The Role Of Histone Demethylase Jmjd3 Regulating Periodontal Ligament Cells Differentiate Into Osteoblasts

Objective: Chronic apical periodontitis is one of the major diseases causing bone destruction in the periapical period.Studies confirm that histone methylation is closely related to cell differentiation.To investigate the role of histone demethylase Jumonji domain-containing 3(Jmjd3)regulating periodontal ligament cells differentiation into osteoblasts.Methods: Periodontal ligament cell line SH-9 cells were cultured in the osteoblast differentiation medium containing 50?g/ml L-ascorbic acid(L-ASA)and 6mM ?-glycerophosphate(?-GP)for different periods(0,3,7,10 days).The expressions of Jmjd3 and bone-related genes including RUNX2,Osterix,and OCN mRNA were detected by real-time RT-PCR.The expression of Jmjd3 protein was examined by Western Blot.SH-9 cells were pretreated with 0,1,10?M GSK-J4,which is the specific inhibitor of Jmjd3,and cultured in the osteoblast differentiation medium for 3 days.The expressions of RUNX2,Osterix,and OCN mRNA were detected by real-time RT-PCR.SH-9 cells were transfected with nonspecific(siCont)or siRNA targeting Jmjd3(siJmjd3)siRNA and cultured for 3 days in the osteoblast differentiation medium,the expressions of Jmjd3,RUNX2,Osterix,and OCN mRNA was detected by real-time RT-PCR.The siCont and siJmjd3 cells were cultured in the osteoblast differentiation medium for 14 days.The cells were stained for mineralization by Alizarin red staining.Statistical analysis was performed using one-way ANOVA and Dunnett t test with SPSS 18.0 software package.Results: The level of Jmjd3 mRNA and the expressions of bone-related genes RUNX2,Osterix,and OCN in SH-9 cells cultured in the osteoblast differentiation medium increased in a time-dependent manner,as determined by real-time RT-PCR(P<0.01).Maximal induction of Jmjd3 mRNA expression was found in SH-9 cells treated with differentiation medium for 7 days(P<0.01).From Western blot analysis,the intensity of a band corresponding to Jmjd3 increased during the SH-9 cells cultured in differentiation medium.The expressions of RUNX2,Osterix,and OCN mRNA decreased significantly after pretreatment with GSK-J4 for 2 hours(P<0.05).Moreover,silencing of Jmjd3 decreased the expression levels of bone-related genes RUNX2,Osterix,and OCN(P<0.01),resulting in the suppression of Periodontal ligament cells osteoblast differentiation and mineralization.Conclusions: Histone demethylase Jmjd3 plays pivotal role in regulating osteoblast differentiation and mineralization in Periodontal ligament cells.