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Amelogenesis involves the coordinated expression of a set of molecules that includes enamel matrix proteins and calcium-binding proteins. Msx2 is a member of the divergent homeobox gene family and is instrumental in dental morphogenesis and biomineralization. This study focused on an EF-hand calcium-binding protein, calbindin-D(28k), which is highly expressed in dental epithelium. In vivo data showed that calbindin-D(28k) levels were higher in ameloblasts from Msx2(+/-) mice than Msx2(+/+) mice. Consistent with this finding, calbindin-D(28k) distribution was affected in transgenic mice with ectopic expression in root epithelium in rests of Malassez in Msx2(+/-) and more clearly in Msx2(-/-) mice. In accordance with these in vivo data, calbindin-D(28k) protein and mRNA levels were decreased in LS8 ameloblast-like cells by exogenous Msx2 overexpression. Furthermore, calbindin-D(28k) promoter activity (nt-1075/+34) was specifically diminished in the presence of Msx2 overexpression, showing that Msx2 behave as a transcriptional repressor for calbindin-D(28k) gene expression. In conclusion, Msx2 may control the spatiotemporally restricted frame of calbindin-D(28k) production in the dental epithelium in relation to enamel mineralization, as previously shown for amelogenin.

Epithelial odontogenic tumors are rare jaw pathologies that raise clinical diagnosis and prognosis dilemmas notably between ameloblastomas and clear cell odontogenic carcinomas (CCOCs). In line with previous studies, the molecular determinants of tooth development-amelogenin, Msx1, Msx2, Dlx2, Dlx3, Bmp2, and Bmp4-were analyzed by RT-PCR, ISH, and immunolabeling in 12 recurrent ameloblastomas and in one case of CCOC. Although Msx1 expression imitates normal cell differentiation in these tumors, other genes showed a distinct pattern depending on the type of tumor and the tissue involved. In benign ameloblastomas, ISH localized Dlx3 transcripts and inconstantly detected Msx2 transcripts in epithelial cells. In the CCOC, ISH established a lack of both Dlx3 and Msx2 transcripts but allowed identification of the antisense transcript of Msx1, which imitates the same scheme of distribution between mesenchyme and epithelium as in the cup stage of tooth development. Furthermore, while exploring the expression pattern of signal molecules by RT-PCR, Bmp2 was shown to be completely inactivated in the CCOC and irregularly noticeable in ameloblastomas. Bmp4 was always expressed in all the tumors. Based on the established roles of Msx and Dlx transcription factors in dental cell fates, these data suggest that their altered expression is a proposed trail to explain the genesis and/or the progression of odontogenic tumors.

Among the four existing isoforms of alkaline phosphatase (AP), the present study is devoted to tissue-nonspecific alkaline phosphatase (TNAP) in mineralized dental tissues. Northern blot analysis and measurements of phosphohydrolase activity on microdissected epithelium and ectomesenchyme, in situ hybridization, and immunolabeling on incisors confirmed that the AP active in rodent teeth is TNAP. Whereas the developmental pattern of TNAP mRNA and protein and the previously described activity were similar in supra-ameloblastic and mesenchymal cells, they differed in enamel-secreting cells, the ameloblasts. As previously shown for other proteins involved in calcium and phosphate handling in ameloblasts, a biphasic pattern of steady-state TNAP mRNA levels was associated with additional variations in ameloblast TNAP protein levels during the cyclic modulation process. Although the association of TNAP upregulation and the initial phase of biomineralization appeared to be a basic feature of all mineralized tissues, ameloblasts (and to a lesser extent, odontoblasts) showed a second selectively prominent upregulation of TNAP mRNA/protein/activity during terminal growth of large enamel crystals only, i.e., the maturation stage. This differential expression/activity for TNAP in teeth vs bone may explain the striking dental phenotype vs bone reported in hypophosphatasia, a hereditary disorder related to TNAP mutation. (J Histochem Cytochem 47:1541-1552, 1999)