Rspo1 Promote Osteoblast Differentiation By Wnt Signaling Pathway

Rheumatoid arthritis(RA) is a Systemic Autoimmune Disease which characterized by chronic erosive arthritis.The chief pathological features are chronic inflammation and pannus formation of synovial membrane lining the joints.Research has showed that Wnt/β-catenin signaling pathway is closely associated with the alternation of bone metabolism caused by RA.In RA patients,the Wnt pathway has been proven to be suppressed,leading to the inhibition of proliferation and differentiation of osteoblast cells,thus promoting the development of osteoporosis and bone erosion.Recently,although R-spondin-1(Rspo1),regarded as the activator of Wnt signaling pathway,has been widely reported,whether it could interact with osteoblast cells remains unclear.Up to date,no effective treatments are available to improve or delay the progress of bone erosion caused by RA.Thus,elucidate the underlying mechanism of bone erosion may provide a scientific basis for the clinical treatment of bone erosion in RA.ObjectiveTo investigate whether Rspo1 could promote osteoblast differentiation via activating Wnt signaling pathway,we constructed C2C12 cells containing TCF luciferase reporter gene and then detected the secretion of osteoprotegerin(OPG),alkaline phosphatase(ALP)and content of intracellular β-catenin under the stimulation of Rspo1 and Wnt3 a.our data establish novel strategies and approaches for the clinical prevention and treatment of bone erosion in RA.MethodC2C12 cells were cultured in regular Dulbecco`s modified Eagle`s medium(DMEM)supplemented with 10% fetal bovine serum,then feeded at a density of 4×104 cells/ml in 48-well palates.Firstly,the xCELLIgence technology of cellular dynamics model analysis system(Roche company)was used to determine whether RSpo1 and Wnt3a could affect the proliferation of C2C12.Secondly,C2C12 cells were co-transected with the plasmid containing TCF luciferase gene and with the pRL plasmid containg SV40 reproter gene via using Lipofectamine 2000 reagent.Then,to verify the effect of Rspo1 on C2C12,the expression of the markers of bone maturation such as OPG and ALP were detected.Cells were treated with 50ng/ml Rspo1,50ng/ml Wnt3 a and 50ng/ml Rspo1 combined with 50ng/ml Wnt3 a respectively and then the culture medium of each experimental group was collected to measure the concentration of OPG through a ELASA detection method,the cell lysates were used to detect the level of ALP.SPSS17.0 statistical software was used and statistical analysis was performed using one-way analysis of variance(ANOVA),followed by the Tukey’s multiple comparison test.β-catenin proteins is a key protein in Wnt signaling pathways,which combined with TCF after transported into nuclear,thus activating the transcription of the downstream target genes.Then,after 6H,the C2C12 co-transfected with plasmids were maintained in fresh DMEM supplemented with Rspo1 and(or)Wnt3a in different concentrations for 24 h.Luciferase reporter gene assays were performed using the Dual Luciferase Reporter Assay System(Promega,USA)according to the manufacturer’s instructions.Finally,Western blotting analysis was performed to examine the expression of β-catenin in the control group and three treatment groups and the data were analyzed statistically.Result1.xCELLIgence system measurement suggest that RSpo1 has no significant effect on proliferation of C2C12 while Wnt3 a notably promotes the expansion of C2C12.2.compared with the control group,the activity of ALP and the secretion of OPG were dramatically upregulated in the group treated with 50ng/ml Rspo1.Results were analyzed by one-way ANOVA and the difference between two groups is significant with a P value < 0.05.3.Compared with the control group,the activity of ALP and the secretion of OPG were significantly increased in the treatment group(50ng/mlRspo1)with P value <0.01.Moreover,Compared with the group treated with 50ng/ml Wnt3 a,the ALP and OPG level were apparently increased in the combined-treatment group(50ng/ml Rspo1+50ng/ml Wnt3a)with P value <0.01.4.Luciferase activity of the reporter gene was detected under the stimulation of the increasing concentrations of Rspo1,Wnt3 a or combined.The data were used to make a curve graph.According to the curve,RSpo1 didn’t remarkably activated the luciferase activity of TCF reporter gene while Wnt3 a obviously activated the luciferase activity in a dose-dependent manner.Combination of RSpo1 and Wnt3 a has a synergistic effect on activating TCF gene.5.western blot analysis was performed to examine the expression level of the intracellular β-catenin in control group and the treatment groups.According to the color of protein electrophoresis stripe,from deep to shallow the order was combined group(50ng/ml Rspo1+50ng/ml Wnt3a),50ng/ml Wnt3 a group,50ng/ml Rspo1 group and the control group respectively.Compared with black control,group treated with 50ng/ml Rspo1 has a deeper protein electrophoresis stripe and a higher bar column without a significant differenc(P>0.05);whereas,the group treated with 50ng/ml Wnt3 a has a deeper protein electrophoresis stripe and a much higher bar column with significant difference(P<0.05),compared with the control group.Similarly,the comparison between the combined-treatment group and 50ng/ml Wnt3 a group was also statistically significant with a P value<0.05.CONCLUSION1.RSpo1 can not promote the proliferation of C2C12;2.RSpo1 promotes C2C12 cells differentiate into Osteoblast cells with expression of ALP and OPG in coordination with Wnt3 a.3.RSpo1 alone has no significant effect on activating Wnt signaling pathway.4.RSpo1 and Wnt3 a collaboratively activate the Wnt/β-catenin signaling pathway,thus participating the bone metabolism and promoting the differentiation of Osteoblast cells.