Nitric oxide transcriptionally down-regulates specific isoforms of divalent metal transporter (DMT1) via NF-kappaB

Studies were performed to examine the affect of nitric oxide (NO) on expression of the divalent metal transporter (DMT1) in undifferentiated P19 embryonic carcinoma cells. DMT1 has four known isoforms which differ in both the N- and C-terminals. Results demonstrate that exposure of P19 cells to the NO precursor, sodium nitro-prusside (SNP), resulted in a decrease in expression of both positive (+) and negative (-) IRE isoforms of DMT1 with no change in the 1A species. Regulation was not as a result of decreased stability of message but was caused by reduction in transcription of the DMT1 1B isoforms. Similar results were observed in other cell lines, including PC12 and SH-SY5Y cells and rat primary sympathetic neurons. Nuclear NF-kappaB was decreased after SNP treatment, suggesting that NF-kappaB may mediate this response. Luciferase reporter assays with normal and NF-kappaB mutated constructs of the 1B promoter confirm that the NF-kappaB site between -23 to -19 upstream from the transcription start site was responsible for regulating expression. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays further demonstrate that the p65 subunit of NF-kappaB and not p50 binding is specifically decreased by NO treatment. Results of these studies provide a general mechanism responsible for regulating DMT1 expression induced by stress-related signaling processes in vivo.