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, SMAD5 or SMAD1 but not SMAD8, knockdown inhibited hepcidin (HAMP) messenger RNA (mRNA) expression. Hepatocyte-specific double-knockout Smad1(fl/fl); Smad5(fl/fl); Cre(+) mice exhibited similar to 90% transferrin saturation and massive liver iron overload, whereas Smad1(fl/fl); Smad5(fl/wt); Cre(+) mice or Smad1(fl/wt); Smad5(fl/fl); Cre(+) female mice with 1 functional Smad5 or Smad1 allele had modestly increased serum and liver iron, and single-knockout Smad5(fl/fl); Cre(+) or Smad1(fl/fl); Cre(+) mice had minimal to no iron loading, suggesting a gene dosage effect. Hamp 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 Hamp at 8 weeks and failed to induce Hamp in response to Bmp6 in primary hepatocyte cultures. Epoetin alfa (EPO) robustly induced bone marrow erythroferrone (Fam132b) mRNA in control and Smad1(fl/fl); Smad5(fl/fl); Cre(+) mice but suppressed hepcidin only in control mice. Likewise, erythroferrone failed to decrease Hamp mRNA in Smad1(fl/fl); Smad5(fl/fl); Cre(+) primary hepatocytes and SMAD1/SMAD5 knockdown Hep3B cells. EPO and erythroferrone reduced liver Smad1/5 phosphorylation in parallel with Hamp mRNA in control mice and Hep3B cells. Thus, Smad1 and Smad5 have overlapping functions to govern hepcidin transcription. Moreover, erythropoietin and erythroferrone target Smad1/5 signaling and require Smad1/5 to suppress hepcidin expression.