Hepatic hepcidin/intestinal HIF-2α axis maintains iron absorption during iron deficiency and overload

Abstract

Iron-related disorders are among the most prevalent diseases worldwide. Systemic iron homeostasis requires hepcidin, a liver-derived hormone that controls iron mobilization through its molecular target ferroportin (FPN), the only known mammalian iron exporter. This pathway is perturbed in diseases that cause iron overload. Additionally, intestinal HIF-2α is essential for the local absorptive response to systemic iron deficiency and iron overload. Our data demonstrate a hetero-tissue crosstalk mechanism, whereby hepatic hepcidin regulated intestinal HIF-2α in iron deficiency, anemia, and iron overload. We show that FPN controlled cell-autonomous iron efflux to stabilize and activate HIF-2α by regulating the activity of iron-dependent intestinal prolyl hydroxylase domain enzymes. Pharmacological blockade of HIF-2α using a clinically relevant and highly specific inhibitor successfully treated iron overload in a mouse model. These findings demonstrate a molecular link between hepatic hepcidin and intestinal HIF-2α that controls physiological iron uptake and drives iron hyperabsorption during iron overload.

Keywords: Gastroenterology; hypoxia.

Figure 1. Temporal disruption of hepatic hepcidin activates intestinal HIF-2α and leads to rapid iron accumulation.

(A) Schematic representation of mice with temporal disruption of hepatocyte hepcidin. (B) qPCR analysis of hepatic hepcidin (Hamp) transcript expression levels (n = 3–8 per group). (C) Representative Prussian blue iron staining and H&E staining of liver tissue from Hamp^ΔLiv mice. Original magnification, ×20 (n = 3 per group). (D–F) Serum (D), heart (E), and pancreatic iron content (F) (n = 3–14 per group). (G) Representative HIF-2α staining of duodenal sections 2 weeks after tamoxifen injection into Hamp^fl/fl and Hamp^ΔLiv mice. Original magnification, ×20 (n = 3 per group). (H) Western blot analysis of FPN, DMT1, DCYTB, and TFR1 expression in duodenal membrane fractions (n = 2–3 per group). (I) qPCR antalysis of duodenal HIF-2α–specific and iron-handling transcripts 2 weeks after tamoxifen injection into Hamp^fl/fl and Hamp^ΔLiv mice (n = 5–8 per group). Data represent the mean ± SEM. Male samples are designated as squares, and female samples are designated as circles. Significance was determined by 1-way ANOVA with Tukey’s post hoc test (B and D–F) or 2-tailed, unpaired t test (I). *P < 0.05, ***P < 0.001, and ****P < 0.0001 versus the Hamp^fl/fl group.

Figure 2. Intestinal epithelial FPN is necessary for the activation of intestinal HIF-2α during systemic iron deficiency.

(A and B) Schematic representation of the experimental design (A) and of intestinal epithelial iron retention following FPN deletion (B). (C) qPCR analysis of duodenal Fpn transcript levels (n = 4–7 per group). (D) Western blot analysis of duodenal FTH1 (n = 3 per group). (E) qPCR analysis of Hamp transcript levels (n = 4–7 per group). (F) Analysis of RBC, HB, and HCT (n = 4–7 per group). (G) Representative HIF-2α staining in duodenal sections. Original magnification, ×20 (n = 3 per group). (H) qPCR analysis of HIF-2α–specific and iron-handling transcripts in duodenal samples (n = 4–6 per group). Male samples are designated as squares, and female samples are designated as circles. Data represent the mean ± SEM. Statistical significance was determined by 2-way ANOVA with Tukey’s post hoc test. ***P < 0.001 and ****P < 0.0001 versus iron-replete Fpn^fl/fl; ^##P < 0.01 and ^####P < 0.0001 versus low-iron Fpn^fl/fl; ^†P < 0.05 versus iron-replete Fpn^ΔIE.

Figure 3. Deletion of intestinal epithelial FPN blocks the intestinal HIF-2α response to erythropoietic demand.

(A) Experimental design for Phz-induced hemolytic anemia model. (B and C) qPCR analysis of kidney Epo (B) and liver Hamp (C) transcript levels (n = 5–9 per group). (D) Western blot analysis of duodenal FTH1 (n = 3 per group). (E) Representative HIF-2α staining of duodenal sections. Original magnification, ×20 (n = 3 per group). (F) qPCR analysis of HIF-2α–specific and iron-handling transcripts in duodenal samples (n = 5–7 per group). Male samples are designated as squares, and female samples are designated as circles. Data represent the mean ± SEM. Statistical significance was determined by 2-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, and ****P < 0.0001 versus vehicle Fpn^fl/fl; ^#P < 0.05 and ^####P < 0.0001 versus vehicle Fpn^fl/fl.

Figure 4. The intestinal HIF-2α response to changes in systemic iron and oxygen is driven by epithelial iron levels.

(A) Schematic of 3-month, inducible iron trapping in animals lacking intestinal epithelial FPN (Fpn^ΔIE) or DMT1 (Dmt1^ΔIE). (B) qPCR analysis of Fpn and Dmt1 transcript levels (n = 4 per group). (C) qPCR analysis of hepatic Hamp transcript expression levels (n = 4 per group). (D) Analysis of RBC, HB, and HCT (n = 3–5 per group). (E) Representative HIF-2α staining of duodenal sections. Original magnification, ×20 (n = 3 per group). (F) qPCR analysis of HIF-2α–specific and iron-handling transcripts in duodenal samples (n = 4 per group). Male samples are designated as squares, and female samples are designated as circles. Data represent the mean ± SEM. Statistical significance was determined by 2-tailed, unpaired t test. *P < 0.05, **P < 0.01, and ****P < 0.0001 compared between Fpn^fl/fl and Fpn^ΔIE cohorts; ^#P < 0.05, ^##P < 0.01, and ^####P < 0.0001 compared between Dmt1^fl/fl and Dmt1^ΔIE cohorts.

Figure 5. The intestinal transcriptome during systemic iron deficiency resembles that of hepcidin deficiency–mediated iron overload.

(A) Experimental design for the samples used in whole-genome RNA-Seq. (B) qPCR analysis of liver Hamp transcript levels in mice on an iron-replete (IR) or low-iron (LI) diet (n = 8–9 per group). (C) Dendrogram comparing genotype-diet interactions following unsupervised hierarchical clustering of genes differentially expressed at a high-stringency FDR of less than 0.01 (n = 3 per group). (D) Heatmap of genes used for unsupervised hierarchical clustering (n = 3 per group). (E) Lower-stringency differential expression analysis at a FDR of less than 0.1 to uncover transcripts in the RNA-Seq data set unique to iron deficiency and hepcidin deficiency. Genes highlighted in red are novel intestinal transcripts regulated by both low iron and hepcidin deficiency (n = 3 per group). Male samples are designated as squares, and female samples are designated as circles. FC, fold change. Data represent the mean ± SEM. Statistical significance was determined by 2-way ANOVA with Tukey’s post hoc test. ****P < 0.0001 versus iron-replete Hamp^fl/fl.

Figure 6. FPN activates HIF-2α in a cell-autonomous manner that is dependent on efflux of the cellular labile iron pool.

(A) Western blot analysis of FPN^GFP HEK293 cells following a 24-hour doxycycline treatment. (B) Western blot analysis of cytosolic and nuclear fractions of FPN^GFP HEK293 cells treated with vehicle (V), 250 ng/ml doxycycline (D), or 100 μM FG4592 (FG) for 24 hours. (C and D) Western blot analysis of cytosolic and nuclear fractions of FPN^GFP HEK293 cells treated with vehicle (V), doxycycline (D), doxycycline and 200 μM FAC (D+F), or doxycycline and 1 mg/ml hepcidin (D+H) for 24 hours (C). Separate doxycycline plus FAC and doxycycline plus hepcidin conditions were also cotreated with FG4592 for 24 hours, as indicated (D). (E) Schematic of the luciferase-based PHD enzyme activity reporter. (F) Fold change of luciferase activity in FPN^GFP HEK293 cells infected with the PHD reporter and treated with vehicle, doxycycline, FG4592, FAC and doxycycline, or doxycycline and hepcidin for 24 hours. (G) Western blot analysis of FPN^GFP HEK293 cells stable for empty lentiCRISPRv2 (Empty) or unique NCOA4 short guide RNAs (NCOA4 sg1 and NCOA4 sg2). Cells were treated with FAC for 24 hours and then with doxycycline for 24 hours. (H) Western blot analysis of FPN^GFP IEC-6 cells treated with vehicle, doxycycline, or doxycycline and hepcidin for 24 hours. (I) ELISA of lysates from FPN^GFP IEC-6 cells treated with vehicle, doxycycline, doxycycline and hepcidin, or DFO for 24 hours. (J) Fold change of luciferase activity in FPN^GFP IEC-6 cells infected with the PHD reporter and treated with vehicle, doxycycline, FAC and doxycycline, or doxycycline and hepcidin for 24 hours. All cell culture experiments were repeated at least 3 times. Data represent the mean ± SEM. Statistical significance was determined by 1-way ANOVA with Tukey’s post hoc test. **P < 0.01 and ****P < 0.0001 versus vehicle; ^#P < 0.05, ^##P < 0.01, and ^####P < 0.0001 versus doxycycline.

Figure 7. Inhibition of HIF-2α using PT2385 reverses iron accumulation in multiple tissues in hepcidin-deficient hemochromatosis.

(A) Experimental design for oral gavage of vehicle or PT2385 in Hamp^ΔLiv mice. (B) qPCR analysis of hepatic Hamp and kidney Epo transcript levels (n = 5–7 per group). (C) Analysis of RBC, HB, and HCT (n = 5–7 per group). (D) Western blot analysis of FPN, DMT1, and DCYTB in duodenal membrane fractions (n = 3 per group). (E) Representative Prussian blue staining for iron in liver tissues. Original magnification, ×20 (n = 3 per group). (F) Serum, liver, heart, and pancreatic iron content (n = 5–7 per group). (G) Schematic representation of hepatic hepcidin/intestinal HIF-2 axis. Male samples are designated as squares, and female samples are designated as circles. Data represent the mean ± SEM. Statistical significance was determined by 1-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus vehicle Hamp^fl/fl; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 versus vehicle Hamp^ΔLiv. PT, PT2385; Veh, vehicle.