Severe iron deficiency anemia in transgenic mice expressing liver hepcidin - PubMed (original) (raw)

Severe iron deficiency anemia in transgenic mice expressing liver hepcidin

Gaël Nicolas et al. Proc Natl Acad Sci U S A. 2002.

Abstract

We recently reported the hemochromatosis-like phenotype observed in our Usf2 knockout mice. In these mice, as in murine models of hemochromatosis and patients with hereditary hemochromatosis, iron accumulates in parenchymal cells (in particular, liver and pancreas), whereas the reticuloendothelial system is spared from this iron loading. We suggested that this phenotypic trait could be attributed to the absence, in the Usf2 knockout mice, of a secreted liver-specific peptide, hepcidin. We conjectured that the reverse situation, namely overexpression of hepcidin, might result in phenotypic traits of iron deficiency. This question was addressed by generating transgenic mice expressing hepcidin under the control of the liver-specific transthyretin promoter. We found that the majority of the transgenic mice were born with a pale skin and died within a few hours after birth. These transgenic animals had decreased body iron levels and presented severe microcytic hypochromic anemia. So far, three mosaic transgenic animals have survived. They were unequivocally identified by physical features, including reduced body size, pallor, hairless and crumpled skin. These pleiotropic effects were found to be associated with erythrocyte abnormalities, with marked anisocytosis, poikylocytosis and hypochromia, which are features characteristic of iron-deficiency anemia. These results strongly support the proposed role of hepcidin as a putative iron-regulatory hormone. The animal models devoid of hepcidin (the Usf2 knockout mice) or overexpressing the peptide (the transgenic mice presented in this paper) represent valuable tools for investigating iron homeostasis in vivo and for deciphering the molecular mechanisms of hepcidin action.

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Figures

Figure 1

Figure 1

Iron content in liver, pancreas, and spleen of Houston _Usf2_−/− mice. Liver, pancreas, and spleen were fixed in formaldehyde and stained with the Perls' stain for iron. Non-heme iron stains blue. Liver, pancreas, and spleen sections from a representative 10-month-old Houston _Usf2_−/− mouse, 8-month-old control mice, and an 8-month-old Paris _Usf2_−/− mouse (×20).

Figure 2

Figure 2

Generation of TTR-HEPC1 transgenic mice. (A) Comparison of the 25-aa peptide sequences from human, mouse, and rat. Conserved cysteine residues are shaded. (B) Schematic representation of the TTR-HEPC1 construct. The murine HEPC1 cDNA was introduced between the transthyretin sequences (consisting of the 3 kb of the mouse TTR regulatory regions 5′ to the cap site, the first exon, first intron, and most of the second exon) and the SV40 small-T poly(A) signal sequence (13). (C) Southern blot analysis of tail DNA from transgenic founders. The founders were distributed among two groups according to their phenotype. Genomic DNA was digested by _Bam_HI and hybridized with the TTR probe represented in B. Two bands of the expected size, 5.3 kbp and 4.7 kbp, representing the endogenous TTR gene (End) and the transgene (Tg), respectively, were detected. (D) Phenotypic features of transgenic mice harboring hepcidin. The arrows indicate the pale newborn THnb10 (arrow) at birth among the nontransgenic littermates and the hairless and crumpled TH5 founder at 4 weeks, as compared with a nontransgenic littermate.

Figure 3

Figure 3

Hematological phenotype of the transgenic mice. Wright-Giemsa-stained blood smears were performed on control (Left) and transgenic mice (Right). Compared with normal erythrocytes, transgenic erythrocytes from THnb10 at birth and TH35 at 4 weeks show marked anisocytosis, poikylocytosis, and hypochromia. Arrows indicate the typical ringed red cells characteristic of iron deficient anemia.

Figure 4

Figure 4

Hepcidin and transthyretin mRNA content during liver development of wild-type C57BL/6 mice as determined by Northern blot analysis. Twenty micrograms of wild-type total liver RNA, from E15.5 and E17.5, birth (B), and during postnatal development from P1 to P56, was electrophoresed, blotted, and hybridized with hepcidin and TTR 32P-labeled probes.

Figure 5

Figure 5

Hepcidin mRNA level in transgenic mice as determined by Northern blot analysis. Twenty micrograms of total liver RNA was electrophoresed, blotted, and hybridized with the 32P-labeled hepcidin probe made by PCR as described in Materials and Methods. This probe reveals both the transgenic hepcidin (Tg-hepc, upper band) and the endogenous hepcidin (End-hepc, lower band). (A) Hepcidin mRNA level in F0 transgenic animals (THnb) at P1, as compared with nontransgenic (NT) littermates. (B) Hepcidin mRNA level in F1 progeny from TH5 at P1, from TH35 at P13, and from TH61 at P1.

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