Thymosin β4 is associated with bone sialoprotein expression via ERK and Smad3 signaling pathways in MDPC-23 odontoblastic cells

Thymosin 4 (T4) regulates the expression of molecules associated with dentinogenesis, including bone sialo-protein (BSP). BSP regulates the initiation of mineralization and the direction of dentin growth. However, the association between T4 signaling and BSP expression in odontoblasts remains unclear. Therefore, the aim of the present study was to investigate T4 mRNA expression in odontoblasts during dentinogenesis and the association between the T4 signaling pathway and BSP expression in MDPC-23 odontoblastic cells. Expression and localization of T4 mRNA was determined by in situ hybridization during mouse tooth development. The effect of T4 signaling on BSP expression was investigated by reverse transcription polymerase chain reaction, western blot analysis, immunofluorescence and a luciferase reporter assay in the presence or absence of specific inhibitors of mitogen activated protein kinase kinase (PD98059) and mothers against decapentaplegic homolog 3 (Smad3; SIS3) in MDPC-23 cells. The expression of T4 mRNA in the odontoblast layer was highest at postnatal day 5, known as the advanced bell stage, when odontoblasts actively secrete dentin matrix proteins. T4 increased BSP mRNA and protein levels in MDPC-23 cells, but this was inhibited by PD98059 or SIS3 treatment. T4 increased levels of phosphorylated (p) extracellular signal-regulated kinase (ERK)1/2, pSmad3, p-catenin, and runt-related transcription factor 2 (Runx2) protein, but these effects were inhibited by PD98059 or SIS3. T4 induced the nuclear translocation of Runx2 and pSmad3, while nuclear translocation of -catenin was decreased. T4 significantly increased BSP promoter activity, which was decreased by PD98059 or SIS3 treatment. T4 induced BSP expression in MDPC-23 cells via ERK and Smad3 signaling pathways, suggesting its role as a signaling molecule in odontoblasts for regulating BSP secretion during dentinogenesis.