Methods: 1) Combined approaches of histology, SEM, micro-CT, x-ray, and double fluorochrome labeling methods were used to characterize the dentin mineralization and tooth root defects in Osx conventional knockout (KO) and conditional knockout (CKO) mice crossed with the 2.3 kb Col 1a1 Cre mice; 2) An odontoblast cell line was transfected with Adv-CMV-Osx and analyzed by real-time RT-PCR and alizarin red S staining; 3) Promoter-luciferase assays were used to determine the effects of Osx on a 5.4-kb DKK1 (a potent Wnt signaling inhibitor) promoter fragment in COS7 and HEK293 cells.
Results: Osx KO, or Osx CKO mice displayed no apparent changes in crown formation at E18.5 and newborn. In contrast, Osx CKO mice developed a profound root phenotype characterized by short roots (>50%) in both incisors and molars; enlarged pulp chambers/root canals, malformed dentinal tubules and decreased dentin mineralization. Furthermore, there were dramatic reductions in levels of Nestin, and DSPP expression, plus a sharp increase of ß-catenin in Osx CKO pulp and odontoblast cells. Transfection of Osx in an odontoblast cell line significantly increased cell differentiation and mineralization. in vitro Promoter studies demonstrated that Osx was able to activate a 5.4 kb DKK1 promoter fragment with a 3-fold increase of luciferase activity.
Conclusions: These findings suggest that Osterix plays distinct roles in root odontoblast differentiation and mineralization through inhibition of Wnt/beta-catenin signaling.
Keywords: Dentin, Odontoblasts, Osterix, beta-catenin and Root
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