Effects Of P60PLAD On Osteoclastogenesis, Differentiation Of Osteoclast And Bone Resorption

The osteolysis around the joint prosthesis and osteoporosis are arisen by the abnormal quantity and function of osteoclasts, which is related to the over activation of osteoclast induced by the accumulation of TNF-αin patho-condition. It has been confirmed that blockade of the TNF-αsignal transduction pathway could inhibit the activation of osteoclast and the related bone resorption. However, this strategy is confined because of some severe immune system complications. Therefore, to search and manufacture the surrogate of TNF-αantagonist has become one of the most important investigation trends to cure bone resorption related diseases.P60PLAD protein is a newly developed small molecular mass protein derived from the extracellular domain of TNFR1. It can highly selectively bind TNFR1, block the binding of TNF-αto TNFR1 and thereby avoid the occurrence of severe immune system complications. Though P60PLAD protein has shown exciting results in a preclinical model of RA, its effect on bone resorption diseases has not been reported. In order to determine the effects of P60PLAD on the osteoclastogenesis, differentiation of osteoclast and bone resorption, a prokaryotic expression vector pGEX-4T-2-P60PLAD was constructed, and recombinant GST-P60PLAD fusion protein was prepared by soluble expression in BL21(DE3) E.coli strain. Meanwhile, in vitro mouse osteoclast culture system induced by TNF-αwas established. At last the GST-P60PLAD fusion protein was applied to this culture system and the conclusions are as below:1. The GST-P60PLAD fusion protein could efficiently suppress the formation and differentiation of mouse osteoclast and inhibit bone absorption induced by TNF-αin a dose-dependent manner. And few osteoclasts could be detected when the concentration of GST-P60PLAD reaches 2×103 ng/ml.2. Pure GST protein can not inhibit the the formation, differentiation of osteoclast and the formation of bone absorption cavity at any concentrations used.3. P60PLAD can inhibit the formation and differentiation of mouse osteoclast induced by TNF-αwith high efficiency. And the functional part of GST-P60PLAD protein is the P60PLAD domain but not the GST fusion tag. In conclusion, P60PLAD could be a novel drug prototype to cure TNF-αmediated bone resorption related diseases in the near future.