Upregulation of DMT1 expression in choroidal epithelia of the blood-CSF barrier following manganese exposure in vitro.

Divalent metal transporter 1 (DMT1), whose mRNA possesses a stem-loop structure in 3'-untranslated region, has been identified in most organs and responsible for transport of various divalent metal ions. Previous work from this laboratory has shown that manganese (Mn) exposure alters the function of iron regulatory protein (IRP) and increases iron (Fe) concentrations in the cerebrospinal fluid (CSF). This study was designed to test the hypothesis that Mn treatment, by acting on protein-mRNA binding between IRP and DMT1 mRNA, altered the expression of DMT1 in an immortalized choroidal epithelial Z310 cell line which was derived from rat choroid plexus epithelia, leading to a compartmental shift of Fe from the blood to the CSF. Immunocytochemistry confirmed the presence of DMT1 in Z310 cell. Following in vitro exposure to Mn at 100 microM for 24 and 48 h, the expression of DMT1 mRNA in Z310 cells was significantly increased by 45.4% (P < 0.05) and 78.1% (P < 0.01), respectively, as compared to controls. Accordingly, Western blot analysis revealed a significant increase of DMT1 protein concentrations at 48 h after Mn exposure (100 microM). Electrophoretic mobility shift assay (EMSA) showed that Mn exposure increased binding of IRP to DMT1 mRNA in cultured choroidal Z310 cells. Moreover, real-time RT-PCR revealed no changes in DMT1 heterogeneous nuclear RNA (hnRNA) levels following Mn exposure. These data suggest that Mn appears to stabilize the binding of IRP to DMT1 mRNA, thereby increasing the expression of DMT1. The facilitated transport of Fe by DMT1 at the blood-CSF barrier may partly contribute to Mn-induced neurodegenerative Parkinsonism.