Alteration at translational but not transcriptional level of transferrin receptor expression following manganese exposure at the blood-CSF barrier in vitro.

Manganese exposure alters iron homeostasis in blood and cerebrospinal fluid (CSF), possibly by acting on iron transport mechanisms localized at the blood-brain barrier and/or blood-CSF barrier. This study was designed to test the hypothesis that manganese exposure may change the binding affinity of iron regulatory proteins (IRPs) to mRNAs encoding transferrin receptor (TfR), thereby influencing iron transport at the blood-CSF barrier. A primary culture of choroidal epithelial cells was adapted to grow on a permeable membrane sandwiched between two culture chambers to mimic blood-CSF barrier. Trace (59)Fe was used to determine the transepithelial transport of iron. Following manganese treatment (100 microM for 24 h), the initial flux rate constant (K(i)) of iron was increased by 34%, whereas the storage of iron in cells was reduced by 58%, as compared to controls. A gel shift assay demonstrated that manganese exposure increased the binding of IRP1 and IRP2 to the stem loop-containing mRNAs. Consequently, the cellular concentrations of TfR proteins were increased by 84% in comparison to controls. Assays utilizing RT-PCR, quantitative real-time reverse transcriptase-PCR, and nuclear run off techniques showed that manganese treatment did not affect the level of heterogeneous nuclear RNA (hnRNA) encoding TfR, nor did it affect the level of nascent TfR mRNA. However, manganese exposure resulted in a significantly increased level of TfR mRNA and reduced levels of ferritin mRNA. Taken together, these results suggest that manganese exposure increases iron transport at the blood-CSF barrier; the effect is likely due to manganese action on translational events relevant to the production of TfR, but not due to its action on transcriptional, gene expression of TfR. The disrupted protein-TfR mRNA interaction in the choroidal epithelial cells may explain the toxicity of manganese at the blood-CSF barrier.