# IRE-dependent Regulation of Intestinal Dmt1 Prevails During Chronic Dietary Iron Deficiency but is Dispensable in Conditions of Acute Erythropoietic Stress

Maria Qatato, Michael Bonadonna, Gaël Palais, Alina Ertl, Gabriele Schmidt, Maria Polycarpou-Schwarz, Zoubida Karim, Bruno Galy

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Maria Qatato, Michael Bonadonna, Gaël Palais, Alina Ertl, Gabriele Schmidt, et al. IRE-dependent Regulation of Intestinal Dmt1 Prevails During Chronic Dietary Iron Deficiency but is Dispensable in Conditions of Acute Erythropoietic Stress. HemaSphere, 2022, 6 (3), pp.e693. 10.1097/HS9.0000000000000693. inserm-03874988

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# IRE-dependent Regulation of Intestinal *Dmt1* Prevails During Chronic Dietary Iron Deficiency but is Dispensable in Conditions of Acute Erythropoietic Stress

Maria Qatato<fnref n="1" />, Michael Bonadonna<fnref n="1" />,<fnref n="2" />, Gaël Palais<fnref n="1" />, Alina Ertl<fnref n="1" />, Gabriele Schmidt<fnref n="3" />, Maria Polycarpou-Schwarz<fnref n="1" />, Zoubida Karim<fnref n="4" />, Bruno Galy<fnref n="1" />

**Correspondence:** Bruno Galy (b.galy@dkfz.de).

Iron must be supplied in adequate amounts to sustain erythropoiesis while avoiding toxic iron accumulation.<fnref n="1" /> Whole body iron levels are largely determined by the rate of dietary iron absorption in the duodenum.<fnref n="2" /> Divalent metal transporter 1 (DMT1, encoded by *Slc11a2*) is an essential iron transporter present at the apical membrane of absorptive enterocytes.<fnref n="3" /> It mediates the influx of nonheme iron from the intestinal lumen into the interior of the cell after reduction of the metal by CYBRD1 (a.k.a. DCYTB); iron is then either stored intracellularly into nanocages made of ferritin-H (FTH1) and -L (FTL1) subunits, or exported into the circulation by the basolaterally expressed transporter ferroportin (FPN, a.k.a. *SLC40A11*) to be loaded onto transferrin after oxidation of the metal by hephaestin (HEPH).<fnref n="4" />

Intestinal iron absorption is systemically modulated by the hormone hepcidin (a.k.a. HAMP), which binds to and inhibits FPN.⁵ Hepcidin production in hepatocytes augments when serum iron levels and body iron stores are elevated; conversely, stimulation of erythropoiesis suppresses hepcidin through the erythroblast-derived hormone erythroferrone (ERFE, encoded by *Fam132b*), allowing for iron export via FPN.<fnref n="1" /> The intestinal iron absorption machinery is also controlled locally by mechanisms acting within duodenal enterocytes. This includes the

regulation of *Dmt1* and *Fpn* transcription and possibly ferritin turnover by hypoxia inducible factor 2 (HIF2).⁶⁻⁸

Four *Dmt1* mRNA isoforms with distinct 5' and 3' ends are transcribed from the *Dmt1* locus.<fnref n="3" /> 3' variants harbor or lack an iron-responsive element (IRE) in their 3' untranslated region (UTR). IREs are cis-regulatory hairpin RNA structures that bind iron regulatory proteins (IRP)-1 and -2 in iron-deficient cells to modulate RNA decay or translation, respectively.<fnref n="1" /> IRE-containing *Dmt1* mRNA variants pre dominate in the intestine, suggesting possible involvement of the IRP/IRE system in local regulation of DMT1.<fnref n="3" /> Recently, we generated a mouse line with targeted disruption of the *Dmt1* 3'IRE (*Dmt1*¹REA).⁹ Our work revealed that the *Dmt1* IRE exerts age-dependent effects on intestinal DMT1 expres sion and body iron homeostasis under standard laboratory conditions.⁹ Here, we set out to determine the role of the *Dmt1* 3'IRE in the regulation of the intestinal iron absorp tion machinery in response to stimuli that heighten the iron demand in adults.

We first studied the role of the *Dmt1* 3'IRE in the response to chronic dietary iron restriction. *Dmt1*¹IRE+/+ (or wild-type, WT) and *Dmt1*¹REAA mice were maintained on an iron-de ficient (<10 ppm Fe) versus control (~200 ppm Fe) diet from postweaning until 12 weeks of age (Figure 1A). Both WT and *Dmt1*¹REAA animals exhibit a marked reduction of serum iron concentration when fed with low iron, associated with an increase in transferrin (Tf) levels and a decrease in Tf sat uration (Figure 1B). Organismal iron stores are depleted, as evidenced by a reduction of serum ferritin, at least in KO mice (Figure 1B), and a diminution of the hepatic iron content (Figure 1C). As expected, the low iron regimen and the ensuing systemic iron deficiency lower hemoglobin levels in both gen otypes (Suppl. Table S1). This is associated with elevation of circulating erythropoietin (EPO) levels (Figure 1D, left), retic ulocyte frequency (Figure 1D, right), and serum ERFE con centration (Figure 1E). The reticulocytosis together with the reduction of body iron levels results in a stark drop in hepcidin (Figure 1F). Collectively, these data reflect a typical situation of iron-deficiency anemia.¹⁰

Histological examination of the duodenum shows enriched iron content deposited in the villi of *Dmt1*¹REAA duodena, in comparison to WT (Figure 1G, bottom), recapitulating formerly

<footnote n="1">¹Division of Virus-Associated Carcinogenesis (F170), German Cancer Research Center (DKFZ), Heidelberg, Germany</footnote>

<footnote n="2">²Biosciences Faculty, University of Heidelberg, Germany</footnote>

<footnote n="3">³Light Microscopy Facility (LMF), German Cancer Research Center (DKFZ), Heidelberg, Germany</footnote>

<footnote n="4">⁴Université de Toulouse, INSERM, CNRS, Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), Université Paul Sabatier (UPS), Toulouse, France</footnote>

<footnote>Supplemental digital content is available for this article.
Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. HemaSphere (2022) 6:3(e693).</footnote>

<footnote>http://dx.doi.org/10.1097/HS9.0000000000000693.</footnote>

<footnote>Received: October 18, 2021 / Accepted: January 28, 2022</footnote>
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**Figure 1.** The *Dmt1* 3'IRE is required for full stimulation of duodenal DMT1 expression during chronic dietary iron deficiency. (A) WT (indicated IRE<sup>+/+</sup>) and *Dmt1*<sup>IRE<sup>Δ</sup>/Δ</sup> (indicated IRE<sup>NΔ</sup>) mice, respectively, were fed with a low (<10 ppm, gray bars) versus a Ctr iron diet (~200 ppm, white bars) from weaning age on until early adulthood (12 weeks of age); (B) serum iron parameters; (C) hepatic iron store; (D) serum EPO levels and percentage of reticulocytes among TER119<sup>+</sup> blood cells. (E, F) Circulating levels of ERFE and hepcidin; the stress hormone ERFE (E) could not be detected under basal conditions (indicated nd). (G) Top histogram: iron content per mg of dry duodenal tissue. Bottom panels: Perl's staining (enhanced with diaminobenzidine) in the duodenum revealing iron deposits in intestinal epithelial cells of *Dmt1*<sup>IRE<sup>Δ</sup>/Δ</sup> fed with the Ctr diet; iron remains below the detection limit in mice receiving the low iron regimen, regardless of genotype (counterstain: hámalaun; scale bar: 200 µm). (H) Top histograms: qRT-PCR analysis of iron uptake molecules in the duodenum. Bottom panels: representative western-blot analysis of DMT1 protein expression in the duodenum, showing blunted DMT1 stimulation in *Dmt1*<sup>IRE<sup>Δ</sup>/Δ</sup> versus *Dmt1*<sup>IRE<sup>+/+</sup></sup> mice. DMT1 runs as a smear, the asterisk indicates a cross-reactive band. (I) Duodenal expression of the *Fth1* and *Ftl1* iron storage molecules was assessed by qRT-PCR (top histograms) and western blotting (lower panels), respectively. The representative western blot shows a marked decrease in ferritin protein levels in iron deficient duodena. (J) Top: qRT-PCR analysis of *Fpn* (left) and *Heph* (right) expression in the duodenum. Bottom panels: western blot analysis *FPN* protein levels. In H to J, qRT-PCR results are presented as fold change over WT mice fed with the Ctr diet. Average mRNA levels of *Actb*, *Tubb5*, and *Gapdh* served as reference except in G, where *Dmt1*-IRE pre-mRNA levels were calibrated to *Actb* pre-mRNA expression. In H to J, *ACTB* served as loading control for western blotting. Statistical significance was determined using 2-way ANOVA with Tuckey's multiple comparisons test, and a *P* value inferior to 0.05 was considered significant. In B to C, n = 8 to 15. In D to G, n = 6 to 10. In H to J, n = 14 to 17. \* *P* < 0.05, \*\* *P* < 0.01, \*\*\* *P* < 0.001, \*\*\*\* *P* < 0.0001. *ACTB* = beta actin; *Ctr* = control; *Cybrd1* = cytochrome b reductase 1; *DMT1* = Divalent metal transporter 1; *EPO* = erythropoietin; *ERFE* = erythroferrone; *FPN* = ferroportin; *FTH1* = ferritin heavy chain 1; *FTL1* = ferritin light chain 1; *GAPDH* = glyceraldehyde-3-phosphate dehydrogenase; *HEPH* = hephaestin; *IRE* = iron-responsive element; *qRT-PCR* = real-time quantitative reverse transcription PCR; *sat.* = saturation; *TUBB5* = tubulin, *beta 5* class I; *WT* = wild-type.
<figure><img src="image_11.png" /><figcaption>**Figure 2.** The *Dmt1* 3′IRE is dispensable for duodenal induction of DMT1 in conditions of stress erythropoiesis. (A) Adult WT (*IRE*<sup>+/+</sup>) and *Dmt1*<sup>IREΔ</sup> (*IRE*<sup>Δ</sup>) mice were subjected to acute stress erythropoiesis. The animals were either treated with EPO (top) or with PHZ (bottom), as indicated. (B) Spleen index (calculated as square root of 100× spleen weight/body weight). (C) Serum iron parameters. (D) Serum levels of ERFE and hepcidin; ERFE was not detected in saline-injected mice (indicated nd). (E) Serum EPO concentration in mice treated with PHZ. (F) Hepatic iron store. (G, H) Duodenal iron metabolism: the bar graphs on the left display the iron content per mg of dry tissue in EPO (G) versus PHZ (H)-injected animals; the histology pictures show iron staining of duodenal villi (Perl's staining enhanced with diaminobenzidine; counterstain: hämalaun; scale bar: 200 µm). (I, J) top histograms: qRT-PCR analysis of *Dmt1* and *Cybrd1* transcripts in the duodenum of animals treated with EPO (I) or with PHZ (J), respectively. Bottom panels: representative western-blot analysis of DMT1 protein expression in the duodenum of EPO (I) versus PHZ (J)-injected mice. The expression of apical iron transport molecules augments in mice exposed to erythropoietic stresses regardless of genotype. In (I) and (J), qRT-PCR results are presented as fold change over WT mice injected with saline. Average mRNA levels of *Actb*, *Tubb5* and *Gapdh* served as reference except for the analysis of *Dmt1*-IRE pre-mRNA levels, which were calibrated to *Actb* pre-mRNA expression. For western blotting, ACTB served as loading control. Statistical significance was determined using 2-way ANOVA with Tuckey's multiple comparisons test, and a *P* value inferior to 0.05 was considered significant. In (B) to (G), n = 9 to 10 (EPO) or n = 8 to 12 (PHZ). In (I) and (J); n = 5 (EPO) or n = 9 to 12 (PHZ). \* *P* < 0.05, \*\* *P* < 0.01, \*\*\* *P* < 0.001, \*\*\*\* *P* < 0.0001. ACTB = beta actin; Cybrd1 = cytochrome b reductase 1; DMT1 = Divalent metal transporter 1; EPO = erythropoietin; ERFE = erythroferrone; IRE = iron-responsive element; PHZ, phenylhydrazine; qRT-PCR = real-time quantitative reverse transcription PCR; sat. = saturation; WT = wild-type.</figcaption></figure>
reported observations.⁹ The low iron regimen, however, strongly
depletes the duodenal iron content in both WT and mutant mice
(Figure 1G, top).

At the molecular level, chronic iron deficiency triggers a
substantial and selective increase in the expression of the
*Dmt1*-*IRE* mRNA isoform in WT animals (Figure 1H, top left
histogram), which translates as increased DMT1 protein lev-
els (Figure 1H, bottom panels). This response is accompanied
by an upregulation of the immature *Dmt1*-*IRE* pre-mRNA
(Figure 1H, bottom left histogram). Most likely, iron deple-
tion in duodenal enterocytes activates HIF2, which in turn
stimulates *Dmt1*-*IRE* transcription.⁷,⁸ This notion is further
supported by the concomitant upregulation of another HIF2-
target, *Cybrd1* (Figure 1H, bottom right histogram).⁷ Similar
to WT, *Dmt1*<sup>IREA/Δ</sup> mice subjected to chronic iron scarcity
display higher expression of both the immature *Dmt1*-*IRE*
pre-mRNA and the *Cybrd1* mRNA (Figure 1H, bottom histo-
grams), consistent with increased HIF2 activity. The augmen-
tation of *Dmt1*-*IRE* pre-mRNA levels is even twice as high
as in WT iron-deficient duodena. However, the expression
of the mature *Dmt1*-*IRE* mRNA is only mildly augmented
and is lower than in WT (Figure 1H, upper left histogram),
and DMT1 protein induction is blunted (Figure 1H, bottom
panels), indicating that disruption of the *Dmt1* 3'IRE impairs
*Dmt1* posttranscriptional stimulation under conditions of
chronic dietary iron deficiency. Despite the blunted DMT1
response, systemic iron metabolism parameters remain glob-
ally within normal range, potentially due to compensatory
effects. In addition to stimulation of apical iron transport
molecules, the low iron regimen and subsequent reduction
of mucosal iron levels lead to diminished expression of the
FTH1 and FTL1 iron storage proteins without concomitant
change in the level of the corresponding mRNAs, both in
WT and *Dmt1*<sup>IREA/Δ</sup> duodena (Figure 1I), possibly reflecting
ferritin degradation⁶ or suppression of ferritin translation by
the IRPs.¹¹ Chronic iron deficiency also augments the expres-
sion of FPN at the protein level, and the response in WT mice
slightly exceeds that of mutant animals (Figure 1J, lower pan-
els). The low iron diet has globally little or no impact on *Fpn
and Heph* mRNA levels (Figure 1J, histograms). Collectively,
these results indicate that stimulation of *Dmt1*-*IRE* tran-
scription by HIF2 does not suffice, and that posttranscrip-
tional stimulation of *Dmt1* through the 3'IRE is additionally
required for full stimulation of intestinal DMT1 expression in
response to chronic dietary iron deficiency.

We next assessed the role of the *Dmt1* 3'IRE in the response to stress erythropoiesis, a condition that acutely increases the erythroid iron demand and thus stimulates the intestinal iron absorption machinery. Mice were injected either with recombinant EPO to stimulate erythropoiesis directly,¹¹ or phenylhydrazine (PHZ) to mimic acute hemolytic anemia, a situation that also enhances erythropoiesis (Figure 2A).¹² As expected, both treatments trigger extramedullary erythropoiesis, as indicated by a marked increase in spleen size in all genotypes (Figure 2B). Accordingly, EPO treatment augments hemoglobin levels (Suppl. Table S1) as well as reticulocyte frequency in peripheral blood, as measured by flow cytometry (WT: 14.3% ± 1.0% versus 5.8% ± 1.2% reticulocytes among TER119⁺ cells in EPO-treated versus control mice; *Dmt1*<sup>IREA/Δ</sup>, 14.1 ± 1.1 and 4.6% ± 0.5%, respectively); reticulocytosis could not be assessed reliably in PHZ-treated mice due to autofluorescence from damaged RBCs (not shown). Expansion of the erythroid compartment in EPO-treated mice increases iron consumption and thus lowers serum iron and transferrin saturation values (Figure 2C), while inducing ERFE and suppressing serum hepcidin (Figure 2D). EPO does not affect hepatic iron levels (Figure 2F), but the amount of iron in duodenal villi tends to be reduced (Figure 2G). Differing from the EPO regimen, the release of iron from damaged RBCs in

PHZ-treated mice results in an elevation of systemic iron indices
in both WT and *Dmt1*<sup>IREA/Δ</sup> animals, with high serum iron and
ferritin concentration, high transferrin saturation values, and
iron accumulation in the liver (Figure 2C,F). Yet, the marked
reduction of RBC counts (Suppl. Table S1) enhances EPO
and ERFE production (Figure 2D,E), albeit to a lesser extent
in *Dmt1*<sup>IREA/Δ</sup> mice. Despite high ERFE levels, the reduction
in hepcidin does not reach statistical significance (Figure 2D).
While EPO treatment tends to decrease the duodenal iron con-
tent (Figure 2G), this is not the case in PHZ-treated animals
(Figure 2H). Consistent with an elevation of iron needs, treat-
ment with either EPO or PHZ significantly increase the levels of
the *Cybrd1* mRNA and the immature *Dmt1*-*IRE* pre-mRNA in
*Dmt1*<sup>IREA/Δ</sup> duodena (Figure 2I). However and in sharp contrast
to chronic dietary iron deficiency (Figure 1H), the absence of
a functional *Dmt1* 3'IRE in *Dmt1*<sup>IREA/Δ</sup> mice does not weaken
the induction of the mature *Dmt1*-*IRE* mRNA nor the stimu-
lation of DMT1 protein expression under conditions of stress
erythropoiesis (Figure 1I,J). Mirroring duodenal iron levels, fer-
ritin protein expression is decreased in EPO, but barely affected
in PHZ-treated mice, regardless of genotype (Suppl. Figure
S1A,B). FPN protein expression is augmented in all genotypes
(Suppl. Figure S1C,D). mRNA levels of *Fth1*, *Ftl1*, and *Heph*
are barely affected, *Fpn* mRNA expression is mildly induced by
EPO (Suppl. Figure S1A–D). Overall, these data show that the
*Dmt1* 3'IRE is largely dispensable for adequate regulation of
key intestinal iron absorption molecules in conditions of acute
erythropoietic stress.

We have formerly reported a strong posttranscriptional
downregulation of *Dmt1*-IRE mRNA expression in *Dmt1*<sup>IREA/Δ</sup>
duodena during the suckling period,⁹ a stage of life character-
ized by high iron needs, low iron stores, and relatively limited
iron supply from maternal milk.¹³,¹⁴ This phenomenon, how-
ever, was not recapitulated in adult mice under steady-state
conditions, uncovering an age-dependent switch in the activity
of the *Dmt1* 3'IRE in the intestine. With the inability to replen-
ish supply from dietary source, WT mice fed with an iron-poor
diet over a long period of time respond by enhancing duode-
nal *Dmt1*-IRE expression. By contrast and despite a marked
stimulation of *Dmt1*-IRE transcription, adult *Dmt1*<sup>IREA/Δ</sup> mice
exhibit low levels of *Dmt1*-IRE both at the mature mRNA
and protein level. Hence, a functional IRE loop in the 3'UTR
of the *Dmt1* mRNA appears to be needed to properly adjust
DMT1 expression during long-term iron scarcity, as observed
in suckling mice⁹ and in animals subjected to dietary iron
deficiency (this study). Conceivably, and by analogy to other
3'IRE motifs, IRP binding to the *Dmt1*-3'IRE stabilizes the
transcript, which together with stimulation of *Dmt1* transcription
by HIF2 is needed to achieve adequate DMT1 protein
expression. In contrast to dietary iron restriction, both EPO
and PHZ treatments represent acute increases in systemic iron
demands in mice already loaded with iron. Our results indi-
cate that the loss of IRE function in *Dmt1* does not affect the
overall response to acute erythropoietic stress. This implies
that the observed upregulation of duodenal DMT1 expres-
sion is largely IRE/IRP-independent and is likely mediated by
HIF2.¹⁵ Among other possibilities, the degree of iron depletion
in absorptive enterocytes (more profound in chronic dietary
iron deficiency than upon EPO or PHZ treatments) might
influence the contribution of IRP/IRE binding and HIF2, to
the regulation of DMT1.

In addition to the age- or tissue-dependent effects on DMT1
expression reported previously,⁹ the *Dmt1* 3'IRE now appears
to also influence DMT1 regulation in a stimulus-dependent
manner. Whether the context-dependent activity of the *Dmt1*
3'IRE involves the luminal or enterocytic iron status, the gut
microbiome, and additional molecules whose activity is also sit-
uation-dependent warrants further research.
## ACKNOWLEDGMENTS

We are grateful to H. Gunshin and N.C. Andrews for sharing the *Dmt1*<sup>IREA</sup> mouse line. We thank the staff of the “Center for Preclinical Research” of the DKFZ for their dedicated care of the animals, and the “Plateforme de Biochimie” at the “Centre de Recherche sur l’Inflammation” (Paris, France) for their measurement of serum parameters.

## AUTHOR CONTRIBUTIONS

MQ and BG designed the research. MQ, MB, GP, AE, GS, MP-S, and BG performed experiments. MQ, MB, ZK, and BG analyzed the data. MQ, ZK, and BG wrote and reviewed the article.

## DISCLOSURES

The authors have no conflicts of interest to disclose.

## SOURCES OF FUNDING

This work was supported by a grant from the Deutsche Forschungsgemeinschaft to BG (GA2075/5-1).

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