Identification of erythroferrone as an erythroid regulator of iron metabolism

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 Hbb(th3/+) mice with thalassemia intermedia, where it contributes to the suppression of hepcidin and the systemic iron overload characteristic of this disease.

Figure 1. Time-course of hepcidin mRNA and protein concentrations after phlebotomy (500 μL) or treatment with erythropoietin (EPO, 200 U)

(A) Hamp mRNA levels were reduced within 9 hours and maximally suppressed 12–15 hours after phlebotomy (solid line) or treatment with EPO (dashed line). (B) Serum hepcidin levels after phlebotomy and treatment with EPO reflect mRNA concentrations. Both graphs show means ± SEM. Log₂ hepcidin mRNA concentration relative to that of a housekeeping gene Hprt, −ΔCt (i.e., Ct Hprt – Ct Hamp) is shown as measured by qRT-PCR and serum hepcidin values were obtained by enzyme-linked immunosorbent assay. Each time-point was compared to the control mice (t=0) by two-tailed Student t-test (n=4 mice per point). ***p<0.001, **p<0.01, *p<0.05 for phlebotomy, ^###p<0.001, ^##p<0.01, ^#p<0.05 for EPO treatment.

Figure 2. Induction of ERFE-encoding Fam132b mRNA after phlebotomy (500 μL) or treatment with erythropoietin (EPO, 200 U)

Fam132b mRNA in the bone marrow (A) and the spleen (B) of 6 week-old C57BL/6 wild-type males is rapidly increased 4–9 hours after phlebotomy (solid line) or EPO treatment (dashed line). Means of −ΔCt (i.e., Ct Hprt – Ct Fam132b) ± SEM are shown as measured by qRT-PCR, and compared for each time-point to the t=0 control by two-tailed Student t-test (n=4). ***p<0.001, **p<0.01, *p<0.05 for phlebotomy, ^###p<0.001, ^##p<0.01, ^#p<0.05 for EPO treatment.

Figure 3. ERFE is produced in erythroblasts and induced by erythropoietic stimuli through the Jak2/Stat5 signaling pathway

(A) Tissue-specific expression of Fam132b mRNA. Fam132b mRNA expression was stimulated only in the bone marrow and in the spleen of 6 weeks-old C57BL/6 mice 15 hours after 200 U EPO treatment (n=6). (B) Fam132b mRNA expression across stages of erythroblast development from proerythroblasts (Pro-E) through basophilic (baso), polychromatic (poly) to orthochromatic (ortho) erythroblasts. Fam132b mRNA was measured by qRT-PCR in erythroid cells isolated from the bone marrows of three controls and three phlebotomized (15 h) 6 week-old C57BL/6 mice. Cells were sorted by size and anti-Ter119 and anti-CD71 staining. (C) Fam132b induction by EPO is dependent on Stat5. Stimulation of Fam132b mRNA expression in erythroblasts by EPO treatment (15 h, 10 U/mL) was inhibited by Stat5 inhibitors pimozide (10 μM) or N′-((4-Oxo-4H-chromen-3-yl)methylene)nicotinohydrazide (200 μM). Erythroferrone (Fam132b) mRNA levels were measured by qRT-PCR. Values shown are means ± SEM of −ΔCt (i.e., Ct Hprt or Rpl4 – Ct Fam132b). Means of −ΔCt values were compared between control and erythropoietic stimulation by two-tailed Student t-test (A, B). Bonferroni correction was applied to the multiple comparisons represented in panel A. (C) Expression ratio ± SEM of Fam132b transcripts in EPO-treated cells relative to controls and normalized to the gene Gypa mRNA was calculated using REST. Normalization by glycophorin A allowed the normalization of Fam132b mRNA expression per red cell. Results of 3 independent experiments with 15 hour treatments in duplicate are shown. Statistical significance was determined using randomization tests. ***p<0.001, **p<0.01

Figure 4. At 6 weeks of age, ERFE-deficient mice have lower hemoglobin and mean corpuscular hemoglobin (MCH) levels than wild type mice

Hemoglobin and MCH were compared between Fam132b^+/+ and Fam132b^−/− littermates at the ages of 3, 6, 12, and 24 weeks. Values shown are means of hemoglobin or MCH ± SEM. Means were compared between genotypes by two-tailed Student t-test (n=8 to 16 mice per time point and genotype). ***p<0.001, **p<0.01, *p<0.05.

Figure 5. Fam132b-deficient mice do not suppress hepcidin in response to phlebotomy and show delayed recovery from anemia

(A) After phlebotomy, hepatic Hamp mRNA expression was measured in Fam132b^+/+ (dashed line), Fam132b^+/− (dotted line) and Fam132b^−/− (solid line) littermates (n=6 to 14 per genotype and time-point). Hepcidin is suppressed after hemorrhage in wild-type mice but not in Fam132b-deficient mice, with Fam132b^+/− presenting an intermediate response between the wild-types and knockouts. (B) After phlebotomy, both Fam132b^+/+ (dashed line) and Fam132b^+/− (dotted line) mice increase Fam132b mRNA expression in the bone marrow, although the maximal levels were about twice as high in Fam132b^+/+ compared to Fam132b^+/−. (C and D) Phlebotomized Fam132b-deficient mice (solid line) compared to wild-type mice (dashed line) showed delayed recovery of hemoglobin and lower mean corpuscular hemoglobin (MCH). Hepcidin (Hamp) (A) and Fam132b (B) mRNA levels were measured by qRT-PCR and shown as −ΔCt (i.e., Ct Hprt – Ct Hamp or Fam132b). For each genotype, means of −ΔCt values were compared at each time-point to the respective control mice t=0 by two-tailed Student t-test. Hematological parameters (C, D) were compared for each measurement between WT (n=17) and KO (n=15) by Student t-test. All graphs show means ± SEM. In the absence of gender differences, the genders were combined for each parameter. ***p<0.001, **p<0.01, *p<0.05

Figure 6. ERFE acts directly on the liver to suppress hepcidin

(A, B, C) Six C57BL/6 male mice were treated intraperitoneally with either mouse recombinant ERFE (2 μg/g) or saline and analyzed 15 hours later. Hepatic hepcidin mRNA (A) and serum hepcidin (B) levels were significantly suppressed by ERFE treatment. (C, D, E) Seven week-old C57BL/6 males were transduced with a lentivirus encoding GFP or mouse ERFE (n=8 per group) and analyzed after 3 weeks. (C) Fam132b mRNA expression was not detectable (ND) in the liver of control mice, and was moderately increased in the liver of mice transduced with the ERFE lentivirus although only 2-fold higher than the baseline bone marrow levels. However, this increase was sufficient to significantly reduce hepatic hepcidin mRNA (D) and serum hepcidin (E) levels. (F) Recombinant mouse ERFE is secreted by HEK293T cells infected with the ERFE-lentivirus, as detected by Western blot with an anti-FLAG antibody. (G) Treatment of mouse primary hepatocytes with supernatants (50% v/v) from HEK293T control cells or HEK293T cells overexpressing ERFE indicated that ERFE acts directly on the liver to suppress hepcidin mRNA expression. Hepcidin (Hamp), serum amyloid 1 (Saa1) and ERFE (Fam132b) mRNA levels were measured by qRT-PCR and are shown as −ΔCt (i.e., Ct Hprt – Ct Hamp, Saa1 or Fam132b). Graphs show means± SEM. Mean values were compared between treated mice and control mice or between ERFE-treated to control samples by two-tailed Student t-test. ***p<0.001, **p<0.01, *p<0.05

Figure 7. In a mouse model of β-thalassemia intermedia, erythroferrone is markedly increased, suppresses hepcidin and contributes to iron overload

Six week-old Hbb^Th3/+ mice had (A) lower hemoglobin and (B) greatly increased serum Epo concentrations compared to their wild-type littermate mice (n=7 mice per genotype). Fam132b mRNA expression (C) was highly increased in the bone marrow and the spleen of Hbb^Th3/+ mice compared to wild-type controls. (D and E) Comparing six week-old littermates WT (n=12), Hbb^Th3/+ (n=12) and Fam132b^−/−Hbb^Th3/+ (n=18), ablation of ERFE reversed hepcidin suppression (D) and significantly decreased hepatic iron overload (E). Values shown are means ± SEM. Statistical comparisons were performed by two-tailed Student t-test for normally distributed variables and Mann-Whitney rank sum test otherwise. ***p<0.001, **p<0.01, *p<0.05.

Figure 8. Proposed role of the erythroid factor erythroferrone (ERFE)

After erythropoietic stimulation, differentiating erythroblasts in the bone marrow and spleen rapidly increase ERFE production in an EPO-Stat5 dependent manner. ERFE is secreted into the circulation and acts directly on the liver to repress hepcidin. ERFE-mediated hepcidin suppression in turn increases iron availability for new red blood cells synthesis.