Zip14 (Slc39a14) mediates non-transferrin-bound iron uptake into cells

Abstract

Zip14 is a member of the SLC39A zinc transporter family, which is involved in zinc uptake by cells. Up-regulation of Zip14 by IL-6 appears to contribute to the hepatic zinc accumulation and hypozincemia of inflammation. At least three members of the SLC39A family transport other trace elements, such as iron and manganese, in addition to zinc. We analyzed the capability of Zip14 to mediate non-transferrin-bound iron (NTBI) uptake by overexpressing mouse Zip14 in HEK 293H cells and Sf9 insect cells. Zip14 was found to localize to the plasma membrane, and its overexpression increased the uptake of both (65)Zn and (59)Fe. Addition of bathophenanthroline sulfonate, a cell-impermeant ferrous iron chelator, inhibited Zip14-mediated iron uptake from ferric citrate, suggesting that iron is taken up by HEK cells as Fe(2+). Iron uptake by HEK and Sf9 cells expressing Zip14 was inhibited by zinc. Suppression of endogenous Zip14 expression by using Zip14 siRNA reduced the uptake of both iron and zinc by AML12 mouse hepatocytes. Zip14 siRNA treatment also decreased metallothionein mRNA levels, suggesting that compensatory mechanisms were not sufficient to restore intracellular zinc. Collectively, these results indicate that Zip14 can mediate the uptake of zinc and NTBI into cells and that it may play a role in zinc and iron metabolism in hepatocytes, where this transporter is abundantly expressed. Because NTBI is commonly found in plasma of patients with hemochromatosis and transfusional iron overload, Zip14-mediated NTBI uptake may contribute to the hepatic iron loading that characterizes these diseases.

Fig. 1.

Overexpression of mZip14 increases iron and zinc accumulation by HEK 293H cells. (A) HEK 293H cells were transfected with either empty vector (pCMV-Sport6) or the same plasmid containing mZip14 and grown for 48 h. Western blots of total cell lysates (15 μg of protein per lane) were prepared by using SDS/PAGE and immunoblotting with anti-mZip14 antibody. (B) Time course of iron accumulation measured with ⁵⁹Fe in medium containing 2 μM ferric citrate (pH 7.4). (C) Time course of zinc accumulation measured with ⁶⁵Zn in medium containing 2 μM ZnCl₂ (pH 7.4), transfected with mZip14 (●) or vector alone (○). Nonspecific ⁵⁹Fe and ⁶⁵Zn binding to the cell surface was estimated by measuring uptake at 4°C. (D) Effect of BPS at 2.5- and 20-fold molar excess on mZip14-dependent iron accumulation after 60 min. Values are mean ± SD (n = 3; ∗, P < 0.05 compared with control) and are representative of multiple experiments.

Fig. 2.

mZip14 localizes to the plasma membrane of Sf9 cells and increases iron and zinc accumulation. (A) Sf9 cells were infected with wild-type baculovirus (control) or baculovirus containing the cDNA for mZip14. After 64 h, total cell lysates (15 μg of protein per lane) were analyzed by Western blotting as above. Lanes represent three independent experiments. (B and C) The same cells were analyzed by immunofluorescence microscopy. Cells were fixed and incubated with an anti-mZip14 antibody followed by goat anti-rabbit IgG-Alexa Fluor 594. Cells were counterstained with DAPI to visualize nuclei. (D) Iron accumulation measured with ⁵⁹Fe in medium containing 2 μM FeCl₂. (E) Zinc accumulation measured with ⁶⁵Zn in medium containing 2 μM ZnCl₂. (F) mZip14-dependent iron accumulation measured with ⁵⁹Fe in medium containing 2 μM FeCl₂ and 200 μM ZnCl₂. Cells were incubated at 27°C for 15 min in uptake medium. Values are the mean ± SD of three separate experiments, each with n = 3. ∗, P < 0.05 compared with control.

Fig. 3.

mZip14 siRNA decreases Zip14 expression in AML12 mouse hepatocytes. (A) QRT-PCR analysis of Zip14 mRNA levels in AML12 cells treated for 96 h with a nontargeting (control) siRNA or siRNA targeted to mZip14. Values are the mean ± SD of three separate experiments, each with n = 3. ∗, P < 0.05 compared with control. (B) Cells in parallel were analyzed for Zip14 protein levels by Western blotting of total cell lysates (50 μg of protein per lane). To confirm equivalent protein loading, the Western blot was stripped and reprobed with anti-tubulin antibody. A representative experiment is shown.

Fig. 4.

Zip14 knockdown in mouse hepatocyte AML12 cells reduces iron and zinc accumulation and MT1 (metallothionein 1) expression without affecting TfR1 mRNA levels. AML12 cells were treated with nontargeting (control) siRNA or Zip14 siRNA for 96 h. (A) Iron accumulation measured with ⁵⁹Fe in medium containing 2 μM ferric citrate. (B) Zinc accumulation measured with ⁶⁵Zn in medium containing 2 μM ZnCl₂. (C and D) QRT-PCR analysis of MT1 (C) and TfR1 (D) mRNA levels. For ⁵⁹Fe and ⁶⁵Zn accumulation, cells were incubated with uptake medium for 15 min. Values are mean ± SD of three separate experiments, each with n = 3. ∗, P < 0.05 compared with control.

Fig. 5.

Tissue-expression profile of mZip14. Individual tissues were excised from two CD-1 mice and pooled before isolating total RNA. Zip14 transcript abundance was determined by QRT-PCR starting with an equal amount of total RNA from each tissue.