Erythropoietin's inhibiting impact on hepcidin expression occurs indirectly

Abstract

Under conditions of accelerated erythropoiesis, elevated erythropoietin (Epo) levels are associated with inhibition of hepcidin synthesis, a response that ultimately increases iron availability to meet the enhanced iron needs of erythropoietic cells. In the search for erythroid regulators of hepcidin, many candidates have been proposed, including Epo itself. We aimed to test whether direct interaction between Epo and the liver is required to regulate hepcidin. We found that prolonged administration of high doses of Epo in mice leads to great inhibition of liver hepcidin mRNA levels, and concomitant induction of the hepcidin inhibitor erythroferrone (ERFE). Epo treatment also resulted in liver iron mobilization, mediated by increased ferroportin activity and accompanied by reduced ferritin levels and increased TfR1 expression. The same inhibitory effect was observed in mice that do not express the homodimeric Epo receptor (EpoR) in liver cells because EpoR expression is restricted to erythroid cells. Similarly, liver signaling pathways involved in hepcidin regulation were not influenced by the presence or absence of hepatic EpoR. Moreover, Epo analogs, possibly interacting with the postulated heterodimeric β common EpoR, did not affect hepcidin expression. These findings were supported by the lack of inhibition on hepcidin found in hepatoma cells exposed to various concentrations of Epo for different periods of times. Our results demonstrate that hepcidin suppression does not require the direct binding of Epo to its liver receptors and rather suggest that the role of Epo is to stimulate the synthesis of the erythroid regulator ERFE in erythroblasts, which ultimately downregulates hepcidin.

Keywords: bone morphogenetic protein 6; erythropoietin receptor; ferroportin; iron; liver.

Fig. 1.

A: HepG2 cells were left untreated (Ctrl) or treated with the indicated concentrations of erythropoietin (Epo) for 24 h. Hepcidin (HAMP) mRNA levels were measured by quantitative RT-PCR. Samples were analyzed in triplicate and normalized to the housekeeping gene 18S RNA. Dots and solid black line represent single animals and the mean, respectively. For better visualization, the y-axis scale has been set up using a logarithmic scale. B–F: data were obtained from ICR CD1 and C57BL/6 wild-type mice (without differences in response between strains) 4 h after injection of saline (Ctrl; n = 3) or 1,200 IU/kg of Epo (Epo; n = 5); 24 h after injection of saline (Ctrl; n = 3) or 1,200 IU/kg Epo (Epo; n = 5). Mice were also treated for four consecutive days with saline (Ctrl; n = 4) or 2,000 IU/kg daily Epo (Epo; n = 4), or 12 μg/kg daily ARA290 (n = 3); and 10 consecutive days with saline (Ctrl; n = 4), 1,200 IU/kg daily Epo (Epo; n = 4), or 10 μg/kg daily carbamylated Epo (CEpo; n = 4). B: hepcidin (HAMP) expression was measured as described above. C: hematocrit was measured using standard methods. D: transferrin receptor (TfR1) and ferroportin (Fpn) levels were detected by immunoblotting in liver extracts. Each panel shows a representative blot obtained with extracts of two animals for each experimental group, as well as the densitometric quantitation of the analysis of the extracts from all of the mice. The values were normalized to β-actin and to controls. E: heavy (H; gray bars) and light (L; black bars) ferritin subunits were measured using an ELISA, and the values were normalized to the protein content. F: BMP6 expression was measured by RT-PCR. Data are presented as means ± SE. *P < 0.05.

Fig. 2.

Data were obtained from wild-type (Wt) controls, as well as from TgEpoR mice (TgEPOR) with EpoR expression restricted to hematopoietic tissue (n = 5) treated for 10 consecutive days with saline (Wt) or 1,200 IU/kg daily Epo. Hepcidin (HAMP) (A) and BMP6 (B) expression was measured by RT-PCR, as described in the legend to Fig 1. Dots and solid black line represent single animals and the mean, respectively. For better visualization, y-axis scale has been set up using a logarithmic scale. The levels of P-Jak2 and total Jak2 (C), P-Erk1/2 and total Erk1/2 (D), and TfR1 (E) were detected by Western blot analysis in liver extracts. Each panel shows a representative blot obtained with extracts of two animals for each experimental group and the densitometric quantitation of the analysis of the extracts from all of the mice. Data are presented as means ± SE. *P < 0.05; **P < 0.01.

Fig. 3.

A: data were obtained from ICR CD1 and C57BL/6 wild-type mice (without differences in response between strains). Mice were treated for four consecutive days with saline (Wt; n = 4) or 2,000 IU/kg daily Epo (Epo; n = 4), 4 h with 1,200 IU/kg of Epo (Epo; n = 5). Moreover, wild-type (Wt) controls and TgEpoR mice (TgEPOR) with EpoR expression restricted to hematopoietic tissue (n = 5) were treated for 10 consecutive days with saline or 1,200 IU/kg daily Epo. Erythroferrone (ERFE) expression was measured by RT-PCR. Samples were analyzed in triplicate and normalized to the housekeeping gene 18S RNA. Dots and solid black line represent single animals and the mean, respectively. For better visualization, the y-axis scale has been set up using a logarithmic scale. B: data were obtained from Wt mice treated for 10 consecutive days with saline (Ctrl; n = 4) or 10 μg/kg CEpo daily (CEpo; n = 4); mice treated for four consecutive days with a control peptide (Ctrl; n = 4); mice 24 h or 4 days after injection of 3 or 12 μg/kg daily of ARA290 (n = 3 for each group). Hepcidin (HAMP) expression was measured by RT-PCR. Samples were analyzed in triplicate and normalized to the housekeeping gene 18S RNA. Dots and solid black line represent single animals and the mean, respectively. For better visualization, y-axis scale has been set up using a logarithmic scale. Data are presented as means ± SE. *P < 0.05; **P < 0.01.