Sl. Wardrop et Dr. Richardson, The effect of intracellular iron concentration and nitrogen monoxide on Nramp2 expression and non-transferrin-bound iron uptake, EUR J BIOCH, 263(1), 1999, pp. 41-49
Recent studies have demonstrated that the protein product (natural resistan
ce associated macrophage protein 2, Nramp2) encoded by the gene Nramp2 acts
as an Fe transporter involved in the uptake of Fe from transferrin (Tf) an
d low M-r Fe complexes. Interestingly, there are two splice variants of Nra
mp2, one with a putative iron-responsive element (IRE) in its 3' untranslat
ed region (UTR) and another without. Due to the importance of Nramp2 in Fe
transport, and the presence of an IRE in its 3'-UTR, we have examined the e
ffect of Fe-deprivation, Fe-loading, and nitrogen monoxide on the expressio
n of Nramp2 mRNA. These results were compared to the expression of transfer
rin receptor (TfR) mRNA which also has IREs in its 3'-UTR and is regulated
by Fe and NO via the binding of iron-regulatory proteins (IRPs) to its IREs
. Our experiments show that the IRE in Nramp2 mRNA does bind the IRPs in ly
sates from a mouse fibroblast cell line (LMTK-). Moreover, reverse transcri
ption-PCR (RT-PCR) demonstrated that both the IRE and non-IRE-containing tr
anscripts were present within these cells. However, there was no change in
Nramp2 mRNA expression in LMTK- cells after a 20-h incubation with either t
he Fe chelator, desferrioxamine (DFO), the Fe donor, ferric ammonium citrat
e (FAC), or the NO generator, S-nitroso-N-acetylpenicillamine (SNAP). In co
ntrast, these agents caused a marked change in the RNA-binding activity of
the IRPs and the expression of TfR mRNA. In addition, both FAC and DFO caus
ed an appropriate change in [Fe-59] uptake from [Fe-59]Tf. viz., an increas
e in Fe uptake after exposure to DFO and a decrease after treatment with FA
C. As Nramp2 can transport Fe from non-Tf-bound Fe, the effect of preincuba
tion with DFO and FAC was also examined on Fe uptake from [Fe-59]nitrilotri
acetate and [Fe-59]citrate. However, in contrast to the results found for [
Fe-59]Tf, incubation with DFO and FAC did not result in appropriate regulat
ion of Fe uptake from [Fe-59]nitrilotriacetate or [Fe-59]citrate. These dat
a demonstrate that non-Trf-bound Fe uptake was not under control of the IRP
-IRE system in these cells. Collectively, the results indicate that in LMTK
-fibroblasts Nramp2 mRNA expression was not regulated like TfR mRNA.