The hepatokine FGL1 regulates hepcidin and iron metabolism during anemia in mice by antagonizing BMP signaling

Abstract

As a functional component of erythrocyte hemoglobin, iron is essential for oxygen delivery to all tissues in the body. The liver-derived peptide hepcidin is the master regulator of iron homeostasis. During anemia, the erythroid hormone erythroferrone regulates hepcidin synthesis to ensure the adequate supply of iron to the bone marrow for red blood cell production. However, mounting evidence suggested that another factor may exert a similar function. We identified the hepatokine fibrinogen-like 1 (FGL1) as a previously undescribed suppressor of hepcidin that is induced in the liver in response to hypoxia during the recovery from anemia, and in thalassemic mice. We demonstrated that FGL1 is a potent suppressor of hepcidin in vitro and in vivo. Deletion of Fgl1 in mice results in higher hepcidin levels at baseline and after bleeding. FGL1 exerts its activity by directly binding to bone morphogenetic protein 6 (BMP6), thereby inhibiting the canonical BMP-SMAD signaling cascade that controls hepcidin transcription.

Graphical abstract

Figure 1.

Recovery from hemorrhage-induced anemia in WT and Erfe^−/− mice. Hemoglobin (A) and hematocrit (B) levels in 7- to 9-week-old WT (blue) and Erfe^−/− (red) male mice 0, 1, 2, 3, 4, 5, and 6 days after phlebotomy (500 μL). (C) Time course of serum ERFE concentration in phlebotomized WT mice. mRNA expression of Hamp (D), Id1 (E), and Smad7 (F) in the liver of phlebotomized WT and Erfe^−/− mice. Data shown are mean ± standard error of the mean (SEM) and were compared for each time point with values for control mice at t = 0 (n = 5-8) for each genotype by 2-way (A,B,D,E,F) or 1-way (C) analysis of variance (ANOVA) and corrected for multiple comparisons by the Holm-Šidák method. ∗∗∗∗P < .0001; ∗∗∗P < .001; ∗∗P < .01; ∗P < .05.

Figure 2.

Fgl1 mRNA expression is induced in the mouse liver during anemia. Representative heat maps of the transcripts induced in the liver (A) and the bone marrow (B) at 24 and 48 hours compared to control mice at t = 0. Time course of Fgl1 mRNA expression in the liver (C) and the bone marrow (D) 1 to 6 days after phlebotomy in WT and Erfe^−/− mice (n = 5-8). WT and Erfe-/- as shown in Figure 1. Data shown are mean ± standard error of the mean (SEM) and were compared for each time point with values for control mice at t = 0 by 2-way ANOVA and were corrected for multiple comparisons by the Holm-Šidák method (C-D). ∗∗∗∗P < .0001; ∗∗∗P < .001; ∗P < .05.

Figure 3.

Fgl1 mRNA expression is induced by hypoxia.Hamp (A) and Fgl1 (B) mRNA expression in the liver, and Erfe and Fgl1 (C) mRNA expression in the bone marrow of 7-week-old mice at t = 0 to 20 hours after a single intraperitoneal injection of EPO (200 U; n = 5). (D) Fgl1 mRNA expression in the liver of 8-week-old WT, Th3/+, and Erfe^−/−; Th3/+ mice (n = 7-9). (E) Fold change of Fgl1 mRNA expression in mouse primary hepatocytes in serum-free media and incubated for 15 hours in low-oxygen condition (2%) or in presence of prolyl-hydroxylases inhibitor DMOG compared with untreated cells. (F) Fgl1 mRNA expression in the liver of Vhl-deficient mice (Albumin-Cre/VHL^flox/flox), and (G) mice treated with prolyl-hydroxylase inhibitor vadadustat. Data shown are mean ± SEM and were compared for each time point with values for control mice at t = 0 (A-C) or to WT mice (D) by 1-way ANOVA and were corrected for multiple comparisons by the Holm-Šidák method or with WT or vehicle-treated mice by the Student t test (F-G). Data shown for the experiment in primary hepatocytes are means of 3 independent experiments and were compared with control cells by the Student t test (E). ∗∗∗∗P < .0001; ∗∗∗P < .001; ∗∗P < .01; ∗P < .05.

Figure 4.

FGL1 is a suppressor of hepcidin in vitro and in vivo.HAMP mRNA expression in Hep3B and HepG2 cells cultured in serum-free medium in response to BMP6 (25 ng/mL, 6 hours) (A) or BMP6 + recombinant mouse FGL1 (1-10 μg/mL) (B). (C) ID1 expression in hepatoma cell lines treated with BMP6 and FGL1. Hepatic Hamp mRNA expression (D), serum hepcidin concentration (E), and liver Id1 (F) and Smad7 (G) mRNA expression, serum (H) and liver iron (I) concentration in mice treated for 6 hours with saline, Fc, or recombinant mouse FGL1 (10 mg/kg; n = 5). Data shown are mean ± SEM of 3 independent experiments (A-C) and were compared with untreated cells using the Student t test (A) or 1-way ANOVA (B-I) and corrected for multiple comparisons using the Holm-Šidák method. ∗∗∗∗P < .0001; ∗∗∗P < .001; ∗∗P < .01; ∗P < .05.

Figure 5.

Fgl1^−/− mice exhibit a blunted response to phlebotomy. (A) Fgl1 mRNA expression in the liver of male WT mice 36 hours after bleeding compared to control mice. Red blood cell count (RBC) (B) and hemoglobin (Hb) (C); Erfe mRNA expression in the bone marrow (D) and the spleen (E); and liver Hamp (F), Id1 (G), and Smad7 (H) mRNA expression in WT and Fgl1^−/− mice at t = 0 or 36 hours after phlebotomy. Data shown are mean ± SEM (n = 5-8) and were compared between control and phlebotomized mice using the Student t test (A) or between WT and Fgl1^−/− mice by 2-way ANOVA and corrected for multiple comparisons using the Holm-Šidák method (B-H). ∗∗∗∗P < .0001; ∗∗∗P < .001; ∗∗P < .01; ∗P < .05.

Figure 6.

The globular domain of FGL1 mediates hepcidin suppression. (A) AlphaFold structure prediction of mouse FGL1. (B) Schematic representation of the recombinant mouse FGL1 protein constructs. HAMP (C) and ID1 (D) expression in Hep3B cells and mouse primary hepatocytes treated for 6 hours with BMP6 and either Fc, FL, or the N-terminal (Nter) or globular (glob) domains of FGL1. Data shown are mean ± SEM of 3 independent experiments (C-D) and were compared with Fc-treated cells using 1-way ANOVA and corrected for multiple comparisons using the Holm-Šidák method. ∗∗∗∗P < .0001; ∗∗∗P < .001; ∗∗P < .01; ∗P < .05.

Figure 7.

FGL1 is a BMP antagonist. Relative expression of Hamp (A), Id1 (B), Smad7 (C) mRNA expression in mouse primary hepatocytes treated with BMP ligands (10 ng/mL) and human Fc immunoglobulin G2 (IgG2; control [CTRL], 10 μg/mL) or Fc-FGL1 (mouse FGL1, 10 μg/mL) for 15 hours relative to untreated cells in serum-free conditions. Data shown are mean ± SEM of 3 independent experiments and were compared for each BMP between FGL1-treated cells and Fc-treated cells using the Student t test. ∗∗∗∗P < .0001. (D) Western blotting of Hep3B cells treated for 6 hours with BMP6 (10 ng/mL), ERFE (1 μg/mL), or FGL1 (10 μg/mL) for P-SMAD5, SMAD5, and GAPDH. (E) Western blotting of pull-down assay of BMP6 and FGL1 (FL, glob, and Nter) or human Fc IgG2.