Regulatory Mechanism Of Sfrp5 On Odontogenic Potential

Tooth loss is a universal public health problem that directly affects patients’ abilities to chew,speak and smile,as well as triggering patients’ mental health issues.Whether it is the natural tooth loss of the elderly or tooth fall-off due to dental caries,periodontal disease,and genetic diseases,both severely affect the patient life.The stem cell-based tissue engineering of whole tooth regeneration is the most ideal way to achieve tooth replacement and regeneration therapy,which requires epithelial and mesenchymal cells,and either of them should have the odontogenic potential to induce tooth formation.At present,due to the apoptosis of the dental epithelium during tooth eruption,adult odontogenic epithelial stem cells cannot be isolated,and a variety of odontogenic mesenchymal stem cells isolated from adult human dental tissue have been evidenced to have only the odontogenic competence but not odontogenic potential.Therefore,one of the biggest challenges of this project is to identify the molecular composition of the odontogenic potential,thus conferring the epithelial or mesenchymal derived cells to possess this potential.Given that the odontogenic mesenchymal cells are more likely to be endowed with odontogenic potential than the non-odontogenic epithelial cells,RNA-Seq has previously been utilized to screen the set of candidate factors for the odontogenic potential in E13.5 mouse dental mesenchymal cells(m MMC).In the current study,we aim to verify the function and understand the regulatory mechanism of Sfrp5 in this set,which is involved in the Wnt pathway that regulates tooth growth,pattern formation,mineralization,and other processes,and is the most important single signal to initiate the development of teeth and other organs.To verify the role of Sfrp5 in regulating the odontogenic potential of m MMCs,exogenous SFRP5 protein was added to the cultured m MMCs to study whether Sfrp5 could maintain or even rescue the odontogenic potential of m MMCs.The results show that exogenous SFRP5 not only effectively maintains but also rescues the ability of m MMC to induce non-dental second branchial arch epithelium to differentiate into enamel-secreting ameloblasts.Moreover,it shows that exogenous SFRP5 is also effective in maintaining and rescuing the expression of essential transcription factors of m MMCs,including Msx1,Pax9,Lhx6,and Lef1,thus maintains and rescues the odontogenic potential of m MMCs.Further investigates the regulation of exogenous SFRP5 in canonical Wnt/β-catenin pathway in m MMCs,and discovers that exogenous SFRP5 performs as an activator rather than an inhibitor for the Wnt/β-catenin pathway in m MMCs,which effectively maintains and rescues the level of β-catenin in m MMCs.These data confirm that exogenous SFRP5 maintains and rescues the odontogenic potential of m MMCs.To study the effect of overexpression of Sfrp5 in vivo on tooth development and odontogenic potential of cultured m MMCs,the conditionally Sfrp5-overexpressing p Mes-Sfrp5 mice were further constructed and crossed with Wnt1-Cre mice to obtain Wnt1-Cre;p Mes-Sfrp5 mice,achieving the overexpression of Sfrp5 in cranial neural crest-derived mesenchymal cells including the dental mesenchymal cell.The results show that augmented Sfrp5 in the dental mesenchyme does not affect tooth morphogenesis as well as the activity of the Wnt/β-catenin signaling pathway,whereas it significantly increased the expression level of essential odontogenic transcription factors,Msx1,Pax9,and Lef1 in the dental mesenchyme.Further studies reveal that enhanced Sfrp5 in the dental mesenchyme does not affect the expression of other Sfrps(Sfrp1,Sfrp2,Sfrp3,and Sfrp4),but induces significant upregulation of other antagonists of the Wnt pathway,Apcdd1,thereby antagonizing the regulation of Wnt/β-catenin and holding its steady-state,thus does not affect tooth morphology and development.Furthermore,E13.5dental mesenchyme of Wnt1-Cre;p Mes-Sfrp5 embryos were dispersed into single cells and cultured in vitro to explore the effect on the odontogenic potential of m MMCs when overexpressed Sfrp5 in vivo.The results are consistent with the effect of addition exogenous SFRP5,enhance Sfrp5 not only effectively maintains the ability of cultured m MMCs to induce the differentiation of second branchial arch epithelial teeth but also restore the expression of essential transcription factors,Msx1 and Pax9,in the dental mesenchyme as well as the activity of canonical Wnt/β-catenin pathway.These results confirm that enhanced mesenchymal Sfrp5 in vivo also maintains the odontogenic potential of cultured m MMC.It has been reported that extracellular Sfrps could enter cells relying on the endocytosis and interact with β-catenin in the cytoplasm and thus enter the nucleus to regulate the transcriptional inhibition or activation of downstream Wnt genes.Based on this,to have an in-depth study of the mechanism of exogenous SFRP5 mediated Wnt/β-catenin in the regulation of the odontogenic potential of m MMC,we examined and found that exogenous SFRP5 effectively increases the content of SFRP5 in the cytoplasm and nucleus of m MMC,which is a common result of increased expression of endogenous Sfrp5 and the entry of exogenous SFRP5 into cells.The results of immunofluorescence show that the localization of increased SFRP5 and β-catenin in m MMCs are partially overlapped,indicating that there may be an interaction between these two proteins in m MMCs.Co IP results further confirm that exogenous SFRP5 andβ-catenin actually combine with each other in m MMCs.Moreover,to screen the downstream regulators of Sfrp5 mediated Wnt/β-catenin in regulating the odontogenic potential of m MMCs,both RNA-Seq and Ch IP-Seq were used to screen the differentially expressed genes in m MMCs treated with exogenous SFRP5.GO analysis shows that these candidates are mainly involved in organ regeneration,osteogenic differentiation,multicellular biological development,promotion of gene expression regulation,cell proliferation,cell cycle regulation,and other biological processes closely related to this study.Venn analysis of the results of RNA-Seq and Ch IP-Seq uncovers 14 common differential genes,of which 6 genes have been proved to be closely related to tooth development.In summary,this study substantiates that both exogenous SFRP5 treatment and the overexpression of Sfrp5 in vivo have a function in maintaining and rescuing the odontogenic potential of cultured m MMCs.Sfrp5 acts as an activator of the Wnt pathway in m MMCs,via interacting with β-catenin and then entering the nucleus to promote the canonical Wnt/β-catenin activity,thereby regulating the downstream target genes.Our data provide an in-depth understanding of the role and mechanism of Sfrp5-mediated Wnt signaling in regulating the odontogenic potential of m MMCs,and supply an important theoretical basis for the future achievement of stem cells-based human tooth regeneration.