We have previously demonstrated that exposure of the human intestinal Caco-2 cell line to zinc stimulates iron uptake via an increase in DMT1 transporter expression (Yamaji et al. 2001). The cellular mechanisms involved in the up regulation of DMT1 by zinc are still unclear, though one possibility is that activation occurs via interaction with putative metal response elements (MRE) residing in the 5′ promoter region of the DMT1 gene (Lee et al. 1998). To test this possibility, we have investigated the effect of zinc on the activity of the DMT1 promoter using a reporter gene assay.
Caco-2 cells were seeded at a density of 6 X 103 cells cm-2 into 24-well plates. After 3 days cells were transfected using the CaPO4 method with pGL3 plasmid (Promega, UK) that contained 1.6 kb of the DMT1 promoter cloned in front of a luciferase reporter gene. Three days following transfection, cells were exposed to zinc (100 µM) for 24 h and luciferase activity in cell lysates was measured by luminescence.
Zinc stimulation of Caco-2 cells transfected with the DMT1 promoter construct significantly increased luciferase activity compared with unstimulated cells (control 928 ± 79 a.u. vs. +Zn 4702 ± 348 a.u., means ± S.E.M., n = 6, P < 0.0001, Student’s unpaired t test). This suggests that zinc may promote the absorption of iron by Caco-2 cell monolayers by activating a transcription factor that can interact with a specific consensus sequence within the DMT1 promoter. The nature of the transcription factor is unknown but the zinc-inducible MTF-1 that binds to MRE sequences in several genes (Andrews, 2001) is a possible candidate. Further studies are underway in our laboratory to further identify the mechanisms involved in this response.
This work was funded by BBSRC grant (90/D13400).