Regulating Effect Of Dishevelled-2 On Bone Metabolism By Crosstalk Between Wnt And NF-κB Signal Pathway

Rheumatoid arthritis(RA),which is charactered with the chonic damage of bone and cartilage caused by consisting intra-articular synovial inflammation,is one of the most common autoimmune diseases.In our country,RA ranked second in disabling disease and the incidence of RA has a rising trend year by year.Although there is so many therapy strategies in treating RA,the molecular mechanism for articular erosions still keeps unclear and the drugs for effectively blocking articular erosions are also rare.Growing evidence has highlighted the role of Wnt and NF-κB signaling pathways in maintaining the dynamic balance of bone metabolism.Inhibition the activation of Wnt and enhancement of NF-κB,which can restrain the proliferation and differentiation of osteoblast and improve the function of osteoclast,then effect the balance of bone metabolism,which are all involved in the onset and progression of RA.Based on the fact that Dishevelled(Dvl)can not only regulate the Wnt signaling pathway,but also regulate NF-κB signaling pathway and our observations that there is a local increased expression in the mice model of RA,we assumed that Dvl may participate in the two pathways to sustain the balance of bone metabolism and the mechanism of Dvl in osteoblast and osteoclast can provide the new experimental proof for the clinical treatment of RA.ObjectiveC2C12 and RAW264.7 are chose for our research.By transfection the expression plasmid and si RNA of Dvl-2,we detected the activity of Wnt and NF-κB signaling pathways and the expression of OPG,RANKL,β-catenin and IκB-α.So we preliminary investigate whether there is a cross-talk between Wnt/β-catenin and NF-κB under the regulation of Dvl-2 to influence the proliferation and differentiation of osteoblast and osteoclast.MethodsFirstly,we use Lipofectamine 2000 to transfect expression plasmid and si RNA of Dvl-2 into the cells,resulting to increase or decrease the expression level of Dvl-2.Fluorescence microscope and Real-time PCR are used to detect the effect of transfection.Secondly,we use CCK8 to explore the role of Dvl-2 in regulating the ability of proliferation of C2C12 and RAW264.7.The ELISA is used to detect the excretion of OPG in supernatant of treated C2C12 cells.Western blot is used to detect the expression level of RANKL and β-catenin in the downstream.Co-transfection of TCF reporter gene plasmid or κB-Luc reporter gene plasmid with PRL-SV40 reporter plasmid is used to detect the activation status of Wnt and NF-κB,which is detecte by Glo Max 20/20.Western blot can investigate the expression of IκB-α.Co-Immunoprecipitation is aimed to detect whether Dvl-2 can bind with p65.Lastly,SPSS18.0 is used to analyze the data.Results1.36 h after the transfection of Dvl-2 overexpression plasmid or si RNA,fluorescence microscope and Real-time PCR have detected the increase or decrease of Dvl-2 expression level compare to controls.2.The results of CCK8 showed that the proliferation of C2C12 cells after overexpression of Dvl-2 has no obviously change,but it could inhibit the proliferation of RAW264.7.3.ELISA is used to detect the concentration of OPG,which is treated after 48 h in C2C12 cells,and the result demonstrated that Dvl-2 and Wnt3 a can enhance the excretion of OPG independently,which showed significantly differences versus controls,and the effect of Dvl-2 was higher than Wnt3 a.When using si RNA,the excretion of OPG had significantly decreased.4.After 48 h of transfection,Western blot showed that overexpression of Dvl-2 could decrease the expression of RANKL in C2C12 cells and downexpression of Dvl-2 could increase the expression of RANKL.5.Glo Max 20/20 showed Dvl-2,Wnt3 a both could activate TCF reporter gene plasmid independently after co-transfection of TCF reporter gene plasmid and Dvl-2 plasmid and the effect of both can enhance when they acted together.The activation of TCF reporter gene plasmid was inhibited after transfect si RNA of Dvl-2.6.Western blot clarified that overexpression of Dvl-2 can upregulate the expression of β-catenin.Downexpression of Dvl-2 showed the opposite results.7.In RAW264.7,dual-luciferase reporter gene demonstrated that the activation of κB-Luc reporter gene had decreased significantly compare to controls when the Dvl-2 overexpressed and vice versa.Meanwhile,Dvl-2 can decrease the expression of IκB-α,which is the NF-κB dependent target gene.8.In order to clarify the mechanism of how Dvl-2 inhibit the NF-κB signaling pathways,we used the Co-IP to detect the specific binding of Dvl-2 and p65.Conclusion1.Dvl-2 has no influence on the proliferation of C2C12 cells.Overexpression of Dvl-2 can inhibit the proliferation of RAW264.7.2.Dvl-2 can enhance the excretion of OPG by the classical Wnt/β-catenin signaling pathway.The ability of Dvl-2,which can increase the excretion of OPG,is higher than Wnt3 a.And the ability can facilitate the differentiation of osteoblast.On the other hand,Dvl-2,decreasing the expression level of RANKL in C2C12 cells and effecting the ratio of OPG/RANKL,can inhibit the mature and differentiation of osteoclast by RANK-RANKL-OPG signaling pathway which is dominated by NF-κB.3.Dvl-2 can activate the classical Wnt/β-catenin signaling pathways to promote the stability of β-catenin in cytoplasmic.4.Dvl-2 can directly inhibit the activation of NF-κB signaling pathways by binding to p65 to inhibit the maturity and differentiation of osteoclast in RAW264.7,and also represent the downexpression of IκB-α.