The molecular basis of ferroportin-linked hemochromatosis

Abstract

Mutations in the iron exporter ferroportin (Fpn) (IREG1, SLC40A1, and MTP1) result in hemochromatosis type IV, a disorder with a dominant genetic pattern of inheritance and heterogeneous clinical presentation. Most patients develop iron loading of Kupffer cells with relatively low saturation of plasma transferrin, but others present with high transferrin saturation and iron-loaded hepatocytes. We show that known human mutations introduced into mouse Fpn-GFP generate proteins that either are defective in cell surface localization or have a decreased ability to be internalized and degraded in response to hepcidin. Studies using co-immunoprecipitation of epitope-tagged Fpn and size-exclusion chromatography demonstrated that Fpn is multimeric. Both WT and mutant Fpn participate in the multimer, and mutant Fpn can affect the localization of WT Fpn, its stability, and its response to hepcidin. The behavior of mutant Fpn in cell culture and the ability of mutant Fpn to act as a dominant negative explain the dominant inheritance of the disease as well as the different patient phenotypes.

Fig. 1.

Disease-inducing Fpn mutations affect localization and response to hepcidin. (A–C) HEK293T cells were transiently transfected with plasmids containing WT Fpn-GFP, Fpn(D157G)-GFP, Fpn(Δ160-162)-GFP, or FpnN144H-GFP, and localization was assessed by epifluorescent microscopy. Eighteen to 24 h after transfection, cells were incubated with 1 μg/ml hepcidin for 4 (B) and 24 (C) h and examined for Fpn-GFP localization. (D) Cells were incubated with or without 1 μg/ml hepcidin for 4 h, and extracts were analyzed by Western blot analysis using antibody to GFP and an antibody to actin as a loading control.

Fig. 2.

Fpn mutations affect intracellular ferritin levels. (A) HEK293T cells were transiently transfected with plasmids containing WT Fpn-GFP or mutant Fpn-GFP. Eighteen hours after transfection, cells were cultured with ferric ammonium citrate (FAC) (20 μM iron). After FAC loading (24 h), cells were incubated with 100 μM cycloheximide (1 h) followed by 1.0 μg/ml hepcidin (4 h), and ferritin levels were determined by ELISA. Error bars are the standard deviation of three experiments. P values were calculated by using a Student t test. (B) ¹²⁵I-hepcidin was added to HEK293T cells expressing WT Fpn-GFP or mutant Fpn-GFP, and cell-associated radioactivity was measured. Each bar represents the average of four to six measurements. The data were normalized to the amount of radioactivity bound to WT Fpn-expressing cells, and the amount of radioactivity bound to untransfected cells was subtracted as background for each point. The relative values were further normalized for the amount of Fpn-GFP expressed in each sample as determined by Western blotting.

Fig. 3.

Immunoprecipitation of Fpn demonstrates that Fpn is a multimer. (A)(Left) HEK293T cells were transiently transfected with plasmids containing WT Fpn-FLAG and Fpn-GFP. Cell extracts (1.5 mg of protein equivalent to one confluent 6.0-cm plate) were obtained after 24 h and immunoprecipitated by using anti-FLAG. EXT, cell extracts before immunoprecipitation; E, FLAG-specific elutions. (Center) Extracts were prepared from cells expressing either Fpn-FLAG or Fpn-GFP. The extracts were mixed and then subjected to immunoprecipitation. (Right) HEK293T cells were transfected with plasmids containing WT Fpn-FLAG and Fpn(Δ160-162)-GFP, and cell extracts were immunoprecipitated by using anti-FLAG. Western blots were analyzed for Fpn-FLAG, Fpn-GFP, and EGF receptor. (B) HEK293T Fpn cells were induced to express Fpn-GFP by the addition of ponasterone for 24 h. Cells were solubilized by the addition of 1.0%Triton X-100. Samples were applied to a Superdex 200 FPLC column that had been equilibrated with Triton X-100. Fractions were collected and analyzed for Fpn-GFP by Western blot analysis.

Fig. 4.

Coexpression of mutant and WT Fpn alters the surface distribution of WT Fpn and affects Fpn response to hepcidin. (A) HEK293T cells were transiently cotransfected with plasmids containing either WT Fpn-FLAG and Fpn-GFP or Fpn-FLAG and Fpn(Δ160-162)-GFP. Localization of different Fpn as well as the Tf-R was assessed by immunofluorescence microscopy. (B) HEK293T cells were transiently transfected with plasmids containing WT Fpn-FLAG and Fpn-GFP or Fpn-FLAG and Fpn(N144H)-GFP. After 24 h, cells were incubated with 1 μg/ml hepcidin (4 h), and localization of Fpn-FLAG/GFP was assessed by immunofluorescence microscopy. (C) Cells were transfected with plasmids containing WT Fpn-FLAG and Fpn-GFP, Fpn-FLAG and Fpn(N144H)-GFP, Fpn-FLAG and Fpn(Δ160-162)-GFP, or Fpn-FLAG and Fpn(Δ162)-GFP. Eighteen hours posttransfection, cells were cultured with ferric ammonium citrate (20 μM iron). After incubation with iron for 24 h, cells were incubated with 100 μM cycloheximide (1 h) followed by 1 μg/ml hepcidin (4 h), and ferritin levels were determined by ELISA. The data are presented as mean ± standard deviation (n = 3).

Fig. 5.

Coexpression of mutant and WT Fpn affects the stability of Fpn and its ability to export iron. (Upper) HEK293T cells were transiently cotransfected with plasmids containing WT Fpn-FLAG, Fpn-GFP, or Fpn(D157G)-GFP. After 24 h, samples were extracted and analyzed by Western blot. The Western blots were probed with antibodies to actin as a control for loading. (Lower) Ferritin levels were assayed in cells transiently transfected with GFP/FLAG vector control, Fpn-FLAG and Fpn-GFP, and Fpn-FLAG and Fpn(D157G)-GFP.