| Objectives:Amelogenesis imperfecta(AI)is a group of inherited diseases with abnormal enamel development.A lot of candidate genes for AI have been reported,the FAM83H gene is considered as the major pathogenic gene of autosomal dominant hypocalcified AI(ADHCAI).All of the mutations reported have been located in the final,largest exon 5 and all but two are nonsense or frameshift mutations lead to premature translation termination.Currently,the mechanism of FAM83H mutation leading to ADHCAI is not clear.The aim of this study was to investigate the effect of Fam83h mutation on the mineralization of mouse ameloblast-cell-line LS8 and to explore the possible pathogenesis of ADHCAI.Methods:1.We constructed four Fam83h mutant plasmid:mutation 1(c.de1706-708GAC,p.de1236D),mutation2(c.860C>A,p.D287X),mutation3(c.1186C>T,p.Q396X),mutation4(c.2431C>T,p.Q811X)using the site-directed mutagenesis technique,then carried the lentivirus package for them and control,named as MUT1,MUT2,MUT3,MUT4 and Control respectively,and the above virus were transfected to the LS8 cells and puromycin were used to screen out stable transfected cells,the MUT3 and Control group cells were used for subsequent experiments,named M3-FLAG and Control;2.Co-immunoprecipitation assay was used to detect the interaction between Fam83h and CKla in the LS8 cells;3.Western blot was used to detect the changes of the expression and localization of Fam83h,CK1? and P-catenin in the cytoplasm,nucleus and whole protein of the M3-FLAG and the Control groups,and verified by immunofluorescence;4.ALP activity and ALP staining were detected at 3d,7d,10d and 14d after rmineralization induced in the M3-FLAG and the Control groups,the mRNA expression of mineralization related indicators(Runx2,Alp and Ocn)was detected by qRT-PCR;5.The LS8 and M3-FLAG cells were exposed to CK1? activator,pyrvinium pamoate,a inhibitor of classical wnt/p-catenin signaling pathway,the mRNA expression of mineralization related indicators(Runx2,Alp and Ocn)were detected in mineralization induced day7 and day 14 respectively.Results:1.We successfully constructed mutant Fam83h plasmids,the PCR products were verified by sequencing.LS8 cells were transfected with lentivirus particles,stable transfectants were screened with puromycin;2.Co-immunoprecipitation results showed that mutant Fam83h interacts with CK1? in LS8 cells;3.Western blot and immunofluorescence showed that mutant Fam83h was mainly expressed in the nucleus,and a small amount was expressed in the cytoplasm.The expression of CKla were inceased in the nucleus and decreased in the cytoplasm,and the expression of ?-catenin were increased in the nucleus and cytoplasm.?-catenin and CK1? were important members of the canonical Wnt/p-catenin signaling pathway;4.The results of mineralization suggested that ALP activity and the mRNA expression level of mineralization related indicators(Runx2,Alp and Ocn)were down-regulated in M3-FLAG group along with the induction of mineralization,ALP staining in M3-FLAG group was lighter than that in Control group;5.qRT-PCR results showed that CKla activator pyrvinium pamoate up-regulated the mRNA expression of mineralization related indicators(Runx2,Alp and Ocn)in LS8 and M3-FLAG cells and rescued the inhibited mineralization in M3-FLAG.Conclusion:Our results indicated that CK1? decreased in the cytoplasm along with the mutant Fam83h entering the nucleus lead to the decrease of phosphorylation of P-catenin,the accumulation of nuclear translocation,the activation of the classical wnt/?-catenirn signaling pathway and the inhibition of LS8 mineralization.However,Classical Wnt/?-catenin signaling pathway inhibitors rescued the mineralization inhibition caused by Fam83h mutations.In brief,the Fam83h mutation altered its protein intracellular localization in LS8 cells and inhibited the mineralization of this cell,the mechanism possibly were associated with the Classical Wnt/p-catenin signaling pathway. |
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