Regulation of divalent metal transporter (DMT) by tumor necrosis factor and lipopolysaccharide is dependent on NF-kappaB in SH-SY5Y neuroblastoma cells

Disturbance in metal homeostasis can lead to a variety of diseases such hemochromatosis, which occurs when systemic iron overwhelms the bodies' transferrin sequestering capacity, and anemia. Neurodegenerative disorders result in iron deposition at the site of the lesion and it is theorized that this may play a role in exacerbating a degenerative cascade. One of the principle proteins involved in maintaining proper metal homeostasis is divalent metal transporter (DMT-1) which has been shown to be the major transporter of metals in the brain.;There are four known variants of DMT-1: The 1A and 1B isoforms, differing at the transcriptional start site and the +IRE and --IRE isoforms, based on the presence or lack of an iron response element (IRE) in the 3' untranslated region of the message. Previous in vitro studies have observed augmentation in the +IRE and --IRE isoforms of DMT-1 in the presence tumor necrosis factor (TNF-alpha), lipopolysacchride (LPS), and interferon-gamma (IFN-gamma). Additional research has revealed that the 1B isoforms of DMT-1 are regulated by the transcription factor NF-kappaB.;Previous studies demonstrated +IRE and --IRE protein induction occurred in the presence of well known NF-kappaB agonists and subsequent mitigation occurred with NF-kappaB inhibition while 1A protein levels were relatively unaffected. It is a reasonable hypothesis that this augmentation that occurred in the presence of TNF-alpha and LPS is mediated by NF-kappaB and that the 1B isoforms of DMT-1 are the most influenced. BEAS-2B lung epithelial were used in an attempt to mimic a previous experiment and confirm results. SH-SY5Y cells are a common model for neuronal tissue and have been used extensively and successfully for DMT-1 research. The SH-SY5Y cells were used to determine whether the +IRE, -IRE, and 1A isoforms of DMT-1 are regulated by TNF-alpha and LPS in a NF-kappaB mediated fashion, via inhibition of NF-kappaB by the cell permeable peptide SN-50. Furthermore, removal of the 1A isoforms by sequential immuno-precipitation theoretically provides a possible way to measure 1B expression under the same conditions.;The results of this study revealed that SH-SY5Y cells experienced an induction of 1B mRNA when exposed to TNF-alpha or LPS. Palliation of this induction occurred when NF-kappaB was inhibited by SN-50. Message level was measured by use of Reverse Transcriptase-PCR. Western blot analysis revealed that exposure to TNF-alpha or LPS significantly augmented +IRE and --IRE protein levels. This increase in +IRE and --IRE protein was mitigated by inhibition of NF-kappaB by SN-50, with the --IRE protein decreasing below basal levels. 1A immuno-depleted/-IRE protein levels confirmed this observation as a significant increase in 1A depleted/--IRE protein was observed with TNF-alpha or LPS challenged cells. Inhibition of NF-kappaB resulted in sub basal expression of 1A depleted/-IRE DMT-1, which is similar to what was observed with the --IRE protein. The 1A isoforms of DMT-1 remained relatively unchanged by NF-kappaB activation or inhibition. BEAS-2B cells experienced augmentation of 1B message level in the presence of TNF-alpha and subsequent palliation of 1B message levels by NF-kappaB inhibition.;Overall, DMT-1 in SH-SY5Y cells was found to be augmented by TNF-alpha and LPS in a NF-kappaB dependent manner. The --IRE form of DMT-1 was the most dramatically influenced by NF-kappaB. Removal of the 1A isoforms via immuno-precipitation revealed that the 1A depleted/-IRE protein is likely a significant component of total DMT-1 levels in SH-SY5Y cells and is the major form mediated by NF-kappaB. It is likely that what is observed in --IRE probing of the 1A depleted lysate is representative of the 1B/-IRE species of DMT-1, as removal of 1A protein means remaining --IRE and +IRE isoforms must be associated with 1B DMT-1.