Expression and Function of NUMB in Ameloblast Differentiation

Objective. To determine whether NUMB plays roles in the differentiation of the dental cells. Background. NUMB has been implicated to play roles in lineage commitment by both gain- and loss-of function approaches. Its function has been attributed to the asymmetric distribution in daughter cells. Methods. RT-PCR, western blot, immunocytochemistry, confocal microscopy and PCR super-array analysis were applied to study the expression and signaling regulatory effects of NUMB. Results. We isolated 2 fulllength clones for NUMB from mouse dental pulp mRNA. One novel sequence contains 200bp insertion in the Phosphotyrosine Binding Domain (PTB) may encode for a new isoform of NUMB. Confocol microscopy detected strong NUMB expression in human dental pulp stem cells (hDPSC), odontoblasts (Od) and pre-ameloblasts (pre-Ab). Western blot analysis indicated NUMB isoforms were differentially expressed in dental tissues. In postnatal mouse tooth germs, NUMB was expressed in the preameloblasts and Odontoblasts, cervical loops, and DPSC in the vicinity of the immature odontoblasts. NUMB overexpression in pre-ameloblasts, significantly reduced activated Notch1 protein expression level. In the super array analysis of Notch1 signaling, NUMB down regulates Shh expression in preamelobasts. Conclusion. New NUMB mRNA was detected in dental pulp cells. NUMB has a temporal expression pattern in developing tooth germs suggesting critical role of NUMB in ameloblasts and odontoblasts differentiation. NUMB negatively regulate activated Notch 1 and SHH signaling in Hat-7 cells. Grants. This work was supported by NIH grant DE 11657 and the Department of Orthodontics at UIC.