Role Of GATA Binding Protein 4 (GATA4) In The Regulation Of Tooth Development Via GNAI3

Objective: Mouse model in vivo and stem cells from apical papilla?SCAPs?in vitro were used to examine the effect of GATA4 on tooth root development.Methods: Wnt1-Cre;GATA4^fl/fl mouse model with conditional knockout of GATA4 in the NCCs-derived dental papilla mesenchymal cells was used in this study.Micro-CT and histological analyses such as Hematoxylin and eosin?HE?staining was conducted to observe tooth root formation.And the expression of GATA4 as well as odonto/osteogenic markers?RUNX2 ? OSX ? OPN ? OCN ? BMP4?,proliferation markers?PCNA?during the development of tooth were detected by immunohistochemistry.Flow cytometric analysis was conducted to identify stem cells from apical papilla.Then cells were transfected with sh GATA4 and sh CTRL respectively,and the expression of GATA4 as well as odonto/osteogenic markers was detected by western blotting.The odonto/osteogenic differentiation capacity of stem cells from apical papilla transfected was observed by ALP staining,alizarin red staining,western blotting and Real-time q PCR.i TRAQ analysis of NCCs after GATA4 knock-down revealed a downregulation of GNAI3 in the sh GATA4 group.We assessed the expression of GNAI3 both in vivo and in vitro.Dual-luciferase and Ch IP assay were carried out to confirm the direct binding of GATA4 to the GNAI3 promoter,both in vitro and in vivo.Then cells were transfected with sh GNAI3 and sh CTRL respectively,and the expression of GNAI3 as well as odonto/osteogenic markers was detected by western blotting.The odonto/osteogenic differentiation capacity of SCAPs transfected was observed by ALP staining,alizarin red staining.Results:1.Micro-CT and histological analyses showed dental papilla mesenchymal cells-specific inactivation of GATA4 leads to short root deformity in both maxillary and mandibular molars.The expression of odonto/osteogenic markers?RUNX2?OSX?OPN?OCN?BMP4?during the development of tooth was reduced in the mutant mice.Absence of GATA4 in dental papilla mesenchymal cells retards the proliferation but has no effect on apoptosis.2.GATA4 knock-down decreased the ability of migration and proliferation compared with control groups.The odonto/osteogenic differentiation ability of SCAPs was decreased in the sh GATA4 group.In the pc DNA-GATA4 group with over-expression of GATA4 in SCAPs,the ALP activity and calcium nodes were markedly increased as observed on ALP assay and ARS staining.3.iTRAQ analysis of NCCs after GATA4 knock-down revealed a downregulation of GNAI3 in the sh GATA4 group.The expression level of GNAI3 in the SCAPs was found markedly decreased in the sh GATA4 group.Dual-luciferase and Ch IP assay confirmed the direct binding of GATA4 to the GNAI3 promoter.In the sh GNAI3 group,the ALP-positive area and the density of calcium nodes decreased significantly.Conclusion: These results suggest that GATA4 modulates odonto/osteogenic differentiation of SCAPs and that this effect is mediated via up-regulation of GNAI3 function.