DDIT3 Regulates Cementoblast Mineralization By Isocitrate Dehydrogenase 1 Through Nuclear Factor-κB Pathway

Objective: DDIT3 is of great importance in endoplasmic reticulum stress and is involved in many inflammatory diseases and mineralization processes.The cementum protects teeth from periodontitis and provides attachment for Sharpey’s fibers of the periodontal ligament.However,the effect of DDIT3 on cementoblast differentiation remains largely unknown.So we wanted to know whether the regulation role of DDIT3 can affect cementoblast process.Materials and methods: Immortalized mouse cementoblast cell line OCCM-30 was maintained in a growth medium for studying cementoblast differentiation in in vitro.Protein levels were measured by western blot.Message RNA levels of related genes were measured by real time q-PCR.Meanwhile,ALP staining and ALP activity assay were used to determine ALP levels during cementoblast differentiation process.Pepmute along with siRNAs were performed to reduce mRNA levels of IDH1 in occm-30 cells.All experiments were repeated at least three times.Data were analyzed using GraphPad Prism Version 5.0.Results: In this study,we found that DDIT3 was suppressed during cementoblast differentiation.Knockdown of DDIT3 increased the messenger RNA(mRNA)and protein levels of several key osteogenic markers in vitro,including alkaline phosphatase,runt‐related transcription factor 2,and osteocalcin(OCN).The results of ALP staining and ALP activity also show the same trend.In addition,isocitrate dehydrogenase 1(IDH1)was increased during cementoblast differentiation,and knockdown of DDIT3 increased the protein and mRNA levels of IDH1.Furthermore,inhibition of IDH1 could partially reduce the negative effect of DDIT3 on cementoblast differentiation.The DDIT3 knockdown activated nuclear factor‐κB(NF‐κB)transcriptional activity and upregulated the expression of p‐p65 and p‐IκBα.The increased osteogenic differentiation ability and IDH1 expression,as induced by the DDIT3 knockdown,could be partially turned over by the addition of NF‐κB inhibitor BAY 11–7082.Overall,our data clarified that DDIT3 suppresses cementoblast differentiation through IDH1,via the NF‐κB pathway.Conclusions: We showed that DDIT3 can suppress cementoblast mineralization.The mechanism might be related to the reduction of IDH1 expression via the NF‐κB pathway.The inhibition of the NF‐κB pathway or IDH1 prevents the effects of DDIT3 on cementoblast differentiation.These findings indicated that DDIT3 and IDH1 have the potential to become a therapeutic target for cementum and periodontal tissue repair.Nevertheless,this conclusion needs to be verified in the future in vivo experiment.