Ferroportin and exocytoplasmic ferroxidase activity are required for brain microvascular endothelial cell iron efflux

The mechanism(s) of iron flux across the brain microvasculature endothelial cells (BMVEC) of the blood-brain barrier remains unknown. Although both hephaestin (Hp) and the ferrous iron permease ferroportin (Fpn) have been identified in BMVEC, their roles in iron efflux have not been examined. Using a human BMVEC line (hBMVEC), we have demonstrated that these proteins are required for iron efflux from these cells. Expression of both Hp and Fpn protein was confirmed in hBMVEC by immunoblot and indirect immunofluorescence; we show that hBMVEC express soluble ceruloplasmin (Cp) transcript as well. Depletion of endogenous Hp and Cp via copper chelation leads to the reduction of hBMVEC Fpn protein levels as well as a complete inhibition of ⁵⁹Fe efflux. Both hBMVEC Fpn protein and ⁵⁹Fe efflux activity are restored upon incubation with 6.6 nM soluble plasma Cp. These results are independent of the source of cell iron, whether delivered as transferrinor non-transferrin-bound ⁵⁹Fe. Our results demonstrate that iron efflux from hBMVEC Fpn requires the action of an exocytoplasmic ferroxidase, which can be either endogenous Hp or extracellular Cp.