Third the ability to form calcified material due
Third, the ability to form calcified material due to the overexpression of TMSB4X may depend on the cell type. The formation of calcified material was induced in the TMSB4X-transfected squamous epithelial cell-derived carcinoma cell lines, MISK81-5 and HSC-3 cells. However, no capacity for calcification was observed in the MKN45 or MKN74 thapsigargin following the TMSB4X transfection, even though the expression level of TMSB4X was upregulated. The tooth germ is generated by segmentation of odontogenic epithelial cells from oral squamous epithelium, resulting in the formation of the dental lamina (Lesot and Brook, 2009). In a recent study, the differentiation of human foreskin keratinocytes into enamel-secreting ameloblasts was induced in a human–mouse chimeric tooth in which keratinocytes were combined with murine embryonic dental mesenchymal cells (Wang et al., 2010). Therefore, the origin of the cell type may be an important factor related to the expression of dental epithelial differentiation marker genes in keratinocytes. We previously demonstrated that Tmsb4x was expressed specifically during the initiation stage of mouse odontogenesis (Yamaza et al., 2001a; Akhter et al., 2005). We have also reported that Runx2 participates in regulating the expression of Amelx, Ambn, Dmp1 and Dspp in mice (Kobayashi et al., 2006). Moreover, our recent data (Ookuma et al., 2013) showed that treatment with a Tmsb4x AS-S-ODN resulted in growth arrest of the tooth germ in cultured E11.0 mouse mandibles and also inhibited the expression of Runx2. Taken together, these findings in this study indicate that TMSB4X may play a role in the upregulation of RUNX2 expression, and could act to induce the secretion of calcification-related enamel matrices. Smart et al. (2011) indicated that Tmsb4x possesses the ability to change the cell characteristics. Tmsb4x upregulation is associated with cell motility to accompany the differentiation of stem cells (Zhou et al., 2013). Following the upregulation of Tmsb4x, however, there is little information regarding the genes/proteins associated with the differentiation of stem cells, as observed in other previous studies. Therefore, it is necessary to provide detailed information concerning the intracellular molecular pathway by which TMSB4X leads to induction of expression of dental epithelial marker genes that are characteristic features of odontogenic epithelial cells, by identifying associated genes/proteins in a future study. The Ca/P ratio is commonly used to determine whether pure hydroxyapatite or a mixture of hydroxyapatite and its precursors is formed. The ratio of Ca/P in hydroxyapatite, Ca10(PO4)6(OH)2, is 1.67 (Keinan et al., 2006). In this study, the Ca/P ratio in the deposits was lower than the ratio of Ca/P in pure hydroxyapatite, thus suggesting that the deposits were a mixture of hydroxyapatite and its precursors. The high concentration of β-GP may induce a supraphysiological P level in the cell culture medium, thereby resulting in the observed lower Ca/P values. Chung et al. (1992) reported that exceeding 2mM β-GP can induce supraphysiological levels of medium phosphate, although medium supplemented with 10mM β-GP is commonly used in studies of osteoblast cultures to promote calcium phosphate deposits in vitro. Alternatively, the deposited component may be hypomineralized during analysis time (Jälevik, 2001). There were no apparent changes in the area of the mineralized regions at three to six weeks in the in vivo implants. Enamel and dentin formation, as well as calcification, are initiated at the late bell stage. At the same time, cell proliferation of the inner enamel epithelial cells ceases in the regions where the cusps are presumably formed (D\'Souza et al., 2013). In addition, secretion of AMBN and calcification may play a role in inhibiting the proliferation of ameloblasts (Fukumoto et al., 2004). In this study, the TMSB4X-transfected HaCaT cells showed decreased cell proliferation activity. In addition, the co-localization of enamel matrix proteins, including ameloblastin, and calcifying material was observed. Thus, the inhibitory effect of enamel matrix proteins on the cell proliferation activity may not cause any apparent changes in the sizes of the mineralized regions observed at three to six weeks in the in vivo implants. In future studies, it will be necessary to clarify the detailed mechanisms underlying this matter.