Divalent metal transporter 1 (Dmt1) mediates copper transport in the duodenum of iron-deficient rats and when overexpressed in iron-deprived HEK-293 cells

Abstract

Intracellular copper-binding proteins (metallothionein I/II) and a copper exporter (Menkes copper-transporting ATPase) are upregulated in duodenal enterocytes from iron-deficient rats, consistent with copper accumulation in the intestinal mucosa. How copper enters enterocytes during iron deficiency is, however, not clear. Divalent metal transporter 1 (Dmt1), the predominant iron importer in the mammalian duodenum, also transports other metal ions, possibly including copper. Given this possibility and that Dmt1 expression is upregulated by iron deprivation, we sought to test the hypothesis that Dmt1 transports copper during iron deficiency. Two model systems were utilized: the Belgrade (b) rat, expressing mutant Dmt1, and an inducible Dmt1-overexpression cell culture system. Mutant rats (b/b) were fed a semipurified, AIN93G-based control diet and phenotypically normal littermates (+/b) were fed control or iron-deficient diets for ~14 wk. An everted gut sleeve technique and a colorimetric copper quantification assay were utilized to assess duodenal copper transport. The control diet-fed +/b rats had normal hematological parameters, whereas iron-deprived +/b and b/b rats were iron deficient and Dmt1 mRNA and protein levels increased. Importantly, duodenal copper transport was similar in the control +/b and b/b rats; however, it significantly increased (~4-fold) in the iron-deprived +/b rats. Additional experiments in Dmt1 overexpressing HEK-293 cells showed that copper ((64)Cu) uptake was stimulated (∼3-fold) in the presence of an iron chelator. Dmt1 transcript stabilization due to a 3' iron-responsive element was also documented, likely contributing to increased transport activity. In summary, these studies suggest that Dmt1 enhances copper uptake into duodenal enterocytes during iron deprivation.

FIGURE 1

Dmt1 mRNA and protein expression and duodenal mutant (b/b) copper transport in control (+/b), iron-deprived (14 wk) control (+/bD), and b/b rats. A representative Western blot from duodenal tissue is shown (A) along with quantitative data (B) from all experimental rats. Band intensities were normalized to total protein on the stained blots (shown below the Western-blot image). The number beside the blot indicates the placement of the closest molecular weight marker (in kDa). Means without a common letter differ, P < 0.005. (C) qRT-PCR analysis of Dmt1 mRNA expression. Means without a common letter differ, P < 0.00005. (D) Copper transport studies in everted gut sacs derived from duodenum of experimental rats. Shown is the amount of copper transported from the mucosal to serosal side. (B–D) Values are means ± SEMs. Means without a common letter differ, P < 0.01, n = 4–5. A.U., arbitrary units; Dmt1, divalent metal transporter 1.

FIGURE 2

DOX induces expression of stably transfected Dmt1 in HEK-293 cells and iron chelation with DFO further potentiates expression. qRT-PCR quantification of rat Dmt1 (A) and hTFR1 (B) mRNA expression levels. (C) A representative Western blot of Dmt1 protein expression and quantitative blot data (D) from all experiments. Band intensities on film were normalized to total protein on stained blots (shown below the Western blot) with that for DOX only set to 1. The number beside the image indicates the placement of the closest molecular weight marker (in kDa). Values are means ± SEMs, n = 4. Means without a common letter differ; P < 0.0005 for a and b and P < 0.00005 for the rest (A); P < 0.001 (B); and P < 0.0005 for b and c and P < 0.00005 for the rest (D). A.U., arbitrary units; DFO, desferrioxamine; Dmt1, divalent metal transporter 1; DOX, doxycycline; hTFR1, human transferrin receptor 1.

FIGURE 3

DOX induction of Dmt1 expression in combination with iron chelation by DFO increases copper transport in HEK293 cells. ⁵⁹Fe (A) or ⁶⁴Cu (B) transport was assessed in cells stably transfected with the rat Dmt1/1A(+IRE) cDNA. Values are means ± SEMs, n = 4. Means without a common letter differ; P < 0.0005 for b and c and P < 0.00005 for the rest (A); P < 0.005 for a and b and P < 0.00005 for the rest (B). DFO, desferrioxamine; Dmt1, divalent metal transporter 1; DOX, doxycycline; IRE, iron-responsive element.

FIGURE 4

The increase in Dmt1 expression in stably transfected HEK-293 cells is mediated by the 3′ IRE. Cells were stably transfected with either the Dmt1(1A+) cDNA (containing the IRE) (A) or the Dmt1(1B-) cDNA (lacking the IRE) (B) and Dmt1 mRNA expression was quantified by qRT-PCR. Values are means ± SEMs, n = 4. Means without a common letter differ, P < 0.00005 (A); P < 0.01 for b and c and P < 0.00005 for the rest (B). A.U., arbitrary units; DFO, desferrioxamine; Dmt1, divalent metal transporter 1; DOX, doxycycline; FAC, ferric ammonium citrate; IRE, iron-responsive element.