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

Abstract

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 (59)Fe efflux. Both hBMVEC Fpn protein and (59)Fe 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 transferrin- or non-transferrin-bound (59)Fe. 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.

Keywords: Blood Brain Barrier; Brain Metabolism; Cell Biology; Ceruloplasmin; Ferroportin; Ferroxidase; Hephaestin; Iron Metabolism; Neurochemistry; Transport Metals.

FIGURE 1.

hBMVEC, which express Hp, sCp, and Fpn, readily accumulate and efflux iron. A, hBMVEC accumulation of 0.5 μm ⁵⁹Fe^II citrate plus ascorbate over a 24-h period. Inset illustrates the first 1 h of accumulation. Time 0 h is subtracted from all data points (n = 6, experimental replicates). B, ⁵⁹Fe efflux assays were performed using hBMVEC cell pellets loaded for 24 h with ⁵⁹Fe^II citrate plus ascorbate (open) or ⁵⁹Fe-Tf (filled). hBMVEC pellets were harvested after allowing 24 h for ⁵⁹Fe efflux. Data are represented as the percent loss of cell-associated ⁵⁹Fe with respect to time 0 h. Data are represented as means ± S.D. (n = 4–8, experimental replicates). C, immunoblots from whole cell lysates of C6 glioma, HepG2, Caco-2 cells, and hBMVEC were probed for either Hp or Fpn. Respective molecular weights are indicated to the right of the blots. Each lane was loaded with 20 μg of total protein. D, total RNA was collected from hBMVEC cells, and RT-PCR was performed to amplify Hp, Fpn, and Cp transcripts. Isoform-specific primers were used to amplify human soluble Cp from hBMVEC total RNA.

FIGURE 2.

Hp and Fpn are co-localized to the cell surface of hBMVEC. A, hBMVEC monolayers were grown on glass coverslips and were processed for indirect immunofluorescence by confocal microscopy probing for Hp and Fpn. DAPI (blue) stains the nucleus. Circles highlight areas of Hp and Fpn co-localization in the merged image of hBMVEC. B, confocal microscopy performed on hBMVEC probed for Hp or Fpn was used to delineate polarized localization of these proteins. Side views demonstrate polarity in the case of Hp but not Fpn. Apical and basal surfaces are indicated to the right of the images. DAPI (blue) stains the nucleus. C, hBMVEC and HepG2 (negative control) monolayers were processed for indirect immunofluorescence probing for Fpn. DAPI (blue) stains the nucleus.

FIGURE 3.

Copper chelation depletes hBMVEC of endogenous Hp. A, indirect immunofluorescence was used to investigate the abundance of Hp protein in BCS-treated and untreated hBMVEC. hBMVEC were treated with 500 μm BCS for 24–48 h. Secondary antibody only controls are shown. DAPI (blue) stains the nucleus. Scale bar represents 10 μm. B, average (Avg) fluorescent intensity from at least five separate fields of view were taken from each condition at each time point and were normalized for their respective secondary only control average fluorescent intensities. A series of paired t tests were used to analyze the data from each time point. *, p < 0.05 and **, p < 0.005. Data are represented as means ± S.D. (n = 5, technical replicates).

FIGURE 4.

Exocytoplasmic ferroxidase activity stabilizes hBMVEC Fpn but not Hp abundance in the presence of BCS. A, indirect immunofluorescence was used to investigate the abundance of Fpn protein in BCS-treated and untreated hBMVEC. hBMVEC were treated with 500 μm BCS for 24–48 h. Additionally, recovery of Fpn protein abundance in BCS-treated hBMVEC by ferroxidase-active sCp (6.6 nm) was investigated. Fresh sCp was added to media during the final 24 h of BCS treatment. Insets depict secondary antibody only controls. DAPI (blue) stains the nucleus. Scale bar represents 10 μm. B, immunoblot depicting Fpn abundance in hBMVEC lysates (20 μg of protein per lane). hBMVEC were incubated for 24 h without BCS, with BCS, or with BCS plus sCp (6.6 nm) for an additional 24 h before lysis. C, ferroxidase assays were used to investigate the abundance of endogenous ferroxidase activity in hBMVEC lysates. hBMVEC were treated for 24 h without BCS, with BCS, or with BCS plus sCp (6.6 nm) for an additional 24 h. Ferroxidase activity is indicated by the fraction of Fe^II remaining as detected by formation of the Fe(II)-ferrozine complex (n = 3, experimental replicates). D and E, samples from A were used to quantify average fluorescent intensities from at least five separate fields of view from each condition at each time point and were normalized for their respective secondary only control average fluorescent intensities. Significance of the data (C) was assessed using one-way analysis of variance parameters. A series of paired t tests were used to analyze the data from each time point in D and E. *, p < 0.005; **, p < 0.001; ***, p < 0.0001. Data are represented as means ± S.D. (A, D, and E, n = 5, technical replicates).

FIGURE 5.

Copper chelation increases hBMVEC iron retention. A, hBMVEC-associated ⁵⁹Fe after loading for 24 h with 0.5 μm ⁵⁹Fe^II citrate plus ascorbate in the presence or absence of 500 μm BCS. B, hBMVEC-associated ⁵⁹Fe after loading for 24 h with ⁵⁹Fe-Tf plus citrate in the presence or absence of 500 μm BCS. A series of paired t tests were used to analyze the data. *, p < 0.05 and **, p < 0.005. Data are represented as means ± S.D. (n = 4–8, experimental replicates).

FIGURE 6.

hBMVEC iron efflux inhibited by BCS treatment can be restored via the addition of exogenous sCp or hBMVEC-conditioned media. A, ⁵⁹Fe efflux assays were performed on hBMVEC loaded with ⁵⁹Fe^II citrate plus ascorbate. ⁵⁹Fe efflux assays were also performed using BCS-treated hBMVEC in the presence or absence of ferroxidase active sCp (6.6 nm) added at the beginning of the 24 h efflux assay. B, ⁵⁹Fe efflux assays were performed on hBMVEC loaded with ⁵⁹Fe-Tf plus citrate. ⁵⁹Fe efflux assays were also performed using BCS-treated hBMVEC in the presence or absence of ferroxidase active sCp (6.6 nm) added at the beginning of the 24 h efflux assay. C, ⁵⁹Fe efflux assays were performed on hBMVEC loaded with ⁵⁹Fe^II citrate plus ascorbate. Efflux activity was partially recovered via the addition of hBMVEC-conditioned media (ECM) to BCS-treated hBMVEC. All BCS values were not statistically different than zero. Significance was determined for each set of data using one-way analysis of variance analysis. *, p < 0.05; **, p < 0.01; and **, p < 0.001. Data are represented as means ± S.D. (n = 4–8, experimental replicates).