Influences Of Osteoprotegerin On The Differentiation,Activation, And Survival Of Osteoclast And The Involved Signalling Pathways

Osteoprotegerin (OPG) which belongs to the tumor necrosis factor (TNF) receptor superfamily. OPG is well known to negatively regulate osteoclast (OC) maturation and activation and promote the development of apoptosis of OC. Clinical trials and animal experiments indicated that it was practicable to prevent and cure human and animal bone disease by the recombinant OPG and OPG gene therapy technology. However, the mechanisms of OPG inhibitory effects to the differentiation, activation and survival of osteoclasts have not been well elucidated. There are many difficulties in drug development related OPG. Isolated primary OC and induced OC were employed in this study, different concentrations of OPG were added to the cells culture in vitro. Morphological observation, bone resorption activity assay, QRT-PCR, and Western blot were all performed to determine the morphological changes, functions, OC releated genes expression, and key signaling proteins change of OC, which were treated by OPG. The aim of this study was to determine the mechanism involved in influence of osteoprotegerin (OPG) on the OC. A series of tests were carried out:1. Influence of osteoprotegerin on differentiation, activation and apoptosis of osteoclasts in vitroThe aim of this study was to determine the influence of osteoprotegerin (OPG) on the differentiation, activation and apoptosis of duck embryo osteoclasts cultured in vitro. Bone marrow cells were harvested from23-day-old duck embryos and cultured in the presence of different concentrations of OPG (group A:no added factors, group B:30ng/mL OPG and group C:100ng/mL OPG). Tartrate-resistant acid phosphatase (TRAP) staining, pit formation assay, and co-staining with TRITC-conjugated phalloidin and Hoechst33258were all performed to determine the number of TRAP-positive cells, bone resorption activity and the level of apoptosis, respectively. The number of TRAP-positive cells and the net expansion of pit formations area peaked on the7th day of culture in all3groups. The number of osteoclasts and the total volume of pit formations in OPG-treated groups were significantly lower when compared to group A (P<0.05). At each time point, the net expansion of pit formations area correlated with the number of TRAP-positive cells. OPG inhibited the de novo formation of F-actin rings and promoted the disruption of existing F-actin rings in mature osteoclasts. In addition, OPG induced apoptosis in mature osteoclasts, as demonstrated by morphological changes in the nuclei. In osteoclast precursors, OPG inhibited differentiation and down regulated the formation of F-actin rings. In mature osteoclasts, OPG suppressed activation and enhanced the development of apoptosis, observed as a decrease in the number of TRAP-positive cells, the disruption of F-actin rings and morphological changes of the nuclei.2. Mechanism involved in osteoprotegerin (OPG) affect differentiation and activation of osteoclastThe aim of this study was to determine the molecular mechanism involved in osteoprotegerin (OPG) affect differentiation and activation of osteoclast in serum-free conditions. RAW264.7cells were incubated with M-CSF+RANKL in serum-free medium for osteoclastogenesis. In the cultivation of the cells,0,10,20,50, and100ng/mL OPG were added to various groups. Osteoclast differentiation and activation were estimated via TRAP staining study, F-actin rings analysis, and bone resorption assay. Furthermore, expression levels of osteoclast related genes, such as TRAP, RANK, MMP-9, CA Ⅱ, and Cathepsin K which were influenced by OPG, were examined using real-time PCR with RAW264.7cells. In summary, these findings suggested that M-CSF+RANKL could promote the differentiation and activation of osteoclast, enhance the expression of TRAP, RANK, MMP-9, CA II, and Cathepsin K mRNA in osteoclast, whereas OPG inhibited them in serum-free conditions.3. Influences of osteoprotegerin on the differentiation, maturation of osteoclast and the involved MAPK signalling pathwayThe aim of this study was to determine the MAPK signalling pathway involved in osteoprotegerin (OPG) affect differentiation and maturation of osteoclast. RAW264.7cells were incubated with M-CSF+RANKL for osteoclastogenesis. In the cultivation of the cells,0,10,20,50, and100ng/mL OPG were added to various groups at defined time points. Cells were collected and total protein were isolated, key signaling proteins involved in the MAPK signalling pathway were monitored via Western blot. Results displayed that phosphorylation of p38-MAPK, JNK-MAPK, ERK-MAPK all were enhanced in M-CSF+RANKL-induced RAW264.7cells. The phosphorylation of p38-MAPK maximized after OPG treated for15min, and the phosphorylation of JNK-MAPK, ERK-MAPK maximized after OPG treated for30min. In addition, various concentrations of OPG could suppress the phosphorylation of all the three proteins in a concentration-dependent manner. The results demonstrated that the MAPK signalling pathway involved in the differentiation and activation of osteoclast, as well as in osteoprotegerin (OPG) affect the differentiation and activation of osteoclast.4. Influences of osteoprotegerin on the differentiation, maturation of osteoclast and the involved Ca2+signalling pathwayThe aim of this study was to determine the Ca2+signalling pathway involved in osteoprotegerin (OPG) affect differentiation and maturation of osteoclast. RAW264.7cells were incubated with M-CSF+RANKL for osteoclastogenesis. In the cultivation of the cells,0,10,20,50, and100ng/mL OPG were added to various groups. Cells were collected at the end of cultivation, the concentrations of [Ca2+]i were determined by flow cytometry, and the phosphorylation of CaMKII was monitored via Western blot. Results displayed that the concentrations of [Ca2+]j were raised significantly (P<0.01), and phosphorylation of CaMKII were enhanced in M-CSF+RANKL-induced RAW264.7cells. However, the concentrations of [Ca2+]i and the phosphorylation of CaMKII were decreased by OPG. The results demonstrated that the Ca2+signalling pathway involved in the differentiation and activation of osteoclast, as well as in OPG affect the differentiation and activation of osteoclast.