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Erythropoietin mediates hepcidin expression in hepatocytes through EPOR signaling and regulation of C/EBPalpha - PubMed

  • ️Tue Jan 01 2008

Erythropoietin mediates hepcidin expression in hepatocytes through EPOR signaling and regulation of C/EBPalpha

Jorge P Pinto et al. Blood. 2008.

Abstract

Hepcidin is the principal iron regulatory hormone, controlling the systemic absorption and remobilization of iron from intracellular stores. Recent in vivo studies have shown that hepcidin is down-regulated by erythropoiesis, anemia, and hypoxia, which meets the need of iron input for erythrocyte production. Erythropoietin (EPO) is the primary signal that triggers erythropoiesis in anemic and hypoxic conditions. Therefore, a direct involvement of EPO in hepcidin regulation can be hypothesized. We report here the regulation of hepcidin expression by EPO, in a dose-dependent manner, in freshly isolated mouse hepatocytes and in the HepG2 human hepatocyte cell model. The effect is mediated through EPOR signaling, since hepcidin mRNA levels are restored by pretreatment with an EPOR-blocking antibody. The transcription factor C/EBPalpha showed a pattern of expression similar to hepcidin, at the mRNA and protein levels, following EPO and anti-EPOR treatments. Chromatin immunoprecipitation experiments showed a significant decrease of C/EBPalpha binding to the hepcidin promoter after EPO supplementation, suggesting the involvement of this transcription factor in the transcriptional response of hepcidin to EPO.

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Figures

Figure 1
Figure 1

EPO modulates hepcidin expression in a dose-dependent manner in HepG2 cells and in freshly isolated mouse hepatocytes. (A) qRT-PCR analysis of HAMP mRNA expression in HepG2 cells treated with rEPO or IL-6. Figure represents the average plus SD of 3 independent experiments; * denotes a statistically significant difference between treated samples and mock (P < .05; one-way ANOVA). (B) qRT-PCR analysis of HAMP mRNA expression in HepG2 cells treated with rEPO (1 or 2 U/mL) and/or with 0.1 to 10 μg/mL anti-EPOR. Bars on the right of each group correspond to HAMP mRNA levels after preabsorption of the anti-EPOR antibody with a specific peptide (+Block). * denotes a significant difference (P < .05) between EPO(+)/anti-EPOR(+)–treated samples and the EPO(+)/anti-EPOR(−) controls (one-way ANOVA) ** stands for a significant difference P < .05 level, between EPO(−)/anti-EPOR(+) or EPO(−)/anti-EPOR(+)/Block(+) and the EPO(−)/anti-EPOR(−) control (2-way ANOVA). Figure represents the average + SD of 3 independent experiments. (C) qRT-PCR analysis of Hamp1 mRNA expression in freshly isolated mouse hepatocytes treated with 0.01 to 2.0 U/mL rEPO (gray bars), and/or 0.1 to 5 μg anti-EPOR (white and black bars, respectively). Untreated HepG2 cells were used as control (hatched bar, left). Treatment with IL-6 was used to assess Hamp1 response to stimulation (gray bar, right). A total of 7 animals were used for each treatment; * denotes a significant difference (P < .05) between EPO(+)/anti-EPOR(−) or EPO(−)/anti-EPOR(+) or IL-6(+) and the nontreated control (one-way ANOVA). ** stands for a significant difference at the 95% confidence level between (EPO(+)/anti-EPOR(+) and the EPO(1 U)/anti-EPOR(−) control (one-way ANOVA).

Figure 2
Figure 2

EPO modulates CEBPA expression in a dose-dependent manner in liver-derived cells. qRT-PCR assessment of CEBPA (A) and Cebpa (B) mRNA expression in HepG2 cells and in mouse hepatocytes treated with rEPO (▒, left) and/or anti-EPOR (□ and ■, respectively) and IL-6 (▒, right). Untreated cells were used as control (▫, left). Figure represents the average + SD of 3 independent experiments for HepG2 cells. A total of 7 animals were used for each mouse hepatocyte treatment; * denotes a significant difference (P < .05) between EPO(+)/anti-EPOR(−) or EPO(−)/anti-EPOR(+) or EPO(−)/anti-EPOR(+)/Block(+) or IL6(+) and the EPO(−)/anti-EPOR(−) control (one-way ANOVA). ** stands for a significant difference (P < .05) between EPO(+)/anti-EPOR(+) or EPO(1 U)/anti-EPOR(+)/Block(+) and the EPO(1 U)/anti-EPOR(−) control (2-way ANOVA).

Figure 3
Figure 3

C/EBPα protein levels and binding to HAMP promoter are modulated by EPO in a dose-dependent manner. (A) Western blot analysis of C/EBPα levels in HepG2 cells after treatment with physiological and supraphysiological doses of rEPO, anti-EPOR antibody, or IL-6. β-Actin was used as a loading control (bottom panel). Numbers on the left of each panel correspond to the sizes of the MW marker bands. Figure shows a representative result of 3 experiments. Vertical lines have been inserted to indicate repositioned gel lanes. (B) Scanning densitometry of C/EBPα Western blot. Data are normalized for β-actin levels and are expressed as arbitrary units (AU). (C) qRT-PCR–ChIP analysis of the effect of rEPO and anti-EPOR on the binding of C/EBPα to the HAMP promoter. Figure shows the averages and SD of 2 experiments, each with 3 replicates. * signals a significant (P < .05) difference between EPO-treated samples and the nontreated control (one-way ANOVA); ** signals a significant (P < .05) difference between anti-EPOR–treated samples and the 2.5 U EPO–treated cells (one-way ANOVA).

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