Krüppel-like factor 6 promotes odontoblastic differentiation through regulating the expression of dentine sialophosphoprotein and dentine matrix protein 1 genes.

Abstract

To investigate the potential role of Krüppel-like factor 6 (KLF6) in the odontoblastic differentiation of immortalized dental papilla mesenchymal cells (iMDP-3) cells. Alizarin Red S (ARS) and Alkaline phosphatase (ALP) staining was used to examine the mineralization effect of iMDP-3 cells after odontoblastic induction. Real-time PCR and Western blotting were employed to analyse dentine sialophosphoprotein (DSPP), dentine matrix protein 1 (DMP1), RUNX family transcription factor 2 (RUNX2), ALP and KLF6 expression during this process. Co-expression of the KLF6 with DMP1, DSPP and RUNX2 was detected by double immunofluorescence staining to explore their local relationship in the cell. To further investigate KLF6 functions, Klf6 gain- and loss-of-function assays followed by ARS and ALP stainings, real-time PCR and Western blotting were performed using Klf6-overexpression plasmids and Klf6 siRNA to investigate whether changes in Klf6 expression affect the odontoblastic differentiation of iMDP-3 cells. Dual-luciferase reporter assays were used to elucidate the mechanistic regulation of Dspp and Dmp1 expression by Klf6. Means were compared using the unpaired t-test and Kruskal-Wallis one-way anova with P<0.05 and P<0.01 defined as statistical significance levels. The expression levels of Klf6 (P<0.01), Dspp (P<0.05), Dmp1 (P<0.01), Runx2 (P<0.01) and Alp (P<0.01) were significantly elevated during odontoblastic differentiation of iMDP-3 cells. KLF6 was co-localized with DSPP, DMP1 and RUNX2 in the cytoplasm and nucleus of iMDP-3 cells. Overexpression of Klf6 promoted the odontoblastic differentiation of iMDP-3, whereas the inhibition of Klf6 prevented this procession. Dual-luciferase assays revealed that Klf6 upregulates Dspp and Dmp1 transcription in iMDP-3 cells during odontoblastic differentiation. Klf6 promoted odontoblastic differentiation by targeting the transcription promoter of Dmp1 and Dspp. This study may offer novel insights into strategies for treating injuries to dental pulp tissue.