Reduced iron export associated with hepcidin resistance can explain the iron overload spectrum in ferroportin disease

Abstract

Background & aims: Ferroportin disease (FD) and hemochromatosis type 4 (HH4) are associated with variants in the ferroportin-encoding gene SLC40A1. Both phenotypes are characterized by iron overload despite being caused by distinct variants that either mediate reduced cellular iron export in FD or resistance against hepcidin-induced inactivation of ferroportin in HH4. The aim of this study was to assess if reduced iron export also confers hepcidin resistance and causes iron overload in FD associated with the R178Q variant.

Methods: The ferroportin disease variants R178Q andA77D and the HH4-variant C326Y were overexpressed in HEK-293T cells and subcellular localization was characterized by confocal microscopy and flow cytometry. Iron export and cytosolic ferritin were measured as markers of iron transport and radioligand binding studies were performed. The hepcidin-ferroportin axis was assessed by ferritin/hepcidin correlation in patients with different iron storage diseases.

Results: In the absence of hepcidin, the R178Q and A77D variants exported less iron when compared to normal and C326Y ferroportin. In the presence of hepcidin, the R178Q and C326Y, but not the A77D-variant, exported more iron than cells expressing normal ferroportin. Regression analysis of serum hepcidin and ferritin in patients with iron overload are compatible with hepcidin deficiency in HFE hemochromatosis and hepcidin resistance in R178Q FD.

Conclusions: These results support a novel concept that in certain FD variants reduced iron export and hepcidin resistance could be interlinked. Evasion of mutant ferroportin from hepcidin-mediated regulation could result in uncontrolled iron absorption and iron overload despite reduced transport function.

Keywords: SLC40A1; ferroportin disease; hemochromatosis; hepcidin resistance.

Figure 1

Hepcidin‐dose dependent ⁵⁹Fe export. (A) ⁵⁹Fe export within 6 hours is expressed as a fractional of total ⁵⁹Fe uptake and relative to the maximal iron exported from cells overexpressing normal ferroportin. Mean and standard deviation are represented. The mock‐treated control cells were transfected with a backbone plasmid with GFP but without ferroportin as described in the methods. The overexpression of normal ferroportin caused a 8‐10 fold increase ⁵⁹Fe export compared to untransfected or mock‐transfected cells. Without incubation with hepcidin, cells overexpressing R178Q and A77D ferroportin exported significantly less iron than normal and C326Y ferroportin expressing cells. Upon incubation with hepcidin, a dose‐dependent decrease on iron export could be observed only in cells expressing normal ferroportin. However, iron export remained similar to baseline in cells expressing R178Q, A77D and C326Y ferroportin, showing therefore resistance to hepcidin (n = 6). (B) Hepcidin‐dose‐dependent intracellular ferritin concentration after 6h incubation. Intracelullar ferritin concentration (ng/mL) is expressed in relation to total protein concentration (mg/mL). Mean and standard deviation are represented. All cells were incubated with 2 mg/mL holotransferrin as described previously. As expected, untransfected cells with holotransferrin showed the highest values and no notable difference under increasing hepcidin concentrations. Cells overexpressing normal ferroportin showed a dose‐dependent increase of intracellular ferritin as an indirect sign of reduced iron export upon hepcidin‐dependent ferroportin deactivation. No such increase could be observed in cells overexpressing the R178Q, A77D and C326Y ferroportin variants (n = 9). Raw p‐values from group comparisons by Student's t‐test are displayed

Figure 2

Relative ¹²⁵I uptake. ¹²⁵I‐Hepcidin uptake within 2 hours of incubation with 1, 10, 100 or 1000 nM non‐labelled hepcidin is expressed in relation to the maximal ¹²⁵I uptake observed in cells overexpressing normal ferroportin. Mean and standard deviation are represented and non‐labelled hepcidin concentrations are shown in a logarithmized axis. Untransfected cells and cells overexpressing the A77D and C326Y ferroportin mutants showed only minimal background (non‐specific) uptake of radioactivity, whereas cells transfected with normal and R178Q ferroportin fitted to a competitor‐response curve. The apparent affinity (IC 50) of hepcidin to normal ferroportin was 88.4 nM and much reduced (444.2 nM) in cells expressing the R178Q variant (n = 6)

Figure 3

Flow cytometry studies. Cells were co‐transfected with a plasmid encoding for a ferroportin‐GFP fusion protein and simultaneously with another vector encoding a HA‐tagged ferroportin, which upon immunostaining allowed cell surface quantification. (A) Ferroportin expression levels as a percentage of gated cells expressing normal and the different ferroportin mutants. Although 23%‐26% of transfected cells expressed the ferroportin‐GFP fusion protein, only 1%‐3% did actually express ferroportin at the cell surface (n = 9). (B) Time‐dependent downregulation of ferroportin by hepcidin. The relative reduction was calculated after a 4h incubation with 1µM hepcidin. Mean and standard deviation are represented. In cells overexpressing normal ferroportin, incubation with hepcidin lead to a 26% reduction of cells expressing ferroportin‐GFP and 23% of cells expressing HA‐tagged ferroportin. In contrast, in cells overexpressing the C326Y ferroportin variant, an increase of 4 and 6% could be measured in cells expressing the GFP fusion and the HA‐tagged ferroportins respectively. R178Q and A77D overexpressing cells showed a differential hepcidin response to internalization (HA‐tagged ferroportin) and degradation (ferroportin‐GFP fusion protein) with higher degradation than internalization (n = 9)

Figure 4

(A) Serum ferritin concentration over 10 years since first observation of hyperferritinemia (July 2007) until last contact on January 2020 in a patient with R178Q variant FD. The time axis is broken, as between October 2008 and June 2012 there is no clinical record. The black upward pointing triangles represent the serum ferritin values expressed in µg/L; the upper cut‐off (200 µg/L) of the normal range is signalized with a dotted line. The white downward pointing triangles represent the 16 phlebotomies (450 mL each) which the patient underwent between November 2007 and October 2008. As shown, ferritin values descent from an initial value of 1149 µg/L, until normal values (52 µg/L) after five venesections. In June 2012, the patient presented again with a high serum ferritin concentration (649 µg/L). After a new increase, the patient underwent a 12‐week therapy with Ombitasvir/Paritaprevir/Ritonavir and Dasabuvir (start with direct‐acting antivirals – DAA – is also marked in the graphic), and had a rapid virological response. Sustained virological response at week 12 after therapy was also achieved. As shown, ferritin levels lowered under and after therapy for chronic HCV infection. (B) Left panel – MRI T2* scan before DAA treatment showing severe splenic iron accumulation (T2* = 8.0 ms, reference range 20 ‐ 80 ms) with normal liver iron concentration (20 µmol/g, reference range 1.5 ‐ 41.5 µmol/g). Right panel – MRI T2* scan after DAA treatment showing less marked iron accumulation in the spleen (T2* = 10.2 ms) and still normal hepatic iron concentration (14 µmol/g)

Figure 5

Regression analysis of ferritin and hepcidin. When compared to a control cohort of 32 patients with HFE‐hemochromatosis (homozygosity for the C282Y polymorphism and wild‐type for the H63D polymorphism) and a cohort of 40 patients with normal HFE genotype (wild‐type for both HFE polymorphisms C282Y and H63D) and proven high iron concentration through MR T2*, our patient with R178Q‐associated FD showed a much steeper correlation between serum ferritin and hepcidin concentrations