Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice

Abstract

We previously reported the disruption of the murine gene encoding the transcription factor USF2 and its consequences on glucose-dependent gene regulation in the liver. We report here a peculiar phenotype of Usf2(-/-) mice that progressively develop multivisceral iron overload; plasma iron overcomes transferrin binding capacity, and nontransferrin-bound iron accumulates in various tissues including pancreas and heart. In contrast, the splenic iron content is strikingly lower in knockout animals than in controls. To identify genes that may account for the abnormalities of iron homeostasis in Usf2(-/-) mice, we used suppressive subtractive hybridization between livers from Usf2(-/-) and wild-type mice. We isolated a cDNA encoding a peptide, hepcidin (also referred to as LEAP-1, for liver-expressed antimicrobial peptide), that was very recently purified from human blood ultrafiltrate and from urine as a disulfide-bonded peptide exhibiting antimicrobial activity. Accumulation of iron in the liver has been recently reported to up-regulate hepcidin expression, whereas our data clearly show that a complete defect in hepcidin expression is responsible for progressive tissue iron overload. The striking similarity of the alterations in iron metabolism between HFE knockout mice, a murine model of hereditary hemochromatosis, and the Usf2(-/-) hepcidin-deficient mice suggests that hepcidin may function in the same regulatory pathway as HFE. We propose that hepcidin acts as a signaling molecule that is required in conjunction with HFE to regulate both intestinal iron absorption and iron storage in macrophages.

Figure 1

Iron accumulation in liver and pancreas of Usf2^−/− mice. Liver and pancreas were fixed in formaldehyde and stained with the Perls' stain for iron. Nonheme iron stains blue. Liver sections are from an 8-month-old wild-type mice (×50) (A), an 8-month-old Usf2^−/− littermate (B), and a 19-month-old Usf2^−/− mouse (×10) (C). Pancreas section in D is from an 8-month-old Usf2^−/− mouse (×12.5). Arrowheads in C indicate iron in the nucleus of the hepatocyte. Arrowheads in D point to islets of Langerhans scattered throughout the exocrine tissue. (E and F) Age-dependent hepatic and pancreatic nonheme iron concentration (micrograms of iron per gram dry tissue) as measured in control (wild-type and heterozygote mice, ▴) and Usf2^−/− mice (□).

Figure 2

Iron content in spleen of Usf2^−/− mice. (A) Age-dependent splenic nonheme iron concentration (micrograms of iron per gram dry tissue) as measured in control (wild-type and heterozygote mice, ▴) and Usf2^−/− mice (□). Spleen section from a representative 8-month-old wild-type mouse (×20) (B) and an 8-month-old Usf2^−/− littermate (×20) (C) stained with the Perls' stain for iron. RP, red pulp; WP, white pulp.

Figure 3

HFE and TFR2 mRNA content in liver of wild-type and Usf2^−/− animals as determined by Northern blot analysis. Twenty micrograms of total liver RNAs from wild-type mice and Usf2^−/− mice (from 3 to 11 months old) were electrophoresed and blotted. Blots were hybridized with a ³²P-labeled probe (made by PCR, as described in Materials and Methods) for HFE (A) and RTf2 (B).

Figure 4

Genomic organization of Usf2 and hepcidin genes. Schematic representation (not to scale) of the locus region encompassing the Usf2 and hepcidin genes. The targeted allele is represented with the betageo cassette insertion in exon 7 (11). Data are the result of genomic RP23–22G9 clone (GenBank). So far, no data are available concerning the orientation and the distance between the two hepcidin genes. The Southern blot in the right of the figure is from tail DNA of wild-type, heterozygote, and homozygote mice digested by BglII and hybridized with the HEPC1 probe. Two bands of the expected size, 12.4 and 5.1 kbp, were detected, whatever the genotype. The same bands were revealed by using the Usf2 probe.

Figure 5

Hepcidin mRNA content in liver of wild-type, Usf2^+/−, and Usf2^−/− animals as determined by Northern blot analysis and RT-PCR. (A) Twenty micrograms of total liver RNAs from wild-type, Usf2^+/−, and Usf2^−/− animals (between 3 and 11 months old) were electrophoresed and blotted. The blot was hybridized with a ³²P-labeled HEPC probe (as described in Materials and Methods), which most likely recognized both HEPC1 and HEPC2 transcripts. (B) Specific HEPC1 and HEPC2 levels were measured by RT-PCR as described in Materials and Methods. Following PCR, the amplified products (171 bp for HEPC1 or HEPC2 and 250 bp for β-actin) were separated by electrophoresis on 1.5% agarose gel. Neither HEPC1 nor HEPC2 specific primers were able to reamplify HEPC2 and HEPC1 PCR products, respectively, demonstrating the high specificity of each pair of primers (not shown).

Figure 6

Hypothetical model for hepcidin as a key regulator of iron homeostasis. In this model, hepcidin prevents iron overload by reducing iron transport in the enterocyte and by programming macrophages to retain iron. In Usf2^−/− mice, the hepcidin defect would be responsible for increased intestinal iron transport and reduced macrophage iron stores.