Gap junctional communication regulates TGF-b1 induced ameloblastin expression

Objectives: Tooth development was regulated by their specific gene expression. We found that Gja1 is specifically expressed in tooth germ. Gja1 is one of the gap junctional proteins that oligomerize to form membrane channels and regulate cell-cell communications. Here, we analyzed the role of Gja1 in tooth using dental epithelial cell line and Gja1-null mice.

Methods: Tooth phenotypes of Gja1-null mice were analyzed by H-E staining. Dental epithelial cell line HAT-7 cells were cultured with D-MED/F-12 supplement with 10%FBS. Ameloblastin expression in HAT-7 cells after stimulation of TGF-b1 with or without Gap junction inhibitor oleamide and 18a-glycyrrherinic acid (18a-GA) was analyzed by RT-PCR. Proliferation was measured by BrdU incorporation method. ERK1/2 and smad phosphorylation were analyzed by western blotting.

Results: Gja1-null mice showed inhibition of tooth germ, salivary gland, and lung morphogenesis, similar to occulodentdigital dysplasia (ODDD). In mutant mice, expression of ameloblastin in ameloblasts was dramatically reduced comparing with wild type. Mutant ameloblasts lose cell polarity as similar to ameloblastin knockout mice. To analyze the regulatory mechanism of ameloblastin expression by Gja1, we used oleamide and 18a-GA. TGF-b1 induced ameloblastin expression transiently in dental epithelial cells. Ameloblastin expression induced by TGF-b1 was inhibited in presence of oleamide. Simillar results were observed in over-expression of mutant Gja1, R76S and R202H, which were gene mutations observed in ODDD patients. On the other hand, over-expression of wild type Gja1 dramatically inhibited proliferation of dental epithelial cells. TGF-b1 induced smad2/3 and ERK1/2 phosphorylation in dental epithelium. However, phosphorylation of ERK1/2, but not smad2/3 was dramatically inhibited in the presence of oleamide and in Gja1 mutant cells.

Conclusion: Gja1 is important for tooth development, especially ameloblast differentiation. Gja1 may regulate proliferation and ameloblastin expression via ERK1/2 signaling in dental epithelium and associated with ODDD phenotype in tooth, especially anodontia, microdontia and amelogenesis imperfecta.