Study On The Function Of Klf4in Mouse Odontoblast Differentiation

1. Tamoxifen-mediated reversible immortalization of mouse dental papilla cell lineObjectives:Odontoblasts are a kind of non-proliferating and terminally differentiated cells which play an important part in pulpo-dentinal complex. Mouse dental papilla cells (mDPCs), which can differentiate into odontoblast-like cells in vitro, have a limited lifespan. To solve this problem, we combined the traditional strategy of "Cre/LoxP-based reversible immortalization" with tamoxifen-regulated Cre recombination system to generate a tamoxifen-mediated reversibly immortalized mouse dental papilla cell line.Methods:mDPCs were sequentially transduced with a floxed SV40Tag-TK and an ERT2CreERT2expressing plasmid. Clonal isolated SV40Tag and Cre positive cells were designated as mDPCET. The reverted cells were acquired by4-hydroxytamoxifen treatment. Primary mDPCs, mDPCET and the reverted cells were examined by investigation of cell proliferation kinetics, expression of odontoblastic-related makers and mineralization ability.Results:mDPCET showed upregulated growth rate and significantly extended lifespan, but retained most of the biochemical and functional characteristics of primary mDPCs. When mDPCET cells were treated with4-hydroxy tamoxifen, ERT2CreERT2was translocated from cytoplasm to nucleus which caused the excision of the SV40Tag-TK and led to the reversion of mDPCET. After the immortalization was reversed, cells underwent replicative senescence, showed higher expression level of odontoblastic-related genes and increased mineral deposition rate.Conclusions:Tamoxifen-mediated reversible immortalization, therefore, allows the expansion of primary mDPCs, leads to production of odontoblast-like cells that retain most odontoblast-specific properties, and can represent as a safe and ready-to-use method due to its simple manipulation. 2. Klf4promoted odontoblastic differentiation of mouse dental papilla cells via DmplObjectives:Odontoblast cells, which derive from dental papilla, are a type of terminally differentiated matrix-secreting cells. Previous studies have identified various transcription factors involved in the differentiation process of odontoblasts. We have recently found that Kriippel-like factor4(Klf4) was expressed in the polarizing and elongating odontoblasts, but the function of Klf4in the differentiation of odontoblasts is still unclear. We hypothesized Klf4promoted the differentiation of odontoblasts by up-regulating some odontoblast-related genes.Methods&Results:In this study, we found that the expression of Klf4increased significantly during the odontoblastic differentiation of primary mouse dental papilla cells and the mouse dental papilla cell line-mDPC6T. Overexpression of Klf4significantly up-regulated odontoblast-related genes, such as Dmpl, Dspp, and Alp, and promoted the accumulation of mineral nodules. Knock-down of Klf4down-regulated expression of Dmpl, Dspp, and Alp, and inhibited mineral deposition. We applied in silico analysis and identified one target gene of Klf4-Dmp1. Based on further analysis of ChIP data, EMSA and dual luciferase activity assays, we confirmed that Klf4was able to specifically bind to the Dmpl promoter and transactivate its expression. Furthermore, forced expression of Dmpl in the Klf4knock-down mDPC6T cell line significantly recovered its odontoblastic differentiation ability. Conclusions:Our data confirmed our hypothesis that Klf4promotes the differentiation of odontoblasts via the up-regulation of Dmpl. 3. Conditional knockout Klf4in dental mesenchyme affected dentin formation.Objectives:Kriippel-like factor4(Klf4) is a member of Klf zink finger transcriptional factor family. The Klf4plays a pivotal role in cellular differentiation. Previously, we have found that Klf4was expressed in the polarizing and elongating odontoblasts. In Part2, we applied in vitro study and showed that Klf4transcriptional activate the expression of Dmpl and promoted odontoblastic differentiation of mouse dental papilla cells. In this study, we hypothesized conditional knockout of Klf4in odontoblasts may affect differentiation of odontoblasts and dentin formation.Methods:Mouse containing inactivated Klf4in their neural crest cells (Wnt1-Cre; Klf4f/f) were obtained by crossing Wntl-Cre; Klf4f/+mice with Klf4mf/f line. Mouse containing activated LacZ in their neural crest cells was obtained by crossing Wntl-Cre mice with R26R-LacZ line. Mouse tail was dissected and the genome DNA was isolated for genotyping. Embryonic head samples were dissected and fixed individually in4%paraformaldehyde (PFA) overnight at4℃, and processed for paraffin section for histological and immunostaining or for X-ray scan.Results:Wntl-cre;R26R-LacZ mouse showed positive X-gal staining in dental mesenchyme. Immunostaining showed that, for the control mice (Klf4f/+), KLF4was expressed in the differentiated odontoblasts and ameloblasts in the cusp of E18.5and PN0.5molar germ. But in conditional knockout mice, expression of Klf4was detected only in the ameloblasts but not the odontoblasts. HE staining showed thicken predentin layer in the conditional knockout mice but not the control mice. Besides conditional knockout mice also showed weaker blue in the dentin for the azan staining. By high resolution X-ray, we have found decrease of the grey scale in the dentin but not the enamel in the conditional knockout mice. More interestingly, we observed caries involve all the mandibular molar in2/4of the3month old conditional knockout mice but none in the control mice.Conclusions:Klf4is essential in the odontoblasts for the dentin mineralization.