The Mechanism Of Exogenous SFRP5 In Regulating β-Catenin Activity Of Mouse Molar Mesenchymal Cells

With the development of tissue engineering and regenerative medicine,dental regeneration can be achieved through stem cell engineering.So far,a variety of dental mesenchymal stem cells have been isolated,but they do not own the odontogenic potential.Hence,finding genes that could endow stem cells the odontogenic potential is one of the key steps in the engineering.Considering that the induction of odontogenic potential is more easily achieved in odontogenic mesenchymal stem cells than in non-odontogenic epithelial stem cells,this study had screened potential odontogenic potential factor,Sfrp5,from mouse molar mesenchymal cells(mMMC)through RNA-Seq in our previously researches.Furthermore,we established two different approaches to add exogenous SFRP5 protein based on the time-node that mMMC lost the odontogenic potential completely,adding exogenous SFRP5 protein to co-culture with mMMC in vitro for 8 hours and supplementing SFRP5-absorbed beads in the recombinants of mMMC(completely lost odontogenic potential)and the second arch epithelium.Both results of two approaches proved that SFRP5 not only maintained but also restored the odontogenic potential of mMMC effectively.Herein,we further delved into the mechanism of exogenous SFRP5 protein in the regulation of odontogenic potential in mMMC,which was poorly investigated before.Due to the N-terminal cysteine-rich domain(CRD)of SFRPs,which was highly similar to the CRD of the Wnt receptor,and the crucial role of endocytosis in the Wnt-receptor mediated signaling transduction,we supposed that SFRPs may also enter the cytoplasm relying on endocytosis.Besides,it had reported that SFRP5 could transfer into HM20 cells with the help of exosomes and promote β-Catenin into the nucleus.In our previous results,we also found that the supplement of exogenous SFRP5 significantly enhanced the β-Catenin expression in the nucleus of mMMC.Therefore,we speculated that the increased β-Catenin activity was closely related to the content of SFRP5 in mMMC.Here,we preferentially examined and found the significantly increased total content of SFRP5 in the mMMC.In order to clarify whether the increase in SFRP5 content is an endogenous change or an exogenous change,the expression of endogenous and exogenous SFRP5 in mMMC were detected by Real-time PCR and immunostaining,respectively.The results showed that both two sources of SFRP5 in mMMC were significantly increased in the maintenance and reactivation approaches,which demonstrate that the increased β-Catenin activity in mMMC results from the entry of exogenous SFRP5 and the enhanced transcription of endogenous Sfrp5.SFRPs possess a bidirectional regulation of Wnt/β-catenin signaling due to its different binding modes with β-Catenin.In the lung carcinoma cell line,SFRPs positively regulate Wnt/β-catenin signaling when β-Catenin bound to its’ C-terminal,while this effect is reversed if the binding site is N-terminal.In order to verify whether the increased Wnt/β-catenin signaling activity in mMMC is related to the direct binding of SFRP5 andβ-Catenin,the co-localization and interaction of these two molecules were performed by immunofluorescence and immunoprecipitation.The results showed that the localization of SFRP5 and β-Catenin not only overlapped in the cytoplasm of Mmmc but also overlapped in the nucleus,and the SFRP5 and β-Catenin did interact.So we believe that the maintenance and reactivation of odontogenic potential in mMMC is asscociated with the increased Wnt/β-catenin activity,which is induced by the interaction of SFRP5 andβ-Catenin.To further elucidate the downstream genes of exogenous SFRP5,we chosedβ-Catenin as the specific primary antibody for ChIP-Seq to identify the key downstream genes.The mMMC that co-cultured with exogenous SFRP5 for 8 hours was lysed and used for ChIP-Seq experient.The qualified ChIP-Seq libraries were constructed for high-throughput sequencing.Subsequently,we filtered the sequencing data and compared them with the whole mouse genome to obtain data about the binding sites.After annotating the information of Peak,the key downstream genes were screened by GO enrichment analysis.We finally obtained 7 key downstream genes regulated by β-Catenin,including Tnc,Bcor,Gli2,Gli3,Inhba,Jag2,Tcf7l2.By reviewing these genes’ function,it showed that Tnccould recruit and concentrate Wnt ligands from the surrounding environment to enhance Wnt signaling and create a suitable micro-environment for tissue regeneration.Bcor was expressed in the tooth mesenchymal cells and it was involved in the dentin regeneration.Deficiency of Bcor resulted in the hypoplasia of tooth.Inhba was a key gene in the development of the mandibular molars,and its absence caused an abnormal phenotype of mandibular molars.Jag2 was expressed in dentin cells,which guided the cell differentiation during dentin formation.Gliswas closely related to the regeneration of dental pulp and periodontal tissues.Tcf7l2 was a co-activator that regulated the transcription of downstream target genes after β-Catenin transferring into the nucleus.This study clarified that the mechanism of exogenous SFRP5 regulated β-Catenin activity in mMMC.The supplement of exogenous SFRP5 protein increased the content of SFRP5(endogenous and exogenous)in mMMC,which bound to and promoted β-Catenin to enter into the nucleus,and may regulate the Tnc to recruit Wnt signal with the assistance of Tcf7l2,thereby maintaining Wnt signal within a stable threshold.Simultaneously,it may also be involved in regulating Bcor,Inhba,Jag2,Gli2/3 and other genes related to tooth development.Our results provided a new data for understanding the development and regeneration of teeth.