Impact of human ferroportin mutations on cellular trace metal homeostasis

Abstract

Ferroportin (Fpn; SLC40A1) plays a role as an exporter in the assimilation of dietary iron (Fe) and manganese (Mn). Recent studies reveal that Fpn deficiency alters body metal levels and promotes brain metal accumulation in the flatiron mouse model of human “ferroportin disease.” This genetic disorder is known as hereditary hemochromatosis (HH) type 4 and arises from mutations in the human Fpn. While clinical manifestations of the disease are quite variable, most mutations are inherited as dominant traits. Ferroportin disease‐causing mutations fall into two functional categories (loss‐versus gain‐of‐function) underlying two distinct clinical entities (HH type 4A and type 4B, respectively). While functional consequences of several human Fpn mutations in Fe metabolism have been analyzed in vitro, little is known about the potential impact of Fpn on cellular Mn homeostasis and how ferroportin disease‐causing mutations might alter Mn metabolism. To explore the effects of these mutations on cellular Mn homeostasis, 17 ferroportin disease‐associated missense mutations were introduced into the human Fpn gene for functional expression in human embryonic HEK293T cells. The effects of wild‐type and mutant Fpn on intracellular Fe levels were determined by looking at the intracellular iron storage protein ferritin. Expression of wild‐type Fpn led to a significant reduction in ferritin compared to control cells. Loss‐of‐function mutants did not cause a significant reduction in ferritin, but gain‐of‐function mutants behaved similarly to wild‐type Fpn. These data are consistent with other studies and agree with differential effects of Fpn mutations on intracellular Fe levels. To determine whether wild‐type and mutant Fpn expression affect cellular Mn homeostasis, intracellular Mn levels were analyzed for trace elements by inductively coupled plasma mass spectrometry (ICP‐MS). Expression of wild‐type Fpn reduced accumulation of intracellular Mn induced by Mn exposure in treated versus control or nontreated cells. While loss‐of‐function mutants had no effect in reducing Mn accumulation, gain‐of‐function mutants were shown to accumulate lower levels of Mn, similar to wild‐type Fpn. To test the physiological relevance of the efflux activity of wild‐type and mutant Fpn, we sought to determine whether expression of wild‐type Fpn conferred protection against Mn‐induced cytotoxicity. Expression of wild‐type Fpn reversed Mn‐induced cytotoxicity. These data are consistent with our previous reports and confirm the cytoprotective effects of wild‐type exporter in the presence of Mn. Similar to wild‐type Fpn, gain‐of‐function mutants protected cells against Mn cytotoxicity, whereas loss‐of‐function mutants failed to confer protection. These combined results demonstrate that Fpn plays a central role in cellular Mn homeostasis and that ferroportin disease‐causing mutations have differential impact on Mn metabolism. These findings highlight the importance of Fpn function in cellular Mn homeostasis and implicate Mn dyshomeostasis in patients with ferroportin disease.Support or Funding InformationNIH R00 ES024340