Abstract
Mutation of distal-less homeobox 3 (DLX3) is responsible for human tricho-dento-osseous syndrome (TDO) with amelogenesis imperfecta, indicating a crucial role of DLX3 in amelogenesis. However, the expression pattern of DLX3 and its specific function in amelogenesis remain largely unknown. The aim of this study was to investigate the effects of DLX3 on enamel matrix protein (EMP) genes. By immunohistochemistry assays of mouse tooth germs, stronger immunostaining of DLX3 protein was identified in ameloblasts in the secretory stage than in the pre-secretory and maturation stages, and the same pattern was found for Dlx3 mRNA using Realtime PCR. In a mouse ameloblast cell lineage, forced expression of DLX3 up-regulated the expression of the EMP genes Amelx, Enam, Klk4, and Odam, whereas knockdown of DLX3 down-regulated these four EMP genes. Further, bioinformatics, chromatin immunoprecipitation, and luciferase assays revealed that DLX3 transactivated Enam, Amelx, and Odam through direct binding to their enhancer regions. Particularly, over-expression of mutant-DLX3 (c.571_574delGGGG, responsible for TDO) inhibited the activation function of DLX3 on expression levels and promoter activities of the Enam, Amelx, and Odam genes. Together, our data show that DLX3 promotes the expression of the EMP genes Amelx, Enam, Klk4, and Odam in amelogenesis, while mutant-DLX3 disrupts this regulatory function, thus providing insights into the molecular mechanisms underlying the enamel defects of TDO disease.
Highlights
Mutation of distal-less homeobox 3 (DLX3) gene, a member of the distal-less homeodomain family (DLX1-6), is responsible for a human autosomal-dominant disease, tricho-dento-osseous syndrome (TDO; OMIM 190320) [1,2]
Consistent with the expression pattern of DLX3 protein, the expression of Dlx3 mRNA was elevated at the early secretory stage (PN3), further increased at late secretory stage (PN7), and decreased at the maturation stage (PN14) (Fig. 2)
We investigated the specific role of the transcriptional factor DLX3, and the expression patterns of Dlx3 mRNA and protein during amelogenesis
Summary
Mutation of distal-less homeobox 3 (DLX3) gene, a member of the distal-less homeodomain family (DLX1-6), is responsible for a human autosomal-dominant disease, tricho-dento-osseous syndrome (TDO; OMIM 190320) [1,2]. Specific Function of DLX3 in Amelogenesis dental findings of enamel hypoplasia and hypomaturation with taurodontism (elongation of the dental pulp chamber), suggesting a specific role of DLX3 in amelogenesis [3]. The process of amelogenesis is divided into three main phases: the pre-secretory, secretory, and maturation stages. During this process, the sequential expression and secretion of enamel matrix proteins (EMPs) are critical and considered to be co-regulated by transcriptional factors, cytokines, growth factors, and signaling molecules [4,5]. Since the hypoplastic and hypomaturation enamel defects of TDO are comparable to those caused by mutations of EMP genes such as AMELX, AMBN, ENAM, MMP20, and KLK4 [6,7,8], a possible link between DLX3 and EMP genes is indicated
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