title: Hyperosmolarity-induced cornification of human corneal epithelial cells is regulated by JNK MAPK.
abstract: OBJECTIVE
  To evaluate the effects of hyperosmolar stress on expression of cornified envelope (CE) precursors and transglutaminases (TGs) by primary cultured human corneal epithelial (PCHCE) cells and the regulatory effects of JNK MAPK on this process.
  
  
  METHODS
  Expression of CE precursors and TGs were evaluated in PCHCE cells exposed to media of increasing osmolarity (350-450 mOsM) for 24, 48, and 72 hours. JNK1 and -2 MAPKs were inhibited by addition of short interfering (si)RNA. Relative levels of mRNA transcripts and proteins were evaluated. TG activity, cell viability, and apoptosis were detected in PCHCE cells, with or without siRNA-JNKs.
  
  
  RESULTS
  Exposure of PCHCE cells to hyperosmolar medium increased TG activity at 3 hours, levels of the CE precursors SPRR1b and -2a and membrane-associated TG1 mRNA at 6 hours, and tissue-type TG2 mRNA at 24 hours. Osmotic stress decreased corneal epithelial cell viability, which was due in part to stimulation of apoptosis and cornification death. Inhibiting JNK2 production by siRNA in osmotically stressed PCHCE cells prevented the stimulation of SPRR and membrane-associated TG1 production and TG activity, and improved cell viability, whereas inhibition of JNK1 prevented early apoptosis.
  
  
  CONCLUSIONS
  Osmotic stress promotes production of certain CE proteins and cross-linking membrane-associated TG1 and decreases cell viability via JNK MAPK-mediated pathways. Strategies that inhibit JNK production downregulate the cornification response of PCHCE cells to osmotic stress. These findings have potential therapeutic implications for preventing cornification of the corneal epithelium in response to the hyperosmolar tear film in dry eye disease.
authors.affiliation: Department of Ophthalmology, The Ocular Surface Center, Cullen Eye Institute, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Chen
authors.forename: Louis
authors.initials: L
authors.lastname: Tong
authors.forename: Zhijie
authors.initials: Z
authors.lastname: Li
authors.forename: Kyung-Chul
authors.initials: KC
authors.lastname: Yoon
authors.forename: Hong
authors.initials: H
authors.lastname: Qi
authors.forename: William
authors.initials: W
authors.lastname: Farley
authors.forename: De-Quan
authors.initials: DQ
authors.lastname: Li
authors.forename: Stephen C
authors.initials: SC
authors.lastname: Pflugfelder
mesh_terms: D000328:Adult; D000368:Aged; D015153:Blotting, Western; D002470:Cell Survival; D002478:Cells, Cultured; D055233:Cornified Envelope Proline-Rich Proteins; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D018613:Microscopy, Confocal; D008875:Middle Aged; D048055:Mitogen-Activated Protein Kinase 8; D048056:Mitogen-Activated Protein Kinase 9; D009994:Osmolar Concentration; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012462:Saline Solution, Hypertonic / Q000633:toxicity*; D014162:Transfection; D011503:Transglutaminases / Q000235:genetics / Q000378:metabolism
chemical_list: D055233:Cornified Envelope Proline-Rich Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C101744:SPRR2A protein, human; D012462:Saline Solution, Hypertonic; C524533:SPRR1B protein, human; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; C105804:transglutaminase 1; D048056:Mitogen-Activated Protein Kinase 9; D048031:JNK Mitogen-Activated Protein Kinases; D048055:Mitogen-Activated Protein Kinase 8; D019204:GTP-Binding Proteins