Effects of histone deacetylase inhibitors on odontoblast differentiation

Abstract

Previous reports demonstrated that histone deacetylase inhibitors (HDACi) enhances osteoblast differentiation in vitro and new bone formation in vivo. However, it has not been elucidated whether HDACi regulate odontoblast differentiation. Nfic and Wnt/β-catenin signaling are involved in odontoblast differentiation and tooth root formation. Smurf1 is an E3 ubiquitin ligase that negatively regulates osteoblast differentiation and bone formation. The purpose of the study was to investigate the effect of HDACi on odontoblast differentiation and to elucidate the regulatory roles of Wnt/β-catenin, Nfic and Smurf1 in HDACi-induced odontoblast differentiation. MDPC23 cell line derived from the mouse pulp cells was treated with SAHA and Trichostatin A. Odontoblast differentiation was evaluated by examining the expression levels of marker genes, including dentin sialophosphoprotein (Dspp), dentin matrix protein 1 (Dmp1) and Nestin using quantitative RT-PCR and Western blot analyses. Matrix mineralization was observed using Alizarin S Red staining. Chromatin immunoprecipitation, biotinylation pull down assays and luciferase reporter assays were performed to examine the regulatory role of Nfic on Dspp transcription. To examine the effect of HDACi on the transcriptional activation of Smurf1 gene, chromatin immunoprecipitation was performed using antibodies to acetylated histone H3 (Ac-H3), methylated histone H3 (H3K4me) and RNA polymerase II. Wnt3a protein levels in conditioned medium obtained from MDPC23 cells were evaluated using ELISA kit. Transcriptional activity of β-catenin/Tcf/Lef complexes was determined by TOP/FOP-flash luciferase reporter assays. Knockdown of Nfic, Smurf1, Wnt3a or β-catenin was induced by transient transfection of small interfering RNA (siRNA) mixtures specific to each gene. HDACi enhanced odontoblast differentiation and matrix mineralization. HDACi increased expression levels of Nfic, and subsequently Nfic directly bound to Dspp promoter and upregulated Dspp transcription. Nfic silencing attenuated HDACi-induced odontoblast differentiation. Overexpression of Smurf1 in MDPC23 cells inhibited odontoblast differentiation, whereas Smurf1 knockdown enhanced odontoblast differentiation. HDACi treatment or overexpression of Nfic downregulated the expression levels of Smurf1 gene. Chromatin immunoprecipitation results demonstrated that increase in Nfic protein level via HDACi treatment or overexpression of Nfic significantly downregulated the levels of Ac-H3, H3K4me and RNA polymerase II associated with the promoter region or transcription start site of the mouse Smurf1 gene. Nfic knockdown attenuated HDACi-mediated downregulation of Smurf1 expression. Activation of Wnt/β-catenin signaling via Wnt3a treatment or β-catenin overexpression enhanced odontoblast differentiation and Nfic expression, whereas knockdown of Wnt3a or β-catenin suppressed odontoblast differentiation and Nfic expression. HDACi increased expression levels of Wnt3a mRNA and protein and enhanced TOP-flash reporter activity in MDPC23 cells. Blockade of Wnt/β-catenin signaling via silencing of Wnt3a suppressed HDACi-mediated odontoblast differentiation and Nfic expression. However, overexpression or knockdown of Nfic did not affect HDACi-mediated induction of Wnt/β-catenin signaling. These indicate that HDACi enhance odontoblast differentiation via activation of Wnt/β-catenin signaling in MDPC23 cells. In turn, increased Nfic protein upregulates odontoblast differentiation marker genes such as Dspp, while downregualting Smurf1, a negative regulator of odontoblast differenation