title: Adenosine monophosphate-activated protein kinase/mammalian target of rapamycin-dependent autophagy protects human dental pulp cells against hypoxia.
abstract: BACKGROUND
  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 immunofluorescence, acridine orange staining, real-time polymerase 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 organelle-positive cells, autophagosome formation, and up-regulation 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.
authors.affiliation: State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine of Ministry of Education (KLOBM), School and Hospital of Stomatology, Wuhan University, Wuhan, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhou
authors.forename: Huan
authors.initials: H
authors.lastname: Liu
authors.forename: Qin
authors.initials: Q
authors.lastname: Sun
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.forename: Heng
authors.initials: H
authors.lastname: Lin
authors.forename: Guohua
authors.initials: G
authors.lastname: Yuan
authors.forename: Lu
authors.initials: L
authors.lastname: Zhang
authors.forename: Zhi
authors.initials: Z
authors.lastname: Chen
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000032:analysis; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D000071192:Autophagy-Related Protein 12; D000071187:Autophagy-Related Protein 5; D000071186:Beclin-1; D018929:Cell Culture Techniques; D015687:Cell Hypoxia / Q000502:physiology*; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D003782:Dental Pulp / Q000166:cytology / Q000201:enzymology*; D005822:Genetic Vectors / Q000235:genetics; D006162:Guanylate Cyclase / Q000494:pharmacology; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000032:analysis / Q000037:antagonists & inhibitors; D007191:Indazoles / Q000494:pharmacology; D008565:Membrane Proteins / Q000032:analysis; D008869:Microtubule-Associated Proteins / Q000032:analysis; D010957:Plasmids / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000032:analysis; D025841:Small Ubiquitin-Related Modifier Proteins / Q000032:analysis; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D025521:Tumor Suppressor Proteins / Q000032:analysis
chemical_list: C466624:ATG12 protein, human; C502778:ATG5 protein, human; D051017:Apoptosis Regulatory Proteins; D000071192:Autophagy-Related Protein 12; D000071187:Autophagy-Related Protein 5; C491997:BECN1 protein, human; C090121:BNIP3 protein, human; D000071186:Beclin-1; D051793:Hypoxia-Inducible Factor 1; D007191:Indazoles; C477012:MAP1LC3A protein, human; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D011518:Proto-Oncogene Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D025521:Tumor Suppressor Proteins; C090937:3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole; C025946:3-methyladenine; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D006162:Guanylate Cyclase; D000225:Adenine