How does hepcidin hinder ferroportin activity?

In this issue of Blood, Aschemeyer et al found that hepcidin bound to the central cavity of ferroportin (Fpn) and occluded its iron export activity, thereby revealing a previously unrecognized mechanism for Fpn regulation.¹ 

Fpn, the sole iron exporter known in vertebrates to date, is highly expressed in intestinal epithelial cells, reticuloendothelial macrophages, and hepatocytes to regulate iron absorption, recycling, and storage. Hepcidin, a small peptide hormone secreted by hepatocytes, regulates the abundance of Fpn in response to iron overload, inflammation, and erythropoiesis to optimize systemic iron homeostasis.²  In the circulation, hepcidin binds to Fpn on the plasma membrane to induce its ubiquitination, internalization, and degradation, which reduces iron influx to the blood. Gene mutations that either disrupt hepcidin expression or change Fpn activity cause hereditary iron disorders, emphasizing the essential role of the hepcidin-Fpn axis in systemic iron hemostasis. Fpn mutations are a common cause of hemochromatosis.³  Gain-of-function mutations that impair Fpn degradation cause high intestinal iron uptake, iron overload in hepatocytes and other tissues, high transferrin saturations, and iron deficiency in macrophages.³  Conversely, loss-of-function mutations that reduce Fpn activity result in iron overload in reticuloendothelial macrophages, hyperferritinemia, and low to normal transferrin saturations.

Aschemeyer et al analyzed gain-of-function Fpn mutations in stably transfected cell lines. They found that these mutations either impaired hepcidin binding to Fpn or interfered with Fpn ubiquitination and degradation following hepcidin treatment. They mapped these mutations onto computational models of human Fpn structure and discovered that 1 class of mutations hindering the hepcidin binding was located in the central cavity, whereas the class of mutations impairing the ubiquitination was positioned at the helix-helix interfaces of the multitransmembrane transporter, which likely interfered with the conformational changes proposed to occur following binding to hepcidin.⁴  Hepcidin binding to the central cavity would likely interfere with the conformational transition of Fpn from an outward- to an inward-facing conformation that permits cytosolic Fe²⁺ ion access to an internal binding site buried in the N-lobe near the central transport cavity, which is essential for the iron export process to initiate.⁴  Accordingly, researchers speculated that hepcidin binding to the central cavity would occlude the iron export activity of Fpn.⁴  To distinguish between the contributions of the well-known Fpn degradation pathway and the effects of occlusion alone to the regulation of Fpn activity, Aschemeyer et al used K8R Fpn, an Fpn mutant known to be resistant to hepcidin-induced degradation; they also used Xenopus oocytes, which likely do not internalize Fpn. Their experiments demonstrated that hepcidin inhibited the iron export activity of Fpn, thereby demonstrating that hepcidin-mediated occlusion plays an important regulatory role.

Recently, our laboratory found that Fpn was highly abundant in mature red blood cells (RBCs).⁵  RBCs lack the ubiquitination and proteasomal degradation machinery needed for Fpn degradation and thus represent an ideal model in which the occlusive effect of hepcidin could be assessed on endogenous Fpn. Using mature human RBCs, Aschemeyer et al showed that hepcidin incubation for 24 hours reduced the non–transferrin-bound iron in the medium in a dose-dependent manner, confirming that hepcidin inhibited the iron export activity of endogenous Fpn. This result was consistent with our observation that hepcidin incubation for 1 hour inhibited the export of iron-55 from preloaded RBCs by 30%.⁵ Aschemeyer et al also showed that minihepcidins, which are more stable and potent than hepcidin, dramatically reduced serum iron levels of mice without changing Fpn protein levels in the spleen and duodenum up to 36 hours after the minihepcidin infusion. Because hepcidin is known to reduce Fpn expression in vivo,^6-8  minihepcidins apparently do not promote Fpn degradation efficiently, an observation that will likely inspire further studies of the hepcidin degradation pathway. Collectively, these observations confirmed the hypothesis that binding of hepcidin to the central cavity of Fpn inhibits its iron export activity (see figure).

These findings have revealed a novel mode of hepcidin in the regulation of Fpn activity that will likely have a significant influence on the understanding of Fpn regulation in normal physiology and pathophysiology. The findings indicate that hepcidin or its mimetics represent promising approaches to treat nonclassical Fpn diseases because they can directly inhibit the iron export activity of Fpn. Fpn is ubiquitously expressed, although expression levels are relatively low in multiple cells, including cardiomyocytes, renal proximal tubules, neurons, pancreatic β cells, endothelial cells, and bronchial epithelial cells. Although the response of Fpn levels to hepcidin in these cells may not be as sensitive as in splenic macrophages, hepcidin binding occludes the iron export activity and may have important and undefined roles in physiology.

These findings also raise some interesting questions. What are the relative contributions of the hepcidin-occlusion effect vs the hepcidin-induced degradation effect on blood iron status in vivo? Aschemeyer et al attempted to assess the significance of the occlusion effect by using minihepcidin infusions, which did not cause the same magnitude of effect as hepcidin in the spleen. The estimated affinity of the hepcidin-Fpn interaction is relatively low (500 nM),⁹  perhaps because the binding between hepcidin and Fpn regulates a highly dynamic process that must be versatile and responsive. Also, once hepcidin has bound, how does it initiate the proposed conformational change, and what causes hepcidin to dissociate to unblock the Fpn export pathway?

In summary, these findings have revealed a new steric mechanism for how hepcidin regulates Fpn activity, which may have significant implications for understanding the regulation of systemic iron homeostasis.