Objective
Dental follicle cell lines with a senescence phenotype have a poor differentiation potential into biomineralizing cells. Previous studies have shown that protein kinase C (PKC) and protein kinase B (AKT) regulate the differentiation of DFCs. This study investigates the extent to which regulation of PKC and AKT can improve the differentiation of dental follicle cells with poor osteogenic potential.
Design
Human senescence dental follicle cells with poor osteogenic differentiation potential were osteogenic differentiated with cell culture media containing dexamethasone or bone morphogenetic protein (BMP) 2 as an inducer. GÖ6976 was used as a PKC inhibitor, and MK-2206 as an AKT inhibitor. The AKT activator SC-79 was also used. Western blot analyses were performed with specific antibodies for the active form of AKT, phosphorylated substrate of PKC and collagen 1. Osteogenic differentiation was quantitatively determined by measuring alkaline phosphatase (ALP) activity and biomineralization using alizarin staining. The gene expression of sclerostin (SOST) and PTHLH was quantitatively determined using real-time RT-PCRs.
Results
The results showed that both the inhibitor MK-2206 inhibits AKT and the activator SC-79 can activate AKT in DFCs. Only inhibition of AKT slightly but significantly enhanced osteogenic differentiation. While inhibition of PKC activity apparently only occurred from day 14 of differentiation using the inhibitor GÖ6976, PKC inhibition promoted osteogenic differentiation and inhibits the expression of SOST and Parathyroid hormone-related protein (PTHLH).
Conclusion
Our results suggest that the addition of GÖ6976 is an efficient method to induce biomineralization in senescent DFCs.