Guanine Nucleotide Binding Protein 3 Promotes The Dental/osteogenic Differentiation Of Apical Dental Papilla Stem Cells Through JNK And ERK Signaling Pathways

In daily clinical practice,there will need some prosthodontic treatment for extraction cases and hypodontia cases,however,even the best prosthodontic tooth is clearly not comparable to natural tooth.The stem cells from apical papilla(SCAPs)are the most important odontoblast precursor cells in the regeneration of the roots and its odonto/osteogenic differentiation is a key process in tooth root formation and development.However,the molecular mechanisms underlying this process remain largely unknown.A previous study in our group showed that GATA-binding protein 4(GATA4)regulated root development through guanine nucleotide binding protein alpha-inhibiting activity polypeptide 3(GNAI3),but the role and mechanism of GNAI3 remained unknown.In this study,we further explored the role and mechanism of GNAI3 in root developmentThis research was divided into three sectionsThe first section:the expression of GNAI3 in mouse root tip,isolation,culture and identification of human SCAPs.Mice were sacrificed 14 days after birth,immunohistochemistry was applied.GNAI3 was detected in Hertwig’s epithelial root sheath(HERS)and the mesenchyme around HERS in mouse molars,indicating that GNAI3 was involved in root development.SCAPs were isolated and cultured Identification of SCAPs was confirmed by flow cytometry by expressing CD90 FITC and CD44 PE,but not CD45 APC and CD 14 PE-CY7,which was accorded with the identification characteristics of SCAPs.Reliable SCAPs were provided as experimental cells for subsequent experimentsThe second section:the expression of GNAI3 during the mineralization of human SCAPs,the effect of GNAI3 knockdown on the proliferation and odonto/osteogenic differentiation of SCAPs.The mineralization of human SCAPs was induced at 0,3,7 and 14 days.The expression of GNAI3 protein was dertermined by Western Blotting which was up-regulated 3 days after mineralization induction,and reached the peak at 7 days(P<0.01),indicating that GNAI3 may play an important role in the process of SCAPs mineralization.Lentiviral particle GFP LV-3[pGLVHl/green fluorescent protein(GFP)+Puro]was applied to continuously knock down GNAI3 expression in SCAPs,knockdown efficiency was confirmed by Western Blotting(P<0.01).We further found that low expression of GNAI3 inhibited the proliferation of SCAPs(P<0.01)by CCK8 assay,cell cycle(P<0.05)by flow cytometry analysis and 12-hour and 24-hour migration(P<0.01)by cell scratch assay.There was no significant difference in cell necrosis and apoptosis Mineralization of SCAPs was detected by alizarin phosphatase(ALP)staining(P<0.01)and alizarin red S(ARS)staining(P<0.01).The mRNA level of odonto/osteogenic specific markers were detected by qPCR,including DMP1(P<0.01),DSPP(P<0.05),OCN(P<0.01),OPN(P<0.01),OSX(P<0.05),and RUNX2(P<0.01).Odonto/osteogenic specific markers were detected by Western Blotting at protein level,including DSPP(P<0.01),RUNX2(P<0.01),OSX(P<0.01),OPN(P<0.01),OCN(P<0.05)and BMP4(P<0.05).The above results indicated low expression of GNAI3 inhibited odonto/osteogenic differentiation of SCAPsThe third section:GNAI3 promote SCAPs mineralization through the mitogen-activated protein kinase(MAPK)pathway.Human SCAPs mineralization was induced for 7 days,Western Blotting showed that low expression of GNAI3 significantly decreased protein expression of p-JNK(P<0.01)and p-ERK(P<0.05),while p-p38 protein expression was not significantly affected,indicating that JNK and ERK,but not p38,were downstream targets of GNAI3.Human SCAPs mineralization was induced at 0,3,7 and 14 days,Western Blotting showed that the phosphorylated JNK protein expression was up-regulated 3 days after mineralization induction,and reached the peak at 7 days(P<0.01),which was consistent with the expression pattern of GNAI3 during SCAPs mineralization,suggesting that GNAI3 may regulate the odonto/osteogenic differentiation of SCAPs through the MAPK-JNK pathway.To further explore the role of JNK and ERK signaling pathways,the JNK inhibitor SP600125 and the ERK inhibitor U0126 were added to SCAPs separately.It was showed by CCK8 test that there was a significant inhibition of proliferation of SCAPs within the beginning 6 days during SCAPs mineralization(SP600125 P<0.01;U0126 P<0.01).Flow cytometry analysis showed cell cycle changes,G1 arrest(SP600125 P<0.01;U0126 P<0.01),G2 phase cells decreased(SP600125 P<0.05;U0126 P<0.05)and S phase cells decreased(SP600125 P<0.05;U0126 P<0.01).Cell scratch test showed 12-hour migration ability inhibition(SP600125 P<0.01;U0126 P<0.01)and 24-hour migration ability inhibition(SP600125 P<0.01;U0126 P<0.01).Significantly,SCAPs treated with the JNK inhibitor SP600125 and the ERK inhibitor U0126 showed highly similar inhibition of proliferation,cell cycle and migration with SCAPs with low expression of GNAI3Summary:These results suggested that GNAI3 may promote odonto/osteogenic differentiation of SCAPs through JNK and ERK signaling pathways,which will provide favorable new ideas for the biological regeneration of dentin and root.