HIF-2alpha, but not HIF-1alpha, promotes iron absorption in mice

Abstract

HIF transcription factors (HIF-1 and HIF-2) are central mediators of cellular adaptation to hypoxia. Because the resting partial pressure of oxygen is low in the intestinal lumen, epithelial cells are believed to be mildly hypoxic. Having recently established a link between HIF and the iron-regulatory hormone hepcidin, we hypothesized that HIFs, stabilized in the hypoxic intestinal epithelium, may also play critical roles in regulating intestinal iron absorption. To explore this idea, we first established that the mouse duodenum, the site of iron absorption in the intestine, is hypoxic and generated conditional knockout mice that lacked either Hif1a or Hif2a specifically in the intestinal epithelium. Using these mice, we found that HIF-1alpha was not necessary for iron absorption, whereas HIF-2alpha played a crucial role in maintaining iron balance in the organism by directly regulating the transcription of the gene encoding divalent metal transporter 1 (DMT1), the principal intestinal iron transporter. Specific deletion of Hif2a led to a decrease in serum and liver iron levels and a marked decrease in liver hepcidin expression, indicating the involvement of an induced systemic response to counteract the iron deficiency. This finding may provide a basis for the development of new strategies, specifically in targeting HIF-2alpha, to improve iron homeostasis in patients with iron disorders.

Figure 1. Intestine-specific deletion of Hif1a and Hif2a.

(A) Immunostaining for the hypoxic marker Hypoxyprobe in WT mouse duodenum (original magnification, ×100) and lung (original magnification, ×200). The control corresponds to the omission of primary antibody. (B) HIF-1α^fl/fl and HIF-2α^fl/fl mice (both floxed at exon 2) were bred with a transgenic strain expressing Cre recombinase under the control of the murine villin promoter. (C) Recombination efficiency for HIF-1α^fl/fl and HIF-2α^fl/fl alleles quantified by real-time PCR of genomic DNA isolated from the duodenum as described previously (30). n = 4 in each group. Hif1a and Hif2a mRNA levels in duodenum scrapings of vil-Cre⁺/HIF-1α^fl/fl and vil-Cre⁺/HIF-2α^fl/fl mice and WT littermates as determined by real-time PCR. n = 4 in each group. **P < 0.01, unpaired Student’s t test. (D) H&E staining of duodenum of a vil-Cre⁺/HIF-2α^fl/fl mouse and a WT littermate (original magnification, ×200).

Figure 2. Deletion of Hif2a in the duodenum decreases the expression of genes involved in iron absorption.

Glut1, DMT1+IRE, Dcytb, and FPN mRNA levels in duodenum scrapings from vil-Cre⁺/HIF-1α^fl/fl and vil-Cre⁺/HIF-2α^fl/fl mice and WT littermates (n ≥ 6 in each group). *P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student’s t test.

Figure 3. HIF-2α binds and trans-activates the human DMT1-1A promoter.

(A) Left: The HIF agonists L-Mim (500 μM), hydralazine (Hydr.; 150 μM), and desferrioxamine mesylate (DFO; 150 μM) induced DMT1-1A promoter–driven luciferase activity. Right: Caco-2/TC7 cells were transiently transfected with pGL3–DMT1-1A or pGL3-6XHRE vectors together with pcDNA3 empty vector as a control (C) or increasing concentrations of pcDNA3–HIF-1α and pcDNA3–HIF-2α. (B) Luciferase assay on Caco-2/TC7 cells transiently transfected with pGL3–DMT1-1A (WT) or serial truncated versions of DMT1-1A promoter (D5 to D1) in the presence of pcDNA3–HIF-2α at a 1:10 ratio. (C) Luciferase assay on Caco-2/TC7 cells transiently transfected with pGL3–DMT1-1A (WT) or pGL3–DMT1-1A mutated in each of the 5 HREs (HRE-1 mut to HRE-5 mut) in the presence of pcDNA3–HIF-2α at a 1:10 ratio. (D) Left: Western blot analysis of Caco-2/TC7 cells incubated or not (–) with L-Mim (500 μM). Lanes were run on the same gel but were noncontiguous, as indicated. Right: Quantification by real-time PCR of a ChIP experiment on Caco-2/TC7 cells incubated with or without L-Mim (500 μM). *P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student’s t test.

Figure 4. Decrease in serum and liver iron and increase in serum EPO levels in intestine-specific Hif2a-knockout mice.

(A) Quantification of serum and liver iron levels in vil-Cre⁺/HIF-1α^fl/fl and vil-Cre⁺/HIF-2α^fl/fl mice and WT littermates (n = 6 in each group). (B) Serum EPO levels in vil-Cre⁺/HIF-1α^fl/fl and vil-Cre⁺/HIF-2α^fl/fl mice and WT littermates. *P < 0.05, ***P < 0.001, unpaired Student’s t test.

Figure 5. Decrease in hepatic hepcidin levels in intestine-specific Hif2a-knockout mice without any significant changes in duodenal FPN protein levels.

(A) Hepcidin mRNA expression in livers of vil-Cre⁺/HIF-1α^fl/fl and vil-Cre⁺/HIF-2α^fl/fl mice and WT littermates by real-time RT-PCR (n = 7). ***P < 0.001, unpaired Student’s t test. (B) FPN expression in duodenum scrapings of WT and vil-Cre⁺/HIF-2α^fl/fl mice from 3 independent littermates, by Western blotting. Results were quantified and normalized (FPN/α-tubulin).