Hepcidin inhibits ferroportin in the Xenopus oocyte expression system via a calcium‐dependent mechanism that does not involve endocytosis

Abstract

The cellular iron‐exporter ferroportin (Fpn), under the control of the hormone hepcidin, serves as a key control point in iron homeostasis. The canonical mechanism by which hepcidin suppresses Fpn activity comprises hepcidin binding by Fpn and the subsequent internalization and ubiquitin‐dependent degradation of the transporter. Here we explored hepcidin inhibition of 55Fe efflux from RNA‐injected Xenopus oocytes expressing human Fpn. Hepcidin pretreatment induced a rapid inhibition (t½ = 8.2 ± [SEM] 0.5 min; r2 > 0.99, P < 0.001) of Fpn‐mediated 55Fe efflux. Hepcidin had no effect on Fpn activity when we removed calcium from the pretreatment medium (0 Ca2+, 0.1 mM EGTA). The action of hepcidin was not dependent on endocytosis, a conclusion based on the following observations: (i) Inhibition of Fpn by hepcidin was only modestly temperature‐dependent (Q10= 2.5 ± SEM 1.4 over the range 17–27 °C) whereas endocytosis is expected to be strongly temperature‐dependent, Q10 in the order of 5–17 [Weigel PH and Oka JA (1981) J Biol Chem256, 2615–17]. (ii) Inhibition of Fpn by hepcidin proceeded even in the presence of blockers of clathrin‐mediated (chlorpromazine, Dynasore) and clathrin‐independent (filipin, genistein) endocytosis. (iii) Hepcidin treatment inhibited 55Fe efflux in oocytes expressing the (K→R)8‐Fpn mutant, in which 8 Lys residues required for ubiquitination and endocytosis of Fpn were mutated to Arg, as much as in wildtype Fpn. (iv) Confocal LSM imaging of wildtype Fpn‐GFP and (K→R)8‐Fpn‐GFP expression in oocytes revealed no detectable endocytosis of either protein after treatment with hepcidin. (v) Hepcidin treatment produced no meaningful change in membrane surface area (as determined by capacitance) of oocytes expressing Fpn, despite reducing Fpn‐mediated iron transport by 65%. Our data reveal that hepcidin can directly block Fpn activity via a Ca2+‐dependent mechanism that does not involve endocytosis. We speculate that the Xenopus oocyte lacks the specific signal by which hepcidin triggers Fpn internalization. The Xenopus oocyte is therefore an ideal model system in which to study the mechanism by which hepcidin directly blocks Fpn activity.Support or Funding InformationNIH grants R01 DK107309, P30 DK078392, and R25 HL115473, and the American Physiological Society