Localization of SUMOylation factors and Osterix in odontoblast lineage cells during dentin formation and regeneration

Small ubiquitin-related modifier (SUMO) conjugation (SUMOylation) is a post-translational modification involved in various cellular processes including the regulation of transcription factors. In this study, to analyze the involvement of SUMOylation in odontoblast differentiation, we examined the immunohistochemical localization of SUMO-1, SUMO-2/3, and Osterix during rat tooth development. At the bud and cap stages, localization of SUMOs and Osterix was hardly detected in the dental mesenchyme. At the bell stage, odontoblasts just beginning dentin matrix secretion and preodontoblasts near these odontoblasts showed intense immunoreactivity for these molecules. However, after the root-formation stage, these immunoreactivities in the odontoblasts decreased in intensity. Next, to examine whether the SUMOylation participates in dentin regeneration, we evaluated the distribution of SUMOs and Osterix in the dental pulp after cavity preparation. In the coronal pulp chamber of an untreated rat molar, odontoblasts and pulp cells showed no immunoreactivity. At 4 days after cavity preparation, positive cells for SUMOs and Osterix appeared on the surface of the dentin beneath the cavity. Odontoblast-like cells forming reparative dentin were immunopositive for SUMOs and Osterix at 1 week, whereas these immunoreactivities disappeared after 8 weeks. Additionally, we further analyzed the capacity of SUMO-1 to bind Osterix by performing an immunoprecipitation assay using C2C12 cells, and showed that Osterix could undergo SUMOylation. These results suggest that SUMOylation might regulate the transcriptional activity of Osterix in odontoblast lineage cells, and thus play important roles in odontoblast differentiation and regeneration.