cAMP signaling up-regulates odonto/osteogenic differentiation of apical papilla-derived stem cells

Abstract

Objectives: This study aims to investigate the underlining interplay of cyclic adenosine monophosphate (cAMP) and transforming growth factor-β1 (TGF-β1) on the odonto/osteogenic differentiation of apical papilla-derived stem cells (SCAPs). Methods: SCAPs were isolated from human immature third molars. Forskolin was used to activate and H89 was used to inhibit cAMP signaling in SCAPs. The protein levels of TGF-β1 signaling downstream molecule (Smad3) and phosphorylation of Smad3 (p-Smad3) were assayed by western blot analysis. Smad3 inhibitor (SIS3) was used to inhibit the effect of TGF-β1 signaling on the odonto/osteogenic differentiation in SCAPs. The amount of calcium mineral deposition was detected by Alizarin red staining. Additionally, quantitative real-time polymerase chain reaction (RT-PCR) were taken to evaluate cAMP signaling on TGF-β1-mediated odonto/osteogenic differentiation potential of SCAPs. Results: Protein expression of p-Smad3 decreased in SCAPs stimulated by the Forskolin (Figure 1A); while H89 increased the level of p-Smad3 (Figure 1B). Alizarin red staining showed that TGF-β1 antagonized the up-regulated effect of Forskolin. Blocking TGF-β1 signaling with SIS3 enhanced the mineralized nodule formation of SCAPs (Figure 2). RT-PCR found that in the presence of TGF-β1, SCAPs with Forskolin displayed markedly down-regulation in the expression of odonto/osteogenic marker compared to Forskolin treated only (Forskolin with TGF-β1:0.552±0.243, Forskolin: 1.439±0.105; p<0.004). Besides, the expression of odonto/osteogenic marker of Forskolin with SIS3 and Forskolin were 1.874±0.161 and 1.439±0.105, respectively (p=0.017). Conclusions: This study demonstrated cAMP signaling induced the odonto/osteogenic differentiation of SCAPs via inhibition of TGF-β1-Smad3 signaling. The cAMP signaling pathway could be a strategy target for dentine regeneration in clinical dentistry.