The prolyl hydroxylase inhibitor dimethyloxalylglycine enhances dentin sialophoshoprotein expression through VEGF-induced Runx2 stabilization.

Saeed Ur Rahman,Kyung Mi Woo,Jeong-Hwa Baek,Hyun-Mo Ryoo,Min-Sun Lee,Srikumar P Chellappan

doi:10.1371/journal.pone.0112078

Abstract

Prolyl hydroxylase (PHD) inhibitors are suggested as therapeutic agents for tissue regeneration based on their ability to induce pro-angiogenic responses. In this study, we examined the effect of the PHD inhibitor dimethyloxalylglycine (DMOG) on odontoblast maturation and sought to determine the underlying mechanism using MDPC-23 odontoblast-like cells. DMOG significantly enhanced matrix mineralization, confirmed by alizarin red staining and by measurement of the calcium content. DMOG dose-dependently increased alkaline phosphatase activity and the expressions of dentin sialophosphoprotein (Dspp) and osteocalcin. To determine the underlying events leading to DMOG-induced Dspp expression, we analyzed the effect of DMOG on Runx2. Knockdown of Runx2 using siRNAs decreased Dspp expression and prevented DMOG-induced Dspp expression. DMOG enhanced the transcriptional activity and level of Runx2 protein but not Runx2 transcript, and this enhancement was linked to the inhibitory effects of DMOG on the degradation of Runx2 protein. The vascular endothelial growth factor (VEGF) siRNAs profoundly decreased the Runx2 protein levels and inhibited the DMOG-increased Runx2 protein. Recombinant VEGF protein treatment significantly and dose-dependently increased the transcriptional activity and level of the Runx2 protein but not Runx2 transcript. Dspp expression was also enhanced by VEGF. Last, we examined the involvement of the Erk mitogen-activated protein kinase and Pin1 pathway in VEGF-enhanced Runx2 because this pathway can regulate the stability and activity of the Runx2 protein. VEGF stimulated Erk activation, and the inhibitors of Erk and Pin1 hampered VEGF-enhanced Runx2 protein. Taken together, the results of this study provide evidence that DMOG can enhance Dspp expression through VEGF-induced stabilization of Runx2 protein, and thus, suggest that DMOG can be used as a therapeutic tool for enhancing odontoblast maturation in dental procedures.

Highlights

In treating the pathological conditions of the dentin-pulp complex, the regenerative dental procedures are a great challenge
Before evaluating the effect of DMOG on odontoblast maturation, we examined whether DMOG shows cytotoxicity in MDPC-23 cells
Consistent with previous reports [19], DMOG increased the level of the hypoxia-inducible factor (HIF)-1a and stimulated the expression of vascular endothelial growth factor (VEGF), which is a pro-angiogenic molecule under the control of HIF-1 (Figs. 1B, 1C)

Summary

Introduction

In treating the pathological conditions of the dentin-pulp complex, the regenerative dental procedures are a great challenge. Odontoblasts reside in dental pulp and responsible for dentin formation. Dentin is a calcified tissue that is similar to bone. The organic matrix of dentin consists of collagenous and non-collagenous proteins. The cleavage products of dentin sialophosphoprotein (Dspp), dentin sialoprotein and dentin phosphoprotein, are contained at high levels in dentin [1]. Mutations of the human DSPP gene are associated with dentinogenesis imperfecta type II [3]. Dspp-deficient mice have teeth that display dentin mineralization defects, which are similar to those in human dentinogenesis imperfecta type III, indicating that Dspp plays a critical role in odontoblast differentiation and dentinogenesis [4]

Methods

Results

Conclusion