Erythroferrone lowers hepcidin by sequestering BMP2/6 heterodimer from binding to the BMP type I receptor ALK3

Erythroferrone (ERFE) is critical for hepcidin suppression by erythropoietic drive to ensure adequate iron supply. However, excessive hepcidin suppression contributes to iron overload in β-thalassemia. ERFE suppresses hepcidin by interfering with bone morphogenetic protein (BMP)-SMAD signaling. To determine whether BMP receptors are required for hepcidin suppression by ERFE, we tested the impact of siRNA knockdown or hepatocyte knockout of BMP receptors on ERFE or erythropoietin (EPO)-mediated hepcidin suppression without or with BMP ligands. Knockdown or knockout of the BMP type I receptor ALK3 inhibited ERFE- and EPO-mediated hepcidin suppression, both in vitro and in mice. ALK3 knockdown also inhibited hepcidin induction by BMP2, but not BMP6; however, ERFE inhibited only BMP6, but not BMP2. To understand these differences, we tested impact of type I receptor knockdown and ERFE on hepcidin induction by the heterodimeric BMP2/6 ligand. BMP2/6 preferentially utilized ALK3 to stimulate hepcidin and was inhibited by ERFE. Moreover, ERFE bound directly to BMP2/6 and BMP6, but not BMP2 or ALK3, in pull-down assays. These data support a model whereby BMP2 and BMP6 function as a heterodimeric ligand in vivo, preferentially utilize ALK3 to regulate hepcidin, and are inhibited by direct binding to ERFE in the context of erythropoietic drive.