Modulation of Dcytb (Cybrd 1) expression and function by iron, dehydroascorbate and Hif-2α in cultured cells

Background: Duodenal cytochrome b (Dcytb) is a mammalian plasma ferric reductase enzyme that catalyses the reduction of ferric to ferrous ion in the process of iron absorption. The current study investigates the relationship between Dcytb, iron, dehydroascorbate (DHA) and Hif-2 alpha in cultured cell lines. Methods: Dcytb and Hif-2 alpha protein expression was analysed by Western blot technique while gene regulation was determined by quantitative PCR. Functional analyses were carried out by ferric reductase and Fe-59 uptake assays. Results: Iron and dehydroascorbic acid treatment of cells inhibited Dcytb mRNA and protein expression. Desferrioxamine also enhanced Dcytb mRNA level after cells were treated overnight. Dcytb knockdown in HuTu cells resulted in reduced mRNA expression and lowered reductase activity. Preloading cells with DMA (to enhance intracellular ascorbate levels) did not stimulate reductase activity fully in Dcytb-silenced cells, implying a Dcytb-dependence of ascorbate-mediated ferrireduction. Moreover, Hif-2 alpha knockdown in HuTu cells led to a reduction in reductase activity and iron uptake. Conclusions: Taken together, this study shows the functional regulation of Dcytb reductase activity by DMA and Hif-2 alpha. General significance: Dcytb is a plasma membrane protein that accepts electrons intracellularly from DHA/ascorbic acid for ferrireduction at the apical surface of cultured cells and enterocytes. (C) 2013 Elsevier B.V. All rights reserved.