Rho/Rho-associated Protein Kinase Signaling Pathway-mediated Downregulation of Runt-related Transcription Factor 2 Expression Promotes the Differentiation of Dental Pulp Stem Cells into Odontoblasts

The present study investigated the role of runt-related transcription factor 2 (Runx2) in regulating the differentiation of human dental pulp stem cells (hDPSCs) into odontoblasts under the mediation of the Rho/Rho-associated protein kinase (ROCK) signaling pathway. hDPSCs and human bone marrow mesenchymal stem cells (hBMSCs) were mineralized to induce differentiation. The expression levels of odontoblast- and osteoblast-specific proteins, dentin sialophosphoprotein (DSPP), osteocalcin (OCN) and Runx2, were measured using western blot analysis. The hDPSCs were treated with Rho/ROCK signaling pathway inhibitor, C3 exoenzyme, and mineralized prior to determining the protein expression levels of RhoA, ROCK, Runx2, OCN, DSPP, and mRNA expression levels of early mineralization genes, including alkaline phosphatase, collagen type I, Msh homeobox 2 and distal-less homeobox 2, and late mineralization genes, including , dentin matrix protein-1 (DMP-1), bone sialoprotein (BSP) and . Flow cytometry data indicated that 95% of the isolated hDPSCs were positive for mesenchymal stem cell markers, including cluster of differentiation (CD)29, CD90 or CD105, and vascular endothelial cell marker, CD146, whereas <5% of the hDPSCs were positive for hematopoietic stem cell markers, CD34 and CD45. The expression levels of DSPP in hDPSCs and OCN in hBMSCs were significantly upregulated with increased time in mineralization medium (P<0.01), which suggested that hDPSCs and hBMSCs were differentiated into odontoblasts and osteoblasts, respectively. During the osteogenic process, Runx2 protein was highly expressed in mesenchymal stem cells following stimulation with mineralization medium compared with cells that received no stimulation. During odontoblast differentiation in hDPSCs, Runx2 protein was highly expressed in the early stage; however, the expression declined in the late stage. Furthermore, treatment with C3 exoenzyme significantly downregulated the expression of RhoA, ROCK and Runx2 compared with the control in hDPSCs (P<0.01). Additionally, in mineralization solution, C3 exoenzyme also significantly downregulated the expression of Runx2 (P<0.01); however, the Rho/ROCK signaling pathway inhibitor did not significantly impact the expression of early mineralization genes. By contrast, C3 exoenzyme significantly upregulated the expression of DSPP and DMP-1, and downregulated the expression of BSP and OCN (P<0.01). The present findings suggested that odontoblast differentiation in hDPSCs may be regulated by Rho/ROCK signaling pathway-mediated downregulation of Runx2.