Study On Nell-1 And APR3 Interaction And Its Biological Significance During Human Dental Pulp Cells Differentiation And Mineralzation

ObjectiveNel-like molecule 1(Nell-1)is an essential positive regulator of bone formation and regeneration.Nell-1 also participates in tooth development and odontoblast differentiation.However,its precise mechanism remains undetermined.This study aims to explore the possible receptor or binding protein of Nell-1.Materials and methodsDouble immunofluorescence staining and Co-immunoprecipitation were performed to detect the expression and co-location of APR3 and Nell-1 in human dental pulp cells.The expression of Nell-1 and APR3 was measured using quantitative real time polymerase chain reaction(RT-PCR)and western blot(WB)during human dental pulp cells differentiation and mineralization.The lentivirus vectors containing Nell-1 and APR3 genes were transfected to human dental pulp cells.Next,alkaline phosphatase activity assay was examined.The expression of related odontoblastic molecular markers was determined by RT-PCR and WB.The capacity of forming mineralized nodules was investigated by alizarin red staining.Moreover,after knocking out APR3,we also evaluated the above experiments.ResultsNell-1 and Apoptosis related protein 3(APR3)expression levels were high in odontoblasts and inversely correlated.Endogenous Nell-1 co-immunoprecipitated with APR3,and this co-IP was reciprocal.Double immunofluorescence staining revealed that Nell-1 and APR3 were colocalized on the nuclear envelope of human dental pulp cells.Nell-1 inhibited the proliferation of these cells co-infected with APR3 through Cyclin D1 downregulation.The interaction of Nell-1 and APR3 stimulated alkaline phosphatase(ALP)activity and promoted the expression and mineralization of DSPP,ALP,OPN,and BSP.The shRNA of APR3 could decrease cell differentiation and mineralization.DiscussionNell-1 and APR3 interaction could positively regulate the differentiation and mineralization of human dental pulp cells,partially mediating through Cyclin D1 downregulation.Future studies should explore the potential functional connection and the molar mechanism of such interaction.