The Effect Of Differentiation Related Transcription Factor Hey1 In The Differentiation Of Odontoblast-like Cell

The effect of differentiation related transcription factor Hey1 in the differentiation of odontoblast-like cellOdontoblasts are highly differentiated cells and aligned in a single layer at the periphery of the dental pulp. The main function of the odontoblasts is to produce dentin that accounts for the largest part of hard tissues of teeth. Severe damages like deep caries and direct injury can destroy the primary odontoblasts, and stimulate the differentiation of progenitor cells in the mature pulp into a kind of “odontoblast-like cells” which share the similar characteristics with the primary odontoblasts. The differentiation of odontoblast-like cell is mediated by many kinds of signaling pathways, including TGF/smads, FGF, Wnt and Notch signaling pathway. Among them, Notch signaling pathway was reported to be effective in cellular differentiation. As a downstream molecule of Notch signaling pathway, Hey1 may be a functional factor in the differentiation of odontoblast-like cell.Hey1(also known as Hesr1, HRT1, and HERP2) is a transcription factor of the HERP(hairy/Enhancer of split-related protein) family whose members function as transcriptional repressors. Hey1 was identified to be a direct target of RBP-Jk and act as a nuclear effector of the Notch signaling pathway and confirmed to be a critical transcription factor in cellular differentiation. Our preliminary study found that Hey1 was expressed in dental pulp tissues. However, the effects and mechanisms of Hey1 in the differentiation of odontoblast-like cell is still need to be revealed.In the present study, we observed the expression of Hey1 in an odontoblast-lineage cell line(OLC) before and after cultured by two kinds of odontogenic differentiation inducer: medium containing β-glycerophosphate(GP), ascorbic acid(AA) and dexamethasone(DEX) or medium containing rh-BMP2. Then the the plamid encoding full-length sequence of Hey1 or Hey1 silencing shRNA were constructed and transfected into OLCs respectively to obtain the Hey1 overexpressing or knockdown cell lines(OLC/Hey1 OP and OLC/hey1 KD). By comparing the differentiation and mineralization capabilities of these cell lines, we concluded that Hey1 may play an important role in the differentiation of odontoblast-like cells. These findings will facilitate the application of Hey1 in pulp or dentin regeneration and vital pulp conservation treatment. Main results of the present study are as the following: 1. The effects of Hey1 overexpression on the differentiation of odontoblast-like cell.The eukaryotic expression plasmid encoding full-length of Hey1, pEF-DEST51-Hey1, was transfected into OLCs. RT-PCR and Western-blot analyses confirmed that the stable Hey1-overexpression cell line(OLC/Hey1 OP) was established. After cultured in two kinds of odontogenic differentiation inducer, the mRNA levels of mineralization-related genes, such as Dspp, Alp were up-regulated in OLC/Hey1 OP cells compared with normal OLCs, as well as DSP protein analyzed by western-blot and immunofluorescence. ALP activities of OLC/Hey1 OP cells were higher than normal OLCs. Alizarin red S staining showed that OLC/Hey1 OP cells formed more mineralized nodules as well. 2. The effects of Hey1 knockdown on the differentiation of odontoblast-like cell.We also established a stable Hey1- knockdown cell line(OLC/hey1 KD) by the RNAi method. OLC/hey1 KD showed lower expression of Hey1 analyzed by real-time PCR. In contrast with Hey1 overexpression, we found that the absence of Hey1 down-regulated the mRNA levels of mineralization-related genes when cultured in these two kinds of odontogenic differentiation inducer. OLC/hey1 KD cells also exhibited lower ALP activities and the cells formed fewer mineralized nodules than OLCs. 3. The effects of Notch signaling pathway inhibitor on the function of Hey1 regulating odontoblast-like cell differentiation.To further investigate whether Notch signaling pathway is responsible for the function of Hey1 regulating odontoblast-like cell differentiation, DAPT was adopted as a Notch signaling pathway inhibitor to survey the change of Hey1 and OLCs differentiation features while Notch signaling pathway was blocked. The results indicated that 1μmol/L DAPT significantly down-regulated the expression of Hey1 in OLCs. Surprisingly, the differentiation activity of odontoblast-like cell was enhanced by DAPT when OLCs were cultured by AA+GP+DEX inducer. But when OLCs were cultured by rh-BMP2 inducer, the differentiation activity was partly suppressed by DAPT and may be rescued by Hey1 overexpression. These findings suggested that Hey1 was not just regulated directly by Notch signaling pathway, and may be a factor in cross-talk of different signaling pathways.