Ectodermal organ development is initiated by inductive tissue interactions. Developing teeth, epidermis, hair, and limbs are classic examples of such inductive processes. Tooth development can be divided into the initiation, bud, cap, and bell stages. In mice, tooth development begins at E11.5 by thickening of the dental epithelium. The dental lamina undergoes further proliferation and subsequently develops into the tooth bud and germ. The tooth bud is formed by the invagination of the placode and the condensation of mesenchyme cells adjacent to the bud. At the cap stage (E14.5), dental epithelial cells differentiate into several cell types, such as the inner dental epithelium and enamel knot cells. Cell death by apoptosis within the enamel knot is critical for cusp formation in molars. At the bell stage (E17.5), the dental mesenchyme differentiates into dentin matrix-secreting odontoblasts, and the inner dental epithelial cells differentiate into enamel matrix-secreting ameloblasts. The goal of the project is to to understand how tooth and craniofacial tissues develop, and to define the molecular defects underlying the anomalies of these tissues. We previously identified ameloblastin (AMBN) as a new member of the enamel matrix. AMBN is secreted by ameloblasts, localizes at the apical region of the cells, and serves as the adhesion molecule for ameloblasts. In AMBN-deficient mice, ameloblasts are detached from the enamel matrix, continue to proliferate and form a multiple cell layer, and often odontogenic tumors develop in the maxilla with age. However, the mechanism of AMBN functions in these biological processes is unclear. In collaboration with a former member of the our section, Dr. Satoshi Fukumoto, we used recombinant AMBN proteins and found that AMBN had heparin-binding domains at the C-terminal half, and that these domains were critical for AMBN binding to dental epithelial cells. We previously reported that about 20% of AMBN-deficient mice developed an odontogenic tumor of dental epithelium origin in the buccal vestibule of the maxilla. The epithelial cells of odontogenic tumors express enamel matrix proteins, including amelogenin, enamelin, and tuftelin. However, they do not express AMBN, which suggests that AMBN deficiency may be the primary cause of tumorigenesis seen in those mice. The appearance of an ameloblastoma in the jaw is the most frequently encountered odontogenic tumor, and is characterized by benign but locally invasive behavior with a high rate of recurrence. Since abnormal proliferation and growth of ameloblastoma cells easily destroy surrounding bony tissues, wide excision is required to treat this disorder. Associations of AMBN mutations were reported in ameloblastomas, adenomatoid odontogenic tumors and squamous odontogenic tumors. These results suggest that AMBN regulates odontogenic tumor formation. We showed that overexpression of recombinant AMBN inhibits proliferation of human ameloblastoma AM-1 cells. This inhibition requires the heparin-binding sites of AMBN and is accompanied by dysregulation of Msx2, p21 and p27. These results suggest that AMBN functions as an odontogenic tumor suppressor. It was previously shown that Msx2, a homeobox-containing transcription factor, was expressed in undifferentiated ameloblasts, while it was downregulated in differentiated ameloblasts. In AMBN-defective ameloblasts, an abnormal upregulation of Msx2 is observed, suggesting that AMBN inhibits the expression of Msx2 in normal tooth development. Our discovery that AMBN transfection dramatically reduced Msx2 expression supports the notion that AMBN negatively regulates Msx2 expression. It has been suggested that Msx homeobox genes inhibit differentiation through upregulation of cyclin D1. In AMBN-transfected AM-1 cells, the cyclin-dependent kinase inhibitors p21 and p27 were upregulated, whereas the expression of CDK1, 4, and 6 were not changed. Thus, downregulation of Msx2 and upregulation of p21 and p27 by AMBN expression likely cause reduced proliferation of AM-1 cells. Furthermore, the overexpression of AMBN lacking three heparin-binding domains did not inhibit proliferation of AM-1 cells, suggesting the crucial role of the heparin-binding domains of AMBN for the inhibition of AM-1 proliferation. In our previous report for AMBN knockout mice (J Cell Biol. 167:973-983, 2004), we described the map of the AMBN targeting vector in which the PGK neo cassette replaced a genomic fragment from the XbaI site within exon 5 to the EcoRI site in intron 6 of the Ambn gene. However, in collaboration with Dr. Antonio Nanci's group, we recently found that the cassette replaced a fragment from the XbaI site within intron 4 to the same EcoRI site in intron 6. This resulted in the removal of a genomic segment from the 5'part of intron 4 to the 3'part of intron 6, and the loss of exons 5 and 6 in the targeted allele. This deletion predicts an in-frame translation product. Indeed, RT-PCR and Western blot analyses revealed the presence of the truncated RNA transcript and protein in enamel organs of the mutant mice.

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Nakamura, Takashi; Jimenez-Rojo, Lucia; Koyama, Eiki et al. (2017) Epiprofin Regulates Enamel Formation and Tooth Morphogenesis by Controlling Epithelial-Mesenchymal Interactions During Tooth Development. J Bone Miner Res 32:601-610
Yoshizaki, Keigo; Hu, Lizhi; Nguyen, Thai et al. (2017) Mediator 1 contributes to enamel mineralization as a coactivator for Notch1 signaling and stimulates transcription of the alkaline phosphatase gene. J Biol Chem 292:13531-13540
Miyazaki, Kanako; Yoshizaki, Keigo; Arai, Chieko et al. (2016) Plakophilin-1, a Novel Wnt Signaling Regulator, Is Critical for Tooth Development and Ameloblast Differentiation. PLoS One 11:e0152206
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Nakamura, Takashi; Yoshitomi, Yasuo; Sakai, Kiyoshi et al. (2014) Epiprofin orchestrates epidermal keratinocyte proliferation and differentiation. J Cell Sci 127:5261-72
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Kaneko, Haruka; Ishijima, Muneaki; Futami, Ippei et al. (2013) Synovial perlecan is required for osteophyte formation in knee osteoarthritis. Matrix Biol 32:178-87
Arakaki, Makiko; Ishikawa, Masaki; Nakamura, Takashi et al. (2012) Role of epithelial-stem cell interactions during dental cell differentiation. J Biol Chem 287:10590-601

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