Ceruloplasmin, the main copper binding protein in blood plasma, has been of particular interest for its role in efflux of iron from cells, but has additional functions. Here we tested the hypothesis that it releases its copper for cell uptake by interacting with a cell surface reductase and transporters, producing apoceruloplasmin. Uptake and transepithelial transport of copper from ceruloplasmin was demonstrated with mammary epithelial cell monolayers (PMC42) with tight junctions grown in bicameral chambers, and purified human 64Cu-labeled ceruloplasmin secreted by HepG2 cells. Monolayers took up virtually all the 64Cu over 16h and secreted half into the apical (milk) fluid. This was partly inhibited by Ag(I). The 64Cu in ceruloplasmin purified from plasma of 64Cu-injected mice accumulated linearly in mouse embryonic fibroblasts (MEFs) over 3-6h. Rates were somewhat higher in Ctr1+/+ versus Ctr1-/- cells, and 3-fold lower at 2°C. The ceruloplasmin-derived 64Cu could not be removed by extensive washing or trypsin treatment, and most was recovered in the cytosol. Actual cell copper (determined by furnace atomic absorption) increased markedly upon 24h exposure to holoceruloplasmin. This was accompanied by a conversion of holo to apoceruloplasmin in the culture medium and did not occur during incubation in the absence of cells. Four different endocytosis inhibitors failed to prevent 64Cu uptake from ceruloplasmin. High concentrations of non-radioactive Cu(II)- or Fe(III)-NTA (substrates for cell surface reductases), or Cu(I)-NTA (to compete for transporter uptake) almost eliminated uptake of 64Cu from ceruloplasmin. MEFs had cell surface reductase activity and expressed Steap 2 (but not Steaps 3 and 4 or dCytB). However, six-day siRNA treatment was insufficient to reduce activity or uptake. We conclude that ceruloplasmin is a circulating copper transport protein that may interact with Steap2 on the cell surface, forming apoceruloplasmin, and Cu(I) that enters cells through CTR1 and an unknown copper uptake transporter.

The blood plasma protein, ceruloplasmin (Cp), is well known for its role in enhancing efflux of iron from certain cells; but it also has other functions, and its copper enters cells all over the mammalian organism, as shown by i.v. infusion of 67Cu‐labeled Cp into rats. In current studies, the ability of Cp to directly donate its Cu to cultured cells was investigated. 64Cu‐labeled human Cp secreted by HepG2 cells was purified and incubated in serum‐free medium with polarized human mammary epithelial cell monolayers with tight junctions (PMC42 cells), applied to the basolateral (“blood” side). Most of the radioactivity entered the cells and 30–50% of what entered was released into the apical (“milk”) fluid. Larger amounts of 64Cu‐Cp were produced in mice and purified for incubation with mouse embryonic fibroblasts (MEF) that did and did not express Ctr1 (kindly provided by Dennis Thiele, Duke University). 64Cu from Cp accumulated in both types of MEFs at a linear rate over 3 h, and was internalized, most being present in the cytosol. Uptake was somewhat less in Ctr1−/− MEFs. Uptake of Cp‐64Cu (which is not exchangeable) was inhibited by an excess of Cu(I) and Cu(II)‐histidine; and the presence of external Cu(II) reductase activity was verified. We conclude that Ctr1 and another (unknown) transporter take up Cu from Cp, and that this is mediated by a Cu reductase. Supported in part by US PHS Grant HD46949.