Mechanism of increased iron absorption in murine model of hereditary hemochromatosis: increased duodenal expression of the iron transporter DMT1

Abstract

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder characterized by tissue iron deposition secondary to excessive dietary iron absorption. We recently reported that HFE, the protein defective in HH, was physically associated with the transferrin receptor (TfR) in duodenal crypt cells and proposed that mutations in HFE attenuate the uptake of transferrin-bound iron from plasma by duodenal crypt cells, leading to up-regulation of transporters for dietary iron. Here, we tested the hypothesis that HFE-/- mice have increased duodenal expression of the divalent metal transporter (DMT1). By 4 weeks of age, the HFE-/- mice demonstrated iron loading when compared with HFE+/+ littermates, with elevated transferrin saturations (68.4% vs. 49.8%) and elevated liver iron concentrations (985 micrograms vs. 381 micrograms). By using Northern blot analyses, we quantitated duodenal expression of both classes of DMT1 transcripts: one containing an iron responsive element (IRE), called DMT1(IRE), and one containing no IRE, called DMT1(non-IRE). The positive control for DMT1 up-regulation was a murine model of dietary iron deficiency that demonstrated greatly increased levels of duodenal DMT1(IRE) mRNA. HFE-/- mice also demonstrated an increase in duodenal DMT1(IRE) mRNA (average 7.7-fold), despite their elevated transferrin saturation and hepatic iron content. Duodenal expression of DMT1(non-IRE) was not increased, nor was hepatic expression of DMT1 increased. These data support the model for HH in which HFE mutations lead to inappropriately low crypt cell iron, with resultant stabilization of DMT1(IRE) mRNA, up-regulation of DMT1, and increased absorption of dietary iron.

Figure 1

Serum transferrin saturations and hepatic iron concentrations and in the HFE^−/− mice and HFE^+/+ littermates. At 4 weeks of age the mice were sacrificed, and blood and liver specimens were obtained. (A) Serum total iron binding capacities and iron concentrations were measured. Transferrin saturations were calculated and expressed as mean ± SEM. ∗, P < 0.001. (B) Liver nonheme iron concentrations (micrograms of iron per gram dry liver) were measured and expressed as mean ± SEM. ∗∗, P < 0.005.

Figure 2

RNA blot analyses of DMT1 expression in duodenum of iron-deficient mice. Three micrograms of poly(A)⁺ RNA from duodenum of a nontransgenic FVB/N control (C, lanes 1, 3, 5) and a transgenic H⁺/K⁺-ATPase β subunit (−1035 to +24)/DT-A iron-deficient littermate (DT-A, lanes 2, 4, 6) were electrophoresed in triplicate and blotted. Lanes 1 and 2 were hybridized with a ³²P-labeled coding-sequence probe [DMT1(total)] to detect all DMT1 transcripts. Lanes 3 and 4 were hybridized with an oligonucleotide probe [DMT1(IRE)] specific to DMT1 transcripts with an IRE, and lanes 5 and 6 were hybridized with an oligonucleotide probe [DMT1(non-IRE)] specific to the DMT1 splice-variant transcripts without an IRE. Blots were exposed to film for 8 h with an intensifying screen and rehybridized with a probe for GAPDH. Positions of RNA size markers are on the left.

Figure 3

RNA blot analysis of DMT1 expression in duodenum of HFE^+/+ and HFE^−/− mice. Poly(A)⁺ RNA (3 μg, lanes 1–4; 10 μg, lanes 5–6) from duodenum of a 4-week-old HFE^+/+ mouse (lanes 1, 3, and 5) and a HFE^−/− littermate (lanes 2, 4, and 6) were electrophoresed in triplicate and blotted. Lanes 1 and 2 were hybridized with a ³²P-labeled coding-sequence probe [DMT1(total)] to detect all DMT1 transcripts. Lanes 3 and 4 were hybridized with an oligonucleotide probe [DMT1(IRE)] specific to DMT1 transcripts with an IRE, and lanes 5 and 6 were hybridized with an oligonucleotide probe [DMT1(non-IRE)] specific to the DMT1 splice-variant transcripts without an IRE. Blots were exposed to film for 18 h (lanes 1–4) or 48 h (lanes 5 and 6) with an intensifying screen, and rehybridized with a probe for GAPDH. Positions of RNA size markers are on the left.

Figure 4

Quantification of duodenal DMT1 expression in HFE^+/+ and HFE^−/− mice. RNA blot analyses were performed on poly(A)⁺ RNA from duodenum of 11 4-week-old HFE^+/+ mice and 14 HFE^−/− littermates by using the DMT1(total) probe. Signals obtained from predominant 4.4-kb transcript were quantified by phosphorimagery and normalized to that obtained by rehybridizing the blots with a probe for GAPDH. Data are presented as mean ± SEM and expressed relative to the mean value obtained from the HFE^+/+ mice. ∗, P = 0.01.

Figure 5

RNA blot analysis of hepatic and duodenal expression of DMT1 in HFE^+/+ and HFE^−/− mice. Pooled poly(A)⁺ RNA (3 μg) from liver (lanes 1 and 2) or duodenum (lanes 3 and4) of five 4-week-old HFE^+/+ mice and five HFE^−/− littermates was electrophoresed, blotted, and hybridized with the DMT1(total) probe. The blot was exposed to film for 18 h with an intensifying screen and rehybridized with a probe for GAPDH.