The Promotional Effect And Mechanism Of TNF-α On RANKL-induced Osteoclastogenesis

Objective:Osteoclast, derived from mononuclear progenitors of pluripotent hematopoietic stem cells, is multinucleated cell and unique in its ability to resorb mineralized tissues like bone. It has been well accepted that two factors are critically required for osteoclastogenesis:macrophage-colony stimulating factor (M-CSF) and ligand of receptor activator of NF-kB (RANKL). This part of experiment is aim to obtain plenty of pure bone marrow mononuclear phagocytes of mouse and to find appropriate concentration of RANKL stimulation for researching the mechanism of osteoclastogenesis in vitro.Methods:Pure bone marrow mononuclear phagocytes of mouse were obtained with the methods of adherence and ficoll density gradient centrifugation. Osteoclasts were induced by bone marrow mononuclear phagocytes of mouse using medium with macrophage colony stimulating factor (M-CSF) and two different concentration of receptor activator of nuclear factor-kappaB ligand (RANKL). We observed the morphology of the cells in different time points. The tartrate resistant acid phosphatase (TRAP)staining was performed in the time point of72h.Results:Pure bone marrow mononuclear phagocytes of mouse were obtained with our method. Two different concentration of RANKL were used in the experiment:1ng/ml and10ng/ml. Two groups of cells showed the same morphology within almost48h-stimulation when the TRAP positive multinuclear cells were appeared. More TRAP positive multinuclear cells were found in the cell group of10ng/ml RANKL stimulation with larger bodies and more nuclei.Conclusion:The BMMs were successfully separated from bone marrow cells. More osteoclasts with larger bodies were induced from mice bone marrow mononuclear phagocytes in the presence of10ng/ml RANKL when stimulated for72h. However, the formation rates of osteoclast were not changed by RANKL concentration. lng/ml RANKL and50ng/ml M-CSF would be used in next experiment. Objective:Higher level of TNF-a have been found in many diseases, such as rheumatoid arthritis, periodontal disease and aseptic periprosthetic osteolysis. These diseases are characterized by bone loss around affected joints or teeth caused by increased osteoclastic bone resorption. The importance of TNF-a in the pathogenesis of various forms of bone loss is supported by both experimental and clinical evidences. While the precise molecular mechanism of how TNF-a promotes osteoclast formation is not clear. Previous reports show that TNF-a targets molecules that regulate calcium signaling, and leads to increased autoamplification of nuclear factor of activated T-cells1(NFATcl) which is dominant in RANKL-induced osteoclastogenesis in vitro. Inositol-1,4,5-trisphosphate receptors (IP3Rs) are important calcium channel responsible for evoking intracellular calcium oscillation. There are three subtypes of IP3R: IP3R1, IP3R2, and IP3R3. The three types are expressed in a tissue and development-specific manner. It was reported that TNF-a promoted IP3R1expression in human neural cells and mesangial cell through phosphatidylcholine-specific phospholipase C (PC-PLC) activation. Whether expression of IP3Rs is affected by stimulation of TNF-a and PC-PLC is involved in osteoclastogenesis induced by RANKL have not been known.Methods:The experimental model of osteoclast differentiation which was established in part1was used to find the promotional effect of TNF-α. Firstly, we speculate that intracellular ROS (reactive oxygen species) level is involved in osteoclastogenesis induced by TNF-a by the detector DCFH-DA. Then we used the inhibitor D609(tricyclodecan-9-yl-xanthogenate) to identify the role of PC-PLC and in TNF-α induction. Calcium oscillation, NFATc1and three subtypes of IP3Rs expression level were checked with different methods.Results:In the present work, we found that TNF-α increased the expression of IP3R1and thus increased cellular calcium oscillation and NFATc1autoamplification, then finnally upregulated the number of osteoclast in RANKL-induced mouse BMMs. PC-PLC specific inhibitor D609eliminated the upregulation of IP3R1by TNF-α, and decreased the autoamplification of NFATc1, thus resulted in less osteoclasts formation. However, D609didn’t inhibit osteoclastogenesis induced by RANKL. The results of using D609reveal that PC-PLC is involved in the promotion of TNF-α on RANKL-induced osteoclastogenesis.Conclusion:In conclusion, we found that D609removed the promotional effect of TNF-α on IP3R1expression during osteoclast formation induced by RANKL The reducing effect of D609on IP3R1expression correlated to the downregulation of NFATcl expression. Exposing BMMs to D609before the cells were conducted to differentiation decreased TRAP-positive MNC number in the culture of RANKL with TNF-α, while moderately increased the number in RANKL without TNF-α. Our data indicate that PC-PLC is involved in osteoclatogenesis induced by TNF-α through upregulating IP3R1expression. The precise mechanism by which TNF-α stimulates osteoclastogenesis needs further discussion.