IADR Abstract Archives
Enamel Hyper-calcification Caused by Histone Methyltransferase Ezh2 Knock-out
Objectives: Enhancer of Zeste Homology 2 (EZH2) catalyzes histone methylation, which is a key factor of cranial neural crest cell development. It has been published that Ezh2 knock-out in uncommitted mesenchymal cells resulted in various defects in skeletal patterning and bone formation. Meantime, relationship between histone methylation and tooth development are less investigated by far. We reveal contribution of histone methylation on tooth development by analyzing Ezh2 knock-out mice teeth.
Methods: Physical characteristics of incisors from Prrx1-Cre:Ezh2fl/fl (Ezh2-KO) mice and their wild type siblings were examined by microCT, scanning electron microscope (SEM), and nano-indentation hardness test. Gene expression of ameloblast makers in both enamel organ culture and incisors from mice, treated or untreated with Ezh2 inhibitor, were analyzed with qPCR. Immunohistochemistry for ameloblast markers were performed using the decalcified maxilla of the Ezh2-KO and wild type mice.
Results: The Ezh2-KO mice showed increased mineral density in the incisor enamel compared to the wild type. Hardness and modulus of the KO mice enamel increased about 47.5% and 40.4%, respectively. Likewise, SEM analyses of incisors from the Ezh2-KO mice showed enamel hyper-calcification which has a high intrinsic electron density whereas the structures of keyhole-shaped enamel rods and dentinal tubules were normal. From ameloblast marker expression analysis, interestingly, we found significant decrease of ameloblastin and increase of KLK4 in Ezh2-KO mice. Moreover, immunohistochemistry of the KO mice ameloblasts showed decreased ameloblastin expression and KLK4 expression started in the earlier stage of ameloblast development.
Conclusions: In this study, we found that Ezh2 knock-out in ameloblasts promotes enamel hyper-calcification. Our analysis suggested that the decreased matrix protein, due to the decreased ameloblastin and hastened KLK4 expression, is cause of the hyper-calcification. These findings about the Ezh2 function on enamel formation could contribute to developing new amelogenesis imperfecta treatments in the future.
Methods: Physical characteristics of incisors from Prrx1-Cre:Ezh2fl/fl (Ezh2-KO) mice and their wild type siblings were examined by microCT, scanning electron microscope (SEM), and nano-indentation hardness test. Gene expression of ameloblast makers in both enamel organ culture and incisors from mice, treated or untreated with Ezh2 inhibitor, were analyzed with qPCR. Immunohistochemistry for ameloblast markers were performed using the decalcified maxilla of the Ezh2-KO and wild type mice.
Results: The Ezh2-KO mice showed increased mineral density in the incisor enamel compared to the wild type. Hardness and modulus of the KO mice enamel increased about 47.5% and 40.4%, respectively. Likewise, SEM analyses of incisors from the Ezh2-KO mice showed enamel hyper-calcification which has a high intrinsic electron density whereas the structures of keyhole-shaped enamel rods and dentinal tubules were normal. From ameloblast marker expression analysis, interestingly, we found significant decrease of ameloblastin and increase of KLK4 in Ezh2-KO mice. Moreover, immunohistochemistry of the KO mice ameloblasts showed decreased ameloblastin expression and KLK4 expression started in the earlier stage of ameloblast development.
Conclusions: In this study, we found that Ezh2 knock-out in ameloblasts promotes enamel hyper-calcification. Our analysis suggested that the decreased matrix protein, due to the decreased ameloblastin and hastened KLK4 expression, is cause of the hyper-calcification. These findings about the Ezh2 function on enamel formation could contribute to developing new amelogenesis imperfecta treatments in the future.