Osteoclasts and osteoblasts are responsible for strict bone maintenance with a balance between bone formation and resorption by interacting with each other. Recently, it has been revealed that osteoblasts/stromal cells regulate differentiation of osteoclasts/hematopoietic cells by two factors, the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) on the plasma membrane, and secreted osteoprotegerin (OPG). However, no factors have yet been reported by which osteoclasts/hematopoietic cells regulate osteoblasts/stromal cells. To elucidate the possibility of signal transduction from osteoclasts to osteoblasts, we studied the conditioned medium of mouse osteoclast-like myeloma cell line RAW264.7 treated with RANKL. We found that this medium contains a factor that inhibits differentiation of mouse osteoblast precursor-like cell line MC3T3-E1 to osteoblasts induced by bone morphogenetic protein 4 (BMP-4) and named this factor osteoblastogenesis inhibitory factor (OBIF). OBIF was purified by successive three-step chromatography by heparin affinity, anion exchange, and reversed-phase columns. Osteoblastogenesis inhibitory activity made one peak in each chromatography step, showing the factor is a single entity. Active fractions were loaded on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and bands of proteins were excised, digested by trypsin, and analyzed by liquid chromatography equipped with tandem mass spectrometry (LC/MS/MS). Consequently, we have identified this factor to be platelet-derived growth factor BB (PDGF BB) homodimer. Furthermore, this identification of PDGF BB as OBIF was confirmed by neutralization of the inhibitory activity of the medium with anti-PDGF antibody. These results show, for the first time, that osteoclasts regulate osteoblasts directly and suggest that PDGF BB is a key factor in bone remodeling.

GroEL encapsulates non-native protein in a folding cage underneath GroES (cis-cavity). Here we report the maximum size of the non-native protein to stay and fold in the cis-cavity. Using total soluble proteins of Escherichia coli in denatured state as binding substrates and protease resistance as the measure of polypeptide held in the cis-cavity, it was estimated that the cis-cavity can accommodate up to ϳ57-kDa non-native proteins. To know if a protein with nearly the maximum size can complete folding in the cis-cavity, we made a 54-kDa protein in which green fluorescent protein (GFP) and its blue fluorescent variant were fused tandem. This fusion protein was captured in the cis-cavity, and folding occurred there. Fluorescence resonance energy transfer proved that both GFP and blue fluorescent protein moieties of the same fused protein were able to fold into native structures in the cis-cavity. Consistently, simulated packing of crystal structures shows that two native GFPs just fit in the cis-cavity. A fusion protein of three GFPs (82 kDa) was also attempted, but, as expected, it was not captured in the cis-cavity.

No abstract