Abstract
Iron is an essential trace metal with many biochemical roles in the body that require its ability to convert between two oxidation states (Fe2+ and Fe3+). It is this property that makes iron so useful, which also causes significant toxicity in iron-overload disorders such as haemochromatosis, while iron-deficiency anaemia is one of the most common dietary deficiencies worldwide. The level of iron within the body is controlled at the point of absorption by regulation of the iron transporters divalent metal transporter 1 (DMT1) at the apical membrane, and Iregl at the basolateral membrane of duodenal enterocytes. One isoform of DMT1 contains an iron response element (IRE) in its mRNA in the 3’untranslated region (UTR), while iregl has an IRE in its 5’UTR. The significance of these regulatory elements remains unclear. The purpose of this study was to investigate how iron transport and transporter expression is regulated in the duodenum. Tissue was obtained from iron-deficient and iron-loaded rats, and Caco-2 cells were used as an in vitro model of intestinal enterocytes. Radioisotope transport studies were used to measure iron transport across Caco-2 monolayers, and protein and mRNA expression of transporters were measured using Western blotting and RT-PCR. Initial studies on rat tissue showed DMT1 (nonIRE) and Iregl mRNA levels were increased in iron-deficient rats, but expression of these transporters was not altered in iron-loaded rats. DMT1 (IRE) mRNA and protein were increased in iron-deficient rats but decreased in iron-loaded rats. The mechanism involved in these responses was investigated further using Caco-2 cells. The response to iron challenge in Caco-2 cells had two phases. There was a rapid response, in which DMT1 (IRE) was internalised within 4 h of iron treatment and subsequently recycled to the membrane within 8 h of the iron challenge being withdrawn. Following longer exposure to iron, there was a specific decrease in cellular expression levels of DMT1 (IRE), with the inverse response observed in cells treated with the iron chelator desferrioxamine (DFO), indicating possible IRE regulation. In contrast to in vivo studies, Iregl expression was not changed in DFO treated cells indicating this transporter may be regulated by endogenous signals (e.g. from iron storage tissues) not present in cell culture. Divalent metals other than iron may also influence expression of iron transporters. Previous studies have shown that cadmium and copper may regulate DMT1 (IRE) via a common mechanism, however zinc induces the opposite response. In this study cobalt was found to induce expression of DMT1 (nonIRE) indicating yet another mechanism for the effects of divalent metals on iron transporters. Recently a liver expressed peptide named hepcidin has been implicated in the central regulation of iron homeostasis. Previous studies using Caco-2 cells have shown that hepcidin can directly target DMT1 (IRE). In this study IL6 stimulated hepatocytes to release a substance, presumably hepcidin, which when applied to Caco-2 cells decreased DMT1 (IRE) expression. This may explain the mechanism that induces anaemia during chronic illness (ACD) where IL6 levels are elevated. A second cytokine implicated in ACD is TNFα, which increases expression of the iron storage protein ferritin but decreases DMT1 (IRE). This effect of TNFα may also have implications for the local regulation of DMT1 as TNFα expression by intraepithelial lymphocytes is stimulated by iron exposure. These findings highlight the complexity of iron homeostasis. Proteins involved in iron metabolism are controlled in response to a range of stimuli including dietary components such as iron levels and the presence of additional divalent metals as well as humoral factors during illness, iron overload or deficiency and hypoxia.