PDGF BB purified from osteoclasts acts as osteoblastogenesis inhibitory factor (OBIF)

The functions of bone-forming osteoblasts and bone-resorbing osteoclasts are intimately linked. Recently it has been revealed that osteoblasts regulate differentiation of osteoclasts by two factors: as a stimulator of osteoclastogenesis, the receptor activator of NF-B ligand (RANKL); and as an inhibitor, osteoprotegerin (OPG). However, no signaling factors from osteoclasts to osteoblasts have yet been identified. In this study, we found that the conditioned medium of mouse osteoclast-like RAW264.7 cells treated with RANKL contains activity that inhibits differentiation of mouse osteoblast-like MC3T3-E1 cells. We named this factor osteoblastogenesis inhibitory factor (OBIF). We partially purified OBIF with successive three-step chromatography by heparin affinity, anion exchange, and reverse-phase columns. This inhibitory activity appeared as one peak in each chromatography step, indicating that the factor was a single entity. Active fractions were loaded on SDS-PAGE, digested in gel by trypsin, and analyzed by liquid chromatography equipped with tandem mass spectrometry (LC/MS/MS). Subsequently, we found platelet-derived growth factor BB homodimer (PDGF BB) to be an OBIF candidate protein, and neutralization of the inhibitory activity of the medium with anti-PDGF antibody confirmed this identification. These results demonstrate, for the first time, that osteoclasts regulate osteoblasts directly and suggest that PDGF BB is a key factor in bone remodeling.