Smad1/5 is required for erythropoietin-mediated suppression of hepcidin in mice.

Anemia suppresses liver hepcidin expression to supply adequate iron for erythropoiesis. Erythroferrone mediates hepcidin suppression by anemia, but its mechanism of action remains uncertain. The bone morphogenetic protein (BMP)-SMAD signaling pathway has a central role in hepcidin transcriptional regulation. Here, we explored the contribution of individual receptor-activated SMADs in hepcidin regulation and their involvement in erythroferrone suppression of hepcidin. In Hep3B cells, or but not knockdown inhibited hepcidin () messenger RNA (mRNA) expression. Hepatocyte-specific double-knockout mice exhibited ∼90% transferrin saturation and massive liver iron overload, whereas mice or female mice with 1 functional or allele had modestly increased serum and liver iron, and single-knockout or mice had minimal to no iron loading, suggesting a gene dosage effect. mRNA was reduced in all Cre mouse livers at 12 days and in all Cre primary hepatocytes. However, only double-knockout mice continued to exhibit low liver at 8 weeks and failed to induce in response to Bmp6 in primary hepatocyte cultures. Epoetin alfa (EPO) robustly induced bone marrow erythroferrone () mRNA in control and mice but suppressed hepcidin only in control mice. Likewise, erythroferrone failed to decrease mRNA in primary hepatocytes and / knockdown Hep3B cells. EPO and erythroferrone reduced liver Smad1/5 phosphorylation in parallel with mRNA in control mice and Hep3B cells. Thus, and have overlapping functions to govern hepcidin transcription. Moreover, erythropoietin and erythroferrone target Smad1/5 signaling and require Smad1/5 to suppress hepcidin expression.