Apical distribution of HFE–β2-microglobulin is associated with inhibition of apical iron uptake in intestinal epithelia cells

Mutations in the HFEgene result in hereditary hemochromatosis, a disorder of iron metabolism characterized by increased intestinal iron absorption. Based on the observation that ectopic expression of HFE strongly inhibits apical iron uptake (Arredondo et al., 2001, FASEB J15, 1276–1278), a negative regulation of HFE on the apical membrane transporter DMT1 was proposed as a mechanism by which HFE regulates iron absorption. To test this hypothesis, we investigated: (i) the effect of HFE antisense oligonucleotides on apical iron uptake by polarized Caco-2 cells; (ii) the apical/basolateral membrane distribution of HFE, β-2 microglobulin and DMT1; (iii) the putative molecular association between HFE and DMT1. We found that HFE antisense treatment reduced HFE expression and increased apical iron uptake, whereas transfection with wild-type HFE inhibited iron uptake. Thus, an inverse relationship was established between HFE levels and apical iron uptake activity. Selective apical or basolateral biotinylation indicated preferential localization of DMT1 to the apical membrane and of HFE and β-2 microglobulin (β2m) to the basolateral membrane. Ectopic expression of HFE resulted in increased distribution of HFE–β2m to the apical membrane. The amount of HFE–β2m in the apical membrane inversely correlated with apical iron uptake rates. Immunoprecipitations of HFE or β2m with specific antibodies resulted in the co-precipitation of DMT1. These results sustain a model by which direct interaction between DMT1 and HFE–β2m in the apical membrane of Caco-2 cells result in down-regulation of apical iron uptake activity.