Nkx3.2 Promotes Primary Chondrogenic Differentiation by Upregulating Col2a1 Transcription 英文参考文献.docVIP

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Nkx3.2 Promotes Primary Chondrogenic Differentiation by Upregulating Col2a1 Transcription 英文参考文献.doc

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Nkx3.2 Promotes Primary Chondrogenic Differentiation by Upregulating Col2a1 Transcription 英文参考文献

Nkx3.2PromotesPrimaryChondrogenicDifferentiation byUpregulatingCol2a1Transcription YoshitakaKawato1,MakotoHirao2,KosukeEbina1*,KenrinShi1,JunHashimoto2,YuiHonjo1, HidekiYoshikawa1,AkiraMyoui3 1DepartmentofOrthopaedics,GraduateSchoolofMedicine,OsakaUniversity,Yamadaoka,Suita,Osaka,Japan,2DepartmentofOrthopaedicSurgery,NationalHospital Organization, Osaka Minami Medical Center, Kawachinagano, Osaka, Japan, 3Medical Center for Translational Research, Osaka University Hospital, Yamadaoka, Suita, Osaka,Japan Abstract Background:TheNkx3.2transcriptionfactorpromoteschondrogenesisbyformingapositiveregulatoryloopwithacrucial chondrogenic transcription factor, Sox9. Previous studies have indicated that factors other than Sox9 may promote chondrogenesis directly, but these factors have not been identified. Here, we test the hypothesis that Nkx3.2 promotes chondrogenesis directly by Sox9-independent mechanisms and indirectly by previously characterized Sox9-dependent mechanisms. Methodology/Principal Findings: C3H10T1/2 pluripotent mesenchymal cells were cultured with bone morphogenetic protein 2 (BMP2) to induce endochondral ossification. Overexpression of wild-type Nkx3.2 (WT-Nkx3.2) upregulated glycosaminoglycan(GAG)productionandexpressionoftypeIIcollagena1(Col2a1)mRNA,andtheseeffectswereevident before WT-Nkx3.2-mediated upregulation of Sox9. RNAi-mediated inhibition of Nkx3.2 abolished GAG production and expressionofCol2a1mRNA.DualluciferasereporterassaysrevealedthatWT-Nkx3.2upregulatedCol2a1enhanceractivityin a dose-dependent manner in C3H10T1/2 cells and also in N1511 chondrocytes. In addition, WT-Nkx3.2 partially restored downregulation of GAG production, Col2 protein expression, and Col2a1 mRNA expression induced by Sox9 RNAi. ChIP assaysrevealedthatNkx3.2boundtotheCol2a1enhancerelement. Conclusions/Significance: Nkx3.2 promoted primary chondrogenesis by two mechanisms: Direct and Sox9-independent upregulationofCol2a1transcriptionandupregulationofSox9mRNAexpressionund