IL-17A Induces Osteoclast Differentiation By Activating TRAF6-mediated C-Fos/NFATc1 Signaling Pathway

Objective Multiple myeloma associated bone disease(MBD)is the most important complication of MBD.The pathogenesis of MBD is still unclear,osteoclast-mediated osteolytic damage is a key link in the development of MBD.Previous studies have found a correlation between serum IL-17 and clinical stage of MBD,but it is not clear whether IL-17 is related to MBD.This study is to explore the role of IL-17 in MBD and its molecular mechanism.Methods1.Morphological observation and CCK-8 assay:The macrophage cells(RAW264.7 cells)were subcultured to logarithmic phase.RAW264.7 cells were treated with different concentrations of IL-17 A to observe the growth and morphological changes.The differentiation of osteoclasts was detected by TRAP staining,and the positive cells of TRAP were counted.The effects of different concentrations of IL-17 A on the growth of RAW264.7 cells were detected by CCK-8assay.2.Expression of key genes in osteoclast differentiation The expressions of TRAF6,c-Fos and NFATc1 in RAW264.7 cells were detected by Real-time Quantitative Polymerase Chain Reaction(q RT-PCR).3.Expression of key proteins in osteoclast differentiation Western blot was used to detect the expression of key node-related proteins in osteoclasts treated with different concentrations of IL-17 A.Results1.The morphological changes of RAW264.7 cells were observed with different concentration of IL-17 A.The number of multinucleated cells with 3 or more nucleus and the number of TRAP positive cells were increased in a dose-dependent manner,the differentiation of osteoclast stimulated by high concentration of IL-17 A is the most significant.The effects of different concentrations of IL-17 A on RAW264.7 cell proliferation were observed by CCK-8 test.The effects of 100ug/L,50ug/L and 0.5ug/L on RAW264.7 cells proliferation were significantly higher than those of the Control Group.2.After IL-17 A was used to interfere RAW264.7 cells,the expressions of osteoclast related transcription factors TRAF6,c-Fos and NFATc1 were detected by q RT-PCR.3.The expression of these proteins was also elevated in RAW264.7 cells after IL-17 A by Western blot.50ug/L IL-17 A highly activated the signal related to osteoclast differentiation in RAW264.7 cells.Conclusion1.IL-17 A directly induced and promoted the differentiation of RAW264.7 cells into osteoclasts in vitro and promoted the proliferation of cells.2.IL-17 A promoted the high expression of TRAF6,and upregulated the expression of c-Fos and NFATc1,which are the key factors of osteoclast differentiation,inducing RAW264.7 cells to differentiate into mature osteoclasts.