Calcium and IL-6 regulate the anterograde trafficking and plasma membrane residence of the iron exporter ferroportin to modulate iron efflux.

Iron is an essential element for proper cell functioning, but unbalanced levels can cause cell death. Iron metabolism is controlled at the blood-tissue barriers provided by microvascular endothelial cells. Dysregulated iron metabolism at these barriers is a factor in both neurodegenerative and cardiovascular diseases. Mammalian iron efflux is mediated by the iron efflux transporter ferroportin (Fpn). Inflammation is a factor in many diseases and correlates with increased tissue iron accumulation. Evidence suggests treatment with interleukin 6 (IL-6) increases intracellular calcium levels and calcium is known to play an important role in protein trafficking. We have shown that calcium increases plasma membrane localization of the iron uptake proteins ZIP8 and ZIP14, but if and how calcium modulates Fpn trafficking is unknown. In this article, we examined the effects of IL-6 and calcium on Fpn localization to the plasma membrane. In HEK cells expressing a doxycycline-inducible GFP-tagged Fpn, calcium increased Fpn-GFP membrane presence by 2 h, while IL-6 increased membrane-localized Fpn-GFP by 3 h. Calcium pretreatment increased Fpn-GFP mediated 55Fe efflux from cells. Endoplasmic reticulum calcium stores were shown to be important for Fpn-GFP localization and iron efflux. Use of calmodulin pathway inhibitors showed that calcium signaling is important for IL-6-induced Fpn relocalization. Studies in brain microvascular endothelial cells in transwell culture demonstrated an initial increase in 55Fe flux with IL-6 that is reduced by 6 h coinciding with upregulation of hepcidin. Overall, this research details one pathway by which inflammatory signaling mediated by calcium can regulate iron metabolism, likely contributing to inflammatory disease mechanisms.

Calcium and the Ca-ATPase SPCA1 modulate plasma membrane abundance of ZIP8 and ZIP14 to regulate Mn(II) uptake in brain microvascular endothelial cells.

Manganese (II) accumulation in human brain microvascular endothelial cells is mediated by the metal-ion transporters ZRT IRT-like protein 8 (ZIP8) and ZRT IRT-like protein 14 (ZIP14). The plasma membrane occupancy of ZIP14, in particular, is increased in cells treated with Mn2+, lipopolysaccharide, or IL-6, but the mechanism of this regulation has not been elucidated. The calcium-transporting type 2C member 1 ATPase, SPCA1, is a Golgi-localized Ca2+-uptake transporter thought to support Golgi uptake of Mn2+ also. Here, we show using surface protein biotinylation, indirect immunofluorescence, and GFP-tagged proteins that cytoplasmic Ca2+ regulates ZIP8- and ZIP14-mediated manganese accumulation in human brain microvascular endothelial cells by increasing the plasma membrane localization of these transporters. We demonstrate that RNAi knockdown of SPCA1 expression results in an increase in cytoplasmic Ca2+ levels. In turn, we found increased cytoplasmic Ca2+ enhances membrane-localized ZIP8 and ZIP14 and a subsequent increase in 54Mn2+ uptake. Furthermore, overexpression of WT SPCA1 or a gain-of-function mutant resulted in a decrease in cytoplasmic Ca2+ and 54Mn2+ accumulation. While addition of Ca2+ positively regulated ZIP-mediated 54Mn2+ uptake, we show chelation of Ca2+ diminished manganese transport. In conclusion, the modulation of ZIP8 and ZIP14 membrane cycling by cytoplasmic calcium is a novel finding and provides new insight into the regulation of the uptake of Mn2+ and other divalent metal ions-mediated ZIP metal transporters.

The iron chelator, PBT434, modulates transcellular iron trafficking in brain microvascular endothelial cells.

Iron and other transition metals, such as copper and manganese, are essential for supporting brain function, yet over-accumulation is cytotoxic. This over-accumulation of metals, particularly iron, is common to several neurological disorders; these include Alzheimer's disease, Parkinson's disease, Friedrich's ataxia and other disorders presenting with neurodegeneration and associated brain iron accumulation. The management of iron flux by the blood-brain barrier provides the first line of defense against the over-accumulation of iron in normal physiology and in these pathological conditions. In this study, we determined that the iron chelator PBT434, which is currently being developed for treatment of Parkinson's disease and multiple system atrophy, modulates the uptake of iron by human brain microvascular endothelial cells (hBMVEC) by chelation of extracellular Fe2+. Treatment of hBMVEC with PBT434 results in an increase in the abundance of the transcripts for transferrin receptor (TfR) and ceruloplasmin (Cp). Western blot and ELISA analyses reveal a corresponding increase in the proteins as well. Within the cell, PBT434 increases the detectable level of chelatable, labile Fe2+; data indicate that this Fe2+ is released from ferritin. In addition, PBT434 potentiates iron efflux likely due to the increase in cytosolic ferrous iron, the substrate for the iron exporter, ferroportin. PBT434 equilibrates rapidly and bi-directionally across an hBMVEC blood-brain barrier. These results indicate that the PBT434-iron complex is not substrate for hBMVEC uptake and thus support a model in which PBT434 would chelate interstitial iron and inhibit re-uptake of iron by endothelial cells of the blood-brain barrier, as well as inhibit its uptake by the other cells of the neurovascular unit. Overall, this presents a novel and promising mechanism for therapeutic iron chelation.

Is brain iron trafficking part of the physiology of the amyloid precursor protein?

The amyloid precursor protein is so named, because a proteolytic fragment of it was found associated with a neuropathic disorder now known as Alzheimer's disease. This fragment, Aß, along with tau makes up the plaques and tangles that are the hallmark of AD. Iron (and other first-row transition metals) is found associated with these proteinaceous deposits. Much research has focused on the relationship of the plaques and iron to the etiology of the disease. This commentary asks another question, one only more recently addressed namely, what is the physiologic function of the amyloid precursor protein (APP) and of its secretase-generated soluble species? Overall, the data make clear that APP and its products have neurotrophic functions and some data indicate one of these may be to modulate the trafficking of iron in the brain.

Fluorescence resonance energy transfer links membrane ferroportin, hephaestin but not ferroportin, amyloid precursor protein complex with iron efflux.

Iron efflux from mammalian cells is supported by the synergistic actions of the ferrous iron efflux transporter, ferroportin (Fpn) and a multicopper ferroxidase, that is, hephaestin (Heph), ceruloplasmin (Cp) or both. The two proteins stabilize Fpn in the plasma membrane and catalyze extracellular Fe3+ release. The membrane stabilization of Fpn is also stimulated by its interaction with a 22-amino acid synthetic peptide based on a short sequence in the extracellular E2 domain of the amyloid precursor protein (APP). However, whether APP family members interact with Fpn in vivo is unclear. Here, using cyan fluorescent protein (CFP)-tagged Fpn in conjunction with yellow fluorescent protein (YFP) fusions of Heph and APP family members APP, APLP1, and APLP2 in HEK293T cells we used fluorescence and surface biotinylation to quantify Fpn membrane occupancy and also measured 59Fe efflux. We demonstrate that Fpn and Heph co-localize, and FRET analysis indicated that the two proteins form an iron-efflux complex. In contrast, none of the full-length, cellular APP proteins exhibited Fpn co-localization or FRET. Moreover, iron supplementation increased surface expression of the iron-efflux complex, and copper depletion knocked down Heph activity and decreased Fpn membrane localization. Whereas cellular APP species had no effects on Fpn and Heph localization, addition of soluble E2 elements derived from APP and APLP2, but not APLP1, increased Fpn membrane occupancy. We conclude that a ferroportin-targeting sequence, (K/R)EWEE, present in APP and APLP2, but not APLP1, helps modulate Fpn-dependent iron efflux in the presence of an active multicopper ferroxidase.

The Ferroxidase Hephaestin But Not Amyloid Precursor Protein is Required for Ferroportin-Supported Iron Efflux in Primary Hippocampal Neurons.

Iron efflux in mammalian cells is mediated by the ferrous iron exporter ferroportin (Fpn); Fpn plasma membrane localization and function are supported by a multicopper ferroxidase and/or the soluble amyloid precursor protein (sAPP). Fpn and APP are ubiquitously expressed in all cell types in the central nervous system including neurons. In contrast, neuronal ferroxidase(s) expression has not been well characterized. Using primary cultures of hippocampal neurons, we examined the molecular mechanism of neuronal Fe efflux in detail. Developmental increases of Fpn, APP, and the ferroxidase hephaestin (Hp) were observed in hippocampal neurons. Iron efflux in these neurons depended on the level of Fpn localized at the cell surface; as noted, Fpn stability is supported by ferroxidase activity, an enzymatic activity that is required for Fe efflux. Iron accumulation increases and iron efflux decreases in Hp knockout neurons. In contrast, suppression of endogenous APP by RNAi knockdown does not affect surface Fpn stability or Fe efflux. These data support the model that the neuronal ferroxidase Hp plays a unique role in support of Fpn-mediated Fe efflux in primary hippocampal neurons. Our data also demonstrate that Hp ferroxidase activity relies on copper bioavailability, which suggests neuronal iron homeostasis will be modulated by cellular copper status.