Rspo1Induce Osteoblast Differentiation Through The Wnt/β-catenin Pathway

Rheumatoid arthritis (RA) is a Systemic Autoimmune Disease which characterizedby chronic erosive arthritis. The chief pathological features are chronic inflammation andpannus formation of synovial membrane lining the joints. Research has showed thatWnt/β-catenin signaling pathway is closely associated with the alternation of bonemetabolism caused by RA. In RA patients, the Wnt pathway has been proven to besuppressed, leading to the inhibition of proliferation and differentiation ofosteoblast cells,thus promoting the development of osteoporosis and bone erosion.Recently, although R-spondin-1(Rspo1), regarded as the activator of Wnt signalingpathway, has been widely reported, whether it could interact with osteoblast cells remainsunclear. Up to date, no effective treatments are available to improve or delay theprogress of bone erosion caused by RA. Thus, elucidate the underlying mechanism ofbone erosion may provide a scientific basis for the clinical treatment of bone erosion inRA.ObjectiveTo investigate whether Rspo1could promote osteoblast differentiation via activatingWnt signaling pathway, we constructed C2C12cells containing TCF luciferase reportergene and then detected the secretion of osteoprotegerin(OPG), alkaline phosphatase(ALP)and content of intracellular β-catenin under the stimulation of Rspo1and Wnt3a. our dataestablish novel strategies and approaches for the clinical prevention and treatment ofbone erosion in RA.MethodC2C12cells were cultured in regular Dulbecco`s modified Eagle`s medium(DMEM) supplemented with10%fetal bovine serum, then feeded at a density of4×104cells/ml in48-well palates. Firstly, the xCELLIgence technology of cellular dynamicsmodel analysis system(Roche company) was used to determine whether Rspo1andWnt3a could affect the proliferation of C2C12. Secondly, C2C12cells were co-transectedwith the plasmid containing TCF luciferase gene and with the pRL plasmid containgSV40reproter gene via using Lipofectamine2000reagent. Then, to verify the effect ofRspo1on C2C12, the expression of the markers of bone maturation such as OPG and ALP were detected. Cells were treated with50ng/ml Rspo1,50ng/ml Wnt3a and50ng/mlRspo1combined with50ng/ml Wnt3a respectively and then the culture medium of eachexperimental group was collected to measure the concentration of OPG through aELASA detection method, the cell lysates were used to detect the level of ALP. SPSS17.0statistical software was used and statistical analysiswas performed using one-way analysis of variance (ANOVA), followed by the Tukey’smultiple comparison test. β-catenin proteins is a key protein in Wnt signaling pathways,which combined with TCF after transported into nuclear, thus activating the transcriptionof the downstream target genes. Then, after6H, the C2C12co-transfected with plasmidswere maintained in fresh DMEM supplemented with Rspo1and(or) Wnt3a in differentconcentrations for24h. Luciferase reporter gene assays were performed using the DualLuciferase Reporter Assay System (Promega, USA) according to the manufacturer’sinstructions. Finally, Western blotting analysis was performed to examine the expressionof β-catenin in the control group and three treatment groups and the data were analyzedstatistically.Result1.xCELLIgence system measurement suggest that Rspo1has no significant effecton proliferation of C2C12while Wnt3a notably promotes the expansion of C2C12.2. compared with the control group, the activity of ALP and the secretion of OPGwere dramatically upregulated in the group treated with50ng/ml Rspo1. Results wereanalyzed by one-way ANOVA and the difference between two groups is significant witha P value <0.05.3. Compared with the control group, the activity of ALP and the secretion of OPGwere significantly increased in the treatment group(50ng/mlRspo1) with P value＜0.01.Moreover, Compared with the group treated with50ng/ml Wnt3a, the ALP and OPGlevel were apparently increased in the combined-treatment group(50ng/mlRspo1+50ng/ml Wnt3a) with P value＜0.01.4. Luciferase activity of the reporter gene was detected under the stimulation of theincreasing concentrations of Rspo1, Wnt3a or combined. The data were used to make acurve graph. According to the curve, Rspo1didn’t remarkably activated the luciferaseactivity of TCF reporter gene while Wnt3a obviously activated the luciferase activity ina dose-dependent manner. Combination of Rspo1and Wnt3a has a synergistic effect on activating TCF gene.5. western blot analysis was performed to examine the expression level of theintracellular β-catenin in control group and the treatment groups. According to the colorof protein electrophoresis stripe, from deep to shallow the order was combinedgroup(50ng/ml Rspo1+50ng/ml Wnt3a),50ng/ml Wnt3a group,50ng/ml Rspo1groupand the control group respectively. Compared with black control, group treated with50ng/ml Rspo1has a deeper protein electrophoresis stripe and a higher bar columnwithout a significant differenc(P＞0.05)； whereas, the group treated with50ng/mlWnt3a has a deeper protein electrophoresis stripe and a much higher bar column withsignificant difference(P＜0.05), compared with the control group. Similarly, thecomparison between the combined-treatment group and50ng/ml Wnt3a group wasalso statistically significant with a P value＜0.05.CONCLUSION1. Rspo1can not promote the proliferation of C2C12;2. Rspo1promotes C2C12cells differentiate into Osteoblast cells with expression ofALP and OPG in coordination with Wnt3a.3. Rspo1alone has no significant effect on activating Wnt signaling pathway.4. Rspo1and Wnt3a collaboratively activate the Wnt/β-catenin signaling pathway,thus participating the bone metabolism and promoting the differentiation ofOsteoblast cells.