Previously uncharacterized isoforms of divalent metal transporter (DMT)-1: implications for regulation and cellular function

Abstract

Divalent metal transporter 1 (DMT1) mediates apical iron uptake into duodenal enterocytes and also transfers iron from the endosome into the cytosol after cellular uptake via the transferrin receptor. Hence, mutations in DMT1 cause systemic iron deficiency and anemia. DMT1 mRNA levels are increased in the duodenum of iron-deficient animals. This regulation has been observed for DMT1 mRNA harboring an iron-responsive element (IRE) in its 3' UTR, but not for a processing variant lacking a 3'UTR IRE, suggesting that the IRE regulates the expression of DMT1 mRNA in response to iron levels. Here, we show that iron regulation of DMT1 involves the expression of a previously unrecognized upstream 5' exon (exon 1A) of the human and murine DMT1 gene. The expression of this previously uncharacterized 5' exon is tissue-specific and particularly prevalent in the duodenum and kidney. It adds an in-frame AUG translation initiation codon extending the DMT1 ORF by a conserved sequence of 29-31 amino acids. In combination with the IRE- and non-IRE variants in the 3'UTR, our results reveal the existence of four DMT1 mRNA isoforms predicting the synthesis of four different DMT1 proteins. We show that two regulatory regions, the 5' promoter/exon 1A region and the IRE-containing terminal exon participate in iron regulation of DMT1 expression, which operate in a tissue-specific way. These results uncover an unexpected complexity of DMT1 expression and regulation, with implications for understanding the physiology, cell biology, and pathophysiology of mammalian iron metabolism.

Fig 1.

Iron regulation of 3′ end variants of DMT1 mRNA in 293 and Caco-2 cells. Semiquantitative RT-PCR was performed by using mRNA from 293 cells (lanes 1–3) or Caco-2 cells (lanes 4–6) that were treated with hemin (H) or desferrioxamine (D), or the remained untreated (C) during 12 h, as described in Materials and Methods. PCR amplification (16 cycles) was performed with hβ-actin primers, and 23 cycles with hTfR, hDMT1-IRE primers, and hDMT1-non-IRE primers. All PCR products have the respective predicted size.

Fig 2.

Identification of a previously uncharacterized isoform of the DMT1 mRNAs. (A) Sequences of the alternative 5′ regions of the human DMT1 mRNA. The sequences of the two DMT1 variants are identical after reaching the sequence corresponding to exon 2. The specific sequences of each isoform are boxed in blue for the newly identified exon 1 (exon 1A) and in green for the previously described exon 1 (exon1B). (B) Revised genomic organization of the human DMT1 gene. The sequence of the exon 1A has been mapped 1.9 kb upstream of exon 1B in the human genomic sequence (NT-009782). The two 5′ DMT1 variants are generated by alternative promoter usage followed by splicing to exon 2. (C) Alignment of the predicted N-terminal extensions encoded by the exon 1A isoforms of human, mouse, and rat () DMT1. Amino acids that are boxed are not conserved in all three species.

Fig 3.

Expression pattern of the DMT1 5′ and 3′ end variants in mouse tissues. Total RNA from organs and tissue obtained from a C57BL6 mouse were reverse-transcribed by using random primers. PCR amplifications were performed by using specific primers of the alternative 5′ exon (DMT1-1A vs. DMT1-1B) or the alternative 3′ terminal exon (DMT1-IRE vs. DMT1-non-IRE). D1 refers to the 1st cm of the duodenum, D2 and D3 refer to the 2nd and the 3rd cm respectively. All depicted PCR products have the respective predicted sizes. Analysis of total RNA from mouse brain homogenate revealed the expression of the 1B isoform as well as of the IRE and non-IRE 3′ variants, but no evidence for the expression of the 1A isoform in brain could be obtained (data not shown).

Fig 4.

Iron regulation of 5′ end variants of DMT1 mRNA in human cell lines. Semiquantitative RT-PCR was performed on total RNA from cells treated as in Fig. 1 by using 16 PCR cycles for hβ-actin and 25 cycles for DMT1 1A and 1B. The forward primers used to amplify the two 5′ variants of DMT1 hybridized to the respective exon 1 (A or B) and the reverse primer to exon 3. The PCR products have a size of 292 and 223 bp, respectively. Shown is one representative experiment of three.

Fig 5.

Iron regulation of the four alternative isoforms of DMT1 mRNA in Caco-2 cells (A and B) as well as mouse kidney and duodenum (C). (A) Caco-2 cells were treated as described in Fig. 1. Total RNA was analyzed by semiquantitative RT-PCR using isoform-specific forward and reverse primers, as described in the text. PCR cycles (16) were used to amplify hβ-actin cDNA, 27 cycles for 1A-IRE, 1B-IRE, and 1B-non-IRE cDNAs, and 31 cycles for 1A-non-IRE cDNA. (B) Quantification of three experiments like the one shown in A by RT-PCR. Gray bars correspond to control, black to hemin, and white to desferrioxamine to treated samples. (C) Quantification of the expression of the four mDMT1 mRNA isoforms in duodenum and kidney from mice fed with an iron deficient diet (white bars) or with a control diet (gray bars). Bars represent average values obtained from four mice each ± SD. No expression of the 1A-non-IRE isoform was detected in kidneys.