ERK/p38Mitogen-activated Protein Kinase Is Involved In MTA-induced Differentiation Of Human Apical Papilla Cells

AimMAPKs (mitogen-activated protein kinases) are serine/threonine-specific protein kinases that respond to extracellular stimuli (mitogens, osmotic stress, heat-shock and proinflammatory cytokines) and regulate various cellular activities, such as gene expression, mitosis, differentiation, proliferation, and cell survival/apoptosis. Human apical papilla cells (HAPCs) have the capacity of multiple differentiation, they are used for tissue engineering and biological root construction. Mineral trioxide aggregate(MTA), as a new bioactive material, has been widely used in the clinical apexification and achieved good results. However the cellular and molecular mechanisms of MTA induced differentiation of HAPCs remain unclear. The purpose of this study was to investigate the effects of MAPKs signal pathway on the differentiation of HAPCs induced by MTA.MethodsPart I Culture and differentiation of human apical papilla cellsHuman apical papilla cells were cultured by using enzyme digestion, cells were in good condition after passaged. HAPCs were cultured in osteogenic induction medium for21days. The alizarin red staining showed that calcium deposition were increased after induction compared with the control group.Part II The effects of MTA on the proliferation and differentiation of HAPCsCCK-8assay was performed to evaluate the proliferation activity of HAPCs induced by MTA of different concentrations. The results showed that MTA with10mg/ml and20mg/ml inhibited the cell proliferation significantly (p<0.05). HAPCs were cultured in a-MEM medium supplemented with MTA of different concentrations (0、0.02mg/ml、0.2mg/ml、2mg/ml).Then alkaline phosphatase (ALP) activity assay and the alizarin red staining assay were performed to detect the early and later osteogenic differentiation. The expression of odonto/osteoblastic genes such as dentin sialophosphoprotein (DSPP), runt-related transcription factor2(Runx2), bone sialoprotein (BSP), osteocalcin (OCN) and Runx2protein were assessed by real-time PCR and Western blot respectively. The results showed that the ALP activity, calcium deposition, the expression of DSPP、Runx2、BSP and OCN mRNA, the expression of Runx2protein were increased after induction by MTA compared with the control group.In summary, MTA can promote odonto/osteoblastic differentiation of HAPCs. MTA at0.2mg/ml as the optimal concentration promoted odonto/osteoblastic differentiation of HAPCs, thus MTA of0.2mg/ml was used in the later experiments.Part Ⅲ The effects of MAPKs signaling pathway on MTA induced odonto/osteoblastic differentiation of HAPCsThe phosphorylation of ERK, p38MAPK and JNK were detected by Western blot in cells treated by MTA. The results showed that MTA can activate the phosphorylation of ERK and p38MAPK.The specific ERK inhibitor U0126and the specific p38MAPK inhibitor SB203580were used to inhibit the activity of ERK and p38MAPK during the induction of MTA on HAPCs. Then the alkaline phosphatase (ALP) activity, the expression of DSPP、Runx2、BSP and OCN mRNA, the expression of Runx2protein were detected on odonto/osteoblastic differentiation of HAPCs induced by MTA combined with treatment of p38MAPK inhibitor and ERK inhibitor. The results demonstrated that the ALP activity, the expression of DSPP、Runx2、BSP and OCN mRNA, the expression of Runx2protein were decreased after inhibition of ERK and p38MAPK. In summary, inhibition of ERK and p38MAPK can reduce odonto/osteoblastic differentiation of HAPCs induced by MTA.ConclusionMTA promote odonto/osteoblastic differentiation of HAPCs. MTA can activate the phosphorylation of ERK and p38MAPK. Inhibition of ERK and p38MAPK reduce odonto/osteoblastic differentiation of HAPCs induced by MTA.