Elevation of cAMP upregulates Cav1.2 expression and promotes odontogenic differentiation of dental pulp stem cells

Abstract

Abstract Human dental pulp stem cells (hDPSCs) are promising cellular sources in dental tissue engineering. Although studies have reported that cyclic adenosine monophosphate (cAMP) and Cav1.2 played important roles in the differentiation of stem cells, the relation between cAMP and Cav1.2 in the odontogenic differentiation of hDPSCs still remained unclear. This study hypothesized that elevating cAMP contributed to the odontogenic differentiation of hDPSCs by regulating Cav1.2 expression. Here, Forskolin was used to activate cAMP and Nimodipine was used to inhibit Cav1.2. This study firstly screened out the safety concentrations of Forskolin and Nimodipine by CCK-8 proliferation assay. Following, Forskolin was used to elevate cAMP during odontogeinic differentiation of hDPSCs. qPCR was performed to compare the odontogenic differentiation-related gene expression between groups. The odontogenic mineralization was evaluated by Alizarin Red Staining. Subsequently, in order to investigate the relation between cAMP and Cav1.2, hDPSCs was treated with Nimodipine in one hour before Forskolin adding. Finally, Alizarin Red Staining and qPCR were performed to observe mineralized deposit formation and the Cav1.2 together with odongenic related gene expression in each group. Results showed that Forskolin promoted the formation of mineralized nodules in hDPSCs. The expression of odontogenic related genes (ALP, RUNX2, OSX, BMP2, DSPP) and Cav1.2 were also upregulated after 14 days of odontogenic induction. Nimodipine inhibited the odontogenic differentiation and attenuated the promoting effect of Forskolin during the odontogenic differentiation of hDPSCs. The above results suggested that the elevation of cAMP could upregulate Cav1.2 expression and significantly promote odongenic differentiation of hDPSCs.