The mechanism of iron release from the placenta is not well understood. We have previously identified a copper oxidase that appears to be intimately involved in the process and have shown that it was regulated by copper and by iron levels in placental cells (BeWo) (Danzeisen et al. 2002). Recently, we identified sequences in the database with a high degree of homology to both hephaestin and serum ceruloplasmin (www.ncbi.nlm.nih.gov accession numbers XP-100543, and XP-146812). Here, we demonstrate, using reverse transcriptase-polymerase chain reaction (PCR), siRNA, Western blotting and real-time PCR, that this gene codes for the placental Cu oxidase.
BeWo cells were grown in Ham’s F12 supplemented with 10 % fetal bovine serum with or without antibiotics as appropriate. Cells were grown to approximately 80 % confluence. For siRNA experiments, the cells were treated according to methods provided on the Ambion web site (www.ambion.com) and using templates designed according to the manufacturer’s instructions (Ambion SilencerTM siRNA Construction Guide). Cells were transfected with the siRNA using siPortAmine (Ambion) according to the manufacturer’s instructions after optimisation for BeWo cells. Following transfection, cells were scraped and solubilised in running buffer before being separated on 7.5 % SDS-PAGE gels. Oxidase protein was visualised using anti-ceruloplasmin antibody (Danzesien et al. 2002). Real-time PCR was performed using the TaqMan system (Perkin Elmer).
As would be expected, the sequences showed a putative membrane-spanning sequence and multi-copper binding site. Reverse transcriptase-PCR reactions gave a single band of the predicted size, which, when sequenced, matched the gene in the data base. We tested the hypothesis that the mRNA coded for the oxidase protein by using siRNA followed by immunoblotting. Transfecting the cells with increasing concentrations of siRNA resulted in a dose-dependent decrease in oxidase protein. This is direct evidence that the gene codes for the placental oxidase.
We have previously shown that the oxidase protein and activity levels are regulated by copper and iron. Here, we demonstrate, using real time PCR, that mRNA levels are decreased by treatment with a copper chelator, but are not altered by iron chelators. We propose calling this member of the Cu oxidase family ‘eleutherin’ after Eleutheria, the sister of Hephaestos and the goddess of midwives.
This work was funded by SEERAD, EUFPV, and the International Copper Association.