Neonatal hemochromatosis

The antimicrobial peptide hepcidin has been implicated in the regulation of iron homeostasis.

Hepcidin is theorized to be a key link between body iron stores and the appropriate modulation of dietary iron absorption.

Key Words: antimicrobial, peptide, hepcidin, iron homeostasis

Maintenance of iron homeostasis entails strict regulation of dietary iron assimilation. As crypt cells mature into absorptive enterocytes, they are believed to be programmed via two independent regulators, the "stores regulator," which transmits information about body iron stores, and the "erythropoeitic regulator," which communicates iron requirements for red cell production.1 Although the latter is believed to have a greater impact on the net flux of iron in the body, the stores regulator does play an essential role in maintaining iron balance and preventing excessive accumulation of this potentially toxic metal. How the absorptive enterocyte is programmed or "tuned" to assimilate the correct amount of dietary iron to reflect body stores has been the subject of much speculation over the years. Recent studies now implicate the small peptide hepcidin as an iron-regulating hormone involved in modulating dietary iron absorption in a manner that reflects body iron status.

Hepcidin was purified from human urine as a small (20-25-residue) peptide with rather broad antifungal and bacteriocidal activity The peptide (Figure 1) has also been isolated from liver as LEAP-1 (liver-expressed antimicrobial peptide).3 A potential role in iron metabolism for this factor emerged in investigations by Pigeon et al.4 when they discovered hepcidin mRNA was overexpressed in livers of mice injected with iron dextran or in iron-loaded mice fed a carbonyl-iron-supplemented diet. Perhaps the most exciting finding from these early studies was that (beta)2-microglobulin knockout mice, which spontaneously iron-load, show decreased expression of hepcidin when placed on a low-iron diet. Hepcidin levels therefore appeared to reflect body iron stores, but how this feature reflected its functional activity remained a mystery.

Clues to solving this puzzle have been recently provided by two genetically modified mouse models. Nicolas et al.5 first explored the unusual phenotype of a Usf2 knockout mouse strain to develop iron overload. USF2 is a transcription factor studied for its role in glucose-dependent gene regulation in liver.6 Whereas other strains of Us)2 knockout mice do not appear affected,7 Nicolas et al.5 found that their homozygous Usf2-/- mice displayed a pattern of iron loading that resembled the human inherited metabolic disorder, hereditary hemochromatosis. This disease is associated with excessive dietary iron absorption leading to iron overload. High levels of tissue iron accumulation were observed in Usf2-/- mice, particularly in the liver and pancreas, and serum iron levels were 1.7-fold higher than controls. Similar to patients with HFE-associated hereditary hemochromatosis8 and HFE-/- knockout mice9,10 (a murine model of the human disease), Usf2-/- macrophages did not accumulate iron and appeared to be resistant to loading. Careful control experiments demonstrated that expression of HFE and TfR2-factors that are known to be mutated in some forms of hemochromatosis-were not altered.8,11 Thus, although the pattern of iron loading observed in the Usf2-/- mice appeared to be identical to that associated with this disorder of iron metabolism, an obvious explanation for this phenotype was still lacking.

Performing suppressive subtractive hybridization between wild-type and Usf2-/- mice to examine differences in mRNA expression that might explain the iron-- loading phenotype, Nicolas et al.5 were able to identify hepcidin cDNA and further found that its mRNA could not be detected in the livers of their knockout mice. Mouse hepcidin genes (HEPC1 and HEPC2) had been previously noted by Pigeon et al.4 to be on chromosome 19 in close proximity to the marine USF2 gene. Thus, Nicolas et al.5 concluded that their fortuitous finding may reflect a cis effect of the insertion of the Neo^sup R^ marker in Usf2 alleles of their knockout model. The key observation from these experiments was that loss of hepcidin expression correlated with the iron overload of these animals.

Because Pigeon et al.4 had reported that hepcidin expression was up-regulated in marine models of iron overload, Nicolas et al.5 proposed that hepcidin might be a regulator of dietary iron absorption, such that excessive intake would occur in its absence. The excessive dietary absorption and iron-loading pattern of hemochromatosis is promoted by defects in three other genes: HFE, (beta)2-- microglobulin, and transferrin receptor-2 (TfR2). Via their interaction with transferrin receptor-1 (TfR1) in intestinal crypt cells, HFE and (beta)2-microglobulin are thought to modulate the iron set-point for absorption relative to body iron stores in response to a soluble plasma component that would signal between liver, bone marrow, intestine, and macrophages.1 Nicolas et al.5 theorized that TfR2 mediates uptake of transferrin-bound iron into the liver and therefore could directly or indirectly modulate expression of hepcidin. Secreted hepcidin (produced under high iron conditions) would then serve as the putative iron-regulating hormone to somehow alter HFE/TfR1 interactions in the crypt cell to decrease the iron set-point (i.e., reduce absorption). In macrophages, similar interactions with hepcidin might increase accumulation or decrease release of iron to maintain iron homeostasis. The relative changes in dietary iron absorption could be mediated by alterations in the expression of the apical transporter DMT1 (also known as DCT1 and Nramp2) and/or the basolateral iron export protein ferroportin-1 (also known as Ireg-1 and MTP1). Whether expression of either of these iron transporters is altered in Usf2-/- mice remains to be tested. As suggested by the authors,5 measurement of hepcidin in hemochromatosis patients with defects in TfR2 would help to examine how this signaling circuit may be provided with a feedback mechanism controlling release of the peptide.

To test the hypothesis of hepcidin as iron regulator, Nicolas et al.12 performed a follow-up investigation to study transgenic mice over-expressing the peptide. Their transgene expression construct used murine HEPC1 (owing to its closer similarity to the human hepcidin ortholog) driven by the transthyretin promoter to direct liver-specific expression. Most F0 transgenic animals over-expressing hepcidin died within hours after birth, with pale skin, decreased body iron levels, and severe microcytic anemia. Three transgenic founders did survive and were rescued with injections of iron-dextran with marked improvements in health, but subsequent mating produced Fl offspring that also suffered perinatal death. Direct measurements of embryonic iron content in 15.5-day-old fetuses (E15.5) generated by one of these founders indicated fourfold less iron compared with control progeny. Except for a brief induction at birth, hepcidin mRNA is not expressed from E15.5 to postnatal day 42 while message for the peptide is high in adult mouse liver (day 56). By contrast, transthyretin is expressed from E15.5 onward. Thus, expression of hepcidin in early development (a time when it is not usually present) results in severe iron deficiency, possibly by altering fetal iron absorption. Unfortunately, only three of the founders survived and they were mosaics, which probably explains why the parents survived but the offspring did not. All of the transgenic animals expressed high levels of hepcidin and were anemic, however, which supports the idea that high levels of hepcidin suppress iron absorption whereas reduced levels promote excess assimilation.

A speculative model to explain the potential role of hepcidin in fetal iron regulation has also been proposed by Nicolas et al.12 Little is known about how iron crosses the placenta, but the major players recognized to function in intestinal iron absorption have been implicated. Iron is taken up by receptor-mediated endocytosis of maternal transferrin by TfR1, which is found associated with HFE and (beta)2-microglobulin in syncytiotrophoblasts.13 DMT1 has been cytolocalized to the maternal face of the syncytiotrophoblastic membrane as well as to the fetal side.14 Mice with a defective DMT1 gene display neonatal anemia,15 further supporting the idea that this transporter is involved in the transfer of iron to the developing fetus. Efflux of iron to the fetus most likely occurs via ferroportin-1, which has also been found in the mouse inner placenta.16,17 Based on the model for hepcidin's signaling in intestinal iron transfer, a similar role in the regulation of maternal iron transfer is easily imagined and helps to explain the severe neonatal anemia of the transgenic mice that over-express hepcidin.

So what is the connection between an antimicrobial peptide (hepcidin) and iron metabolism? Hepcidin expression is increased in mice injected with lipopolysaccharide, which induces an inflammatory response.4 Inflammation and infection are associated with the anemia of chronic disease, prompting Fleming and Sly18 to advance the idea that hepcidin may also play a role in this disorder of iron metabolism. Very much like hemochromatosis, signaling between iron stores and the intestine appears to be disrupted in the anemia of chronic disease, except that uptake is reduced while storage iron remains high. Thus, increased hepcidin expression during inflammation would diminish intestinal iron absorption while promoting iron loading by macrophages, both of which are hallmarks of anemia of chronic disease. This interesting hypothesis warrants further investigation to explore the full dimensions of hepcidin's function as an iron-regulating hormone. Whereas much more work remains to be done to define the peptide's role in iron metabolism, it seems likely that future research on hepcidin will help to illuminate how iron homeostasis is maintained.

Note added in proof: Nicolas et al. have more recently discovered that hepcidin expression decreases under experimentally-induced anemia, further implicating its function in maintaining iron homeostasis [Nicolas G, Chauvet C, Viatte L, Danan JL, Bigard X, Devaux I, et al. The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia and inflammation. J Clin Invest. 2002;110:1037-10441.

1. Roy CN, Enns CA. Iron homeostasis: new tales from the crypt. Blood. 2000;96:4020-4027.

2. Park CH, Valore EV, Waring AJ, Ganz T. Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem. 2001;276:7806-7810.

3. Krause A, Neitz S, Magert H-J, Schulz A, Forssmann WG, Schulz-Knappe P, et al. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity. FEBS Lett. 2000;480:147-150.

4. Pigeon C, Ilyin G, Courselaud B, Leroyer P, Turlin B, Brissot P, et al. A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem. 2001;276:7811-7819.

5. Nicolas G, Bennoun M, Devaux I, Beaumont C, Grandchamp B, Kahn A, et al. Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice. Proc Natl Acad Sci U S A. 2001;98:8780-8785.

6. Vallet VS, Henrion AA, Bucchini D, Cassado M, Raymondjean M, Kahn A, et al. Glucose-dependent liver gene expression in Upstream Stimulatory Factor 2 mice. J Biol Chem. 1997;272:21994-1999.

7. Sirito M, Lin Q, Deng JM, Behringer RR, Sawadogo M. Overlapping roles and asymmetrical cross-regulation of the USF proteins in mice. Proc Natl Acad Sci USA. 1998;95:3758-3763.

8. Feder JN, Gnirke A, Thomas W, Tsuchihashi Z, Ruddy DA, Basava A, et al. A novel MHC class I-like gene is mutated in patients with hereditary hemochromatosis. Nat Genet. 1996;13:399-408.

9. Levy JE, Montross LK, Cohen DE, Fleming MD, Andrews NC. The C282Y mutation causing hemochromatosis does not produce a null allele. Blood. 1999;94:9-11.

10. Zhou XY, Tomatsu S, Fleming RE, Parkkila S, Waheed A, Jiang J, et al. HFE gene knockout produces

mouse model of hemochromatosis. Proc Natl Acad Sci U S A. 1998;95:2492-2497.

11. Camaschella C, Roetto A, Cali A, DeGobbi M, Garozzo G, Carella M, et al. The gene TFR2 is mutated in a new type of haemochromatosis mapping to 7q22. Nat Genet. 2000;25:14-15.

12. Nicolas G, Bennoun M, Porteu A, Mativet S, Beaumont C, Grandchamp B, et al. Severe iron deficiency anemia in transgenic mice expressing liver hepcidin. Proc Natl Acad Sci U S A. 2002;99:45964601.

13. Parkkila S, Waheed A, Britton RS, Bacon BR, Zhao XY, Tomatsu S, et al. Association of the transferrin receptor in human placenta with HFE, the protein defective in hereditary hemochromatosis. Proc Natl Acad Sci U S A. 1997;9:13198-13202.

14. Georgieff MK, Wobken JK, Welle J, Burdo JR, Connor JR. Identification and localization of divalent metal transporter-1 (DMT-1) in term human placenta. Placenta. 2000;21:799-804.

15. Fleming MD, Trenor CC, Su MA, Foernzler D, Beier DR, Dietrich WF, et al. Microcytic aenemia mice have a mutation in Nramp2, a candidate iron transporter gene. Nat Genet. 1997;16:383-386.

16. Donovan A, Brownlie A, Zhou Y, Shepard J, Pratt SJ, Moynihan J, et al. Positional cloning of zebrafish ferroportin 1 identifies a conserved vertebrate iron exporter. Nature. 2000;403:776-781.

17. McKie AT, Marciani P, Rolfs A, Brennan K, Wehr K, Barrow D, et al. A novel duondenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation. Mol Cell. 2000;5: 299-309.

18. Fleming R, Sly WS. Hepcidin: a putative iron-regulatory hormone relevant to hereditary hemochromatosis and the anemia of chronic disease. Proc Natl Acad Sci U S A. 2001;98:8160-8162.

This review was prepared by Marianne Wessling-- Resnick, Ph.D., Department of Nutrition, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115.

Copyright International Life Sciences Institute and Nutrition Foundation Nov 2002
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