Hepcidin-25 inhibits TfR up-regulation and ferritin reduction caused by wt FPN but not by mutant FPN. (A-B) 293T cells were transiently transfected with either c-Myc–tagged wt FPN (A) or c-Myc–tagged Y64N FPN (B) for 2 days with or without 0.5 μM hepcidin-25 added. Cells were then stained simultaneously for surface expression of c-Myc and TfR and analyzed by flow cytometry. Transfected cells (c-Myc⁺ cells) were gated on and their TfR expression is displayed relative to the TfR expressed by control CD8-transfected cells. The wt FPN expression caused an increase in TfR expression compared with control cells (compare red line with gray histogram in panel A) consistent with FPN causing iron deficiency; coculture with hepcidin-25 reverses the effect of wt FPN (green line is similar to gray filled histogram in panel A). In contrast the up-regulation of TfR by Y64N (compare red line with gray filled histogram in panel B) is not counteracted by coculture with hepcidin-25 (green line is similar to red line in panel B). The blue-filled histogram represents the fluorescence of cells stained with an isotype control antibody (anti–rabbit IgG). (C) Quantitation of inhibition of FPN and FPN mutant-mediated up-regulation of TfR by hepcidin-25. Cells were transfected with c-Myc–tagged FPN and FPN mutants with or without added hepcidin-25 and stained for surface c-Myc and TfR then analyzed as described in panels A and B. The MFI of the different populations was calculated using CellQuest software and is displayed with the MFI of the TfR expressed by control CD8-transfected cells subtracted. The increase of TfR expression induced by wt human, wt murine, and Q248H FPN is counteracted by hepcidin-25, whereas the TfR increase mediated by C326Y and Y64N FPN was resistant to hepcidin-25 inhibition, and the raised TfR due to N144D and N144H expression was partially reduced by hepcidin-25. (D) 293T cells were transfected with wt FPN or C326Y FPN in the presence of 1 mg/mL human holo-Tf to increase background ferritin levels, and with or without added hepcidin-25. Cells were transfected with CD8 as a control. After 2 days cells were analyzed for ferritin levels by ELISA. Cells were lysed in NP40 at 10⁷cells/mL, doubling dilutions were made, and 20 μL transferred to ELISA plate in triplicate. Graph shows mean total ferritin in nanograms per 10⁷ cells (± 95% CI). Cells transfected with wt FPN have around 40 ng ferritin/10⁷cells, a 3-fold reduction compared with control cells transfected with CD8. Coculture with hepcidin-25 completely reverses the effect of wt FPN, but hepcidin-25 has no effect on the reduction of ferritin mediated by C326Y FPN. These results are representative of 3 experiments. Horizontal bars indicate mean values.