Reduced sensitivity of the ferroportin Q248H mutant to physiological concentrations of hepcidin

Abstract

Ferroportin Q248H mutation has an allele frequency of 2.2-13.4% in African populations and is associated with a mild tendency to increased serum ferritin in the general population. Some investigators have reported that ferroportin Q248H is degraded after exposure to hepcidin in exactly the same manner as wild-type ferroportin, but supraphysiological concentrations of hepcidin were used. The aim of our study was to determine whether ferroportin Q248H may have reduced sensitivity to physiological concentrations of hepcidin. The sensitivity of ferroportin Q248H to hepcidin was determined in 293T cells transiently expressing ferroportin using immunoblotting and fluorescence analysis. Ferritin concentrations were measured in these cells and also in human primary monocytes derived from humans with different ferroportin genotypes. The effect of Q248H on serum iron measures was examined in patients with sickle cell anemia. Immunoblotting and fluorescence analysis showed decreased sensitivity of ferroportin Q248H to physiological concentrations of hepcidin. Lower ferritin concentrations were observed after incubation with iron and hepcidin in 293T cells expressing ferroportin Q248H and in primary monocytes from ferroportin Q248H subjects. In sickle cell anemia, ferroportin Q248H heterozygotes had lower serum ferritin concentrations than wild-type subjects, consistent with enhanced iron release by macrophage ferroportin Q248H. A clinical benefit of ferroportin Q248H was suggested by lower echocardiographic estimates of pulmonary artery pressure in patients carrying mutant alleles. In conclusion, our results suggest that ferroportin Q248H protein is resistant to physiological concentrations of hepcidin and that this mutation has discernible effects on iron metabolism-related clinical complications of sickle cell anemia. They provide a mechanistic explanation for the effect of ferroportin Q248H on iron status in individuals of African descent and suggest that these changes in iron metabolism may be beneficial under certain disease-specific circumstances. (ClinicalTrials.gov Identifier:NCT00011648).

Figures

Figure 1.

Ferroportin Q248H is less sensitive to hepcidin than WT ferroportin. (A) Effect of hepcidin on ferroportin Q248H expression. 293T cells were transfected with vectors expressing ferroportin Q248H mutant, WT ferroportin, or ferroportin C326Y (a mutant known to have reduced sensitivity to hepcidin). Ferroportin was expressed as a fusion with a c-myc–derived tag. The transfected cells were treated with the indicated concentrations of hepcidin for 1 h. Expression of ferroportin (Fpn) was analyzed by immunoblotting with anti-c-myc antibody and compared to the loading control with anti-tubulin antibody. (B) Quantification of WT and Q248H mutant ferroportin expression. WT ferroportin and Q248H mutant ferroportin were expressed in 293T cells and analyzed as in panel A in untreated cells and cells treated with 0.03 or 0.3 μM hepcidin. Results from three independent experiments are shown. (C) Effect of ferroportin Q248H expression on cellular ferritin levels. 293T cells were transfected as in panel A and incubated with ferric ammonium citrate for 16 h. The cells were then treated with 100 μM cycloheximide for 1 h followed by treatment with 0.01 μM hepcidin for 18 h. Ferritin was measured in the cell lysates by ELISA and adjusted to total protein concentrations measured by the Bradford assay.

Figure 2A,B.

Ferroportin Q248H-EGFP is resistant to hepcidin. Ferroportin WT and mutants Q248H or C326Y EGFP-fused proteins were expressed in 293T cells and treated with hepcidin at 37°C for 4 h. EGFP fluorescence was measured in live cells as described in the Design and Methods section. (A) Measurement of EGFP fluorescence showed significantly increased expression of ferroportin (FPN) C326Y-EGFP compared to FPN WT-EGFP at hepcidin concentrations of 0.01, 0.03 and 0.1 μM. FPN Q248H-EGFP was also increased at these hepcidin concentrations but less so than FPN C326Y. (B) Expression of ferroportin was analyzed by immunoblotting with anti-EGFP antibody and compared to the loading control with anti-α-tubulin antibody. Compared to ferroportin WT, ferroportin Q248H had reduced sensitivity to 0.03 μM hepcidin but not to 0.3 μM hepcidin.

Figure 3.

Lower ferritin increase in response to hepcidin in primary human macrophages expressing ferroportin Q248H. Human primary macrophages were treated with ferric ammonium citrate for 18 h and then with 0.01-0.5 μM hepcidin concentrations for 6 h. The cells were lysed and ferritin concentrations were measured using an ELISA and adjusted to the total protein concentration determined by the Bradford assay. (A) Results are presented as fold increase in ferritin levels after hepcidin treatment relative to the levels in untreated controls. (0), macrophages expressing WT ferroportin. (1), macrophages expressing heterozygous ferroportin Q248H. (2), macrophages expressing homozygous ferroportin Q248H. (B) Ferritin concentrations are shown in untreated macrophages and macrophages treated with 0.5 μM hepcidin.