Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron

Genetic studies in animal models of microcytic anemia and biochemical studi es of transport have implicated the Nramp2 gene in iron transport. Nramp2 g enerates two alternatively spliced mRNAs that differ at their 3' untranslat ed region by the presence or absence of an iron-response element (IRE) and that encode two proteins with distinct carboxy termini. Antisera raised aga inst Nramp2 fusion proteins containing either the carboxy or amino termini of Nramp2 and that can help distinguish between the two Nramp2 protein isof orms (IRE: isoform I; non-IRE: isoform II) were generated. These antibodies were used to identify the cellular and subcellular localization of Nramp2 in normal tissues and to study possible regulation by dietary iron deprivat ion. Immunoblotting experiments with membrane fractions from intact organs show that Nramp2 is expressed at low levels throughout the small intestine and to a higher extent in kidney. Dietary iron starvation results in a dram atic upregulation of the Nramp2 isoform I in the proximal portion of the du odenum only, whereas expression in the rest of the small intestine and in k idney remains largely unchanged in response to the lack of dietary iron. In proximal duodenum, immunostaining studies of tissue sections show that Nra mp2 protein expression is abundant under iron deplete condition and limited to the villi and is absent in the crypts. In the villi, staining is limite d to the columnar absorptive epithelium of the mucosa (enterocytes), with n o expression in mucus-secreting goblet cells or in the lamina propria. Nram p2 expression is strongest in the apical two thirds of the villi and is ver y intense at the brush border of the apical pole of the enterocytes, wherea s the basolateral membrane of these cells is negative for Nramp2. These res ults strongly suggest that Nramp2 is indeed responsible for transferrin-ind ependent iron uptake in the duodenum, These findings are discussed in the c ontext of overall mechanisms of iron acquisition by the body. (C) 1999 by T he American Society of Hematology.