: Sp6 is a member of the Sp family of transcription factors that regulate a wide range of cellular functions, such as cell growth and differentiation. Sp6, also called epiprofin, is specifically expressed in tooth germ, limb bud, and hair follicle, but there is little information on its function.To investigate the possible role of Sp6 in tooth development, first we established an Sp6-overproducing clone, CHA9, and analyzed the features of the cell, including cell proliferation and gene expression. The parental cells of CHA9 are the ameloblast-lineage G5 cells that we previously established from rat dental epithelia of lower incisor. Sp6 overproduction accelerated cell proliferation and induced the expression of ameloblastin mRNA, a marker of ameloblast differentiation. Second, we performed genome-wide screening of Sp6 target genes by microarray analysis. Out of a total 20,450 genes, 448 genes were up-regulated and 500 genes were down-regulated by Sp6. We found the expression of follistatin, a BMP antagonist, to be 22.4-fold lower in CHA9 than in control cells. Transfection of the Sp6-antisense construct into CHA9 cells restored follistatin expression back to equivalent levels seen in control cells, indicating that Sp6 regulates follistatin gene expression in ameloblasts.Our findings demonstrate that the follistatin gene is one of the Sp6 target genes in ameloblasts and suggest that Sp6 promotes amelogenesis through inhibition of follistatin gene expression. J. Med. Invest. 55 : 87-98, February, 2008

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SUMMARYBmp4 expression is tightly regulated during embryonic tooth development, with early expression in the dental epithelial placode leading to later expression in the dental mesenchyme. Msx1 is among several transcription factors that are induced by epithelial Bmp4 and that, in turn, are necessary for the induction and maintenance of dental mesenchymal Bmp4 expression. Thus, Msx1 −/− teeth arrest at early bud stage and show loss of Bmp4 expression in the mesenchyme. Ectopic expression of Bmp4 rescues this bud stage arrest. We have identified Tbx2 expression in the dental mesenchyme at bud stage and show that this can be induced by epithelial Bmp4. We also show that endogenous Tbx2 and Msx1 can physically interact in mouse C3H10T1/2 cells. In order to ascertain a functional relationship between Msx1 and Tbx2 in tooth development, we crossed Tbx2 and Msx1 mutant mice. Our data show that the bud stage tooth arrest in Msx1 −/− mice is partially rescued in Msx1;Tbx2 +/− compound mutants. This rescue is accompanied by formation of the enamel knot (EK) and by restoration of mesenchymal Bmp4 expression. Finally, knockdown of Tbx2 in C3H10T1/2 cells results in an increase in Bmp4 expression. Together, these data identify a novel role for Tbx2 in tooth development and suggest that, following their induction by epithelial Bmp4, Msx1 and Tbx2 in turn antagonistically regulate odontogenic activity that leads to EK formation and to mesenchymal Bmp4 expression at the key bud-to-cap stage transition.KEY WORDS: Bmp4, Msx1, Tbx2, Protein-protein interaction, Tooth

Background: Ameloblasts are epithelially derived cells responsible for enamel formation through a process known as amelogenesis. Amongst the several transcription factors that are expressed during amelogenesis, both Msx2 and Sp6 transcription factors play important role. Msx2 and Sp6 mouse mutants, exhibit similar amelogenesis defects, namely enamel hypoplasia, while humans with amelogenesis imperfecta (AI) carry mutations in the human homologues of MSX2 or SP6 genes. These across species similarities in function indicate that these two transcription factors may reside in the same developmental pathway. In this paper, we test whether they work in a coordinated manner to exert their effect during amelogenesis. Methods: Two different dental epithelial cell lines, the mouse LS8 and the rat G5 were used for either overexpression or silencing of Msx2 or Sp6 or both. Msx2 mutant mouse embryos or pups were used for in vivo studies. In situ hybridization, semiquantitative and quantitative real time PCR were employed to study gene expression pattern. MatInspector was used to identify several potential putative Msx2 binding sites upstream of the murine Sp6 promoter region. Chromatin Immunoprecipitation (chIP) was used to confirm the binding of Msx2 to Sp6 promoter at the putative sites. Results: Using the above methods we identified that (i) Msx2 and Sp6 exhibit overlapping expression in secretory ameloblasts, (ii) Sp6 expression is reduced in the Msx2 mouse mutant secretoty ameloblasts, and (iii) that Msx2, like Sp6 inhibits follistatin expression. Specifically, our loss-of function studies by silencing Msx2 and/or Sp6 in mouse dental epithelial (LS8) cells showed significant downregulation of Sp6 but upregulation of Fst expression. Transient transfection of Msx2 overexpression plasmid,

Thrombospondin 1 (TSP1) is a multifunctional extracellular glycoprotein present mainly in the fetal and adult skeleton. Although an inhibitory effect of TSP1 against pathological mineralization in cultured vascular pericytes has been shown, its involvement in physiological mineralization by osteoblasts is still unknown. To determine the role of TSP1 in biomineralization, mouse osteoblastic MC3T3-E1 cells were cultured in the presence of antisense phosphorothioate oligodeoxynucleotides complementary to the TSP1 sequence. The 18- and 24-mer antisense oligonucleotides caused concentration-dependent increases in the number of mineralized nodules, acid-soluble calcium deposition in the cell/matrix layer, and alkaline phosphatase activity within 9 days, without affecting cell proliferation. The corresponding sense or scrambled oligonucleotides did not affect these parameters. In the antisense oligonucleotide-treated MC3T3-E1 cells, thickened extracellular matrix, well-developed cell processes, increased intracellular organelles, and collagen fibril bundles were observed. On the other hand, the addition of TSP1 to the culture decreased the production of a mineralized matrix by MC3T3-E1 cells. Furthermore, MC3T3-E1 clones overexpressing mouse TSP1 were established and assayed for TSP1 protein and their capacity to mineralize. TSP1 dose-dependently inhibited mineralization by these cells both in vitro and in vivo. These results indicate that TSP1 functions as an inhibitory regulator of bone mineralization and matrix production by osteoblasts to sustain bone homeostasis.

Dental pulp cells have a capacity to differentiate into mineralization-inducing cells. To clarify the molecular mechanism, we established an in vitro mineralization-inducing system by rat clonal dental pulp cell line, RPC-C2A, and tried to purify a mineralization-inducing factor in conditioned medium (CM) from pre-osteoblastic MC3T3-E1 cells. The active factor was impermeable to an ultrafiltration membrane, and sedimented by ultracentrifugation. The sedimented factor was found as a needle-like structure about 1.3 microm in average length as observed by transmission electron microscopy. The factor contained type I collagen, suggesting not a matrix vesicle, but a soluble matrix. The mineralization-inducing activity was also detected in CM from primary culture of rat calvaria (RC) cells. These results suggested that the soluble matrices from osteoblastic cells serve, at least in part, as differentiation-inducing agents.

Background: 1,25-dihydroxyvitamin D3 has been proven to be able to control the formation and biomineralization of tissue through a regulatory gene. A previous research even showed that a cell responsible for the formation of the enamel, dentin and bone was the target of 1,25-dihydroxivitamin D3. Purpose: This research was aimed to determine the role of 1,25-dihydroxyvitamin D3 in vivo in the development of teeth and alveolar bone tissue by analyzing MSX2 gene expression as a gene marker responsible for the growth and development of enamel, dentin, tooth root and alveolar bone. Methods: Samples used for RT-PCR analysis were total RNA of insisivus teeth and alveolar bone derived from mice. RT-PCR analysis was conducted by using primer-specific gene, MSX2. Primer gene, GAPDH, was also used as an internal control. Five hundred nanograms of total RNA were used as a template for PCR. Semi quantitative results of PCR were quantified by using ImageJ software. results: RT-PCR analysis showed that the expression level of MSX2 was enhanced in the samples of teeth and alveolar bone treated with 1,25 dihydroxyvitamin D3. The increasing of MSX2 expression significantly occurred in alveolar bone samples. Conclusion: It can be concluded that 1,25 dihydroxyvitamin D3 could enhance MSX2 expression as a marker of the development of teeth and alveolar bone tissue. Therefore, 1,25-D3 dihydroxyvitamin is expected to be used as an agent to help the regeneration of teeth and bone tissue.

BackgroundThe precise formation of mineralized dental tissues such as enamel and/or dentin require tight transcriptional control of the secretion of matrix proteins. Here, we have investigated the transcriptional regulation of the second most prominent enamel matrix protein, enamelin, and its regulation through the major odontogenic transcription factor, MSX2.ResultsUsing in vitro and in vivo approaches, we identified that (a) Enam expression is reduced in the Msx2 mouse mutant pre‐secretory and secretory ameloblasts, (b) Enam is an early response gene whose expression is under the control of Msx2, (c) Msx2 binds to Enam promoter in vitro, suggesting that enam is a direct target for Msx2 and that (d) Msx2 alone represses Enam gene expression.ConclusionsCollectively, these results illustrate that Enam gene expression is controlled by Msx2 in a spatio‐temporal manner. They also suggest that Msx2 may interact with other transcription factors to control spatial and temporal expression of Enam and hence amelogenesis and enamel biomineralization.