| Runx2is a transcription factor expressed in odontogenic and osteogenicmesenchymal cells during the embryogenesis. Runx2expression in odontogenicdifferentiation is different from osteogenic, which suggests that the specific expression ofRunx2may be a key regulator of osteoblasts and odontoblasts differentiation. Runx2expression is regulated by controlling Runx2protein stability through ubiquitination andthe proteasome pathway. Smurf1is an important member of E3ubiquitin ligases andmediates Runx2degradation in an ubiquitin-and proteasome-dependent manner. Little isknown about Smurf1-mediated Runx2ubiquitination and proteasome degradation plays animportant role in odontogenic differentiation of dental pulp stem cells （DPSCs）.Objective: To compare Runx2expression during odontoblast and osteoblastdifferentiation and reveal that Smurf1regulates Runx2expression and plays a role in odontogenic differentiation of DPSCs via Smurf1shRNA.Methods:1. Isolation, cultivation and identification of HDPSCsSingle-colony-derived DPSCs were isolated from dental pulp tissues by enzymaticdissociation and limiting dilution, and ex vivo expanded. Flow cytometry was adopted toidentify if HDPSCs express mesenchymal stem cell markers. Then after being induced todifferentiate into odontoblasts and adipocytes, the multi-lineage differentiation capacity ofHDPSCs was identified.2. Compare of Runx2expression in odontoblast differentiation with osteoblastHBMSCs and HDPSCs were induced for3weeks with the mineralizing culturemedium including dexamethasone, β-glicerophosplale and vitamin C. Western blot andReal-time PCR technique were adopted to evaluate Runx2expression at differentinduction time points.3. Investigating that Smurf1mediates Runx2ubiquitination and proteasomedegradationLentiviral transfection technology was adopted to enable Smurf1knocked down inHDPSCs. Then, the Smurf1-/- cells were cultured in mineralizing culture medium. Westernblot and Real-time quantitative PCR were used to detect Runx2expression. Proteasomeinhibitor MG132and protein synthesis inhibitor cycloheximide were respectively used totreat undifferentiated HDPSCs and Smurf1-/- HDPSCs. And western blot was used todetect Runx2expression.4. Study about the role of Smurf1in odontogenic differentiation of DPSCsSmurf1-/- HDPSCs were cultured in mineralizing culture medium for7days and14days, Western blot was used to detect expression of Runx2, Real-time quantitative PCR tomeasure expression of odontogenic phenotypic genes and alizarin red S staining tomeasure mineralized nodules formation.Results:1. Our cells were identified as HDPSCs, which possessed stem cell properties.DPSCs cultured in mineralizing culture medium could differentiate into odontoblasts and cells exposed to adipogenic medium could differentiate into adipocytes.2. During the osteogenic differentiation of HBMSCs, Runx2expression increasedpersistently. But in the odontogenic differentiation of HDPSCs, Runx2showed high to lowexpression trend. It showed increasing positive expression in early differentiation and withthe process of mineralization, Runx2expression reduced gradually.3. In undifferentiated Smurf1-/- HDPSCs and the cells cultured in mineralizing culturemedium for7days, Runx2protein expression significantly increased. But Runx2mRNAlevels were not changed. Proteasome inhibitor MG132enhanced Runx2proteinexpression in undifferentiated HDPSCs. The rate of reduction in the Runx2protein aftertreatment with cycloheximide, a protein synthesis inhibitor, was slowed significantly inundifferentiated Smurf1-/- HDPSCs.4. After odontogenic induction, Smurf1-/- HDPSCs formed more mineralized nodulesas indicated by alizarin red S staining. After7days odontogenic induction, earlymineralization genes expression did not significantly chang. In late differentiation （14d）,Runx2protein expression was significantly increased. DSPP and DMP-1, genes associatedwith odontogenic differentiation, were up-regulated, while OCN and BSP, genesassociated with osteoblastic differentiation, down-regulated.Conclusion: Smurf1mediates Runx2ubiquitination and degradation through UPS inodontogenic differentiation of DPSCs, and mainly plays a role in late differentiation.Smurf1-regulated Runx2expression may be involved in odontogenic differentiation as akey regulatory mechanism of cells differentiation.To further study the directional differentiation mechanism of DPSCs, Smurf1overpression lentiviral vector will be used to validate the results, and Runx2shRNA tofurther study whether or not Smurf1-mediated Runx2ubiquitination and proteasomedegradation plays an important role in odontogenic differentiation of HDPSCs. Thefollow-up study will focus on nude mice transplantation experiment which will be used toresearch into the applications in the field of tissue engineering. |
