Identification, Distribution, and Expression of Nucleobindin During Tooth Formation

Objective: Nucleobindin, a Ca²⁺- binding protein, was first identified as an intracellular DNA-binding protein, detected in the Golgi apparatus and endoplasmic reticulum. Moreover, nucleobindin has been identified as a secreted protein in bone extracellular matrix. The structure of nucleobindin has been shown to contain two EF hands corresponding to Ca²⁺-binding sites, a signal sequence, a nuclear targeting sequence and a leucine zipper. A variety of functions have been suggested for nucleobindin, which includes Ca²⁺- binding in the Golgi, interaction with G-proteins and has been postulated to posses an important role in Ca²⁺- regulated signal transduction events. The aim of the present study was to examine the distribution and gene expression of nucleobindin in rat tooth. Methods: Western blot analysis was performed, whereby total rat dentin protein extracts were probed with a synthetic peptide antibody specific for the C- terminal of nucleobindin. The distribution of nucleobindin within the tooth was examined by immunohistochemistry and immuno-electron microscopy and furthermore, transcript for nucleobindin was studied in tooth by Northern blot analysis and in situ hybridisation. Results: Nucleobindin was identified at a molecular weight of 63 kDa in extracts of rat dentin by Western blot analysis. Immuno-electron microscopy markers observed nucleobindin within odontoblasts, in the ER and mitochondria. In agreement, immunohistochemistry localised nucleobindin in the entire odontoblast layer and also in ameloblasts. By in situ hybridisation a specific signal for nucleobindin mRNA was detectable in odontoblasts and ameloblasts. Northern blot analysis of total RNA from rat tooth identified one band at 2.4 kb, supporting the finding that nucleobindin is produced in tooth. Conclusion: The present study localised nucleobindin within the odontoblasts, indicating that nucleobindin is synthesised in odontoblasts and thereby may play a role in mineralisation of dentin.