Role Of Sclerostin In Odontogenic Differentiation Of Human Odontoblast-like Cells Under Tensile Stress&Clinical Case Reports

Non-carious sclerotic dentin is a typical one of reactive dentin beneath non-carious cervical lesions,which serves as a natural barrier to prevent external stimuli from entering the pulp cavity attributed to closed tubules and over-mineralized matrix.Its formation mechanism remains unclear but mechanical stress might be a potential inducing factor.Sclerostin(SOST)is a mechanosensory protein and serves as an inhibitor of dentin formation.However,its function on mechanotransduction in dentine-pulp complex has not been elucidated yet.Odontoblasts,which secret dentin matrix and promote mineralization in the stage of terminal differentiation,are the first cells exposed to and transduce external stimuli in dentine-pulp complex.In this study,taking non-carious sclerotic dentin as a pointcut,we established in vitro model treating human odontoblast-like cells(HODLCs)with tensile stress to investigate the role and mechanisms of SOST on reactive dentin formation under mechanical stimulation.Part Ⅰ.SOST inhibits odontogenic differentiation of HODLCs under tensile stressAim: The aim of this study is to investigate the function of SOST in odontogenic differentiation of HODLCs under tensile stress,for exploring potential role of SOST in the formation of noncarious sclerotic dentin.Materials and methods: Teeth with non-carious cervical lesions and teeth without defect or caries were collected and made into sections,then SOST expression was detected by immunohistochemical staining.HODLCs were cultured in vitro and treated with tensile stress for 24 h by FX-5000 Tension System,then the expressions of SOST,dentin sialophosphoprotein(DSPP),osteopontin(OPN),osteocalcin(OCN)and runt-related transcription factor 2(Runx2)were detected by q PCR and Western blot.SOST was overexpressed in HODLCs by lentiviral transduction.Then SOST-overexpressed HODLCs were treated with tensile stress for 24 h,and the expressions of DSPP,OPN,OCN and Runx2 were detected by q PCR and Western blot.Results: Compared with normal dentin from same-aged donors,SOST expression was decreased in odontoblasts beneath non-carious sclerotic dentin.Tensile stress downregulated SOST expression in HODLCs,whereas the expressions of odontogenic differentiation makers(DSPP,OPN,OCN and Runx2)were upregulated.Besides,SOST overexpression attenuated the upregulation of DSPP,OPN,OCN and Runx2.Conclusion: SOST downregulation was a reactive change of odontoblasts beneath non-carious sclerotic dentin.and was indispensable for the odontogenic differentiation of HODLCs under tensile stress.SOST might participate in the formation of reactive dentin under mechanical stimulation.Part Ⅱ.Tensile stress downregulates the expression of SOST via ERK1/2 pathway and proteasome pathwayAim: The aim of this study was to investigate the relationship between SOST,extracellular signal-regulated kinase(ERK)1/2 signaling pathway and P38 signaling pathway,and to verify whether SOST downregulation in HODLCs under tensile stress was a result of posttranslational regulation,for exploring the mechanisms on the decline of SOST in HODLCs under tensile stress.Materials and methods: HODLCs were treated with tensile stress for 24 h by FX-5000 Tension System,then the expressions of phosphorylated ERK1/2,total ERK1/2,phosphorylated P38 and total P38 were detected by Western blot.HODLCs were pretreated with P38 inhibitor SB203580 and ERK1/2 inhibitor PD98059 respectively,then treated with tensile stress for 24 h,and SOST expression was detected by q PCR and Western Blot.SOST-overexpressed HODLCs were treated with tensile stress for 24 h,then the expressions of phosphorylated ERK1/2,total ERK1/2,phosphorylated P38 and total P38 were detected by Western blot.SOSToverexpressed HODLCs were treated with tensile stress for 24 h by FX-5000 Tension System,then SOST expression was detected by q PCR and Western blot.HODLCs and SOSToverexpressed HODLCs were pretreated with proteasome inhibitor MG132,then SOST expression was detected by Western blot.Results: Tensile stress activated ERK1/2 pathway and P38 pathway in HODLCs.PD98059 eliminated the decrease of SOST,whereas SB203580 had no effect on SOST expression in HODLCs.SOST overexpression inhibited the activity of ERK1/2 pathway but had no effect on the activity of P38 pathway in HODLCs under tensile stress.After treated by tensile stress,there was an evident decline of SOST in SOST-overexpressed HODLCs at protein level but no significant difference at m RNA level.Addition of MG132 rescued the decrease of SOST induced by tensile stress in both HODLCs and SOST-overexpressed HODLCs.Conclusion: ERK1/2 signaling pathway and proteasome pathway mediate the decrease of SOST in HODLCs under tensile stress.Part Ⅲ.STAT3 pathway mediates the regulation of SOST on odontogenic differentiation of HODLCs under tensile stressAim: The aim of this study was to investigate the relationship between SOST,Wnt/β-catenin pathway and signal transducer and activator of transcription 3(STAT3)pathway,for exploring the downstream mechanisms on SOST to regulate odontogenic differentiation of HODLCs under tensile stress.Materials and methods: HODLCs were treated with tensile stress for 24 h by FX-5000 Tension System,then the expressions of phosphorylated β-catenin,total β-catenin,phosphorylated STAT3 and total STAT3 were detected by Western blot.SOST-overexpressed HODLCs were treated with tensile stress for 24 h,then the expressions of phosphorylated STAT3 and total STAT3 were detected by Western blot.SOST-overexpressed HODLCs were pretreated with STAT3 inhibitor MG132,then treated with tensile stress for 24 h,and the expressions of SOST,DSPP,OPN,OCN and Runx2 were detected by q PCR and Western blot.Results: Tensile stress reduced the activity of Wnt/β-catenin pathway and STAT3 pathway.SOST overexpression ameliorated the reduction of STAT3 activity.Addition of Stattic resulted in restoration of DSPP,OPN,OCN and Runx2 expressions downregulated by SOST overexpression.And Stattic failed to rescue the decline of SOST in SOST-overexpressed HODLCs induced by tensile stress.Conclusion: Under tensile stress,SOST regulates the odontogenic differentiation of HODLCs through STAT3 signaling pathway.Summary:Tensile stress downregulates the expression of SOST via the ERK1/2 and proteasome signaling pathways to accelerate odontogenic differentiation of HODLCs through the STAT3 signaling pathway.SOST participates in reactive dentin formation under mechanical stimulation by negatively regulating odontoblasts differentiation,which might provide a novel perspective of non-carious sclerotic dentin formation.