The Mechanism Of Bmi1 And P27 On Tooth And Mandible Development

To explore the mechanism of action of the polycomb repressor Bmi1 in the tooth development and intramembranous ossification.After weaning,Bmil gene knockout(Bmi1-1-)mice were fed on a normal diet with or without supplied with antioxidant N-acetylcysteine(NAC)for 2 weeks and phenotypes of the tooth and mandibles were compared each other and their wild-type littermates on the normal diet by radiograph,histochemistry and immunohistochemistry.Alterations of oxidative stress,DNA damage,cell proliferation and cell cycle-related parameters were also examined in mandibles.Results showed that the tooth volume and the dentin sialoprotein immunopositive areas,the cortical thickness,alveolar bone volume,osteoblast number and activity,and mRNA expression levels of Runx2,alkaline phosphotase and type I collagen were all reduced significantly in Bmil-/-mice compared with their wild-type littermates,whereas these parameters were increased significantly in NAC-treated Bmil1/-mice compared with untreated Bmi1-/-mice,although they were not normalized.The activities of superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px)were reduced,DNA damage markers including y-H2AX and 8-oxoguanine levels were increased,the number of Ki67 positive cells was decreased,whereas protein expression levels of p16,p19,p21,p27 and p53 were up-regulated in teeth and mandibles from Bmi1-/-mice compared with those from wild-type mice;alterations of these antioxidative enzyme activities,DNA damage markers,cell proliferation and cell cycle-related parameters were all partially rescued by the treatment with antioxidant NAC in Bmi1 deficient mice.These results demonstrated that Bmi1 deficiency contributes to defects in dental and alveolar bone formation,while treated with antioxidant could improve these defects obviously.Therefore,our results indicate that Bmi1 plays an important role in stimulating tooth formation and alveolar bone formation by maintaining redox homeostasis,preventing DNA damage and inhibiting cyclin-dependent kinase inhibitors.To determine whether the deletion of p16 can correct tooth and mandible growth retardation caused by Bmi1,we compared the tooth and mandible phenotypes of homozygous p16-deficient(p16-/-)mice,homozygous Boil-deficient(Bmi1-/-)mice,double homozygous Bmi1-and p16-deficient(Bmi1-/-p16-/-)mice to those of their wild-type littermates at 4 weeks of age by radiograph,histochemistry and immunohistochemistry.Results showed that dental volume and dentin sialoprotein immunopositive areas,the ratio of the predentin area to total dentin area and that of biglycan immunopositive area to dentin area had no significant difference in p16-t-mice compared to their wild-type littermates.Compared to Bmil-/-mice,the dental mineral density,dental volume and dentin sialoprotein immunopositive areas were increased whereas the ratio of the predentin area to total dentin area and that of biglycan immunopositive area to dentin area were decreased in Bmi1-/-p16-/-mice.These results indicate that the deletion of p16 can improve tooth development in Bmi1 knockout mice.Compared to their wild-type littermates,the mandible mineral density,cortical thickness,alveolar bone volume,osteoblast number and activity,alkaline phosphotase positive area were all increased significantly in the mandibles;while TRAP-positive osteoclast number and surface area were also increased in the trabecular bone of the mandibles in p16-/-mice.Compared to Bmil-/-mice,the mandible mineral density,cortical thickness,alveolar bone volume,osteoblast number and activity,alkaline phosphotase positive area were all increased significantly in the mandibles;while TRAP-positive osteoclast number and surface area were also increased in the trabecular bone of the mandibles in.These results indicate that the deletion of p16 can improve mandible growth in Bmi1 knockout mice.Furthermore,the protein expression levels of cyclin D,CDK4 and p53 were increased significantly in p16-/-mice compared with those from wild-type mice;the protein expression levels of cyclin D and CDK4 were decreased significantly,whereas that of p27 and p53 were increased significantly;these parameters were partly rescued in Bmi1-/-p16-/-mice compared with those from Bmi1-/-mice.Our results indicate that Bmil plays an important role in stimulating tooth formation and alveolar bone formation by maintaining redox homeostasis,preventing DNA damage and inhibiting cyclin-dependent kinase inhibitors.These results demonstrated that the deletion of p16 can improve defects in dental and alveolar bone formation,which is induced by Bmi1 deficiency.Therefore,our results indicate that Bmi1 plays roles in regulating tooth and mandible development by inhibiting p16 signal pathway which initiated entry into cell cycle to stimulate cell proliferationAccording to our study,p27 as one of cyclin-dependent kinase inhibitors is crucial to the tooth and mandible formation.To assess the roles of p27 in hard tissue formation in oro-facial tissues,we examined the effect of p27 deficiency on dental formation and alveolar bone development in mice.At 2 weeks of age,the dental mineral density,dental volume and dentin sialoprotein immunopositive areas were increased whereas the ratio of the predentin area to total dentin area and that of biglycan immunopositive area to dentin area were decreased in p27-/-mice compared to their wild-type littermates.The mandible mineral density,cortical thickness,alveolar bone volume,type ? collagen immunopositive areas,osteoblast number and activity,and mRNA expression levels of Runx2,alkaline phosphotase,osteocalcin and Bmp2 were all increased significantly in the mandibles;while TRAP-positive osteoclast number and surface area were also increased in the trabecular bone of the mandibles in p27-/-mice.Furthermore,the PCNA positive cell number in Hertwig's epithelial root sheath and the protein expression levels of cyclin E and CDK2 were increased significantly in p27-/-mice.These results demonstrated that p27 deficiency resulted in the upregulation of Cyclin E and CDK2 expression and cell proliferation,increasing of dentin formation and avelar bone volum,therefore accelerated the development of tooth and mandible.Results from this study indicate that p27 plays a negative regulatory role in dentin formation and alveolar bone development.This study not only illustrated mechanisms of Bmil and p27 on tooth and mandible development,but also provide experimental and theoretical basis for Bmi1 and p27 as new targets for prevention and treatment on diseases of dental and mandibular formation.