BMP2 and BMP6 prodomains are essential to generate heterodimers in liver endothelial cells and associate with ligands after cleavage and secretion

Liver endothelial cell (LEC)-derived bone morphogenetic proteins (BMP)2 and BMP6 coordinately regulate hepatocyte hepcidin production to control systemic iron homeostasis, but the mechanisms are uncertain. BMPs are homodimeric or heterodimeric proteins that are generated as precursors comprised of a non-signaling prodomain and an active ligand released by proteolytic cleavage. Although prodomains do not signal, they play pleiotropic roles in ligand generation and activity. Here, we used LEC- and hepatocyte-derived cell cultures to explore how BMP2 and BMP6 prodomains and proteolytic cleavage impact homodimer formation, heterodimer formation, and hepcidin regulation. We show that when co-expressed in LECs, BMP2 and BMP6 form heterodimers, which more potently induce hepcidin versus homodimers. Proteolytic cleavage of BMP2 precursors at two furin consensus sites is required to generate fully active BMP2/6 heterodimers, although cleavage at either site generates partially active heterodimers. Uncleavable BMP6 precursors fail to generate fully active BMP2/6 heterodimers and dominant negatively inhibit BMP2 homodimer formation. We also show that in LECs, cognate BMP2 and BMP6 prodomains are essential for homodimer and heterodimer formation. Finally, both prodomains remain noncovalently associated with BMP2/6 heterodimers after cleavage and secretion. BMP6 prodomains also remain associated with BMP6 homodimers, whereas the association of BMP2 prodomains with BMP2 homodimers is influenced by the presence of the linker region between the cleavage sites. Our findings suggest that BMP2/6 heterodimers are likely the primary endogenous ligand that regulates hepcidin and iron homeostasis, and both BMP2 and BMP6 prodomains are critical to functional BMP2/6 formation in LECs.