Mediator 1 contributes to enamel mineralization as a coactivator for Notch1 signaling and stimulates transcription of the alkaline phosphatase gene

Keigo Yoshizaki,Lizhi Hu,Thai Nguyen,Kiyoshi Sakai,Masaki Ishikawa,Ichiro Takahashi,Satoshi Fukumoto,Pamela K Denbesten,Daniel D Bikle,Yuko Oda,Yoshihiko Yamada

doi:10.1074/jbc.m117.780866

Abstract

Tooth enamel is mineralized through the differentiation of multiple dental epithelia including ameloblasts and the stratum intermedium (SI), and this differentiation is controlled by several signaling pathways. Previously, we demonstrated that the transcriptional coactivator Mediator 1 (MED1) plays a critical role in enamel formation. For instance, conditional ablation of Med1 in dental epithelia causes functional changes in incisor-specific dental epithelial stem cells, resulting in mineralization defects in the adult incisors. However, the molecular mechanism by which Med1 deficiency causes these abnormalities is not clear. Here, we demonstrated that Med1 ablation causes early SI differentiation defects resulting in enamel hypoplasia of the Med1-deficient molars. Med1 deletion prevented Notch1-mediated differentiation of the SI cells resulting in decreased alkaline phosphatase (ALPL), which is essential for mineralization. However, it does not affect the ability of ameloblasts to produce enamel matrix proteins. Using the dental epithelial SF2 cell line, we demonstrated that MED1 directly activates transcription of the Alpl gene through the stimulation of Notch1 signaling by forming a complex with cleaved Notch1-RBP-Jk on the Alpl promoter. These results suggest that MED1 may be essential for enamel matrix mineralization by serving as a coactivator for Notch1 signaling regulating transcription of the Alpl gene.

Highlights

Tooth enamel is mineralized through the differentiation of multiple dental epithelia including ameloblasts and the stratum intermedium (SI), and this differentiation is controlled by several signaling pathways
These results indicate that enamel matrix mineralization is inhibited in Med1 KO teeth, whereas enamel matrix proteins are present
We examined the expression levels of Notch receptors (Notch1 and Notch2) and Notch ligands (Jag1 and Jag2) and its target gene, Hes1, by qPCR using mRNA from dental epithelial tissues dissected from postnatal day 7 (P7) molars of Med1 KO and CON mice (Fig. 3). qPCR analyses showed that mRNA levels of Notch1 and Hes1 were down-regulated in KO molars compared with CON molars (Fig. 3A)

Summary

To whom correspondence may be addressed

Using the dental epithelial SF2 cell line, we demonstrated that MED1 directly activates transcription of the Alpl gene through the stimulation of Notch signaling by forming a complex with cleaved Notch1–RBP-Jk on the Alpl promoter. These results suggest that MED1 may be essential for enamel matrix mineralization by serving as a coactivator for Notch signaling regulating transcription of the Alpl gene. Our study demonstrates that MED1 promotes SI differentiation and activates the gene transcription of Alpl via Notch signaling, which is required for enamel matrix mineralization

Results

Discussion

Experimental procedures