Mechanisms Of Osteocytic Smad4 Regulating Osteogenic Differentiation Through Notch Signaling

Objective:To establish the MLO-Y4 cells stably expressing exogenous Dll1,Dll3 and Dll4 genes to study the effects of Notch signaling Delta-like ligands Dll1,Dll3 and Dll4 on osteogenic differentiation of bone marrow stromal cells.And this is to determine whether Delta-like ligands of Notch signaling have osteogenic effects,in order to reveal the molecular mechanism of osteocytic Smad4 promoting bone formation.Methods:1.Gene cloning technology was used to prepare lentiviral vectors over-expressing Dll1,Dll3 and Dll4 genes,and three target genes were cloned into the lentiviral overexpression vector pLent-GFP-PURO-cmv(Flag).2.293 T cells were co-transfected with the recombinant lentiviral expression vector and the lentiviral packaging plasmid system(psPAX2,pMD2.G),and MLO-Y4 cells were infected with the obtained recombinant lentiviral solution.Puromycin was used to select MLO-Y4 cells stably expressing Dll1,Dll3 and Dll4 genes.The expression of target genes Dll1,Dll3,and Dll4 in the selected MLO-Y4 cells were identified by real-time fluorescence quantitative(QPCR).3.The three cell lines were co-cultured with wild-type mouse bone marrow stromal cells(BMSC),and Alkaline phosphatase(ALP)staining and quantitative determination were used to detect the changes of ALP activity in co-culture after 3 days.The expressional levels of osteogenesis-related marker genes and Notch signaling target genes in co-culture were detected by QPCR for 3 days.The above indicators were measured after we added DAPT,a Notch signal inhibitor.Results:1.Agarose gel electrophoresis and sequencing results showed that the three target genes Dll1,Dll3 and Dll4 were successfully cloned into the lentiviral overexpression vector pLent-GFP-PURO-cmv(Flag),respectively.2.Compared with the negative control group,Dll1,Dll3 and Dll4 mRNA levels in the three transfection groups were significantly increased(P<0.05).3.Compared with the negative control group,the overexpression of Dll4 in osteocytes promoted the ALP activity of BMSCs after co-culture for 3 days(P<0.05).QPCR results illustrated that the expression of osteogenesis-related marker genes such as ALP,Runx2,OPN andCollagen1 in the MLO-Y4-Dll4 group were all risen(P<0.05),and Notch signaling target genes Hey1,Hey L,Hes1 and Hes7 were also risen(P<0.05).After DAPT was added,ALP activity in the MLO-Y4-Dll4 group decreased,and the expression of osteogenesis-related marker genes and Notch signaling target genes both decreased(P <0.05).Conclusions:1.MLO-Y4 cell lines stably expressing the exogenous Dll1,Dll3 or Dll4 gene were established.2.Overexpression of Dll4 in osteocytes can promote early osteogenic differentiation of BMSC.The classic Notch signaling pathway play an important role in this process,revealing that Dll4 may play a role in promoting bone formation by Smad4 in osteocytes through Notch signaling.