Calcium ion-induced formation of β-sheet/-turn structure leading to alteration of osteogenic activity of bone morphogenetic protein-2

Preserving bioactivity of bone morphogenetic protein 2 (BMP-2) still remains a challenge in protein-based therapy. It is not known how Ca(2+) released from extracellular matrix or existing in physiological environment influences bioactivity in situ till now. Here, effects of extracellular Ca(2+) on conformation and osteogenic bioactivity of recombinant human BMP-2 (rhBMP-2) were investigated systematically. In vitro results indicated that Ca(2+) could bind rhBMP-2 rapidly and had no obvious effect on cell behaviors. Low concentration of Ca(2+) (0.18 mM) enhanced rhBMP-2-induced osteogenic differentiation, while high Ca(2+) concentration (>1.80 mM) exerted negative effect. In vivo ectopic bone formation exhibited similar trend. Further studies by circular dichroism spectroscopy, fluorescence spectroscopy, together with cell culture experiments revealed at low concentration, weak interaction of Ca(2+) and rhBMP(-)2 slightly increased β-sheet/-turn content and facilitated recognition of BMP-2 and BMPRIA. But, high Ca(2+) concentration (>1.8 mM) induced formation of Ca-rhBMP-2 complex and markedly increased content of β-sheet/-turn, which led to inhibition binding of rhBMP-2 and BMPRIA and thus suppression of downstream Smad1/5/8, ERK1/2 and p38 mitogen-associated protein kinase signaling pathways. Our work suggests osteogenic bioactivity of BMP-2 can be adjusted via extracellular Ca(2+), which should provide guide and assist for development of BMP-2-based materials for bone regeneration.