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Identification of erythroferrone as an erythroid regulator of iron metabolism

Nature Genetics volume 46, pages 678–684 (2014)Cite this article

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An Author Correction to this article was published on 27 February 2020

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Abstract

Recovery from blood loss requires a greatly enhanced supply of iron to support expanded erythropoiesis. After hemorrhage, suppression of the iron-regulatory hormone hepcidin allows increased iron absorption and mobilization from stores. We identified a new hormone, erythroferrone (ERFE), that mediates hepcidin suppression during stress erythropoiesis. ERFE is produced by erythroblasts in response to erythropoietin. ERFE-deficient mice fail to suppress hepcidin rapidly after hemorrhage and exhibit a delay in recovery from blood loss. ERFE expression is greatly increased in Hbbth3/+ mice with thalassemia intermedia, where it contributes to the suppression of hepcidin and the systemic iron overload characteristic of this disease.

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Figure 1: Time course of hepcidin mRNA and protein concentrations after phlebotomy (500 μl) or treatment with EPO (200 U).
Figure 2: Induction of ERFE-encoding Fam132b mRNA levels after phlebotomy (500 μl) or treatment with EPO (200 U).
Figure 3: ERFE is produced in erythroblasts and induced by erythropoietic stimuli through the JAK2-STAT5 signaling pathway.
Figure 4: ERFE-deficient mice have lower hemoglobin and MCH levels than wild-type mice at 6 weeks of age.
Figure 5: Fam132b-deficient mice do not suppress hepcidin in response to phlebotomy and show delayed recovery from anemia.
Figure 6: ERFE acts directly on the liver to suppress hepcidin.
Figure 7: ERFE expression is markedly increased, suppresses hepcidin and contributes to iron overload in a mouse model of β-thalassemia intermedia.
Figure 8: Proposed role of the erythroid factor erythroferrone (ERFE).

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  • 13 June 2014

    In the version of this article initially published online, the key in Figure 5b was incorrect. The dotted line with open squares represents data from Fam132b+/- mice. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

The authors thank V. Gabayan for her invaluable work with mouse breeding. Flow cytometry was performed in the University of California, Los Angeles (UCLA) Jonsson Comprehensive Cancer Center (JCCC) and the Center for AIDS Research Flow Cytometry Core Facility, which is supported by US National Institutes of Health (NIH) awards CA-16042 and AI-28697 and by the JCCC, the UCLA AIDS Institute and the David Geffen School of Medicine at UCLA. The UCLA Vector Core is supported by the JCCC from US NIH grant P30-CA-016042 and the CURE: Digestive Disease Research Core Center from US NIH grant P30-DK-041301. This research was supported by US NIH grant R01-DK-065029 to T.G. and grants R01-DK-090554 and 5R01-DK-095112 to S.R. and by the American Society of Hematology scholar award to L.K.

Author information

Authors and Affiliations

  1. Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA

    Léon Kautz, Grace Jung, Erika V Valore, Elizabeta Nemeth & Tomas Ganz

  2. Department of Pediatrics, Division of Hematology-Oncology, Weill Cornell Medical College, New York, New York, USA

    Stefano Rivella

  3. Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, New York, USA

    Stefano Rivella

  4. Department of Pathology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA

    Tomas Ganz

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  1. Léon Kautz
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Contributions

L.K. designed and performed the experiments, analyzed the data and wrote the manuscript. G.J. assisted with experiments. S.R. provided Hbbth3/+ mouse samples for preliminary experiments and E.V.V. prepared a key reagent. E.N. and T.G. conceived the project and wrote the manuscript.

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Correspondence to Tomas Ganz.

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Competing interests

E.N. and T.G. are shareholders and scientific advisors of Intrinsic LifeSciences and Merganser Biotech and are consultants for Xenon Pharmaceuticals. S.R. receives laboratory support from Isis, Bayer and Merganser Biotech and is a shareholder of Merganser Biotech. The remaining authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Serum iron levels after erythropoietic stimulation.

(a,b) Changes in serum iron levels between paired samples from the initial phlebotomy and terminal analysis (blue line) (a) and serum iron concentrations after EPO injection (red line) (b) in 6-week-old C57BL/6 mice. No significant changes in serum iron concentration were observed. Values shown are means ± s.d. For each time point, means of serum iron changes were compared to the initial value (zero) (a), or serum iron concentrations were compared to untreated mice at t = 0 (b) by two-tailed Student's t test (n = 4 mice per group).

Supplementary Figure 2 Smad5 phosphorylation and Id1 mRNA expression in response to erythropoietic stimulation.

(a) Smad5 phosphorylation was compared by protein blotting in the livers of wild-type Sv129/C57BL/6 mice at 0 and 15 h after phlebotomy. (b) Densitometric ratio of phosphorylated Smad5 to total Smad5 for phlebotomized mice. Means ± s.e.m. are shown. (c) Smad5 phosphorylation was compared by protein blotting in the livers of wild-type C57BL/6 mice at 0 and 15 h after EPO injection. (d) Densitometric ratio of phosphorylated Smad5 to total Smad5 for the EPO-treated mice. Means ± s.e.m. are shown. (e) Inhibitor of DNA binding 1 (Id1) mRNA was measured in 6-week-old C57BL/6 wild-type males after phlebotomy (blue line) or EPO injection (red line) and was only slightly decreased within 9 h of phlebotomy but not significantly after EPO injection. Id1 mRNA levels were measured by qRT-PCR. Values shown are the means ± s.e.m. of –ΔCt values (i.e., CtHprt – CtId1) and were compared for each time point to control mice by two-tailed Student's t test (n = 4). *P < 0.05.

Supplementary Figure 3 Hepcidin suppression by increased erythropoietic activity is independent of the effect of the BMP pathway.

(a,b) Hepcidin (Hamp) mRNA response to bleeding in 6-week-old male Tfr2Y245X mice on a standard diet with 336 ppm iron (Tfr2 ko SD; red line) or an iron-deficient diet containing 4 ppm iron for 2 weeks after weaning (Tfr2 ko ID; blue line), Hjv-deficient mice (Hjv ko; green line), 7-week-old wild-type C57BL/6 mice treated with 50 mg of iron-dextran (WT+Fe; black line) and 6-week-old wild-type C57BL/6 mice (also shown in Fig. 1) as a reference (dashed black line). (a) Hamp mRNA levels are shown on a logarithmic scale as –ΔCt values (i.e., Ct Hprt – Ct Hamp) measured by qRT-PCR. (b) Relative Hamp transcript abundance was calculated as 2–ΔCt(Hprt –Hamp) and represented on a linear scale. (c) Liver iron content and (d) Fam132b mRNA levels in the bone marrow were measured in the same groups of mice. Values shown are means ± s.e.m. and were compared for each time point to control mice at t = 0 by two-tailed Student's t test (n = 4 mice per group and time point). ***P < 0.001, **P < 0.01, *P < 0.05.

Supplementary Figure 4 Serum EPO levels after erythropoietic stimulation.

Serum EPO levels were measured in 6-week-old wild-type C57BL/6 males after phlebotomy and in control mice. EPO levels are rapidly increased within 4 h and start to decrease after 15 h. Values shown are means ± s.e.m. Mean serum EPO values were compared for each time point to those for control mice at t = 0 by two-tailed Student's t test (n = 4 mice per group and time point). ***P < 0.001, *P < 0.05.

Supplementary Figure 5 Erythroferrone is induced by EPO in human erythroblasts.

(a,b) FAM132B mRNA expression was induced by between 16- and 40-fold in human fetal erythroblasts (a) and by 16-fold in erythroblasts from human adult bone marrow (b) 15 h after treatment with EPO (10 U/ml). Cells from three different donors were analyzed individually. Mean values ± s.e.m. are shown from six (donor 1), three (donor 2) or two (bone marrow) replicates where –ΔCt values (i.e., Ct HPRT – Ct FAM132B) were compared between untreated and treated cells by two-tailed Student's t test. ***P < 0.001.

Supplementary Figure 6 Erythroferrone Fam132b mRNA expression is not influenced by the hypoxia pathway.

Vascular endothelial growth factor α (Vegfa) and Fam132b mRNA levels were compared in mouse bone marrow 15 h after treatment with 10 U/ml EPO or 1 mM DMOG (dimethyloxalylglycine, N-(methoxyoxoacetyl)-glycine methyl ester). Vegfa mRNA expression (a marker of hypoxia) was induced 15 h after treatment with DMOG, while Fam132b mRNA levels remained unchanged. EPO treatment is shown as a positive control to demonstrate cell responsiveness. Mean values ± s.e.m. of –ΔCt values (i.e., Ct Gypa – Ct Vegfa or Fam132b) were compared between control and EPO- or DMOG-treated cells by two-tailed Student's t test. Results are shown from three independent experiments, each performed in triplicate. ***P < 0.001, **P < 0.01.

Supplementary Figure 7 Erythroferrone FAM132B mRNA expression is not influenced by systemic inflammation.

Liver Hamp, Il6 and Tnfa mRNA levels and bone marrow Fam132b mRNA levels in 6-week-old C57BL/6 mice (n = 3 mice per group) were compared between mice injected with saline or with LPS (Sigma, 055:B5; 1 μg/g body weight) 4 h after treatment. Hepcidin, Il6 and Tnfa expression was induced 4 h after LPS injection, while bone marrow Fam132b levels remained unchanged. Mean values ± s.e.m. of –ΔCt values (i.e., Ct Hprt – Ct Hamp, Il6, Tnfa or Fam132b) were compared between saline- and LPS-treated mice by Student's t test. ***P < 0.001, **P < 0.01.

Supplementary Figure 8 Six-week-old ERFE-deficient mice have normal iron stores and hepcidin expression but have reduced hemoglobin levels compared to wild-type mice.

Hemoglobin levels (a), liver iron content (b) and liver hepcidin (Hamp) mRNA levels (c) were measured in 6-week-old Fam132b+/+, Fam132b+/– and Fam132b–/– littermates. Spleen iron levels (d) were compared between Fam132b+/+ and Fam132b–/– mice. Graphs show means ± s.e.m. Because no differences between the sexes were observed, the sexes were combined for each parameter. All comparisons were carried out by two-tailed Student's t test (n = 11–14). ***P < 0.001, *P < 0.05.

Supplementary Figure 9 ERFE-deficient mice have normal mean corpuscular volume (MCV) and serum iron concentrations.

MCV (a) and serum iron concentrations (b) were compared at various ages in Fam132b–/– mice (red line) and wild-type mice (black line). Graphs show mean values ± s.e.m., with n = 8–16 mice per time point and genotype. No statistically significant difference was observed between wild-type and knockout mice by Student's t test.

Supplementary Figure 10 ERFE-deficient mice have normal hemoglobin, iron stores and hepcidin at 3 and 24 weeks of age.

Hemoglobin levels (a,d), liver iron content (b,e) and liver hepcidin (Hamp) mRNA levels (c,f) were measured in 3-week-old (a–c) and 6-month-old (d–f) Fam132b+/+, Fam132b+/– and Fam132b–/– littermates. No significant differences were observed between genotypes. Values shown for hepcidin expression are means ± s.e.m. of –ΔCt values (i.e., Ct Hprt – Ct Hamp). Mean values for hemoglobin levels, liver iron content and hepcidin levels were compared between each genotype by two-tailed Student's t test. In the absence of differences between the sexes at 3 weeks of age, data for both sexes were combined for each parameter (n = 9–12). A sex-separated study was performed at 6 months of age, as females have higher liver stores than males (n = 3–11).

Supplementary Figure 11 Serum iron concentrations in ERFE-deficient mice in response to phlebotomy.

Changes in serum iron concentrations from the initial phlebotomy levels to the end time point were compared for each mouse in Fam132b+/+ (black line) and Fam132b–/– (red line) littermates (n = 8–14 per genotype and time point). Serum iron levels were significantly decreased at all time points in Fam132b–/– mice but not in Fam132b+/+ mice, confirming that the lack of hepcidin suppression after phlebotomy leads to impaired iron regulation. Values shown are means ± s.d. Means of serum iron values were compared for each time point to the values in control mice by two-tailed Student's t test. ***P < 0.001, **P < 0.01, *P < 0.05.

Supplementary Figure 12 ERFE is not required for erythroblast differentiation during stress erythropoiesis.

Bone marrow erythroblasts from Fam132b+/+ and Fam132b–/– mice were analyzed by flow cytometry in control mice and mice 72 h after phlebotomy (two mice per group are shown). No significant difference in erythroblast differentiation was observed between Fam132b+/+ and Fam132b–/– mice. Flow cytometry data were analyzed using FCS express software (DeNovo software).

Supplementary Figure 13 ERFE-deficient mice show lower MCV and hematocrit levels during recovery from anemia.

Eight-week-old phlebotomized Fam132b–/– mice (solid line) had lower MCV (a) and hematocrit (b) levels compared to wild-type mice (dashed line). Hematological parameters (a,b) were compared for each measurement between wild-type and knockout mice by two-tailed Student's t test (n = 15–17). Graphs show means ± s.e.m. In the absence of differences between the sexes, data for both sexes were combined for each parameter. **P < 0.01, *P < 0.05 (see also Fig. 4).

Supplementary Figure 14 Saa1 and Id1 expression in ERFE-treated mice.

Liver Saa1 and Id1 mRNA expression (a,c) was measured in mice treated with recombinant ERFE (n = 6) or saline (n = 6) for 15 h. Liver Saa1 and Id1 mRNA levels (b,d) were measured in mice expressing ERFE lentivirus (n = 8) or control lentivirus (n = 7) for 3 weeks. Mean values ± s.e.m. of –ΔCt values (i.e., Ct Hprt – Ct Id1 or Saa1) were compared between control and ERFE-treated mice by two-tailed Student's t test.

Supplementary Figure 15 Ablation of ERFE in thalassemic mice decreases serum iron concentrations, erythrocyte mean corpuscular volume and mean corpuscular hemoglobin concentrations.

Serum iron (a), hemoglobin (b), MCV (c) and MCH (d) levels were determined in wild-type (n = 12), Hbbth3/+ (n = 12) and Fam132b–/–; Hbbth3/+ (n = 18) littermate mice at 6 weeks of age. Consistent with decreased iron overload, lower serum iron, MCV and MCH levels were observed in Fam132b–/–; Hbbth3/+ mice compared to their Hbbth3/+ counterparts. Hemoglobin concentrations were similar in Fam132b–/–; Hbbth3/+ and Hbbth3/+ mice. Mean values ± s.e.m. for serum iron, hemoglobin, MCV and MCH levels were compared for each genotype by two-tailed Student's t test.

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Mouse Fam132b promoter analysis 3k. (XLSX 91 kb)

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Kautz, L., Jung, G., Valore, E. et al. Identification of erythroferrone as an erythroid regulator of iron metabolism. Nat Genet 46, 678–684 (2014). https://doi.org/10.1038/ng.2996

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