Control of systemic iron homeostasis by the 3’ iron-responsive element of divalent metal transporter 1 in mice

Abstract

Tight control of intestinal iron absorption is required to avoid both iron insufficiency and excess.1 Dietary nonheme iron is taken up by absorptive enterocytes via the apical iron transporter DMT1 (a.k.a. SLC11A2),2–4 and transferred into the circulation by ferroportin (FPN, a.k.a. SLC40A1), with the help of a ferroxidase, hephaestin (HEPH).1 FPN activity is controlled by the liver hormone hepcidin,1 but DMT1 seems regulated locally via mechanisms operating within enterocytes.5,6DMT1 messenger ribonucleic acid (mRNA) exists in four isoforms that differ in their 5’ and 3’ ends.7 3’ end diversity results from alternative usage of splicing and polyadenylation sites and yields isoforms that either contain or lack a conserved iron responsive element (IRE) in their 3’ untranslated region (UTR). IRE-containing isoforms are predominant in duodenal enterocytes. IREs are stem-loop structures that interact with iron regulatory proteins (IRPs, a.k.a. ACO1 and IREB2) in iron-depleted cells.1 IRP binding to multiple IREs in the 3’-UTR of the transferrin receptor 1 (TFRC) mRNA limits its degradation by Regnase-1 (a.k.a. ZC3H12A).8 The presence of an IRE-like motif in DMT1 suggests that DMT1 could be regulated by IRPs, similar to TFRC. However, the single DMT1 IRE contains an additional 3’-bulge in its upper stem, and DMT1 mRNA seems to lack a Regnase-1 binding site.9 Importantly, DMT1 expression only responds to iron fluctuation in a subset of cell lines,10,11 and the DMT1 3’IRE failed to exhibit iron-dependent regulation in reporter assays.11 Furthermore, Dmt1 transcription is controlled by HIF2α (a.k.a. EPAS1),5,6 which itself is regulated by IRPs,1 confounding the study of specific functions of the Dmt1 3’IRE.12 Here, we address the role of this ribonucleic acid (RNA) motif using a mouse model with selective disruption of the Dmt1 3’IRE. We established a mouse line lacking the 5’ stem, the apical loop and part of the 3’ stem of the Dmt1 3’IRE (Fig. 1A–D and Supplemental Materials and Methods, https://links.lww.com/HS/A91). Mutagenesis of the Dmt1 3’IRE impairs IRP binding. Importantly, all four DMT1 transcripts are adequately expressed in homozygous mutant mice, including those that bear the dysfunctional IRE (Supplemental Figure 1, https://links.lww.com/HS/A91). The resulting allele (designated Dmt1IREΔ) is inherited in Mendelian proportions (Supplemental Table 1, https://links.lww.com/HS/A91). Both Dmt1IREΔ/Δ males and females are viable and fertile and exhibit normal posture and activity patterns. Blood cell parameters (Supplemental Table 2, https://links.lww.com/HS/A91) are globally preserved during postnatal growth (2 weeks of age, during a period of high iron demand), early adulthood (3 months of age) and advanced age (9 months). A flow cytometry analysis did not reveal any abnormality of terminal erythroid differentiation in young adults (Supplemental Figure 2, https://links.lww.com/HS/A91). The mean weight is slightly higher in 2-week-old Dmt1IREΔ/Δ pups but later is comparable to Dmt1IRE+/+ littermates (Supplemental Table 3, https://links.lww.com/HS/A91). Spleen, liver, kidney, and heart weights were unchanged (Supplemental Table 3, https://links.lww.com/HS/A91). These data show that while Dmt1 is essential during perinatal life and critical for erythroid iron acquisition,2,3 its 3’IRE is not required under standard laboratory conditions and appears to be dispensable for normal hematopoiesis.