Regulation of divalent metal transporter (DMT) 1 by post-translational and transcriptional mechanisms

Disturbance in metal homeostasis can lead to a variety of diseases including anemia, hemochromatosis and other related disorders. One of the major proteins involved in maintaining this metal homeostasis is the Divalent Metal Transporter (DMT1). DMT1 is a 12 transmembrane protein which can transport a variety of metals including iron, manganese, cadmium and nickel. There are at least four known isoforms of DMT1, two differing at the transcription start site and other two differing at the 3' end, based on presence or absence of iron response element (IRE) in the 3' untranslated region.; Studies have shown that DMT1 is the major transporter of metals in brain. It is reasonable to assume that iron requirements of cells are altered during various stages of neural differentiation. P19 mouse embryonic carcinoma cells are a model for neuro-ectodermal differentiation, since they can be differentiated into neuron and glial cells by retinoic acid treatment. These cells were used to determine whether isoforms of DMT1 are differentially regulated during early stages of differentiation. The results of these studies reveal that protein and message expression for the +/-IRE forms of DMT1, but not that of the 1A isoform, were down-regulated within the first few hrs upon removal of RA, at which time the cells begin to differentiate. The turnover of the +/-IRE isoforms of DMT1 protein during this period was found to be dependent on both the proteasomal and lysosomal pathways. Changes in mRNA levels were shown to be regulated by nitric oxide produced by the induction of neuronal NOS after removal of RA. Nitric oxide functions by inhibiting NF-kappaB nuclear translocation and the subsequent binding to the putative NF-kappaB response element (at -19 to -23) within the 1B promoter. Gel-shift analysis and ChIP assay indicated that nuclear NF-kappaB is capable of binding to this response element and that binding decreases during early stages of differentiation. Luciferase reporter gene assay demonstrated that a mutation in this binding domain leads to decreased activity. These data demonstrate that during early stages of neuronal differentiation of P19 cells, there is a decrease in specific isoforms of DMT1 via both post-translational and transcriptional mechanisms.; Nitric oxide treatment has shown to decrease transferrin receptor mRNA and increase ferritin mRNA via post-transcriptional mechanism. One of the C-terminal isoforms of DMT1 contains an IRE, which is thought to play a role in post-transcriptional regulation of the isoform. (Abstract shortened by UMI.)...