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.

Ceruloplasmin (Cp) is an abundant multifunctional copper‐binding protein in the blood plasma best known for its ferroxidase activity. Using purified 64Cu‐labeled Cp, we have now established that it also delivers copper directly to cultured cells. Here, we further examined whether this process occurs at the cell surface, involving a reductase, and attempted to identify a new copper transporter and overexpress human Cp. Uptake of copper from 64Cu‐Cp (purified from mouse plasma) by mouse embryonic fibroblasts that do and do not express Ctr1, was not prevented by endocytosis inhibitors; and holoCp appeared to convert to apoCp during uptake. The only known copper reductase expressed was identified as Steap2 by qPCR. Successive siRNA treatments did not reduce cell surface reductase activity, and other approaches are being applied. Identification of copper uptake transporter in the Ctr1‐/‐ fibroblasts was sought using biotinylation‐targeting with mouse Cp, and cross‐linking with formaldehyde. No clear candidates have as yet emerged. To provide a reliable source of 64Cu‐Cp for future studies, attempts were made to clone and express human Cp in Sf9 insect cells using baculovirus (which did not succeed); overexpression in human cell lines using either a conventional or lentivirus construct is now in the process. We conclude that uptake of copper from Cp occurs at the cell surface and may require a reductase that delivers Cu1+ not just to Ctr1 but also to another transporter.

The main Cu‐binding protein of blood plasma, ceruloplasmin (Cp), has mainly been studied for its role in mediating cellular iron efflux but also has other functions, and as shown by i.v. infusion of 67Cu‐Cp, its Cu enters most major organs. To determine whether it delivers Cu directly to cells and the mechanisms involved, we incubated purified radio‐copper‐labeled and unlabeled Cp with cells and measured rates of Cu uptake and accumulation. Uptake was demonstrated for human mammary epithelial cell monolayers (PMC42 cells) with tight junctions, and with mouse embryonic fibroblasts expressing/not expressing Ctr1. Cp‐Cu was internalized. It could not be washed from the cell surface even with acidic buffers, and was found in the cytosol. Inhibitors of endocytosis did not impair uptake. Cu deficient cells exposed to Cp increased their cellular Cu contents; and holo‐Cp was converted to apo‐Cp during cell treatment, with no loss of total Cp protein. Uptake was almost the same in cells not expressing Ctr1. Excess ionic Cu(II) and Cu(I) virtually eliminated Cp‐Cu uptake. The presence of Cu(II) and Fe(III) reductase activities on the cell surface was verified. We conclude that Cp‐Cu delivers Cu to cells by a process involving cell surface reduction of Cp‐Cu(II), release of apo‐Cp, and uptake of the resulting Cu(I) by Ctr1 and an additional as yet unidentified Cu(I) transporter. Grant Funding Source: Supported by PHS Grant R15 GM100464