Abstract
BMP9, a member of the TGFβ superfamily, is a homodimer that forms a signaling complex with two type I and two type II receptors. Signaling through high-affinity activin receptor-like kinase 1 (ALK1) in endothelial cells, circulating BMP9 acts as a vascular quiescence factor, maintaining endothelial homeostasis. BMP9 is also the most potent BMP for inducing osteogenic signaling in mesenchymal stem cells in vitro and promoting bone formation in vivo. This activity requires ALK1, the lower affinity type I receptor ALK2, and higher concentrations of BMP9. In adults, BMP9 is constitutively expressed in hepatocytes and secreted into the circulation. Optimum concentrations of BMP9 are essential to maintain the highly specific endothelial-protective function. Factors regulating BMP9 stability and activity remain unknown. Here, we showed by chromatography and a 1.9 Å crystal structure that stable BMP9 dimers could form either with (D-form) or without (M-form) an intermolecular disulfide bond. Although both forms of BMP9 were capable of binding to the prodomain and ALK1, the M-form demonstrated less sustained induction of Smad1/5/8 phosphorylation. The two forms could be converted into each other by changing the redox potential, and this redox switch caused a major alteration in BMP9 stability. The M-form displayed greater susceptibility to redox-dependent cleavage by proteases present in serum. This study provides a mechanism for the regulation of circulating BMP9 concentrations and may provide new rationales for approaches to modify BMP9 levels for therapeutic purposes.
Highlights
Bone morphogenetic protein 9 (BMP9) circulates at low concentrations maintaining endothelial integrity
We demonstrated that BMP9 stability and activity was regulated by redox potential as well as proteolysis
Because BMP9 binds to activin receptor-like kinase 1 (ALK1) extracellular domain (ECD) with high affinity and circulates as a complex with its prodomain, we examined whether the medium; D-form (M-form) could form a complex with ALK1 ECD and BMP9 prodomain using native PAGE followed by SDSPAGE (Fig. 3, A and B)
Summary
Bone morphogenetic protein 9 (BMP9) circulates at low concentrations maintaining endothelial integrity. BMP9 is a circulating vascular quiescence factor [1], one of only two BMP5 ligands that activate the endothelial ALK1/bone morphogenetic protein receptor type II (BMPR-II) pathway [2]. We hypothesized that there may be mechanisms in place to regulate circulating BMP9 at the optimum levels and activities for ALK1-specific signaling We addressed this question by characterizing recombinant BMP9 produced in mammalian cells and purified under native, non-denaturing conditions. The redox-dependent cleavage of the non-covalently linked BMP9 dimer would provide a controlled natural degradation pathway for BMP9 Such redox-dependent cleavage would suggest that there is a constant degradation of the BMP9 from the circulation, a fraction of BMP9 (covalently linked BMP9 dimer) remains stable and resistant to proteolysis, ensuring the constitutive activation of the endothelial ALK1/BMPRII pathway to maintain the homeostasis of the vascular endothelium
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