Is the Iron Donor Lipocalin 2 Implicated in the Pathophysiology of Hereditary Hemochromatosis?

Abstract

Under normal conditions, iron is taken up by the cells through the transferrin-mediated pathway. However, in hereditary hemochromatosis, a common iron-overloading disorder associated with mutations in the HFE gene, iron in plasma exceeds transferrin-binding capacity, and non–transferrin-bound iron (NTBI) appears in the circulation of patients with iron overload. NTBI can be taken up by hepatocytes through a transferrin-independent pathway. Lipocalin 2 (Lcn2), a secreted protein of the lipocalin family, has emerged as the mediator of an alternative, transferrin-independent pathway for cellular iron delivery. To evaluate the importance of Lcn2 in the pathogenesis of hepatic iron loading in Hfe knockout mice, we generated HfeLcn2 double-deficient mice. Our studies revealed that deletion of Lcn2 in Hfe-knockout mice does not influence hepatic iron accumulation in Hfe^−/− mice, or their response to iron loading, as the phenotype of HfeLcn2^−/− mice remained indistinguishable from that of Hfe^−/− mice.

Conclusion

Lcn2 is not essential for iron delivery to hepatocytes in hemochromatosis.

Under normal conditions, the major pathway for cellular iron uptake is through internalization of the complex of iron-bound transferrin and the transferrin receptor. Thereafter, iron is released from transferrin as the result of acidic pH in endosomes and is transported to the cytosol by divalent metal ion transporter 1 (also known as SLC11A2).^1,2 However, there is convincing evidence that, in situations of disrupted iron homeostasis, iron can also be delivered to cells by alternative, transferrin-independent mechanisms. For example, mice and humans lacking transferrin, while anemic, develop iron overload in the liver, suggesting that iron is being delivered to hepatocytes by transferrin-independent pathways.^3,4 Likewise, in HFE-related hereditary hemochromatosis (HH), a common disorder of iron homeostasis characterized by excessive iron deposition principally in the liver,^5,6 iron overload exceeds the iron-binding capacity of serum transferrin,^7,8 and this non–transferrin-bound iron (NTBI) is cleared from the circulation and deposited into hepatocytes.⁹ This alternate iron uptake pathway is further brought into evidence by studies in Dmt1^−/− knockout mice.¹⁰ In fact, despite the presence of a disrupted transferrin cycle, Dmt1^−/− mice have abnormally high iron liver stores. Moreover, iron loading with iron dextran leads to significant accumulation of iron in Dmt1^−/− hepatocytes, further indicating that the liver has alternate mechanisms than the transferrin cycle for iron uptake.¹⁰ The identification of the components of NTBI, however, remains elusive.

Lipocalin 2 (Lcn2), also called neutrophil gelatinase-associated lipocalin, or 24p3,¹¹ has been proposed to be a mediator of the transferrin-independent iron delivery pathway¹² after it was found that Lcn2 can bind bacterial ferric siderophores,¹³ and that Lcn2–siderophore–iron complexes can transport iron into cells during kidney development.¹⁴

Lcn2 has been found to exert a broad range of biological activities, which seems to depend on whether Lcn2 is bound to iron-laden siderophores or not, implicating iron-loaded Lcn2-siderophore as an iron donor and, conversely, iron-free Lcn2-siderophore as an iron chelator.^15,16 As such, iron-loaded Lcn2-siderophore (Holo-Lcn2) has been shown to be required for mesenchymal–epithelial transition of embryonic kidney¹⁴ and oncogene Ras-transformed epithelial cells,¹⁷ as well as for kidney protection from renal failure,^18,19 and delivery of ferric ion to mouse spermatozoa.²⁰ In contrast, the Lcn2–siderophore complex without iron²¹ and Lcn2 (Apo-Lcn2)²² chelates iron from cells and, through iron deprivation, can induce apoptosis of pro-B cells²² and inhibit bacterial growth^13,23,24 and erythropoiesis.^25,26 Additional evidence that Lcn2 may assist cellular iron trafficking is provided by the demonstration that iron delivered through Lcn2 has been shown to regulate iron-sensitive genes²¹ and the identification of one of its receptors as being megalin.²⁷ Megalin is also known to bind another iron-binding protein, namely lactoferrin.^28,29 Thus, Lcn2 has emerged as a possible candidate involved in NTBI uptake under iron overload conditions and, as such, is possibly implicated in the pathophysiology of HH.^{12, 30}

In this study, we investigated whether Lcn2, as a component of an alternative iron delivery system, may contribute to the pathophysiology of HH. For this purpose, we generated and then characterized iron metabolism in HfeLcn2 double mutant mice.

Materials and Methods

Animals

All procedures were performed in accordance with Canadian Council on Animal Care guidelines and approved by our institution’s Animal Care Committee. Control, wild-type mice were C57BL/6 female mice purchased from Charles River Laboratories, Inc. (Wilmington, MA). Hfe^−/− mice were kindly provided by Dr. Nancy C. Andrews, Howard Hughes Medical Institute and Harvard Medical School, Children’s Hospital (Boston, MA)³¹ in the 129/SvEvTac background and were back-crossed onto the C57BL/6 (B6) background for 10 generations.³² Lcn2^−/− mice generated in the C57BL/6 (B6) background have been described.²³ Compound mutants (HfeLcn2^−/−) were obtained by interbreeding Hfe^−/− and Lcn2^−/− mice and were genotyped via polymerase chain reaction (PCR) (Supplementary Fig. 1A,B). The following primers were used: Hfe, 5′-AGTTGG-GAGTGGTGTCCGA-3′, 5′-TGGCTACAGTGTGAG-AGGC-3′, and 5′CTAGCTTCGGCCGTGACG-3′; Lcn2, 5′-CCTCAAGGACGACAACATCA-3′, 5′-ACCCATT-CAGTTGTCAATGC-3′, 5′-TTGGGT- GGAGAGGC-TATTC-3′, and 5′-AGGTGAGATGACAGGAGATC-3′. All mice used in the experiments were genotyped via PCR assay performed on DNA prepared from mouse tails. Absence of Lcn2 expression in Lcn2^−/− and HfeLcn2^−/− mice was confirmed via western blotting using a monoclonal antimouse Lcn2/neutrophil gelatinase-associated lipocalin antibody (R&D Systems, Inc., Minneapolis, MN) (Supplementary Fig. 1C).

Diets

Mice were given a commercial diet (Harlan Teklad, Madison, WI) or, when indicated, an iron-supplemented diet containing 2.5% (wt/wt) carbonyl iron (Sigma Immunochemicals, St. Louis, MO).

Measurement of Serum Iron, Transferrin Saturation, and Tissue Iron Concentration

Serum iron, total iron-binding capacity, and transferrin saturation were assessed via colorimetric assay.³³ Iron levels in the liver, spleen, heart, and kidneys were measured via acid digestion of tissue samples followed by iron quantification via atomic absorption spectroscopy.³³

Quantitative Reverse-Transcription PCR

Total RNA was isolated with Trizol reagent (Invitrogen, Burlington, Ontario, Canada), and reverse-transcription was performed with the Thermoscript RT-PCR system (Invitrogen). Hepcidin 1 (Hamp1) and β-actin messenger RNA levels were measured via real-time PCR using a Rotor Gene 3000^™ Real Time DNA Detection System (Montreal Biotech, Inc., Kirkland, Quebec, Canada) with the QuantiTect SYBRGreen I PCR kit (Qiagen, Mississauga, Ontario, Canada) as described,³³ and expression levels of hepcidin were normalized to the housekeeping gene β-actin. The primers employed were as follows: β-actin, 5′-TGTTACCAACTGGGACGACA-3′ and 5′-GGTGTTGAAGGTCTCAAA-3′; Hamp 1, 5′AGAGC-TGCAGCCTTTGCAC3′ and 5′GAAGATGCAGATG-GGGAAGT3′.

Statistical Analysis

All statistics were calculated using SigmaStat 3.1 (Systat Software, Richmond, CA). All values are expressed as the mean ± standard deviation. Multiple comparisons were evaluated statistically via one-way analysis of variance, followed by the Bonferroni multiple comparison test.

Results

Hfe^−/− mice develop iron overload characterized by high circulating iron levels and deposition of excess iron in the liver, but with resistance to iron loading in the spleen due to deficient iron storage in macrophages.³¹ To determine whether Lcn2 participates in iron delivery to the liver in Hfe-deficient mice, we interbred Hfe^−/− and Lcn2^−/− mice. Circulating iron levels, assessed by measuring serum iron and transferrin saturation as well as iron deposition in the liver and spleen, were analyzed at 10 and 20 weeks of age (Fig. 1). Iron parameters in Lcn2 single knockout mice were similar to those in wild-type mice (B6), whereas Hfe^−/− and HfeLcn2 double mutants had higher amounts of circulating and liver iron than B6 mice (≈1.5- to 2-fold higher [P < 0.05]) (Fig. 1A–C). In contrast, spleen iron content, though slightly increased with age, was lower in 10-week-old Hfe^−/− and HfeLcn2^−/− mice compared with B6 (31% lower [P < 0.05]) or Lcn2^−/− mice (Fig. 1D). No significant differences were found in heart iron (Fig. 1E), whereas iron levels in kidneys were slightly increased in both Hfe^−/− and HfeLcn2^−/− mice compared with B6 mice (Fig. 1F). Thus, we found no significant differences between Hfe^−/− and HfeLcn2^−/− mice regarding iron parameters. These results indicate that iron accumulation in the livers of Hfe^−/− mice did not improve in the absence of Lcn2, suggesting that Lcn2-mediated cellular iron delivery is not essential in iron uptake by the liver in Hfe^−/− mice.

Fig. 1.

Iron parameters in 10- and 20-week-old wild-type (B6) and mutant mice. (A) Serum iron. (B) Transferrin saturation. (C–F) Iron concentration in the (C) liver, (D) spleen, (E) heart, and (F) kidney. The results are expressed as the mean ± standard deviation (n = 6 to 8 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001 (mutant mice versus B6 mice of the same age).

To further investigate the responses to dietary iron loading in compound mutants, we challenged the mice with a 2.5% wt/wt carbonyl iron-supplemented diet for 2 weeks (Fig. 2). B6 and Lcn2^−/− mice fed the iron-enriched diet showed significant increments of serum iron, transferrin saturation (>50% increase [P < 0.01]), hepatic iron content (>4-fold rise [P < 0.001]) and spleen iron content (>2.5-fold elevation [P < 0.001]) compared with mice on the control diet (Fig. 2A–C). Whereas Hfe^−/− and HfeLcn2^−/− double knockout mice also manifested heightened liver iron content over the already-elevated levels observed on the control diet in response to dietary iron loading (>2-fold rise; P < 0.001), the increase in iron loading of the spleen was considerably more modest than what was seen in B6 (P < 0.01) or Lcn2^−/− mice on the iron-supplemented diet (Fig. 2D).

Fig. 2.

Response to iron-loading in wild-type (B6) and mutant mice. (A) Serum iron. (B) Transferrin saturation. (C) Iron concentration in the liver. (D) Iron concentration in the spleen. (E) Hepatic hepcidin 1 messenger RNA expression (Hamp 1) in mice on a standard diet (gray bars) and mice on a diet supplemented with 2.5% carbonyl iron for 2 weeks (black bars). The results are expressed as the mean ± standard deviation (n = 6 to 8 mice per group). ^##P < 0.001 (mutant mice versus B6 mice). *P < 0.01, **P < 0.001 (iron-supplemented versus standard diet).

Because augmented iron absorption in Hfe^−/− mice has been related to inappropriate expression levels of hepcidin,³⁴ the principal regulator of systemic iron homeostasis, we also measured hepatic hepcidin 1 messenger RNA. As illustrated in Fig. 2E, we found lower amounts of hepcidin in both Hfe^−/− and HfeLcn2 double mutants than in B6 and Lcn2^−/− mice on the control diet (≈30% decrease). Hepcidin levels rose approximately 2.5- to 3.5-fold in response to iron loading in all mouse strains (P < 0.01) but remained significantly lower in Hfe^−/− and HfeLcn2^−/− mice compared with B6 (P < 0.05) or Lcn2^−/− mice. Because the response to iron loading was indistinguishable between Hfe^−/− and HfeLcn2^−/− mice, these results further confirm that Lcn2 is not essential for iron delivery to the liver and the regulation of hepcidin in HH.

Discussion

Although under normal conditions hepatocytes acquire iron mostly through the transferrin-receptor pathway, the existence of uptake of NTBI in HH is now well established. Several candidates have been proposed, including Lcn2, but clear demonstration of its participation and importance in HH has not been yet provided.

We set out to determine whether Lcn2 represents a physiologically relevant mechanism of iron uptake by the liver in HH, an iron-overloading disease. For this purpose, we generated and characterized iron metabolism in HfeLcn2 double knockout mice. The Hfe^−/− mice have increased plasma NTBI levels and, importantly, hepatocytes from Hfe^−/− mice have been shown to uptake significantly more NTBI than control mice.⁸ We found that basal iron status and iron metabolism changes induced by oral iron supplementation were indistinguishable between Hfe single knockout and HfeLcn2 double knockout mice. Our results indicate that Lcn2 is dispensable for NTBI uptake by hepatocytes in HH. However, they do not exclude that the Lcn2-mediated iron delivery pathway may be involved in other pathologies.

The identification of the components of NTBI delivery pathways remains important for the understanding of the patophysiology of HH and other iron overload diseases such as hypotransferrinemia and thalassemia, because in these situations plasma iron exceeds the binding capacity of transferrin and uptake of NTBI significantly contributes to iron accumulation in the liver, pancreas, and heart.³⁵

NTBI uptake mechanisms have been described in a variety of cell lines,^36,37 including hepatocytes.³⁸ In hepatocytes, this system requires the reduction of Fe³⁺ to Fe²⁺.³⁸ Iron salts (such as iron ascorbate, citrate, and nitrilotriacetate) have been suggested as candidates for low molecular-weight NTBI carriers, but the transporters of NTBI have not yet been identified. In addition to Lcn2, which the present study suggests is redundant, potential candidates include L-type voltage-dependent Ca2+ channels (LVDCCs)³⁹ and Zip14.⁴⁰

LVDCCs have been identified as key transporters of iron into cardiomyocytes and neuronal cells under iron overload conditions.^{39, 41} Further support for a role for cardiac LVDCCs in myocardial NTBI uptake under conditions of iron overload comes from the demonstration that LVDCC blockers are protective and able to attenuate myocardial iron accumulation in iron-overloaded mice.⁴²

Zip14 is a zinc transporter and member of the SLC39A metal ion transporter family⁴³ that is highly expressed in hepatocytes. Recent studies have shown that mouse Zip14 transports both iron and zinc in cultured hepatocytes.⁴⁰

In conclusion, the work presented here shows that Lcn2 is dispensable for iron delivery to hepatocytes in the context of Hfe deficiency. Further studies will be necessary to establish whether other candidates—namely LVDCCs and Zip14 —are involved in the uptake of NTBI by hepatocytes under iron overload conditions such as HH.

Abbreviations

HH

hereditary hemochromatosis

Lcn2

lipocalin 2

LVDCC

L-type voltage-dependent Ca2+ channel

NTBI

non–transferrin-bound iron

PCR

polymerase chain reaction