title: Hyperosmotic stress-induced corneal epithelial cell death through activation of Polo-like kinase 3 and c-Jun.
abstract: OBJECTIVE
  Hyperosmotic stress causes cell shrinkage, perturbs cell function, and damages DNA, resulting in cell cycle arrest and apoptosis. In the present study, the authors explore the mechanism involving hyperosmotic stress-induced activation of c-Jun/AP-1 through a novel Plk3 pathway in human corneal epithelial cells.
  
  
  METHODS
  Human primary corneal epithelial cells and cell line were cultured in a serum-free keratinocyte medium and DMEM/F12 medium containing 10% FBS in a 37°C incubator supplied with 5% CO(2), respectively. Western blot analysis was used to determine protein expression and phosphorylation levels. Protein kinase activities were measured by immunocomplex kinase assay. Cell viability and apoptosis were determined by MTT assay and caspase-3 (DEVDase) activity.
  
  
  RESULTS
  It was found that hyperosmotic stress-induced increases in the phosphorylation of c-Jun, resulting in apoptosis through the activation of Plk3 in human corneal epithelial cells. Plk3 was activated by extracellular hyperosmotic stress to directly phosphorylate c-Jun in the serine 63 and 73 residues. Hyperosmotic stress-induced c-Jun phosphorylation was enhanced by overexpression of constitutively positive Plk3 mutants and suppressed by the knockdown of Plk3 mRNA with Plk3-specific siRNA. Further studies indicated that the phosphorylation of c-Jun by Plk3 was responsible for hyperosmotic stress-induced apoptosis, which was independent from activation of the JNK signaling pathway in human corneal epithelial cells.
  
  
  CONCLUSIONS
  These results, for the first time, provide a novel and alternative signaling mechanism that involves hyperosmotic stress-induced activation of the Plk3 pathway in addition to JNK/p38 MAPK pathways to regulate the c-Jun/AP-1 transcriptional complex and human corneal epithelial cell fate.
authors.affiliation: Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Torrance, California 90502, USA.
authors.forename: Ling
authors.initials: L
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Dai
authors.forename: Luo
authors.initials: L
authors.lastname: Lu
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D019573:Epithelium, Corneal / Q000201:enzymology* / Q000473:pathology*; D006801:Humans; D006982:Hypertonic Solutions / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D009997:Osmotic Pressure; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D014162:Transfection; D025521:Tumor Suppressor Proteins; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D000097868:Polo-like Kinases
chemical_list: D053148:Caspase 3; D006982:Hypertonic Solutions; D048031:JNK Mitogen-Activated Protein Kinases; D017346:Protein Serine-Threonine Kinases; D016755:Proto-Oncogene Proteins c-jun; D013778:Tetrazolium Salts; D013844:Thiazoles; D025521:Tumor Suppressor Proteins; D048051:p38 Mitogen-Activated Protein Kinases; C486256:PLK3 protein, human; D000097868:Polo-like Kinases; C022616:thiazolyl blue