Adenosine Monophosphate-activated Protein Kinase/Mammalian Target of Rapamycin-dependent Autophagy Protects Human Dental Pulp Cells against Hypoxia

Introduction: Human dental pulp cells (HDPCs) are recalcitrant to hypoxic stress. We investigated whether hypoxia-induced autophagy of HDPCs offered these cells a survival advantage and the underlying mechanism of this resistance. Methods: The viability and apoptosis of HDPCs were examined after exposure to hypoxia by Vi-CELL cell viability analyzer and flow cytometry. Autophagy was assessed by using inimunofluorescence, acridine orange staining, real-time polynnerase chain reaction, and Western blotting. Either 3-methyladenine or expression vectors encoding dominant negative ATG5 were used to inhibit autophagy. Rapamycin was used as an autophagic inducer. To explore the mechanisms of autophagy, adenosine monophosphate activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway and hypoxia-inducible transcription factor-1 were suppressed by chemical inhibitors Compound C and YC-1, respectively. Results: The exposure of HDPCs to hypoxia had no effect on viability and resulted in increasing acidic vesicular organellepositive cells, autophagosonne formation, and upregulation of autophagy genes. Inhibition of autophagy with 3- methyladenine or expression vectors encoding dominant negative ATG5 abrogated the protective effects of HDPCs. The phosphorylation of AMPK was up-regulated; whereas the phosphorylation of mTOR was down-regulated in hypoxia-treated HDPCs, which were both attenuated by Compound C. Furthermore, treatment with Compound C rather than YC-1 reduced the autophagy. Conclusions: Our results suggested that autophagy of HDPCs might be cytoprotective against hypoxic stress via the AMPK/mTOR signaling pathway.