| Objectives : Odontoblast differentiation is an important process during tooth development and dentin formation,in the process dental papiila cells differentiate into pre-odontoblasts,then pre-odontoblasts undergo organelle and cytoskeleton rearrangement,cell elongation,polarization,and finally become mature secretory odontoblasts.Many factors have been found to regulate the process.Histone deacetylase 6(HDAC6)is a member of the histone deacetylase family,its substrates includes acetylated tubulin,cortactin and so on.It plays important roles in cell shape maintain,cell polarity,cell migration,proliferation and differentiation and so on.Recently studies found it was involved in modulation of osteoblast differentiation.But researches of HDAC6 on odontoblast differentiation are rare,so in our study we aim to reveal the expression and function of HDAC6 in odontoblast differentiation.Methods: PN2.5 mice mandibles were dissected and prepared for tissue section of mice molar and incisor.Then immunohistochemistry and immunofluorescence staining were used for detecting the expression of HDAC6 and colocalization of HDAC6 and DSP in vivo.Human dental papilla cells(h DPCs)were isolated from human healthy third molar germ and induced by odontoblastic induction medium in vitro.Expression levels of HDAC6 and odontoblast differentiation marker proteins were assessed by western blot during odontoblastic differentiation of h DPCs in vitro.Knocking down and enzymatic activity inhibition of HDAC6 were performed to investigate its function in odontoblastic differentaition.After that,western blot was used to detect the expression of HDAC6 and odontoblast differentiation marker proteins.What is more,immunofluorescence staining was used to detect the expression of HDAC6 in cells and the change of DMP1.Mineralized nodules and ALPase activity were also detected.Results: We found HDAC6 was up regulated during odontoblast differentiation in mice molar and incisor in vivo and odontoblastic differentiation of h DPCs in vitro,and it showed colocalization with DSP in the cytoplasm of mature odontoblasts in mice.Knockdown or inhibition of HDAC6 in h DPCs led to the decrease of the expression of odontoblast marker proteins DSP,DMP1,OSX,the decline of mineralized nodules number and ALPase activity,indicating its positive role in odontoblast differentiation.Conclusion: HDAC6 expressed in mice molar and incisor odontoblasts and h DPCs,and it expressed increasingly during odontoblast differentiation in vivo and in vitro,what is more,it was able to promote odontoblastic differentiation of h DPCs.Objectives:Autophagy is a process regulated by autophagy related genes.The process includes the formation of a double membrane vesicles,the autophagosomes,which encapsulated cargos and transport them to lysosomes for degradation.It is involved in degradation and recycling of proteins and organelles and supplies energy to maintain cell homeostasis.Previous studies have shown that autophagy played important roles in tooth development and odontoblastic differentiation in inflammatory environment.What is more,HDAC6 could regulate autophagy through several mechanisms in neurodegenerative disease and cancer.However,the relationship of HDAC6 and autophagy in odontoblasts have not been investigated before.In this study,we aim to investigate the relationship of HDAC6 and autophagy during odontoblast differentiation and whether HDAC6 promotes odontoblastic differentiation of h DPCs through autophagy.Methods: PN2.5 mice mandibles were dissected and prepared for tissue section of mice incisor.Then immunohistochemistry was used for detecting the expression of ATG5 and LC3 in PN2.5 mice incisor.Double immunofluorescence staining and western blot were used for detecting the expression pattern of HDAC6 and autophagy related proteins during odontoblast differentiation in vivo and odontoblastic differentiation of h DPCs in vitro.si RNA was used to knock down HDAC6 in h DPCs,then immunofluorescence staining and western blot were used for detecting the change of HDAC6 substrates and proteins involved in autophagy process.At the same time,the m Cherry-GFP-LC3 plasmid was used to monitor autophagy flux.Results: ATG5 and LC3 expressd increasingly during odontoblast differentiation in vivo.HDAC6 showed colocalization with ATG5 and LC3 during odontoblast differentiation in PN2.5 mice incisor,they showed low expression in undifferentiated cells and high expression in mature polarized odontoblasts.They also similarly expressed increasingly during odontoblastic differentiation of h DPCs in vitro.When HDAC6 was knocked down in h DPCs,the expression of LC3 puncta was increased,the proteins involved in autophagy process were also changed,which indicaing that HDAC6 regulated autophagy in h DPCs.During odontoblastic differentiation of h DPCs,when HDAC6 was knocked down,the expression of autophagy related proteins also changed,which was that ATG5 of autophagy elongation stage showed no change,while LC3 II and P62 involved in the fusion and degradation stages of autophagy increased,indicating that autophagy degradation was blocked.Meanwhile the m Cherry-GFP-LC3 plasmid also showed that autolysosomes were decreased while autophagosomes were accumulated when HDAC6 was knocked down,indicating the fusion of autophagosomes and lysosomes was blocked.Finally,when HDAC6 was knocked down,the expression of its substrate ac-Tubulin was up regulated,and its immunofluorescence intensity and distribution in cells were increased,while LC3 puncta were located among ac-Tubulin.Conclusion: HDAC6 regulates the fusion of autophagosomes and lysosomes through the microtubule to maintain autophagy flux and autophagy activity,and thus it is involved in odontoblast differentiation. |
