Inactivation of PI3K/Akt promotes the odontoblastic differentiation and suppresses the stemness with autophagic flux in dental pulp cells

Abstract

Background/purpose Autophagy is involved in controlling differentiation of various cell types. The present study aimed to investigate the mechanism related to autophagy in regulating odontogenic differentiation of dental pulp cells. Materials and methods Human dental pulp cells (HDPCs) were cultured in differentiation inductive medium (DM) and odontoblastic differentiation and mineralization were evaluated by alkaline phosphatase (ALP) staining and Alizarin red S staining, respectively. Tooth cavity preparation was made on the mesial surface of lower first molars in rat. The expression of autophagy-related signal molecules was detected using Western blot analysis and Immunohistochemistry. Results HDPCs cultured in DM showed increased autophagic flux and declined phosphorylation of phosphoinositide 3-kinases (PI3K), protein kinase B (Akt), and mTOR. Dentin matrix protein-1 (DMP-1) and dentin sialoprotein (DSP), markers of odontoblastic differentiation, were upregulated and autophagic activation showing increased LC3-II and decreased p62 levels was observed during odontogenic differentiation of HDPCs. However, PI3K blocker 3-methyladenine (3MA), lentiviral shLC3 and Akt activator SC79 attenuated the expression of LC3II as well as DMP-1, ALP activity and mineralization enhanced in HDPCs under DM condition. In addition, 3MA, shLC3 and SC79 recovered the expression of pluripotency factor CD146, Oct4 and Nanog downregulated in DM condition. In rat tooth cavity preparation model, the expression of LC3B and DMP-1 was elevated near odontoblast-dentin layer during reparative dentin formation, whereas 3MA significantly reduced the expression of LC3B and DMP-1. Conclusion These findings indicated autophagy promotes the odontogenic differentiation of dental pulp cells modulating stemness via PI3K/Akt inactivation and the repair of pulp.

An overview of autophagy in the differentiation of dental stem cells

“…During odontoblastic differentiation, the expression of dentin matrix protein-1 (DMP-1) and dentin sialoprotein (DSP) is steadily increased, paralleled by autophagosome and autophagolysosome formation. Furthermore, there is a notable shift from cytosolic LC3-I to the autophagosome marker LC3-II, albeit with diminished levels of the selective autophagy cargo receptor p62 [105]. A similar trend was observed in the osteoblast differentiation of DPSCs.…”

“…Additionally, autophagy interfaces with signaling pathways to amplify cell differentiation and mineralization. In this respect, research has shown that autophagy fosters odontoblastic differentiation via the phosphatidylinositol 3kinase (PI3K)/Akt signaling while simultaneously attenuating stemness in DPCs under odontogenic induction conditions [105]. AMPK activation has been reported to promote osteogenic differentiation of DPSCs through different approaches: early mTOR inhibition-mediated autophagy and later activation of the Akt/mTOR signaling axis [114] suggesting that the differentiation ability of DSCs is closely associated with the regulatory effects of autophagy.…”