Alternative splicing of exon 16 of the DMT1 gene leads to mRNA variants with differing 3â untranslated regions (Lee et al. 1998) – one contains an iron-responsive element (IRE) whereas the other does not. The relative roles of these splice variants in mediating the intestinal absorption of divalent metals is still unclear. DMT1 can potentially transport a number of metals, including iron (its preferred substrate; Tandy et al. 2000), and copper and cobalt (Gunshin et al. 1997). Previous studies in our laboratory have shown that both iron and copper regulate transporter protein levels and transport function in Caco-2 cells by selectively down-regulating the expression of the IRE-containing splice variant (Tennant et al. 2002; Yamaji et al. 2002). To determine whether reduced expression of DMT1(IRE) is a common mechanism by which all potential substrates regulate transporter function we have examined the effects of cobalt on DMT1 protein levels in human intestinal Caco-2 cells using isoform-specific antibodies.
Caco-2 cells were grown for 21 days in 75 cm2 flasks at which time they are fully differentiated. For the final 24 h cells were incubated in the presence or absence of cobalt (100 µM). Cells were removed from the flask using a cell scraper and used to prepare plasma membranes (Tennant et al. 2002) that were subjected to Western blotting using commercially available antibodies (Alpha Diagnostics Inc., USA) against human DMT1 isoforms. The effects of cobalt on a housekeeper protein, villin, were also measured. Cross-reacting bands were semiquantified using Scion Image software and statistical analysis was performed using Student’s unpaired t test. Data are means ± S.E.M. of 4-6 experiments.
Exposure of Caco-2 cells to cobalt did not alter the expression of the housekeeper protein villin (control 81.4 ± 6.4 a.u.; Co 71.8 ± 10.7 a.u.; P = 0.54). Interestingly, the expression of DMT1(IRE) was not significantly different in control and cobalt-treated cells (control 80.0 ± 7.1 a.u.; Co 66.5 ± 7.9 a.u.; P = 0.28), whereas the non-IRE isoform was significantly increased following exposure to cobalt (control 35.8 ± 6.6 a.u.; Co 86.8 ± 8.1 a.u.; P = 0.001).
Our previous studies with iron and copper suggested that the regulation of DMT1 by its substrates occurred at the post-transcriptional level, perhaps via the interaction of cytosolic iron regulatory proteins with the IRE in the 3â untranslated region of DMT1. The mechanism by which cobalt regulates DMT1 expression is unclear and we are currently investigating whether it exerts its influence at the transcriptional or post-translational level.
D.M.J. is supported by a BBSRC Quota Studentship.