron transporter genes in the duodenum.

Two opposing models have been discussed for the role of HFE in HH. According to the first, HFE function in duodenal enterocytes is required for normal intestinal iron absorption, possibly involving the interaction of HFE with TfR1.^13–15  According to the second, HFE function in a nonintestinal cell (possibly hepatocytes or hepatic macrophages) enables appropriate hepatic hepcidin expression, which in turn determines physiologic iron absorption.

To address these models, we directly investigated the in vivo function of intestinal Hfe. We generated Hfe^VillinCre mice that lack Hfe exclusively in the intestine. Using laser capture microdissection and genomic PCR analysis we demonstrate that the floxed Hfe allele is efficiently recombined (close to 100%) in isolated crypt and villi enterocytes (Figure 2). We consider this to be particularly important because (1) in crypt enterocytes, where TfR1 is predominantly expressed,⁹  the Hfe-TfR1 complex has been proposed to serve as part of a sensor mechanism of systemic iron status¹⁶ ; and (2) transgenic expression of Hfe in villi enterocytes has been reported to mediate hepatic iron deposition.¹²  Our data show that Hfe^VillinCre mice (Cre⁺) do not accumulate iron in the liver (Table 2); in addition, they display unaltered UIBC, serum iron, transferrin saturation, and plasma ferritin levels (Table 2) as well as unchanged hepatic hepcidin mRNA expression (Figure 3). These data demonstrate that duodenal Hfe is dispensable for normal systemic iron homeostasis in steady state condition. These findings hence refute the hypothesis according to which intestinal Hfe function is disturbed in HH.

The transferrin saturation is surprisingly high in C57BL/6 wild-type mice, a finding consistent with data obtained in a previous study.²⁷  The high degree of transferrin saturation in C57BL/6 mice may be related to the iron content of the diet and/or the genetic background of the mice. In addition, and as shown before,²⁸  female mice, compared with their male counterparts, display a higher degree of transferrin saturation (Table 2), correlating with increased serum and hepatic iron levels. The high degree of transferrin saturation observed in C57BL/6 mice may explain the only marginal increase in transferrin saturation in male and the lack of increase in female mice with constitutive Hfe deficiency.

To assess whether intestinally expressed Hfe contributes to the maintenance of local, enterocyte-specific iron homeostasis, we investigated the expression of duodenal iron transporters. Of relevance, increased mRNA expression of DMT-1, ferroportin, and/or Dcytb has been reported in the duodenal mucosa of patients with HH and some mouse lines with constitutive Hfe deficiency.^18,29–31  Here, we demonstrate that DMT-1 and ferroportin mRNA expression is unaltered both in constitutive Hfe^−/− mice as well as in Hfe^VillinCre (Cre⁺) mice compared with their respective controls that were all maintained on a C57BL/6 genetic background. These findings are in line with our previous data showing that DMT-1 mRNA as well as ferroportin mRNA and protein levels remained unchanged in constitutive Hfe^−/− mice on a mixed C57BL/6 × 129P2/OlaHsd genetic background. In contrast, Dcytb mRNA expression was significantly increased in Hfe^−/− males on a mixed C57BL/6 × 129P2/OlaHsd,^7,31  in Hfe^−/− females on the 129S6/SvEvTac¹⁸  and in Hfe^−/− mice (both male and female) on the C57BL/6 (Figure 4A) genetic backgrounds. Hfe^VillinCre (Cre⁺) mice do not exhibit a significant increase in Dcytb mRNA expression compared with their Cre⁻ littermates. This finding indicates that regulatory cues induced by the lack of Hfe in tissues other than the duodenum (possibly the liver) are responsible for the increased Dcytb mRNA expression in Hfe^−/− mice. We speculate that the low hepatic hepcidin expression resulting from general Hfe deficiency increases basolateral iron export from enterocytes via ferroportin. As a consequence the intracellular iron level in enterocytes may be lowered and the increase in Dcytb mRNA may reflect a compensatory response. Although increased Dcytb expression seems to be a consequence of Hfe deficiency, targeted disruption of the Dcytb gene in the mouse does not affect systemic and local iron homeostasis parameters.³²  To test whether indeed elevated Dcytb activity confers the increased iron uptake observed in HH, cross-breeding of Hfe^−/− and Dcytb-deficient mice would be informative.

Contribution: M.V.S., J.K., and T.H. participated in designing and performing the research; M.V.S. controlled and analyzed the data; R.K. and J.S. collected data; B.G., B.R., and E.W. centralized the mouse work; W.S., M.W.H., and M.U.M. initiated and conceptualized the work; M.V.S., M.W.H., and M.U.M. wrote the paper; and all authors checked the final version of the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

M.V.S., J.K., and T.H. contributed equally to this study. W.S., M.W.H., and M.U.M. also contributed equally to this study.

Correspondence: Martina U. Muckenthaler, University of Heidelberg, Department of Pediatric Oncology, Hematology, and Immunology, Im Neuenheimer Feld 153, 69120 Heidelberg, Germany; e-mail: martina.muckenthaler@med.uni-heidelberg.de; and Matthias W. Hentze, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany; e-mail: hentze@embl.de.

We thank Vladimir Benes and members of the EMBL Genomics Core Facility for support with qRT-PCR, Dr Gröne and M. Bonrouhi (Deutsches Krebsforschungszentrum [DKFZ]) for help with laser dissection microscopy, and M. Hrabe de Angelis and the German Mouse Clinic for their work. We thank the EMBL transgenic mouse service and the staff of the EMBL animal house for their contribution to this work.

This work was supported by a research grant from the Deutsche Forschungsgemeinschaft (M.U.M.), by a PhD fellowship from the Boehringer Ingelheim Fonds (J.K.), and by the Bundesministerium für Bildung und Forschung (BMBF)/Nationales Genomforschungsnetz (NGFN) (01GR0430) (B.R.).