Pax2 Is Essential For Proliferation And Osteogenic Differentiation Of Mouse Mesenchymal Stem Cells Via Runx2

Objective: This study was aimed to investigate the effects of Paired box 2(Pax2)on osteogenesis of mouse mesenchymal stem cells(MSCs).Methods: The mouse pluripotent stem cell line C3H/10T1/2 and mouse compact bone-derived mouse MSCs(mMSCs)were used in this study.Western blotting analysis and immunofluorescent staining were used to investigate the primary expression of Pax2 during osteogentic differentiation.In vivo,immunohistochemistry was used to detect the expression of Pax2 in the tibial sections of C57BL/6 mice at embryonic stage 17.5(E17.5)and post-natal 21(P21)days.Pax2 was knocked down in C3H/10T1/2 cells and mMSCs with lentivirus expressing short hairpins directed at Pax2 mRNA.Next,cell proliferation,cell cycle and cell apoptosis were analyze with the CCK-8 assay and flow cytometry.The potential effects of Pax2 on osteogentic differentiation of C3H/10T1/2 cells and mMSCs were analyzed by measuring the mRNA and protein level expression of several osteogenic genes with qRT-PCR and Western blotting analysis.Mineralization analyses were performed by ALP and ARS staining.Furthermore,bioinformatics analysis(Genomatix program)was used to predict the binding site of Pax2 in the Runx2 promoter region,and the dual luciferase assay was carried out to detect the transcriptional activity of Runx2.The protein expression levels of ERK,JNK and p38 were detected using Western blotting analysis and immunofluorescence staining.Last but not least,a heterotopic bone regeneration model based on mMSCs was established for the studies in vivo.Statistical analyses were assessed by SPSS 19.0 software.Results: Here,we reported for the first time that the expression of Pax2 gradually increased during osteogenic differentiation of mouse MSCs and osteoprogenitor cells.However,detected in osteoblastic cells of mouse tibia,the expression of Pax2 in the embryonic stage was higher than that in adulthood.In C3H/10/T1/2 cells and mMSCs,Pax2 knock-down inhibits the cell proliferation,down-regulates the mRNA and protein levels of osteogenic genes such as Runx2,Osterix,Col1a1,Opn,and OC,as well as represses the ALP activity and mineralization.In addition,Pax2 enhanced the transcriptional activity of Runx2,and activated the MAPK pathway genes(ERK,JNK and p38).Furthermore,knock-down of Pax2 repressed the mMSCs-mediated bone regeneration in an ectopic bone formation model.Conclusion: Pax2 promotes the osteogenesis of mouse MSCs,suggesting that Pax2 has a role in the pathophysiology of bone related diseases,and has potential application in cell therapy and bone tissue regeneration.