Study On Effect And Mechanism Of Dlx2 Overexpression In Osteogenesis And Chondrogenesis Of Mouse Mesenchymal Cells

[Purpose]Congenital cranio-maxillofacialdefects and abnormities are one of major challenges for oral and maxillofacial surgeons.Therefore it is of great significance to search for the causes and understand the mechanisms of these cranio-maxillofacial anomalies for better birth and health.However,only a few are known about accurate positioning of the anomalies.For example,some syndromes in which specific gene mutations have been identified include van der Woude syndrome(IRF6),popliteal pterygium syndrome(IRF6),autosomal-recessive cleft palate/ectodermal dysplasia(PVRL1),hypodontia/clefting(MSX1).Most of cranio-maxillofacial anomalies are of epigenetics without obvious genetic characteristics.One of the most important part of epigenetics is transcriptional regulation,which plays crucial roles in development,tumorigenesis and inflammation.In addition,homobox genes,key component of transcriptional factors,play critical roles in develpment of craio-maxillofacial tissues.Dlx2,one of homobox gene family,plays a crucial role in the craniofacial development which mainly originatese from cranial neural crest cells.Previous finds revealed that Dlx2^-/-nude mice,as well as Dlx2 overexpression transgenic mice,exhibited serious cranio-maxillofacial deformities.However,their exact role of Dlx2in the osteogenesis and chondrogenesis is controversial,and the detailed molecular mechanisms are greatly unclear.In this study,we try to explore the effect and mechanism of Dlx2 in osteogenenic and chondrogenic differentiation of mouse mesenchymal cells.[Materials and methods]This study was divided into two parts.Part 1,lentivirus was constructed to upregulate expression level of Dlx2 gene in osteogenesis of Mc3T3 E1 from cranial bone and m BMSCs from mouse.Quantitative RT-PCR and western blotting were carried out to measure the expression level of genes in Dlx2-overexpressed cell lines;Dlx2-overesprssion cell lines and control groups were stained with Alizarin red and alkaline phosphatase to measure protein expression level.q PCR was used to find out possible target genes.Several experimental methods:q PCR,Western Blot,immunofluorescence,immunoprecipitation and chromatin immunoprecipitation assay were performed to explore the effect of Dlx2 overexpression in osteogenenic differentiation of mouse mesenchymal cells.Part 2,TMC-23 and chondrocytes from basicraial cartilage were chosen to evaluate the effect of Dlx2 overexpression in chondrogenic differentiation of mesenchymal cells.Quantitative RT-PCR and western blotting were carried out to measure the expression level of genes in Dlx2-overexpressed cell lines;Overexpressed cell lines and control groups were stained with alician blue to measure protein level.Ch IP and luciferase were performed to testify possible moleculars and possible pathways for understanding the exact role of Dlx2in chondrogenesis.[Results]Part I 1.Dlx2-overexpressed mouse osteogenic precursor cells(Mc3T3 E1 cell)was constructed successfully.2.Both ALP staining and alizard staining in Dlx2overexpressed cells were significantly enhanced.Quantative analysis of ALP staining indicated hyperactive osteogenesis.3.Expression level of Osteocalcin were significantly increased with overexpression of Dlx2 in Mc3T3 E1,C3H10 T1/2 and m BMSCs by means of q RT-PCR(p<0.01).,4.Expression level of 2000 bp promoter of Osteocalcin were significantly increased with overexpression of Dlx2 by means of Luciferase assay(p<0.01).Ch IP analysis showed section 8 of osteocalcin promoter was more obvious than other sections.There was no significant luciferase strength difference after site-directed mutagenesis of section 8 of osteocalcin promoter.5.Msx2 could be detected after overexpression of Dlx2 in Mc3T3 E1 cells,indicating that Msx2 could be a co-transcription factor during osteogenesis in Mc3T3 E1 cells with overexpression of Dlx2.Part II 1.Dlx2-overexpressed mouse osteogenic precursor cells(TMC23 cell)was constructed successfully and cells of Dlx2 overexpression and control TMC23 cells were accumulated for microarray analysis.2.Alician Blue staining of Dlx2 overexpressed TMC23 cells were significantly enhanced(p<0.01).3.Genes of chondrogenesis from lieratures and genes of chondrogenesis screened by microarrayed in this study were intersected,showing possible genes in chondrogenesis of Dlx2 overexpressed TMC23 cells as follows,MMP2,MMP13,MMP14,VEGFa,VCAM1,ICAM1,Sox6,Fibin,Adam12,Adam15,Dlx-5,ECM.4.Expression level of 2000 bp promoter of MMP13 were significantly decreased through overexpression of Dlx2 by means of Luciferase assay.Meanwhile,accumulation of section 4 of MMP13promoterwere significantly enhanced by means of Ch IP.And there was no significant difference of luciferase strength after site-directed mutagenesis of section 4 of MMP13promoter.[Conclusion]In osteogenesis,Dlx2 could increase transcription level of osteocalcin through directly binding to promoter of osteocalcin,leading to promotion of osteogenic differentiation of Mc3T3 E1 cells and m BMSCs.While in chondrogenesis,Dlx2 could decrease transcription and translation level of MMP13 through directly binding to its promoter,followed by slowed degradeation of extracellular matrix of TMC23 cells and mouse skull base chondrocytes,finallythe cease of differentiation of chondrogenesis at hypertrophyic stage.