The Experimental Study Of Transcription Factor FOXO1 On Osteoclast Differentiation And Function

Objective1.To study the role of transcription factor FOXO1 in osteoclast differentiation and function.2.To study the possible molecular mechanism of the effect of FOXO1 on the differentiation and function of osteoclast.Methods1.In BMMCs and RAW264.7 cells,the expression of FOXO1 in RANKL-induced osteoclast differentiation was detected by Q-PCR.The expression of FOXO1 was also detected by Western blot in RANKL-induced RAW264.7 cells.The influence of FOXO1 overexpression on osetoclast differentiation was detected by TRAP staining.The expression levels of TRAP,CK,MMP9 and ATP6v0d2 was detected by Q-PCR afer FOXO1 overexprssion.The influence of FOXO1 overexpression on osetoclast function was detected by Corning OsteoAssay Surface.2.The effect of FOXO1 inhibiton(a small molecule inhibitor AS 1842856)on osteoclast and function was detected.The influence of FOXO1 inhibiton on osetoclast differentiation was detected by TRAP staining.The expression levels of TRAP,CK,MMP9 and ATP6v0d2 was detected by Q-PCR afer FOXO1 inhibition.The influence of FOXO1 inhibiton on osetoclast function was detected by Corning OsteoAssay Surface.3.The effect of FOXO1 on MAPKs and NF-?B signaling pathway was detected by Western blot and EMSA.The small molecule inhibitor 10058-F4 and ROS scavenger N-acetylcysteine were employed to detect whether they can rescue the effect of FOXO1 inhibition on osteoclast differentiation and function.Results1.The expression of FOXO1 in RAW264.7 and BMMCs cells was decreased after RANKL induction.Overexpression of FOXO1 in RAW264.7 cells decreased the osteoclast formation and function,the expression of osteoclast marker genes(TRAP,CK,MMP9 and ATP6v0d2)were also decreased.2.The expression levels of TRAP,CK,MMP9 and ATP6v0d2 were increased by FOXO1 inhibition(AS1842856 treatment),and the resorption area of osteoclasts was also increased.The number of TRAP(+)cells was increased by AS 1842856 treatment both in RAW264.7 cells and BMMCs.3.In the molecular mechanism,we found that the phosphorylation of JNK and P38 were increased by FOXO1 inhibition,the transcriptional activity of NF-?B and AP-1 were also enhanced.The expression of MYC was increased after FOXO1 inhibition,10058-F4(a small molecular inhibitor of MYC)rescued the promotion of osteoclast differentiation and function after FOXO1 inhibition;N-acetylcysteine,a ROS scavenger,also rescued the promotion of osteoclast differentiation and function after FOXO1 inhibition.Conclusion1.The transcription factor FOXO1 has an inhibitory effect on osteoclast formation and function.2.MYC,ROS,MAPKs and NF-?B signaling pathways mediate the inhibitory effect of FOXO1,and the transcription factor FOXO1 is expected to be a new target for osteoporosis therapy.