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

Abstract

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.

Combination of polyetherketoneketone scaffold and human mesenchymal stem cells from temporomandibular joint synovial fluid enhances bone regeneration

“…61 reported that the pattern of RUNX2 expression fluctuated during osteogenic differentiation of MSCs. The expression of RUNX2 was found to increase initially and then to decrease in human dental pulp MSCs, while it remained consistently high in human bone marrow MSCs during their osteogenic/odontogenic differentiation 61 . It was also reported that the mRNA level of RUNX2 correlate positively with the level of OCN during osteogenesis 62 .…”

Suppression of AKT-mTOR signal pathway enhances osteogenic/dentinogenic capacity of stem cells from apical papilla

“…Indeed, although the expression of Runx2 , an essential transcriptional factor for the differentiation and maturation of osteoblasts [ 29 ], is constantly increasing during the differentiation of osteoblasts, the expression of Runx2 is known to exhibit a stage-specific expression level of Runx2 during the differentiation of odontoblast; the increased expression of Runx2 in the early stage is suppressed in the terminal stage associated with the enhanced expression of DSPP [ 30 , 31 ], suggesting that inhibition of PI3-AKT-mTOR signal pathway might accelerate the ability of osteogenic/dentinogenic differentiation of SCAP. Recent studies clarified that stage-specific regulation of the Runx2-targeting molecules, including microRNA 338-3p, microtubule-associated protein tau, small heterodimer partner, and Rho/Rho-associated protein kinase, transcriptionally suppresses Runx2 expression, resulting in DSPP upregulation at the terminal stage of odontoblast differentiation [ 32 – 35 ]. Current studies report that AKT and its underlying signal FOXO1 negatively regulate Runx2 transcription in osteosarcoma and prostate cancer cells [ 36 , 37 ].…”

Stem Cell Mechanobiology and the Role of Biomaterials in Governing Mechanotransduction and Matrix Production for Tissue Regeneration

“…Disruption of the cytoskeleton and the Rho/ROCK pathway of MSCs on rope-like silk scaffolds diminish the expression of tendon differentiation markers and lead to a loss of spindle morphology ( Maharam et al, 2015 ). Furthermore, downregulation of osteogenic marker RUNX2, mediated via the Rho/ROCK signaling pathway promotes the differentiation of dental pulp stem cells into odontoblasts ( Huang et al, 2018 ). Rho and the actin cytoskeleton have also been shown to be necessary to maintain nuclear YAP/TAZ in MSCs ( Dupont et al, 2011 ).…”