SLC39A14 Is Required for the Development of Hepatocellular Iron Overload in Murine Models of Hereditary Hemochromatosis

Abstract

Nearly all forms of hereditary hemochromatosis are characterized by pathological iron accumulation in the liver, pancreas, and heart. These tissues preferentially load iron because they take up non-transferrin-bound iron (NTBI), which appears in the plasma during iron overload. Yet, how tissues take up NTBI is largely unknown. We report that ablation of Slc39a14, the gene coding for solute carrier SLC39A14 (also called ZIP14), in mice markedly reduced the uptake of plasma NTBI by the liver and pancreas. To test the role of SLC39A14 in tissue iron loading, we crossed Slc39a14(-/-) mice with Hfe(-/-) and Hfe2(-/-) mice, animal models of type 1 and type 2 (juvenile) hemochromatosis, respectively. Slc39a14 deficiency in hemochromatotic mice greatly diminished iron loading of the liver and prevented iron deposition in hepatocytes and pancreatic acinar cells. The data suggest that inhibition of SLC39A14 may mitigate hepatic and pancreatic iron loading and associated pathologies in iron overload disorders.

Figure 1. Hepatic and Pancreatic Uptake of Intravenously Administered ⁵⁹Fe-NTBI is Impaired in Slc39a14^−/− Mice

(A) In vivo ⁵⁹Fe-NTBI uptake by various tissues of 6-wk-old wild-type (WT, n = 5) and Slc39a14^−/− mice (n = 10). Tissue uptake of ⁵⁹Fe from NTBI was calculated as a percentage of whole-body cpm. (B) Immunoblot analysis of SLC39A14 in liver homogenates from WT, Slc39a14^+/−, and Slc39a14^−/− mice (n = 4 per group). (C) In vivo ⁵⁹Fe-NTBI uptake by the liver in WT (n = 10), Slc39a14^+/− (n = 9), and Slc39a14^−/− (n = 15) mice. (D) In vitro ⁵⁹Fe-NTBI uptake by cultures of primary hepatocytes isolated from 13-wk-old WT and Slc39a14^−/− mice. Data are presented as pmol ⁵⁹Fe per mg of protein (n = 3 per group). (E) In vivo ⁵⁹Fe-TBI uptake by various tissues of 6-wk-old WT and Slc39a14^−/− mice (n = 4–5 per group). Tissue uptake of ⁵⁹Fe from TBI was calculated as a percentage of whole-body cpm. Data are presented as percent of total body cpm. Significance was calculated by using Student’s t-test. All data are shown as the mean ± SEM. ***p < 0.001, *p < 0.05.

Figure 2. Ablation of Slc39a14 in Hfe^−/− Mice Impairs Hepatic Iron Loading

(A) Tissue non-heme iron concentrations in WT, Slc39a14^−/−, Hfe^−/−, and Hfe^−/−;Slc39a14^−/− mice at 4 wk of age (n = 5–9 per group). (B) Iron absorption (% of dose) was measured after intragastric gavage of ⁵⁹Fe (n = 5–13 per group). (C) Hepatic ⁵⁹Fe accumulation (% of absorbed ⁵⁹Fe) was measured after intragastric gavage of ⁵⁹Fe (n = 5–6 per group). (D–F) Representative images of Perls’ iron stain in paraffin-embedded liver sections of mice at 4 wk of age (n = 3 per group). Branches of the portal (P) and central (C) veins are indicated. (D, E) Liver sections processed by using standard Perls’ iron stain (blue stain) and hematoxylin counterstain. Scale bars, 200 and 100 μm, respectively. (F) Serial liver sections processed by using DAB-enhanced Perls’ stain (black stain) and neutral red counterstain. Scale bars, 100 μm. (G–J) Plasma iron indices. (G) Plasma iron concentrations (n = 5–6 per group). (H) Total iron-binding capacity, TIBC (n = 5–6 per group). (I) Transferrin saturation (n = 5–6 per group). (J) Plasma NTBI concentrations (n = 8–13 per group). (K) Body weights of mice at 4 wk of age (n= 6–12 per group). All data are shown as the mean ± SEM. Means without a common superscript differ significantly (p < 0.05). See also Figure S1.

Figure 3. Ablation of Slc39a14 in the Hfe2^−/− Mouse Model of Juvenile Hemochromatosis Prevents Hepatic Iron Overload

(A) Tissue non-heme iron concentrations in WT, Slc39a14^−/−, Hfe2^−/−, and Hfe2^−/−;Slc39a14^−/− mice at 6 wk of age (n = 4–11 per group). (B) Iron absorption (% of dose) was measured after intragastric gavage of ⁵⁹Fe (n = 4–10 per group). (C–E) Hepatic, pancreatic, and splenic ⁵⁹Fe accumulation (% of absorbed ⁵⁹Fe) was measured after intragastric gavage of ⁵⁹Fe (n = 4–11 per group). (F–H) Representative images of Perls’ iron stain in paraffin-embedded liver sections of mice at 6 wk of age (n = 3 per group). (F, G) Liver sections processed by using standard Perls’ iron stain (blue stain) and hematoxylin counterstain. Scale bars, 200 and 100 μm, respectively. (H) Serial liver sections processed by using DAB-enhanced Perls’ stain (black stain) and neutral red counterstain. Arrows indicate iron deposits in non-parenchymal cells. Scale bars, 100 μm. (I–L) Plasma iron indices. (I) Plasma iron concentrations (n = 4–6 per group). (J) Total iron-binding capacity, TIBC (n = 4–6 per group). (K) Transferrin saturation (n = 4–6 per group). (L) Plasma NTBI concentrations (n = 5–9 per group). (M) Body weights of mice at 6 wk of age (n = 7–11 per group). All data are shown as the mean ± SEM. Means without a common superscript differ significantly (p < 0.05). See also Figure S1, Figure S2, Figure S3, Figure S5, and Table S1.

Figure 4. Hfe2^−/−;Slc39a14^−/− Mice Display Altered Iron Loading in the Pancreas

(A) Representative images of DAB-enhanced Perls’ iron stain in paraffin-embedded pancreas sections of WT, Slc39a14^−/−, Hfe2^−/−, and Hfe2^−/−;Slc39a14^−/− mice at 6 wk of age (n = 4 per group). Serial sections of pancreas processed in parallel by using DAB-enhanced Perls’ iron stain (black stain) with or without neutral red counterstain (+NR and −NR respectively). Black arrowheads indicate pancreatic islets. Arrows indicate iron deposits in Hfe2^−/−/Slc39a14^−/− mice. Scale bar, 200 μm. (B) Higher magnification of the boxed regions indicated in the Hfe2^−/−;Slc39a14^−/− sections in (A) and representative images of serial sections from Hfe2^−/−;Slc39a14^−/− mice showing immunostaining for markers of ductal epithelium (PCK-26), blood vessel endothelium (CD-31), lymphatic endothelium (LYVE-1) and connective tissue (collagen I), respectively (n = 4 per group). Slides processed for immunostaining were counterstained with hematoxylin. Arrows indicate iron deposits; arrowheads indicate areas of positive immunostaining (brown stain). Scale bar, 100 μm. See also Figure S4.

Figure 5. Slc39a14^−/− Mice Display Altered Hepatic Iron Loading in Response to Dietary Iron Overload

(A) Tissue non-heme iron concentrations in weanling WT and Slc39a14^−/− mice fed a normal-iron control diet (WT-FeC and Slc39a14^−/−-FeC) or an iron-overloaded diet (WT-FeO and Slc39a14^−/−-FeO) for 4 wk (n = 6–10 per group). (B–D) Representative images of Perls’ iron stain in paraffin-embedded liver sections of mice at 7 wk of age (n = 3–4 per group). Branches of the portal (P) and central (C) veins are indicated. (B, C) Liver sections processed by using standard Perls’ iron stain (blue stain) and hematoxylin counterstain. Scale bars, 200 μm and 100 μm, respectively. (D) Serial liver sections processed by using DAB-enhanced Perls’ stain (black stain) and neutral red counterstain. Arrows indicate iron deposits in non-parenchymal cells. Scale bar, 100 μm. (E–H) Plasma iron indices. (E) Plasma iron concentrations (n = 6–11 per group). (F) Total iron-binding capacity, TIBC (n = 6–11 per group). (G) Transferrin saturation (n = 6–11 per group). (H) Plasma NTBI concentrations (n = 5–6 per group). (I) Body weights of mice at the end of the 4-wk feeding study (n= 11–16 per group). All data are shown as the mean ± SEM. Means without a common superscript differ significantly (p < 0.001). See also Figure S4 and Figure S5.

Figure 6. Hepatic Expression of Hamp and Bmp6 mRNA in Slc39a14^−/− Mice with Genetic or Dietary Iron Overload

Relative expression of hepcidin (Hamp) and Bmp6 mRNA was measured in (A) WT, Slc39a14^−/−, Hfe^−/−, and Hfe^−/−;Slc39a14^−/− mice at 4 wk of age (n = 7–9 per group), (B) WT, Slc39a14^−/−, Hfe2^−/−, and Hfe2^−/−;Slc39a14^−/− mice at 6 wk of age (n = 4–10 per group), and (C) WT-FeC, Slc39a14^−/−-FeC, WT-FeO and Slc39a14^−/−-FeO mice at 7 wk of age (n = 6 per group). Transcript levels were normalized to those of Rpl13a. All data are shown as the mean ± SEM. Means without a common superscript differ significantly (p < 0.01). (D) Proposed role of SLC39A14 in iron loading of the liver and pancreas and effect of SLC39A14 deficiency on hepatic BMP6 regulation. In iron overload, the iron-binding capacity of plasma transferrin (gray ovals) becomes exceeded, giving rise to NTBI, which loads into hepatocytes and pancreatic acinar cells via SLC39A14. In iron overload with SLC39A14 deficiency, iron does not load in hepatocytes or acinar cells, but can accumulate in nonparenchymal cells of the liver (endothelial cells (EC), hepatic stellate cells (HSC), and Kupffer cells (KC)) and collagen fibers in the pancreas. That hepatic BMP6 expression can be upregulated in iron overload with SLC39A14 deficiency (A, B, C) suggests that BMP6 expression is regulated by SLC39A14-independent iron loading of nonparenchymal cells. See also Figure S6.