Modulation of the HIF2α-NCOA4 axis in enterocytes attenuates iron loading in a mouse model of hemochromatosis

Intestinal iron absorption is regulated by hypoxia inducible factor-2α (HIF2α).¹ Intestinal HIF2α signaling is integrated into systemic cues for iron requirements via the hepatic hormone hepcidin and the iron exporter ferroportin (Fpn1).² During increased systemic iron requirements, iron is released from ferritin (FTN)³ by ferritinophagy via lysosomal degradation mediated by nuclear receptor coactivator 4 (NCOA4).^4,5 Ferritinophagy is also regulated by intracellular iron.⁶ The significance of intestinal iron handling in systemic demand and erythropoiesis and the role of intestinal FTN in iron absorption are well known.^7,8 However, questions remain regarding how ferritinophagy is integrated into systemic iron regulation. In this work, using genetic mouse models of HIF2α and NCOA4 perturbation, we showed that the intestinal HIF2α/ferritinophagic axis is essential for systemic iron regulation, and intestinal NCOA4 can be therapeutically targeted to treat iron overload disorders.

Whole-body NCOA4-knockout (KO) (NCOA4^ΔWB) mice and intestine-specific lines HIF-2αKO: HIF2α^ΔIE, VhlKO: Vhl^ΔIE, and Vhl-HIF2αKO: Vhl/HIF2α^ΔIE; and tamoxifen-inducible, liver-specific hepcidin KO mice (HAMP^ΔLiv: Alb^CreERT2; HAMP^fl/fl) have been described.^2,9 The intestine-specific NCOA4-KO (NCOA4^ΔIE) line was generated by crossing NCOA4^F/F10 with villin-Cre mice of the C57BL/6J background. For baseline studies, a standard diet (iron, 240 ppm; 5001; Laboratory Diet), and for iron studies, an iron-enriched diet (350 ppm; 115180), or low-iron (<5 ppm; 115072) diet (Dyets Inc) were used. Phenylhydrazine (Phz)-induced hemolysis (intraperitoneal injections; 60 mg/kg body weight on 2 consecutive days), followed by euthanasia within 0, 3, or 6 days of the second injection and oral administration of FG-4592 (12.5 mg/kg per day, 5 days; Cayman Chemicals). Mucosal scrapings to isolate enterocytes were used in all experiments.

Two guide RNAs targeting mouse hepcidin (supplemental Table 1) were cloned in a CRISPR-Cas9–containing adenovirus dual gRNA expression vector (pAV[CRISPR]-hCAS9; VectorBuilder, Inc) (supplemental Figure 5A). The adenovirus (AV) injection contained 3 × 10¹¹ particles per animal via tail vein (supplemental Figure 5B).

NCOA4 antibodies included anti-rabbit A302‐272A (30968; Bethyl Laboratories [BL]); anti-goat (1:200, sc‐30968; Santa Cruz Biotechnology); anti-rabbit ferritin H (Cell Signaling Technology [CST]; 2998); anti-rabbit HIF-2α (BL; A700-003); anti-rabbit IRP2 (CST; 37135); anti-rabbit DMT1 (Alpha Diagnostic International [ADI]; NRAMP21-A); anti-rabbit ferroportin 1(ADI; MTP11-A); anti-mouse actin, (60008-1-Ig; 1:10 000; Proteintech;); anti-rabbit LAMP1 (sc-20011; Santa Cruz Biotechnology); and anti-rabbit LAMP2 (7125116; Invitrogen). Dilutions were 1:1000, unless otherwise specified.

Swiss-rolled, frozen, 7-μm sections fixed in 4% paraformaldehyde in phosphate-buffered saline and incubated overnight at 4°C with rabbit anti-NCOA4 antibody (1:100; BL), were incubated for 1 hour at room temperature with Alexa Fluor 488–labeled goat anti-rabbit IgG (1:500; Molecular Probes, Inc).

A 1-kb (relative to the transcription start site) mouse NCOA4 promoter region was cloned in pGL3 basic vector between NheI and XhoI (Promega). HIF1α, HIF2α overexpressing plasmids, and the luciferase assay have been described.⁹ 

Iron and transferrin saturation (K392; Biovision), alanine aminotransferase and aspartate aminotransferase (MAK052 and MAK055, respectively; Sigma Aldrich), and erythropoietin (EPO; MEP00B; R&D Systems).

Hematology, iron staining, quantitative reverse transcription-polymerase chain reaction¹¹; human intestinal organoid generation¹²; enterocyte membrane¹³ and lysosome isolation¹⁴; chromatin immunoprecipitation assay¹; and iron quantification by inductively coupled plasma mass spectrometry¹⁵ have been described.

The PCR primers are listed in supplemental Table 1.

Results are expressed as the mean ± standard error of the mean. The significance between 2 groups was determined by a 2-tailed, unpaired Student's t test. Significance among multiple groups was calculated by 1-way analysis of variance followed by Tukey's post hoc test or by 2-way analysis of variance followed by Sidak's multiple-comparison test. GraphPad Prism 8.0 was used for statistical analyses.

NCOA4 is highly expressed in the small intestine (Figure 1A). Duodenal NCOA4 RNA and protein expression are induced in dietary iron deficiency and Phz-mediated hemolytic anemia (Figure 1B-C; supplemental Figure 1A-E). Single-cell transcriptomic analysis has revealed that NCOA4 expression is more abundantly expressed in differentiated enterocytes than in other intestinal epithelial or stem cell lineages.^16,17 NCOA4 (Figure 1D) and divalent metal transporter 1 (DMT1) RNA expression is significantly upregulated in differentiated human intestinal organoids by iron chelator deferoxamine or hypoxia (1% O₂; supplemental Figure 1F). The induction observed in NCOA4 and DMT1 RNA and NCOA4 protein levels during iron deficiency were abolished in the intestine-specific HIF2α-KO (HIF2α^ΔIE) mouse duodenum (Figure 1E-G). Furthermore, duodenal NCOA4 RNA and protein expressions were induced in pharmacological (FG4592, a PHD inhibitor) and genetic (intestine-specific HIF overexpression [Vhl^ΔIE]) models of HIF activation (Figure 1G-I). A robust increase in FTN heavy chain (FTN H) expression in the Vhl^ΔIE duodenum (Figure 1I) is suggestive of an increase in HIF2α-dependent iron absorption and a corresponding downregulation of iron regulatory protein 2 (IRP2) is indicative of an active iron response element-IRP feedback system (Figure 1I). Increased lysosomal FTN in the Vhl^ΔIE duodenum indicates that NCOA4-mediated lysosomal FTN enrichment is HIF dependent (Figure 1J). Indeed, duodenal NCOA4 and DMT1 induction is completely abolished in the intestine-specific Vhl-HIF2α double-KO mice (Figure 1K-L; supplemental 1H). Subsequent promoter activity analysis of the human intestinal cell lines HCT116 and Caco-2 demonstrated that intestinal NCOA4 was selectively HIF2α dependent (Figure 1M). An NCOA4 promoter analysis revealed the presence of a canonical hypoxia response element (supplemental Figure 1I) and chromatin immunoprecipitation assay of the Vhl^ΔIE duodenum confirmed the binding of HIF2α at the NCOA4 promoter (Figure 1N).

In agreement with previous reports, whole-body NCOA4-KO (NCOA4^ΔWB) mice (generated by CRISPR/CAS9; supplemental Figures 2A-E and 3A-B) basally exhibited hypochromia with microcytosis (normal hemoglobin, low mean corpuscular height [MCH], and low mean corpuscular volume [MCV]) (supplemental Figure 3C).^6,10 Interestingly, no changes were observed in hepatic hepcidin. Lower renal erythropoietin (EPO) RNA expression (supplemental Figure 3D), but unaltered serum EPO (supplemental Figure 3E) were also noted. Phz-treated NCOA4^ΔWB mice exhibited higher tissue FTN H expression (supplemental Figure 4A). NCOA4^ΔWB mice exhibited lowered hemoglobin and hematocrit after Phz treatment, suggesting a possible role of ferritinophagy in acute iron demand (supplemental Figure 4B). Phz-treated NCOA4^ΔWB mice had significantly higher serum EPO on day 3, but no difference on day 6 (supplemental Figure 4C), reflected in the improved MCV and MCH values (supplemental Figure 4B). In the NCOA4^ΔWB mice receiving an iron-deficient diet (1 week), liver and splenic FTN H expression was significantly higher than in the NCOA4^F/F cohort (supplemental Figure 5A). Intriguingly, FTN was significantly reduced in both NCOA4^F/F and NCOA4^ΔWB duodenum during dietary iron deficiency (supplemental Figure 5A). This result suggests an active IRP-mediated FTN translational inhibition in iron-deficient enterocytes, and/or NCOA4 independent FTN turnover via bulk- or ATG5-dependent autophagy. NCOA4^ΔWB mice on normal (350-ppm) iron diet basally show microcytosis (low MCV) and hypochromia (low MCH), but NCOA4^ΔWB mice on iron-deficient diet did not show further alterations (supplemental Figure 5B). Tissue FTN H and FTN light chain (FTN L) RNA expression analyses did not show a significant difference among all groups of the experiment (supplemental Figure 5C).

To determine the role of intestinal ferritinophagy in systemic iron metabolism, intestine-specific NCOA4-KO (NCOA4^ΔIE) mice were generated (Figure 2A). NCOA4^ΔIE duodenum showed increased FTN H expression (Figure 2B), but unaltered hematology compared with NCOA4^F/F mice (supplemental Figure 6A). Phz-treated NCOA4^ΔIE mice did not show major hematological changes except a significant increase in MCV (supplemental Figure 6B), and no changes in hepatic hepcidin RNA expression, both basally and after Phz treatment was observed (supplemental Figure 6C). NCOA4^ΔIE mice revealed significant intestinal iron retention (supplemental Figure 6D), suggesting the possible role of NCOA4 in intestinal iron mobilization. However, disruption of intestinal NCOA4 did not alter systemic iron homeostasis in basal or anemic conditions. The intestinal response to increased systemic iron demand was similar during both deficiency and overload (primary and secondary). Intestinal HIF2α was activated, and intestinal iron absorption was increased.^1,2,11,13,18 Consistent with this, a genetic iron overload model (hepatic hepcidin disruption (HAMP^ΔLiv) demonstrated a significant increase in intestinal NCOA4 (Figure 2C).² To understand genetically whether intestinal ferritinophagy alters iron overload, we would have to cross the NCOA4^ΔIE and HAMP^ΔLiv mice, with possible confounding intestinal and hepatic disruption of both NCOA4 and hepcidin. Therefore, we generated an AV-mediated CRISPR/CAS9-based hepcidin KO mouse model, an efficient gene-silencing approach with broad tropism¹⁹ (Figure 2D-F; supplemental Figure 7A-B). In agreement with Figure 2C, duodenal NCOA4 mRNA expression was increased after hepcidin disruption by AV (Figure 2G). NCOA4^ΔIE or littermate controls were treated with control or CRISPR/CAS9-based hepcidin KO AV (Figure 2H). After 8 weeks, hepatic hepcidin RNA expression was equally suppressed in both AV-treated groups (supplemental Figure 7C). The induction observed in the hepatic FTN H protein levels of the AV-treated NCOA4^F/F mice was completely abolished in the corresponding NCOA4^ΔIE cohort (supplemental Figure 7D). Furthermore, assessment of liver, pancreas, serum iron, and transferrin saturation revealed that NCOA4^ΔIE mice are significantly protected from developing iron overload (Figure 2I-J). Hepcidin KO in NCOA4^ΔIE mice exhibited increased intestinal iron retention as evidenced by duodenal enterocyte iron and FTN protein expression analyses (Figure 2K-L). However, duodenal DMT1 and Fpn1 proteins were increased similarly after hepcidin disruption in NCOA4^F/F or NCOA4^ΔIE mice (supplemental Figure 7E). Serum transaminase levels (alanine aminotransferase and aspartate aminotransferase) were not altered, suggesting that iron overload–mediated hepatic damage has yet to set in (supplemental Figure 7F). These data suggest that hyperabsorption of iron can be attenuated by inhibition of intestinal NCOA4.

Our work suggests that enterocyte iron and FTN levels are critical determinants of the necessity for NCOA4 in intestinal iron absorption. In hemochromatosis, relative to iron deficiency, the increased iron flux led to high FTN turnover and enhanced requirement of ferritinophagy in intestinal iron absorption (Figure 2M). Therefore, genetic and pharmacological approaches to specifically target intestinal NCOA4 may be beneficial for managing iron overload disorders. Targeting intestine-specific ferritinophagy by bulk autophagy inhibitors²⁰ or intestine targeting of NCOA4 via gene-silencing methods²¹ carries enormous therapeutic potential, either as a single agent or combination therapy with other investigational agents such as DMT1 inhibitors or DMT1/transmembrane protease serine 6 antisense oligonucleotides.

The visual abstract and schematic diagram (Figure 2M) were created with BioRender.com.

This work was supported by National Institutes of Health, National Cancer Institute grants R01CA148828 and R01CA245546; National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) grants R01DK095201 (Y.M.S.) and R01DK124384 (J.D.M.); a Burroughs Wellcome Fund Career Award for Medical Scientists (J.D.M.); and the University of Michigan Comprehensive Cancer Center (UMCCC) Core Grant P30CA046592, the University of Michigan GI SPORE Molecular Pathology and Biosample Core (P50CA130810), and the Center for Gastrointestinal Research (DK034933).

Contribution: N.K.D. and Y.M.S. conceived and designed the study; N.K.D., C.J., A.S., A.J.S., N.S.-C., S.S., Z.Z., X.M., and S.P. developed the methodologies; N.K.D., C.J., A.S., A.J.S., N.S.-C., S.S., Z.Z., X.M., and S.P. acquired the data; N.K.D., C.J., L.R., J.D.M., and Y.M.S. analyzed and interpreted the data; J.D.M. provided the key reagents; N.K.D. and Y.M.S. supervised the study and wrote the manuscript; and all authors edited and provided inputs to the manuscript.

Conflict-of-interest disclosure: J.D.M. holds a patent pertaining to the autophagic control of iron metabolism. The remaining authors declare no competing financial interests.

Yatrik M. Shah, Department of Molecular and Integrative Physiology, University of Michigan, 1301 East Catherine St, Ann Arbor, MI 48109; e-mail: shahy@umich.edu.