The Role And Mechanism Of Homeobox Gene Distal-less3in Proliferation And Odontoblastic Differentiation Of Human Dental Pulp Cells

The human dental pulp is made of ectomesenchymal components with neural crest-derived cells, which have plasticity and multipotential capabilities. The human dental pulp contains mixed population of cells including fibroblasts, odontoblasts, as well as undifferentiated mesenchymal cells and plays important roles in dentinogenesis.The differentiation of odontoblasts is the crucial for tooth germ development and self-renewal of dental pulp. This differentiation process is orchestrated by hundreds of genes and signalling molecules. Distal-less3(Dlx3), as a member of the Dlx vertebrate family, is mapped to17q21.33and has an essential role in the development of the epidermis as well as ectodermal appendages such as glands, teeth, and hair follicles. During tooth morphogenesis, Dlx3is initially expressed in the dental epithelium from which ameloblasts differentiate to form enamel and is later expressedin the dental epithelium as well as the dental mesenchyme. Some studies have evaluated Dlx3function in osteoblast development. Odontoblasts and osteoblasts share many similarities. Therefore, we hypothesized that Dlx3may exert effects on human dental pulp cells (hDPCs) during tooth development.The objectives of this study was to investigate the effects of Dlx3on proliferation and odontoblastic differentiation of human dental pulp cells (hDPCs), and to explore the molecular mechanism of Dlx3action during proliferation. The present study consists of two parts:Part Ⅰ:Effects of Homeobox Gene Distal-less3on Proliferation and Odontoblastic Differentiation of Human Dental Pulp Cells1Dlx3Expression during odontoblastic differentiation of Human Dental Pulp CellsDental pulp cells were obtained from the premolars undergoing tooth extraction of5young orthodontic patients. The isolated cells were cultured in odontoblasticinduction medium containing50μg/mL ascorbic acid,10nM dexamethasone, and10mM b-glycerolphosphate. To detect the expression of Dlx3in odontoblastic cell cultures, the mRNA and protein levels of Dlx3and DSPP were measured by semiquantitative RT-PCR and Western blot analysis. Results:Dlx3mRNA and protein were increasingly expressed in coincidence with DSPP. Conclusions:Dlx3expression progressively increased during odontoblast differentiation2Stable Overexpression of Dlx3in hDPCsFull-length Dlx3cDNA along with a cytomegalovirus promoter was inserted into the Hpal-Xhol site of the lentiviral expression vector pLL3.7to construct pLL3.7-Dlx3. To generate the lentivirus, the expression vectors pLL3.7or pLL3.7-Dlx3were transfected into293FT packaging cells along with psPAX2and pMD2.G plasmids. The supernatants were harvested at48and72hours to collect the produced lentivirus. To obtained stable cell populations of hDPC/Dlx3and hDPC/pLL3.7, the human detal pulp cells were incubated with lentivirus for72hours. HDPC/Dlx3can simultaneously express both Dlx3and GFP. GFP expression from the pLL3.7vector allows the measurement of infection efficiency. Results:High infection efficiency was observed in these cells on day0—day14under fluorescent microscopy. Further verification by flowcytometer demonstrated that more than90%cells expressed GFP on day14. The expression of Dlx3mRNA and Dlx3protein was higher in hDPC/Dlx3than those in hDPC/wt and hDPC/pLL3.7until day14. Conclusions:Stable cell population overexpressed Dlx3was generated.3Cell Proliferation AssayWe measured cell proliferation by direct cell counting and EdU labeling assay. Direct cell counting showed that the proliferation rate of Dlx3-infected cells decreased, whereas the hDPC/wt and hDPC/pLL3.7groups had normal and similar cell growth curves, and there was no significant difference between them. EdU labeling was performed on days0,7, and14. Fewer cells showed red fluorescence indicating EdU labeling in the hDPC/Dlx3group than those in the hDPC/wt and hDPC/pLL3.7groups. Conclusions:Dlx3inhibited the proliferation of hDPCs.4Detection of cell differentiationTo directly address the functional role of Dlx3in odontoblastic differentiation, mineralization assay and ALP activity assays were performed. After28days of culture in mineralization medium, formation of cell nodules was observed by phase-contrast microscopy. Obvious calcified nodules were found in the hDPC/Dlx3group compared with in the hDPC/wt and hDPC/pLL3.7groups. ALP activity in the hDPC/Dlx3group was also significantly elevated; it was significantly higher in the Dlx3-treated group than in the control groups. To further document the functional role of endogenous Dlx3in promoting odontoblast differentiation, we selected mineralization-related genes and proteins for these studies. The results demonstrates that Dlx3up-regulated the endogenous expression of all mineralization genes. Notably, ALP was robustly induced by Dlx3(15-fold on day14), whereas DSPP, DMP1, and Nes showed a1.9-fold to3.0-fold response to Dlx3on day14. The Western blot analysis showed higher DSP and DMP1expression levels in the hDPC/Dlx3group than those in hDPC/wt and hDPC/pLL3.7groups. All these results indicated that Dlx3promoted the odontoblastic differentiation of hDPCs.Conclusion: Dlx3functioned in the up-regulation of mineralization-related genes to promote odontoblast differentiation.Part Ⅱ:Characterization of the mechanism of Dlx3modulated proliferation in human dental pulp cells1The inhibition of Wnt/β-catenin signaling activity by Dlx3constitutes a possible mechanism for the inhibition by Dlx3of hDPCs proliferation.To explore possible mechanisms by which Dlx3controls human dental pulp cells proliferation, we used immunofluorescence to detect the expression of Dlx3and DKK1.We carried out quantitative real time RT-PCR to compare RNA levels of Dkkl,c-myc and clyclin D1between hDPC/Dlx3and hDPCs/pLL3.7. To further confirm the effect of Dlx3on Dkkl expression, we used siRNA transfection to knockdown DKK1expression in hDPC/Dlx3and hDPCs/pLL3.7to determine the possible change of cell proliferation. Results:Dlx3and DKK1were expressed in the human dental pulp cells. Among the differentially expressed genes, the expression of the canonical Wnt signaling inhibitor Dkkl was upregulated. In contrast, expression of several Wnt target genes, such as c-Myc and cyclin Dl, was lower in hDPC/Dlx3than in hDPCs/pLL3.7. when DKK1mRNA expression was decreased by90%by siRNA against DKK1, the proliferation rate of Dlx3-infected cells were increased. Conclusion:The marked increase in Dkkl RNA level in Dlx3overexpressed cells suggests that Dlx3controls Dkkl gene expression.2Identification of the Dlx3binding site in the promoter of Dkkl geneIn order to identify putative transcriptional factor binding sites, we analyzed a2000bp region upstream of translational start site of DKKL To determine the minimal region required for basal activity of the promoter, a series of constructs were generated, and luciferase activity were measured after transient transfecting these constructs into293cells. Results:we found12Dlx3potential binding sites in the2000bp region upstream of DKK1. According to the luciferase activity of these constructs, it is found that the proximal transcription regulator is located in Dkkl promoter region-1656to-1245. Conclusion:Dlx3stimulates Dkkl promoter activity.3Dlx3associates with native Dkkl promoter in vivoChIP assay was performed to determine whether Dlx3could associate with Dkkl promoter in vivo. Chromatin DNA was extracted and immunoprecipitated with an anti-Dlx3antibody. Immunoprecipitated DNA was amplified by PCR using primers specific for the region-1656to-1245. Nonimmune human IgG was used as negative control for immunoprecipitation. Results:Dlx3was found to associate with the Dkkl promoter region containing-1656to-1245regeion compared with IgG control group. Conclusion:The data suggest that Dlx3associates with Dkkl promoter in vivo.4Test the role of putative Dlx3-binding sites with site-specific mutationsTo define which DNA elements bind to the core promoter region of DKK1, we generated four point mutants of Dkkl-1656promoter reporter using Dkk1-1656as a template. A series of5’-deletion mutants were tested in293cells co-transfected with h-Dlx3-pcDNA3.1+.36h after transfection,cell lysates were prepared for luciferase assay. P<0.05versus p1656group. Results:Deletion of Dlx3-binding sites I or IV did not affect the transcriptional induction by Dlx3, whereas disruption of site II or III reduced significantly the Dlx3’S transcriptional response. Conclusion:Measurements of luciferase activity suggested that Dlx3responsive elements are located in the Dlx3Ⅱ and Dlx3III binding sites in DKK1promter.