Cellular and transgenic mouse studies of mutant DLX3

Objectives: A 4bp deletion mutation in the DLX3 gene is associated with Tricho-Dento-Osseous (TDO) syndrome characterized by abnormality in hair, tooth and bone development such as taurodontism, enamel hypoplasia, and increased bone thickness and density. To examine the effects of 4bp deleted DLX3 (MT-DLX3) gene, we have investigated the roles of 4bp deleted DLX3 gene on tooth and bone formation in vitro and in vivo. Methods: We have transduced MT-DLX3 into preosteoblastic MC3T3E1 and pluripotent mesenchymal C2C12 cells and tested AP activity, mineral deposition, collagen1A1 and osteocalcin promoter activities. To study the in vivo roles of MT-DLX3 on tooth and bone development, we have generated 4bp deleted DLX3 transgenic mice driven by a 2.4kbp mouse collagen1A1 promoter. Results: Transduction of MT-DLX3 into MC3T3E1 and C2C12 cells enhanced AP activity, mineral deposition, and osteocalcin promoter activities compared to those of WT-DLX3 or EV transduced cells (p <0.05), but did not enhance Col1A1 promoter activity in these cells. Transduction of MT-DLX3 into C2C12 cells abolished desmin gene expression, which is an early myoblastic differentiation marker. Primary osteoblastic cells isolated from TG mice showed the enhanced mineral deposition by stimulation of osteogenic factors. Bone marrow and spleen cells showed decreased osteoclast formation by osteoclastic factors compared to those of control mice. Furthermore, immunohistochemical studies and faxitron results have demonstrated that TG mice showed markedly decreased dentine formation with abnormal odontoblastic cell organization and enhanced bone formation as similarly seen in TDO patients. Conclusion: These results demonstrate that the 4bp deleted MT-DLX3 associated with TDO syndrome enhances osteoblastic bone formation and decreases dentine formation in vitro and in vivo. TG mice could be a useful tool to understand the in vivo roles and mechanisms of a 4bp deleted DLX3 gene on tooth and bone development as observed in TDO patient phenotype.