Nonclassical ferroportin disease (FD) is a form of hereditary hemochromatosis caused by mutations in the iron transporter ferroportin (Fpn), resulting in parenchymal iron overload. Fpn is regulated by the hormone hepcidin, which induces Fpn endocytosis and cellular iron retention. We characterized 11 clinically relevant and 5 nonclinical Fpn mutations using stably transfected, inducible isogenic cell lines. All clinical mutants were functionally resistant to hepcidin as a consequence of either impaired hepcidin binding or impaired hepcidin-dependent ubiquitination despite intact hepcidin binding. Mapping the residues onto 2 computational models of the human Fpn structure indicated that (1) mutations that caused ubiquitination-resistance were positioned at helix-helix interfaces, likely preventing the hepcidin-induced conformational change, (2) hepcidin binding occurred within the central cavity of Fpn, (3) hepcidin interacted with up to 4 helices, and (4) hepcidin binding should occlude Fpn and interfere with iron export independently of endocytosis. We experimentally confirmed hepcidin-mediated occlusion of Fpn in the absence of endocytosis in multiple cellular systems: HEK293 cells expressing an endocytosis-defective Fpn mutant (K8R), oocytes expressing wild-type or K8R Fpn, and mature human red blood cells. We conclude that nonclassical FD is caused by Fpn mutations that decrease hepcidin binding or hinder conformational changes required for ubiquitination and endocytosis of Fpn. The newly documented ability of hepcidin and its agonists to occlude iron transport may facilitate the development of broadly effective treatments for hereditary iron overload disorders.
Introduction: The iron-regulatory hormone hepcidin regulates the body iron stores and its expression is repressed when erythropoietic activity intensifies to meet the iron requirements for erythropoiesis (e.g. during anemia). Under the influence of erythropoietin (EPO), the hormone erythroferrone (ERFE) is secreted by erythroid precursors in the bone marrow and the spleen, and suppresses hepcidin synthesis to facilitate the recovery from anemia. However, the mechanism by which ERFE suppresses hepcidin is unknown. In contrast with forms of anemia in which hepcidin is suppressed, patients with mutations in transmembrane serine protease 6 (TMPRSS6) have iron-refractory iron deficiency anemia (IRIDA) but increased hepcidin production despite a severe anemia and elevated EPO levels. Recently, it has been suggested that matriptase-2 activity facilitates ERFE-mediated suppression of hepcidin. We therefore investigated the potential crosstalk between ERFE and Matriptase 2. Methods: We first measured serum ERFE concentration in Tmprss6-/- mice. To assess the contribution of ERFE to the phenotype of Tmprss6-/-mice, we next generated Tmprss6-/-mice with disrupted Erfe (Erfe+/- Tmprss6-/-; Erfe-/- Tmprss6-/- and Erfe+/+ Tmprss6-/-). To determine whether ERFE requires TMPRSS6 to regulate hepcidin production, we treated freshly isolated hepatocytes from wild-type (WT) or Tmprss6-/- mice with conditioned medium from cells expressing recombinant ERFE or not. Results: While wild-type mice have undetectable plasma ERFE (below the 500 pg/ml limit of detection), plasma ERFE concentration was elevated in Tmprss6-/- to levels comparable to those of WT animals 24 hours after phlebotomy (~3 ng/ml) but was lower than ERFE levels in thalassemic mice (~10 ng/ml). Ablation of Erfe in Tmprss6-/- mice did not result in any change in hematological parameters, hepcidin expression and iron levels compared to Tmprss6-/- animals at 6 weeks of age. However, treatment of WT and Tmprss6-/-hepatocytes with ERFE resulted in a comparable suppression of hepcidin mRNA expression. Conclusion: Although matriptase-2 may dampen the BMP signaling under the influence of EPO, it is not part of the ERFE signaling pathway. Disclosures Ganz: Intrinsic Lifesciences: Other: shareholder and scientific advisor; Merganser Biotech: Other: shareholder and scientific advisor; Silarus therapeutics: Other: shareholder and scientific advisor; Keryx Biopharmaceuticals: Consultancy. Nemeth:Intrinsic Lifesciences: Other: shareholder and scientific advisor; Merganser Biotech: Other: shareholder and scientific advisor; Silarus therapeutics: Other: shareholder and scientific advisor.
The cellular iron‐export protein ferroportin (Fpn), expressed in enterocytes and macrophages, is essential for iron homeostasis. Fpn operates under the control of the iron‐regulatory hormone hepcidin. The canonical mechanism by which hepcidin suppresses Fpn activity comprises hepcidin binding by Fpn and the subsequent internalization and ubiquitin‐dependent degradation of the transport protein [Drakesmith H et al (2015) Cell Metab 22, 777–787]. Here we explored hepcidin inhibition of 55Fe efflux from RNA‐injected Xenopus oocytes expressing human Fpn. Hepcidin treatment induced a rapid inhibition (t½ = 8.2 ± [SEM] 0.5 min; r2 > 0.99, P < 0.001) of Fpn‐mediated 55Fe efflux that was not dependent on endocytosis, a conclusion based on the following four 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 substantially more temperature‐dependent, Q10 in the order of 5–17 [Weigel PH and Oka JA (1981) J Biol Chem 256, 2615–2617]. (ii) Inhibition of Fpn by hepcidin proceeded even in the presence of blockers of clathrin‐mediated (Dynasore) and clathrin‐independent (filipin, genistein) endocytosis. (iii) Hepcidin treatment inhibited 55Fe efflux in oocytes expressing the K8R mutant, in which 8 Lys residues required for ubiquination and endocytosis of Fpn were mutated to Arg, to the same degree as in wildtype Fpn. (iv) Confocal LSM imaging of wildtype Fpn‐GFP and K8R‐Fpn‐GFP expression in oocytes revealed no detectable endocytosis of either protein after 30‐min treatment with hepcidin. Our data reveal that hepcidin can directly block Fpn activity independent of Fpn internalization. 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–NIDDK grant R01 DK107309This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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