title: Adenosine triphosphate-induced rabbit corneal endothelial cell proliferation in vitro via the P2Y2-PI3K/Akt signaling axis.
abstract: OBJECTIVE
  To investigate the effect of the ATP-P2Y2-PI3K/Akt signaling axis on promoting rabbit corneal endothelial cell (RCEC) proliferation in vitro.
  
  
  METHODS
  Five concentrations of adenosine triphosphate (ATP; 1, 10, 25, 50 and 100 μM) were added to RCECs, and the cell proliferation was detected using Cell Counting Kit-8 (CCK8) and Ki67 immunohistochemical staining. Other P2Y2 receptor agonists and antagonists were added to the cells, and the proliferation effect was evaluated using CCK8 to determine the involvement of the P2Y2 receptor. Changes in the expression of phosphorylated Akt in RCECs treated with different concentrations of extracellular ATP and the duration of extracellular ATP on Akt phosphorylation were investigated using Western blotting. The pharmacological profiles with or without the PI3K/Akt pathway inhibitors were also determined using Western blotting.
  
  
  RESULTS
  We found that 10 μM ATP strongly promoted RCEC proliferation in vitro. Additionally, 25 μM ATP had a proliferation effect, whereas other concentrations (1, 50 and 100 μM) had no effect compared with the control group. Selective P2Y2 receptor agonists (UTP, ATPγS and Ap4A) showed the same promotion effect, while P2Y2 antagonists and PI3K/Akt inhibitors inhibited the effect of ATP. Moreover, phosphorylated Akt could be induced by the addition of extracellular ATP at all five concentrations and lasted for 1 h. This phosphorylation was prevented by PI3K/Akt inhibitors and a P2Y2 antagonist.
  
  
  CONCLUSIONS
  These findings showed that 10 μM ATP markedly promoted RCEC proliferation via the P2Y2-PI3K/Akt signaling axis.
authors.affiliation: Department of Ophthalmology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China.
authors.forename: Junzhao
authors.initials: J
authors.lastname: Chen
authors.forename: Chunyi
authors.initials: C
authors.lastname: Shao
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Lu
authors.forename: Chenxi
authors.initials: C
authors.lastname: Yan
authors.forename: Qinke
authors.initials: Q
authors.lastname: Yao
authors.forename: Mengyu
authors.initials: M
authors.lastname: Zhu
authors.forename: Ping
authors.initials: P
authors.lastname: Chen
authors.forename: Ping
authors.initials: P
authors.lastname: Gu
authors.forename: Yao
authors.initials: Y
authors.lastname: Fu
authors.forename: Xianqun
authors.initials: X
authors.lastname: Fan
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D003315:Cornea / Q000166:cytology*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D011817:Rabbits; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D000255:Adenosine Triphosphate; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt