Lack of Hepcidin Gene Expression and Severe Tissue Iron Overload in Upstream Stimulatory Factor 2 (USF2) Knockout Mice

Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice Gae¨ l Nicolas*, Myriam Bennoun*, Isabelle Devaux†, Carole Beaumont†, Bernard Grandchamp†, Axel Kahn*, and Sophie Vaulont*‡ *Institut National de la Santeét de la Recherche Me´dicale 129, Departement Genetique Developpement et Pathologie Molećulaire, Institut Cochin de Genetique Molećulaire, Faculte´deMe´ decine Cochin-Port Royal, 75014 Paris, France; and †Institut National de la Santeét de la Recherche Me´dicale 409, Faculte´deMe´ decine Xavier Bichat, 75018 Paris, France Edited by William S. Sly, Saint Louis University School of Medicine, St. Louis, MO, and approved May 10, 2001 (received for review April 11, 2001) We previously reported the disruption of the murine gene encoding dietary iron leading to an iron overload in plasma and multiple the transcription factor USF2 and its consequences on glucose-depen- organs. Hemochromatosis is usually due to a mutation in the dent gene regulation in the liver. We report here a peculiar phenotype HLA-linked hemochromatosis gene (named HFE) located on of Usf2؊/؊ mice that progressively develop multivisceral iron over- chromosome 6p, and most patients are homozygous for the load; plasma iron overcomes transferrin binding capacity, and non- C282Y mutation in HFE (8). In addition, other loci have been transferrin-bound iron accumulates in various tissues including pan- involved in different HH families; a nonsense mutation in the creas and heart. In contrast, the splenic iron content is strikingly lower transferrin receptor 2 gene (TFR2) on 7q has been reported in in knockout animals than in controls. To identify genes that may two HH non-HLA-linked families (9), and a locus for juvenile account for the abnormalities of iron homeostasis in Usf2؊/؊ mice, we hemochromatosis has recently been mapped to chromosomal used suppressive subtractive hybridization between livers from arm1q(HFE2). Finally, although it has long been known that Usf2؊/؊ and wild-type mice. We isolated a cDNA encoding a peptide, iron absorption is regulated in response to the level of body iron stores and to the amount of iron needed for erythropoiesis (10), hepcidin (also referred to as LEAP-1, for liver-expressed antimicrobial the molecular nature of the signals that program the intestinal peptide), that was very recently purified from human blood ultrafil- cells to adjust iron absorption still remains to be identified. trate and from urine as a disulfide-bonded peptide exhibiting anti- We previously reported the disruption of the murine gene microbial activity. Accumulation of iron in the liver has been recently encoding the transcription factor USF2 and its consequences on reported to up-regulate hepcidin expression, whereas our data clearly glucose-dependent gene regulation in the liver (11). We now show show that a complete defect in hepcidin expression is responsible for that Usf2Ϫ/Ϫ mice develop multivisceral iron overload that spares progressive tissue iron overload. The striking similarity of the alter- only the spleen and whose iron content is strikingly lower in ations in iron metabolism between HFE knockout mice, a murine knockout animals than in controls. Although these iron metabolic ,model of hereditary hemochromatosis, and the Usf2؊/؊ hepcidin- disorders resemble those observed in hereditary hemochromatosis deficient mice suggests that hepcidin may function in the same we demonstrate that they are not because of an alteration in genes regulatory pathway as HFE. We propose that hepcidin acts as a previously identified for their implication in this pathology—e.g., signaling molecule that is required in conjunction with HFE to regu- HFE or TFR2. Therefore, to identify new candidate genes that may Ϫ Ϫ late both intestinal iron absorption and iron storage in macrophages. account for the abnormalities of iron homeostasis in Usf2 / mice, we used suppressive subtractive hybridization between livers from Usf2Ϫ/Ϫ mice and wild-type mice. We isolated a cDNA encoding the ron is an essential element required for growth and survival of peptide hepcidin. Hepcidin (also referred to as LEAP-1, for Ialmost every organism. In mammals, the iron balance is liver-expressed antimicrobial peptide) was very recently purified primarily regulated at the level of duodenal absorption of dietary from human blood ultrafiltrate and from urine and was found to be iron. Following absorption, ferric iron is loaded into apo- a disulfide-bonded peptide exhibiting antimicrobial activity (12, 13). transferrin in the circulation and transported to the tissues, The protein is synthesized in the liver in the form of a propeptide including erythroid precursors, where it is taken up by trans- that contains 83 amino acids and is converted into mature peptides ferrin receptor-mediated endocytosis. Reticuloendothelial mac- of 20, 22, and 25 amino acids (13, 14). Hepcidin was recently rophages play a major role in the recycling of iron from the reported to be highly synthesized in livers of experimentally or degradation of hemoglobin of senescent erythrocytes, whereas spontaneously iron-overloaded mice (14). Here, we demonstrate hepatocytes contain most of the iron stores of the organism in that hepcidin gene expression is totally silent in the iron overload Ϫ Ϫ ferritin polymers. Over the past 5 years, an important body of Usf2 / mice model. Taken together, these results suggest that information concerning the proteins involved in iron absorption hepcidin can act as a signaling molecule involved in the mainte- and in the regulation of iron homeostasis has arisen from the nance of iron homeostasis. study of inherited defects, both in humans and mice, leading to Materials and Methods distinct iron disorders (for review see ref. 1). In the case of iron ؊/؊ deficiency, the pathophysiological consequences of gene defects Generation and Genotyping of Usf2 Mice. Disruption of the Usf2 identified are well understood because they usually result in loss gene has been described (11). The mutated allele contains the of function of proteins directly involved in the pathway of iron absorption. The proteins include the iron transporters DMT1 This paper was submitted directly (Track II) to the PNAS office. (also called Nramp2 or DCT1) (2, 3), ferroportin (also called Abbreviations: HH, hereditary hemochromatosis; HFE, the protein defective in heredi- IREG1 or MTP1) (4), and copper oxidases coupled to ferro- tary hemochromatosis; HEPC, hepcidin; USF, upstream stimulatory factor; RT, reverse portin, namely ceruloplasmin (5, 6) and haephastin (7). In transcription. contrast, several abnormalities associated with genetic iron See commentary on page 8160. overload have identified various proteins whose functional role ‡To whom reprint requests should be addressed. E-mail: [email protected]. in the control of iron homeostasis remains poorly understood. In The publication costs of this article were defrayed in part by page charge payment. This humans, hereditary hemochromatosis (HH) is a common auto- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. somal recessive genetic disease caused by hyperabsorption of §1734 solely to indicate this fact. 8780–8785 ͉ PNAS ͉ July 17, 2001 ͉ vol. 98 ͉ no. 15 www.pnas.org͞cgi͞doi͞10.1073͞pnas.151179498 Downloaded by guest on October 2, 2021 promoterless IRES␤geo cassette in exon 7 of the murine USF2 SDS at 68°C for 20 min and two times in 0.2 ϫ SSC͞0.1% SDS at gene. All studied mice have a mixed genetic background that 68°C for 20 min. included contributions from C57BL͞6 and 129͞Sv strains. Mice were maintained on a standard laboratory mouse chow (AO3, Reverse Transcription (RT) and RT-PCR. Double-stranded cDNA was Usine d’Alimentation Rationnelle, France) containing 280 mg of synthesized in 20 ␮l, with 2 ␮g total RNA (or polyA RNA for the ferric carbonate per kg. Mice were killed from the ages of 2.5 subtracted library), in the presence of 0.25 mM of each dNTP, 200 months up to 19 months. Genotyping on mouse-tail DNA was ng of random hexanucleotide primers, 20 units of RNasin (Pro- performed by using a single PCR reaction to identify wild-type and mega), 10 mM DTT, and 200 units Moloney murine leukemia virus USF2 knockout alleles. Genomic DNA (0.5–1 ␮g) was used in a reverse transcriptase (GIBCO). After denaturation of RNA at 70°C 50-␮l reaction that included three primers; the wild-type USF2 for 10 min in a therman cycler (Perkin–Elmer), the reaction was allele was amplified by using forward GCGAAGCCCTGGGT- performedfor1hat42°Cbefore reverse transcriptase was inacti- TCAATC (annealing in intron 6) and reverse GGGGTCCAC- vated for 6 min at 96°C. At the end of the reaction, 80 ␮lof10mM CACTTCAAGAGG (annealing in intron 7) primers, and the Tris⅐HCl (pH 8.0) and 0.1 mM EDTA (pH 8.0) were added. PCR knockout USF2 allele was amplified by using the forward GC- amplification was performed with 5 ␮l of reverse transcriptase GAAGCCCTGGGTTCAATC and reverse GAATTCTCTA- reaction mixture in 50 ␮lof20mMTris⅐HCl (pH 8.4)͞50 mM ͞ ͞ ͞ ͞ GAGCGGCCGGAC (annealing in the Neo selection marker of KCl 2 mM MgCl2 0.05% (vol/vol) W-1 0.2 mM of each dNTP 1 the targeting construct) primers. PCR was performed as follows: 37 pmol of forward and reverse specific primers (listed below)͞1 pmol cycles (each cycle consisting of 30 s at 94°C, 30 s at 56°C, and 40 s of forward and reverse control ␤-actin primers͞2 units of Taq at 72°C) with an initial denaturation step at 94°C for 4 min, and a polymerase (GIBCO). PCR conditions were 25 cycles of denatur- final elongation step at 72°C for 5 min in 20 mM Tris⅐HCl (pH ation at 94°C for 20 s, annealing at 50°C for 20 s, and primer ͞ ͞ ͞ ͞ ͞ 8.4) 50 mM KCl 0.05% W-1 2 mM MgCl2 5% glycerol 0.04% extension at 72°C for 20 s.

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